Recombinant turkey herpesvirus vectors expressing avian pathogen antigens and their use

Abstract

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 rival vectors for protection against a variety of pathogens. The present invention also relates to methods for making and using such recombinant viral vectors.

Description

[Technical Field]

[0001] Cross-reference of related applications This application asserts the benefit under Section 119(e) of the U.S. Patent Act to U.S. Provisional Application No. 62 / 898651, filed September 11, 2019, which is incorporated herein by reference in its entirety.

[0002] This invention relates to recombinant viral vectors for insertion and expression of exogenous genes for use in safe immunization for protection against various pathogens. The invention also relates to a polyvalent composition or vaccine comprising one or more recombinant viral vectors for protection against various pathogens. Furthermore, this invention relates to a method for preparing and using such recombinant viral vectors. [Background technology]

[0003] Marek's disease is a highly contagious lymphoproliferative disorder and one of the most prevalent avian diseases, primarily affecting young chickens. Marek's disease is caused by the Marek's disease virus. Marek's disease virus (MDV) is a herpesvirus and a member of the Mardivirus genus, possessing three serotypes (species): MDV-1 (Gallid herpesvirus 2), MDV-2 (Gallid herpesvirus 3), and MDV-3 (Meleagrid herpesvirus 1, turkey herpesvirus (HVT)). MDV-1 is the most virulent of these three serotypes and causes widespread disease in unvaccinated poultry. Birds infected with MDV-1 exhibit neurological, visceral, and cutaneous clinical symptoms, 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. The incidence rate in affected birds is 10–50%, and the mortality rate can reach up to 100%. Marek's disease can affect birds of any age, but acute Marek's disease causes death in large numbers of young, unvaccinated birds between 4 and 8 weeks of age. Marek's disease spreads through direct or indirect exposure to chicken scales from infected chickens, and the virus is taken in by inhalation. MDV-2 and MDV-3 represent asymptomatic virus strains and have been used in the preparation of vaccines against the associated, more virulent MDV-1.

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

[0005] Recombinant vectorized vaccines have been developed to provide simultaneous immunity against multiple pathogens. These vaccines are constructed by removing several non-essential gene sections from 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 pathogen. The newly produced vector is then used to infect the host, which replicates and expresses the antigens of the highly virulent organism, thereby inducing an immune response. Recombinant vectorized vaccines combine the advantages of both live and dead vaccines. Recombinant vectors provide longer-lasting immunity, similar to live vaccines, while simultaneously causing a milder reaction after vaccination, like dead vaccines. In addition, both the vector and the inserted genes can provide immunity that protects birds from two or more diseases.

[0006] Marek's disease viruses are among the most effective vectors for multivalent vaccines to confer immunity against poultry diseases because these viruses induce lifelong protection with just one vaccination. In addition, these viruses are limited to avian hosts, so there is no risk of infection to other animals and people working on poultry farms. Of the Marek's disease viruses, turkey herpesvirus (HVT) has been used more widely, 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, was soon found to be protective against MDV, and was licensed as a vaccine in 1971. Turkey herpesvirus (HVT) has similar antigenic characteristics to Marek's disease virus (MDV-1) but is not pathogenic to chickens. In addition, because HVT is not susceptible to maternal antibodies against MDV or HVT, live HVT vaccines have been used to effectively vaccinate against MDV-1 in ovov or in the early pre-hatch stage. Furthermore, the HVT genome has been used as a vaccine vector to carry exogenous DNA sequences of other avian pathogens. [Overview of the project]

[0007] This invention provides recombinant viral vectors for insertion and expression of exogenous genes for use in safe immunization to protect birds from various pathogens. The invention also provides a polyvalent composition or vaccine comprising one or more recombinant HVT viral vectors for protection against various pathogens. In addition, the invention provides methods for preparing and using recombinant viral vectors alone or in combination with other vaccines or pharmaceutical compositions.

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

[0009] In one embodiment, the present invention provides a recombinant turkey herpesvirus (HVT) genome comprising one or more nucleotide sequences encoding one or more heterogeneous antigens or multiple antigens inserted into the intergenetic locus UL35 / UL36 in the unique long region of the HVT genome, and one or more nucleotide sequences encoding one or more heterogeneous 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 recombinant HVT in which one or more heterologous antigens provide protection against avian pathogens or multiple pathogens selected from the group consisting of infectious bursal disease virus (IBDV), Newcastle disease virus (NDV), infectious bronchitis virus (IBV), infectious laryngotracheitis virus (ILTV), avian anemia virus (CAV), and avian influenza virus (AIV).

[0011] In one or more embodiments, the present invention provides recombinant HVT in which one or more heterologous antigens are selected from the group consisting of the following: VP2, VP3, or VP4 protein of infectious bursal disease virus (IBDV); VP1 or VP2 protein of avian 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); gB, gC, gD, gE, gH, gI, or gL protein of infectious laryngotracheitis virus (ILTV); and 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 that one or more heterologous antigens are protective against IBDV. In one embodiment, the recombinant HVT of the present invention is provided that one or more heterologous antigens are the VP2 protein of IBDV. In one embodiment, the recombinant HVT of the present invention is provided that the VP2 protein sequence is encoded by a nucleotide sequence having at least 80% sequence identity with respect to a nucleotide sequence containing 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 containing either SEQ ID NO: 5 or SEQ ID NO: 10.

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

[0014] In one or more embodiments, the recombinant HVT of the present invention is provided that one or more heterologous antigens are protective against NDV and IBDV. In one or more embodiments, the recombinant HVT of the present invention is provided 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 F protein of NDV encoded by a nucleotide sequence having at least 80% sequence identity with respect to the nucleotide sequence containing SEQ ID NO. 3, and a VP2 protein of IBDV encoded by a nucleotide sequence having at least 80% sequence identity with respect to the nucleotide sequence containing SEQ ID NO. 5 or SEQ ID NO. 10.

[0016] In one or more embodiments, the recombinant HVT of the present invention is provided that the F protein of NDV, encoded by a nucleotide sequence including SEQ ID NO: 3, and the VP2 protein of IBDV, are encoded by a nucleotide sequence including 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 multiple cassettes comprising one or more nucleotide sequences or multiple sequences encoding one or more heterologous antigens or multiple antigens. In one embodiment, the recombinant HVT comprises a recombinant HVT genome comprising an expression cassette comprising a nucleotide sequence encoding a promoter operably ligated to one or more nucleotides encoding the antigen to be expressed. In one embodiment, the antigen to be expressed comprises the F protein of NDV. In one embodiment, the antigen to be expressed comprises the VP2 protein of IBDV. In one embodiment, the antigen to be expressed comprises 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 that one or more promoters are selected from the group consisting of the pre-initial cytomegalovirus human (hCMV) promoter, the guinea pig pre-initial CMV promoter, the mouse pre-initial CMV promoter, the Pec promoter, the β-chicken triactin promoter, the SV40 promoter, the pseudorabies virus promoter with glycoprotein X promoter, the herpes simplex virus-1 alpha-4 promoter, the Marek's disease virus promoter with glycoprotein gA, gC, gB, gE, or gI promoter, the infectious laryngotracheitis virus promoter with glycoprotein gB, gE, gI, gD promoter, and the bovine herpesvirus 1 / 1 VP8 promoter. In one embodiment, the recombinant HVT comprises the human CMV promoter. In one embodiment, the recombinant HVT comprises the mouse CMV promoter. In one embodiment, the recombinant HVT comprises the hCMV promoter and the 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 SV40 polyA sequence (SEQ ID NO: 12). In one embodiment, the polyA signal is the BGH polyA signal. In one embodiment, the polyA signal is the 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 further comprising a nucleotide sequence encoding a polyadenylation signal, wherein all parts of the VP2 expression cassette are inserted into the non-coding region of the HVT genome. In one embodiment, the CMV promoter comprises the 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 poly A 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 inserted into the HVT genome at the UL55 / gene3 site in that order.

[0021] In one aspect, the recombinant HVT of the present invention 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, and all parts of the NDV F cassette are inserted at non-coding positions within the HVT genome. In one embodiment, the CMV promoter comprises the mCMV (SEQ ID NO: 2) promoter. In one embodiment, the nucleotide sequence encoding the F protein of NDV 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 inserted in order 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, and all of the VP2 expression cassette is inserted at non-coding positions 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 the 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 the 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 inserted into the HVT genome in the UL35 / 36 non-coding region (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); and, in order, a nucleotide sequence encoding an mCMV promoter inserted into the HVT genome in the UL55 / gene3 non-coding region (SEQ ID NO: 2), a nucleotide sequence encoding an F protein from NDV (SEQ ID NO: 3), and a nucleotide sequence encoding an SV40 polyadenylation signal (SEQ ID NO: 12). In one embodiment, the recombinant HVT of the present invention comprises a promoter operably ligated to a nucleotide sequence encoding an infectious laryngotracheitis virus antigen, further comprising 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 infectious laryngotracheitis virus (ILTV) gB, gC, gD, gE, gH, gI, gL, or ILT antigens. 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, avian 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 ligated 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 avian 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, each comprising a nucleotide sequence encoding a polyadenylation signal, each comprising a nucleotide sequence encoding a promoter operably ligated to a nucleotide encoding an ILT antigen, 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 ligated 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 avian 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), 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 immunogenic compositions comprising recombinant HVT of the present invention, further comprising a pharmaceutically acceptable carrier, excipient, or adjuvant.

[0027] In one or more embodiments, the present invention provides a vaccine composition comprising recombinant HVT of the present invention, 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 the naturally attenuated MDV-1 strain Rispens (CVI-988) or three Gallid herpesvirus strains SB-1 virus. In one embodiment, the vaccine of the present invention is provided that the additional MDV comprises a recombinant genome. In one embodiment, the vaccine of the present invention is provided 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 use in vaccination of 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 use in protection of birds against clinical symptoms caused by one or more avian pathogens. In one or more embodiments, the vaccine of the present invention provides use in protection of 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 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), avian anemia virus (CAV), and avian influenza virus (AIV). In one embodiment, the vaccine of the present invention is provided that one or more avian pathogens include Newcastle disease virus. In one embodiment, the vaccine of the present invention is provided that one or more avian pathogens include infectious bursal disease virus (IBDV). In one embodiment, the vaccine of the present invention is provided that one or more avian pathogens include Newcastle disease virus and bursal disease virus.

[0030] In one or more embodiments, the vaccine of the present invention provides use in avian vaccination, wherein the vaccine is administered by at least one dose of the vaccine by spray administration, in ovo administration, subcutaneous administration, intramuscular administration, oral administration, nasal administration, or a combination thereof. In one embodiment, the vaccine of the present invention is provided that the vaccine is administered by in ovo administration. In one embodiment, the vaccine of the present invention is provided that the in ovo administration is performed on an embryonated egg between approximately 16 and 22 days after development. In one or more embodiments, the vaccine of the present invention is provided that the in ovo administration is performed on an embryonated egg around 18 days after development. In one embodiment, the vaccine of the present invention is provided that the administration of the vaccine includes in ovo administration followed by spray administration. In one embodiment, the vaccine of the present invention is provided that the administration of the vaccine includes spray administration.

[0031] In one embodiment, the present invention provides a method for vaccinating birds to treat or prevent Marek's disease and one or more avian diseases caused by one or more avian pathogens, comprising the step of administering an effective amount of the vaccine composition of the present invention. In one embodiment, the method of the present invention is provided that 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), avian anemia virus (CAV), and avian influenza virus (AIV). In one embodiment, the method of the present invention is provided that one or more avian pathogens include infectious bursal disease virus (IBDV). In one embodiment, the method of the present invention is provided that one or more avian pathogens include Newcastle disease virus (NDV). In one embodiment, the method of the present invention is provided that 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 animal avian to Marek's disease virus and one or more avian pathogens, comprising the step of administering an effective amount of the immunogenic composition or vaccine composition of the present invention to the bird. In one embodiment, the method of the present invention is provided that 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), avian anemia virus (CAV), and avian influenza virus (AIV). In one embodiment, the method of the present invention is provided that one or more avian pathogens include infectious bursal disease virus (IBDV). In one embodiment, the method of the present invention is provided that one or more avian pathogens include Newcastle disease virus (NDV). In one embodiment, the method of the present invention is provided that one or more avian pathogens include infectious bursal disease virus (IBDV) and Newcastle disease virus (NDV). In one or more embodiments, the method of the present invention is provided that the administration is carried out by spray administration, in oval administration, subcutaneous administration, intramuscular administration, oral administration or nasal administration. In one embodiment, the method includes in oval administration. In one embodiment, the method is provided that the in oval administration is performed on brooding eggs between approximately 16 and 22 days after development. In one or more embodiments, the method is provided that the in oval administration is performed on brooding eggs around 18 days after development. In one or more embodiments, the method is provided that the administration route includes in oval administration followed by spray administration. In one embodiment, the method is provided that the administration route includes spray administration. In one or more embodiments, the method is provided that the birds are selected from a group consisting of chickens, turkeys, geese, ducks, pheasants, ostriches, pigeons and quail. In one embodiment, the method is provided that the birds include chickens.

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

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

[0035] A brief explanation 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] [Modes for carrying out the invention]

[0036] The following is a detailed description provided to assist those skilled in the art. Those skilled in the art may modify and alter the embodiments described herein without departing from the spirit or scope of the invention.

[0037] The present invention relates to a vaccine for use in birds based on a biological recombinant turkey herpesvirus, namely, Marek's disease virus (MDV), more specifically, HVT virus (turkey herpesvirus), in which one or more nucleotide sequences are inserted that encode and express the antigenic polypeptide of the aforementioned pathogenic drug, under conditions that provide immunization resulting in effective protection of vaccinated animals against the pathogenic drug or multiple drugs. Marek's disease (MD) is a common lymphoproliferative disease of chickens caused by Marek's disease virus (MDV), which can cause significant losses in the poultry industry. Currently, MD is controlled in poultry using a vaccine with serotype 3 of MDV, which is the associated turkey herpesvirus (HVT). By introducing genes from poultry viruses other than MDV into the HVT genome at specific genetic locations, the inventors of the present invention have been able to develop a novel recombinant viral vaccine that enables simultaneous protection of poultry against MD and one or more additional diseases with a single dose of the viral vaccine.

[0038] This invention provides recombinant viral vectors for insertion and expression of exogenous genes for use in safe immunization to protect birds from various pathogens. The invention also provides a polyvalent composition or vaccine comprising one or more recombinant HVT viral vectors for protection against various pathogens. In addition, the invention provides methods for preparing and using recombinant viral vectors alone or in combination with other vaccines or pharmaceutical compositions.

[0039] In one embodiment, the present invention provides a recombinant turkey herpesvirus (HVT) genome comprising one or more nucleotide sequences encoding one or more heterologous antigens inserted into the intergenetic locus UL35 / UL36 in the unique long (UL) region of the HVT genome.

[0040] In one embodiment, the present invention provides a recombinant turkey herpesvirus (HVT) genome comprising one or more nucleotide sequences encoding one or more heterogeneous antigens or multiple antigens inserted into the intergenetic locus UL35 / UL36 in the unique long region of the HVT genome, and one or more nucleotide sequences encoding one or more heterogeneous 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 relates to recombinant HVT, wherein one or more heterologous antigens or multiple antigens are protective against avian pathogens or multiple pathogens selected from the group consisting of infectious bursal disease virus (IBDV), Newcastle disease virus (NDV), infectious bronchitis virus (IBV), infectious laryngotracheitis virus (ILTV), avian anemia virus (CAV), and avian influenza virus (AIV).

[0042] In one or more embodiments, the present invention provides recombinant HVT in which one or more heterologous antigens are selected from the group consisting of the following: VP2, VP3, or VP4 protein of infectious bursal disease virus (IBDV); VP1 or VP2 protein of avian 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); gB, gC, gD, gE, gH, gI, or gL protein of infectious laryngotracheitis virus (ILTV); and 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 that one or more heterologous antigens are protective against IBDV. In one embodiment, the recombinant HVT of the present invention is provided that one or more heterologous antigens are the VP2 protein of IBDV. In one embodiment, the recombinant HVT of the present invention is provided that the VP2 protein sequence is encoded by a nucleotide sequence having at least 80% sequence identity with respect to a nucleotide sequence containing 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 containing either SEQ ID NO: 5 or SEQ ID NO: 10.

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

[0045] In one or more embodiments, the recombinant HVT of the present invention is provided that one or more heterologous antigens are protective against NDV and IBDV. In one or more embodiments, the recombinant HVT of the present invention is provided 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 provides an F protein of NDV encoded by a nucleotide sequence having at least 80% sequence identity with respect to the nucleotide sequence containing SEQ ID NO. 3, and a VP2 protein of IBDV encoded by a nucleotide sequence having at least 80% sequence identity with respect to the nucleotide sequence containing SEQ ID NO. 5 or SEQ ID NO. 10.

[0047] In one or more embodiments, the recombinant HVT of the present invention is provided that the F protein of NDV, encoded by a nucleotide sequence including SEQ ID NO: 3, and the VP2 protein of IBDV, are encoded by a nucleotide sequence including 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 multiple cassettes comprising one or more nucleotide sequences or multiple sequences encoding one or more heterologous antigens or multiple antigens. In one embodiment, the recombinant HVT comprises a recombinant HVT genome comprising an expression cassette comprising a nucleotide sequence encoding a promoter operably ligated to one or more nucleotides encoding the antigen to be expressed. In one embodiment, the antigen to be expressed comprises the F protein of NDV. In one embodiment, the antigen to be expressed comprises the VP2 protein of IBDV. In one embodiment, the antigen to be expressed comprises both the F protein of NDV and the VP2 protein of IBDV.

[0049] In one embodiment, the recombinant HVT of the present invention is provided that one or more promoters are selected from the group consisting of the pre-initial cytomegalovirus human (hCMV) promoter, the guinea pig pre-initial CMV promoter, the mouse pre-initial CMV promoter, the Pec promoter, the β-chicken triactin promoter, the SV40 promoter, the pseudorabies virus promoter with glycoprotein X promoter, the herpes simplex virus-1 alpha-4 promoter, the Marek's disease virus promoter with glycoprotein gA, gC, gB, gE, or gI promoter, the infectious laryngotracheitis virus promoter with glycoprotein gB, gE, gI, gD promoter, and the bovine herpesvirus 1 / 1 VP8 promoter. In one embodiment, the recombinant HVT comprises the human CMV promoter. In one embodiment, the recombinant HVT comprises the mouse CMV promoter. In one embodiment, the recombinant HVT comprises the hCMV promoter and the 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 SV40 polyA sequence (SEQ ID NO: 12). In one embodiment, the polyA signal is the BGH polyA signal. In one embodiment, the polyA signal is the SV40 polyA signal.

