BOVINE HERPESVIRUS TYPE 1 (BOHV-1) VECTOR AGAINST BOVINE RESPIRATORY DISEASE COMPLEX

Abstract

The application describes recombinant BoHV-1 triple mutant viruses that express protective antigens of other bovine respiratory viruses associated with Bovine respiratory disease complex (BRDC).

Claims

1. A BoHV-1 recombinant vector comprising at least one heterologous antigen inserted therein, where the BoHV-1 vector has a deletion of a cytoplasmic tail of envelope glycoprotein gE (gE-CT), a deletion of an entire envelope protein, a deletion of envelope protein UL49.5 residues 30-32, a deletion of UL49.5 cytoplasmic tail residues 80-96, or a combination thereof.

2. The BoHV-1 recombinant vector of claim 1, wherein the at least one heterologous antigen is from BVDV, BRSV, or a combination thereof.

3. The BoHV-1 recombinant vector of claim 1, wherein at least one heterologous antigen is a BVDV E2 antigen having at least 95% amino acid sequence identity to SEQ ID NO:2, 3, 4, 5, 7, 10, 12, 55, or 57.

4. The BoHV-1 recombinant vector claim 1, wherein at least one heterologous antigen is a BVDV E2 antigen encoded by a nucleic acid segment with at least 95% nucleotide sequence identity to SEQ ID NO:6, 8, 9, 11, 13, 14, 56, or 58.

5. The BoHV-1 recombinant vector of claim 1, wherein at least one heterologous antigen is a BVDV Ems antigen having at least 95% amino acid sequence identity to SEQ ID NO:39 or 42.

6. The BoHV-1 recombinant vector of claim 1, wherein at least one heterologous antigen is a BVDV Ems antigen encoded by a nucleic acid segment with at least 95% nucleotide sequence identity to SEQ ID NO:40 or 42.

7. The BoHV-1 recombinant vector of claim 1, wherein at least one heterologous antigen is a BRSV F protein antigen having at least 95% amino acid sequence identity to SEQ ID NO:15, 16, 17, 20, 22, or 24.

8. The BoHV-1 recombinant vector of claim 1, wherein at least one heterologous antigen is a BRSV F protein antigen encoded by a nucleic acid segment with at least 95% nucleotide sequence identity to SEQ ID NO: 18, 19, 21, 23, or 25.

9. The BoHV-1 recombinant vector of claim 1, wherein at least one heterologous antigen is a BRSV G protein antigen having at least 95% amino acid sequence identity to SEQ ID NO:26, 28, 29, 31, 34, or 36.

10. The BoHV-1 recombinant vector of claim 1, wherein at least one heterologous antigen is a BRSV G protein antigen encoded by a nucleic acid segment with at least 95% nucleotide sequence identity to SEQ ID NO:30, 32, 33, 35, 37, or 38.

11. The BoHV-1 recombinant vector of claim 1, wherein at least one heterologous antigen is expressed from a heterologous promoter.

12. The BoHV-1 recombinant vector of claim 1, wherein at least one heterologous antigen is expressed from a heterologous promoter selected from a viral promoter, a bacterial promoter or a mammalian promoter.

13. The BoHV-1 recombinant vector of claim 1, wherein at least one heterologous antigen is expressed from an elongation factor 1 promoter

14. The BoHV-1 recombinant vector of claim 1, wherein at least one heterologous antigen is expressed as a fusion protein.

15. The BoHV-1 recombinant vector of claim 1, wherein at least one heterologous antigen is expressed as a fusion protein with a fusion partner selected from a gD signal sequence, a V5 epitope, a histidine tail comprising 2-10 histidine residues, GM-CSF, or any combination thereof.

16. A composition comprising a carrier and at least one BoHV-1 recombinant vector of claim 1.

17. The composition of claim 16, further comprising at least one antigenic polypeptide or peptide.

18. The composition of claim 16, further comprising a GM-CSF polypeptide.

19. The composition of claim 16, formulated for administration by an intranasal route.

20. A method comprising administering at least one BoHV-1 recombinant vector of claim 1 to a mammal.

21. The method of claim 20, which reduces the incidence or severity of respiratory symptoms in the mammal.

22. The method of claim 20, wherein the mammal is a bovine animal or an experimental animal.

23. The method of claim 20, which reduces the incidence or severity of respiratory symptoms of bovine respiratory disease complex (BRDC) in a bovine mammal to which the composition is administered.

Description

DESCRIPTION OF THE FIGURES

[0009] FIG. 1 shows a schematic diagram of the BVDV genome illustrating the location of various BVDV encoded proteins, including the E2 coding region, which is enlarged and shows the location of the Transmembrane region.

[0010] FIG. 2 schematically illustrates the structure of a BRSV genome showing various BRSV encoded proteins. The BRSV F and G protein regions are enlarged. Furin cleavage sites (FCS 1 and FCS2) and the pep 27 of BRSV F are also shown. In addition, a cysteine noose region within the BRSV G protein (residues 157-188) is shown.

[0011] FIG. 3A-3I illustrate schematic diagrams of BoHV-1 vector development. FIG. 3A shows a schematic diagram of BoHV-1 U.sub.L49.5 30-32 CT-null/gE-CT/Us9 virus genome (BoHV-1 tmv) showing (1) a schematic of the BoHV-1 genome with the locations of U.sub.L49.5 (gN), gE, Us9 and bICP22 open reading frames; (2) a schematic of the BoHV-1 U.sub.L49.5 30-32 CT-null genome; and (3) a schematic of pBoHV-1 gE-CT/Us9 plasmid showing the location and sizes of deleted sequences (gE CT, Us9 and Us9-bICP22 intergenic region), the flanking upstream gE (ectodomain and transmembrane) and downstream bICP22 sequences. Arrows indicate open reading frames of corresponding genes. FIG. 3B shows a schematic diagram of a wild type BoHV-1 genome showing the locations of UL 49.5 (gN), gE, Us9 and bICP 22 open reading frames (ORFs). FIG. 3C shows a schematic diagram of a BoHV-1 gE CT/Us9 deletion (pBoHV-1 gE CT/Us9) vector. FIG. 3D shows a schematic diagram illustrating the BoHV-1 tmv (U.sub.L49.5 30-32 CT-null/gECT/Us9 virus) genome. FIG. 3E shows a schematic diagram illustrating BHV-1 tmv virus vector organization showing UL49.5, gE CT/Us9 deletion sites. Arrows indicate open reading frames of corresponding genes. The asterisks (*) identifies a deletion of 3.44 kb nucleotides spanning the gE CT, Us9. The gE CT/Us9 deletion locus is flanked by gE ecto domain on the left and Us9-bICP22 intergenic region and carboxy terminal bICP22 gene sequence on the right. FIG. 3F shows a schematic diagram illustrating a pBHV-1 gECT US9 deletion vector showing the gECT/U.sub.S9 deletion site (nucleotides 122989-123993) and the location of an U.sub.S8 (gE) partial gene (nucleotides 121637 . . . 122989; gE Ecto) as well as the downstream 1 Kb Us9/bICP22 intergenic and partial carboxy terminal bICP22 (nucleotides 123993-124996). Nucleotide numbers refer to gene locations. FIG. 3G shows a schematic diagram of a BVDV1 E2 expression cassette. FIG. 3H shows a schematic diagram of a BVDV2 E2 expression cassette. FIG. 3I shows a schematic diagram of a BRSV G expression cassette. FIG. 3J shows a schematic diagram of a BRSV F expression cassette. As illustrated, FIGS. 3G-3J illustrate the insertion site of respective sequences. Nucleotide numbers are based on GenBank accession #JX898220).

[0012] FIG. 4A-4C illustrate characterization of the BoHV-1 tmv mutant virus. FIG. 4A illustrates an immunoblot of Mock-infected, BoHV-1 wild type-infected, and BoHV-1 tmv-infected cell lysates probed with an anti-BoHV-1 UL49.5 specific antibody. FIG. 4B illustrates an immunoblot of Mock-infected, BoHV-1 wild type-infected, and BoHV-1 tmv-infected cell lysates probed with an anti-BoHV-1 gE-specific antibody. FIG. 4C illustrates an immunoblot of Mock-infected, BoHV-1 wild type-infected, and BoHV-1 tmv-infected cell lysates probed with a BoHV-1 Us9-specific antibody. Note that UL49.5 30-32 CT-null (FIG. 4A) and gE CT-specific bands (FIG. 4B) of BoHV-1 tmv mutant virus are smaller than their respective corresponding bands of wild type BoHV-1. In addition, the Us9 band is lacking in the BoHV-1 tmv-infected cell lysates.

[0013] FIG. 5 graphically illustrates growth of BHV-tmv virions. Growth of BHV-tmv viruses compared with BoHV-1 gE-deleted (i.e., complete deletion of gE) and wild type BoHV-1 viruses was performed in Madin-Darby bovine kidney (MDBK) cells obtained from the American Type Culture Collection (Manassas, Va.). Confluent MDBK cells were infected with the respective viruses at a multiplicity of infection of five plaque forming units (PFU) per cell. After 1 h of adsorption at 4 C., residual input viruses were removed. The cultures were washed three times with phosphate-buffered saline, and 5 ml of medium was added to each flask before further incubation (37 C.). At the indicated time intervals, replicate cultures were frozen. Virus yields were determined by plaque assay. Each data point represents the average of duplicate samples obtained from separate infections.

[0014] FIG. 6A-6D graphically illustrate the pathogenicity and vaccine efficacy of the BoHV-1 tmv. FIG. 6A graphically illustrates the clinical scores of calves that were subjected to primary intranasal infection/immunization with BoHV-1 tmv viruses or BoHV-1 gE-deleted viruses, followed by virulent wild type BoHV-1 viral challenge. As a control, sham-infected calves were also tested with mock primary infection/immunization, followed by actual challenge with virulent wild type BoHV-1. FIG. 6B graphically illustrates nasal virus shedding following primary intranasal infection/immunization with BoHV-1 tmv viruses or BoHV-1 gE-deleted viruses followed by challenge with virulent wild type BoHV-1 virus. As a control, sham-infected calves were also tested during primary infection and after challenge with virulent wild type BoHV-1 viruses. FIG. 6C graphically illustrates virus neutralizing antibody titers in calves following primary intranasal infection/immunization with BoHV-1 tmv or BoHV-1 gE-deleted viruses followed by challenge with virulent wild type BoHV-1. As a control, sham-infected calves were also tested by mock-primary infection/immunization followed by challenge with virulent wild type BoHV-1 virus. FIG. 6D illustrates serum interferon levels in calves sham infected/immunized (control), or infected/immunized with BoHV-1 tmv or BoHV-1 gE-deleted viruses followed by virulent wild type BoHV-1 challenge. High binding EIA plate (Costar, Corning, N.Y.) wells coated with anti-bovine IFN-specific rabbit polyclonal antibodies (10 g/ml; Endogen, Rockford, Ill.) were used to capture IFN- in serially diluted calf sera (100 l) from different treatment groups. As a control, 100 l of the serial diluted recombinant bovine IFN (Thermo, Pierce, Ill.) were added instead of sample sera. After incubation (for 1 hour), the test wells were washed and incubated (1 hr) with biotinylated rabbit anti-bovine IFN polyclonal Ab (Endogen, Rockford, Ill.). After washing, the test wells were incubated (for 1 hour) further with avidin-HRP (eBioscience, San Diego, Calif.) and developed with substrate 2,2-azino-bis(3-ethylbenzothiazoline-6-sulphonic acid) (ABTS, Sigma). The test results were measured at OD.sub.405 by using an ELISA reader.

[0015] FIG. 7 shows an immunoblot illustrating a BoHV-1 gE CT-specific mouse monoclonal antibody. Note that the antibody recognized the 92 kD gE-specific band in the wild type BoHV-1-infected cell lysate but not the gE CT-deleted BoHV-1 tmv-infected cell lysate.

[0016] FIG. 8 shows a schematic diagram illustrating construction of a BVDV E2.1 chimeric gene and a BoHV-1 E2.1 insertion plasmid (pBoHV-1 INS/E2.1). Panel A shows the locations of PCR primers P1 Forward (P1 F) and P2 Reverse (P2 R) for PCR amplification of an E2.1 chimeric expression cassette within a pEF6/v5-His-TOPO vector. Panel B shows the location of KpnI site in pBoHV-1 gECT/Us9 for insertion of chimeric E2.1 gene. Panel C shows the pBoHV-1 INS/E2.1 plasmid containing BVDV E2.1 expression cassette flanked by gE CT upstream gE Ecto and transmembrane (Tm) nucleic acid segments, and the Us9 downstream bICP 22 nucleic acid segments.

[0017] FIG. 9A-9B shows immunoblots of proteins expressed by a BoHV-1 tmv vector encoding the BVDV type 1 chimeric E2 (E2.1) protein. FIG. 9A shows an immunoblot of lysates of mock-infected control cells and cells infected with BoHV-1 tmv or the modified BoHV-1 tmv that encodes a chimeric E2 (E2.1) protein, after probing with an anti-V5 monoclonal antibody. FIG. 9B shows an immunoblot of lysates of mock-infected control cells, BVDV-infected cells, and cells infected with BoHV-1 tmv or the modified BoHV-1 tmv that encodes a chimeric E2 (E2.1) protein, after probing with a polyclonal anti-BVDV antibody. As shown FIG. 9B, there is a 43 kD protein visible in BVDV-infected lysates, which is a precursor endoplasmic reticulum-processed form of E2.1. Also, shown in FIG. 9B, there is a 53-54 kD protein band in the BVDV lysates, which is the Golgi processed EndoH resistant mature E2.1. The molecular masses of both the ER and Golgi processed forms of the BoHV-1 tmv expressed chimeric E2.1 shown in FIG. 9B are 5 kD larger (48 kD and 58/59 kD, respectively), due to the additional V5 and His tag residues (see also FIG. 9A).

[0018] FIG. 10 illustrates a nucleotide sequence (SEQ ID NO: 1) of a portion of an expression cassette that can be part of a vector (e.g., BoHV-1 tmv vector) and that can be used for insertion of a BVDV E2 protein antigen, BRSV F protein antigen, BRSV G protein antigen, or a combination thereof. This sequence is referred to as a pPreBVDV-E2/BRSV-F or G sequence. The SEQ ID NO: 1 sequence is composed in the 5-3 direction of the following: (i) human elongation factor 1 promoter sequence flanked by KpnI/ClaI restriction sites (underlined) (ii) 12 bp spacer and NsiI restriction site (iii) V5-epitope coding sequence (lower case, italic) (iv) 6His coding sequence (bolded) (v) a stop codon (TGA) (vi) bovine growth hormone polyadenylation signal sequence (BGH poly A)(underlined, italic) and (vii) KpnI site.

[0019] FIG. 11A-11B show schematic diagrams illustrating the strategy of the construction of chimeric BVDV E2 1 or 2 expression cassette, and BoHV-1 gECTUs9/BVDV E2-1 or 2 insertion vector. FIG. 11A shows a schematic diagram of a chimeric BVDV E2-1 or 2 gene expression cassette. FIG. 11B shows a schematic diagram illustrating a BoHV-1 gE CT and Us9 deletion and chimeric BVDV E2-1 or 2 nucleic acid insertion sites.

[0020] FIG. 12A-12B illustrate expression of BVDV E2 antigens from a recombinant BoHV-1 tmv vector. FIG. 12A shows an immunoblot of MDBK cell lysates that were either mock-transfected or transfected with BoHV-1tmv recombinant vectors expressing either BVDV type 1 (BoHV-1 tmv E2-1 clone 8 and clone 2) or type 2 E2 proteins (BoHV-1 tmv E2-2) as detected by a BVDV type 1 E2-specific monoclonal antibody (VMRD #157). FIG. 12B shows an immunoblot of MDBK cell lysates that were either mock-transfected or transfected with BoHV-1tmv recombinant vectors expressing either BVDV type 1 (BoHV-1 tmv E2-1 clone 8 and clone 2) or type 2 E2 proteins (BoHV-1 tmv E2-2) as detected by a BVDV type 2 E2-specific monoclonal antibody (VMRD # BA-2). As control, the reactivity of the monoclonal antibody Ab 157 and the monoclonal antibody mAb BA-2 is illustrated for mock and BVDV-1 type 1 (strain sanger) and BVDV type 2 (strain 125) infected MDBK cell lysates.

[0021] FIG. 13 graphically illustrates single step growth kinetics of BoHV-1tmv, BoHV-1tmv/E2-1 and BoHV-1tmv/E2-2. Samples were collected at 0 h, 3 h, 7 h, 11 h, 17 h, 23 h, 29 h, 35 h, 41 h and 47 hours post infection and titrated by plaque assay. The titration values of each time point were used to make the growth curve for comparison.

[0022] FIG. 14 graphically illustrates the plaque morphology of BoHV-1 tmv/E2-1 and BoHV-1tmv/E2. Plaque sizes produced by parental BoHV-1tmv, BoHV-1 tmv/E2-1 and BoHV-1tmv/E2 viruses were measured at 48 hours post-infection. Average plaque diameters of 50 randomly selected plaques are shown as meanstandard deviation.

[0023] FIG. 15A-15D illustrate a nucleotide sequence of a chimeric BVDVE2-1 expression cassette (SEQ ID NO:9) and the encoded amino acid sequence. FIG. 15A shows a nucleotide sequence for a human elongation factor 1 promoter flanked by KpnI/ClaI restriction sites (KpnI sequence underlined), followed by a Kozak sequence, and part of a coding region for a BHV-1 gD signal sequence (GenBank accession # AFV53430.1, amino acid 1-19). The sequence continues in FIG. 15B. FIG. 15B continues from FIG. 15A and shows the 3 end of the coding region for a BHV-1 gD signal sequence (GenBank accession # AFV53430.1, aa 1-19) followed by a Bos taurus codon optimized, nucleotide sequence for BVDV E2-1 (GenBank accession # NP_776263.1). Hence, the sequence shown in FIG. 15A-15B is a nucleotide sequence for a BVDVE2-1 chimeric coding region contained within ClaI and NsiI sites. The sequence includes the following in the 5-3 direction: Kozak sequence, coding sequence for a BHV-1 gD signal sequence (GenBank accession # AFV53430.1, amino acid 1-19), Bos taurus codon optimized nucleotide sequence for BVDV E2-1 coding region (GenBank accession # NP_776263.1). The 1.235 bp ClaI/NsiI fragment was cloned into the corresponding ClaI/NsiI sites of the pPreBVDV-E2 chimeric vector (FIG. 10; SEQ ID NO: 1) resulting in BVDVE2-1 chimeric gene expression cassette. BVDV E2-1 sequence is indicated by uppercase letters without underlining. FIG. 15C shows the amino acid sequence (SEQ ID NO:55) and nucleotide sequence (SEQ ID NO:56) of a fusion protein that has a BHV1 gD signal sequence (amino acid 1-19)(GenBank accession # AFV53430.1) fused to a portion of the BVDVE2-1 sequence (GenBank accession # NP_776263.1). The gD-BVDVE2-1 fusion protein nucleotide and amino acid sequences continue in FIG. 15D. Hence, FIG. 15D shows nucleotide and amino acid sequences for the C-terminal 235-373 amino acids of the gD signal sequence-BVDVE2-1 fusion protein.

[0024] FIG. 16A-16D illustrate a nucleotide sequence of a chimeric BVDVE2-2 expression cassette (SEQ ID NO: 14). FIG. 16A shows a nucleotide sequence for a human elongation factor 1 promoter flanked by KpnI/ClaI restriction sites (KpnI sequence underlined), followed by a Kozak sequence, and part of a coding region for a BHV-1 gD signal sequence (GenBank accession # AFV53430.1, amino acid 1-19). FIG. 16B continues from FIG. 16A and shows the 3 end of the coding region for a BHV-1 gD signal sequence (GenBank accession # AFV53430.1, amino acid 1-19) followed by a Bos taurus codon-optimized, nucleotide sequence for BVDV E2-2 (GenBank accession # AAC72814.1). The 1.195 bp ClaI/NsiI fragment was cloned into the corresponding ClaI/NsiI sites of the pPreBVDV-E2 chimeric vector (SEQ ID NO: 1) resulting in BVDVE2-2 chimeric gene expression cassette. FIG. 16C shows the amino acid sequence (SEQ ID NO:57) and nucleotide sequence (SEQ ID NO:58) of a fusion protein that has a BHV1 gD signal sequence (aa 1-19) (GenBank accession # AFV53430.1) fused to a portion of the BVDVE2-2 sequence (GenBank accession # AAC72814.1). The gD-BVDVE2-2 fusion protein nucleotide and amino acid sequences continue in FIG. 15D. Hence, FIG. 15D shows nucleotide and amino acid sequences for the C-terminal 235-390 amino acids of the gD signal sequence-BVDVE2-1 fusion protein.

[0025] FIG. 17A-17F show a nucleotide sequence for a BRSV F-version 1 expression cassette (SEQ ID NO: 19) as well as amino acid and nucleotide sequences for a BRSV-F1-V5/6His fusion protein sequences (SEQ ID NOs: 20 and 21, respectively). FIG. 17A-17C show a nucleotide sequence for a BRSV F version 1 expression cassette (SEQ ID NO:19) that includes a BRSV F version 1 sequence (GenBank accession # GI: 3451386/CAA 76980.1) contained within ClaI and NsiI sites that were cloned in to the corresponding ClaI/NsiI sites of the pPreBVDV-E2/BRSV-F or G chimeric vector (SEQ ID NO:1) resulting in the BRSV F-version 1 expression cassette. The BRSV F version-1 sequence is indicated by uppercase. The BRSV-F protein has separate Furin cleavage sites (FCS 1 and FCS 2). BRSV-F version 1 protein is generated by mutating two arginine residues of FCS 1 (RAKR) to alanine (AAKA) and all three arginine residues of FCS2 (RKRR) to alanine (AKAA). The BRSV-F version 1 nucleotide sequence is codon optimized for Bos taurus, and has an upstream Kozak sequence as well as a BHV-1 gD signal sequence. FIG. 17A shows a nucleotide sequence for a human elongation factor 1 promoter flanked by KpnI/ClaI restriction sites (KpnI sequence underlined), followed by a Kozak sequence, and part of a coding region for a BHV-1 gD signal sequence (GenBank accession # AFV53430.1, amino acid 1-19). FIG. 17B continues from FIG. 17A and shows the 3 end of the coding region for a BHV-1 gD signal sequence (GenBank accession # AFV53430.1, aa 1-19) followed by a Bos taurus codon-optimized, nucleotide sequence for BRSV F version 1 sequence (GenBank accession # GI: 3451386/CAA 76980.1). FIG. 17C continues from FIG. 17B and shows the V5 epitope, histidine tail, and polyA sequences. FIG. 17D-17F show amino acid and nucleotide sequences for a BRSV-F1-V5/6His fusion protein sequences (SEQ ID NOs: 20 and 21, respectively). FIG. 17D shows the first 216 amino acids of the fusion protein, and the corresponding nucleotide sequence therefor. FIG. 17E is a continuation of the fusion protein sequence, showing amino acids 217-432 of the fusion protein, and the corresponding nucleotide sequence therefor. FIG. 17F is a continuation of the fusion protein sequence, showing amino acids 433-619 of the fusion protein, and the corresponding nucleotide sequence therefor. The SEQ ID NO:21 nucleotide sequence is codon optimized for Bos taurus. The SEQ ID NOs: 20 and 21 sequences include the following: BHV1 gD signal sequence (amino acid 1-20)(GenBank accession #AFV53430.1) fused to the BRSV-F version 1 sequence (GenBank accession # GI: 3451386/CAA 76980.1), followed by a V5 epitope and a string of six histidines (6His). The BRSV-F protein has two separate Furin cleavage sites (FCS 1 and FCS 2). In the BRSV-F version 1 chimeric protein, two arginine residues of FCS 1 (RAKR; SEQ ID NO:51) are exchanged with alanine (AAKA: SEQ ID NO:52) and three arginine residues of FCS 2 (RKRR; SEQ ID NO:53) are exchanged with alanine (AKAA; SEQ ID NO:54). The gD signal, BRSV-F1, V5 epitope and 6His sequence are identified.

[0026] FIG. 18A-18C show a fusion protein BRSV-F version 2 protein sequence (SEQ ID NO:24) aligned with the corresponding nucleotide sequence (SEQ ID NO:25). FIG. 18A shows amino acids 1-216 of the fusion protein. FIG. 18B is a continuation of the fusion protein sequence, showing amino acids 217-414 of the fusion protein, and the corresponding nucleotide sequence therefor. FIG. 18C is a continuation of the fusion protein sequence, showing amino acids 415-594 of the fusion protein, and the corresponding nucleotide sequence therefor. The fusion protein nucleotide sequence (SEQ ID NO:25) is codon optimized for Bos taurus expression. The fusion protein includes a BHV1 gD signal sequence (amino acid 1-20)(GenBank accession # AFV53430.1) fused to the BRSV-F version 2 sequence (GenBank accession # GI: 3451386/CAA 76980.1). BRSV-F protein has two separate Furin cleavage sites (FCS 1 and FCS 2). The BRSV-F version 2 protein shown has a FCS 1 (usually RAKR; SEQ ID NO:51) sequence that has been modified to include alanine residues (AAAA; SEQ ID NO:52) and a FCS 2 (usually RKRR; SEQ ID NO:53) sequence modified to include alanine (AAAA: SEQ ID NO:54). The codon optimized chimeric BRSV-F version 2 sequence shown has an upstream Kozak sequence and BHV-1 gD signal sequence at the N-terminus.

[0027] FIG. 19A-19B illustrate a nucleotide sequence of a chimeric BRSV G version 1 expression cassette (SEQ ID NO:33). FIG. 19A shows a nucleotide sequence for a human elongation factor 1 promoter flanked by KpnI/ClaI restriction sites (KpnI sequence underlined), followed by a Kozak sequence, and part of a coding region for a BHV-1 gD signal sequence (GenBank accession # AFV53430.1, aa 1-19). FIG. 19B continues from FIG. 19A and shows the 3 end of the coding region for a BHV-1 gD signal sequence (underlined, GenBank accession # AFV53430.1, aa 1-19) followed by a Bos taurus codon-optimized, nucleotide sequence for BRSV G version 1 (not underlined; GenBank accession # GI: 17939989/AAL49398.1). The ClaI/NsiI fragment was cloned into the corresponding ClaI/NsiI sites of an expression system (e.g., a vector having SEQ ID NO: 1) resulting in a BRSV G version 1 chimeric gene expression cassette.