[0051] In one embodiment, the recombinant HVT of the present invention comprises a CMV promoter operably ligated to a nucleotide sequence encoding a VP2 protein from an IBDV, further comprising a nucleotide sequence encoding a polyadenylation signal, wherein all portions 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 from the 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 poly-A 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 includes, in order, SEQ ID NO: 1, SEQ ID NO: 5, or SEQ ID NO: 10 and SEQ ID NO: 6, which are inserted into the HVT genome at the UL55 / gene3 site.

[0052] In one embodiment, the recombinant HVT of the present invention comprises a CMV promoter operably ligated to a nucleotide sequence encoding the F protein of an NDV, further comprising a nucleotide sequence encoding a polyadenylation signal, wherein all portions of the NDV F cassette are inserted at a non-coding position in the HVT genome. In one embodiment, the CMV promoter comprises the mCMV (SEQ ID NO: 2) promoter. In one embodiment, a nucleotide sequence encoding the F protein of an NDV, comprising 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 poly-A 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 in the genome. In one embodiment, the expression cassette comprises, 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.

[0053] In one embodiment, the recombinant HVT of the present invention comprises a CMV promoter operably ligated to a nucleotide sequence encoding the VP2 protein of IBDV, further comprising a nucleotide sequence encoding a polyadenylation signal, and all of the VP2 expression cassette are inserted at a non-coding site in the HVT genome. In one embodiment, the recombinant HVT of the present invention further comprises a CMV promoter operably ligated to a nucleotide sequence encoding the F protein of NDV, further comprising 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 ligated to a nucleotide sequence encoding the F protein of NDV, further comprising 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 inserted into the HVT genome in the UL35 / 36 non-coding region (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); and, in order, a nucleotide sequence encoding an mCMV promoter inserted into the HVT genome in the UL55 / gene3 non-coding region (SEQ ID NO: 2), a nucleotide sequence encoding an F protein from NDV (SEQ ID NO: 3), and a nucleotide sequence encoding an SV40 polyadenylation signal (SEQ ID NO: 12). 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, avian 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 further provides a promoter operably ligated 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 avian 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).

[0055] In one embodiment, the recombinant HVT of the present invention comprises one or more ILT antigens as part of an expression cassette, each comprising a nucleotide sequence encoding a polyadenylation signal, each comprising a nucleotide sequence encoding a promoter operably ligated to a nucleotide encoding an ILT antigen, 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 ligated 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 avian 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), 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 immunogenic compositions comprising recombinant HVT of the present invention, further comprising a pharmaceutically acceptable carrier, excipient, or adjuvant.

[0058] In one or more embodiments, the present invention provides a vaccine composition comprising recombinant HVT of the present invention, 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 the naturally attenuated MDV-1 strain Rispens (CVI-988) or three Gallid herpesvirus strains SB-1 virus. In one embodiment, the vaccine of the present invention is provided that the additional MDV comprises a recombinant genome. In one embodiment, the vaccine of the present invention is provided 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 use in vaccination of 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 use in protection of birds against clinical symptoms caused by one or more avian pathogens. In one or more embodiments, the vaccine of the present invention provides use in protection of 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 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), avian anemia virus (CAV), and avian influenza virus (AIV). In one embodiment, the vaccine of the present invention is provided that one or more avian pathogens include Newcastle disease virus. In one embodiment, the vaccine of the present invention is provided that one or more avian pathogens include infectious bursal disease virus (IBDV). In one embodiment, the vaccine of the present invention is provided that one or more avian pathogens include Newcastle disease virus and bursal disease virus.

[0061] In one or more embodiments, the vaccine of the present invention provides use in avian vaccination, wherein the vaccine is administered by at least one dose of the vaccine by spray administration, in oval administration, subcutaneous administration, intramuscular administration, oral administration, nasal administration, or a combination thereof. In one embodiment, the vaccine of the present invention is provided that the vaccine is administered by in oval administration. In one embodiment, the vaccine of the present invention is provided that the in oval administration is performed on brooding eggs between approximately 16 and 22 days after development. In one or more embodiments, the vaccine of the present invention is provided that the in oval administration is performed on brooding eggs around 18 days after development. In one embodiment, the vaccine of the present invention is provided that the administration of the vaccine includes in oval administration followed by spray administration. In one embodiment, the vaccine of the present invention is provided that the administration of the vaccine includes spray administration.

[0062] In one embodiment, the present invention provides a method for vaccinating birds to treat or prevent Marek's disease and one or more avian diseases caused by one or more avian pathogens, comprising the step of administering an effective amount of the vaccine composition of the present invention. In one embodiment, the method of the present invention is provided that 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), avian anemia virus (CAV), and avian influenza virus (AIV). In one embodiment, the method of the present invention is provided that one or more avian pathogens include infectious bursal disease virus (IBDV). In one embodiment, the method of the present invention is provided that one or more avian pathogens include Newcastle disease virus (NDV). In one embodiment, the method of the present invention is provided that 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 animal avian to Marek's disease virus and one or more avian pathogens, comprising the step of administering an effective amount of the immunogenic composition or vaccine composition of the present invention to the bird. In one embodiment, the method of the present invention is provided that 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), avian anemia virus (CAV), and avian influenza virus (AIV). In one embodiment, the method of the present invention is provided that one or more avian pathogens include infectious bursal disease virus (IBDV). In one embodiment, the method of the present invention is provided that one or more avian pathogens include Newcastle disease virus (NDV). In one embodiment, the method of the present invention is provided that one or more avian pathogens include infectious bursal disease virus (IBDV) and Newcastle disease virus (NDV). In one or more embodiments, the method of the present invention is provided that the administration is carried out by spray administration, in oval administration, subcutaneous administration, intramuscular administration, oral administration or nasal administration. In one embodiment, the method includes in oval administration. In one embodiment, the method is provided that the in oval administration is performed on brooding eggs between approximately 16 and 22 days after development. In one or more embodiments, the method is provided that the in oval administration is performed on brooding eggs around 18 days after development. In one or more embodiments, the method is provided that the administration route includes in oval administration followed by spray administration. In one embodiment, the method is provided that the administration route includes spray administration. In one or more embodiments, the method is provided that the birds are selected from a group consisting of chickens, turkeys, geese, ducks, pheasants, ostriches, pigeons and quail. In one embodiment, the method is provided that the birds include chickens.

[0064] General methodology: It should be understood that the present invention is not limited to the specific methodologies, protocols, and reagents described herein, and that these are subject to change. The terminology used herein is intended solely to describe specific embodiments 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 inventions described herein shall have meanings generally understood by those skilled in the art. Furthermore, unless otherwise required by the context, singular terms shall include plural forms and plural terms shall include singular forms. Generally, the terminology and techniques used in connection with cell and tissue culture, molecular biology, and the chemistry and hybridization of proteins and oligonucleotides or polynucleotides described herein are well known and commonly used in the art.

[0066] Standard techniques are used for recombinant DNA and oligonucleotide synthesis, as well as for tissue culture and transfection, as are well known to those skilled in the art. Enzymatic reactions and purification techniques are carried out according to the manufacturer's specifications, as commonly achieved in the art, or as described herein. The aforementioned techniques and procedures are generally carried out according to conventional methods well known in the art and as described herein, but are not limited to the various general and more specific references cited and discussed throughout this specification. 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 Protocols in Immunology(E.Coligan et al.,eds., 1991), Short Protocols in Molecular Biology (Wiley and Sons, 1999), Immunobiology (CA Janeway and P. Travers, 1997), Antibodies (P. Finch, 1997), Antibodies: a practical approach (D. Catty., ed., IRL Press, 1988-1989), Monoclonal antibodies: a practical 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 JDCapra, eds., Harwood Academic Publishers, 1995), and Cancer: Principles and Practice of Oncology (YTDeVita et al. al.,eds.,JBLippincott See Company, 1993.

[0067] Except in the operating examples, or where otherwise indicated, all figures representing amounts of ingredients or reaction conditions used herein should be understood in all cases to be modified by the term "approximately".

[0068] All specified patents and other publications are expressly incorporated herein by reference for the purpose of describing and disclosing methodologies described in such publications that may be used in connection with the present invention. These publications are provided only for their disclosures prior to the filing date of this application.

[0069] definition Before describing the present invention in detail, we define some terms used in the context of the present invention. 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 the meanings recognized in their respective art.

[0070] In this disclosure, terms such as “comprises,” “comprised,” “comprising,” “contains,” “containing,” “consisted of,” “essentially consisted of,” “includes,” and “included” are defined in accordance with standard U.S. patent law and international patent law practices.

[0071] The term “approximately” is used herein to indicate that a value includes the standard deviation of errors in the device or method used to determine that value. The use of the term “or” in the claims is used to mean “and / or” unless it is expressly indicated that it refers only to substitutes, or that the substitutes are mutually exclusive; however, this disclosure supports the definitions of substitutes only and the definitions of “and / or.” Unless used in conjunction with non-inclusive language in the claims, or unless otherwise specifically noted, the words “a” and “an” indicate “one or more.” Thus, the term “conferred by a transgene” includes, 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 mimes that function in a similar manner to naturally occurring amino acids. Naturally occurring amino acids are those encoded by the genetic code, as well as those that are later modified, such as hydroxyproline, γ-carboxyglutamate, and O-phosphoserine. Amino acid analogs refer to compounds that have the same basic chemical structure as naturally occurring amino acids (i.e., hydrogen, carboxyl group, amino group, and α-carbon bonded to an R group, e.g., homoserine, norleucine, methionine sulfoxide, methionine methylsulfonium). Such analogs may have a modified R group (e.g., norleucine) or a modified peptide skeleton, but retain the same basic chemical structure as naturally occurring amino acids. Amino acid mimes refer to chemical compounds that have a different structure from the general chemical structure of amino acids, but function in a similar manner to naturally occurring amino acids.

[0073] Amino acids may be referred 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 by their commonly accepted one-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 the locally regular three-dimensional structure within a polypeptide. These structures are commonly known as domains, e.g., enzyme domains, extracellular domains, transmembrane domains, pore domains, or cytoplasmic tail domains. Domains are parts of a polypeptide that form compact units of the polypeptide. Exemplary domains include those with enzymatic activity. Domains may consist of elongated β-sheets and smaller organized sections such as α-helices. "Tertiary structure" refers to the complete three-dimensional structure of a polypeptide monomer. "Quaternary structure" refers to a three-dimensional structure formed by the non-covalent 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 fragment thereof that specifically binds to and recognizes an antigen. The recognized immunoglobulin genes may include kappa, lambda, alpha, gamma, delta, epsilon, and muon constant region genes, as well as a multitude of immunoglobulin variable region genes. The light chain may be classified as either kappa or lambda. The heavy chain may be classified as gamma, muon, alpha, delta, or epsilon, which then define the immunoglobulin classes IgY, IgG, IgM, IgA, IgD, and IgE, respectively.

[0075] Exemplary immunoglobulin (antibody) structural units may include tetramers, 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 involved in 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 by 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 chemically or de novo using recombinant DNA methodologies. Therefore, as used herein, the term “antibody” also includes any antibody fragment produced by modification of a whole antibody, or an antibody fragment de novo synthesized using recombinant DNA methodologies, or an antibody fragment identified using other methods known in the art.

[0076] Many techniques known in the art can be used for the preparation of antibodies, such as recombinant antibodies, monoclonal antibodies, or polyclonal antibodies. Genes encoding the heavy and light chains of the 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 combinations of heavy and light chain gene products generate a large pool of antibodies with different antigen specificities. Techniques for producing single-chain or recombinant antibodies have been found in the art and can be adapted to produce antibodies against polypeptides according to the present invention. Phage display techniques can also be used to identify antibodies and heteromer fragments that specifically bind to selected antigens. Antibodies may also be bispecific (i.e., able to recognize two different antigens) or heteroconjugate, for example, two covalently bound antibodies, or immunotoxins.

[0077] As used herein, “antigen” refers to viral proteins or polypeptides, such as viral polypeptides, as well as viral particles. In some embodiments, the antigen according to the present invention may also be 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 an entire organism, a weakened organism, a dead organism, or a living 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, that can induce an immune response.

[0078] As used herein, the term “bird” includes poultry, such as members of the order Galliformes. More specifically, it refers to classes of birds of economic and / or agricultural interest, such as chickens, turkeys, geese, ducks, pheasants, ostriches, pigeons, and quail.

[0079] As used herein, “biological specimen” or “sample” may include, but is not limited to, blood and portions of blood, serum, plasma, platelets, or erythrocytes, sputum, cloacal swabs, mucous membranes, tissues, cultured cells (including primary cultures), explants, and transformed cells, biological fluids, feces, and urine. Biological specimens may also include tissue sections such as biopsy specimens and autopsy specimens, as well as frozen sections taken for histological purposes. Biological specimens may be obtained from eukaryotes, for example, birds, but not limited to birds from the order Galliformes, for example, chickens, quail, and turkeys. Any tissue suitable for use in accordance with the present invention may be used, for example, skin, brain, spinal cord, adrenal gland, pectoralis muscle, lung, heart, liver, crop, fore-stomach, gizzard, duodenum, small intestine, large intestine, cloaca, kidney, bursa of Fabricius, spleen, pancreas, adrenal gland, 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 very similar to that of the given residue, and therefore does not result in a substantial decrease in polypeptide function (e.g., enzyme activity). Conservative amino acid substitutions are commonly known in the art, and examples are described, for example, in U.S. Patents 6,790,639, 6,774,107, 6,194,167, or 5,350,576. In preferred embodiments, a conservative amino acid substitution is any one occurring within one of the following six groups: ●1. Small aliphatic substantially nonpolar residues: Ala, Gly, Pro, Ser, and Thr, ●2. Large aliphatic nonpolar residues: lie, Leu, and Val, Met, ●3. Negatively charged polar residues and their amides: Asp and Glu, ●4. Amides of negatively charged polar 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 a preferred embodiment, a conserved amino acid substitution is one of the following listed as native residue (conserved 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] In the context of assays for testing compounds that modulate the activity of viruses as described herein, the term “functional effect” includes determining a parameter, indirect or direct, under the influence of such virus, e.g., a phenotypic effect or a chemical effect. “Functional effect” may include in vitro, in vivo, and ex vivo activity and can be measured by any means known to those skilled in the art, such as changes in the spectral characteristics, shape, chromatography, or solubility properties of a protein; measuring inducible markers or transcriptional activation of a protein; measuring binding activity or binding assays, e.g., measuring 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; and identifying downstream or reporter gene expression, e.g., identification via chemiluminescence, fluorescence, colorimetric reactions, antibody binding, and / or inducible markers.

[0083] The term "gene" refers to a component that includes viral DNA or RNA, cDNA, viral introns and exons, artificial viral DNA polynucleotides, or other DNA encoding viral peptides, viral polypeptides, viral proteins, or viral RNA transcription molecules, as well as genetic elements that may be adjacent to coding sequences involved in regulating expression, such as promoter regions, 5' leader regions, and 3' untranslated regions, which may exist as native or transgenes in the viral genome. A polynucleotide sequencing method can be used to determine the order of nucleotides containing the gene for a gene or a fragment thereof.

[0084] The term "turkey herpesvirus (HVT)" is defined as a non-pathogenic virus of domesticated turkeys and is classified as the third serotype within the group of Marek's disease viruses, which are antigenically and genetically related lymphophosphorus turkey herpesviruses.

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

[0086] The term “host cell” means any cell of any organism that is selected, modified, transformed, grown, or used or manipulated in any way to produce a substance, for example, by the expression of genes, DNA or RNA sequences, proteins or enzymes by the cell. Host cells are intended to include any individual cell or cell culture that may or may have been a recipient for a vector or for the incorporation of exogenous nucleic acid molecules, polynucleotides, and / or proteins. They are also intended to include single-cell offspring. Offspring may not necessarily be exactly identical to the original parent cell (in morphology or genomic or whole DNA complement) due to spontaneous, accidental, or intentional mutations. Cells may be prokaryotes or eukaryotes.

[0087] As used herein, the terms “host,” “subject,” “patient,” or “organism” may include animals, in particular birds, in particular poultry. In veterinary use, birds may be from the order Galliformes, including chickens, quail, and turkeys. The term “living host” refers to the host or another living organism as described above. This term may also refer to the host or the entire organism and not merely a part (e.g., brain or other organ) removed from a living host. These terms also include individuals at all stages of development, including the embryonic and fetal stages.

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

[0089] For sequence comparison, one sequence typically functions as a reference sequence, against which other sequences are compared. When using a sequence comparison algorithm, the reference sequence and the comparison sequence may be input into a computer, and, if desired, sequence algorithm program parameters are selected. Then, based on the selected parameters, a sequence identity percentage is generated for the comparison sequence relative to the reference sequence. Examples of algorithms that may be suitable for determining sequence identity percentage and sequence similarity are the BLAST and BLAST 2.0 algorithms, described in Altschul et al., (Nuc Acids Res 25:3389-3402, 1977) and Altschul et al., (J Mol Biol 215:403-410, 1990), respectively. 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,” “pharmaceutical composition,” or “vaccine” means a composition containing an antigen suitable for administration to a subject, e.g., a bird subject. The compositions described above generally mean that they induce an immune response in the subject. The immune response may include a T-cell response, a B-cell response, or both T-cell and B-cell responses. The composition may play a role in sensitizing the subject by presenting an antigen that associates with MHC molecules on the cell surface. In addition, it may generate antigen-specific T lymphocytes or antibodies to enable future protection of the immunized host. An “immunogenic composition” may contain a live vaccine, an attenuated vaccine, or a dead / inactivated vaccine containing an immunogenic portion of a whole organism or derived therefrom that induces either or both a cell-mediated (T-cell) immune response or an antibody-mediated (B-cell) immune response, and may protect an animal from one or more symptoms associated with a microbial infection or from death resulting from a microbial infection. Generally, an "immunogenic composition" is sterile and, preferably, free from contaminants that could induce an undesirable response in the subject (for example, the compound(s) in the immunogenic composition are pharmaceutical grade). Immunogenic compositions may be designed for administration to subjects requiring it via several different routes of administration, including inovo, oral, intravenous, oral cavity, rectal, parenteral, intraperitoneal, intradermal, intratracheal, intramuscular, subcutaneous, and inhalation.