[0028] FIG. 20A-20B illustrate a nucleotide sequence of a chimeric BRSV G version 2 expression cassette (SEQ ID NO:38). FIG. 38A shows a nucleotide sequence for a human elongation factor 1 promoter flanked by KpnI/ClaI restriction sites (KpnI sequence underlined), followed by a Kozak sequence, and part of a coding region for a BHV-1 gD signal sequence (GenBank accession # AFV53430.1, aa 1-19). FIG. 20B continues from FIG. 20A and shows the 3 end of the coding region for a BHV-1 gD signal sequence (underlined) followed by a Bos taurus codon-optimized, nucleotide sequence for BRSV G version 2 (not underlined: GenBank accession # AFV53430.1). The ClaI/NsiI fragment was cloned into the corresponding ClaI/NsiI sites of an expression system (e.g., a vector having SEQ ID NO: 1) resulting in a BRSV G version 1 chimeric gene expression cassette.

[0029] FIG. 21A-21B illustrate examples of BVDV ERNS expression cassettes. FIG. 21A illustrates a nucleotide sequence (SEQ ID NO:40) an expression module encoding a BVDV1 ERNS fusion protein. Expression cassettes for BVDV1 ERNS can include, for example, the following components in a 5-3 direction: i) Restriction site for PacI (lower case, bold, underlined) ii) Kozak sequence (upper case, italicized) iii) 57 bps BHV1gD signal (lower case, underlined; GenBank accession #AFV53430.1) iv) 681 bps BVDV1E.sup.rns coding sequence (upper case, bold; GenBank accession #NP_776261.1) v) 429 bps Bos taurus GM-CSF coding sequence (uppercase, underlined; GenBank accession # NP_776452.1) vi) 42 bps V5 epitope coding sequence (upper case, italicized) vii) a stop codon (TAA) (uppercase, bold, italicized) viii) Restriction site for NotI (lower case, bold, underlined). FIG. 21B illustrates a nucleotide sequence (SEQ ID NO:41) of an expression cassette for BVDV2 ERNS that includes the following in a 5-3 direction: i) Restriction site for PacI (lower case, bold, underlined) ii) Kozak sequence (upper case, italicized) iii) 57 bps BHV1gD signal (lower case, underlined; GenBank accession #AFV53430.1) iv) 681 bps BVDV2E.sup.rns coding sequence (upper case, bold; GenBank accession #NP_777483.1) v) 429 bps Bos taurus GM-CSF coding sequence (uppercase, underlined: GenBank accession # NP_776452.1) vi) 42 bps V5 epitope coding sequence (upper case, italicized) vii) a stop codon (TAA) (uppercase, bold) viii) Restriction site for NotI (lower case, bold, underlined).

[0030] FIG. 22 shows schematic diagram of vectors and antigenic inserts for such vectors. Panel A is a schematic diagram of BHV-1 tmv (BHV-1 U.sub.L49.5 30-32 CT-null/gECT/Us9) viral vector that can express BVDV1 E2 or BVDV2 E2 (BHV-1 tmv BVDV1 E2 or BVDV2 E2). Panel B schematically illustrates a construct of U.sub.S4/gG gene (nucleotides 117260 . . . 118645) and surrounding genes that can be located in BHV-1 tmv BVDV E2 or BVDV2 E2. Nucleotide numbers refer to gene locations. Arrows indicate open reading frames of corresponding genes. The asterisks symbol (*) identifies a polyadenylation signal of U.sub.S3 and U.sub.S4. Panel C shows a schematic diagram of BHV-1 tmv containing an additional gG gene deletion, showing the gG deletion site (nucleotides 117260-118594) (BHV-1 tmv-gG BVDV1/2). Panel D is a schematic diagram of BHV-1 tmv-gG BVDV1 E2/E.sup.rns/GM-CSF. Panel E is a schematic diagram of BVDV2 E2/E.sup.rns/GM-CSF. Panels D and E illustrate the gG and the respective E.sup.rns/GM-CSF insertion sites. Nucleotide numbers are based on GenBank accession #JX898220.

[0031] FIG. 23A-B illustrate a nucleotide sequence (SEQ ID NO:49) of BHV-1 gG deletion vector. The vector has the following in the 5-3 direction: (i) An EcoRV restriction site, a 1 kb US3 sequence (upper case underlined, GenBank accession # JX898220.1, protein ID-AFV53428.1, nucleotides 116260-117125) followed by 588 bps CMV immediate early promoter sequence (lower case underlined), PacI and NotI sites with a eight base pair spacer (GTGTGTGTGT (SEQ ID NO:50) in between, followed by a 1 kb US6 downstream sequence (underlined italic, GenBank accession #JX898220.1, protein ID-AFV53430.1, nucleotides 118595-119595), 225 bps BGH poly A (uppercase) and a HindIII restriction site at the 3 end.

[0032] FIG. 24A-24C illustrate expression of BRSV F and G proteins from recombinant BHV-1 tmv. FIG. 24A illustrates expression of BRSV F protein from recombinant BHV-1 tmv as determined by a rabbit anti-BRSV F specific antibody (GenScript) staining of an immunoblot of infected Botur cell lysates. Lysate from BRSV Nebraska 236-652 strain infected Botur cells served as positive control. FIG. 24B illustrates expression of BRSV G protein from recombinant BHV-1 tmv as determined by anti-V5 specific monoclonal antibody (Invitrogen, #R960-25) staining of immunoblots of infected MDBK cell lysate. FIG. 24C illustrates expression of BRSV G protein from recombinant BHV-1 tmv as determined by rabbit anti-BRSV G specific antibody (GenScript) staining of an immunoblot of infected Botur cell lysates.

DETAILED DESCRIPTION

[0033] For many years, bovine respiratory disease complex (BRDC), a multifactorial disease in cattle, has been the most costly disease of the beef cattle industry in North America, costing the US cattle industry more than a billion dollars per year (31, 32). BRDC is also a significant problem for dairy cattle and cow-calf operations. Economic losses due to BRDC arise from a combination of mortality, metaphylactic, and therapeutic use of antibiotics, and reduced growth performance of affected cattle. BRDC typically involves an initial viral respiratory infection followed by a secondary bacterial infection, where the bacterial infection can involve Mannheimia haemolytica (M. haemolytica). It is generally accepted that initial respiratory viral infection(s) creates a favorable condition for colonization of the lungs by bacteria, commonly by M. haemolytica, resulting in severe pneumonia and death of infected cattle, especially in the feed lots (85).

[0034] Five viruses, bovine herpesvirus type 1 (BoHV-1), bovine respiratory syncytial virus (BRSV), bovine viral diarrheal virus (BVDV), respiratory bovine coronavirus (RBCoV), and parainfluenza 3 virus (PI-3) are involved in BRDC (19, 22, 23, 40, 48, 69, 73, 85). Based on the severity, pathogenicity and prevalence of the diseases caused by these viruses in cattle, BoHV-1, BRSV and BVDV are more significant. All the BRDC associated viruses, except BoHV-1, are RNA viruses and BoHV-1, BVDV, and BRSV are often immunosuppressive (8, 12, 38, 43, 44, 62, 64, 69, 72).

[0035] Traditional modified live virus (MLV) vaccines against these viruses are efficacious as judged by virus shedding and clinical scores following experimental vaccination and challenge (24, 31, 32, 93). However, recent reports indicate that multivalent vaccines against BoHV-1, BVDV and BRSV can be associated with outbreaks of respiratory disease and abortions under field conditions (21, 65, 66, 89).

[0036] The problems associated with modified live virus vaccines in the field are due to the following:

[0037] i) Like wild-type (wt) BoHV-1 virions, modified live virus vaccine strains are immunosuppressive, they establish latency in the trigeminal ganglia following intranasal infection, and following reactivation from latency they are shed in the nasal secretions (31, 32, 60). Consequently, the modified live virus vaccine strains may circulate in the cattle population and therefore may have the potential to revert to wild type (39, 60).

[0038] ii) Both BVDV and BRSV have RNA genomes that have high mutation rates and that can cause problems both in vaccine production and vaccine use. Therefore, deliberate and regular use of modified live virus vaccines in cattle population could be a major contributing factor in the emergence of mutant variants in the field (17). Such problems with modified live BRSV and BVDV vaccine viruses are further complicating the epidemiology of the diseases in the field and raising questions on the long-term benefits of modified live virus vaccines (12, 28, 39, 48).

[0039] iii) Notably, BVDV is highly immunosuppressive and this property is maintained in BVDV modified live virus vaccine strains (12, 70, 72). Therefore, the combined immunosuppressive effects of BoHV-1 and BVDV in the multivalent vaccine formulations described herein can synergistically interfere further with the protective immune response against the vaccine viruses.

[0040] iv) BRSV has a role in immunopathogenesis especially after reinfection and/or challenge infection (3, 27).

[0041] A substantial improvement in vaccine technology is described herein.

[0042] Previous work by the inventor shows that calves latently infected with envelope proteins gE cytoplasmic tail truncated (gE CT-null) or Us9-deleted BoHV-1 mutant viruses do not shed virus in the nose following reactivation and that both gE CT and Us9 are required for anterograde neuronal transport of the virus (11, 15, 16, 55). In addition, the inventor has determined that relative to calves infected with wild type BoHV-1, calves infected with a recombinant BoHV-1 virus lacking immunosuppressive domain of envelope protein U.sub.L49.5 (BoHV-1 UL49.530-32 CT-null) have higher BoHV-1-specific serum neutralizing titers and earlier cellular immune responses (90). Hence the inventor has incorporated the gE CT and Us9 sequence deletions into the BoHV-1UL49.530-32 CT-null virus and thereby constructed a BoHV-1UL49.530-32 CT-null/gECT/Us9 virus (BoHV-1 triple mutant virus).

[0043] Results obtained by the inventor show that the BoHV-1 triple mutant virus (BoHV-1 tmv) is significantly more efficacious than a gE-deleted BoHV-1. The triple gene mutated BoHV-1 tmv is not only a superior vaccine against BoHV-1, but it can be used as a vector for expressing carefully engineered protective antigens of other respiratory RNA viruses such as BVDV and BRSV. Information on the BoHV-1 triple mutant virus (BoHV-1 tmv, also called BoHV-1 tmv) is also provided in PCT Application PCT/US2015/043112 (published Feb. 4, 2016 as WO 2016/019244, and incorporated herein by reference in its entirety).

[0044] As described herein, a BoHV-1 tmv vector can be used to express the BVDV type 1 or type 2 envelope protein, E2, and/or the BRSV envelope proteins G or F. The BoHV-1 tmv vectored subunit vaccines are efficacious in calves against the respective subunit vaccine-specific virulent virus challenges (BVDV types 1 or 2, or BRSV). Methods and immunological compositions described herein can vaccinate beef and dairy cattle against BoHV-1, BVDV and BRSV, the three most significant bovine respiratory viruses that predispose to fatal secondary bacterial pneumonia.

Bovine Respiratory Disease Complex

[0045] Bovine respiratory disease complex (BRDC) is a multifactorial disease condition clinically characterized by acute bronchopneumonia. The causes of BRDC are multiple and complex, but in most cases stress and/or viral infection followed by bacterial infection can be involved (31, 32, 85). Five viruses, bovine herpesvirus type (BoHV-1), bovine respiratory syncytial virus (BRSV), bovine viral diarrheal virus (BVDV), respiratory bovine corona virus (RBCoV) and parainfluenza 3 virus (PI-3) can be associated with BRDC (5, 22, 23, 26, 40, 48, 69, 73, 85). These viruses generally produce mild respiratory signs by themselves but may produce severe respiratory signs when combined with stress or other viral and bacterial agents.

[0046] Four bacterial agents, M. hemolytica, Pasteurella maltocida, Histophilus somni and Mycoplasma bovis, have been implicated in BRDC (7, 23, 85). M. hemolytica has traditionally been the most common bacterial isolate. However, there is an apparent increase in prevalence of other agents such as M. bovis (85). All of these bacterial pathogens are normal flora in the upper respiratory tracts. However, due to stress and/or viral respiratory infection(s), these bacteria may colonize the lungs and cause severe bronchopneumonia.

[0047] BRD-like symptoms have been induced experimentally upon exposure of animals to M. haemolytica following infection by BoHV-1 (30). Similar results were obtained with endobronchial instillation of BVDV followed by M. hemolytica by five days later (68). BRD outbreaks have also been linked serologically to BRSV and BVDV (19, 29). Occasionally, viral agents may produce clinical syndromes consistent with BRDC in the absence of bacterial co-infection, but their involvement is generally considered as antecedent to, or concurrent with, bacterial infection. Recent reports also linked RBCoV to BRDC, although it has received considerably less attention than other viral agents (49, 59, 67, 80).

[0048] In summary, initial respiratory viral infection plays a crucial role in triggering severe bacterial pneumonia.

Bovine Herpesvirus Type 1 (BoHV-1)

[0049] BoHV-1 is an important viral pathogen of cattle that can cause severe respiratory tract infections known as infectious bovine rhinotracheitis (IBR), abortion in pregnant cows, and is an important component of the BRDC (31, 32). The ability of BoHV-1 to immunosuppress the infected cattle (43, 64), establish a lifelong latent infection in the trigeminal ganglia (TG) of infected animals, reactivate from latency upon stress, and be transported anterogradely from neuron cell bodies in the TG to axon termini in the nasal epithelium followed by replication and nasal virus shedding allows the virus to circulate in susceptible cattle populations (31, 32).

[0050] A variety of mechanisms have been proposed to explain the role of BoHV-1 in viral-bacterial synergism in BRDC. One mechanism may involve BoHV-1 induction of inflammatory cytokines, which activate lymphocyte function-associated antigen-1 (LFA-1) on bovine leukocytes, thereby increasing susceptibility to M. haemolytic leukotoxin (LKT) (50). Such susceptibility contributes to the recruitment and activation of neutrophils which in turn amplifies the detrimental effects of LKT resulting in erosive lesions in the upper respiratory tract (71). Another mechanism can involve BoHV-1 induction of immunosuppression by causing abortive infection and subsequent apoptosis of CD4+ T lymphocytes (87). A further mechanism can involve BoHV-1 down-regulation of MHC class I molecule cell surface expression (43), which consequently suppresses the development of cytotoxic T lymphocytes. Therefore, during both the primary infection and latency reactivation, the immunosuppressive effects of virus replication combined with erosive lesions in the respiratory epithelium promote the establishment of bacterial pathogens, for example M. haemolytica, in the lower respiratory tract (31, 32, 85).

[0051] A variety of different vaccine approaches have been utilized over the years with variable success to prevent and control BoHV-1 dissemination in cattle. These approaches include: traditional modified live virus (MLV), traditional inactivated vaccines, entire gE gene-deleted MLV that allows differentiation between vaccinated and infected animals (DIVA, marker vaccine), inactivated marker vaccine, and protein/DNA based subunit vaccines (31, 32, 62).

[0052] Traditional MLV vaccines can in some cases be efficacious in reducing virus shedding and clinical disease in the vaccinated animals. However, there are important problems associated with traditional modified live virus vaccines. Traditional modified live virus vaccines cannot be distinguished from the wild type virus based on serological markers in a vaccinated animal when compared with an infected animal. Like wild type viruses, traditional modified live virus vaccines are shed following latency reactivation of the virus. Multivalent modified live virus BRD vaccines have been linked to recent outbreaks of respiratory infection in vaccinated feedlot cattle (89) and abortions in pregnant heifers (65, 66).

[0053] Currently, a gE-deleted marker vaccine is favored over traditional modified MLV because the virus is significantly attenuated, the virus does not shed following latency reactivation and it has a serological marker (10, 31, 32, 62). However, gE-deleted viruses induce significantly less serum neutralizing antibody relative to traditional modified live virus gC-deleted and TK-deleted viruses (31, 32, 34, 55) while the gC-deleted virus retains some degree of virulence and both gC-deleted and TK-deleted viruses are shed following latency reactivation (34, 35). Most importantly, traditional modified live virus, live gC-deleted viruses, gE-deleted viruses, and TK-deleted viruses have intact immunosuppressive properties (31, 32, 62).

[0054] Problems associated with inactivated and protein-based subunit viral vaccines include that they require at least two vaccinations and, more importantly, they do not induce a cellular immune response (31, 32, 62). Experimentally, DNA based envelope glycoprotein gD subunit vaccine induced protective serum neutralizing antibodies and cellular immune response (58), however it required two immunizations, which may not be cost effective for the cattle industry.

[0055] BoHV-1 envelope glycoprotein U.sub.L49.5 interferes with transporter associated with antigen processing (TAP) functions required for peptide transport through the ER and subsequent loading into MHC-I. As a consequence, MHC-I surface expression is down regulated (43, 44). We have identified that the U.sub.L49.5 luminal domain residues 30-32 together with U.sub.L49.5 CT residues 80-96 promote efficient TAP inhibition and MHC-I down regulation functions (91). Following intranasal infection in calves, a U.sub.L49.5 subdomain deletion mutant (BoHV-1U.sub.L 49.530-32 CT-null virus) lacking these sequences induced better serum neutralizing and cellular immune responses (90).

[0056] BoHV-1 envelope proteins gE and Us9 are not essential for retrograde transport from sensory nerve endings of the trigeminal ganglia neurons in the nasal epithelium to neuronal cell bodies in the trigeminal ganglia. However, they are needed for viral anterograde transport from trigeminal ganglia neurons to their processes in the nose. As a result, the deletion mutants are not shed from the nose following latency reactivation (10, 11, 16, 55). BoHV-1 gE-deleted viruses are highly attenuated in calves infected intranasally (34).

[0057] The inventor and coworkers have also determined that like a currently available gE-deleted virus, the BoHV-1 tmv virus with the gE cytoplasmic tail (CT) deletion is also highly attenuated and is not shed following latency reactivation (55). In microfluidic chambers of primary rabbit dorsal root ganglionic neuron cultures, the inventor and coworkers have confirmed that both Us9-deleted and gE CT-null (gE Am453) viruses have anterograde spread defects, but their retrograde transport is not affected (16, 55).

[0058] To construct a novel BoHV-1 tmv vaccine vector that lacks the immunosuppressive property and at the same time is not shed following latency reactivation, the inventor and coworkers have combined the individual deletions/mutations described above with respect to U.sub.L49.5, gE CT and Us9 in a single virus, BoHV-1U.sub.L 49.5 30-32 CT-null/gECT/Us9 (BoHV-1 tmv). This novel triple mutant virus can be a more efficacious vaccine than the gE-deleted virus (current marker vaccine). By deleting the gE CT and entire Us9, the virus can be attenuated, and at the same time its safety can be maximized with respect to latency reactivation.

[0059] A recent comparative vaccine efficacy study comparing the BoHV-1 tmv with a gE-deleted virus demonstrates the benefits of the BoHV-1 tmv and the results are presented in the Examples section. These results demonstrate that the calves vaccinated with the BoHV-1 tmv generated significantly better protective immune response against virulent BoHV-1 challenge compared with the gE-deleted virus-infected calves.

Bovine Viral Diarrhea Virus (BVDV)

[0060] BVDV, a Pestivirus, is a significant pathogen of cattle that causes acute respiratory and enteric infections as well as reproductive disease of varying severity depending on the BVDV strain (69). Acute BVDV infections caused by type 1 strains result in mild disease characterized by fever, respiratory signs, diarrhea and leukopenia whereas infections caused by type 2 strains are more severe, usually characterized by high fever, hemorrhage, diarrhea, leukopenia and death. Regardless of type 1 or type 2, all BVDV infections in cattle, including MLV vaccine strains, are accompanied by immunosuppression due, at least in part, to the death of B and T cells within lymph nodes and gut associated lymphoid tissues as well as reduction in numbers of circulating white blood cells (12, 70, 72). In addition, BVDV regulates MHC-II expression and T helper 2 (Th2) responses (8). Consequently, the immunosuppression leaves the infected animal vulnerable to secondary and opportunistic infections, even after recovery, resulting in BRDC/shipping fever (8, 85).

[0061] BVDV has a positive stranded 12.3 Kb RNA genome which encodes a single polyprotein of approximately 4000 amino acids that is co- and post-translationally cleaved by the host and viral proteases to produce mature structural and nonstructural proteins of the virus (63). The order of proteins in the polyprotein is Npro (a nonstructural auto-protease), the capsid protein (C), the envelope glycoproteins (Erns, E1, and E2), and the nonstructural proteins (p7, NS/NS3, NS4A, NS4B, NS5A, and NS5B) (see FIG. 1).

[0062] Both MLV and killed vaccines have been used extensively in the US over the years, however the incidence of BRD and epidemiological situation in the vaccinated herds has not improved noticeably (31, 32, 56). The killed vaccines do not provide adequate protection against subsequent infection in the vaccinated population (31, 32). BVDV MLV vaccines are usually administered as multivalent (BoHV-1, BRSV, BVDV and PI3) vaccines and can sometimes be efficacious under experimental conditions (93), however they may serve as a source of in utero infection and immunosuppression (12, 70, 72). While inactivated vaccines are not desirably efficacious, the immunosuppressive effects of modified live BVDV alone could facilitate secondary bacterial infections (8, 23, 62). In addition, there is a concern that MLV BVDV vaccine virus may confound the BVD problem in the field further because of higher mutation rates associated with RNA viruses (9). Because of these problems associated with BVDV vaccines, Europeans have adopted a test and cull policy that does not permit BVDV MLV vaccination in cattle (74).

Protective Antigens of BVDV

[0063] The E2 protein, also known as gP 53, plays a major role in virus attachment and entry of BVDV. In addition, BVDV E2 is important for the induction of neutralizing antibodies and protection against BVDV challenge in cattle. The E2 protein of the NADL strain consists of about 370-410 amino acids and has a predicted molecular weight of about 40-55 kD.

[0064] A sequence of a BVDV strain NADL polyprotein, which includes the E2 protein, is available in the NCBI database (GI:7960753) and is shown below as SEQ ID NO:2.