[0091] As used herein, the term “immunogenic” protein or peptide includes polypeptides that are immunologically active in the sense that, when administered to a host, they can induce humoral and / or cellular immune responses directed toward the protein. Preferably, the protein fragments are such that they have substantially the same immunological activity as the full-length protein. Thus, the protein fragments according to the present invention include, or consist of, at least one epitope or antigenic determinant. “Immunogenic” protein or polypeptide, as used herein, includes the full-length sequence of a protein, its analogues, or an immunogenic fragment thereof. “Immunogenic fragment” means a fragment of a protein that includes one or more epitopes and thus induces the immune response described above.

[0092] The term “immunogenic protein or peptide” further intends deletions, additions, and substitutions to a sequence insofar as the polypeptide functions to produce an immune response as defined herein. The term “conservative mutation” refers to replacing an amino acid residue with another biologically similar residue, or replacing a nucleotide in a nucleic acid sequence such that the encoded amino acid residue remains unchanged or is another biologically similar residue. In this regard, particularly preferred substitutions are generally those that are essentially conserved, i.e., substitutions that occur within a family of amino acids. For example, amino acids are generally divided into the following four families: (1) acidic – aspartates and glutamates, (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 the substitution of one hydrophobic residue, such as isoleucine, valine, leucine, or methionine, with another hydrophobic residue, or the substitution of one polar residue with another polar residue, such as arginine being replaced with lysine, glutamic acid with aspartic acid, or glutamine with asparagine, or similar conservative substitutions of amino acids by structurally related amino acids that do not significantly affect bioactivity. Therefore, proteins that have substantially the same amino acid sequence as the reference molecule but have only minor amino acid substitutions that do not substantially affect the immunogenicity of the protein are within the definition of a reference polypeptide. All polypeptides produced by these modifications are included herein. The term "conservative mutation" also includes the use of a substituted amino acid instead of an unsubstituted parent amino acid, however antibodies listed for substituted polypeptides will also react immunologically with unsubstituted polypeptides.

[0093] As used herein, “immunologically effective dose” refers to the amount of antigen or vaccine sufficient to induce an immune response, either cellular (T cell) or humoral (B cell or antibody) response, as measured by standard assays known to those skilled in the art. For example, in relation to the present invention, “immunologically effective dose” is the minimum protective dose (potency). The effectiveness of an antigen as an immunogen can be measured by proliferation assays, by cell lysis assays such as chromium-releasing assays that measure the ability of T cells to lyse its particular target cells, by measuring the level of B cell activity by measuring the level of antigen-specific circulating antibodies in serum, or by any other assay known to and used by those skilled in the art. Furthermore, the protective level of the immune response can be measured by challenging a host immunized with the injected antigen. For example, if the antigen for which an immune response is desired is a virus or tumor cells, the protective level induced by an “immunologically effective dose” of the antigen is measured by detecting the survival or mortality rate of an animal after viral or tumor cell challenge.

[0094] Determining the immunological effective dose of a vaccine according to the present invention is within the reach of those skilled in the art, for example, by monitoring the immunological response after vaccination or challenge infection (e.g., by re-isolation of the pathogen), or by monitoring the clinical signs or serological parameters of the target disease and comparing them to the response observed in mock-vaccinated animals. The administration scheme for applying the vaccine according to the present invention to a target organism may be a single dose or multiple doses, and may be administered simultaneously or sequentially in an amount compatible with the vaccine formulation and immunologically effective.

[0095] The terms “inhibitor,” “activator,” and “modulator” of viral nucleic acids and polypeptide sequences are used to refer to the activation, inhibition, or modulation of molecules identified using in vitro and in vivo assays of viral nucleic acids and polypeptide sequences. Inhibitors are compounds that can bind to a virus, partially or completely block its activity, reduce or prevent viral activity or expression, delay activation, inactivate, desensitize, or downmodulate it. Activators are compounds that increase, open, activate, promote, enhance, sensitize, antagonize, or upmodulate viral activity. Inhibitors, activators, or modulators also include the genetically modified forms of viruses described herein, e.g., altered forms of activity, as well as naturally occurring and synthetic ligands, substrates, antagonists, agonists, antibodies, peptides, cyclic peptides, nucleic acids, antisense molecules, ribozymes, small chemical molecules, etc. Assays for inhibitors and activators include, for example, expressing the virus of the present invention in vitro, intracellularly, or at the cell membrane, applying the 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 the virus of the present invention treated with a potential activator, inhibitor, or modifier may be compared to a control sample lacking the inhibitor, inhibitor, or modifier. The control sample on which the test sample or assay is compared may be assigned a relative protein activity value of 100%. Inhibition of the virus 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 intergeneric locus is defined, as used herein, as a region of DNA sequence located between genes, including untranslated regions, 5' and 3' flanking regions, introns, and the like. Intergeneric regions are portions of non-coding DNA that may contain gene regulatory elements such as promoters and enhancers.

[0098] The term "isolated" means a substance that has been substantially separated from other naturally occurring substances or concentrated compared to other substances. Isolated substances are typically of a purity of at least about 80% by weight, at least 90% by weight, at least 98% by weight, or at least about 99% by weight.

[0099] A “label” or “detectable portion” is a composition detectable by spectroscopic, photochemical, biochemical, immunochemical, chemical, or other physical means. For example, useful labels include: 32 Examples include phosphorus (P), fluorescent dyes, high electron-density reagents, enzymes (such as those commonly used in ELISA), biotin, digoxigenin, or haptens, as well as proteins that can be detected, for example, by incorporating a radiolabel into the peptide, or antibodies that react specifically with the peptide.

[0100] As used herein, “Marek’s disease virus” or “MDV” refers to any alphaherpesvirus of the genus Mardivirus, including turkey herpesvirus (HVT), as described herein. In certain embodiments, the present invention relates to Marek’s disease virus, its genetic components, genes, and proteins produced thereby. As used herein, such a virus may include the genetic components of the virus, i.e., its genome and transcripts, proteins encoded by the genome (including structural and non-structural proteins), and functional or non-functional viral particles. Polynucleotide and polypeptide sequences encoding such viruses are well known in the art and will be readily apparent to those skilled in the art.

[0101] The terms “variant” and “mutation” mean any detectable change in genetic material (e.g., DNA), or any process, mechanism, or result of such 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 mutation process; and any expression product (e.g., protein or enzyme) expressed by a modified gene or DNA sequence. The term “variant” may also be used to refer to any type of variant, such as a modified or altered gene, DNA sequence, enzyme, or cell.

[0102] As used herein, the term “nucleic acid” refers to a single-stranded or double-stranded polymer of deoxyribonucleotide or ribonucleotide bases read from the 5' end to the 3' end. “Nucleic acid” may also optionally contain non-naturally occurring or altered nucleotide bases that allow for correct misreading by polymerase and do not reduce the expression of the polypeptide encoded by that nucleic acid. The term “nucleotide sequence” or “nucleic acid sequence” refers to both the sense and antisense strands of a nucleic acid as individual single-stranded or double-stranded 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 discharged with the corresponding acylated amino acid), 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 functional terms encompassing genome sequences, ribosomal RNA sequences, transfer RNA sequences, messenger RNA sequences, operon sequences, and smaller, manipulated nucleotide sequences that express or can be adapted to express proteins, polypeptides, or peptides. The nomenclature used herein is required by Title 37 of the United States Code of Federal Regulations §1.822 and is set forth in the tables of 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, which are configured or assembled to perform their normal functions. Thus, flanking sequences operably linked to a coding sequence may be able to duplicate, transcribe, and / or translate the coding sequence. For example, a promoter or enhancer is operably linked to a coding sequence if it affects the transcription of the sequence. Flanking sequences do not need to be contiguous with a coding sequence in order to function correctly. For example, an intervening untranslated but transcribed sequence may exist between a promoter sequence and a coding sequence, and the promoter sequence can still be considered “operably linked” to the coding sequence.

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

[0105] As used herein, “poultry” refers to domesticated 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, but may also include geese, ducks, swans, guinea fowl, pigeons, and the like.

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

[0107] Certain nucleic acid sequences may also encompass “splice variants.” Similarly, a particular protein encoded by a nucleic acid implicitly encompasses any protein encoded by a splice variant of that nucleic acid. Splice variants are the products of alternative splicing of genes. After transcription, the initial nucleic acid transcript may be spliced ​​so that a different (alternative) nucleic acid splice product encodes a different polypeptide. Mechanisms for producing splice variants vary, but include alternative splicing of exons. Alternative polypeptides derived from the same nucleic acid by over-reading transcription are also included in this definition. Any product of a splicing reaction (including recombinant forms of splice products) is included in this definition.

[0108] The terms “polypeptide,” “peptide,” and “protein” are used interchangeably herein to refer to polymers of amino acid residues. This term applies to amino acid polymers in which one or more amino acid residues are artificial chemical mimics of corresponding naturally occurring amino acids, as well as to naturally occurring amino acid polymers and naturally occurring amino acid polymers.

[0109] The terms “polyvalent vaccine,” “combination or combo vaccine,” and “multivalent vaccine” are used interchangeably to refer to vaccines containing more than one antigen. A polyvalent vaccine may contain two, three, four, or more antigens. A polyvalent vaccine may contain a recombinant viral vector, an active virus or a weakened or dead wild-type virus, or a mixture of a recombinant viral vector and a wild-type virus in an active, weakened, or dead form.

[0110] As used herein, "promoter" refers to a DNA sequence that defines the site where RNA polymerase transcription of a gene begins. Promoters are typically located upstream of the transcription start site. Promoters may 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 strands are transcribed. Promoters may originate from sources including viruses, bacteria, fungi, plants, insects, and animals. Promoters can constitutively or differentially regulate the expression of gene components in relation to the cell, tissue, or organ in which expression occurs, or in relation 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 triatin 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 terms “to treat preventively” or “to treat preventively” mean to completely or partially prevent a disease or its symptoms, and / or may be therapeutic in the sense of a partial or complete cure of the disease and / or adverse effects resulting therefrom.

[0112] The term "recombinant," when used, for example, in reference to a cell, 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 by the modification of a native nucleic acid or protein, or that the cell originates from such a modified cell. Therefore, for example, a recombinant cell may express genes not found in the cell's native (non-recombinant) form, or native genes that are otherwise abnormally expressed, underexpressed, or not expressed at all. In some embodiments, the recombinant sequence may also include nucleic acids, proteins, or recombinant genomes (e.g., viral genomes). Recombinant viral vectors described herein may contain a transgene operably ligated to a heterologous promoter for transcription of the transgene.

[0113] The term "stringent hybridization conditions" refers to conditions under which a probe hybridizes to its target sequence, typically in a complex mixture of nucleic acids, but not to other sequences. Stringent conditions may be sequence-dependent and will vary depending on the context. Longer sequences hybridize specifically at higher temperatures. Stringent conditions can be achieved by adding destabilizers such as formamide.

[0114] Appropriate stringency conditions for promoting DNA hybridization are well known to those skilled in the art and may include, for example, washing with 6x sodium chloride / sodium citrate (SSC) at about 45°C, followed by washing with 2x SSC at 50°C. The salt concentration in the washing step can be selected from low stringency of about 2x SSC at about 50°C to high stringency of 0.2x SSC at 50°C. The temperature in the washing step can be increased from low stringency conditions at room temperature and about 22°C to high stringency at about 65°C. The temperature and / or salt conditions may be varied as appropriate for optimal results. According to the present invention, nucleic acids may exhibit at least about 80% to about 100% sequence identity with one or more nucleic acid molecules described herein, e.g., at least about 80%, about 85%, about 90%, about 95%, about 98%, about 99%, or about 100% sequence identity.

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

[0116] As used herein, the terms “therapeutic effective dose,” “effective dose,” or “therapeutic effective dose” refer to the dose that produces an effect when administered. Such a dose or amount may also refer to an embodiment of the drug administered that alleviates to some extent one or more symptoms of a disease, i.e., one or more of the infections being treated, and / or an amount that prevents to some extent one or more symptoms of a disease (i.e., infection) that the treated host has developed or is at risk of developing. The exact dose will vary depending on the therapeutic purpose, and a person skilled in the art will be able to determine such a dose using the art known in the art.

[0117] As used herein, “transgene” refers to a segment of DNA containing a heterologous coding sequence or other genetic material for introduction from one organism to another. For example, in certain embodiments, the transgene according to 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,” “to treat,” and “to treat” are defined as acting on a disease, disorder, or condition by using an agent to reduce or improve the pharmacological and / or physiological effects of a 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) and includes (a) reducing the risk of developing the disease in a subject that has been determined to be predisposed to the disease but has not yet been diagnosed as being infected with the disease, (b) preventing the development of the disease, and (c) mitigating the disease, i.e., causing a regression of the disease, and / or reducing one or more symptoms of the disease. “Treatment” also means the delivery of an inhibitor to provide a pharmacological effect even when the disease or condition is not present. For example, “treatment” includes the delivery of a disease or pathogen inhibitor that results in an enhanced or desired effect in a subject (e.g., a reduction in the pathogen load, a reduction in disease symptoms, etc.).

[0119] The term "unit dosage form," as used herein, refers to a physically distinct unit suitable as a unit dose 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 the desired effect in conjunction with a pharmaceutically acceptable diluent, carrier, or vehicle. The specifications of a unit dosage form depend on the specific compound used, the route and frequency of administration, the effect to be achieved, and the pharmacodynamics of each compound in the host.

[0120] The terms “vaccine” or “vaccine composition” are used interchangeably herein and refer to a pharmaceutical composition comprising at least one immunogenic composition of the present invention that induces an immune response in a subject. The vaccine or vaccine composition can protect a subject from disease or possible death and may or may not contain 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 comprise a recombinant HVT virus having one or more heterologous antigens encoding a gene inserted into the HVT genome at the intergenetic region UL35 / 36. In some embodiments, the compositions of the present invention comprise a recombinant HVT virus having one or more heterologous antigens encoding a gene inserted into the HVT genome at UL35 / 36 and one or more antigens encoding a gene inserted into the HVT genome at UL55. In some embodiments, the antigen-coding genes are antigens derived from poultry pathogens such as Newcastle disease virus, bursal disease virus, infectious bronchitis virus, avian influenza virus, infectious laryngotracheitis virus and / or avian anemia virus. In some embodiments, recombinant HVT is combined with another recombinant Marek's disease virus vaccine that elicits a protective immune response in poultry. The vaccine or vaccine composition of the present invention may also include further components typical of a vaccine or vaccine composition, such as adjuvants or immunomodulators. The vaccine may include one or more of the above-mentioned elements 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, antimicrobial and antifungal agents, isotonic agents and absorption retarders in the host. The term “carrier” refers to a diluent, adjuvant, excipient, or vehicle on which the pharmaceutical composition is administered. Such pharmaceutical carriers may be sterile liquids, such as water and oil, and may be of petroleum, animal, plant, or synthetic origin, such as peanut oil, soybean oil, mineral oil, and sesame oil. Water is a preferred carrier when the pharmaceutical composition is administered intravenously. Saline solutions, dextrose aqueous solutions, and glycerol aqueous solutions can also be used as liquid carriers, particularly in injectable solutions. Suitable pharmaceutical excipients include starch, glucose, lactose, sucrose, gelatin, malt, rice, wheat flour, chalk, silica gel, sodium stearate, glycerol monostearate, talc, sodium chloride, dried skim milk, glycerol, propylene glycol, water, and ethanol. If desired, the compositions may also contain small amounts of wetting or emulsifying agents or pH buffers. These compositions may take the form of solutions, suspensions, emulsions, tablets, pills, capsules, powders, or sustained-release formulations. Depending on the method of administration, the compositions may be formulated with conventional binders and carriers such as triglycerides. Certain formulations may contain pharmaceutical-grade standard carriers such as mannitol, lactose, starch, magnesium stearate, sodium saccharin, cellulose, and magnesium carbonate. Examples of suitable pharmaceutical carriers are described by E.W. Martin in "Remington's Pharmaceutical Sciences." The formulations must be suitable for the mode of administration. Suitable carriers will be obvious to those skilled in the art and will largely depend on the route of administration. Additional components that may be present in the present invention include adjuvants, preservatives, surfactants, chemical stabilizers, suspending agents, or dispersants. Typically, stabilizers, adjuvants, and preservatives are optimized to determine the best formulation for efficacy in the target area.

[0122] A “variant” peptide, as used herein, refers to a peptide whose amino acid sequence differs from the “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, and which retains at least one desired activity of the parent vaccine peptide. For example, a variant may 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 the vaccine of the present invention. Typically, a variant has an amino acid sequence having 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 95% sequence identity. This sequence identity or homology is defined herein as the percentage of amino acid residues in a candidate sequence that are identical to parent peptide residues after aligning the sequences and, if necessary, introducing gaps to achieve the maximum sequence identity percentage. Any N-terminal, C-terminal, or internal extension, deletion, or insertion into the peptide sequence should not be interpreted as affecting sequence identity or homology. The variant retains the ability to induce an immune response and preferably has a desired activity superior to that 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 conserved mutations or mutations in non-essential residues or regions. Functional variants may also contain similar amino acid substitutions that do not alter function or alter it only slightly. Alternatively, such substitutions may have a positive or negative effect on function to some extent. Non-functional variants typically contain one or more non-conserved amino acid substitutions, deletions, insertions, inversions, or cleavages, or substitutions, insertions, inversions, or deletions in essential residues or regions.

[0124] Furthermore, polypeptides often contain amino acids other than the 20 "naturally occurring" amino acids. Moreover, many amino acids, including terminal amino acids, can be modified by natural processes such as processing and other post-translational modifications, or by chemical modification techniques well known in the art. Known modifications include, but are not limited to, acetylation, acylation, ADP-ribosylation, amidation, covalent bonding of flavins, covalent bonding of heme moieties, covalent bonding of nucleotides or nucleotide derivatives, covalent bonding of lipids or lipid derivatives, covalent bonding of phosphotidylinositol, crosslinking, cyclization, disulfide bond formation, demethylation, covalent crosslinking, cystine formation, pyroglutamate formation, formylation, gammacarboxylation, glycosylation, GPI anchor formation, hydroxylation, iodization, methylation, myristoylation, oxidation, proteolysis, phosphorylation, prenylation, racemization, selenoylation, sulfation, arginylation, and other transfer-RNA mediated additions of amino acids to proteins, as well as ubiquitination. Such modifications are well known to those skilled in the art and are described in detail in the scientific literature. Several particularly common modifications, such as glycosylation, lipid attachment, sulfated proteins, gamma-carboxylation, hydroxylation, and ADP-ribosylation of glutamate residues, are described, for example, in the most basic literature, e.g., Proteins - Structure and Molecular Properties (2nd ed., TECreighton, WH Freeman & Co., NY, 1993). Many detailed reviews on this subject are available, for example, Wold, Posttranslational Covalent Modification of proteins, 1-12 (Johnson, ed., Academic Press, NY, 1983), Seifter et al. 182 Meth. Enzymol. 626-46 (1990), and Rattan et al. 663 Ann. NY Acad. Sci. 48-62 (1992).

[0125] Therefore, the peptides of the present invention also include derivatives or analogues in which the substituted amino acid residues are 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 the antigen and / or its epitope or peptide, and are therefore included in the present invention.

[0126] In this specification, a “variant” nucleic acid refers to a molecule whose sequence differs from that of the “parent” nucleic acid. Deviations from a polynucleotide sequence can result from mutational changes such as the deletion, substitution, or addition of one or more nucleotides. Each of these changes may occur one or more times in a given sequence, either individually or in combination.

[0127] Just as polypeptides can contain conserved amino acid substitutions, their polynucleotides can contain conserved codon substitutions. A codon substitution is considered conserved if, when expressed, it produces the aforementioned conserved amino acid substitutions. Modified codon substitutions that do not result in amino acid substitutions are also useful in the polynucleotides according to the present invention. Therefore, for example, a polynucleotide encoding a selected polypeptide useful in one embodiment of the present invention can be mutated by a modified codon substitution to approximate the codon usage frequency shown by the expressing host cell transformed with it, or to improve its expression in other ways.

[0128] As used herein, “vector” means 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, cosmid or phage vectors, DNA or RNA expression vectors associated with cationic condensers, liposome-encapsulated DNA or RNA expression vectors, and certain eukaryotic cells such as producer cells. A vector has an expression regulatory sequence, meaning a nucleic acid sequence that directs the transcription of the nucleic acid, as described herein. The expression regulatory sequence may be a promoter, such as a constitutive or inductive promoter, or an enhancer. The expression regulatory sequence is “operatably ligated” to the nucleic acid sequence to be transcribed. A nucleic acid is “operatably ligated” when it is positioned in a functional relationship with another nucleic acid sequence. For example, if a pre-sequence or secretion leader DNA is expressed as a preprotein involved in polypeptide secretion, it is operatively ligated to the polypeptide DNA; if a promoter or enhancer affects the transcription of the sequence, it is operatively ligated to the coding sequence; or if a ribosome binding site is positioned to facilitate translation, it is operatively ligated to the coding sequence. Generally, "operatively ligated" means that the DNA sequence to be ligated is contiguous, and in the case of a secretion leader, it is contiguous and in the reading phase. However, enhancers do not need to be contiguous. Ligation is achieved by ligation at a convenient restriction site. If such a site is not present, synthetic oligonucleotide adapters or linkers are used according to conventional practice.

[0129] As used herein, “viral protein” or “viral polypeptide” refers to a protein encoded by a virus described herein, including structural and non-structural proteins. Such proteins may 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 may also include naturally occurring or non-naturally occurring viral proteins from ILTV, such as gB, gC, gD, gE, gH, gI, or gL, S1, S2, or M proteins from Infectious Bronchitis Virus (IBV), VP1 or VP2 proteins from Avian Anemia Virus (CAV), and / or HA, NA, NP, or M proteins from Avian Influenza Virus (AIV).

[0130] According to the present invention, the recombinant viral vectors described herein may enable protection of poultry from two or more different viral pathogens by providing recombinant viral vectors that express genes from viral pathogens. In some embodiments, the recombinant viral vectors of the present invention may be provided to poultry in the immunogenic compositions described herein. Genes from any viral pathogen suitable for use with the recombinant viral vectors described herein may be used. For example, in some embodiments, the recombinant viral vectors may express genes from Newcastle disease virus (NDV), infectious bursal disease virus (IBDV), avian influenza virus (AIV), avian anemia virus (CAV), infectious bronchitis virus (IBV), and infectious laryngotracheitis virus (ILTV).

[0131] According to the present invention, a transgene conferring protection or resistance from a specific virus or multiple viruses may be inserted into the viral genome at a specific location. For example, in some embodiments, the transgene described herein may be inserted into the viral genome in an intergeneric region adjacent to HVT UL35 / UL36 in a unique long region of the genome. In another embodiment of the present invention, the transgene is described herein as comprising one or more heterogenes inserted into the viral genome in an intergeneric region adjacent to HVT UL35 / UL36 in the HVT genome, in addition to the second site used, and one or more heterogenes being 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, the recombinant viral vector may express multiple genes derived from a single viral species, or it may express genes derived from more than one viral species to obtain resistance to multiple viruses. For example, in one embodiment, the present invention provides a recombinant viral vector comprising an HVT genome and at least one transgene from a different viral pathogen, and thus providing protection in birds such as poultry against Marek's disease and at least one other viral disease. For example, in one embodiment, the recombinant viral vector according to the present invention may provide protection in poultry against MDV and NDV, or protection against MDV and IBDV, or protection against MDV, NDV and IBDV.

[0133] The viral antigens for expression in poultry using the recombinant viral vector of the present invention may be encoded by viral genes, such as the viral genes described herein. Those skilled in the art will understand that, in this regard, it may not be necessary to incorporate the entire particular viral gene to obtain the desired viral resistance. Rather, only a portion of such a gene may be used. It may be desirable to select a specific portion of the desired gene that is specific to any given targeted virus or multiple 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 that are suitable for use with the present invention. Those skilled in the art will understand that modifications can be made to viral genes or multiple genes, or the proteins encoded therein, to increase the activity of the viral proteins when introduced into a target. Modifications made to viral genes or proteins may increase or decrease the host's response to a particular virus.

[0134] In certain embodiments, the recombinant Marek's disease virus or recombinant viral vector of the present invention may have a transgene encoding an IBDV viral protein or gene product, for example, an IBDV VP2 protein or gene product. In another embodiment, such recombinant virus or viral vector may have a transgene encoding an NDV viral protein or gene product, for example, an NDV F or HN protein or gene product. In another embodiment, such recombinant virus or viral vector may have a transgene encoding an avian influenza virus (AIV) viral protein or gene product, for example, an AIV HA or N protein or gene product. In another embodiment, such recombinant virus or viral vector may have a transgene encoding an infectious bronchitis virus (IBV) viral protein or gene product, for example, an IBV S1 or S2 protein or gene product. The transgene of the present invention may have more than one gene, including a gene fusion protein or gene product such as an NDV F-HN fusion protein, chimera, or gene product. In some embodiments, the complete coding sequence of such gene may be used to produce a full-length or fully functional protein or polypeptide. Alternatively, a portion or fragment of a viral protein or polypeptide may be sufficient to provide protection against or resistance to a specific virus or multiple viruses.

[0135] In certain embodiments, the recombinant Marek's disease virus or recombinant viral vector of the present invention may have a transgene encoding an immunomodulatory substance 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 of the genomic sites described herein. In some embodiments, the encoding transgene may be inserted into one of the sites described herein, and the transgene encoding a viral protein may be inserted into the other site. Other immunomodulatory substances such as interferons, chemokines, glucans, and granulocyte colony-stimulating factors may also be useful, and oligodeoxyribonucleotides 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 may 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 according to the present invention. Alternatively, viruses, their polymorphic variants, orthologues, or alleles may be cloned by immunological detection of homologs using an expression library with antiserum or purified antibodies directed against viruses from another species or part thereof.

[0137] Methods for preparing and screening cDNA libraries are well known in the art. For example, to prepare a cDNA library for cloning viral genes expressed by a genome, mRNA can be reverse transcribed into cDNA using reverse transcriptase. The cDNA may then be ligated into a vector, such as a recombinant vector, and introduced into host cells or organisms for propagation, screening, and cloning.

[0138] In the case of a genome library, DNA may be extracted from a desired tissue, digested using biological enzymes, or mechanically sheared. The resulting DNA fragments may then be isolated from undesirable fragments and constructed into a suitable vector, which may then be packaged in vitro. The recombinant vector can be analyzed by any method known in the art.

[0139] Nucleic acid sequences may be amplified from mRNA, from cDNA, or directly from a genomic library or cDNA library using methods such as polymerase chain reaction (PCR and RT-PCR) and ligase chain reaction (LCR). Denatured 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 may also be useful, for example, for cloning nucleic acid sequences encoding proteins to be expressed, for producing nucleic acids to be used as probes for detecting the presence of targeted diseases (e.g., MDV, NDV, and / or IBDV), for nucleic acid sequencing, or for encoding mRNA in biological samples for other purposes. Genes amplified by PCR can be purified from agarose and cloned into a suitable 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 poly(A) RNA, Northern blotting, dot blotting, insight hybridization, RNase protection, and high-density polynucleotide array techniques.

[0141] The viral genome or nucleic acid encoding a protein can be identified using high-density oligonucleotide array technology (e.g., GeneChip®) to identify the viral gene, ortholog, allele, its variants, and polymorphic variants in this invention. The selected genes can be cloned into intermediate vectors before being transformed into prokaryotic or eukaryotic cells for replication and / or expression. These intermediate vectors may be prokaryotic vectors, such as plasmids or shuttle vectors.

[0142] Nucleic acid modification Any number of methods well known to those skilled in the art can be used to isolate and manipulate DNA molecules. For example, polymerase chain reaction (PCR) techniques can be used to amplify a particular 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 the direct synthesis of fragments by chemical means. Thus, all or part of the nucleic acids described herein may be synthesized.

[0143] As used herein, the term “complementary nucleic acid” refers to two nucleic acid molecules that can specifically hybridize with each other to form a non-parallel double-stranded nucleic acid structure. In this view, one nucleic acid molecule is said to be a complement to another 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 with each other, at least under conventional low-stringent conditions. Similarly, these molecules are said to be complementary if they can hybridize with each other with sufficient stability to allow them to remain annealed with each other, under conventional high-low stringent conditions. Conventional stringent conditions are described by Sambrook, et al. (1989) and Haymes et al. (1985).

[0144] Deviations from perfect complementarity are permissible as long as the molecules retain the ability to form a double-stranded structure. Therefore, for a nucleic acid molecule or fragment of a nucleic acid molecule to function as a primer or probe, such a molecule or fragment only needs to be sufficiently complementary in sequence so that it can form a stable double-stranded structure at the specific 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 certain number of nucleotides, and some of the sequences 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. Nucleotide sequences observed from 5' to 3' are said to be “complementary” to a second nucleotide sequence observed from 3' to 5', or complementary to a second nucleotide sequence observed from 3' to 5', if the first nucleotide sequence exhibits complete complementarity with the second sequence or reference sequence. As used herein, a nucleic acid sequence molecule is said to exhibit “complete complementarity” if all nucleotides in one of the 5' to 3' segments of a sequence read are complementary to all nucleotides in the other sequence when read from 3' to 5'. A nucleotide sequence complementary to a reference nucleotide sequence exhibits the same sequence as its reverse complement.

[0146] Recombinant vectors and host cells Recombinant DNA vectors may be, for example, linear or circular plasmids. A vector system may be a single vector or plasmid, or two or more vectors or plasmids containing together the total DNA to be introduced into the genome of a host cell. Recombinant vectors described herein may be, for example, expression vectors for enabling the production of a desired protein in a host cell, such as a bacterial cell. Nucleic acid molecules, or their complements or fragments, described herein may be inserted into a vector under the control of a suitable promoter that functions in one or more microbial hosts and drives the expression of a linked coding sequence or other DNA sequence. Many vectors are available and known in the art for this purpose, and the selection of a suitable 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 may contain a variety of components depending on its function (e.g., DNA amplification or DNA expression) and the specific host cell to which it is suited. Vector components for bacterial transformation generally include, but are not limited to, one or more of the following: a signal sequence, an origin of replication, one or more selectable marker genes, and an inducible promoter that enables the 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 viral particle that has been genetically modified by the incorporation of one or more heterologous nucleic acid sequences (i.e., DNA encoding a viral gene or fragment or part thereof that is not identical to the nucleic acid sequence of a gene naturally present in the virus) into the viral genome. In infection of cells with recombinant Marek’s disease virus, the recombinant virus expresses heterologous genes in the form of heterologous polypeptides.

[0148] As used herein, “recombinant viral vector” or “viral vector” refers to a recombinant construct inserted into a virus for introduction into a host cell. Such vectors according to the present invention may be derived from any HVT strain. Where appropriate, a viral gene or protein-coding sequence may be incorporated into such recombinant viral vector described herein for introduction into chickens or other poultry for protection from 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 intergeneric region. The intergeneric region according to the present invention may be flanked by HVT UL35 and HVT UL36 in a unique long region of the genome. In some embodiments of the present invention, one or more heterologous nucleotides encoding an antigen may also be inserted into a region defined by the UL55 locus of the HVT genome. In some embodiments, the insertion site of the present invention may comprise all or part of a flanking gene on either side of the intergeneric region. By inserting one or more transgenes into one of these regions, the production of a recombinant viral vector can be enabled, which can then be introduced into chickens or other poultry for protection against one or more diseases.

[0150] As used herein, the term “operatably linked” means, when used in relation to regulatory sequences and nucleotide sequences, that the regulatory sequence causes the regulated expression of the linked structural nucleotide sequence. The terms “regulatory sequence,” “regulatory element,” or “control element” refer to nucleotide sequences located upstream (5' sequence), internally, or downstream (3' sequence) of a structural nucleotide sequence. Such sequences affect the timing and level or amount of transcription, RNA processing or stability, or translation of the associated structural nucleotide sequence. Examples of regulatory sequences include, but are not limited to, promoters, leader sequences, introns, enhancers, stem-loop structures, repressor-binding sequences, and polyadenylation-recognition sequences, and include, but are not limited to, the bovine growth hormone poly(A) signaling, Simian virus 40 (SV40) poly(A) signaling, Autographa californica nuclear polyhedron disease virus (AcNPV) 1629 ORF poly(A) signaling, and herpes simplex virus (HSV) thymidine kinase (TK) poly(A) signaling. Those skilled in the art will recognize that different combinations of promoters and / or regulatory elements may be used to increase or decrease the expression of the transgenes described herein.

[0151] Promoters that function in different species are also well known in the art. Promoters useful for polypeptide expression include inductive, viral, synthetic, or constitutive promoters, and / or tissue-specific, temporally regulated, spatially regulated, and spatially and temporally regulated promoters. For example, useful promoters according to the present invention include, but are not limited to, the pre-early (IE) cytomegalovirus human (CMV) promoter, the guinea pig CMV promoter, the SV40 promoter, the pseudorabies virus promoter, e.g., the pseudorabies virus promoter for the glycoprotein X promoter, herpes simplex virus-1, e.g., the alpha-4 promoter, the Marek's disease virus promoter (including any isolate or strain of MDV, such as MDV-1, MDV-2, etc.), and HVT, e.g., promoters that control the expression of glycoproteins, e.g., gC, gB, gE, or gI, the infectious laryngotracheitis virus promoter, e.g., the infectious laryngotracheitis virus promoter for the glycoprotein gB, gE, gI, gD genes, or any other suitable promoter. Those skilled in the art will recognize a method for identifying a useful promoter according to the present invention.

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

[0153] Vector construction and selection The 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 skilled in the art and may be carried out using standard recombinant DNA techniques. Recombinant DNA vectors or constructs may include selectable markers that confer a selectable phenotype to cells. Alternatively, cells containing exogenous nucleic acids encoding polypeptides or proteins described herein may be selected using the selectable markers. Such markers may encode, for example, biocide resistance or antibiotic resistance (e.g., kanamycin, G418, bleomycin, hygromycin, etc.). The selectable markers may include any markers that are well known to those skilled in the art and suitable for use in accordance with the present invention.

[0154] Recombinant vectors or constructs may also contain screenable markers that can be used to monitor expression but do not have the potential to induce cell death. Suitable screenable markers may include, for example, one or more of the various fluorescent protein genes such as β-glucuronidase or uidA genes (GUS), 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 with various chromogenic substrates, luciferase genes, xylE genes encoding catechol dioxygenase that converts chromogenic catechols, β-amylase genes, tyrosinase genes encoding enzymes that can oxidize tyrosine to DOPA and dopaquinone and subsequently condense into melanin, or α-galactosidases that catalyze chromogenic α-galactose substrates.

[0155] Protein expression in host cells To obtain high levels of expression of the cloned viral genes described herein, nucleic acids may be subcloned into an expression vector containing a strong promoter to direct transcription and a transcription / translation terminator. In the case of encoded proteins, 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 expressing proteins are available in several prokaryotic and eukaryotic species known in the art. Commercial kits for such expression systems are readily available. Eukaryotic expression systems for mammalian cells, yeast, and insect cells are well known in the art and are also commercially available.

[0156] The selection of an appropriate promoter for directing the expression of heterologous nucleic acids varies depending on the specific application. Such promoters may be located at a distance from the heterologous transcription start site similar to the distance in their natural setting, but those skilled in the art will understand that some variation in this distance may be tolerated without loss of promoter function.

[0157] In addition to promoters, expression vectors typically contain a transcription or expression cassette that includes all the elements necessary for nucleic acid expression in host cells. Genetic information can be transported into cells using any conventional vector known in the art that can be used for expression in eukaryotic or prokaryotic cells. Thus, a typical expression cassette contains a promoter operably ligated to a nucleic acid sequence encoding a selected nucleic acid, as well as corresponding signals necessary for efficient processing (e.g., ribosome binding sites, polyadenylation, and translation termination). Additional elements may include enhancers, and in the case of genomic DNA as a structural gene, introns having functional splice donor and acceptor sites.