TABLE-US-00001 1 MELITNELLYKTYKQKPVGVEEPVYDQAGDPLFGERGAVH 41 PQSTLKLPHKRGERDVPTNLASLPKRGDCRSGNSRGPVSG 81 IYLKPGPLFYQDYKGPVYHRAPLELFEEGSMCETTKRIGR 121 VTGSDGKLYHIYVCIDGCIIIKSATRSYQRVFRWVHNRLD 161 CPLWVTSCSDTKEEGATKKKTQKPDRLERGKMKIVPKESE 201 KDSKTKPPDATIVVEGVKYQVRKKGKTKSKNTQDGLYHNK 241 NKPQESRKKLEKALLAWAIIAIVLFQVTMGENITQWNLQD 281 NGTEGIQRAMFQRGVNRSLHGIWPEKICTGVPSHLATDIE 321 LKTIHGMMDASEKTNYTCCRLQRHEWNKHGWCNWYNIEPW 361 ILVMNRTQANLTEGQPPRECAVTCRYDRASDLNVVTQARD 401 SPTPLTGCKKGKNTSFAGILMRGPCNFEIAASDVLFKEHE 441 RISMFQDTTLYLVDGLTNSLEGARQGTAKLTTWLGKQLGI 481 LGKKLENKSKTWFGAYAASPYCDVDRKIGYIWYTKNCTPA 521 CLPKNTKIVGPGKFDTNAEDGKILHEMGGHLSEVLLLSLV 561 VLSDFAPETASVMYLILHFSIPQSHVDVMDCDKTQLNLTV 601 ELTTADVIPGSVWNLGKWVCIRPNWWPYETTVVLAFEEVS 641 QVVKLVLRALRDLTRIWNAATTTAFLICLVKIVRGQMVQG 681 ILWLLLITGVQGHLDCKPEFSYAIAKDERIGQLGAEGLTT 721 TWKEYSPGMKLEDTMVIAWCEDGKLMYLQRCTRETRYLAI 761 LHTRALPTSVVFKKLFDGRKQEDVVEMNDNFEFGLCPCDA 801 KPIVRGKFNTTLLNGPAFQMVCPIGWTGTVSCTSFNMDTL 841 ATTVVRTYRRSKPFPHRQGCITQKNLGEDLHNCILGGNWT 881 CVPGDQLLYKGGSIESCKWCGYQFKESEGLPHYPIGKCKL 921 ENETGYRLVDSTSCNREGVAIVPQGTLKCKIGKTTVQVIA 961 MDTKLGPMPCRPYEIISSEGPVEKTACTFNYTKTLKNKYF 1001 EPRDSYFQQYMLKGEYQYWFDLEVTDHHRDYFAESILVVV 1041 VALLGGRYVLWLLVTYMVLSEQKALGIQYGSGEVVMMGNL 1081 LTHNNIEVVTYFLLLYLLLREESVKKWVLLLYHILVVHPI 1121 KSVIVILLMIGDVVKADSGGQEYLGKIDLCFTTVVLIVIG 1161 LIIARRDPTIVPLVTIMAALRVTELTHQPGVDIAVAVMTI 1201 TLLMVSYVTDYFRYKKWLQCILSLVSGVFLIRSLIYLGRI 1241 EMPEVTIPNWRPLTLILLYLISTTIVTRWKVDVAGLLLQC 1281 VPILLLVTTLWADFLTLILILPTYELVKLYYLKTVRTDIE 1321 RSWLGGIDYTRVDSIYDVDESGEGVYLFPSRQAKQGNFSI 1361 LLPLIKATLISCVSSKWQLIYMSYLTLDFMYYMHRKVIEE 1401 ISGGTNIISRLVAALIELNWSMEEEESKGLKKFYLLSGRL 1441 RNLIIKHKVRNETVASWYGEEEVYGMPKIMTIIKASTLSK 1481 SRHCIICTVCEGREWKGGTCPKCGRHGKPITCGMSLADFE 1521 ERHYKRIFIREGNFEGMCSRCQGKHRRFEMDREPKSARYC 1561 AECNRLHPAEEGDFWAESSMLGLKITYFALMDGKVYDITE 1601 WAGCQRVGISPDTHRVPCHISFGSRMPFRQEYNGFVQYTA 1641 RGQLFLRNLPVLATKVKMLMVGNLGEEIGNLEHLGWILRG 1681 PAVCKKITEHEKCHINILDKLTAFFGIMPRGTTPRAPVRF 1721 PTSLLKVRRGLETGWAYTHQGGISSVDHVTAGKDLLVCDS 1761 MGRTRVVCQSNNRLTDETEYGVKTDSGCPDGARCYVLNPE 1801 AVNISGSKGAVVHLQKTGGEFTCVTASGTPAFFDLKNLKG 1841 WSGLPIFEASSGRVVGRVKVGKNEESKPTKIMSGTQTVSK 1881 NTADLTEMVKKITSMNRGDFKQITLATGAGKTTELPKAVI 1921 EEIGRHKRVLVLIPLRAAAESVYQYMRLKHPSISFNLRIG 1961 DMKEGDMATGITYASYGYFCQMPQPKLRAAMVEYSYIFLD 2001 EYHCATPEQLAIIGKIHRFSESIRVVAMTATPAGSVTTTG 2041 QKHPIEEFIAPEVMKGEDLGSQFLDIAGLKIPVDEMKGNM 2081 LVFVPTRNMAVEVAKKLKAKGYNSGYYYSGEDPANLRVVT 2121 SQSPYVIVATNAIESGVTLPDLDTVIDTGLKCEKRVRVSS 2161 KIPFIVTGLKRMAVTVGEQAQRRGRVGRVKPGRYYRSQET 2201 ATGSKDFHYDLLQAQRYGIEDGINVTKSFREMNYDWSLYE 2241 EDSLLITQLEILNNLLISEDLPAAVKNIMARTDHPEPIQL 2281 AYNSYEVQVPVLFPKIRNGEVTDTYENYSFLNARKLGEDV 2321 PVYIYATEDEDLAVDLLGLDWPDPGNQQVVETGKALKQVT 2361 GLSSAENALLVALFGYVGYQALSKRHVPMITDIYTIEDQR 2401 LEDTTHLQYAPNAIKTDGTETELKELASGDVEKIMGAISD 2441 YAAGGLEFVKSQAEKIKTAPLFKENAEAAKGYVQKFIDSL 2481 IENKEEIIRYGLWGTHTALYKSIAARLGHETAFATLVLKW 2521 LAFGGESVSDHVKQAAVDLVVYYVMNKPSFPGDSETQQEG 2561 RRFVASLFISALATYTYKTWNYHNLSKVVEPALAYLPYAT 2601 SALKMFTPTRLESVVILSTTIYKTYLSIRKGKSDGLLGTG 2641 ISAAMEILSQNPVSVGISVMLGVGAIAAHNAIESSEQKRT 2681 LLMKVFVKNFLDQAATDELVKENPEKIIMALFEAVQTIGN 2721 PLRLIYHLYGVYYKGWEAKELSERTAGRNLFTLIMFEAFE 2761 LLGMDSQGKIRNLSGNYILDLIYGLHKQINRGLKKMVLGW 2801 APAPFSCDWTPSDERIRLPTDNYLRVETRCPCGYEMKAFK 2841 NVGGKLTKVEESGPFLCRNRPGRGPVNYRVTKYYDDNLRE 2881 IKPVAKLEGQVEHYYKGVTAKIDYSKGKMLLATDKWEVEH 2921 GVITRLAKRYTGVGFNGAYLGDEPNHRALVERDCATITKN 2961 TVQFLKMKKGCAFTYDLTISNLTRLIELVHRNNLEEKEIP 3001 TATVTTWLAYTFVNEDVGTIKPVLGERVIPDPVVDINLQP 3041 EVQVDTSEVGITIIGRETLMTTGVTPVLEKVEPDASDNQN 3081 SVKIGLDEGNYPGPGIQTHTLTEEIHNRDARPFIMILGSR 3121 NSISNRAKTARNINLYTGNDPREIRDLMAAGRMLVVALRD 3161 VDPELSEMVDFKGTFLDREALEALSLGQPKPKQVTKEAVR 3201 NLIEQKKDVEIPNWFASDDPVFLEVALKNDKYYLVGDVGE 3241 VKDQAKALGATDQTRIIKEVGSRTYAMKLSSWFLQASNKQ 3281 MSLTPLFEELLLRCPPATKSNKGHMASAYQLAQGNWEPLG 3321 CGVHLGTIPARRVKIHPYEAYLKLKDFIEEEEKKPRVKDT 3361 VIREHNKWILKKIRFQGNLNTKKMLNPGKLSEQLDREGRK 3401 RNIYNHQIGTIMSSAGIRLEKLPIVRAQTDTKTFHEAIRD 3441 KIDKSENRQNPELHNKLLEIFHTIAQPTLKHTYGEVTWEQ 3481 LEAGINRKGAAGFLEKKNIGEVLDSEKHLVEQLVRDLKAG 3521 RKIKYYETAIPKNEKRDVSDDWQAGDLVVEKRPRVIQYPE 3561 AKTRLAITKVMYNWVKQQPVVIPGYEGKTPLFNIFDKVRK 3601 EWDSFNEPVAVSFDTKAWDTQVTSKDLQLIGEIQKYYYKK 3641 EWHKFIDTITDHMTEVPVITADGEVYIRNGQRGSGQPDTS 3681 AGNSMLNVLTMMYAFCESTGVPYKSFNRVARIHVCGDDGF 3721 LITEKGLGLKFANKGMQILHEAGKPQKITEGEKMKVAYRF 3761 EDIEFCSHTPVPVRWSDNTSSHMAGRDTAVILSKMATRLD 3801 SSGERGTTAYEKAVAFSFLLMYSWNPLVRRICLLVLSQQP 3841 ETDPSKHATYYYKGDPIGAYKDVIGRNLSELKRTGFEKLA 3881 NLNLSLSTLGIWTKHTSKRIIQDCVAIGKEEGNWLVNADR 3921 LISSKTGHLYIPDKGFTLQGKHYEQLQLRTETNPVMGVGT 3961 ERYKLGPIVNLLLRRLKILLMTAVGVSS
Any antigenic segment or fragment of the NADL polyprotein can be used as an antigen for expression by the expression cassettes and vectors described herein.

[0065] The E2 protein of the NADL strain can include about amino acids 660-1066 of the SEQ ID NO:2 polyprotein (see UniProt database sp|P19711|660-1066); the E2 protein with amino acids 660-1066 is shown below as SEQ ID NO:3.

TABLE-US-00002 1 ATTTAFLVCLVKIVRGQMVQGILWLLLITGVQGHLDCKPE 41 FSYAIAKDERIGQLGAEGLTTTWKEYSPGMKLEDTMVIAW 81 CEDGKLMYLQRCTRETRYLAILHTRALPTSVVFKKLFDGR 121 KQEDVVEMNDNFEFGLCPCDAKPIVRGKFNTTLLNGPAFQ 161 MVCPIGWTGTVSCTSFNMDTLATTVVRTYRRSKPFPHRQG 201 CITQKNLGEDLHNCILGGNWTCVPGDQLLYKGGSIESCKW 241 CGYQFKESEGLPHYPIGKCKLENETGYRLVDSTSCNREGV 281 AIVPQGTLKCKIGKTTVQVIAMDTKLGPMPCRPYEIISSE 321 GPVEKTACTFNYTKTLKNKYFEPRDSYFQQYMLKGEYQYW 361 FDLEVTDHHRDYFAESILVVVVALLGGRYVLWLLVTYMVL 401 SEQKALG

[0066] The C-terminus of E2 includes about 30 hydrophobic amino acids (FIG. 1), which can serve as a transmembrane anchor for the full length E2, which remains cell associated in virus-infected cells (53).

[0067] In another example, BVDV E2 protein with an amino acid sequence provided by GenBank accession# GI 3859502 (1.18 kb) (shown below as SEQ ID NO:4) can be incorporated into the vectors and compositions described herein.

TABLE-US-00003 1 FPECKEGFQYAISKDRKIGPLGPFSLTTTWHLPTKKIVDS 41 MVQVWCDGKNLKILETCTKEERYLVAVHERALSTSAEFMQ 81 ISSGTKGPEVIDMHDDFEFGLCPCDSKPVMRGKFNASLLN 121 GPAFQMVCPQGWTGTIECILANQDTLDTTVVRTYRRTTPF 161 QRRKWCTYEKIIGEDIHECILGGNWTCITGDHSKLKDGPI 201 KKCKWCGYDFFDSEGLPHYPIGKCMLSNESGYRIVDDTSC 241 DRGGVAIVPTGTLKCRIGKATVQVIATNTDLGPMPCSPDE 281 VIASEGPVEKTACTFNYSKTLPNKYYEPRDRYFQQYMLKG 321 EWQYWFDLDTVDHHKDYFSEFIVIAVVALLGGKYVLWLLV 361 TYMILSEQMAMG

[0068] An example of a BVDV E2-1 protein with an amino acid sequence provided below as SEQ ID NO:5 can be encoded within the expression cassettes, vectors and compositions described herein.

TABLE-US-00004 1 HLDCKPEFSYAIAKDERIGQLGAEGLTTTWKEYSPGMKLE 41 DTMVIAWCEDGKLMYLQRCTRETRYLAILHTRALPTSVVF 81 KKLFDGRKQEDVVEMNDNFEFGLCPCDAKPIVRGKFNTTL 121 LNGPAFQMVCPIGWTGTVSCTSFNMDTLATTVVRTYRRSK 161 PFPHRQGCITQKNLGEDLHNCILGGNWTCVPGDQLLYKGG 201 SIESCKWCGYQFKESEGLPHYPIGKCKLENETGYRLVDST 241 SCNREGVAIVPQGTLKCKIGKTTVQVIAMDTKLGPMPCRP 281 YEIISSEGPVEKTACTFNYTKTLKNKYFEPRDSYFQQYML 321 KGEYQYWFDLEVTDHHRDYFAESILVVVVALLGGRYVLWL 361 LVTYMVLSEQKALG

[0069] A Bos taurus codon optimized nucleic acid segment encoding the BVDV E2-1 protein with SEQ ID NO:5, for example, can have the following nucleotide sequence, shown below as SEQ ID NO:6.

TABLE-US-00005 1 CACCTGGATTGCAAGCCTGAGTTCTCATACGCCATCGCTA 41 AAGACGAGAGAATTGGCCAGCTGGGGGCCGAAGGACTGAC 81 CACAACTTGGAAGGAGTATTCTCCAGGCATGAAACTGGAA 121 GATACCATGGTCATCGCTTGGTGCGAGGACGGGAAGCTGA 161 TGTACCTGCAGCGGTGCACAAGAGAAACTCGATATCTGGC 201 CATTCTGCATACTCGAGCTCTGCCCACCAGTGTGGTCTTC 241 AAGAAACTGTTTGACGGACGGAAGCAGGAGGATGTGGTCG 281 AAATGAACGACAATTTCGAGTTTGGCCTGTGCCCCTGTGA 321 TGCCAAGCCTATCGTGAGGGGAAAATTCAACACCACACTG 361 CTGAATGGCCCAGCTTTTCAGATGGTGTGCCCCATTGGCT 401 GGACCGGGACAGTCTCATGTACCAGCTTCAACATGGACAC 441 TCTGGCCACTACCGTGGTCCGCACTTACCGGAGGTCTAAG 481 CCCTTTCCTCACAGACAGGGCTGCATCACCCAGAAAAACC 521 TGGGGGAGGATCTGCATAACTGCATTCTGGGAGGAAATTG 561 GACCTGCGTGCCAGGGGACCAGCTGCTGTACAAGGGAGGC 601 TCCATCGAATCTTGCAAGTGGTGTGGCTACCAGTTCAAAG 641 AGAGCGAAGGGCTGCCTCACTATCCAATTGGARAGTGTAA 721 ACTGGAGAACGAAACCGGCTATCGGCTGGTGGATTCTACA 761 AGTTGCAATAGGGAGGGAGTGGCTATCGTCCCTCAGGGGA 801 CACTGAAGTGTAAAATCGGAAAGACAACTGTGCAGGTCAT 841 TGCTATGGACACTAAACTGGGGCCAATGCCCTGCAGACCT 881 TACGAGATCATTAGCTCCGAGGGACCAGTGGAAAAGACCG 921 CCTGTACCTTCAACTACACTAAAACCCTGAAGAACAAGTA 961 TTTCGAACCCCGAGATTCCTACTTTCAGCAGTATATGCTG 1001 AAGGGCGAGTACCAGTATTGGTTCGACCTGGAAGTGACAG 1041 ACCACCATAGGGATTACTTTGCCGAGAGCATCCTGGTGGT 1081 CGTGGTCGCTCTGCTGGGAGGACGCTACGTGCTGTGGCTG 1121 CTGGTGACCTATATGGTCCTGTCCGAGCAGAAGGCCCTGG 1161 GC

[0070] An example of an amino acid sequence of a BVDV E2-1 protein with an N-terminal gD signal sequence is provided below as SEQ ID NO:7. This fusion protein can be encoded within the expression cassettes, vectors and compositions described herein.

TABLE-US-00006 1 MQGPTLAVLGALLAVAVSLHLDCKPEFSYAIAKDERIGQL 41 GAEGLTTTWKEYSPGMKLEDTMVIAWCEDGKLMYLQRCTR 81 ETRYLAILHTRALPTSVVFKKLFDGRKQEDVVEMNDNFEF 121 GLCPCDAKPIVRGKFNTTLLNGPAFQMVCPIGWTGTVSCT 161 SFNMDTLATTVVRTYRRSKPFPHRQGCITQKNLGEDLHNC 201 ILGGNWTCVPGDQLLYKGGSIESCKWCGYQFKESEGLPHY 241 PIGKCKLENETGYRLVDSTSCNREGVAIVPQGTLKCKIGK 281 TTVQVIAMDTKLGPMPCRPYEIISSEGPVEKTACTFNYTK 321 TLKNKYFEPRDSYFQQYMLKGEYQYWFDLEVTDHHRDYFA 361 ESILVVVVALLGGRYVLWLLVTYMVLSEQKALG
A Bos taurus codon optimized nucleic acid segment encoding the gD-BVDV E2-1 fusion protein with SEQ ID NO:7, for example, can have the following nucleotide sequence, shown below as SEQ ID NO:8.

TABLE-US-00007 1 ATCGATGCCGCCACCATGCAGGGACCAACACTGGCCGTGC 41 TGGGGGCTCTGCTGGCTGTGGCTGTCTCCCTGTTCCCCGA 81 GTGCAAGGAAGGATTTCAGTACGCCATCAGCAAGGACCGG 121 AAAATTGGACCACTGGGACCAGAGTCCCTGACCACAACTT 161 GGCACCTGCCCACCAAGAAAATCGTGGACTCTATGGTGCA 201 GGTCTGGTGCGATGGCAAGAACCTGAAAATTCTGGAGACA 241 TGTACTAAGGAGGAGAGATACCTGGTGGCTGTCCACGAGC 281 GCGCTCTGTCTACCAGTGCCGAGTTCATGCAGATCAGCTC 321 CGGAACAAAGGGCCCTGAAGTGATCGACATGCACGACGAT 361 TTCGAATTTGGCCTGTGCCCCTGTGATAGTAAGCCTGTGA 401 TGCGCGGAAAATTCAACGCTTCACTGCTGAATGGCCCTGC 441 CTTTCAGATGGTGTGCCCACAGGGGTGGACCGGAACAATC 481 GAGTGTATTCTGGCTAACCAGGACACACTGGATACCACAG 521 TGGTCCGGACTTACCGGAGGACTACCCCTTTTCAGCGCAG 561 AAAGTGGTGCACCTATGAGAAAATCATTGGCGAGGACATC 601 CACGAGTGCATCCTGGGCGGGAATTGGACCTGTATCACAG 641 GCGACCATTCTAAGCTGAAAGATGGGCCAATTAAGAAATG 681 CAAGTGGTGTGGCTACGACTTCTTTGATAGTGAGGGACTG 721 CCTCATTATCCAATCGGCAAATGTATGCTGTCAAACGAAA 761 GCGGGTACAGATATGTGGACGATACTAGCTGCGATCGAGG 801 AGGAGTGGCTATCGTCCCAACTGGGACCCTGAAGTGTAGG 841 ATCGGAAAAGCTACCGTGCAGGTCATTGCCACAAATACTG 881 ACCTGGGACCAATGCCTTGCTCCCCAGATGAAGTGATCGC 921 TTCTGAGGGACCTGTCGAAAAGACTGCCTGTACCTTCAAC 961 TACTCCAAGACACTGCCAAACAAGTACTATGAGCCCCGAG 1001 ACCGGTACTTCCAGCAGTATATGCTGAAGGGGGAATGGCA 1041 GTACTGGTTTGACCTGGATACCGTGGACCACCATAAGGAT 1081 TACTTCTCAGAGTTTATCGTGATTGCCGTGGTCGCTCTGC 1121 TGGGGGGAAAGTACGTGCTGTGGCTGCTGGTCACCTATAT 1161 GATCCTGAGTGAACAGATGGCCATGGGCATGCAT

[0071] An expression cassette with a ClaI-NsiI nucleic acid segment that includes the SEQ ID NO:7 BVDV E2-1 nucleotide sequence has been made. This BVDV E2-1 expression cassette includes the following in the 5-3 direction: a Kozak sequence, nucleic acid segment for BHV-1 gD signal sequence (GenBank accession # AFV53430.1, aa 1-19), a Bos taurus codon-optimized nucleotide sequence for BVDV E2-1 (GenBank accession # NP_776263.1), and is provided below as SEQ ID NO:9 (and shown in FIG. 15A-15B).

TABLE-US-00008 1 GGTACCCTCGTGAGGCTCCGGTGCCCGTCAGTGGGCAGAG 41 CGCACATCGCCCACAGTCCCCGAGAAGTTGGGGGGAGGGG 81 TCGGCAATTGAACCGGTGCCTAGAGAAGGTGGCGCGGGGT 121 AAACTGGGAAAGTGATGTCGTGTACTGGCTCCGCCTTTTT 161 CCCGAGGGTGGGGGAGAACCGTATATAAGTGCAGTAGTCG 201 CCGTGAACGTTCTTTTTCGCAACGGGTTTGCCGCCAGAAC 241 ACAGGTAAGTGCCGTGTGTGGTTCCCGCGGGCCTGGCCTC 281 TTTACGGGTTATGGCCCTTGCGTGCCTTGAATTACTTCCA 321 CCTGGCTGCAGTACGTGATTCTTGATCCCGAGCTTCGGGT 361 TGGAAGTGGGTGGGAGAGTTCGAGGCCTTGCGCTTAAGGA 401 GCCCCTTCGCCTCGTGCTTGAGTTGAGGCCTGGCCTGGGC 441 GCTGGGGCCGCCGCGTGCGAATCTGGTGGCACCTTCGCGC 431 CTGTCTCGCTGCTTTCGATAAGTCTCTAGCCATTTAAAAT 521 TTTTGATGACCTGCTGCGACGCTTTTTTTCTGGCAAGATA 561 GTCTTGTAAATGCGGGCCAAGATCTGCACACTGGTATTTC 601 GGTTTTTGGGGCCGCGGGCGGCGACGGGGCCCGTGCGTCC 641 CAGCGCACATGTTCGGCGAGGCGGGGCCTGCGAGCGCGGC 681 CACCGAGAATCGGACGGGGGTAGTCTCAAGCTGGCCGGCC 721 TGCTCTGGTGCCTGGCCTCGCGCCGCCGTGTATCGCCCCG 761 CCCTGGGCGGGAAGGCTGGCCCGGTCGGCACCAGTTGCGT 801 GAGCGGAAAGATGGCCGCTTCCCGGCCCTGCTGCAGGGAG 841 CTCAAAATGGAGGACGCGGCGCTCGGGAGAGCGGGCGGGT 881 GAGTCACCCACACAAAGGAAAAGGGCCTTTCCGTCCTCAG 921 CCGTCGGTTCATGTGACTCCACGGAGTACCGGGCGCCGTC 961 CAGGCACCTCGATTAGTTCTCGAGCTTTTGGAGTACGTCG 1001 TCTTTAGGTTGGGGGGAGGGGTTTTATGCGATGGAGTTTC 1041 CCCACACTGAGTGGGTGGAGACTGAAGTTAGGCCAGCTTG 1081 GCACTTGATGTAATTCTCCTTGGAATTTGCCCTTTTTGAG 1121 TTTGGATCTTGGTTCATTCTCAAGCCTCAGACAGTGGTTC 1161 AAAGTTTTTTTCTTCCATTTCAGGTGTCGTGAGGAATTAG 1201 CTTGGTACTAATACGACTCACTATAGGGAGACCCAAGCTG 1241 GCTAGGTAAGTGTACGAGCTCGATCACTAGTCCAGTGTGG 1281 ATCGATCGCCGCCACCATGCAGGGACCCACCCTGGCCGTG 1321 CTGGGCGCTCTGCTGGCTGTGGCTGTCAGTCTGCACCTGG 1361 ATTGCAAGCCTGAGTTCTCATACGCCATCGCTAAAGACGA 1401 GAGAATTGGCCAGCTGGGGGCCGAAGGACTGACCACAACT 1441 TGGAAGGAGTATTCTCCAGGCATGAAACTGGAAGATACCA 1481 TGGTCATCGCTTGGTGCGAGGACGGGAAGCTGATGTACCT 1521 GCAGCGGTGCACAAGAGAAACTCGATATCTGGCCATTCTG 1561 CATACTCGAGCTCTGCCCACCAGTGTGGTCTTCAAGAAAC 1601 TGTTTGACGGACGGAAGCAGGAGGATGTGGTCGAAATGAA 1641 CGACAATTTCGAGTTTGGCCTGTGCCCCTGTGATGCCAAG 1681 CCTATCGTGAGGGGAAAATTCAACACCACACTGCTGAATG 1721 GCCCAGCTTTTCAGATGGTGTGCCCCATTGGCTGGACCGG 1761 GACAGTCTCATGTACCAGCTTCAACATGGACACTCTGGCC 1801 ACTACCGTGGTCCGCACTTACCGGAGGTCTAACCCCTTTC 1841 CTCACAGACAGGGCTGCATCACCCAGAAAAACCTGGGGGA 1881 GGATCTGCATAACTGCATTCTGGGAGGAAATTGGACCTGC 1921 GTGCCAGGGGACCAGCTGCTGTACAAGGGAGGCTCCATCG 1961 AATCTTGCAAGTGGTGTGGCTACCAGTTCAAAGAGAGCGA 2001 AGGGCTGCCTCACTATCCAATTGGAAAGTGTAAACTGGAG 2041 AACGAAACCGGCTATCGGCTGGTGGATTCTACAAGTTGCA 2081 ATAGGGAGGGAGTGGCTATCGTCCCTCAGGGGACACTGAA 2121 GTGTAAAATCGGAAAGACAACTGTGCAGGTCATTGCTATG 2161 GACACTAAACTGGGGCCAATGCCCTGCAGACCTTACGAGA 2201 TCATTAGCTCCGAGGGACCAGTGGAAAAGACCGCCTGTAC 2241 CTTCAACTACACTAAAACCCTGAAGAACAAGTATTTCGAA 2281 CCCCGAGATTCCTACTTTCAGCAGTATATGCTGAAGGGCG 2321 AGTACCAGTATTGGTTCGACCTGGAAGTGACAGACCACCA 2361 TAGGGATTACTTTGCCGAGAGCATCCTGGTGGTCGTGGTC 2401 GCTCTGCTGGGAGGACGCTACGTGCTGTGGCTGCTGGTGA 2441 CCTATATGGTCCTGTCCGAGCAGAAGGCCCTGGGCATGCA 2481 TGGTAAGCCTATCCCTAACCCTCTCCTCGGTCTCGATTCT 2521 ACGCGTACCGGTCATCATCACCATCACCATTGAGTTTAAA 2561 CCCGCTGATCAGCCTCGACTGTGCCTTCTAGTTGCCAGCC 2601 ATCTGTTGTTTGCCCCTCCCCCGTGCCTTCCTTGACCCTG 2641 GAAGGTGCCACTCCCACTGTCCTTTCCTAATAAAATGAGG 2681 AAATTGCATCGCATTGTCTGAGTAGGTGTCATTCTATTCT 2721 GGGGGGTGGGGTGGGGCAGGACAGCAAGGGGGAGGATTGG 2761 GAAGACAATAGCAGGCATGCTGGGGATGCGGTGGGCTCTA 2801 TGGCTTGGTACC.

[0072] The 1.235 bp ClaI/NsiI fragment encoding the gD signal sequence-BVDV E2-1 fusion protein was cloned into the corresponding ClaI/NsiI sites of the pPreBVDV-E2 chimeric vector (which includes SEQ ID NO: 1) resulting in a BVDVE2-1 chimeric gene expression cassette (SEQ ID NO:9).

[0073] An example of a BVDV E2-2 protein with an amino acid sequence provided below as SEQ ID NO: 10 can be encoded within the expression cassettes, vectors and compositions described herein.

TABLE-US-00009 1 FPECKEGFQYAISKDRKIGPLGPESLTTTWHLPTKKIVDS 41 MVQVWCDGKNLKILETCTKEERYLVAVHERALSTSAEFMQ 81 ISSGTKGPEVIDMHDDFEFGLCPCDSKPVMRGKFNASLLN 121 GPAFQMVCPQGWTGTIECILANQDTLDTTVVRTYRRTTPF 161 QRRKWCTYEKIIGEDIHECILGGNWTCITGDHSKLKDGPI 201 KKCKWCGYDFFDSEGLPHYPIGKCMLSNESGYRYVDDTSC 241 DRGGVAIVPTGTLKCRIGKATVQVIATNTDLGPMPCSPDE 281 VIASEGPVEKTACTFNYSKTLPNKYYEPRDRYFQQYMLKG 321 EWQYWFDLDTVDHHKDYFSEFIVIAVVALLGGKYVLWLLV 361 TYMILSEQMAMG
A Bos taurus codon optimized nucleic acid segment encoding the BVDV E2-2 protein with SEQ ID NO: 10, for example, can have the following nucleotide sequence, shown below as SEQ ID NO:11.

TABLE-US-00010 1 TTCCCCGAGTGCAAGGAAGGATTTCAGTACGCCATCAGCA 41 AGGACCGGAAAATTGGACCACTGGGACCAGAGTCCCTGAC 81 CACAACTTGGCACCTGCCCACCAAGAAAATCGTGGACTCT 121 ATGGTGCAGGTCTGGTGCGATGGCAAGAACCTGAAAATTC 161 TGGAGACATGTACTAAGGAGGAGAGATACCTGGTGGCTGT 201 CCACGAGCGCGCTCTGTCTACCAGTGCCGAGTTCATGCAG 241 ATCAGCTCCGGAACAAAGGGCCCTGAAGTGATCGACATGC 281 ACGACGATTTCGAATTTGGCCTGTGCCCCTGTGATAGTAA 321 GCCTGTGATGCGCGGAAAATTCAACGCTTCACTGCTGAAT 361 GGCCCTGCCTTTCAGATGGTGTGCCCACAGGGGTGGACCG 401 GAACAATCGAGTGTATTCTGGCTAACCAGGACACACTGGA 441 TACCACAGTGGTCCGGACTTACCGGAGGACTACCCCTTTT 481 CAGCGCAGAAAGTGGTGCACCTATGAGAAAATCATTGGCG 521 AGGACATCCACGAGTGCATCCTGGGCGGGAATTGGACCTG 561 TATCACAGGCGACCATTCTAAGCTGAAAGATGGGCCAATT 601 AAGAAATGCAAGTGGTGTGGCTACGACTTCTTTGATAGTG 641 AGGGACTGCCTCATTATCCAATCGGCAAATGTATGCTGTC 631 AAACGAAAGCGGGTACAGATATGTGGACGATACTAGCTGC 721 GATCGAGGAGGAGTGGCTATCGTCCCAACTGGGACCCTGA 761 AGTGTAGGATCGGAATAGCTACCGTGCAGGTCATTGCCAC 801 AAATACTGACCTGGGACCAATGCCTTGCTCCCCAGATGAA 841 GTGATCGCTTCTGAGGGACCTGTCGAAAAGACTGCCTGTA 881 CCTTCAACTACTCCAAGACACTGCCAAACAAGTACTATGA 921 GCCCCGAGACCGGTACTTCCAGCAGTATATGCTGAAGGGG 961 GAATGGCAGTACTGGTTTGACCTGGATACCGTGGACCACC 1001 ATAAGGATTACTTCTCAGAGTTTATCGTGATTGCCGTGGT 1041 CGCTCTGCTGGGGGGAAAGTACGTGCTGTGGCTGCTGGTC 1081 ACCTATATGATCCTGAGTGAACAGATGGCCATGGGC

[0074] An example an amino acid sequence of a BVDV E2-2 protein with an N-terminal gD signal sequence is provided below as SEQ ID NO: 12. This fusion protein can be encoded within the expression cassettes, vectors and compositions described herein.

TABLE-US-00011 1 MQGPTLAVLGALLAVAVSLFPECKEGFQYAISKDRKIGPL 41 GPESLTTTWHLPTKKTVDSMVQVWCDGKNLKILETCTKEE 81 RYLVAVHERALSTSAEFMQISSGTKGPEVIDMHDDFEFGL 121 CPCDSKPVMRGKFNASLLNGPAFQMVCPQGWTGTIECILA 121 NQDTLDTTVVRTYRRTTPFQRRKWCTYEKIIGEDIHECIL 121 GGNWTCITGDHSKLKDGPIKKCKWCGYDFFDSEGLPHYPI 121 GKCMLSNESGYRYVDDISCDRGGVAIVPTGTLKCRIGKAT 121 VQVIATNTDLGPMPCSPDEVIASEGPVEKTACTFNYSKTL 121 PNKYYEPRDRYFQQYMLKGEWQYWFDLDTVDHHKDYFSEF 121 IVIAVVALLGGKYVLWLLVTYMILSEOMAMGMH
A Bos taurus codon optimized nucleic acid segment encoding the gD-BVDV E2-2 fusion protein with SEQ ID NO: 12, for example, can have the following nucleotide sequence, shown below as SEQ ID NO: 13.

TABLE-US-00012 1 ATGCAGGGACCAACACTGGCCGTGCTGGGGGCTCTGCTGG 41 CTGTGGCTGTCTCCCTGTTCCCCGAGTGCAAGGAAGGATT 81 TCAGTACGCCATCAGCAAGGACCGGAAAATTGGACCACTG 121 GGACCAGAGTCCCTGACCACAACTTGGCACCTGCCCACCA 161 AGAAAATCGTGGACTCTATGGTGCAGGTCTGGTGCGATGG 201 CAAGAACCTGAAAATTCTGGAGACATGTACTAAGGAGGAG 241 AGATACCTGGTGGCTGTCCACGAGCGCGCTCTGTCTACCA 281 GTGCCGAGTTCATGCAGATCAGCTCCGGAACAAAGGGCCC 321 TGAAGTGATCGACATGCACGACGATTTCGAATTTGGCCTG 361 TGCCCCTGTGATAGTAAGCCTGTGATGCGCGGAAAATTCA 401 ACGCTTCACTGCTGAATGGCCCTGCCTTTCAGATGGTGTG 441 CCCACAGGGGTGGACCGGAACAATCGAGTGTATTCTGGCT 481 AACCAGGACACACTGGATACCACAGTGGTCCGGACTTACC 521 GGAGGACTACCCCTTTTCAGCGCAGAAAGTGGTGCACCTA 561 TGAGAAAATCATTGGCGAGGACATCCACGAGTGCATCCTG 601 GGCGGGAATTGGACCTGTATCACAGGCGACCATTCTAAGC 641 TGAAAGATGGGCCAATTAAGAAATGCAAGTGGTGTGGCTA 681 CGACTTCTTTGATAGTGAGGGACTGCCTCATTATCCAATC 721 GGCAAATGTATGCTGTCAAACGAAAGCGGGTACAGATATG 761 TGGACGATACTAGCTGCGATCGAGGAGGAGTGGCTATCGT 801 CCCAACTGGGACCCTGAAGTGTAGGATCGGAAAAGCTACC 841 GTGCAGGTCATTGCCACAAATACTGACCTGGGACCAATGC 881 CTTGCTCCCCAGATGAAGTGATCGCTTCTGAGGGACCTGT 921 CGAAAAGACTGCCTGTACCTTCAACTACTCCAAGACACTG 961 CCAAACAAGTACTATGAGCCCCGAGACCGGTACTTCCAGC 1001 AGTATATGCTGAAGGGGGAATGGCAGTACTGGTTTGACCT 1041 GGATACCGTGGACCACCATAAGGATTACTTCTCAGAGTTT 1081 ATCGTGATTGCCGTGGTCGCTCTGCTGGGGGGAAAGTACG 1201 TGCTGTGGCTGCTGGACACCTATATGATCCTGAGTGAACA 1261 GATGGCCATGGGCATGCAT

[0075] An expression cassette with a ClaI-NsiI nucleic acid segment that includes the SEQ ID NO: 13 nucleotide sequence of the BVDV E2-2 fusion protein has been made. This BVDV E2-2 expression cassette includes the following in the 5-3 direction: a Kozak sequence, nucleic acid segment for BHV-1 gD signal sequence (GenBank accession # AFV53430.1, aa 1-19), a Bos taurus codon-optimized nucleotide sequence for BVDV E2-2 (GenBank accession # AAC72814.1), and is provided below as SEQ ID NO: 14 (and shown in FIG. 16A-16B).

TABLE-US-00013 1 GGTACCCTCGTGAGGCTCCGGTGCCCGTCAGTGGGCAGAG 41 CGCACATCGCCCACACTCCCCGAGAAGTTGGGGGGAGGGG 81 TCGGCAATTGAACCGGTGCCTAGAGAAGGTGGCGCGGGGT 121 AAACTGGGAAAGTGATGTCGTGTACTGGCTCCGCCTTTTT 161 CCCGAGGGTGGGGGAGAACCGTATATAAGTGCAGTAGTCG 201 CCGTGAACGTTCTTTTTCGCAACGGGTTTGCCGCCAGAAC 241 ACAGGTAAGTGCCGTGTGTGGTTCCCGCGGGCCTGGCCTC 281 TTTACGGGTTATGGCCCTTGCGTGCCTTGAATTACTTCCA 321 CCTGGCTGCAGTACGTGATTCTTGATCCCGAGCTTCGGGT 361 TGGAAGTGGGTGGGAGAGTTCGAGGCCTTGCGCTTAAGGA 401 GCCCCTTCGCCTCGTGCTTGAGTTGAGGCCTGGCCTGGGC 441 GCTGGGGCCGCCGCGTGCGAATCTGGTGGCACCTTCGCGC 431 CTGTCTCGCTGCTTTCGATAAGTCTCTAGCCATTTAAAAT 521 TTTTGATGACCTGCTGCGACGCTTTTTTTCTGGCAAGATA 561 GTCTTGTAAATGCGGGCCAAGATCTGCACACTGGTATTTC 601 GGTTTTTGGGGCCGCGGGCGGCGACGGGGCCCGTGCGTCC 641 CAGCGCACATGTTCGGCGAGGCGGGGCCTGCGAGCGCGGC 681 CACCGAGAATCGGACGGGGGTAGTCTCAAGCTGGCCGGCC 721 TGCTCTGGTGCCTGGCCTCGCGCCGCCGTGTATCGCCCCG 761 CCCTGGGCGGCAAGGCTGGCCCGGTCGGCACCAGTTGCGT 801 CACCCGAAAGATGGCCGCTTCCCGGCCCTGCTGCAGGGAG 841 CTCAAAATGGAGGACGCGGCGCTCGGGAGAGCGGGCGGGT 831 GAGTCACCCACACAAAGGAAAAGGGCCTTTCCGTCCTCAG 921 CCGTCGCTTCATGTGACTCCACGGAGTACCGGGCGCCGTC 961 CAGGCACCTCGATTAGTTCTCGAGCTTTTGGAGTACGTCG 1001 TCTTTAGGTTGGGGGGAGGGGTTTTATGCGATGGAGTTTC 1041 CCCACACTGAGTGGGTGGAGACTGAAGTTAGGCCAGCTTG 1081 GCACTTGATGTAATTCTCCTTGGAATTTGCCCTTTTTGAG 1121 TTTGGATCTTGGTTCATTCTCAAGCCTCAGACAGTGGTTC 1161 AAAGTTTTTTTCTTCCATTTCAGGTGTCGTGAGGAATTAG 1201 CTTGGTACTAATACGACTCACTATAGGGAGACCCAAGCTG 1241 GCTAGGTAAGTGTACGAGCTCGATCACTAGTCCAGTGTGG 1281 ATCGATCGCCGCCACCATGCAGGGACCAACACTGGCCGTG 1321 CTGGGGGCTCTGCTGGCTGTGGCTGTCTCCCTGTTCCCCG 1361 AGTGCAAGGAAGGATTTCAGTACGCCATCAGCAAGGACCG 1401 GAAAATTGGACCACTGGGACCAGAGTCCCTGACCACAACT 1441 TGGCACCTGCCCACCAAGAAAATCGTGGACTCTATGGTGC 1431 AGGTCTGGTGCGATGGCAAGAACCTGAAAATTCTGGAGAC 1521 ATGTACTAAGGAGGAGAGATACCTGGTGGCTGTCCACGAG 1561 CGCGCTCTGTCTACCAGTGCCGAGTTCATGCAGATCAGCT 1601 CCGGAACAAAGGGCCCTGAAGTGATCGACATGCACGACGA 1641 TTTCGAATTTGGCCTGTGCCCCTGTGATAGTAAGCCTGTG 1681 ATGCGCGGAAAATTCAACGCTTCACTGCTGAATGGCCCTG 1721 CCTTTCAGATGGTGTGCCCACAGGGGTGGACCGGAACAAT 1761 CGAGTGTATTCTGGCTAACCAGGACACACTGGATACCACA 1801 GTGGTCCGGACTTACCGGAGGACTACCCCTTTTCAGCGCA 1841 GAAAGTGGTGCACCTATGAGAAAATCATTGGCGAGGACAT 1881 CCACGAGTGCATCCTGGGCGGGAATTGGACCTGTATCACA 1921 GGCGACCATTCTAAGCTGAAAGATGGGCCAATTAAGAAAT 1961 GCAAGTGGTGTGGCTACGACTTCTTTGATAGTGAGGGACT 2001 GCCTCATTATCCAATCGGCAAATGTATGCTGTCAAACGAA 2041 AGCGGGTACAGATATGTGGACGATACTAGCTGCGATCGAG 2081 GAGGAGTGGCTATCGTCCCAACTGGGACCCTGAAGTGTAG 2121 GATCGGAAAAGCTACCGTGCAGGTCATTGCCACAAATACT 2161 GACCTGGGACCAATGCCTTGCTCCCCAGATGAAGTGATCG 2201 CTTCTGAGGGACCTGTCGAAAAGACTGCCTGTACCTTCAA 2241 CTACTCCAAGACACTGCCAAAGAAGTACTATGAGCCCCGA 2281 GACCGGTACTTCCAGCAGTATATGCTGAAGGGGGAATGGC 2321 AGTACTGGTTTGACCTGGATACCGTGGACCACCATAAGGA 2361 TTACTTCTCAGAGTTTATCGTGATTGCCGTGGTCGCTCTG 2401 CTGGGGGGAAAGTACGTGCTGTGGCTGCTGGTCACCTATA 2441 TGATCCTGAGTGAACAGATGGCCATGGGCATGCATGGTAA 2481 GCCTATCCCTAACCCTCTCCTCGGTCTCGATTCTACGCGT 2521 ACCGGTCATCATCACCATCACCATTGAGTTTAAACCCGCT 2561 GATCAGCCTCGACTGTGCCTTCTAGTTGCCAGCCATCTGT 2601 TGTTTGCCCCTCCCCCGTGCCTTCCTTGACCCTGGAAGGT 2641 GCCACTCCCACTGTCCTTTCCTAATAAAATGAGGAAATTG 2681 CATCGCATTGTCTGAGTAGGTGTCATTCTATTCTGGGGGG 2721 TGGGGTGGGGCAGGACAGCAAGGGGGAGGATTGGGAAGAC 2761 AATAGCAGGCATGCTGGGGATGCGGTGGGCTCTATGGCTT 2801 GGTACC

[0076] Some studies have reported that immunization with E2 glycoproteins cloned in live recombinant virus vectors induced BVDV-specific neutralizing antibody (6, 18, 77). Subunit E2 protein based vaccines, expressed in yeast, have been tested against classical swine fever, a related pestivirus of swine (54). Even though a protective serum neutralizing response may in some cases have been observed for a subunit E2 protein vaccine, more than one injection was required for such a response in animals, and such treatment did not provide a cellular immune response (54). In addition, the cost of protein expression and purification may not be economically feasible for cattle (62).

[0077] Vaccination of mice with an experimental DNA plasmid encoding the E2 glycoprotein of BVDV type 1 (E2.1) produced some humoral and cellular immune responses (53). In cattle, animals vaccinated with DNA plasmid encoding E2.1 were partially protected against BVDV type 2 challenge, whereas animals vaccinated with plasmids encoding both E2.1 and E2.2 (BVDV type 2 E2) were well protected from challenge with BVDV type 2 when they were subsequently boosted with E2.2 encoding DNA or E2 protein (52). Therefore, such DNA based subunit E2 vaccines require at least two injections, which may not be cost effective for the cattle industry.

[0078] The inventor and coworkers have successfully incorporated a chimeric gene cassette encoding BVDV type 1 E2 ORF (BVDV E2.1) into the novel BoHV-1 tmv vector. Such a vector can achieve good immunological reactions in animals after just one administration.

Erns Antigens

[0079] Another BVDV antigen that can be encoded within and expressed by the expression systems described herein is an envelope Ems (about 227 amino acids) glycoprotein. Ems and E1 are synthesized early as a heterodimer (Ems-E1 precursor), but at later stages of polypeptide processing, Ems forms a disulfide bonded homodimer (Rumenapf et al., 1993; Konig et al., 1995), whereas E2 forms a disulfide-linked homodimer and also a heterodimer with E1 (Weiland et al., 1990: Rumenapf et al., 1991). Ems and E2 are located at the surface of infected cells (Weiland et al., 1999), and may induce virus-neutralizing antibodies. Ems is secreted from infected cells but it is also a structural component of the virus particle (Rumenapf et al., 1993; Weiland et al., 1992). Some studies indicate that Ems may be a ribonuclease.

[0080] Ems has a mass of 44-48 kDa (Ahn et al. 1996), and its C-terminus functions as (1) a membrane anchor: (2) a retention/secretion signal; (3) a binding site for cell surface glycosaminoglycans (GAGs); and (4) a signal peptidase cleavage site. In one example, a BVDV1 Ems can have the following amino acid sequence (SEQ ID NO:39).

TABLE-US-00014 1 ENITQWNLQDNGTEGIQRAMFQRGVNRSLHGIWPEKICTG 41 VPSHLATDIELKTIHGMMDASEKTNYTCCRLQRHEWNKHG 81 WCNWYNIEPWILVMNRTQANLTEGQPPRECAVTCRYDRAS 121 DLNVVTQARDSPTPLTGCKKGKNFSFAGILMRGPCNFEIA 161 ASDVLFKEHERISMFQDTTLYLVDGLTNSLEGARQGTAKL 201 TTWLGKQLGILGKKLENKSKTWFGAYA

[0081] A fusion protein of the BVDV1 envelope Ems protein with a gD signal sequence and a GMCSF (GenBank accession # NP_776452.1) fusion partner can have a nucleotide sequence, for example, as shown below as SEQ ID NO:40 (see also FIG. 21A).

TABLE-US-00015 1 TTAATTAACGCCACCATGCAGGGACCTACTCTGGCTGTGC 41 TGGGGGCTCTGCTGGCTGTCGCCGTCAGTCTGGAAAACAT 81 CACTCAGTGGAATCTGCAGGATAACGGCACCGAGGGGATC 121 CAGCGCGCCATGTTCCAGCGAGGCGTGAACCGGTCACTGC 161 ACGGGATCTGGCCAGAAAAGATTTGCACCGGAGTCCCAAG 201 CCACCTGGCTACCGACATCGAGCTGAAGACAATTCATGGA 241 ATGATGGATGCTAGCGAAAAAACCAACTACACATGCTGTC 281 GGCTGCAGAGGCACGAGTGGAATAAGCATGGCTGGTGTAA 321 CTGGTATAATATCGAACCCTGGATCCTGGTCATGAACAGA 361 ACACAGGCCAATCTGACTGAGGGACAGCCACCTCGAGAAT 401 GCGCTGTCACTTGTCGCTACGACAGAGCTAGCGATCTGAA 441 CGTGGTCACACAGGCTCGAGACTCCCCAACTCCTCTGACC 481 GGCTGCAAGAAAGGGAAGAACTTCTCCTTTGCTGGGATCC 521 TGATGCGCGGACCCTGTAATTTTGAGATTGCCGCTTCTGA 561 TGTGCTGTTCAAAGAGCACGAAAGAATCAGTATGTTTCAG 601 GACACCACACTGTACCTGGTGGATGGCCTGACCAACTCCC 641 TGGAGGGAGCTAGGCAGGGAACAGCTAAGCTGACTACCTG 681 GCTGGGGAAACAGCTGGGAATTCTGGGCAAGAAACTGGAA 721 AACAAGTCTAAAACCTGGTTCGGAGCCTATGCTATGTGGC 761 TGCAGAATCTGCTGCTGCTGGGCACAGTGGTCTGCTCTTT 801 TAGTGCCCCTACTAGGCCACCCAATACAGCTACTCGCCCA 841 TGGCAGCACGTGGACGCCATCAAGGAGGCTCTGAGTCTGC 881 TGAACCATAGCTCCGACACTGATGCCGTGATGAATGACAC 921 CGAGGTGGTCTCCGAAAAATTTGATTCTCAGGAGCCCACC 961 TGTCTGCAGACACGGCTGAAGCTGTACAAAAACGGGCTGC 1001 AGGGATCACTGACCAGCCTGATGGGAAGCCTGACTATGAT 1041 GGCCACCCACTATGAGAAGCATTGCCCTCCAACACCTGAA 1081 ACTAGTTGTGGGACCCAGTTCATCAGCTTCAAGAATTTCA 1121 AAGAAGACCTGAAAGAGTTCCTGTTTATCATTCCATTTGA 1181 CTGTTGGGAGCCAGCCCAGAAAGGTAAGCCTATCCCTAAC 1201 CCTCTCCTCGGTCTCGATTCTACGTAAGCGGCCGC

[0082] Another example of a BVDV2 Ems that has the following amino acid sequence (SEQ ID NO:42).

TABLE-US-00016 1 ENITQWNLMDNGTEGIQQAMFLRGVNRSLHGIWPEKICTG 41 VPTHLATDYELKEIVGMMDASEKTNYTCCRLQRHEWNKHG 81 WCNWFHIEPWIWLMNKTQNNLTEGQPLRECAVTCRYDKET 121 ELNIVTQARDRPTTLTGCKKGKNFSFAGVILDGPCNFKVS 161 VEDVLFKEHDCGNMLQETAIQLLDGATNTIEGARVGTAKL 201 TTWLGKQLGILGKELENKSKAWFGAHA

[0083] An example of a fusion protein of the BVDV2 envelope Ems protein with a gD signal sequence and a GMCSF (GenBank accession # NP_776452.1) fusion partner can have a nucleotide sequence, for example, provided as SEQ ID NO:41 (see also FIG. 21B).

[0084] A gG-deletion BoHV-1 virus was generated to attenuate the virus with respect to immune evasion and to provide a vector for insertion of an Ems-GMSF chimeric gene cassette at the gG locus. BHV-1 gG is secreted after proteolytic processing. In addition, gG is present on the virus envelope and is associated with infected cell membranes (Bryant et al., 2003). Like other alpha herpesvirus gG homologues, the BHV-1 gG has chemokine binding activity and it blocks the interaction of chemokines with cellular receptors and glycol-aminoglycans (GAGs). The chemokine binding activity of gG also occurs on cell membranes because membrane-anchored forms of gG bind to various chemokines (Bryant et al., 2003). Chemokines are small proteins (8-10 kd) that function as cytokines, and thus regulate trafficking and effector functions of leukocytes Baggiolini (1998). As such, chemokines are important regulators of inflammation, immune surveillance, and they have potent anti-viral functions. Functionally, chemokines can be divided into two groups: pro-inflammatory chemokines that are inducible and housekeeping chemokines that are constitutively expressed. Activation of chemokine functions are dependent on selective recognition and activation of chemokine receptors belonging to the seven-membrane domain. G protein-coupled receptor super family. Chemokines can also bind to glycosamino-glycans (GAGS). Chemokine binding to GAGS on cells, in particular endothelial cells, results in chemotactive chemokine gradients that allow the correct presentation of chemokines to leukocytes and therefore enable target cells to cross the endothelial barrier and migrate to tissues. BoHV-1, BoHV-5, and equine herpesvirus 1 encode a glycoprotein (gG) that is secreted from infected cells, and can bind to a broad range of chemokines (Bryant et al. (2003). Interactions between gG and chemokines block chemokine activity by preventing their interactions with specific receptors and GAGS. By preventing chemokine-GAG interactions, gG disrupts chemokine gradients, which controls the local environment surrounding an infected cell. A BoHV-1 gG deletion mutant was reported to have reduced virulence [78] suggesting gG is a viral immune evasion gene. Deletion of the BoHV-1 gene encoding gG leads to viral attenuation in calves because the mutant virus is more immunogenic (Kaashock et al., 1998). The chemokine binding activity encoded by BoHV-1 gG is responsible for the attenuated phenotype following infection of calves (Bryant et al. 2003). Removal of the gG locus reduces many of negative effects that could otherwise occur.

[0085] FIG. 21B illustrates an example of a nucleotide sequence of a fusion protein of the BVDV2 envelope Ems protein with a gD signal sequence and a GMCSF (GenBank accession # NP_776452.1) fusion partner can have (SEQ ID NO:41) that can be inserted in the gG-deleted BoHV vector. An example of a nucleotide sequence of a BoHV-1 gG deletion vector is shown below as SEQ ID NO:49 (see also FIG. 23).