[0158] In addition to promoter sequences such as the promoters described herein, expression cassettes may also contain transcription termination regions downstream of structural genes to provide efficient transcription termination. Termination regions may originate 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, and CAT may be included in the vector as markers for vector transduction. Epitope tags or sequence tags may also be added to recombinant proteins to provide a convenient isolation method.

[0159] Expression vectors containing regulatory elements derived from eukaryotic viruses are typically used in eukaryotic expression vectors, such as SV40 vectors, papillomavirus vectors, retrovirus vectors, and vectors derived from Epstein-Barr virus. Other exemplary eukaryotic vectors include pMSG, pAV009 / A+, pMTO10 / A+, pMAMneo-5, baculovirus pDSVE, and any other vectors that enable protein expression under the direction of the CMV promoter, SV40 early promoter, SV40 late promoter, metallothionein promoter, mouse mammary cancer virus promoter, Rous sarcoma virus promoter, polyhedrin promoter, or any 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 inductive promoters. In inductive promoters, expression levels are linked to the concentration of an inducer, such as tetracycline or ecdysone, by incorporating the response elements of these drugs into the promoter. High levels of expression can be obtained from inductive 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 include a replicon that functions in E. coli, an antibiotic resistance gene for selecting bacteria that possess the recombinant plasmid, and a specific restriction site in the non-essential region of the plasmid to allow insertion of a eukaryotic sequence. Any antibiotic resistance gene suitable for use with the present invention may be used.

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

[0163] After the expression vector or transgene has been introduced into host cells, the cells may be cultured under conditions optimal for the expression of the desired protein, which may be recovered using standard techniques known in the art. Viral pathogens or viral proteins, such as those described herein, may then be purified for use in diagnostic assays, for the preparation of antibodies and immunogenic compositions, and for the identification of antiviral compounds. Naturally occurring proteins may be purified from biological samples, such as tissue samples from birds infected with viruses as described herein, and recombinant proteins may 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 may be selectively adsorbed onto a purification column and then released from the column in a relatively pure form using a suitable ligand or substrate. The fusion protein may then be removed by enzymatic activity. The protein may 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 quantities by bacteria, for example, using inductive or constitutive promoters. Promoter induction using IPTG is an example of an inductive promoter system. Bacteria may be grown from fresh or frozen cultures according to standard procedures known in the art.

[0166] Proteins expressed in bacteria may form insoluble aggregates called inclusion bodies. Suitable protocols for purifying protein inclusion bodies are known in the art. Lysis of bacteria for recovery of expressed proteins can be performed using any method known in the art, including the introduction of chemical buffers, sonication, and mechanical disruption. Inclusion bodies may also be solubilized, and the lysed cell suspension may be centrifuged to remove unwanted cell debris. Inclusion body proteins can be regenerated by dilution or dialyzing with a suitable buffer.

[0167] Recombinant proteins can also be obtained from bacterial periplasm. After lysing of bacterial cells, the bacterial periplasm fraction can be isolated by any method known in the art. Recombinant proteins present in the supernatant can be isolated 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 difference filtration, which isolates proteins based on molecular weight using filtration through membranes of different pore sizes. Column chromatography can be used to isolate proteins from other proteins based on size, net surface charge, hydrophobicity, or affinity for a ligand or substrate. In addition, the protein may be immunopurified by conjugating an antibody against the protein of interest to a column. All of these methods are well known in the art. It will be obvious to those skilled in the art that chromatographic techniques can be carried out at any scale 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 may include antibody preparation by selecting antibodies from recombinant antibody libraries in phages or other vectors, as well as preparation of polyclonal and monoclonal antibodies by immunization of rabbits or mice.

[0170] Antibodies that specifically react to a desired viral pathogen can be produced using viral proteins or portions thereof, viral particles, and / or several antigens or antigenic regions, including nucleic acids. For example, recombinant viral proteins or their antigenic fragments can be isolated using methods described herein or any method 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) or recombinant proteins using methods known in the art. Synthetic peptides derived from viral sequences may also be used to generate antibodies, conjugate them 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 mouse or rabbit strains can be immunized with a protein using a standard adjuvant, such as the adjuvant described herein, using a standard immunization protocol known in the art. If a sufficiently high titer of antibody against that protein is obtained, an antiserum may be prepared and concentrated to obtain an antibody reactive against that protein.

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

[0173] Monoclonal antibodies and polyclonal serum may be collected and titrated against the desired antigen or protein using an immunoassay, such as a solid-phase immunoassay using a protein immobilized on a solid support. Antibodies specific to only a particular viral protein may be produced by removing other cross-reactive proteins. In this way, antibodies that bind only to selected proteins may be obtained.

[0174] When specific antibodies against desired viral antigens, such as proteins, viruses, and / or nucleic acids, become available, the desired antigens can be detected using various immunoassay methods. Antibodies can also be used therapeutically.

[0175] Proteins associated with or distinct from the viral particles described herein may 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 presented in the viral particles and / or epitopes defined by viral proteins isolated from the viral particles (e.g., which may be present in infected cells). The immunological assay may use antibodies that specifically bind to selected proteins or antigens. Antibodies may be produced by any of several methods well known to those skilled in the art. The immunoassay may also use labeling agents to specifically bind to the complex formed by the antibody and antigen for detection purposes. The labeling agent may itself be one of the parts containing 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 part, such as a secondary antibody that specifically binds to the antibody / antigen complex. The secondary antibody may be specific to the species of antibody from which the primary antibody originates. The labeling agent may be modified with a detectable part, such as biotin, to which another molecule (e.g., streptavidin) can specifically bind. Various detectable parts are well known to those skilled 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 competitive or non-competitive, and quantitative or non-quantitative. Non-competitive immunoassays are assays in which the antigen can be detected directly, and in some cases, the amount of antigen is measured directly. In competitive assays, viral antigens present in a sample are detected indirectly by a detectable signal associated with a known added (exogenous) viral antigen that has been replaced by an antiviral 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 in which any cross-reactive antibody can be removed from pooled antiserum. Additional assay types, including but not limited to Western blotting or liposome immunoassays, may also be used in accordance with the present invention.

[0177] Those skilled in the art will understand 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 include labels or detectable groups that do not significantly interfere with the specific binding of the antibody used in the assay. The detectable groups may be any material having detectable physical or chemical properties. Such detectable labels are known in the art, and generally, any label useful in this manner may be applied to the present invention. Therefore, as used herein, “label” may be any composition detectable by spectroscopic, photochemical, biochemical, immunochemical, electrical, optical, or chemical means. Useful labels in the present invention include magnetic beads (e.g., DYNABEADS®), fluorescent dyes (e.g., fluorescein isothiocyanate, Texas red, rhodamine, etc.), and radiolabels (e.g., 3 H, 125 1. 35 S, 14 C, or 32 P) may include an enzyme (e.g., horseradish peroxidase, alkaline phosphatase, and / or any other known in the art and used in ELISA), and a colorimetric label such as colloidal gold or colored glass or plastic beads (e.g., polystyrene, polypropylene, latex, etc.).

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

[0180] Non-radioactive labels can be attached by indirect means. Generally, ligand molecules (e.g., biotin) are covalently bound to the molecule. The ligand may then be conjugated to another molecule (e.g., streptavidin), which may be inherently detectable or covalently bound to a signaling system such as a detectable enzyme, fluorescent compound, or chemiluminescent compound. Ligands and their corresponding targets can be used in any suitable combination with antibodies that recognize viral antigens or secondary antibodies that recognize antiviral antigens. The molecules may also be directly conjugated to signaling compounds, for example, by conjugation to an enzyme or fluorophore. The enzymes of interest used as labels may be hydrolases such as phosphatases, esterases, and glycosidases, or oxidotases such as peroxidases. Fluorescent compounds may include fluorescein and its derivatives, rhodamine and its derivatives, dansyl, umbelliferone, and the like. Examples of chemiluminescent compounds include luciferin, 2,3-dihydrophthalazinedione, for example, luminol, or other compounds known in the art.

[0181] The means of 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 can be detected visually, for example, by an electron detector such as a charge-coupled device (CCD) or a photomultiplier tube. Similarly, enzyme labels can be detected by providing a suitable substrate for the enzyme and detecting the resulting reaction product. Colorimetric or chemiluminescent labels can 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 and may rather 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 as described herein may 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 as described herein comprises, for example, a recombinant vector having one or more transgenes as described herein inserted into a viral genome in an intergeneric region adjacent to the intergeneric locus UL35 / UL36 in the unique long (UL) region of the HVT genome. In one embodiment, the present invention provides a recombinant turkey herpesvirus (HVT) genome comprising one or more nucleotide sequences encoding one or more heterogeneous antigens inserted into the intergeneric locus UL35 / UL36 in the unique long region of the HVT genome, and one or more nucleotide sequences encoding one or more heterogeneous antigens inserted into the UL55 site in the unique long region (UL) of the HVT genome.

[0183] In other embodiments, proteins or peptides, their immunogenic fragments, and / or polynucleotides, as well as antiviral antibodies and / or T cells, may be incorporated into a pharmaceutical composition or immunogenic composition (e.g., a vaccine). In another embodiment, the immunogenic composition according to the present invention may include at least a third, fourth, and the like, which may encode additional viral proteins. In such a method, 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 vaccines, and attenuated vaccines, or non-replicating vaccines, or dead vaccines) or subunit vaccines, such as structural or non-structural viral proteins or their immunogenic fragments, can be used to treat or prevent viral infection by inducing an immune response in a subject. Alternatively, the pharmaceutical composition may include antigen-presenting cells transfected with viral polynucleotides so that the antigen-presenting cells express viral peptides.

[0184] The immunogenic compositions according to the present invention may be designed to generate antibody immunity and / or cellular immunity in a subject. Such compositions may comprise one or more such compounds together with pharmaceutically acceptable carriers that do not exist naturally. In other embodiments, the immunogenic compositions according to the present invention may comprise one or more adjuvants or pharmaceutically acceptable carriers, such that at least one of them does not exist naturally. The pharmaceutically acceptable carrier or adjuvant may 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. The pharmaceutically acceptable carrier or adjuvant may contain a substance designed to protect the antigen from rapid catabolism, e.g., aluminum hydroxide or mineral oil, or an immune response stimulant, e.g., a protein derived from Bortadella pertussis or Mycobacterium tuberculosis. Examples of commercially available adjuvants include Freund's incomplete and complete adjuvants, 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. Those skilled in the art will be able to identify suitable pharmaceutically acceptable carriers for use with the present invention.

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

[0186] Nucleic acid vaccines encoding viral genomes, structural proteins, non-structural proteins, or fragments thereof as 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. A suitable nucleic acid expression system may contain the DNA sequence necessary for expression in the target (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, reproducible virus.

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

[0188] Where described herein, immunogenic compositions may be combined with pharmaceutically acceptable carriers. The selection of a suitable carrier may be determined in part by the specific composition to be administered (e.g., nucleic acids, proteins, regulatory compounds, or transduced cells), as well as the specific method used to administer the composition. Thus, a wide variety of suitable formulations of the pharmaceutically or immunogenic compositions that can be used in the present invention are available. Administration may be by any convenient method, for example, by injection, oral administration, inhalation, transdermal application, or rectal administration. Injections of recombinant vectors or immunogenic compositions described herein may be provided to subjects such as poultry in single doses or in multiple doses, or in more than one dose, such as in repeated doses.

[0189] For example, suitable formulations for parenteral administration via intra-articular, intravenous, intramuscular, intradermal, intraperitoneal, inovo, and subcutaneous routes include aqueous and non-aqueous isotonic sterile injection solutions that may contain antioxidants, buffers, bacteriostatic agents, and solutes to make the formulation isotonic with the blood of the intended target, as well as aqueous and non-aqueous sterile suspensions that may contain suspending agents, solubilizers, thickeners, stabilizers, and preservatives. In the embodiment of the present invention, the composition may be administered, for example, by intravenous infusion, orally, topically, intraperitoneally, intravesically, or subarachnoidally.

[0190] Such compositions may also comprise a buffer (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 to make the formulation isotonic, hypotonic, or weakly hypertonic with respect to the target blood, suspending agents, thickeners, and / or preservatives. Alternatively, the compositions of the present invention may be formulated as lyophilized products. The compounds may also be encapsulated in liposomes using methods known in the art.

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

[0192] Formulations suitable for oral administration may consist of (a) a liquid solution, e.g., a diluent, e.g., an effective amount of packaged viral protein or nucleic acid suspended in water, saline or PEG400; (b) capsules or tablets, each containing a predetermined amount of the active ingredient, in liquid, solid, granule or gelatin form; (c) a suspension in a suitable liquid; or (d) a suitable emulsion. Tablet forms may contain one or more of the following: 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, flavoring agents, dyes, disintegrants, and pharmaceutically suitable carriers. The lozenge form may include aromatic tablets containing an active ingredient in a flavor (e.g., sucrose), as well as the active ingredient in addition to the active ingredient, in an inert base such as gelatin and glycerin, or sucrose and acacia emulsion, gel, which contains a carrier known in the art.

[0193] The selected compound can be prepared, alone or in combination with other suitable components, into an aerosol formulation administered by inhalation. The aerosol formulation may be placed in a pressurized, acceptable propellant such as dichlorodifluoromethane, propane, or nitrogen.

[0194] In connection with the present invention, the dose administered to a subject should be sufficient to influence 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 body weight and / or surface area of ​​the patient being treated. The size of the dose may also be determined by the presence, 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 comprising the vectors described herein, the effective amount of vector administered may be determined in part on the circulating plasma level of the vector, vector toxicity, the health of the subject, and the production of anti-vector antibodies.

[0195] With regard to administration, the compounds of the present invention and transduced cells can be administered at a rate determined by the LD-50 of the inhibitor, vector, or transduced cell type, as well as by the side effects of the inhibitor, vector, or cell type at various concentrations, such as that applicable to the subject's body weight and overall health condition. 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 may 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 of antibodies against viral proteins and viral antigens In one embodiment of the present invention, the presence of the virus, viral nucleic acid, or viral protein described herein in a sample can be determined by an immunoassay. Enzyme-mediated immunoassays such as immunofluorescence assays (IFA), enzyme-linked immunosorbent assays (ELISA), capture assays, microagglutination assays, and immunoblotting assays (e.g., Western blotting) can be readily adapted to achieve detection of the virus or viral protein. The ELISA method may be effective for the detection of the virus or viral protein described herein. Such an ELISA may include, for example, the steps of: (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; (3) contacting the biological sample with an antibody bound to a detectable portion (e.g., horseradish peroxidase enzyme or alkaline phosphatase enzyme); (4) contacting the biological sample with a substrate for the enzyme; (5) contacting the biological sample with a detection reagent such as a color reagent; and (6) observing the detectable result. In some embodiments, suitable biological samples for use in such ELISAs may be blood or other fluids. In another embodiment, the ELISA described herein may detect viruses or viral proteins in tissue samples. Such methods can be readily modified by those skilled in the art to detect the presence of antiviral antibodies or specific viral proteins, as well as viruses, in a sample. In certain embodiments, the ELISA according to the present invention may detect the presence of antiviral antibodies.

[0198] The ELISA assay described herein may include a nitrocellulose strip impregnated with the viral protein described herein. The nitrocellulose strip may produce a visual result when in contact with a test sample containing an antiviral nucleoprotein antibody. Such a test may identify subjects that already have antibodies against the viral protein, and thus subjects may have immunity to the virus. Administration of the immunogenic composition to prevent viral infection described herein may be unnecessary in such subjects, and therefore, identifying subjects that already have immunogenic antibodies can prevent unnecessary administration of immunogenic compounds to such subjects. In this view, one embodiment of the present invention may include identifying subjects lacking antiviral antibodies using an assay described herein, such as an ELISA assay, and then providing those subjects with the immunogenic composition described herein to prevent viral infection. In another embodiment, the nitrocellulose strip for use in the ELISA according to the present invention may be impregnated with an antibody, such as an antiviral antibody, and may produce a visual result when in contact with a test sample containing a viral protein. Such a test may identify subjects infected with the virus described herein.

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

[0200] As will be readily apparent to those skilled in the art, the biological sample for use in the above assay may be taken directly from the subject or in a partially purified form. Antibodies specific to a particular virus react by binding to the virus as a primary reaction. Subsequently, a secondary reaction with an antibody bound to or labeled to a detectable portion may be added to enhance the detection of the primary reaction. Generally, in the secondary reaction, antibodies or other ligands that are specifically or nonspecifically reactive to 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 portion can be detected by visual inspection of precipitates or color changes, visual inspection by microscopy, or automated detection by methods such as spectroscopy or radiometric measurement. Examples of detectable portions include fluorescein and rhodamine (for fluorescence microscopy), horseradish peroxidase (for optical or electron microscopy and biochemical detection), biotin-streptavidin (for optical or electron microscopy), and alkaline phosphatase (for biochemical detection by color change). The detection methods and portions used can be selected from, for example, those disclosed herein or any available in the art.

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

[0203] Next, the amplified nucleotides, such as cDNA, may be separated and detected using techniques well known in the art, such as gel electrophoresis. Similarly, the presence of viral polynucleotides in a biological sample may be detected using an oligonucleotide probe that specifically hybridizes with viral polynucleotides in a hybridization assay.

[0204] The virus-specific nucleic acid probes or primers described herein may be generated using the polynucleotide sequences disclosed herein. The probes are preferably fragments of at least about 12, 15, 16, 18, 20, 22, 24, or 25 nucleotides, or other polynucleotide sequences encoding viral nucleic acid or polypeptides. The nucleic acid probes may be 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 less than 0.05 kb in length. The probes may be generated, for example, by chemical synthesis, PCR amplification, generation from longer polynucleotides using restriction enzymes, or by other methods known in the art. The polynucleotides described herein may also be used in methods or assays involving the use of solid substrates, such as arrays. Such arrays may have one or more different polynucleotides that can be immobilized on the array using methods known in the art.

[0205] In some embodiments, the polynucleotides of the present invention may be detectably labeled. Detectable labels include, but are not limited to, radioactive labels, fluorescent dyes such as fluorescein isothiocyanate (FITC), rhodamine, Texas Red, phycoerythrin, allophycocyanin, 6-carboxyfluorescein (6-FAM), 2’,7’-dimethoxy-4’,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 radioactive labels such as H, etc. In some embodiments, the detectable label 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 a specific viral nucleic acid (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 well known in the art. For the detection of viral polynucleotides in blood or blood-derived samples, methods that enable the detection of single base pair mismatches can be used.

[0207] Since 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 prior to performing a hybridization assay.

[0208] Nucleic acid probes may be prepared using viral genomes described herein. Such probes may contain at least about 8 nucleotides or more and may be prepared synthetically or by excision from recombinant polynucleotides. The probes described herein may be hybridized with viral nucleic acids and therefore may be useful for detecting specific viruses in biological samples. The probes described herein may also be useful for identifying infected objects and for further characterization of viral genomes. Probes for detecting viral polynucleotides (natural or derived) may be of a specific length or may have sequences that allow for the detection of unique viral sequences by hybridization. About 6 to 8 nucleotides may be useful, but longer sequences, such as sequences of about 10 to 12 nucleotides or about 20 or more nucleotides, may be preferred. Those skilled in the art will recognize methods for preparing and using 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 the preparation of such probes may include complements to any specific portion of a viral genome, for example, a portion of the viral genome that allows a particular virus to be distinguished from other viruses that may be present in the sample. The probes described herein may have perfect complementation to the target sequence of interest, or they may have one or more mismatches. A useful probe according to the present invention having one of many mismatches will still hybridize to the target sequence of interest. To use such probes as diagnostic agents, the biological sample to be analyzed may be treated prior to analysis, if desired, to extract the nucleic acids contained therein. The nucleic acids obtained from the sample may be subjected to gel electrophoresis or other size separation techniques. The probes may be labeled with detectable labels described herein. Suitable labels and methods for labeling probes may include any labels known in the art and described herein, or other labels useful in the present invention.

[0210] The probe may be perfectly complementary to the viral genome or a portion thereof (e.g., all or part of the sequences encoding the viral proteins described herein). High stringency conditions may be desirable to prevent or at least minimize false-positive results. The stringency of hybridization may be determined by several factors during hybridization and washing, including temperature, ionic strength, duration, and reagent concentration. The probe or nucleic acid from the sample may be provided in solution for such assays or attached to a support (e.g., a solid or semi-solid support). Examples of supports that may be used include, but are not limited to, nitrocellulose (e.g., in membrane or microtiter well form), polyvinyl chloride (e.g., sheet or microtiter well), and polystyrene latex (e.g., beads or microtiter plates, polyvinylidine fluoride, diazotized paper, nylon membranes, activated beads, and protein A beads).

[0211] In one embodiment, a probe or sample nucleic acid may be provided on an array for detection. The array can be constructed, for example, by spotting polynucleotide probes onto a two-dimensional matrix or a substrate in the array (e.g., glass, nitrocellulose, etc.). The probe may be bound to the substrate by either covalent bonding or nonspecific interactions such as hydrophobic interactions. A sample of polynucleotide can be detected by labeling (e.g., using radioactive or fluorescent labeling) and then hybridized to the probe. A double-stranded polynucleotide containing the labeled sample polynucleotide bound to the probe polynucleotide can be detected after the unbound portion of the sample is removed. Techniques for constructing arrays and methods for using these arrays are known in the art. The array 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, the array facilitates rapid and convenient analysis.

[0212] Diagnostic tests and kits The present invention further provides diagnostic reagents and kits comprising one or more such reagents for use in a variety of diagnostic assays, including, for example, immunoassays such as ELISA and "sandwich" type immunoassays, and nucleic acid assays (e.g., PCR assays). In relevant embodiments, the assay may be performed in flow-through or strip test form, and the binder is immobilized on a membrane such as nitrocellulose. Such a kit may preferably comprise 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 comprise an immunogenic composition such as a recombinant virus as described herein. Other compounds such as reagents and pharmaceutically acceptable carriers may be included in the kit. When provided in such a kit, the immunogenic composition may be a solution, such as a pre-measured dose or volume, or 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 applied to the surface of the solid support when the kit is used. The signal generation means may be pre-associated with the antibody or nucleic acid of the present invention, or may require a combination with one or more components, such as a buffer, nucleic acid, antibody-enzyme complex, enzyme substrate, etc., before use.

[0213] The kit may also include additional reagents, such as blocking reagents to reduce nonspecific binding to the solid-phase surface, washing reagents, enzyme substrates, and enzymes. The solid-phase surface may be in the form of a microtiter plate, microspheres, or other material suitable for immobilizing nucleic acids, proteins, peptides, or polypeptides. An enzyme that catalyzes the formation of a chemiluminescent or chromogenic product or the reduction of a chemiluminescent or chromogenic substrate is one such component of the signal-generating means. Such enzymes are well known in the art. If radioactive labels, chromogenic labels, fluorescent labels, or 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 in a second separate container in which the second composition is placed and appropriately divided. Alternatively, the detection reagent and label may be prepared in a single container.

[0214] All specified patents and other publications are expressly incorporated herein by reference for the purpose of describing and disclosing methodologies described in such publications that may be used in connection with the present invention. These publications are provided only for their disclosures prior to the filing date of this application.

[0215] The present invention is further illustrated and supported by the following embodiments. However, these embodiments should not be considered in any way to further limit the scope of the invention. Rather, those skilled in the art will readily understand that other embodiments, modifications, and equivalents of the invention exist without departing from the spirit and / or the accompanying claims of the invention. The following is a description of the claims as they were at the time of filing the application. [Claim 1] A recombinant turkey herpesvirus (HVT) genome comprising one or more nucleotide sequences encoding one or more heterologous antigens inserted into the intergenetic locus UL35 / UL36 in the unique long (UL) region of the HVT genome. [Claim 2] A recombinant turkey herpesvirus (HVT) genome comprising one or more nucleotide sequences encoding one or more heterologous antigens inserted into the intergenetic 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. [Claim 3] Recombinant HVT according to claim 1 or 2, wherein the one or more heterologous antigens are protective against 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), avian anemia virus (CAV), and avian influenza virus (AIV). [Claim 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 the chicken anemia virus (CAV) c) The F / HN chimeric protein of the Newcastle disease virus (NDV), or the F, NP, P, M, HN, or L protein, d) S1, S2, or M protein of infectious bronchitis virus (IBV) e) The gB, gC, gD, gE, gH, gI or gL proteins of the infectious laryngotracheitis virus (ILTV), and f) Recombinant HVT according to any one of the prior claims, selected from the group consisting of any of the HA, NA, NP, or M proteins of the avian influenza virus (AIV). [Claim 5] Recombinant HVT according to any one of the prior claims, wherein one or more heterologous antigens are protective against IBDV. [Claim 6] Recombinant HVT according to claim 5, wherein one or more heterologous antigens are the VP2 protein of IBDV. [Claim 7] The recombinant HVT according to claim 6, wherein the VP2 protein sequence is encoded by a nucleotide sequence having at least 80% sequence identity with respect to the nucleotide sequence comprising SEQ ID NO: 5 or SEQ ID NO: 10. [Claim 8] The recombinant HVT according to claim 6, wherein the VP2 protein is encoded by a nucleotide sequence comprising SEQ ID NO: 5 or SEQ ID NO: 10. [Claim 9] Recombinant HVT according to any one of claims 1 to 4, wherein one or more heterologous antigens or multiple antigens are protective against Newcastle disease virus (NDV). [Claim 10] The recombinant HVT according to claim 9, wherein one or more heterologous antigens comprise the F protein of NDV. [Claim 11] The recombinant HVT according to claim 10, wherein the F protein of the NDV is encoded by a nucleotide sequence having at least 80% sequence identity with respect to the sequence containing SEQ ID NO: 3. [Claim 12] The recombinant HVT according to claim 10, wherein the F protein of NDV is encoded by a nucleotide sequence including SEQ ID NO: 3. [Claim 13] Recombinant HVT according to any one of the prior claims, wherein one or more heterologous antigens are protective against NDV and IBDV. [Claim 14] Recombinant HVT according to claim 13, wherein the at least one heterologous antigen comprises the F protein of NDV and the VP2 protein of IBDV. [Claim 15] Recombinant HVT according to claim 14, wherein the F protein of NDV is encoded by a nucleotide sequence having at least 80% sequence identity with respect to the nucleotide sequence containing SEQ ID NO: 3, and the VP2 protein of IBDV is encoded by a nucleotide sequence having at least 80% sequence identity with respect to the nucleotide sequence containing SEQ ID NO: 5 or SEQ ID NO: 10. [Claim 16] The recombinant HVT according to claim 14 or 15, wherein the F protein of NDV is encoded by a nucleotide sequence including SEQ ID NO: 3, and the VP2 protein of IBDV is encoded by a nucleotide sequence including SEQ ID NO: 5 or SEQ ID NO: 10. [Claim 17] Recombinant HVT according to any one of the prior claims, comprising a genome including one or more expression cassettes each containing one or more nucleotide sequences encoding one or more heterologous antigens. [Claim 18] The recombinant HVT genome according to claim 17, wherein the expression cassette comprises one or more promoters. [Claim 19] Recombinant HVT according to claim 18, wherein one or more promoters are selected from the group consisting of the pre-initial cytomegalovirus human (hCMV) promoter, guinea pig pre-initial CMV promoter, mouse pre-initial CMV promoter, Pec promoter, β-chicken triactin promoter, SV40 promoter, pseudorabies virus promoter with glycoprotein X promoter, herpes simplex virus-1 alpha-4 promoter, Marek's disease virus promoter with glycoprotein gA, gC, gB, gE, or gI promoter, infectious laryngotracheitis virus promoter with glycoprotein gB, gE, gI, gD promoter, and bovine herpesvirus 1 / 1 VP8 promoter. [Claim 20] Recombinant HVT according to claim 19, wherein the one or more promoters include the human CMV promoter. [Claim 21] Recombinant HVT according to claim 19, wherein the one or more promoters include the mouse CMV promoter. [Claim 22] The recombinant HVT according to claim 19, wherein the one or more promoters include the hCMV promoter and the mCMV promoter. [Claim 23] Isolated DNA encoding a recombinant HVT genome according to any one of claims 1 to 22. [Claim 24] An immunogenic composition comprising recombinant HVT according to any one of claims 1 to 23, further comprising a pharmaceutically acceptable carrier, excipient, or adjuvant. [Claim 25] A vaccine composition comprising recombinant HVT according to any one of claims 1 to 23, further comprising a pharmaceutically acceptable carrier, excipient, or adjuvant. [Claim 26] The vaccine according to claim 25, further comprising an additional Marek's disease virus (MDV) selected from the group consisting of a naturally attenuated MDV-1 strain Rispens (CVI-988) or three Gallid herpesvirus strains SB-1 virus. [Claim 27] The vaccine according to claim 26, wherein the additional MDV comprises a recombinant genome. [Claim 28] The vaccine according to 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. [Claim 29] The vaccine according to any one of claims 25 to 28, for use in the vaccination of birds against one or more diseases caused by one or more avian pathogens. [Claim 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. [Claim 31] The vaccine according to 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. [Claim 32] The vaccine according to 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). [Claim 33] The vaccine according to claim 32, wherein the one or more avian pathogens include the Newcastle disease virus. [Claim 34] The vaccine according to claim 32, wherein the one or more avian pathogens include the infectious bursal disease virus (IBDV). [Claim 35] The vaccine according to claim 32, wherein the one or more avian pathogens include the Newcastle disease virus and the infectious bursal disease virus. [Claim 36] Use of the vaccine according to any one of claims 25 to 35 in vaccination of birds, wherein the vaccine is administered by at least one administration of the vaccine by spray administration, in ovo administration, subcutaneous administration, intramuscular administration, oral administration, nasal administration, or a combination thereof. [Claim 37] The vaccine according to claim 36, wherein the vaccine is administered by in ovo administration. [Claim 38] The vaccine according to claim 37, wherein the in ovo administration is performed on embryonated eggs between about 16 and 22 days after hatching. egg) is performed. [Claim 39] The vaccine according to any one of claims 36 to​​​​​​​The vaccine according to claim 36, wherein the administration of the vaccine includes spray administration. [Claim 42] A method for vaccinating birds to treat or prevent Marek's disease and one or more avian diseases caused by one or more avian pathogens, comprising the step of administering an effective amount of a vaccine composition according to any one of claims 25 to 35. [Claim 43] The method according to 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), avian anemia virus (CAV), and avian influenza virus (AIV). [Claim 44] The method according to claim 43, wherein the one or more avian pathogens include the infectious bursal disease virus (IBDV). [Claim 45] The method according to claim 43, wherein the one or more avian pathogens include the Newcastle disease virus (NDV). [Claim 46] The method according to claim 43, wherein the one or more avian pathogens include the infectious bursal disease virus (IBDV) and the Newcastle disease virus (NDV). [Claim 47] A method for inducing an immune response in an animal avian to Marek's disease virus and one or more avian pathogens, comprising the step of administering to the bird an effective amount of an immunogenic composition or vaccine composition according to any one of claims 24 to 35. [Claim 48] The method according to 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), avian anemia virus (CAV), and avian influenza virus (AIV). [Claim 49] The method according to claim 48, wherein the one or more avian pathogens include the infectious bursal disease virus (IBDV). [Claim 50] The method according to claim 48, wherein the one or more avian pathogens include the Newcastle disease virus (NDV). [Claim 51] The method according to claim 48, wherein the one or more avian pathogens include the infectious bursal disease virus (IBDV) and the Newcastle disease virus (NDV). [Claim 52] The method according to any one of claims 42 to 51, wherein the administration is carried out by spray administration, inovo administration, subcutaneous administration, intramuscular administration, oral administration or nasal administration. [Claim 53] The method according to claim 52, wherein the route of administration includes inovactor administration. [Claim 54] The method according to claim 53, wherein the inovo administration is performed on an ovum between approximately 16 and 22 days after development. [Claim 55] The method according to any one of claims 52 to 54, wherein the inovo administration is performed on a developing egg approximately 18 days after development. [Claim 56] The method according to any one of claims 52 to 55, wherein the administration route includes inovo administration followed by spray administration. [Claim 57] The method according to claim 52, wherein the administration route includes spray administration. [Claim 58] The method according to 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 quail. [Claim 59] The method according to claim 58, wherein the bird includes a chicken. [Examples]

[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 using GeneArt (ThermoFisher). 2.5 µg of the 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 into 0.006 moi (1.5 x 10⁻¹⁶) cells. 4 pfu / 2.5x10 6 Cells were infected with HVT. Three days later, these cells were placed in T75 (1x10) containing fresh CEF. 7 Cells (T75) were passaged at a 1:15 ratio. These cells were then plated at a 1:50 ratio on 24-well plates after 3 days. Cells from wells containing green fluorescent foci were plated on 96-well plates using fresh cells (6 x 10^4 cells / well) at dilutions of 1:200, 1:500, and 1:1000. Each well containing a single green focus was purified three times using the limiting dilution method on a 96-well plate. The purified virus was expanded using CEF cells to create a frozen stock, which was named "HVT-gfp-B".

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

[0218] Construction of HVT-gfp-A modified transfer plasmids The modified transfer plasmid HVT-gfp-A (SEQ ID NO: 16) was created by applying site-directed mutagenesis using two pairs of primers (upper primer pair for generating SbfI upstream of the gfp gene: SEQ ID NOs: 40 and 41, lower primer for generating SbfI downstream of the gfp gene: SEQ ID NO: 42, 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 into secondary CEF cells on a 6-well plate using 7.5 μL of PEI (polyethyleneimine). Green fluorescent foci became apparent at the first passage. After three rounds of purification by the limiting dilution method, one clone of HVT-gfp-A was further expanded to create a frozen stock.

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

[0220] Example 2 Construction of HVT-IBD Construction of HVT-IBD #1 The HVT-IBD #1 transfer plasmid (SEQ ID NO: 20) was chemically synthesized by GeneArt, ThermoFisher). 2.5 μg of the above plasmid was transfected into secondary CEF cells using the 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 containing fresh CEF (1x10 7Cells (T75) were passaged at a ratio of 1:7.5. These cells were then plated into 10 of a 96-well plate, and a replica plate was prepared after 3 days. One set of plates was fixed and stained with anti-IBDV chicken serum. Two wells containing foci positively stained for IBD were identified. The corresponding wells containing positively stained foci were purified in 3 rounds using the limiting dilution method with a 96-well plate. The purified virus was expanded using CEF cells, and a frozen stock was prepared. This was named "HVT-IBD #1".

[0221] PCR analysis of different clones using primers immediately outside the UL55-Gene3 integration site (upper primer: SEQ ID NO: 46, lower primer: SEQ ID NO: 47, Panel A) yielded a 2.414 kb band, while the PCR band of the original vector was 1.922 kb. Correct integration was further confirmed by using primers around the junction downstream of the insertion site (upper primer SEQ ID NO: 48 localized within the IBDV VP2 coding region, lower primer SEQ ID NO: 49 localized 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 performed using primers around the junction upstream of the insertion site (upper primer SEQ ID NO: 50 localized upstream of the transfer plasmid, lower 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] HVT-IBD #5 Construction The HVT-IBD #5 transfer plasmid (SEQ ID NO: 21) was chemically synthesized by GeneArt (ThermoFisher). JBJ-1 cells (chicken fibroblast cell line) in a 6-well plate were transfected with 2.5 ug of the aforementioned plasmid using LTX transfection reagent (Invitrogen). Transfected cells were infected with HVT at a dose of 0.05 moi approximately 5 hours after transfection. Transfected / infected cells were amplified by serial passage (1:4 to 1:10), and a portion was then seeded in a 96-well plate at limiting dilution. IBDV VP2 antigen expression was evaluated by staining a monolayer of live cells with antibody without fixation. See Figures 4A and 4B. Stained foci were collected by cell trypsination using cloning cylinders placed around the positive foci. This "live cell staining" and subsequent passage with cloning cylinders were repeated four times to obtain a pure VP2-positive culture. This culture was amplified by serial passage on JBJ-1 cells, followed by final amplification on primary CEF cells in a roller bottle. A frozen cell stock was prepared using the collected CEF cells and named "HVT-IBD #5".