TABLE-US-00017 1 GATATCAGGCAACGGGGCCTGCTCCCGCGCAGGAGCACGT 41 GGTGCTCAAGATCGGGGCCTCGGCCTCTACGCTGGCCGAG 81 GCTATGCTACTGCGAACCTTGGACCACGCCAACGTGGTCA 121 AGCTGAAGGCCGTGCTCTTCCACGGGGAGCTGGTGTGCGT 161 GGTGCTGGCGCGCTACCGCGAGGACCTGCACACGCACCTC 201 TGGAGAATCAACCGCCCGCTGGCGCTCCCCGCGGCGCTGG 241 CGGTGACGCGGGCCGTGCTGCGGGGCCTCGCGTACCTGCA 281 CTCCCGCCGGATCGCTCACCGGGACGTCAAAACGGAAAAC 321 GTCTTCCTCAACGGCCCAGGCGACGTGTGCCTGGGCGACT 361 TTGGCGCGGCACACGGGCCGGTCACCGAGCCCCGCTACTA 401 CGGCCTGGCCGGCACCCTGGAGACGAACTCGCCAGAGCTG 441 CTGGCGCGCGCGCGCTACGACTGCCGCACGGACGTGTGGA 481 GCGCGGGCGTCGTCGCGTACGAGATGCTGGCATACCCGCG 521 CGCGCTGTTCGACAGCCCCGCGGGCCCGCAGGGCGAGGAC 561 GCCGAGGCATCGGGCCCGCCGACGATCTTGGGCGACCGCG 601 ACTGCGCCCGGCAGCTGCTCCGCGTGATTCGCCGGCTGGC 641 CGTGCACGCCGAAGAGTTTCCACCCAGCCCCACTGACCGG 681 CTGACCCGCAACTTCAAGCGCCACGCGAGCACGCGCCGAG 721 AGCCGCACAGCCCGTACCGCTGCCTGGCGGTGCTCCGGCT 761 GCCCTGCGACGCCGACCGCCTCCTACACCAGATGCTGACC 801 TTTGACTTTCGCGCGCGCCCCACCGCCGCGGAGCTGCTGG 841 AGCACCCCGTCTTCGGTGCGGCCTCGGGGTAGCCCCGGGG 881 GTTTCCCGCAAAACTGAGGCATATAAGGCGCGGGCACCGG 921 CAAGTTTGGCATCCACACTTCGCGCTGTGGACACGAGAGC 961 GAACGCGAGCGAACGCGAGCGGAAGCGCGAGCACACGACT 1001 GCGATCGTTGACATTGATTATTGACTAGTTATTAATAGTA 1041 ATCAATTACGGGGTCATTAGTTCATAGCCCATATATGGAG 1081 TTCCGCGTTACATAACTTACGGTAAATGGCCCGCCTGGCT 1121 GACCGCCCAACGACCCCCGCCCATTGACGTCAATAATGAC 1161 GTATGTTCCCATAGTAACGCCAATAGGGACTTTCCATTGA 1201 CGTCAATGGGTGGAGTATTTACGGTAAACTGCCCACTTGG 1241 CAGTACATCAAGTGTATCATATGCCAAGTACGCCCCCTAT 1281 TGACGTCAATGACGGTAAATGGCCCGCCTGGCATTATGCC 1321 CAGTACATGACCTTATGGGACTTTCCTACTTGGCAGTACA 1361 TCTACGTATTAGTCATCGCTATTACCATGGTGATGCGGTT 1401 TTGGCAGTACATCAATGGGCGTGGATAGCGGTTTGACTCA 1441 CGGGGATTTCCAAGTCTCCACCCCATTGACGTCAATGGGA 1481 GTTTGTTTTGGCACCAAAATGAACGGGACTTTCCAAAATG 1521 TCGTAACAACTCCGCCCCATTGACGCAAATGGGCGGTAGG 1561 CGTGTACGGTGGGAGGTCTATATAAGCAGAGCTCTTAATT 1601 AAGTGTGTGTGTGCGGCCGCCTGTGCCTTCTAGTTGCCAG 1641 CCATCTGTTGTTTGCCCCTCCCCCGTGCCTTCCTTGACCC 1681 TGGAAGGTGCCACTCCCACTGTCCTTTCCTAATAAAATGA 1721 GGAAATTGCATCGCATTGTCTGAGTAGGTGTCATTCTATT 1761 CTGGGGGGTGGGGTGGGGCAGGACAGCAAGGGGGAGGATT 1801 GGGAAGACAATAGCAGGCATGCTGGGGATGCGGTGGGCTC 1841 TATGGTCGCCGGCACCCCACGCCGCCCCGACCCCGCTGTC 1881 CCGCGTTTACAATAAACAGTTATTCTTACCAACGTTGGTG 1921 CGCCTGTCGCGTGTCTATTGCGAGTTAAACCGAGTGCCCC 1961 ACCCAGGCAGGGCGGGGGTTGGGCCGGGCCGCAGCCCCGG 2001 CTGGGTATATATCCCCGACGGGCGACTAGAGATACACTCG 2041 CCCCGCGCGGCTGCTGCGAGCGGGCGAACATGCAAGGGCC 2081 GACATTGGCCGTGCTGGGCGCGCTGCTCGCCGTTGCGGTG 2121 AGCTTGCCTACACCCGCGCCGCGGGTGACGGTATACGTCG 2161 ACCCGCCGGCGTACCCGATGCCGCGATACAACTACACTGA 2201 ACGCTGGCACACTACCGGGCCCATACCGTCGCCCTTCGCA 2241 GACGGCCGCGAGCAGCCCGTCGAGGTGCGCTACGCGACGA 2281 GCGCGGCGGCGTGCGACATGCTGGCGCTGATCGCAGACCC 2321 GCAGGTGGGGCGCACGCTGTGGGAAGCGGTACGCCGGCAC 2361 GCGCGCGCGTACAACGCCACGGTCATATGGTACAAGATCG 2401 AGAGCGGGTGCGCCCGGCCGCTGTACTACATGGAGTACAC 2441 CGAGTGCGAGCCCAGGAAGCACTTTGGGTACTGCCGCTAC 2481 CGCACACCCCCGTTTTGGGACAGCTTCCTGGCGGGCTTCG 2521 CCTACCCCACGGACGACGAGCTGGGACTGATTATGGCGGC 2561 GCCCGCGCGGCTCGTCGAGGGCCAGTACCGACGCGCGCTG 2601 TACATCGACGGCACGGTCGCCTATACAGATTTCATGGTTT 2641 CGCTGCCGGCCGGGGACTGCTGGTTCTCGAAACTCGGCGC 2681 GGCTCGCGGGTACACCTTTGGCGCGTGCTTCCCGGCCCGG 2721 GATTACGAGCAAAAGAAGGTTCTGCGCCTGACGTATCTCA 2761 CGCAGTACTACCCGCAGGAGGCACACAAGGCCATAGTCGA 2801 CTACTGGTTCATGCGCCACGGGGGCGTCGTTCCGCCGTAT 2841 TTTGAGAAGCTT

[0086] Expression of granulocyte-macrophage colony-stimulating factor (GM-CSF) can stimulate the immune response against antigens such as those described herein. Accordingly, GM-CSF can be expressed separately or as a fusion partner with one or more of the antigens described herein. The expression module with the nucleotide sequence shown as SEQ ID NO:40 is just one example of a use for GM-CSF.

[0087] An example of a Bos taurus GM-CSF amino acid sequence that can be encoded in any of the expression cassettes or vectors described herein, or that can serve as a fusion partner with any of the antigens described herein, is shown below as SEQ ID NO: 41.

TABLE-US-00018 1 MNLQNLLLLGTVVCSFSAPTRPPNTATRPWQHVDAIKEAL 41 SLLNHSSDTDAVMNDTEVVSEKFDSQEPTCLQTRLKLYKN 81 GLQGSLTSLMGSLTMMATHYEKHCPPTPETSCGTQFISFK 121 NFKEDLKEFLFIIPFDCWEPAQK

Bovine Respiratory Syncytia Virus (BRSV)

[0088] BRSV, a pneumovirus, is a major etiological agent of respiratory tract disease in calves, and has been frequently seen in conjunction with H. somnus in BRDC (7). BRSV seasonal outbreaks in young calves are common due to stress of weaning, crowding and temperature changes. Persistent BRSV infections in cattle have been suspected and may serve as a source of virus transmission to susceptible animals (7, 47).

[0089] BRSV has a negative stranded RNA genome of approximately 15.2 Kb, which encodes two non-structural proteins (NS1 and NS2) and eight virion-associated structural proteins: nucleocapsid, phosphoprotein (P), matrix protein (M), small hydrophobic protein (SH), glycoprotein (G), fusion protein (F), matrix protein 2 (M2) and large or RNA dependent RNA polymerase protein (L) (47) (FIG. 2).

[0090] Of the eight structural proteins, G, F and SH are surface/envelope glycoproteins (47). Neither G nor SH is required for BRSV infectivity in cell culture (37). Even though G and SH are not essential for virus replication in vitro, recombinant BRSV lacking G and SH are highly attenuated in vivo indicating that the G and SH proteins are important for BRSV pathogenesis (47). The F protein is indispensable for virus replication (37). Transfection of cells with BRSV F gene results in formation of multinucleated syncytia (37, 42) and co-expression of F with G enhances fusion activity in the transfected cells (75). Taken together, such data indicate that the BRSV G protein is needed for enhanced fusogenic and infective property of the virus.

[0091] Most anti BRSV neutralizing antibodies have been mapped to the fusion glycoprotein F (83, 92). The BRSV F protein is a type I membrane protein. The BRSV F mRNA (e.g., 1899 nucleotides long excluding the poly A tail) can contain a single ORF that is predicted to encode a protein of 574 amino acids with a molecular weight of 63.8 kD. BRSV F protein is synthesized as an inactive precursor F0 (70 kD), which is not fusogenic. The F0 of BRSV contains two Furin cleavage sequence (FCS) motifs FCS 1 (133RKKR136) and FCS 2 (106RARR109) (88, 96) (FIG. 2). Cleavage at both sites by Furin leads to the release of pep27 and generation of the fusogenic form F1/F2 complex (88, 96). Following cleavage, F1 and F2 are linked by a disulfide bridge and an N-glycosylated peptide of 27 amino acids (pep 27) is released (88, 96).

[0092] In BRSV infected cells in vitro, pep27 can be subjected to post-translational modifications and can be converted into virokinin, a member of the tachykinin family. The tachykinin family of peptides can have pro-inflammatory and immunomodulatory properties (88). Some data indicate that BRSV virokinin may cause bronchoconstriction, for example, because it induces smooth muscle contraction (96). A recombinant BRSV lacking the pep27 induced less pulmonary inflammation in calves without affecting the induction of protective immunity in calves (88). A DNA vaccine encoding the F gene of BRSV induced significant protection against BRSV infection in young calves (81).

BRSV F Protein Antigens

[0093] An example of a BRSV F protein sequence lacking its signal sequence (approximately 1.76 Kb) and encoded by a 1700 nucleotide nucleic acid segment is available as GenBank accession GI 210828, and provided below as SEQ ID NO: 15.

TABLE-US-00019 1 MAATAMRMIISIIFISTYMTHITLCQNITEEFYQSTCSAV 41 SRGYLSALRTGWYTSVVTIELSKIQKNVCKSTDSKVKLIK 81 QELERYNNAVIELQSLMQNEPASFSRAKRGIPELIHYTRN 121 STKRFYGLMGKKRKRRFLGFLLGIGSAIASGVAVSKVLHL 161 EGEVNKIKNALLSTNKAVVSLSNGVSVLTSKVLDLKNYID 201 KELLPKVNNHDCRISNIETVIEFQQKNNRLLEIAREFSVN 241 AGITTPLSTYMLTNSELLSLINDMPITNDQKKLMSSNVQI 281 VRQQSYSIMSVVKEEVIAYVVQLPIYGVIDTPCWKLHTSP 321 LCTTDNKEGSNICLTRTDRGWYCDNAGSVSFFPQAETCKV 361 QSNRVFCDTMNSLTLPTDVNLCNTDIFNTKYDCKIMTSKT 401 DISSSVITSIGAIVSCYGKTKCTASNKNRGIIKTFSNGCD 441 YVSNKGVDTVSVGNTLYYVNKLEGKALYIKGEPIINYYDP 481 LVFPSDEFDASIAQVNAKINQSLAFIRRSDELLHSVDVGK 521 STTNVVITTIIIVIVVVILMLIAVGLLFYCKTRSTPIMLG 561 KDQLSGINNLSFSK

[0094] BRSV-F proteins typically have two separate Furin cleavage sites (FCS 1 and FCS 2). In some cases, BRSV F protein can be designed such that both the FCS 1 (106RAKR109) and FCS 2 (133RKRR136) highlighted above can be mutated to 106AAKA 109 and 133AKAA 136, respectively. The rational for the changes to the F protein sequence is twofold. First, without Furin cleavage the fusogenic form of the F1/F2 complex will not be generated and second, a functional pep27 virokinin associated with pulmonary inflammation will not be released (96). A modification of the SEQ ID NO: 15 BRSV F protein sequence with these changes is shown below as SEQ ID NO:16.

TABLE-US-00020 1 MAATAMRMIISIIFISTYMTHITLCQNITEEFYQSTCSAV 41 SRGYLSALRTGWYTSVVTIELSKIQKNVCKSTDSKVKLIK 81 QELERYNNAVIELQSLMQNEPASFSAAKAGIPELIHYTRN 121 STKRFYGLMGKKAKAAFLGFLLGIGSAIASGVAVSKVLHL 161 EGEVNKIKNALLSTNKAVVSLSNGVSVLTSKVLDLKNYID 201 KELLPKVNNHDCRISNIETVIEFQQKNNRLLEIAREFSVN 241 AGITTPLSTYMLTNSELLSLINDMPITNDQKKLMSSNVQI 281 VRQQSYSIMSVVKEEVIAYVVQLPIYGVIDTPCWKLHTSP 321 LCTTDNKEGSNICLTRTDRGWYCDNAGSVSFFPQAFTCKV 361 QSNRVFCDTMNSLTLPTDVNLCNTDIFNTKYDCKIMTSKT 401 DISSSVITSIGAIVSCYGKTKCTASNKNRGIIKTFSNGCD 441 YVSNKGVDTVSVGNTLYYVNKLEGKALYIKGEPIINYYDP 481 LVFPSDEFDASIAQVNAKINQSLAFIRRSDELLHSVDVGK 521 STTNVVITTIIIVIVVVILMLIAVGLLFYCKTRSTPIMLG 561 KDQLSGINNLSFSK

[0095] In another example, a BRSV-F version 1 protein is shown below as SEQ ID NO: 17.

TABLE-US-00021 1 MATTAMTMIISIIFISTYVTHITLCQNITEEFYQSTCSAV 41 SRGYLSALRTGWYTSVVTIELSKIQKNVCKSTDSKVKLIK 81 QELERYNNAVVELQSLMQNEPASFSAAKASIPELIHYTRN 121 STKKFYGLMGKKAKAAFLGFLLGIGSAIASGVAVSKVLHL 161 EGEVNKIKNALLSTNKAVVSLSNGVSVLTSKVLDLKNYID 201 KELLPKVNNHDCRISNIATVIEFQQKNNRLLEIAREFSVN 241 AGITTPLSTYMLTNSELLSLINDMPITNDQKKLMSSNVQI 281 VRQQSYSIMSVVKEEVIAYVVQLPIYGVIDTPCWKLHTSP 321 LCTTDNKEGSNICLTRTDRGWYCDNAGSVSFFPQAETCKV 361 QSNRVFCDTMNSLTLPTDVNLCNTDIFNTKYDCKIMTSKT 401 DISSSVITSIGAIVSCYGKTKCTASNKNRGIIKTFSNGCD 441 YVSNKGVDTVSVGNTLYYVNKLEGKALYIKGEPIINYYDP 481 LVFPSDEFDASIAQVNAKINQSLAFIRRSDELLHSVDVGK 521 STTNVVITTIIIVIVVVILMLIAVGLLFYCKTRSTPIMLG 561 KDQLSGINNLSFSK
A codon optimized chimeric BRSV F version 1 nucleic acid segment encoding the SEQ ID NO: 17 protein can, for example, have the nucleotide sequence shown below as SEQ ID NO: 18.

TABLE-US-00022 1 ATGGCTACAACCGCTATGACTATGATTATCACCATCATCT 41 TCATCTCCACCTACGTGACCCACATCACCCTGTGCCAGAA 81 CATCACCGAGGAGTTCTACCAGTCCACCTGCAGCGCTGTG 121 TCCAGGGCCTACCTGTCCGCTCTGAGAACCGGCTGGTACA 161 CCTCCGTGGTGACCATCGAGCTGAGCAAGATCCAGAAGAA 201 CGTGTGCAAGAGCACCGACTCCAAGGTGAAGCTGATCAAG 241 CAGGAGCTGGAGCGGTACAACAACGCCGTGGTGGAGCTGC 281 AGAGCCTGATGCAGAACGAGCCTGCTTCCTTCAGCGCTGC 321 TAAGGCCTCCATCCCTGAGCTGATCCACTACACCAGGAAC 361 AGCACCAAGAAGTTCTACGGCCTGATGGGCAAGAAGGCCA 401 AGGCCGCCTTCCTGGGCTTCCTGCTGGGAATCGGCAGCGC 441 TATCGCTTCCGGAGTGGCTGTGTCCAAGGTGCTGCACCTG 481 GAGGGCGAGGTGAACAAGATCAAGAACGCCCTGCTGAGCA 521 CCAACAAGGCCGTGGTGTCCCTGAGCAACGGCGTGAGCGT 561 GCTGACCTCCAAGGTGCTGGACCTGAAGAACTACATCGAC 601 AAGGAGCTGCTGCCTAAGGTGAAGAACCACGACTGCCGGA 641 TCTCCAACATCGCCACCGTGATCGAGTTCCAGCAGAAGAA 681 CAACCGGCTGCTGGAGATCGCCAGGGAGTTCTCCGTGAAC 721 GCCGGCATCACCACCCCTCTGAGCACCTACATCCTGACCA 761 ACAGCGAGCTGCTGTCCCTGATCAACGACATGCCCATCAC 801 CAACGACCAGAAGAAGCTGATGAGCTCCAACGTGCAGATC 841 GTGAGGCAGCAGTCCTACAGCATCATGAGCGTGGTGAAGG 881 AGGAGGTCATCGCCTACGTGGTGCAGCTGCCCATCTACGC 921 CGTGATCGACACCCCTTGCTGGAAGCTGCACACCTCCCCC 961 CTGTGCACCACCGACAACAAGGAGGGCAGCAACATCTGCC 1001 TGACCCGCACCGACAGAGGCTGGTACTGCGACAACGCCGG 1041 CTCCGTGAGCTTCTTCCCTCAGGCCGAGACCTGCAAGGTG 1081 CAGTCCAACCGCGTGTTCTGCGACACCATGAACAGCCTGA 1121 CCCTGCCCACCGACGTGAACCTGTGCAACACCGACATCTT 1141 CAACACCAAGTACGACTGCAAGATCATGACCAGCAAGACC 1181 GACATCAGCTCCAGCGTGATCACCAGCATCGGCGCCATCG 1201 TGTCCTGCTACGGCAAGACCAAGTGCACCGCCTCCAACAA 1241 GAACAGAGGCATCATCAAGACCTTCTCCAACGGCTGCGAC 1281 TACGTGAGCAACAAGGGCGTGGACACCGTGAGCGTGGGCA 1321 ACACCCTGTACTACGTGAACAAGCTGGAGGGCAAGGCCCT 1361 GTACATCAAGGGCGAGCCCATCATCAACTACTACGACCCC 1401 CTGGTGTTCCCTAGCGACGAGTTCGACGCCTCCATCGCCC 1441 AGGTGAACGCCAAGATCAACCAGTCCCTGGCCTTCATCCG 1481 GAGGAGCGACGAGCTGCTGCACTCCGTGGACGTGGGCAAG 1521 AGCACCACCAACGTGGTGATCACCACCATCATCATCGTGA 1561 TCGTGGTGGTGATCCTGATGCTGATCGCCGTGGGCCTGCT 1601 CTTCTACTGCAAGACCCGCAGCACACCTATTATGCTGGGC 1641 AAGGACCAGCTGAGCGGAATCAACAATCTGTCATTTTCTA 1681 AG

[0096] FIG. 17A-17C show an example of a nucleotide sequence for a BRSV F version 1 expression cassette. FIG. 17D-17F show examples of amino acid and nucleotide protein sequences (SEQ ID NOs: 20 and 21, respectively) of a fusion protein that includes the foregoing SEQ ID NO: 17 and SEQ ID NO: 18 BRSV F version 1 amino acid and nucleotide sequences. The BRSV F version 1 fusion protein (FIG. 17) includes a gD signal sequence at the N-terminus, and at the C-terminus a V5 epitope and a series of six histidines. In some cases, the BRSV F version 1 protein can be encoded in an expression cassette without the gD signal sequence and without the C-terminal V5 epitope and histidine tail.

[0097] In another example, a BRSV-F version 2 protein sequence is shown below as SEQ ID NO:22.

TABLE-US-00023 1 MATTAMTMIISIIFISTYVTHITLCQNITEEFYQSTCSAV 41 SRGYLSALRTGWYTSVVTIELSKIQKNVCKSTDSKVKLIK 81 QELERYNNAVVELQSLMQNEPASFSAAAASIPELIHYTRN 121 STKKFYGLMGKKAAAAFLGFLLGIGSAIASGVAVSKVLHL 161 EGEVNKIKNALLSTNKAVVSLSNGVSVLTSKVLDLKNYID 201 KELLPKVNNHDCRISNIATVIEFQQKNNRLLEIAREFSVN 241 AGITTPLSTYMLTNSELLSLINDMPITNDQKKLMSSNVQI 281 VRQQSYSIMSVVKEEVIAYVVQLPIYGVIDTPCWKLHTSP 321 LCTTDNKEGSNICLTRTDRGWYCDNAGSVSFFPQAETCKV 361 QSNRVFCDTMNSLTLPTDVNLCNTDIFNTKYDCKIMTSKT 401 DISSSVITSTGAIVSCYGKTKCTASNKNRGIIKTFSNGCD 441 YVSNKGVDTVSVGNTLYYVNKLEGKALYIKGEPIINYYDP 481 LVFPSDEFDASIAQVNAKINQSLAFIRRSDELLHSVDVGK 521 STTNVVITTIIIVIVVVILMLIAVGLLFYCKTRSTPIMLG 561 KDQLSGINNLSFSK
A codon optimized chimeric BRSV F version 2 nucleic acid segment encoding the SEQ ID NO:22 protein can, for example, have the nucleotide sequence shown below as SEQ ID NO:23.

TABLE-US-00024 1 ATGGCTACAACCGCTATGACTATGATTATCAGCATCATCT 41 TCATCTCCACCTACGTGACCCACATCACCCTGTGCCAGAA 81 CATCACCGAGGAGTTCTACCAGTCCACCTGCAGCGCTGTG 121 TCCAGGGGCTACCTGTCCGCTCTGAGAACCGGCTGGTACA 161 CCTCCGTGGTGACCATCGAGCTGAGCAAGATCCAGAAGAA 201 CGTGTGCAAGAGCACCGACTCCAAGGTGAAGCTGATCAAG 241 CAGGAGCTGGAGCGGTACAACAACGCCGTGGTGGAGCTGC 281 AGAGCCTGATGCAGAACGAGCCTGCTTCCTTCAGCGCTGC 321 TGCCGCCTCCATCCCTGAGCTGATCCACTACACCAGGAAC 361 AGCACCAAGAAGTTCTACGGCCTGATGGGCAAGAAGGCCG 401 CCGCCGCCTTCCTGGGCTTCCTGCTGGGAATCGGCAGCGC 441 TATCGCTTCCGGAGTGGCTGTGTCCAAGGTGCTGCACCTG 481 GAGGGCGAGGTGAACAAGATCAAGAACGCCCTGCTGAGCA 521 CCAACAAGGCCGTGGTGTCCCTGAGCAACGGCGTGAGCGT 561 GCTGACCTCCAAGGTGCTGGACCTGAAGAACTACATCGAC 601 AAGGAGCTGCTGCCTAAGGTCAACAACCACGACTGCCGGA 641 TCTCCAACATCGCCACCGTGATCGAGTTCCAGCAGAAGAA 681 CAACCGGCTGCTGGAGATCGCCAGGGAGTTCTCCGTGAAC 721 GCCGGCATCACCACCCCTCTGAGCACCTACATGCTGACCA 761 ACAGCGAGCTGCTGTCCCTGATCAACGACATGCCCATCAC 801 CAACGACCAGAAGAAGCTGATGAGCTCCAACGTGCAGATC 841 GTGAGGCAGCAGTCCTACAGCATCATGAGCGTGGTGAAGG 881 AGGAGGTGATCGCCTACGTGGTGCAGCTGCCCATCTACGG 921 CGTGATCGACACCCCTTGCTGGAAGCTGCACACCTCCCCC 961 CTGTGCACCACCGACAACAAGGAGGGCAGCAACATCTGCC 1001 TGACCCGCACCGACAGAGGCTGGTACTGCGACAACGCCGG 1041 CTCCGTGAGCTTCTTCCCTCAGGCCGAGACCTGCAAGGTG 1081 CAGTCCAACCGCGTGTTCTGCGACACCATGAACAGCCTGA 1121 CCCTGCCCACCGACGTGAACCTGTGCAACACCGACATCTT 1161 CAACACCAAGTACGACTGCAAGATCATGACCAGCAAGACC 1201 GACATCAGCTCCAGCGTGATCACCAGCATCGGCGCCATCG 1241 TGTCCTGCTACGGCAAGACCAAGTGCACCGCCTCCAACAA 1281 GAACAGAGGCATCATCAAGACCTTCTCCAACGGCTGCGAC 1321 TACGTGAGCAACAAGGGCGTGGACACCGTGAGCGTGGGCA 1361 ACACCCTGTACTACGTGAACAAGCTGGAGGGCAAGGCCCT 1401 GTACATCAAGGGCGAGCCCATCATCAACTACTACGACCCC 1441 CTGGTGTTCCCTAGCGACGAGTTCGACGCCTCCATCGCCC 1481 AGGTGAACGCCAAGATCAACCAGTCCCTGGCCTTCATCCG 1521 GAGGAGCGACGAGCTGCTGCACTCCGTGGACGTGGGCAAG 1561 AGCACCACCAACGTGGTGATCACCACCATCATCATCGTGA 1601 TCGTGGTGGTGATCCTGATGCTGATCGCCGTGGGCCTGCT 1641 GTTCTACTGCAAGACCCGCAGCACACCTATTATGCTGGGC 1681 AAGGACCAGCTGAGCGGAATCAACAATCTGTCATTTTCTA 1721 AG

[0098] FIG. 18A-18C show examples of amino acid and nucleotide protein sequences (SEQ ID NOs: 24 and 25, respectively) of a fusion protein that includes the foregoing SEQ ID NO:22 and 23 BRSV F version 2 amino acid and nucleotide sequences. The BRSV F version 2 fusion protein (FIG. 18A-18C) includes a gD signal sequence at the N-terminus. In some cases, a BRSV F version 2 fusion protein can have a V5 epitope and/or a series of histidines at the C-terminus. Such BRSV F version 2 fusion protein sequences can be encoded in an expression in a manner that is similar to the BRSV F version 1-containing expression cassette shown in FIG. 17A-17C.

[0099] BRSV F nucleic acid segments can also be obtained by amplification from various BRSV genomic sources or from plasmids that encode BRSV sequences. For example, plasmid pEF6/V5-His-TOPO (FIG. 8A) can be a source of BRSV F nucleic acid segments. The BRSV F nucleic acids obtained can be verified by sequencing and then inserted in an expression cassette, for example, in a manner that is similar to the BRSV F version 1-containing expression cassette shown in FIG. 17A-17C.

BRSV G Protein Antigens

[0100] BRSV G protein is type II integral membrane protein. The BRSV G protein mRNA (e.g., 838 nucleotides long excluding the poly A tail) contains a single ORF which encodes a polypeptide of 257 amino acids (47). The amino terminal 37 aa are predicted to be within the cytoplasm which is followed by a membrane spanning domain (aa 38-66) and a large ectodomain (aa 67-257) (FIG. 2). The ectodomain has a central conserved folded region also known as a cysteine noose (aa 158-189) which is flanked by two highly glycosylated, mucin like regions (46, 47)(FIG. 2). The BRSV G protein, especially the cysteine noose domain, contributes to immunopathogenesis of the virus by inducing unbalanced T cell responses (26). A peptide spanning amino acids 149 to 200 of G protein has recently been shown to prime nave inbred mice for polarized type 2 T-cell responses and pulmonary eosinophilia (20).

[0101] In spite of the problems associated with the G protein in the epizootiology and disease pathogenesis, antibodies and CD8+ T cells directed against the G protein-specific B cell and T cell epitopes are important for virus clearance and protection against the disease (19, 82-84).

[0102] An example, of a BRSV G protein amino acid sequence is available from GenBank (accession # GI 1912278; and encoded by a 729 nucleotide nucleic acid) segment. An example of such a BRSV G protein amino acid sequence is shown below as SEQ ID NO:26.