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

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

[0225] Lysates of infected cells were prepared and Western blot analysis was performed using a monoclonal antibody against IBDV R63. Protein bands of approximately 50 kD were observed in all lanes except for the lane containing the lysate of the HVT-gfp-A vector. See Figure 6.

[0226] PCR analysis of clones using two sets of primers to confirm correct integration. First primer set targeting the upstream integration site: upper primer 5'-sequence number 56 localized upstream of the UL35-UL36 integration site, lower primer sequence number 57 localized within the Pec promoter. As expected, a 0.911 kb PCR band was obtained from this primer set. Second primer set targeting the downstream integration site: upper primer 5'-sequence number 58 localized within the IBDV VP2 coding region, lower primer 5'-sequence number 59 localized downstream of the UL35-UL36 insertion site. As expected, a 1.244 kb PCR band was obtained. See Figures 7A and 7B.

[0227] HVT-IBD #9 Construction The HVT-IBD #9 transfer plasmid (SEQ ID NO: 23) was chemically synthesized by GeneArt (ThermoFisher). 2.5 ug of the 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 a concentration of 0.075 moi. Three days later, these cells were passaged three times at a 1:10 ratio against fresh CEF cells in T75 (1 x 10^7 cells / T75). These cells were then plated into 10 of a 96-well plate, yielding 90 non-green foci. Three of these were positively stained with anti-IBDV chicken serum. Two clones were purified in three rounds using the limiting dilution method in a 96-well plate. The purified virus was expanded using CEF cells to create frozen stocks. We named this "HVT-IBD #9".

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

[0229] HVT-IBD #30 Construction The HVT-IBD #30 transfer plasmid (SEQ ID NO: 24) was chemically synthesized by GeneArt, ThermoFisher. Secondary CEF cells were co-transfected with 0.1 ug of the aforementioned plasmid and 2.5 ug of HVT in a 6-well plate using PEI (polyethyleneimine) transfection reagent. After 3 days, these cells were passaged at a 1:12 ratio on fresh CEF cells. Focuses expressing IBD VP2 were visualized by staining the unfixed cultures with chicken polyclonal serum for IBDV, and these foci were marked using a fluorescence microscope. A total of 16 positive foci were passaged on fresh CEF cells by trypsinization using a cloning cylinder, and the foci were separated from the foci that did not express VP2. Four of these cultures were cloned three times using the same procedure and then amplified on primary CEF cells in a roller bottle. A frozen cell stock was established and named "HVT-IBD #30".

[0230] PCR analysis of four different clones using primers for the upstream region of the UL55-Gene3 integration site (upper primer: SEQ ID NO: 66, lower primer: SEQ ID NO: 67, Panel A) yielded a 1.673 kb band. Correct integration was further confirmed by using primers around the 3' junction of the insertion site (upper primer SEQ ID NO: 68 localized within the IBDV VP2 coding region, lower primer SEQ ID NO: 69 localized 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 (upper primer SEQ ID NO: 70, lower 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 The HVT-IBD #31 transfer plasmid (SEQ ID NO: 25) was chemically synthesized using GeneArt and ThermoFisher. 0.01 ug of linear transfer plasmid (digested using EcoR1 and HindIII) was co-transfected in a 6-well plate with 2.5 ug of HVT-gfp-A digested in secondary CEF cells with Sbf1 using PEI (polyethyleneimine) transfection reagent. Four days after transfection, one non-green focus was observed, while no focus was observed when HVT-gfp-A digested with Sbf1 alone was used. After passage, the two non-green focuses were purified three times using the limiting dilution method in a 96-well plate. The purified virus was expanded using CEF cells to create a frozen stock, which was named "HVT-IBD #31".

[0232] Lysates of infected cells were prepared and Western blot analysis was performed using a monoclonal antibody against IBDV R63. A protein band of approximately 50 kD was observed in all lanes that lacked mAbs for IBDV R63 and had anti-IBDV chicken serum as the sole probe. See Figures 10A and B.

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

[0234] HVT-IBD #34 Construction The HVT-IBD #34 transfer plasmid (SEQ ID NO: 28) was chemically synthesized by GeneArt, ThermoFisher. 2.5 ug of the 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-GFP-B at 0.05 moi. After 3 days, these cells were placed in a T75 (1x10) container with fresh CEF cells. 7 Cells (T75) were passaged at a 1:15 ratio. Infected cells were plated onto 10 × 96 well plates, grown for 3 days, and then passaged in 96-well replicate plates. One replicate was fixed and stained with anti-IBDV chicken serum, and three wells containing foci positively stained for IBDV were identified. The corresponding wells from the live cell replicate plates were purified using 96-well plates by 3 rounds of limiting dilution cloning. One of the purified viruses was expanded using CEF cells to create a frozen stock, which was named "HVT-IBD #34".

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

[0236] HVT-ND #38 Construction The 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 co-transfected with Sbf1-digested HVT-gfp-A DNA using PEI (polyethyleneimine, 7.5 μL) in a 6-well plate containing secondary CEF cells. Six days after transfection, the transfected cells were plated on a 96-well plate and stained 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 named "HVT-ND #38".

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

[0238] HVT-ND #39 Construction The HVT-IBD #39 transfer plasmid (SEQ ID NO: 30) was chemically synthesized by BioBasic Inc. Secondary CEF cells were co-transfected with 0.01 ug of the aforementioned plasmid and 2.5 ug of HVT in a 6-well plate using PEI (polyethyleneimine) transfection reagent. After 6 days, these cells were passaged at 1:24 on 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 fluorescence microscope. A total of four positive foci were passaged on fresh CEF cells by trypsinization using a cloning cylinder, and the foci were separated from foci that did not express F protein. Four of these cultures were cloned three times using the same procedure and then amplified on primary CEF cells in a roller bottle. A frozen cell stock (clone 2 in the figure) was set aside and named "HVT-ND #39".

[0239] PCR analysis of three different clones using primers for the upstream region of the UL35-UL36 integration site (upper primer: SEQ ID NO: 90, lower primer: SEQ ID NO: 91 localized within the chicken beta-actin promoter, Panel A) yielded a 0.835 kb band. Correct integration was further confirmed by using primers around the junction downstream of the insertion site (upper primer SEQ ID NO: 92 localized within the poly-A region, lower primer SEQ ID NO: 93 localized downstream of the UL35-UL36 insertion site, Panel B). As expected, a 0.856 kb PCR band was obtained. The correct construct was further confirmed by using primers outside the expression cassette (upper primer SEQ ID NO: 94, lower 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 The HVT-IBD #40 transfer plasmid (SEQ ID NO: 31) was chemically synthesized by BioBasic Inc. The HindIII-digested transfer plasmid for HVT-ND #40 was co-transfected with Sbf1-digested HVT-gfp-A DNA using PEI (polyethyleneimine, 7.5 μL) in a 6-well plate containing secondary CEF cells. Seven days after transfection, the transfected cells were plated on a 24-well plate and stained with NDV chicken serum. 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 named "HVT-ND #40".

[0241] PCR analysis of four different clones using primers for the upstream region of the UL35-UL36 integration site (upper primer: SEQ ID NO: 96, lower primer: SEQ ID NO: 97 localized within the chicken beta-actin promoter, Panel A) yielded a 0.835 kb band. Correct integration was further confirmed by using primers around the junction downstream of the insertion site (upper primer SEQ ID NO: 98 localized within the NDV F coding region, lower primer SEQ ID NO: 99 localized downstream of the UL35-UL36 insertion site, Panel B). As expected, a 0.856 kb PCR band was obtained. The correct construct was further confirmed by using primers outside the expression cassette (upper primer SEQ ID NO: 100, lower 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. Cloning plasmids were chemically synthesized by DNA2.0. PCR amplification of the NDV F gene expression cassette of the HVT-ND #42 transfer plasmid was performed using the following primers: upper primer, SEQ ID NO: 102; lower primer, 5'-SEQ ID NO: 103. The amplified PCR fragments were cloned to the AscI and NheI sites UL55 / gene3 to construct the final transfer plasmid HVT-ND #42 (SEQ ID NO: 35).

[0243] In 6-well plates, HVT-infected CEF cells were transfected with a transfer plasmid using Lipofectamine LTX. Three days after transfection, the transfected / infected cells were plated into 6-well replicate plates and then plated into 96-well plates to screen for ND-expressing foci by staining with NDV antiserum. Wells corresponding to those containing ND-expressing 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 named "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 3.597kb 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. Primer set 2: upper primer: SEQ ID NO: 106, lower primer: SEQ ID NO: 107, yielded a 2.243kb band. Primer set 3: upper primer: SEQ ID NO: 108, lower primer: SEQ ID NO: 109, yielded a 2.356kb PCR band. Primer set 4: upper primer: SEQ ID NO: 110, lower primer: SEQ ID NO: 111, yielded a 2.424kb PCR band. Primer set 5: upper primer: SEQ ID NO: 112, lower primer: SEQ ID NO: 113, yielded a 2.170kb PCR band. Correct insertion was further confirmed by using primers around the junction downstream of the insertion site (Primer Set 6: Upper primer SEQ ID NO: 114 localized within the NDV F gene coding sequence, lower primer SEQ ID NO: 115 localized 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 The 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. The resulting digested DNA (10 ng) was used 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 in a 1:6 ratio with fresh secondary cells and stained with chicken anti-NDV polyclonal serum. Focuses expressing NDV were identified 3-4 days after passage. Three positively stained foci were isolated by trypsinization using cloning. The isolated 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 cycles. The cloned cultures were then amplified and frozen stocks were prepared. The frozen stock was named "HVT-ND #44".

[0246] A 0.71 kb band was obtained from PCR analysis of one final clone using primers for the upstream region of the UL55-Gene3 integration site (upper primer: SEQ ID NO: 116, localized upstream of UL55; lower primer: SEQ ID NO: 117, localized within the chicken beta-actin promoter, Panel A). A 0.965 kb PCR band was obtained from a similarly localized primer pair: upper primer: SEQ ID NO: 118; lower primer: SEQ ID NO: 119 (Panel B). Correct integration was further confirmed by using primers around the junction downstream of the insertion site (upper primer: SEQ ID NO: 120, localized within the NDV F gene coding sequence; lower primer: SEQ ID NO: 121, localized 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 the primers on the outside of the expression cassette (upper primer SEQ ID NO: 122, lower 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:) was chemically synthesized by BioBasic Inc. This plasmid was transfected into CEF cells infected with HVT-GFP-B using Lipofectamine LTX in a 6-well plate. Three days after transfection, transfected / infected 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 stained with chicken NDV serum using IFA to confirm NDV F gene expression. One of the purified viruses was expanded using CEF cells to create a frozen stock, which was named "HVT-ND #45".

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

[0249] HVT-ND #46 Construction The HVT-ND #46 transfer plasmid (SEQ ID NO: 38) was chemically synthesized by BioBasic Inc. This plasmid was transfected into CEF cells with HVT-GFP-B viral DNA using PEI (polyethyleneimine, 7.5 μL) 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 stained with chicken NDV serum using IFA to confirm NDV F gene expression. One of the purified viruses was expanded using CEF cells to create a frozen stock, which was named "HVT-ND #46".

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

[0251] Construction of HVT-ND #48 A linear transfer plasmid for HVT-ND #48 (SEQ ID NO: 39) was co-transfected with HVT-gfp-B DNA in a 6-well plate containing secondary CEF cells using PEI (polyethyleneimine, 7.5 μL). Four days after transfection, the transfected cells were plated on a 96-well plate and stained with NDV chicken serum. Four non-green foci were found, two of which were positive and stained positively with 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 to create a frozen stock, which was named "HVT-ND #48".

[0252] The 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. The resulting digested DNA (10 ng) was used 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 in a 1:6 ratio with fresh secondary cells and stained with NDV chicken polyclonal serum. Four days after passage, NDV-expressing foci were identified. Three positively stained foci were collected by trypsination using a cloning cylinder. The collected 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 cycles. The cloned cultures were then transferred from a 6-well plate to 75 cm³. 2 225 cm to the flask 2 After amplified to flask size, primary CEF cells were used, and 850 cm³ 2 Final amplification was performed in a roller bottle. The final culture was collected and named "HVT-ND #48".

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

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

[0255] Example 4 In vivo IBDV efficacy trial 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 for their in vivo efficacy against highly toxic IBDV challenge (STD strain, provided by the USDA) in SPF birds. A positive control of the commercially available vaccine Vaxxitek (Merial) was used in this study. 1500 pfu of each recombinant virus was injected in vivo at E18. The inverse 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. The inventors of this application observed 100% protection for HVT-IBD #9, 96% protection for HVT-IBD #1, #30, and #34, and 92% protection for HVT-IBD #6a, while the inventors' positive control, Vaxxitek, provided 92% protection. [Table 3]

[0256] Example 5: IBDV serological response of HVT-IBD#1, #5, #9, and #15 in commercially available broiler birds Serological responses to IBDV antigens were measured using the commercially available Elisa ProFlok ND plus kit. 1500 pfu (0.2 mL) of each recombinant (HVT-IBD #1, #5, #9, #15) was subcutaneously (SC) injected into 1-day-old chicks. Serum samples were isolated on days 12, 19, 26, 33, 39, 47, and 54, and these are shown 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 response of HVT-IBD#6a, #30, and #31 in commercially available broiler birds Serological responses to IBDV antigens were measured using the commercially available ELISA kit ProFlok IBD plus. 1500 pfu (0.2 mL) of each recombinant (HVT-IBD #61, #30, #31) was subcutaneously injected 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 study of SPF birds HVT-ND #38, #39, #44, and #48 Four HVT-ND recombinants, HVT-IBD#38, #39, #44, and #48, were tested for their in vivo efficacy against a highly toxic NDV challenge (Texas GB strain, provided by the USDA) in SPF birds. A positive control of the commercially available vaccine Vectormune ND(Ceva) was used in this study. 1500 pfu of each recombinant virus was injected in vivo with 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, the inventors found that only 95-96% of HVT-IBD#39 and #44 expressed this antigen. The NDV Texas GB challenge was performed on day 28 according to USDA instructions. All birds were observed two weeks after the challenge. The inventors observed 90% protection for HVT-ND#38 and #48, 50% protection for HVT-ND#39, and 60% protection for HVT-ND#44, while their positive control, Vectormune ND, provided 90% protection. See Table 5 below. [Table 6]

[0259] 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 the kit. The percentage of birds with positive ND titers is shown in Table 6 below. [Table 7]

[0260] Example 8: In vivo NDV efficacy trial of HVT-ND#40, #42, #45, and #46 in SPF birds. Four HVT-ND recombinants, HVT-IBD#40, #42, #45, and #46, were tested for their in vivo efficacy against a highly toxic NDV challenge (Texas GB strain, provided by the USDA) in SPF birds. A positive control of the commercially available vaccine Vectormune ND(Ceva) was used in this study. 1500 pfu of each recombinant virus was injected in vivo 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 expressed the NDV F antigen, the inventors found that only 94–99% of HVT-IBD#40 expressed this antigen. The NDV Texas GB challenge was performed on day 28 according to USDA instructions. All birds were observed two weeks after the challenge. The inventors of this application 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] 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 the kit. The percentage of birds with positive ND titers is shown in Table 8 below. [Table 9]

[0262] Example 9 In vivo MDV efficacy trial of SFP birds HVT-ND#38, #42, and #45 Three HVT-ND recombinants, HVT-IBD#38, #42, and #45, were tested for their in vivo efficacy against highly toxic MDV challenge (GA22) in SPF birds. A positive control of the commercially available vaccine Vectormune ND (Ceva) was used in this study. 1500 pfu of each recombinant virus was injected in vivo with E18. The inverse 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 observed 54 days after challenge. The inventors 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 efficacy study of HVT-ND #38, #42, and #45 in broiler chickens Three HVT-ND recombinants, HVT-IBD#38, #42, and #45, were tested in vivo against a highly toxic NDV challenge (Texas GB strain, provided by the USDA) in broiler birds. A positive control of the commercially available vaccine Vectormune ND (Ceva) was used in this study. 4000 pfu of each recombinant virus was injected in vivo with E18. The inverse titer of the vaccine virus was determined for each recombinant after vaccination. The NDV Texas GB challenge was performed on day 28 according to USDA instructions. All birds were observed two weeks after the challenge. The inventors observed 100% protection for HVT-ND#42 and #45, and 37% protection for HVT-ND#38, while the inventors' positive control, Vectormune ND, provided 50% protection. The inverse 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 trial of HVT-ND#38, #42, and #45 SFP birds on day 20 of the challenge. Three HVT-ND recombinants, HVT-IBD#38, #42, and #45, were tested for their in vivo efficacy against a highly toxic NDV challenge (Texas GB strain, provided by the USDA) in SPF birds at 20 days of challenge. A positive control of the commercially available vaccine Vectormune ND (Ceva) was used in this study. 2000 pfu of each recombinant virus was injected in vivo with E18. The inverse titer of the vaccine virus was determined for each recombinant after vaccination. The NDV Texas GB challenge was performed at 20 days according to USDA instructions. All birds were observed 2 weeks after the challenge. The inventors observed 87.5% protection for HVT-ND#42, 70% protection for HVT-ND#45, and 65% protection for HVT-ND#38, while the inventors' positive control, Vectormune ND, provided 87.5% protection. Please refer to Table 11 below. [Table 12]

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

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

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

[0269] Example 15 ND immunogenicity study of HVT-ND (#42, MSV+5) in SPF birds Three HVT-ND recombinant HVT-ND(#42, MSV+5) were tested for immunogenicity against a highly toxic NDV challenge (Texas GB strain, provided by the USDA) in SPF birds on day 28. All birds were observed two weeks after the challenge. The inventors observed 100% (30 / 30) protection for both inovocine and subcutaneous vaccination on the day of hatching. See Table 15 below. [Table 16]

[0270] Example 16 MD immunogenicity study of SPF avian HVT-ND (#42, MSV+5) Three HVT-ND recombinant strains, HVT-ND (#42, MSV+5), were tested for immunogenicity against a highly toxic MDV challenge (GA22 strain) in SPF birds on day 5. All birds were observed 54 days after the challenge. The inventors observed 100% (30 / 30) protection for both inovocine and subcutaneous vaccination on the day of hatching. See Table 16 below. [Table 17]

[0271] Example 17 In vitro growth experiment In vitro growth experiments were conducted on HVT-ND#38, #42, and #45. The resulting height was 490 cm. 2 5 x 10 8 Primary CEF cells were seeded. HVT-ND#38, #42, and #45 were inoculated into each roller bottle at three different MOIs: 0.001, 0.003, and 0.008. Infected cells were harvested 48 hours after infection and titrated on CEF cells. Both HVT-ND#42 and #45 grew well, reaching 2.86 x 10⁶ cells, respectively. 6 and 2.97x10 6It has a titer of pfu / mL. HVT-ND#38 is 1.67 x 10 6 It had a titer of pfu / mL. See Table 17 below. [Table 18]

[0272] Example 18 HVT-IBD-ND #42-#30LP C2 Construction 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 of the HVT-ND #42 transfer plasmid was performed 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 construct the final transfer plasmid #42. This plasmid was used for transfection / infection to produce HVT-ND#42.