TABLE-US-00025 1 MSNHTHHLKFKTLKRAWKASKYFIVGLSCLYKFNLKSLVQ 41 TALTTLAMITLTSLVITAIIYISVGNAKAKPTSKPTIQQT 81 QQPQNHTSPFFTEHNYKSTHTSIQSTTLSQLPNTDTTRET 121 TYSHSINETQNRKIKSQSTLPATRKPPINPSGSNPPENHQ 161 DHNNSQTLPYVPCSTCEGNLACLSLCQIGPERAPSRAPTI 201 TLKKTPKPKTTKKPTKTTIHHRTSPEAKLQPKNNTAAPQQ 241 GILSSPEHHTNQSTTQI
The BRSV G protein employed in the expression can be modified by deletion of a cysteine noose domain (173 CSTCEGNLACLSLC 186 (SEQ ID NO:27 highlighted above). An example of a modified BRSV G protein with such a deletion has the sequence shown below as SEQ ID NO:28.

TABLE-US-00026 1 MSNHTHHLKFKTLKRAWKASKYFIVGLSCLYKFNLKSLVQ 41 TALTTLAMITLTSLVITAIIYISVGNAKAKPTSKPTIQQT 81 QQPQNHTSPFFTEHNYKSTHTSIQSTTLSQLPNTDTTRET 121 TYSHSINETQNRKIKSQSTLPATRKPPINPSGSNPPENHQ 161 DHNNSQTLPYVPQIGPERAPSRAPTITLKKTPKPKTTKKP 201 TKTTIHHRTSPEAKLQPKNNTAAPQQGILSSPEHHTNQST 241 TQI
This deletion can be made by deleting the 42 nucleotides that encode the CSTCEGNLACLSLC (SEQ ID NO:27) peptide. The rational for this deletion is that removal of the central core of the cysteine noose will eliminate the protein region associated with immunopathogenesis (20).

[0103] An example, of a BRSV G version 1 protein amino acid sequence is available from GenBank (accession # GI: 17939989/AAL49398.1) and shown below as SEQ ID NO:29.

TABLE-US-00027 1 MSNHTHHLKFKTLKRAWKASKYFIVGLSCLYKFNLKSLVQ 41 TALTTLAMITLTSLVITAIIYISVGNAKAKPTSKPTIQQT 81 QQPQNHTSPFFTEHNYKSTHTSIQSTTLSQLPNTDTTRET 121 TYSHSINETQNRKIKSQSTLPATRKPPINPSGSNPPENHQ 161 DHNNSQTLPYVPSSTSEGNLASLSLSQIGPERAPSRAPTI 201 TLKKTPKPKTTKKPTKTTIHHRTSPEAKLQPKNNTAAPQQ 241 GILSSPEHHTNQSTTQI
A Bos taurus codon optimized nucleic acid segment encoding the BRSV G version 1 protein with SEQ ID NO:29, for example, can have the following nucleotide sequence, shown below as SEQ ID NO:30.

TABLE-US-00028 1 ATGTCTAACCATACTCACCATCTGAAGTTCAAGACCCTGA 41 AGCGGGCCTGGAAGGCCTCCAAGTACTTCATCGTGGGCCT 81 GAGCTGCCTGTACAAGTTCAACCTGAAGAGCCTGGTGCAG 121 ACCGCTCTGACCACCCTGGCCATGATCACCCTGACCTCCC 161 TGGTGATCACCGCCATCATCTACATCAGCGTGGGCAACGC 201 CAAGGCCAAGCCCACCTCCAAGCCTACCATCCAGCAGACC 241 CAGCAGCCTCAGAACCACACCAGCCCCTTCTTCACCGAGC 281 ACAACTACAAGTCCACCCACACCTCCATCCAGAGCACCAC 321 CCTGTCCCAGCTGCCTAACACCGACACCACCCGCGAGACC 361 ACCTACAGCCACTCCATCAACGAGACCCAGAACCGCAAGA 401 TCAAGAGCCAGTCCACCCTGCCTGCCACCAGAAAGCCCCC 441 TATCAACCCCAGCGGCTCCAACCCCCCTGAGAACCACCAG 481 GACCACAACAACAGCCAGACCCTGCCCTACGTGCCTAGCT 521 CCACCTCCGAGGGAAACCTGGCTAGCCTGTCCCTGAGCCA 561 GATCGGACCTGAGAGGGCTCCTAGCAGGGCTCCCACCATC 601 ACCCTGAAGAAGACCCCCAAGCCTAAGACCACCAAGAAGC 641 CCACCAAGACCACCATCCACCACAGGACCTCCCCTGAGGC 681 TAAGCTGCAGCCCAAGAACAACACCGCCGCCCCCCAGCAG 721 GGAATCCTGAGCAGCCCCGAACACCACACAAACCAGAGCA 761 CTACCCAGATC

[0104] An example of a fusion protein sequence that includes a gD signal sequence with the BRSV G version 1 protein amino acid sequence having SEQ ID NO:29, is shown below with SEQ ID NO:31.

TABLE-US-00029 1 MQGPTLAVLGALLAVAVSLPMSNHTHHLKFKTLKRAWKAS 41 KYFIVGLSCLYKFNLKSLVQTALTTLAMITLTSLVITAII 81 YISVGNAKAKPTSKPTIQQTOQPQNHTSPFFTEHNYKSTH 121 TSIQSTTLSQLPNTDTTRETTYSHSINETQNRKIKSQSTL 161 PATRKPPINPSGSNPPENHQDHNNSQTLPYVPSSTSEGNL 201 ASLSLSQIGPERAPSRAPTITLKKTPKPKTTKKPTKTTIH 241 HRTSPEAKLQPKNNTAAPQQGILSSPEHHTNQSTTQI
A Bos taurus codon optimized nucleic acid segment encoding the SEQ ID NO:31 fusion protein that includes the gD signal sequence and the BRSV G version 1 protein can, for example, have the following nucleotide sequence, shown below as SEQ ID NO:32.

TABLE-US-00030 1 ATGCAGGGACCAACTCTGGCTGTGCTGGGGGCTCTGCTGG 41 CTGTCGCTGTGTCACTGCCTATGTCTAACCATACTCACCA 81 TCTGAAGTTCAAGACCCTGAAGCGGGCCTGGAAGGCCTCC 121 AAGTACTTCATCGTGGGCCTGAGCTGCCTGTACAAGTTCA 161 ACCTGAAGAGCCTGGTGCAGACCGCTCTGACCACCCTGGC 201 CATGATCACCCTGACCTCCCTGGTGATCACCGCCATCATC 241 TACATCAGCGTGGGCAACGCCAAGGCCAAGCCCACCTCCA 281 AGCCTACCATCCAGCAGACCCAGCAGCCTCAGAACCACAC 321 CAGCCCCTTCTTCACCGAGCACAACTACAAGTCCACCCAC 361 ACCTCCATCCAGAGCACCACCCTGTCCCAGCTGCCTAACA 401 CCGACACCACCCGCGAGACCACCTACAGCCACTCCATCAA 441 CGAGACCCAGAACCGCAAGATCAAGAGCCAGTCCACCCTG 481 CCTGCCACCAGAAAGCCCCCTATCAACCCCAGCGGCTCCA 521 ACCCCCCTGAGAACCACCAGGACCACAACAACAGCCAGAC 561 CCTGCCCTACGTGCCTAGCTCCACCTCCGAGGGAAACCTG 601 GCTAGCCTGTCCCTGAGCCAGATCGGACCTGAGAGGGCTC 641 CTAGCAGGGCTCCCACCATCACCCTGAAGAAGACCCCCAA 681 GCCTAAGACCACCAAGAAGCCCACCAAGACCACCATCCAC 721 CACAGGACCTCCCCTGAGGCTAAGCTGCAGCCCAAGAACA 761 ACACCGCCGCCCCCCAGCAGGGAATCCTGAGCAGCCCCGA 801 ACACCACACAAACCAGAGCACTACCCAGATC

[0105] An expression cassette that encodes the fusion protein with the gD signal sequence and the BRSV G version 1 (GenBank accession # GI: 17939989/AAL49398.1) is shown below as SEQ ID NO:33 (and in FIG. 19A-19B).

TABLE-US-00031 1 GGTACCCTCGTGAGGCTCCGGTGCCCGTCAGTGGGCAGAG 41 CGCACATCGCCCACAGTCCCCGAGAAGTTGGGGGGAGGGG 81 TCGGCAATTGAACCGGTGCCTAGAGAAGGTGGCGCGGGGT 121 AAACTGGGAAAGTGATGTCGTGTACTGGCTCCGCCTTTTT 161 CCCGAGGGTGGGGGAGAACCGTATATAAGTGCAGTAGTCG 201 CCGTGAACGTTCTTTTTCGCAACGGGTTTGCCGCCAGAAC 241 ACAGGTAAGTGCCGTGTGTGGTTCCCGCGGGCCTGGCCTC 281 TTTACGGGTTATGGCCCTTGCGTGCCTTGAATTACTTCCA 321 CCTGGCTGCAGTACGTGATTCTTGATCCCGAGCTTCGGGT 361 TGGAAGTGGGTGGGAGAGTTCGAGGCCTTGCGCTTAAGGA 401 GCCCCTTCGCCTCGTGCTTGAGTTGAGGCCTGGCCTGGGC 441 GCTGGGGCCGCCGCGTGCGAATCTGGTGGCACCTTCGCGC 481 CTGTCTCGCTGCTTTCGATAAGTCTCTAGCCATTTAAAAT 521 TTTTGATGACCTGCTGCGACGCTTTTTTTCTGGCAAGATA 561 GTCTTGTAAATGCGGGCCAAGATCTGCACACTGGTATTTC 601 GGTTTTTGGGGCCGCGGGCGGCGACGGGGCCCGTGCGTCC 641 CAGCGCACATGTTCGGCGAGGCGGGGCCTGCGAGCGCGGC 681 CACCGAGAATCGGACGGGGGTAGTCTCAAGCTGGCCGGCC 721 TGCTCTGGTGCCTGGCCTCGCGCCGCCGTGTATCGCCCCG 761 CCCTGGGCGGCAAGGCTGGCCCGGTCGGCACCAGTTGCGT 801 GAGCGGAAAGATGGCCGCTTCCCGGCCCTGCTGCAGGGAG 841 CTCAAAATGGAGGACGCGGCGCTCGGGAGAGCGGGCGGGT 881 GAGTCACCCACACAAAGGAAAAGGGCCTTTCCGTCCTCAG 921 CCGTCGCTTCATGTGACTCCACGGAGTACCGGGCGCCGTC 961 CAGGCACCTCGATTAGTTCTCGAGCTTTTGGAGTACGTCG 1001 TCTTTAGGTTGGGGGGAGGGGTTTTATGCGATGGAGTTTC 1041 CCCACACTGAGTGGGTGGAGACTGAAGTTAGGCCAGCTTG 1081 GCACTTGATGTAATTCTCCTTGGAATTTGCCCTTTTTGAG 1121 TTTGGATCTTGGTTCATTCTCAAGCCTCAGACAGTGGTTC 1161 AAAGTTTTTTTCTTCCATTTCAGGTGTCGTGAGGAATTAG 1201 CTTGGTACTAATACGACTCACTATAGGGAGACCCAAGCTG 1241 GCTAGGTAAGTGTACGAGCTCGATCACTAGTCCAGTGTGG 1281 ATCGATGCCGCCACCATGCAGGGACCAACTCTGGCTGTGC 1121 TGGGGGCTCTGCTGGCTGTCGCTGTGTCACTGCCTATGTC 1161 TAACCATACTCACCATCTGAAGTTCAAGACCCTGAAGCGG 1201 GCCTGGAAGGCCTCCAAGTACTTCATCGTGGGCCTGAGCT 1241 GCCTGTACAAGTTCAACCTGAAGAGCCTGGTGCAGACCGC 1281 TCTGACCACCCTGGCCATGATCACCCTGACCTCCCTGGTG 1321 ATCACCGCCATCATCTACATCAGCGTGGGCAACGCCAAGG 1361 CCAAGCCCACCTCCAAGCCTACCATCCAGCAGACCCAGCA 1401 GCCTCAGAACCACACCAGCCCCTTCTTCACCGAGCACAAC 1441 TACAAGTCCACCCACACCTCCATCCAGAGCACCACCCTGT 1481 CCCAGCTGCCTAACACCGACACCACCCGCGAGACCACCTA 1521 CAGCCACTCCATCAACGAGACCCAGAACCGCAAGATCAAG 1561 AGCCAGTCCACCCTGCCTGCCACCAGAAAGCCCCCTATCA 1601 ACCCCAGCGGCTCCAACCCCCCTGAGAACCACCAGGACCA 1641 CAACAACAGCCAGACCCTGCCCTACGTGCCTAGCTCCACC 1681 TCCGAGGGAAACCTGGCTAGCCTGTCCCTGAGCCAGATCG 1721 GACCTGAGAGGGCTCCTAGCAGGGCTCCCACCATCACCCT 1761 GAAGAAGACCCCCAAGCCTAAGACCACCAAGAAGCCCACC 1801 AAGACCACCATCCACCACAGGACCTCCCCTGAGGCTAAGC 1841 TGCAGCCCAAGAACAACACCGCCGCCCCCCAGCAGGGAAT 1881 CCTGAGCAGCCCCGAACACCACACAAACCAGAGCACTACC 1921 CAGATCATGCATGGTAAGCCTATCCCTAACCCTCTCCTCG 1961 GTCTCGATTCTACGCGTACCGGTCATCATCACCATCACCA 2001 TTGAGTTTAAACCCGCTGATCAGCCTCGACTGTGCCTTCT 2041 AGTTGCCAGCCATCTGTTGTTTGCCCCTCCCCCGTGCCTT 2081 CCTTGACCCTGGAAGGTGCCACTCCCACTGTCCTTTCCTA 2121 ATAAAATGAGGAAATTGCATCGCATTGTCTGAGTAGGTGT 2161 CATTCTATTCTGGGGGGTGGGGTGGGGCAGGACAGCAAGG 2201 GGGAGGATTGGGAAGACAATAGCAGGCATGCTGGGGATGC 2241 GGTGGGCTCTATGGCTTGGTACC

[0106] An example of a BRSV G version 2 protein amino acid sequence is available from GenBank (accession # GI: 17939989/AAL49398.1) and shown below as SEQ ID NO:34.

TABLE-US-00032 1 MSNHTHHLKFKTLKRAWKASKYFIVGLSCLYKFNLKSLVQ 41 TALTTLAMITLTSLVITAIIYISVGNAKAKPTSKPTIQQT 81 QQPQNHTSPFFTEHNYKSTHTSIQSTTLSQLPNTDTTRET 121 TYSHSINETQNRKIKSQSTLPATRKPPINPSGSNPPENHQ 161 DHNNSQTLPYVPQIGPERAPSRAPTITLKKTPKPKTTKKP 201 TKTTIHHRTSPEAKLQPKNNTAAPQQGILSSPEHHTNQST 241 TQI
A Bos taurus codon optimized nucleic acid segment encoding the BRSV G version 2 protein with SEQ ID NO:34, for example, can have the following nucleotide sequence, shown below as SEQ ID NO:35.

TABLE-US-00033 1 ATGTCTAACCATACTCACCATCTGAAGTTCAAGACCCTGA 41 AGCGGGCCTGGAAGGCCTCCAAGTACTTCATCGTGGGCCT 81 GAGCTGCCTGTACAAGTTCAACCTGAAGAGCCTGGTGCAG 121 ACCGCTCTGACCACCCTGGCCATGATCACCCTGACCTCCC 161 TGGTGATCACCGCCATCATCTACATCAGCGTGGGCAACGC 201 CAAGGCCAAGCCCACCTCCAAGCCTACCATCCAGCACACC 241 CAGCAGCCTCAGAACCACACCAGCCCCTTCTTCACCGAGC 281 ACAACTACAAGTCCACCCACACCTCCATCCAGAGCACCAC 321 CCTGTCCCAGCTGCCTAACACCGACACCACCCGCGAGACC 361 ACCTACAGCCACTCCATCAACGAGACCCAGAACCGCAAGA 401 TCAAGAGCCAGTCCACCCTGCCTGCCACCAGAAAGCCCCC 441 TATCAACCCCAGCGGCTCCAACCCCCCTGAGAACCACCAG 481 GACCACAACAACAGCCAGACCCTGCCCTACGTGCCTCAGA 521 TCGGACCTGAGAGGGCTCCTAGCAGGGCTCCCACCATCAC 561 CCTGAAGAAGACCCCCAAGCCTAAGACCACCAAGAAGCCC 601 ACCAAGACCACCATCCACCACAGGACCTCCCCTGAGGCTA 641 AGCTGCAGCCCAAGAACAACACCGCCGCCCCCCAGCAGGG 681 AATCCTGAGCAGCCCCGAACACCACACAAACCAGAGCACT 721 ACCCAGATC

[0107] An example of a fusion protein sequence that includes a gD signal sequence with the BRSV G version 2 sequence having SEQ ID NO:34, is shown below with SEQ ID NO:36.

TABLE-US-00034 1 MQGPTLAVLGALLAVAVSLPMSNHTHHLKFKTLKRAWKAS 41 KYFIVGLSCLYKFNLKSLVQTALTTLAMITLTSLVITAII 81 YISVGNAKAKPTSKPTIQQTQQPQNHTSPFFTEHNYKSTH 121 TSIQSTTLSQLPNTDTTRETTYSHSINETQNREIKSQSTL 161 PATRKPPINPSGSNPPENHQDHNNSQTLPYVPQIGPERAP 201 SRAPTITLKKTPKPKTTKKPTKTTIHHRTSPEAKLQPKNN 241 TAAPQQGILSSPEHHTNQSTTQI
A Bos taurus codon optimized nucleic acid segment encoding the SEQ ID NO:36 fusion protein that includes the gD signal sequence and the BRSV G version 2 protein can, for example, have the following nucleotide sequence, shown below as SEQ ID NO:37.

TABLE-US-00035 1 ATGCAGGGACCAACTCTGGCTGTGCTGGGGGCTCTGCTGG 41 CTGTCGCTGTGTCACTGCCTATGTCTAACCATACTCACCA 81 TCTGAAGTTCAAGACCCTGAAGCGGGCCTGGAAGGCCTCC 121 AAGTACTTCATCGTGGGCCTGAGCTGCCTGTACAAGTTCA 161 ACCTGAAGAGCCTGGTGCAGACCGCTCTGACCACCCTGGC 201 CATGATCACCCTGACCTCCCTGGTGATCACCGCCATCATC 241 TACATCAGCGTGGGCAACGCCAAGGCCAAGCCCACCTCCA 281 AGCCTACCATCCAGCAGACCCAGCAGCCTCAGAACCACAC 321 CAGCCCCTTCTTCACCGAGCACAACTACAAGTCCACCCAC 361 ACCTCCATCCAGAGCACCACCCTGTCCCAGCTGCCTAACA 401 CCGACACCACCCGCGAGACCACCTACAGCCACTCCATCAA 441 CGAGACCCAGAACCGCAAGATCAAGAGCCAGTCCACCCTG 481 CCTGCCACCAGAAAGCCCCCTATCAACCCCAGCGGCTCCA 521 ACCCCCCTGAGAACCACCAGGACCACAACAACAGCCAGAC 561 CCTGCCCTACGTGCCTCAGATCGGACCTGAGAGGGCTCCT 601 AGCAGGGCTCCCACCATCACCCTGAAGAAGACCCCCAAGC 641 CTAAGACCACCAAGAAGCCCACCAAGACCACCATCCACCA 681 CAGGACCTCCCCTGAGGCTAAGCTGCAGCCCAAGAACAAC 721 ACCGCCGCCCCCCAGCAGGGAATCCTGAGCAGCCCCGAAC 761 ACCACACAAACCAGAGCACTACCCAGATC

[0108] An expression cassette that encodes the fusion protein with the gD signal sequence and the BRSV G version 2 (GenBank accession # GenBank accession # GI: 17939989/AAL49398.1) is shown below as SEQ ID NO:38 (and in FIG. 20).

TABLE-US-00036 1 GGTACCCTCGTGAGGCTCCGGTGCCCGTCAGTGGGCAGAG 41 CGCACATCGCCCACAGTCCCCGAGAAGTTGGGGGGAGGGG 81 TCGGCAATTGAACCGGTGCCTAGAGAAGGTGGCGCGGGGT 121 AAACTGGGAAAGTGATGTCGTGTACTGGCTCCGCCTTTTT 161 CCCGAGGGTGGGGGAGAACCGTATATAAGTGCAGTAGTCG 201 CCGTGAACGTTCTTTTTCGCAACGGGTTTGCCGCCAGAAC 241 ACAGGTAAGTGCCGTGTGTGGTTCCCGCGGGCCTGGCCTC 281 TTTACGGGTTATGGCCCTTGCGTGCCTTGAATTACTTCCA 321 CCTGGCTGCAGTACGTGATTCTTGATCCCGAGCTTCGGGT 361 TGGAAGTGGGTGGGAGAGTTCGAGGCCTTGCGCTTAAGGA 401 GCCCCTTCGCCTCGTGCTTGAGTTGAGGCCTGGCCTGGGC 441 GCTGGGGCCGCCGCGTGCGAATCTGGTGGCACCTTCGCGC 481 CTGTCTCGCTGCTTTCGATAAGTCTCTAGCCATTTAAAAT 521 TTTTGATGACCTGCTGCGACGCTTTTTTTCTGGCAAGATA 561 GTCTTGTAAATGCGGGCCAAGATCTGCACACTGGTATTTC 601 GGTTTTTGGGGCCGCGGGCGGCGACGGGGCCCGTGCGTCC 641 CAGCGCACATGTTCGGCGAGGCGGGGCCTGCGAGCGCGGC 681 CACCGAGAATCGGACGGGGGTAGTCTCAAGCTGGCCGGCC 721 TGCTCTGGTGCCTGGCCTCGCGCCGCCGTGTATCGCCCCG 761 CCCTGGGCGGCAAGGCTGGCCCGGTCGGCACCAGTTGGGT 801 GAGCGGAAAGATGGCCGCTTCCCGGCCCTGCTGCAGGGAG 841 CTCAAAATGGAGGACGCGGCGCTCGGGAGAGCGGGCGGGT 881 GAGTCACCCACACAAAGGAAAAGGGCCTTTCCGTCCTCAG 921 CCGTCGCTTCATGTGACTCCACGGAGTACCGGGCGCCGTC 961 CAGGCACCTCGATTAGTTCTCGAGCTTTTGGAGTACGTCG 1001 TCTTTAGGTTGGGGGGAGGGGTTTTATGCGATGGAGTTTC 1041 CCCACACTGAGTGGGTGGAGACTGAAGTTAGGCCAGCTTG 1081 GCACTTGATGTAATTCTCCTTGGAATTTGCCCTTTTTGAG 1121 TTTGGATCTTGGTTCATTCTCAAGCCTCAGACAGTGGTTC 1161 AAAGTTTTTTTCTTCCATTTCAGGTGTCGTGAGGAATTAG 1201 CTTGGTACTAATACGACTCACTATAGGGAGACCCAAGCTG 1241 GCTAGGTAAGTGTACGAGCTCGATCACTAGTCCAGTGTGG 1281 ATCGATGCCGCCACCATGCAGGGACCAACTCTGGCTGTGC 1321 TGGGGGCTCTGCTGGCTGTCGCTGTGTCACTGCCTATGTC 1361 TAACCATACTCACCATCTGAAGTTCAAGACCCTGAAGCGG 1401 GCCTGGAAGGCCTCCAAGTACTTCATCGTGGGCCTGAGCT 1441 GCCTGTACAAGTTCAACCTGAAGAGCCTGGTGCAGACCGC 1481 TCTGACCACCCTGGCCATGATCACCCTGACCTCCCTGGTG 1521 ATCACCGCCATCATCTACATCAGCGTGGGCAACGCCAAGG 1561 CCAAGCCCACCTCCAAGCCTACCATCCAGCAGACCCAGCA 1601 GCCTCAGAACCACACCAGCCCCTTCTTCACCGAGCACAAC 1641 TACAAGTCCACCCACACCTCCATCCAGAGCACCACCCTGT 1681 CCCAGCTGCCTAACACCGACACCACCCGCGAGACCACCTA 1721 CAGCCACTCCATCAACGAGACCCAGAACCGCAAGATCAAG 1761 AGCCAGTCCACCCTGCCTGCCACCAGAAAGCCCCCTATCA 1801 ACCCCAGCGGCTCCAACCCCCCTGAGAACCACCAGGACCA 1841 CAACAACAGCCAGACCCTGCCCTACGTGCCTCAGATCGGA 1881 CCTGAGAGGGCTCCTAGCAGGGCTCCCACCATCACCCTGA 1921 AGAAGACCCCCAAGCCTAAGACCACCAAGAAGCCCACCAA 1961 GACCACCATCCACCACAGGACCTCCCCTGAGGCTAAGCTG 2001 CAGCCCAAGAACAACACCGCCGCCCCCCAGCAGGGAATCC 2041 TGAGCAGCCCCGAACACCACACAAACCAGAGCACTACCCA 2081 GATCATGCATGGTAAGCCTATCCCTAACCCTCTCCTCGGT 2121 CTCGATTCTACGCGTACCGGTCATCATCACCATCACCATT 2161 GAGTTTAAACCCGCTGATCAGCCTCGACTGTGCCTTCTAG 2201 TTGCCAGCCATCTGTTGTTTGCCCCTCCCCCGTGCCTTCC 2241 TTGACCCTGGAAGGTGCCACTCCCACTGTCCTTTCCTAAT 2281 AAAATGAGGAAATTGCATCGCATTGTCTGAGTAGGTGTCA 2321 TTCTATTCTGGGGGGTGGGGTGGGGCAGGACAGCAAGGGG 2361 GAGGATTGGGAAGACAATAGCAGGCATGCTGGGGATGCGG 2401 TGGGCTCTATGGCTTGGTACC

[0109] Other BRSV G nucleic acid sequences can be obtained from various genomic, cDNA, and plasmid sources by PCR using primer pairs specific for amino terminal BRSV G sequence and verified by sequencing before or after insertion into an expression cassette or vector.

[0110] Live attenuated vaccines are currently available for BRSV as multivalent BoHV-1, BVDV, BRSV and PI3 vaccines. However, reduced clinical disease and lung lesions (i.e., criteria for the vaccine efficacy) following experimental challenge with such vaccines, have not been achieved consistently for treatment of BRSV. Vaccine development against BRSV has been hampered by enhanced BRSV disease in vaccinated animals after subsequent infection (3, 20, 26, 36, 47, 76). In addition, the virus replicates poorly in cell cultures. DNA plasmid and vaccinia virus-based subunit vaccines carrying envelope proteins G and F proteins have been tested and are reported to be protective against BRSV challenge (4, 78, 79, 81).

[0111] Protection against respiratory viruses is better when animals are vaccinated intranasally (natural route). However, vaccinia virus is administered intradermally and the DNA plasmid vaccine is administered in the muscle. Such routes of administration are not optimal for immunization of cattle. In addition, the DNA vaccine may not be cost effective for use in cattle because two injections are needed.

[0112] Chimeric BRSV F and G gene expression cassettes that lack the sequences associated with immune mediated pathogenesis have been prepared that can exhibit efficacy and protective immune responses against virulent BRSV challenge.

[0113] In summary, current vaccines against the viruses associated with BRDC are not highly efficacious for several reasons: [0114] i) In the case of BoHV-1, MLV vaccine viruses, including the gE gene-deleted marker vaccine, have intact immunosuppressive properties, while the killed inactivated vaccines are not adequately protective because they do not generate cellular immunity (31, 32). [0115] ii) ii) For the modified live RNA vaccine strains, their immunosuppressive properties combined with a higher mutation rate complicates successful immunization/protection and safety in the field (9, 13, 25, 38, 62, 69, 73, 94). Taken together, a different vaccine approach against BoHV-1 as well as against BRDC associated respiratory RNA viruses is essential.

Antigen Sequence Variants

[0116] The antigen that can be encoded into the recombinant BoHV-1 tmv viruses described herein include variants of the specific nucleic acids, polypeptides or peptides that are described herein. A variant nucleic acid or a polypeptide is substantially the same as an antigen (e.g., an E2, F, or F) nucleic acid or polypeptide sequence defined by any of the SEQ ID NOs described herein but has at least one nucleotide or amino acid difference relative an antigen (e.g., an E2, F, or F) nucleic acid or polypeptide sequence defined by SEQ ID NO herein. For example, the variant nucleic acid or a polypeptide can have at least 80%, 90%, 92%, 95%, 97% or 99% sequence identity to a SEQ ID NO provided herein. The range of percent sequence identity can include at least any percent between 80 and 100 relative an antigen (e.g., an E2, F, or F) nucleic acid or polypeptide sequence defined by SEQ ID NO herein. The percent identical nucleotides or amino acids can be contiguous or dispersed across the sequences. Such variant nucleic acids or polypeptides can, for example, have substitutions that are mostly conservative.

[0117] Conservative amino acid substitutions refer to the interchangeability of residues having similar side chains. For example, a group of amino acids having aliphatic side chains is glycine, alanine, valine, leucine, and isoleucine; a group of amino acids having aliphatic-hydroxyl side chains is serine and threonine: a group of amino acids having amide-containing side chains is asparagine and glutamine: a group of amino acids having aromatic side chains is phenylalanine, tyrosine and tryptophan; a group of amino acids having basic side chains is lysine, arginine and histidine; and a group of amino acids having sulfur-containing side chain is cysteine and methionine. In one embodiment, conservative amino acid substitution groups are: valine-leucine-isoleucine; phenylalanine-tyrosine: lysine-arginine; alanine-valine; glutamic-aspartic; and asparagine-glutamine. In one embodiment, the antigen (e.g., an E2, Ems, F, or G) polypeptide has one or more, for instance, at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 12, 14, 15, or 20, nonconservative amino acid substitutions, relative to a polypeptide defined by one of SEQ ID NOs described herein. In one embodiment, the antigen (e.g., an E2, Ems, F, or G) polypeptide has one or more, for instance, at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 12, 14, 15, or 20, conservative amino acid substitutions, relative to a polypeptide defined by one of SEQ ID NOs described herein. In some cases, the antigen (e.g., an E2, Ems, F, or G) polypeptide has less than 100, 95, 90, 80, 75, 60, 50, 40, 35, 30, or 25, nonconservative amino acid substitutions, relative to a polypeptide defined by one of SEQ ID NOs described herein. In some cases, the antigen (e.g., an E2, Ems, F, or G) polypeptide has less than 100, 95, 90, 80, 75, 60, 50, 40, 35, 30, or 25, conservative amino acid substitutions, relative to a polypeptide defined by one of SEQ ID NOs described herein.

Construction of BoHV-1 Tmv Vector Encoding BVDV and/or BRSV Antigens

[0118] The selected antigens for insertion into BoHV-1 tmv vector can be encoded by nucleic acid segments that are synthesized by various procedures. As discussed above, these antigens can include a BVDV E2 protein, BVDV Ems protein, a BRSV F protein, a BRSV G protein, or any combination thereof. In some cases, the nucleic acids encoding such antigen can be codon-optimized, with a bias for expression in bovine cells.

[0119] The nucleic acids encoding such antigens can have a consensus Kozak sequence (e.g., GCCATGG) at the 5 end of the coding region. In some cases, the nucleic acids encoding such antigens can also encode signal sequence at the amino terminal end of the coding region of the protein. For example, such a signal sequence can be a BoHV-1 gD signal sequence. In some cases, the BRSV G protein may not have a signal sequence, because the absence of such a signal sequence can help the G protein to retain its type II membrane topography. The nucleic acids encoding each antigen can in some cases be cloned separately into a convenient plasmid or vector and then combined into the BoHV-1 tmv vector.

[0120] The nucleic acid segments encoding the selected protein antigens for insertion into BoHV-1 tmv vector can be inserted into an expression cassette that includes a promoter to facilitate expression of the protein antigens. Host cells can be transformed by the expression cassette or expression vector. Some procedures for making such expression cassettes, expression vectors, and genetically modified host cells are described below.

[0121] The nucleic acid segments encoding protein antigens can be operably linked to a promoter, which provides for expression of an mRNA encoding the protein antigens, polypeptides or peptides. The promoter can be a promoter functional in a host cell such as a viral promoter, a bacterial promoter or a mammalian promoter. The promoter can be a heterologous promoter. As used herein, heterologous when used in reference to a gene or nucleic acid refers to a gene or nucleic acid that has been manipulated in some way. For example, a heterologous promoter can be a promoter that contains sequences that are not naturally linked to an associated coding region. Thus, for example, a heterologous promoter may not in some cases be the same as the natural viral promoter that drives expression of BVDV E2 protein, BVDV Ems, a BRSV F protein, or a BRSV G protein.

[0122] Nucleic acid segments encoding protein antigens are operably linked to the promoter when so that the coding region(s) of the protein antigen(s) is located downstream from the promoter. The operable combination of the promoter with a protein antigen coding region is a key part of the expression cassette or expression vector.

[0123] Promoter regions are typically found in the flanking DNA upstream from the coding sequence in both prokaryotic and eukaryotic cells. A promoter sequence provides for regulation of transcription of the downstream gene sequence and typically includes from about 50 to about 2,000 nucleotide base pairs. Promoter sequences also contain regulatory sequences such as enhancer sequences that can influence the level of gene expression. Some isolated promoter sequences can provide for gene expression of heterologous DNAs, that is a DNA different from the native or homologous DNA.

[0124] Promoter sequences are also known to be strong or weak, or inducible. A strong promoter provides for a high level of gene expression, whereas a weak promoter provides a very low level of gene expression. An inducible promoter is a promoter that provides for the turning on and off of gene expression in response to an exogenously added agent, or to an environmental or developmental stimulus. For example, a bacterial promoter such as the P.sub.tac promoter can be induced to vary levels of gene expression depending on the level of isothiopropylgalactoside added to the transformed cells. Promoters can also provide for tissue specific or developmental regulation. An isolated promoter sequence that is a strong promoter for heterologous DNAs is advantageous because it provides for a sufficient level of gene expression for easy detection and selection of transformed cells and provides for a high level of gene expression when desired. In some embodiments, the promoter is an inducible promoter and/or a tissue-specific promoter.

[0125] Examples of promoters that can be used include, but are not limited to, a human elongation factor 1 promoter, a T7 promoter (e.g., optionally with the lac operator), the CaMV 35S promoter (Odell et al., Nature. 313:810-812 (1985)), the CaMV 19S promoter (Lawton et al., Plant Molecular Biology. 9:315-324 (1987)), nos promoter (Ebert et al., Proc. Natl. Acad. Sci. USA. 84:5745-5749 (1987)), Adh1 promoter (Walker et al., Proc. Natl. Acad. Sci. USA. 84:6624-6628 (1987)), sucrose synthase promoter (Yang et al., Proc. Natl. Acad. Sci. USA. 87:4144-4148 (1990)), -tubulin promoter, ubiquitin promoter, actin promoter (Wang et al., Mol. Cell. Biol. 12:3399 (1992)), cab (Sullivan et al., Mol. Gen. Genet. 215:431 (1989)), PEPCase promoter (Hudspeth et al., Plant Molecular Biology. 12:579-589 (1989)), the CCR promoter (cinnamoyl CoA:NADP oxidoreductase, EC 1.2.1.44) isolated from Lollium perenne, (or a perennial ryegrass) and/or those associated with the R gene complex (Chandler et al., The Plant Cell. 1:1175-1183 (1989)).

[0126] Other constitutive or inducible promoters can be used with or without associated enhancer elements. Examples include a baculovirus derived promoter, the p10 promoter. Typically, a viral or mammalian promoter is employed, but in some cases, plant or yeast promoters can also be used.

[0127] Alternatively, novel tissue specific promoter sequences may be employed in the practice of the present invention. Coding regions from a particular cell type or tissue can be identified and the expression control elements of those coding regions can be identified using techniques available to those of skill in the art.

[0128] The nucleic acid encoding the protein antigens or peptide therefrom can be combined with the promoter by available methods to yield an expression cassette, for example, as described in Sambrook et al. (Molecular Cloning: A Laboratory Manual. Second Edition (Cold Spring Harbor, N.Y.: Cold Spring Harbor Press (1989); Molecular Cloning: A Laboratory Manual. Third Edition (Cold Spring Harbor, N.Y.: Cold Spring Harbor Press (2000)). For example, a plasmid containing a promoter such as the T7-lac promoter can be constructed or obtained from Snap Gene (see, e.g., website at snapgene.com/resources/plasmid_files/pet_and_duct_vectors_%28novagen%29/pET-43.1a%28+%29/). These and other plasmids are constructed to have multiple cloning sites having specificity for different restriction enzymes downstream from the promoter. The nucleic acid encoding the BoHV-1 protein antigens or peptide therefrom can be subcloned downstream from the promoter using restriction enzymes and positioned to ensure that the DNA is inserted in proper orientation with respect to the promoter so that the DNA can be expressed as sense RNA.

[0129] Expression cassettes that include a promoter operably linked to a protein antigen, polypeptide or peptide coding region can include other elements such as a segment encoding 3 nontranslated regulatory sequences, and restriction sites for insertion, removal and manipulation of segments of the expression cassettes. The 3 nontranslated regulatory DNA sequences can act as a signal to terminate transcription and allow for the polyadenylation of the resultant mRNA. The 3 nontranslated regulatory DNA sequence preferably includes from about 300 to 1,000 nucleotide base pairs and contains prokaryotic or eukaryotic transcriptional and translational termination sequences. Various 3 elements that are available to those of skill in the art can be employed. These 3 nontranslated regulatory sequences can be obtained as described in An (Methods in Enzymology. 153:292 (1987)). Many such 3 nontranslated regulatory sequences are already present in plasmids available from commercial sources such as Clontech, Palo Alto, Calif. The 3 nontranslated regulatory sequences can be operably linked to the 3 terminus of a BoHV-1 protein antigens polypeptide or peptide coding region by available methods.

[0130] Once the nucleic acid encoding the protein antigens or peptide therefrom is operably linked to a promoter (e.g., and other selected elements), the expression cassette so formed can be subcloned into a plasmid or other vector (e.g., an expression vector). Such expression vectors can have a prokaryotic or eukaryotic replication origin, for example, to facilitate episomal replication in bacterial, vertebrate and/or yeast cells.

[0131] Examples of vectors that provide for easy selection, amplification, and transformation of the expression cassette in prokaryotic and eukaryotic cells include pET-43.1a(+), pUC-derived vectors such as pUC8, pUC9, pUC18, pUC19, pUC23, pUC119, and pUC120, pSK-derived vectors, pGEM-derived vectors, pSP-derived vectors, or pBS-derived vectors. The additional DNA sequences include origins of replication to provide for autonomous replication of the vector, additional selectable marker genes, such as antibiotic or herbicide resistance, unique multiple cloning sites providing for multiple sites to insert DNA sequences, and/or sequences that enhance transformation of prokaryotic and eukaryotic cells.

[0132] In order to improve identification of transformed cells, a selectable or screenable marker gene can be employed in the expression cassette or expression vector. Marker genes are genes that impart a distinct phenotype to cells expressing the marker gene and thus allow such transformed cells to be distinguished from cells that do not have the marker. Such genes may encode either a selectable or screenable marker, depending on whether the marker confers a trait which one can select for by chemical means, i.e., through the use of a selective agent (e.g., an antibiotic), or whether it is simply a trait that one can identify through observation or testing, i.e., by screening (e.g., the R-locus trait). Of course, many examples of suitable marker genes are known to the art and can be employed in the practice of the invention.

[0133] Included within the terms selectable or screenable marker genes are genes which encode a secretable marker whose secretion can be detected as a means of identifying or selecting for transformed cells. Examples include markers which encode a secretable antigen that can be identified by antibody interaction, or secretable enzymes that can be detected by their catalytic activity. Secretable proteins fall into a number of classes, including small, diffusible proteins detectable, e.g., by ELISA; and proteins that are inserted or trapped in the cell wall (e.g., proteins that include a leader sequence such as that found in the expression unit of extensin or tobacco PR-S).

[0134] Possible selectable markers for use in connection with the present invention include, but are not limited to, an ampicillin gene, which codes for the ampicillin antibiotic. Other examples include a neo gene (Potrykus et al., Mol. Gen. Genet. 199:183-188 (1985)) which codes for kanamycin resistance and can be selected for using kanamycin, G418, and the like; a mutant acetolactate synthase gene (ALS) which confers resistance to imidazolinone, sulfonylurea or other ALS-inhibiting chemicals (European Patent Application 154,204 (1985)); a methotrexate-resistant DHFR gene (Thillet et al., J. Biol. Chem. 263:12500-12508 (1988)); a dalapon dehalogenase gene that confers resistance to the herbicide dalapon: a mutated anthranilate synthase gene that confers resistance to 5-methyl tryptophan: a -galactosidase gene, which encodes an enzyme for which there are chromogenic substrates: a luciferase (lux) gene (Ow et al., Science. 234:856-859, 1986), which allows for bioluminescence detection: or an aequorin gene (Prasher et al., Biochem. Biophys. Res. Comm. 126:1259-1268 (1985)), which may be employed in calcium-sensitive bioluminescence detection, or a green or yellow fluorescent protein gene (Niedz et al., Plant Cell Reports. 14:403 (1995).

[0135] One example of an expression vehicle that can be modified to include one or more nucleic acid segment encoding one or more protein antigens (e.g., a BVDV E2 protein, BVDV Ems protein, a BRSV F protein, a BRSV G protein, or any combination thereof) is a pPreBVDV-E2/BRSV-F or G vehicle which includes a sequence shown in FIG. 10, also provided below as SEQ ID NO: 1.

TABLE-US-00037 1 GGTACCCTCGTGAGGCTCCGGTGCCCGTCAGTGGGCAGAG 41 CGCACATCGCCCACAGTCCCCGAGAAGTTGGGGGGAGGGG 81 TCGGCAATTGAACCGGTGCCTAGAGAAGGTGGCGCGGGGT 121 AAACTGGGAAAGTGATGTCGTGTACTGGCTCCGCCTTTTT 161 CCCGAGGGTGGGGGAGAACCGTATATAAGTGCAGTAGTCG 201 CCGTGAACGTTCTTTTTCGCAACGGGTTTGCCGCCAGAAC 241 ACAGGTTAGTGCCGTGTGTGGTTCCCGCGGGCCTGGCCTC 281 TTTACGGGTTATGGCCCTTGCGTGCCTTGAATTACTTCCA 321 CCTGGCTGCAGTACGTGATTCTTGATCCCGAGCTTCGGGT 361 TGGAAGTGGGTGGGAGAGTTCGAGGCCTTGCGCTTAAGGA 401 GCCCCTTCGCCTCGTGCTTGAGTTGAGGCCTGGCCTGGGC 441 GCTGGGGCCGCCGCGTGCGAATCTGGTGGCACCTTCGCGC 481 CTGTCTCGCTGCTTTCGATAAGTCTCTAGCCATTTAAAAT 521 TTTTGATGACCTGCTGCGACGCTTTTTTTCTGGCAAGATA 561 GTCTTGTAAATGCGGGCCAAGATCTGCACACTGGTATTTC 601 GGTTTTTGGGGCCGCGGGCGGCGACGGGGCCCGTGCGTCC 641 CAGCGCACATGTTCGGCGAGGCGGGGCCTGCGAGCGCGGC 681 CACCGAGAATCGGACGGGGGTAGTCTCAAGCTGGCCGGCC 721 TGCTCTGGTGCCTGGCCTCGCGCCGCCGTGTATCGCCCCG 761 CCCTGGGCGGCAAGGCTGGCCCGGTCGGCACCAGTTGCGT 801 GAGCGGAAAGATGGCCGCTTCCCGGCCCTGCTGCAGGGAG 841 CTCAAAATGGAGGACGCGGCGCTCGGGAGAGCGGGCGGGT 881 GAGTCACCCACACAAAGGAAAAGGGCCTTTCCGTCCTCAG 921 CCGTCGCTTCATGTGACTCCACGGAGTACCGGGCGCCGTC 961 CAGGCACCTCGATTAGTTCTCGAGCTTTTGGAGTACGTCG 1001 TCTTTAGGTTGGGGGGAGGGGTTTTATGCGATGGAGTTTC 1041 CCCACACTGAGTGGGTGGAGACTGAAGTTAGGCCAGCTTG 1081 GCACTTGATGTAATTCTCCTTGGAATTTGCCCTTTTTGAG 1121 TTTGGATCTTGGTTCATTCTCAAGCCTCAGACAGTGGTTC 1161 AAAGTTTTTTTCTTCCATTTCAGGTGTCGTGAGGAATTAG 1201 CTTGGTACTAATACGACTCACTATAGGGAGACCCAAGCTG 1241 GCTAGGTAAGTGTACGAGCTCGATCACTAGTCCAGTGTGG 1281 ATCGATGATATCTCTAGAATGCATGGTAAGCCTATCCCTA 1321 ACCCTCTCCTCGGTCTCGATTCTACGCGTACCGGTCATCA 1361 TCACCATCACCATTGAGTTTAAACCCGCTGATCAGCCTCG 1241 ACTGTGCCTTCTAGTTGCCAGCCATCTGTTGTTTGCCCCT 1281 CCCCCGTGCCTTCCTTGACCCTGGAAGGTGCCACTCCCAC 1321 TGTCCTTTCCTAATAAAATGAGGAAATTGCATCGCATTGT 1361 CTGAGTAGGTGTCATTCTATTCTGGGGGGTGGGGTGGGGC 1401 AGGACAGCAAGGGGGAGGATTGGGAAGACAATAGCAGGCA 1441 TGCTGGGGATGCGGTGGGCTCTATGGCTTGGTACC
For example, a BVDV E2 protein, a BVDV Ems protein, a BRSV F protein, a BRSV G protein, or any combination thereof, can be inserted into a pPreBVDV-E2/BRSV-F or G vehicle at a KpnI site as illustrated in FIGS. 8 and 11.

[0136] The expression cassettes and/or expression vectors can be introduced into a recipient host cell to create a transformed cell by available methods. As illustrated in the Examples, many of the vectors can infect some mammalian cells types, for example, the Madin-Darby bovine kidney (MDBK) cell line obtained from the American Type Culture Collection (Manassas, Va.) can be used for growth, culturing, and analysis of expression cassettes, expression vectors, and viruses that may express the protein antigens. Such MDBK cells can be maintained in Dulbecco's modified Eagle's medium (DMEM) supplemented with 5-10% heat-inactivated fetal bovine serum (FBS) (HyClone Laboratories, Inc., South Logan, Utah).

[0137] The frequency of occurrence of cells taking up exogenous (foreign) DNA some transformation procedures can be low, and it is likely that not all recipient cells receiving DNA segments or sequences will result in a transformed cell. Some may show only initial and transient gene expression. However, cells from virtually any species can be stably transformed, and those cells can be utilized to generate antigenic polypeptides or peptides.

[0138] Transformation of the host cells with expression cassettes or expression vectors can be conducted by any one of a number of methods available to those of skill in the art. Examples are: transformation by direct DNA transfer into host cells by electroporation, direct DNA transfer into host cells by PEG precipitation, direct DNA transfer to plant cells by microprojectile bombardment, and calcium chloride/heat shock.

[0139] Methods such as microprojectile bombardment or electroporation can be carried out with naked DNA where the expression cassette may be simply carried on any E. coli-derived plasmid cloning vector. In the case of viral vectors, it is desirable that the system retain replication functions, but lack functions for disease induction.

[0140] For example, the Madin-Darby bovine kidney (MDBK) cell line obtained from the American Type Culture Collection (Manassas, Va.) can be used for growth, culturing, and analysis of expression cassettes, expression vectors, and viruses that may express the protein antigens. Such MDBK cells can be maintained in Dulbecco's modified Eagle's medium (DMEM) supplemented with 5-10% heat-inactivated fetal bovine serum (FBS) (HyClone Laboratories, Inc., South Logan, Utah).

[0141] Once the expression cassette or vector encoding one or more protein antigens has been constructed and introduced into a host cell, the host cells can be screened for the ability to express the encoded protein antigens by available methods. For example, when the protein antigens has a poly-histidine tag, and the His-tagged protein antigens can be detected or isolated by use of anti-His tag antibodies. In another example, protein antigens can be detected using antibodies that bind to the polypeptides or peptides (e.g., via western blot or ELISA). Nucleic acids encoding the protein antigens can be detected by Southern blot, or nucleic acid amplification using complementary probes and/or primers. Sequencing of the nucleic acids can be performed to confirm the expression cassette or vector sequences.

[0142] For example, protein antigens (e.g., a BVDV E2 protein, a BRSV F protein, a BRSV G protein, or any combination thereof) within an expression cassette that includes V5 epitope (e.g., an expression cassette that includes portions of SEQ ID NO:1) can be expressed transiently in 293T transfected cells and verified by immuno-fluorescence using V5 epitope specific antibody (Invitrogen, R960-25). For example, expression of a BVDV E2 protein, a BRSV F protein, a BRSV G protein, or any combination thereof can be detected by use of protein-specific antibodies such as BVDV type 2 E2-specific MAb (VMRD BA-2); or BRSV F specific MAb 8G12 (41). Other antibodies can also be used such as a BRSV F specific rabbit polyclonal ab45478 (abcam) or a BRSV antibody (Rabbit anti BRSV polyclonal antibody PAB 14559, Abnova corporation; MAb RSV 133/cat# ab94966, abcam).

[0143] Protection of animals against viral infection, or the symptoms thereof can be compared to infection and symptoms of a virulent viral infection, for example, an infection by a BHV-1 Cooper (Colorado-1) strain, which can be obtained from the American Type Culture Collection (Cat # CRL-1390; Manassas, Va.). Propagation of such a virulent BHV-1 strain can be performed as described by Chowdhury (Microbiol 52(1-2): 13-23 (1996)).

Vaccines

[0144] A vaccine includes at least one isolated recombinant BoHV-1 tmv virus that can express one or more heterologous antigens, peptides or polypeptides, and optionally one or more other isolated viruses including other isolated recombinant BoHV-1 tmv viruses, one or more antigens, immunogenic proteins or glycoproteins of one or more isolated viruses or one or more other pathogens, e.g., an immunogenic protein from one or more bacteria, non-BoHV viruses, yeast or fungi, or isolated nucleic acid encoding one or more viral proteins (e.g., DNA vaccines). For example, the BoHV-1 tmv viral construct may be a vaccine for pathogens that contribute to bovine respiratory disease complex (BRDC). In some cases, the vaccines can in GM-CSF either as protein within the vaccine composition, or expressed from an expression cassette or expression vector.

[0145] A complete vaccine may be concentrated (e.g., by ultrafiltration) and then at least partially purified (e.g., by zonal centrifugation or by chromatography). Viruses other than the BoHV-1 tmv virus, such as a multivalent vaccine, can also be included in the vaccine compositions. The vaccine can be inactivated before or after purification, for example, using formalin or beta-propiolactone, for instance.

[0146] A subunit vaccine can include purified peptides and/or polypeptides (e.g., glycoproteins) as antigens or as immunological stimulants. Such a vaccine may be prepared as follows: using viral suspensions fragmented by treatment with detergent, the antigenic (e.g., surface) polypeptides and/or peptides can be purified, by ultracentrifugation, for example. A detergent may be used such as a cationic detergent. For example, a detergent such as hexadecyl trimethyl ammonium bromide (Bachmeyer, 1975), an anionic detergent such as ammonium deoxycholate (Laver & Webster, 1976): or a nonionic detergent such as that commercialized under the name TRITON X100 can be employed. The subunit vaccine can, for example, be combined with a BoHV-1 tmv virus of the invention in a multivalent vaccine.

[0147] A split vaccine can include virions that have been subjected to treatment with agents that dissolve lipids. A split vaccine can be prepared as follows: an aqueous suspension of purified virus, inactivated or not, is treated, under stirring, by lipid solvents such as ethyl ether or chloroform, associated with detergents. The dissolution of the viral envelope lipids results in fragmentation of the viral particles. Then the residual infectious particles are inactivated if this has not already been done. The split vaccine may be combined with an attenuated virus of the invention in a multivalent vaccine.

Inactivated Vaccines

[0148] BoHV-1 tmv replicates like wild type BoHV-1 virions and expresses antigens that reduce the incidence of disease. However, there may be cases where it may be useful for a recombinant BoHV-1 tmv to be inactivated. Inactivated recombinant BoHV-1 tmv viruses and other viral vaccines constructs can be provided by inactivating replicated virus using available methods, such as, but not limited to, treatment of viral solutions at temperatures from 60 C. to 80 C., or at low pH (1.7-4.0), or by use of ethanol, methanol, guanidine hydrochloride, formalin or -propiolactone. Inactivated viral constructs and/or vaccines that can be used can include whole-virus (WV) vaccines or subvirion (SV) (split) vaccines. Vaccines can contain intact, inactivated viruses, or purified viruses that have been disrupted with detergents that solubilize the lipid-containing viral envelope, followed by chemical inactivation of residual virus.