[0274] Generation of transfer plasmid-#30 The initial transfer plasmid HVT-IBD #30 was chemically synthesized by BioBasic Inc. Cloning plasmids were chemically synthesized by DNA2.0. PCR amplification of the IBD gene expression cassette of plasmid #30 was performed 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 construct the final transfer plasmid #30. This plasmid was used for transfection / infection to produce HVT-IBD-ND #42-#30 LP C2.

[0275] Building HVT-ND #42: Co-infection / transfection: CEF cells were seeded in a 6-well plate, and the following day, transfection was performed using Lipofectamine® LTX Reagent (ThermoFisher) with HVT working seed infection (140 ul) + plasmid-#42 (linearized by SpeI+SbfI digestion). Two days after transfection, transfected cells were collected. Screened positive foci in the 6 wells were plated with IFA using chicken anti-NDV polyclonal antibody (live cell staining, approximately 1:250 dilution), and then further purified once in a 96-well plate by limiting dilution (by viability staining) to obtain a single clone. The purified clone was passaged twice in a 6-well plate with two copies, and the purity of the clone was confirmed by IFA (fixation and staining). This 6-well sample 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 the 6 wells were plated with IFA using chicken anti-IBD polyclonal antibody (live cell staining, approximately 1:250 dilution), and then further purified once in a 96-well plate by limiting dilution (by viability staining) to obtain a single clone. Two purified clones were taken out and passaged in 6-well plates with two replicas, 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. Recombinant viruses were collected, divided equally into 1 mL vials, frozen overnight at -80°C, and then transferred to an LN tank.

[0277] Example 19: In vivo NDV efficacy trial of HVT-IBD-ND#42-#30, #42-#32, and #104 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 toxic NDV challenges in SPF birds. The NDV Texas GB challenge was performed on day 28. Approximately 1500 PFU of each recombinant virus was injected in vivo with E18. All birds were observed two weeks after the challenge. See Table 18 below. [Table 19]

[0278] Example 20: In vivo efficacy study of SPF birds HVT-IBD-ND#42-#30, #42-#32, and #104 for IBD 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 toxic IBDV challenge in SPF birds at days 14 and 21. Approximately 2000 PFU of each recombinant virus were injected in vivo with E18. All birds were necropsied 5 days after challenge. See Table 19 below. [Table 20]

[0279] Example 21 In vivo MDV efficacy trial 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 highly toxic MDV challenge (GA22) in SPF birds. Approximately 1500 PFU of each recombinant virus was injected in vivo with 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 study of SPF birds HVT-IBD-ND#42-#30, #42-#32, and #104 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 vivo with E18. vvIBDV challenge was performed on days 14 and 21. All birds were observed 10 days after challenge. Histological examination of the bursa was performed on each bird at the end of the study. See Table 21 below. [Table 22]

[0281] Example 23 Immunotherapy for IBD duration in SPF birds with HVT-IBD-ND (#42-#30, X+5) up to day 63 of the challenge. HVT-IBD-ND recombinants #42-#30 (MSV+5) were tested for the duration of immunity to highly toxic classical IBDV challenge in SPF birds on day 63 of challenge. All birds were observed 4 days after challenge and subsequently necropped. See Table 22. [Table 23]

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

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

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

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

[0286] Example 28 Compatibility of Bursaplex with HVT-ND IBDV effectiveness BURSAPLEX® (Zoetis, US5871748, incorporated herein by reference) is a vaccine against bursal disease (IBD) comprising a vaccine conjugate consisting of a live, attenuated infectious bursal disease (IBD) strain 2512 conjugated to the virus and the neutralizing antibody BDA. Bursaplex generates active immunity against IBD in poultry, particularly chickens. E18 eggs were inovically injected with either the control or the test vaccine (HVT-ND containing Bursaplex in a 1:1 ratio) and transferred, along with uninjected eggs, to incubators assigned as specified by Biometrics. On the day of hatching, 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 classically virulent IBDV, and on day 38, all birds were necropsied for the presence of cystic lesions. Chickens in the T01-negative group did not develop significant observable lesions, while 100% of chickens in the T02 challenge control group developed significant observable lesions. Both T03 (HVT-ND + Bursaplex, inovoc) and T04 (HVT-ND + Bursaplex, subcutaneous) were 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 inovoc or subcutaneously.

[0287] NDV effectiveness E18 eggs were inovically injected with either a control or test vaccine (HVT-ND with Bursaplex in a 1:1 ratio) and transferred to incubators assigned as specified by Biometrics, along with uninjected eggs. On the day of hatching, T04 birds were subcutaneously vaccinated. Blood samples were collected on days 6, 13, 20, and 27 for NDV serology. On day 28, designated birds were challenged with short-latency NDV, and on day 42, all surviving birds were killed. Chickens in the T01 negative group did not develop clinical signs, while 100% of chickens in the T02 challenged control group developed clinical signs of Newcastle disease, including mortality. T03 (HVT-ND + Bursaplex, inovically) and T04 (HVT-ND + Bursaplex, SC) were protected at 92.5% and 95%, respectively. It can be concluded that Poulvac Procerta HVT-ND and Poulvac Bursaplex are compatible when administered together and remain effective against NDV challenge whether administered in ovarian or subcutaneously.

[0288] Example 29 Compatibility of HVT-ND with Magniplex IBD effectiveness MAGNIPLEX® (Zoetis) is a vaccine against bursal disease (IBD) containing a vaccine conjugate consisting of a live, attenuated infectious bursal disease (IBD) strain V877 conjugated to the virus and the neutralizing antibody BDA. Bursaplex generates active immunity against IBD in poultry, particularly chickens. E18 eggs were inovically injected with either the control or the test vaccine (HVT-ND Pre-license serial containing Magniplex in a 1:1 ratio) and transferred, along with uninjected eggs, to incubators assigned as specified by Biometrics. On the day of hatching, 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 classically virulent IBDV, and on day 38, all birds were necropsied for the presence of cystic lesions. Chickens in the T01-negative group did not develop significant observable lesions, while 100% of chickens in the T02 challenge control group developed significant observable lesions. Both T03 (HVT-ND + Magniplex, inovoc) and T04 (HVT-ND + Magniplex, subcutaneous) were 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 inovoc or subcutaneously. [Table 28] [Table 29]

[0289] ND effectiveness E18 eggs were inovically injected with either a control or a test vaccine (HVT-ND containing Magniplex in a 1:1 ratio) and transferred to incubators assigned as specified by Biometrics, along with uninjected eggs. On the day of hatching, T04 birds were subcutaneously vaccinated. Blood samples were collected on days 6, 13, 20, and 27 for NDV serology. On day 28, designated birds were challenged with short-latency NDV, and on day 42, all surviving birds were killed. Chickens in the T01 negative group did not develop clinical signs, while 100% of chickens in the T02 challenged control group developed clinical signs of Newcastle disease, including mortality. T03 (HVT-ND + Magniplex, inovically) and T04 (HVT-ND + Magniplex, SC) were protected at 92.5% and 95%, respectively. It can be concluded that Poulvac Procerta HVT-ND and Poulvac Magniplex are compatible when administered together and remain effective against NDV challenge whether administered inovically or subcutaneously. [Table 30] [Table 31]

Claims

1. A recombinant turkey herpesvirus (HVT) genome comprising one or more nucleotide sequences encoding one or more heterologous antigens inserted into the intergenetic locus UL35 / UL36 in the unique long (UL) region of the HVT genome, The one or more heterologous antigens mentioned above a) VP2, VP3, or VP4 protein of infectious bursal disease virus (IBDV), b) VP1 or VP2 protein of chicken anemia virus (CAV), c) F / HN chimeric protein of Newcastle disease virus (NDV), or F, NP, P, M, HN, or L protein, d) S1, S2, or M protein of infectious bronchitis virus (IBV) e) gB, gC, gD, gE, gH, gI or gL proteins of infectious laryngotracheitis virus (ILTV), and f) Any of the HA, NA, NP, or M proteins of the avian influenza virus (AIV) A recombinant HVT genome selected from a group consisting of the following.

2. A recombinant turkey herpesvirus (HVT) genome comprising one or more nucleotide sequences encoding one or more heterologous antigens inserted into the intergenetic 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, The one or more heterologous antigens mentioned above a) VP2, VP3, or VP4 protein of infectious bursal disease virus (IBDV), b) VP1 or VP2 protein of chicken anemia virus (CAV), c) F / HN chimeric protein of Newcastle disease virus (NDV), or F, NP, P, M, HN, or L protein, d) S1, S2, or M protein of infectious bronchitis virus (IBV) e) gB, gC, gD, gE, gH, gI or gL proteins of infectious laryngotracheitis virus (ILTV), and f) Any of the HA, NA, NP, or M proteins of the avian influenza virus (AIV) A recombinant HVT genome selected from a group consisting of the following.

3. The recombinant HVT genome according to claim 1 or 2, wherein one or more heterologous antigens are protective against 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), avian anemia virus (CAV), and avian influenza virus (AIV).

4. The recombinant HVT genome according to any one of claims 1 to 3, wherein one or more heterologous antigens are protective against IBDV.

5. The recombinant HVT genome according to claim 4, wherein one or more heterologous antigens are the VP2 protein of IBDV.

6. The VP2 protein sequence is encoded by a nucleotide sequence that has 90% or more sequence identity with respect to the nucleotide sequence containing SEQ ID NO: 5 or SEQ ID NO:

10. The recombinant HVT genome according to claim 5.

7. The recombinant HVT genome according to claim 5, wherein the VP2 protein is encoded by a nucleotide sequence including SEQ ID NO: 5 or SEQ ID NO:

10.

8. The recombinant HVT genome according to any one of claims 1 to 3, wherein one or more heterologous antigens or multiple antigens are protective against Newcastle disease virus (NDV).

9. The recombinant HVT genome according to claim 8, wherein one or more heterologous antigens include the F protein of NDV.

10. The recombinant HVT genome according to claim 9, wherein the F protein of NDV is encoded by a nucleotide sequence having 90% or more sequence identity with respect to the sequence containing SEQ ID NO:

3.

11. The recombinant HVT genome according to claim 9, wherein the F protein of NDV is encoded by a nucleotide sequence including SEQ ID NO:

3.

12. The recombinant HVT genome according to any one of claims 1 to 11, wherein one or more heterologous antigens are protective against NDV and IBDV.

13. The recombinant HVT genome according to claim 12, wherein one or more heterologous antigens include the F protein of NDV and the VP2 protein of IBDV.

14. The recombinant HVT genome according to claim 13, wherein the F protein of NDV is encoded by a nucleotide sequence having 90% or more sequence identity with respect to the nucleotide sequence containing SEQ ID NO: 3, and the VP2 protein of IBDV is encoded by a nucleotide sequence having 90% or more sequence identity with respect to the nucleotide sequence containing SEQ ID NO: 5 or SEQ ID NO:

10.

15. The recombinant HVT genome according to claim 13 or 14, wherein the F protein of NDV is encoded by a nucleotide sequence including SEQ ID NO: 3, and the VP2 protein of IBDV is encoded by a nucleotide sequence including SEQ ID NO: 5 or SEQ ID NO:

10.

16. The recombinant HVT genome according to any one of claims 1 to 15, comprising a genome including one or more expression cassettes containing one or more nucleotide sequences encoding one or more heterologous antigens.

17. The recombinant HVT genome according to claim 16, wherein the expression cassette comprises one or more promoters.

18. The recombinant HVT genome according to claim 17, wherein one or more promoters are selected from the group consisting of the pre-early cytomegalovirus human (hCMV) promoter, guinea pig pre-early CMV promoter, mouse pre-early CMV promoter, Pec promoter, β-chicken triactin promoter, SV40 promoter, pseudorabies virus promoter with glycoprotein X promoter, herpes simplex virus-1 alpha-4 promoter, Marek's disease virus promoter with glycoprotein gA, gC, gB, gE, or gI promoter, infectious laryngotracheitis virus promoter with glycoprotein gB, gE, gI, gD promoter, and bovine herpesvirus 1 / 1 VP8 promoter.

19. The recombinant HVT genome according to claim 18, wherein one or more promoters include a human CMV promoter.

20. The recombinant HVT genome according to claim 18, wherein one or more promoters include a mouse CMV promoter.

21. The recombinant HVT genome according to claim 18, wherein the one or more promoters include the hCMV promoter and the mCMV promoter.

22. Isolated DNA encoding a recombinant HVT genome according to any one of claims 1 to 21.

23. An immunogenic composition comprising recombinant HVT according to any one of claims 1 to 22, further comprising a pharmaceutically acceptable carrier, excipient, or adjuvant.

24. A vaccine composition comprising recombinant HVT according to any one of claims 1 to 22, further comprising a pharmaceutically acceptable carrier, excipient, or adjuvant.

25. The vaccine according to claim 24, further comprising an additional Marek's disease virus (MDV) selected from the group consisting of the naturally attenuated MDV-1 strain Rispens (CVI-988) or three Gallid herpesvirus strains SB-1 virus.

26. The vaccine according to claim 25, wherein the additional MDV comprises a recombinant genome.

27. The vaccine according to claim 26, 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.

28. A vaccine according to any one of claims 24 to 27 for use in vaccinating birds against one or more diseases caused by one or more avian pathogens, The one or more avian pathogens mentioned above are infectious bursal disease virus (IBDV), New A vaccine selected from the group consisting of Castle disease virus (NDV), infectious bronchitis virus (IBV), infectious laryngotracheitis virus (ILTV), avian anemia virus (CAV), and avian influenza virus (AIV).

29. A vaccine according to any one of claims 24 to 27 for use in the protection of birds against clinical symptoms caused by one or more avian pathogens, A vaccine in which 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), avian anemia virus (CAV), and avian influenza virus (AIV).

30. A vaccine according to any one of claims 24 to 27 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, A vaccine in which 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), avian anemia virus (CAV), and avian influenza virus (AIV).

31. The vaccine according to any one of claims 28 to 30, comprising one or more avian pathogens, including the Newcastle disease virus.

32. The vaccine according to any one of claims 28 to 30, wherein the one or more avian pathogens include the infectious bursal disease virus (IBDV).

33. The vaccine according to any one of claims 28 to 30, wherein the one or more avian pathogens include the Newcastle disease virus and the bursal disease virus.

34. The vaccine according to any one of claims 24 to 33 for use in avian vaccination, wherein the vaccine is administered by spray, in ovo, subcutaneous, intramuscular, oral, nasal, or a combination thereof, by at least one administration of the vaccine.

35. The vaccine according to claim 34, wherein the vaccine is administered inovo.

36. The vaccine according to claim 35, wherein the inovo administration is performed in an embryonated egg between 16 and 22 days after development.

37. The vaccine according to any one of claims 34 to 36, wherein the inovo administration is performed on an ovum on the 18th day after development.

38. The vaccine according to any one of claims 34 to 37, wherein the administration of the vaccine includes inovo administration and subsequent spray administration.

39. The vaccine according to claim 34, wherein the administration of the vaccine includes spray administration.

40. A method for vaccinating birds to treat or prevent Marek's disease and one or more avian diseases caused by one or more avian pathogens, comprising the step of administering an effective amount of a vaccine composition according to any one of claims 24 to 33, The one or more avian pathogens mentioned above are infectious bursal disease virus (IBDV), New A method selected from the group consisting of Castle disease virus (NDV), infectious bronchitis virus (IBV), infectious laryngotracheitis virus (ILTV), avian anemia virus (CAV), and avian influenza virus (AIV).

41. The method according to claim 40, wherein the one or more avian pathogens include the infectious bursal disease virus (IBDV).

42. The method according to claim 40, wherein the one or more avian pathogens include the Newcastle disease virus (NDV).

43. The method according to claim 40, wherein the one or more avian pathogens include the infectious bursal disease virus (IBDV) and the Newcastle disease virus (NDV).

44. A method for inducing an immune response in an animal animal against Marek's disease virus and one or more avian pathogens, comprising the step of administering to the bird an effective amount of an immunogenic composition or vaccine composition according to any one of claims 23 to 33, A method in which 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), avian anemia virus (CAV), and avian influenza virus (AIV).

45. The method according to claim 44, wherein the one or more avian pathogens include the infectious bursal disease virus (IBDV).

46. The method according to claim 44, wherein the one or more avian pathogens include the Newcastle disease virus (NDV).

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

48. The method according to any one of claims 40 to 47, wherein the administration is carried out by spray administration, inovo administration, subcutaneous administration, intramuscular administration, oral administration or nasal administration.

49. The method according to claim 48, wherein the route of administration includes inovactor administration.

50. The method according to claim 49, wherein the inovo administration is performed on a developing egg between 16 and 22 days after development.

51. The method according to any one of claims 48 to 50, wherein the inovo administration is performed on an ovum on the 18th day after development.

52. The method according to any one of claims 48 to 51, wherein the route of administration includes inovo administration followed by spray administration.

53. The method according to claim 48, wherein the route of administration includes spray administration.

54. The method according to any one of claims 41 to 53, wherein the bird is selected from the group consisting of chickens, turkeys, geese, ducks, pheasants, ostriches, pigeons, and quail.

55. The method according to claim 54, wherein the bird includes a chicken.

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