Live Attenuated Virus Vaccines

[0149] Live, attenuated recombinant BoHV-1 tmv virus vaccines, such as those including a recombinant BoHV-1 tmv virus can be used for preventing or treating various viral infections of cattle or other animals. Attenuation may be achieved in a single step by transfer of attenuated genes from an attenuated donor virus to a replicated isolate or reassorted virus according to available methods. The attenuated genes are derived from an attenuated parent virus.

Pharmaceutical Compositions

[0150] Pharmaceutical compositions include one or more BoHV-1 tmv viral vector isolates, e.g., one or more recombinant BoHV-1 tmv viral vector, a subunit thereof, isolated protein(s) thereof, and/or isolated nucleic acid encoding one or more proteins thereof, optionally further comprising sterile aqueous or non-aqueous solutions, suspensions, and emulsions. The compositions can include other viral vaccines, and polypeptides and/or peptides that are either antigenic or that can stimulate an immune response. The compositions can further comprise auxiliary agents or excipients, available in the art. The compositions can be presented in the form of individual doses (unit doses).

[0151] Conventional vaccines generally contain about 0.1 to 200 g, e.g., 30 to 100 g, of each recombinant BoHV-1 tmv virus type within the composition. The vaccine forming the main constituent of the vaccine composition of the invention may comprise a single recombinant BoHV-1 tmv virus, or a combination of recombinant BoHV-1 tmv viruses, for example, two or three recombinant BoHV-1 tmv viruses.

[0152] Preparations for parenteral administration include sterile aqueous or non-aqueous solutions, suspensions, and/or emulsions, which may contain auxiliary agents or excipients known in the art. Examples of non-aqueous solvents are propylene glycol, polyethylene glycol, vegetable oils such as olive oil, and injectable organic esters such as ethyl oleate. Carriers or occlusive dressings can be used to increase skin permeability and enhance antigen absorption. Liquid dosage forms for oral administration may generally comprise a liposome solution containing the liquid dosage form. Suitable forms for suspending liposomes include emulsions, suspensions, solutions, syrups, and elixirs containing inert diluents available in the art, such as purified water. Besides the inert diluents, such compositions can also include adjuvants, wetting agents, emulsifying and suspending agents, or sweetening, flavoring, or perfuming agents.

[0153] When a composition is used for administration to an animal, it can further comprise salts, buffers, adjuvants, or other substances which are desirable for improving the efficacy of the composition. For vaccines, adjuvants, substances which can augment a specific immune response, can be used. Normally, the adjuvant and the composition are mixed prior to presentation to the immune system, or presented separately, but into the same site of the organism being immunized.

[0154] Heterogeneity in a vaccine may be provided by mixing at least two recombinant BoHV-1 tmv virus types, such as 2-20 types, or any range or value between 2-20. Vaccines can be provided for variations in a single strain of a recombinant BoHV-1 tmv virus, using techniques available in the art.

[0155] A pharmaceutical composition can further or additionally include additional ingredients, for example, for gene therapy, anti-inflammatory agents or immune enhancers, and for vaccines, GM-CSF, gamma globulin, amantadine, guanidine, hydroxybcnzimidazole, interferon-, interferon-, interferon-, tumor necrosis factor-alpha, thiosemicarbarzones, methisazone, rifampin, ribavirin, a pyrimidine analog, a purine analog, foscarnet, phosphonoacetic acid, acyclovir, dideoxynucleosides, a protease inhibitor, or ganciclovir.

[0156] The composition can also contain variable but small quantities of endotoxin-free formaldehyde, and preservatives, which have been found safe and not contributing to undesirable effects in the organism to which the composition is administered.

Pharmaceutical Purposes

[0157] The administration of the composition (or the antisera that it elicits) may be for either a prophylactic or therapeutic purpose. When provided prophylactically, the compositions are provided before symptoms or clinical signs of a pathogen (e.g., viral) infection becomes manifest. The prophylactic administration of the composition serves to prevent or attenuate any subsequent infection. The prophylactic administration of the composition serves to prevent or attenuate one or more symptoms or clinical signs associated with the disease.

[0158] When provided therapeutically, a viral vaccine is provided upon the detection of a symptom or clinical sign of actual infection. The therapeutic administration of the compositions serves to attenuate infection.

[0159] Thus, a recombinant BoHV-1 tmv viral composition can be provided either before the onset of infection (so as to prevent or attenuate an anticipated infection) or after the initiation of an actual infection.

[0160] A composition or component thereof is said to be pharmacologically acceptable if its administration can be tolerated by a recipient mammal. Such a composition or component thereof is said to be administered in a therapeutically effective amount if the amount administered is physiologically significant. A composition is physiologically significant if its presence results in a detectable change in the physiology of a recipient patient, e.g., enhances at least one primary or secondary humoral or cellular immune response against at least one strain of an infectious virus.

[0161] Any protection provided by the compositions described herein need not be absolute, i.e., the viral infection need not be totally prevented or eradicated, if there is a statistically significant improvement compared with a control population or set of mammals. Protection may be limited to mitigating the severity or rapidity of onset of symptoms or clinical signs of the viral infection.

Pharmaceutical Administration

[0162] Compositions are described herein that can include any of the expression cassettes, expression vectors, or recombinant viruses described herein. In some cases the compositions can include a carrier, for example, a carrier formulated to facilitate administration of any of the compositions containing any such expression cassettes, expression vectors, or recombinant viruses described herein. Although the compositions can include any expression cassettes, expression vectors, or recombinant viruses described herein, in some cases the compositions include one or more recombinant BoHV-1 tmv virus, for example, one or more recombinant BoHV-1 tmv virus that can express an E2 protein, BVDV Ems protein, BRSV F protein, BRSV G protein, or a combination thereof.

[0163] A composition may confer resistance to one or more pathogens, e.g., one or more virus strains, by either passive immunization or active immunization. In active immunization, an attenuated live vaccine composition is administered prophylactically to a host (e.g., a mammal), and the host's immune response to the administration protects against infection and/or disease. For passive immunization, the elicited antisera can be recovered and administered to a recipient suspected of having an infection caused by at least one influenza virus strain.

[0164] Hence, methods are provided herein that involve administering at least one of the BoHV-1 recombinant vectors described herein to an animal. Such administration can reduces the incidence or severity of respiratory symptoms in the mammal. While various types of animals or mammals can be administered the compositions described herein, in some cases, the mammal is a wild or domesticated bovine animal. In some cases, the animal administered is an experimental animal. For example, the compositions can be administered to wild or domesticated cattle, buffalo, oxen, pigs, horses, poultry, goats, rats, mice, and/or any animal that may be susceptible to respiratory diseases. In some cases the compositions are administered to mammals that may be susceptible to bovine respiratory disease complex (BRDC).

[0165] In some cases, the compositions containing recombinant BoHV-1 tmv virus can be administered to young cattle (calves). In other cases, the compositions containing recombinant BoHV-1 tmv virus are administered to a female cow (at or prior to pregnancy or parturition), under conditions of time and amount sufficient to cause the production of an immune response which serves to protect both the female and the fetus or newborn (via passive incorporation of the antibodies across the placenta or in the mother's milk).

[0166] Methods for preventing or attenuating a disorder or disease, e.g., an infection by at least one strain of pathogen, are also described herein. As used herein, a vaccine is said to prevent or attenuate a disease if its administration results either in the total or partial attenuation (i.e., suppression) of a clinical sign or condition of the disease, or in the total or partial immunity of the individual to the disease.

[0167] For example, administration of the compositions described herein can reduce the incidence of shedding viruses (e.g., in nasal secretions) by the animals to which the composition was administered by at least 50%, or at least 60%, or at least 70%, or at least 80%, or at least 85%, or at least 90%, or at least 95%, or at least 97%, or at least 98%, or at least 99%. Such reduction in virus shedding can be in comparison to an animal of the same species that has not received a composition described herein and that can be infected by BoHV, BVDV, BRSV, or a combination thereof.

[0168] For example, administration of the compositions described herein can increase the titer of neutralizing antibodies against BoHV, BVDV, or BRSV in an animal administered a composition described herein by at least two-fold, at least five-fold, at least seven-fold, at least ten-fold, at least fifteen-fold, at least twenty-fold, or at least twenty five-fold. Such a fold increase in neutralizing antibodies against BoHV, BVDV, or BRSV can be in comparison to an animal of the same species that has not received a composition described herein. In some cases, the fold increase in neutralizing antibodies can in comparison to animals that are infected by BoHV, BVDV, BRSV, or a combination thereof, and that have not been administered a composition described herein.

[0169] A composition having at least one recombinant BoHV-1 tmv virus, can in some cases be is combined with one or more other isolated viral compositions (e.g., other types of viral vaccines), or can be combined with one or more isolated viral proteins, or can be combined with one or more isolated nucleic acid molecules encoding one or more viral proteins, or a combination thereof. Such compositions and combinations of compositions, may be administered by any means that achieve the intended purposes.

[0170] For example, administration of such a composition may be by various parenteral routes such as intranasal, subcutaneous, intravenous, intradermal, intramuscular, intraperitoneal, oral, nasal, or transdermal routes. Parenteral administration can be accomplished by bolus injection or by gradual perfusion over time.

[0171] A typical regimen for preventing, suppressing, or treating viral disease includes administration of an effective amount of an immunological composition (e.g., a vaccine composition) as described herein, administered as a single treatment, or repeated as enhancing or booster dosages, over a period up to and including between one week and about 24 months, or any range or value therein.

[0172] According to the present invention, an effective amount of a composition is one that is sufficient to achieve a desired effect. It is understood that the effective dosage may be dependent upon the species, age, sex, health, and weight of the recipient, kind of concurrent treatment, if any, frequency of treatment, and the nature of the effect wanted. The ranges of effective doses provided below are not intended to limit the invention and represent dose ranges.

[0173] The dosage of a live, attenuated or killed recombinant virus composition for an animal such as a mammalian adult organism may be from about 10.sup.2-10.sup.15, 10.sup.3-10.sup.12, 10.sup.3-10.sup.11, 10.sup.4-10.sup.11, plaque forming units (PFU)/kg, or any range or value therein. The dose of inactivated viral proteins may range from about 0.1 to 1000, e.g., 10 to 100 g, such as about 15 g, of viral protein. However, the dosage should be a safe and effective amount as determined by conventional methods, using existing vaccines as a starting point.

[0174] The dosage of immunoreactive viral protein in each dose of replicated virus vaccine may be standardized to contain a suitable amount, e.g., 30 to 100 g or any range or value therein, such as about 15 g, or the amount recommended by government agencies or recognized professional organizations. The quantity of protein or recombinant virus can also be standardized.

Definitions

[0175] Within this application, a wild type or a naturally-occurring BoHV virus means a BoHV virus which has not been genetically engineered, and includes, but is not limited to, wild-type BoHV viruses and BoHV viruses selected from BoHV viruses that exist in nature and have spontaneous deletions.

[0176] As used herein, nonessential gene means a gene that is not essential for viral replication.

[0177] As used herein, an infectious, biologically contained virus means that the virus is incapable of producing progeny in normal cells or incapable of significant replication in vitro or in vivo, e.g., titers of less than about 10.sup.2 to 10.sup.3 PFU/mL, in the absence of helper virus or a viral protein stably supplied in trans.

[0178] A used herein, replication-deficient virus means that the virus can replicate to a limited extent in vitro or in vivo, e.g., titers of at least about 10.sup.2 to 10.sup.3 PFU/mL, in the absence of helper virus or a viral protein supplied in trans.

[0179] As used herein, the term isolated refers to in vitro preparation and/or isolation of a nucleic acid molecule, e.g., vector or plasmid, peptide or polypeptide (protein), or virus of the invention, so that it is not associated with in vivo substances, or is substantially purified from in vitro substances. An isolated virus preparation is generally obtained by in vitro culture in cells and propagation, and is substantially free from other infectious agents.

[0180] As used herein, substantially purified means the object species is the predominant species, e.g., on a molar basis it is more abundant than any other individual species in a composition, and preferably is at least about 80% of the species present, and optionally 90% or greater, e.g., 95%, 98%, 99% or more, of the species present in the composition.

[0181] As used herein, substantially free means below the level of detection for a particular infectious agent using standard detection methods for that agent.

[0182] A recombinant virus is one which has been manipulated in vitro, e.g., using recombinant DNA techniques, to introduce changes to the viral genome. Reassortant viruses can be prepared by recombinant or non-recombinant techniques.

[0183] As used herein, the term recombinant nucleic acid or recombinant DNA sequence or segment refers to a nucleic acid, e.g., to RNA or DNA, that has been derived or isolated from a source, that may be subsequently chemically altered in vitro so that its sequence is not naturally occurring or does not identically correspond to naturally occurring sequences. For example, a recombinant nucleic acid can have segments that are not positioned as they would be positioned in a native genome. An example of DNA derived from a source, would be a DNA sequence that is identified as a useful fragment, and which is then chemically synthesized in essentially pure form. An example of such DNA isolated from a source would be a useful DNA sequence that is excised or removed from said source by chemical means, e.g., by the use of restriction endonucleases, so that it can be further manipulated, e.g., amplified, or modified, by genetic engineering methods.

[0184] As used herein, a heterologous nucleic acid segment is from a source other than a parent virus or genome. For example, a heterologous nucleic acid segment can be a segment that includes a promoter sequence, a nucleotide sequence encoding a peptide (e.g., an antigenic peptide) from another virus or organism, a nucleotide sequence encoding a polypeptide (e.g., an antigenic polypeptide) from another virus or organism, or a reporter gene or a gene from another virus or organism. Such a heterologous nucleic acid segment can encode a heterologous peptide or polypeptide.

[0185] The terms isolated polypeptide, isolated peptide or isolated protein include a polypeptide, peptide or protein encoded by cDNA or recombinant RNA including one of synthetic origin, or some combination thereof.

[0186] The term recombinant protein or recombinant polypeptide as used herein refers to a protein molecule expressed from a recombinant DNA molecule. In contrast, the term native protein is used herein to indicate a protein isolated from a naturally occurring (i.e., a non-recombinant) source. Molecular biological techniques may be used to produce a recombinant form of a protein with identical properties as compared to the native form of the protein.

[0187] Methods of alignment of sequences for comparison are well known in the art. Thus, the determination of percent identity between any two sequences can be accomplished using a mathematical algorithm. Conserved and non-conserved sequence domains can be identified by alignment of sequences.

[0188] Computer implementations of these mathematical algorithms can be utilized for comparison of sequences to determine sequence identity. Alignments using these programs can be performed using the default parameters. Software for performing BLAST analyses is publicly available through the National Center for Biotechnology Information (see website at www.ncbi.nlm.nih.gov/). The algorithm may involve first identifying high scoring sequence pairs (HSPs) by identifying short words of length W in the query sequence, which either match or satisfy some positive-valued threshold score T when aligned with a word of the same length in a database sequence. T is referred to as the neighborhood word score threshold. These initial neighborhood word hits act as seeds for initiating searches to find longer HSPs containing them. The word hits are then extended in both directions along each sequence for as far as the cumulative alignment score can be increased. Cumulative scores are calculated using, for nucleotide sequences, the parameters M (reward score for a pair of matching residues; always >0) and N (penalty score for mismatching residues; always <0). For amino acid sequences, a scoring matrix is used to calculate the cumulative score. Extension of the word hits in each direction are halted when the cumulative alignment score falls off by the quantity X from its maximum achieved value, the cumulative score goes to zero or below due to the accumulation of one or more negative-scoring residue alignments, or the end of either sequence is reached.

[0189] In addition to calculating percent sequence identity, the BLAST algorithm may also perform a statistical analysis of the similarity between two sequences. One measure of similarity provided by the BLAST algorithm may be the smallest sum probability (P(N)), which provides an indication of the probability by which a match between two nucleotide or amino acid sequences would occur by chance. For example, a test nucleic acid sequence is considered similar to a reference sequence if the smallest sum probability in a comparison of the test nucleic acid sequence to the reference nucleic acid sequence is less than about 0.1, more preferably less than about 0.01, and most preferably less than about 0.001.

[0190] The BLASTN program (for nucleotide sequences) may use as defaults a wordlength (W) of 11, an expectation (E) of 10, a cutoff of 100, M=5, N=4, and a comparison of both strands. For amino acid sequences, the BLASTP program may use as defaults a wordlength (W) of 3, an expectation (E) of 10, and the BLOSUM62 scoring matrix. See website at www.ncbi.nlm.nih.gov. Alignment may also be performed manually by inspection.

[0191] For sequence comparison, typically one sequence acts as a reference sequence to which test sequences are compared. For example, any of the sequences defined by SEQ ID NO herein can be a reference sequence. When using a sequence comparison algorithm, test and reference sequences are input into a computer, subsequence coordinates are designated if necessary, and sequence algorithm program parameters are designated. The sequence comparison algorithm then calculates the percent sequence identity for the test sequence(s) relative to the reference sequence, based on the designated program parameters.

[0192] As used herein, individual (as in the subject of the treatment) means a mammal. Mammals include, for example, humans; non-human primates, e.g., apes and monkeys, and non-primates, e.g., dogs, cats, rats, mice, pigs, cattle, horses, sheep, and goats. Non-mammals include, for example, fish and birds. In some cases, the mammal is a bovine animal (e.g., a calf, cow, steer, or any type of cattle).

[0193] The term disease or disorder are used interchangeably, and are used to refer to diseases or conditions caused by viral or microbial infection.

[0194] Substantially as the term is used herein means completely or almost completely; for example, a composition that is substantially free of a component either has none of the component or contains such a trace amount that any relevant functional property of the composition is unaffected by the presence of the trace amount, or a compound is substantially pure is there are only negligible traces of impurities present.

[0195] Treating or treatment within the meaning herein refers to an alleviation of symptoms associated with a disorder or disease, inhibiting means inhibition of further progression or worsening of the symptoms associated with the disorder or disease, and preventing refers to prevention of the symptoms associated with the disorder or disease.

[0196] As used herein, an effective amount or a therapeutically effective amount of an agent of the invention e.g., a recombinant BoHV-1 tmv encoding a gene product, refers to an amount of the agent that alleviates, in whole or in part, symptoms associated with the disorder or condition, or halts or slows further progression or worsening of those symptoms, or prevents or provides prophylaxis for the disorder or condition, e.g., an amount that is effective to prevent, inhibit or treat in the individual one or more respiratory (e.g., bovine respiratory disease complex (BRDC)) symptoms.

[0197] In particular, a therapeutically effective amount refers to an amount effective, at dosages and for periods of time necessary, to achieve the desired therapeutic result. A therapeutically effective amount is also one in which any toxic or detrimental effects of compounds of the invention are outweighed by the therapeutically beneficial effects.

[0198] A vector as used herein refers to a macromolecule or association of macromolecules that comprises or associates with a polynucleotide and which can be used to mediate delivery of the polynucleotide to a cell, either in vitro or in vivo. Illustrative vectors include, for example, plasmids, viral vectors, liposomes and other gene delivery vehicles. The polynucleotide to be delivered, sometimes referred to as a target polynucleotide or transgene, may comprise a coding sequence of interest in gene therapy (such as a gene encoding a protein of therapeutic interest) and/or a selectable or detectable marker.

[0199] Where transcription of the heterologous nucleic acid segment is desired in the intended target cell, it can be operably linked to its own or to a heterologous promoter, depending for example on the desired level and/or specificity of transcription within the target cell, as is known in the art. Various types of promoters and enhancers are suitable for use in this context. Constitutive promoters provide an ongoing level of gene transcription, and may be preferred when it is desired that the therapeutic or prophylactic polynucleotide be expressed on an ongoing basis. Inducible promoters generally exhibit low activity in the absence of the inducer, and are up-regulated in the presence of the inducer. They may be preferred when expression is desired only at certain times or at certain locations, or when it is desirable to titrate the level of expression using an inducing agent. Promoters and enhancers may also be tissue-specific: that is, they exhibit their activity only in certain cell types, presumably due to gene regulatory elements found uniquely in those cells.

[0200] Illustrative examples of promoters are the human elongation factor 1 (hEF-1) promoter, SV40 late promoter from simian virus 40, the Baculovirus polyhedron enhancer/promoter element. Herpes Simplex Virus thymidine kinase (HSV tk), the immediate early promoter from cytomegalovirus (CMV) and various retroviral promoters including LTR elements. Inducible promoters include heavy metal ion inducible promoters (such as the mouse mammary tumor virus (mMTV) promoter or various growth hormone promoters), and the promoters from T7 phage which are active in the presence of T7 RNA polymerase. By way of illustration, examples of tissue-specific promoters include various surfactin promoters (for expression in the lung), myosin promoters (for expression in muscle), and albumin promoters (for expression in the liver). A large variety of other promoters are known and generally available in the art, and the sequences of many such promoters are available in sequence databases such as the GenBank database.

[0201] Where translation is also desired in the intended target cell, the heterologous polynucleotide will preferably also comprise control elements that facilitate translation (such as a ribosome binding site or RBS and a polyadenylation signal). Accordingly, the heterologous polynucleotide generally comprises at least one coding region operatively linked to a suitable promoter, and may also comprise, for example, an operatively linked enhancer, ribosome binding site and poly-A signal. The heterologous polynucleotide may comprise one encoding region, or more than one encoding regions under the control of the same or different promoters. The entire unit, containing a combination of control elements and encoding region, is often referred to as an expression cassette.

[0202] The following non-limiting Examples illustrate some of the materials and methods employed in development of the invention.

Example 1: Construction of a BoHV-1gE CT/Us9 Deletion/Chimeric Gene Insertion Plasmid

[0203] This Example illustrates construction of a pBoHV-1 gECT/Us9 plasmid.

[0204] As depicted in FIG. 3A-3B, the Us9 gene is located immediately downstream of the gE cytoplasmic tail (gE CT residues 451-575) coding region. The pBoHV-1 gECT/Us9 plasmid contains a deletion of the BHV gE cytoplasmic tail and Us9 coding sequences, leaving the 1.4 kB region upstream of the gE and CT coding regions. The 1.4 kB region upstream region contains the gE ecto and transmembrane (Tm) domains. A downstream region contains a partial 1.1 Kb Us9 region containing partial bICP22 sequences flanking a unique KpnI site incorporated at the deletion locus (FIG. 3C).

[0205] In the resulting pBoHV-1 gE CT/Us9 plasmid, the KpnI site created in the gE CT and Us9 deletion locus allows for the insertion of a KpnI/KpnI fragment containing the BVDV E2, BRSV F and G ORF coding chimeric gene sequences (see FIG. 8).

Example 2: Construction and Characterization of a BoHV-1 UL49.5 30-32 CT-Null/gECT/Us9 Virus (BHV-1 Tmv Vector)

[0206] This Example describes construction and characterization of a BoHV-1 vector that lacks the gE cytoplasmic tail (gE CT residues 451-575), which is associated with virulence function, and that lacks the entire 435 base pair long Us9 open reading frame. This vector is called the BoHV-1 UL49.5 30-32 CT-null/gECT/Us9 virus, or the BoHV-1 tmv vector.

[0207] To construct the BoHV-1 tmv vector, the pBoHV-1 gE CT/Us9 plasmid was cotransfected with a full-length BoHV-1U.sub.L49.5 30-32 CT-null parental viral DNA generated earlier (91). One putative triple mutant virus (BoHV-1 tmv) harboring the intended deletion was plaque purified and the nucleotide sequence spanning the U.sub.L49.5, gE and Us9 genes was identified by such procedures.

[0208] Immunoblotting and one-step growth curve analyses of the identified BoHV-1 tmv were performed. Growth of BHV-tmv viruses compared with BoHV-1 gE-deleted and wild type BoHV-1 viruses in MDBK cells is shown as a one-step growth curve. Confluent MDBK cells were infected with the respective viruses at a multiplicity of infection of five plaque forming units (PFU) per cell. After 1 h of adsorption at 4 C., residual input viruses were removed. The cultures were washed three times with phosphate-buffered saline, and 5 ml of medium was added to each flask before further incubation (37 C.). At the indicated time intervals, replicate cultures were frozen. Virus yields were determined by plaque assay. Each data point represents the average of duplicate samples obtained from separate infections.

[0209] The nucleotide sequence data and immunoblotting data (FIG. 4) confirmed that the intended deletions were present while the growth curve results demonstrated that BoHV-1 tmv replicates like wild type BoHV-1 virions (FIG. 5).

Example 3: Pathogenicity and Vaccine Efficacy of BoHV-1 Tmv Relative to BoHV-1 gE-Deleted Virus Against Virulent Wild Type BoHV-1 Challenge

[0210] To determine pathogenicity and vaccine efficacy of the BoHV-1 tmv was compared to a gE-deleted virus (where the entire gE gene is deleted, not just the gE cytoplasmic tail (CT)-truncated BoHV-1 virus prepared by the inventor), followed by challenge with virulent wild type BoHV-1 virus.

[0211] Fifteen BoHV-1 and BVDV negative, 4-month-old cross-bred bull calves were selected and randomly assigned into six rooms: three calves each into three rooms and two calves each into another three rooms at the LSU Ag-center large animal isolation facility. Animal infection, handling, sample collection and euthanasia protocols were previously approved by the LSU Institutional Animal Care and Use Committee.

[0212] Five calves of a triple mutant group were inoculated with 1 ml/nostril of BoHV-1 tmv (in DMEM) containing 110.sup.7 PFUs for a total of 210.sup.7 PFUs/animal. Five calves in gE-deleted group were similarly inoculated with 110.sup.7 PFUs/nostril of the gE-deleted virus, and five calves in the control group were similarly inoculated with cell culture media (Table 1).

[0213] Following primary infection, calves were observed for clinical signs of BoHV-1 infection daily for nine days. Nasal swabs were collected daily. Subsequently, calves were observed on alternate days and nasal swabs collected once a week until 28 days post-infection with the BoHV-1 tmv virus or the gE-deleted virus.

[0214] On day 28, calves in all three groups were challenged with 1 ml/nostril of virulent wild type BoHV-1 Cooper strain virus containing 210.sup.7 PFUs/ml for a total of 410.sup.7 PFUs/animal.

[0215] Following challenge, the animals were observed and nasal swabs collected similarly as above daily for 12 days. On day 15 post challenge, all the infected calves were euthanized and necropsied (Table 1).

TABLE-US-00038 TABLE 1 Summary of Viral Vaccination and Challenge Procedure BHV-1 tmv BHV-1 gE -del Control 5 calves 5 calves 5 calves Day 0 Day 0 Day 0 1 10.sup.7 1 10.sup.7 Sham-infected with PFUs/ml/nostril PFUs/ml/nostril 1 ml media/nostril Day 1, 2, 3, 4, 5, 6, 7, 8, 9, 12, 15, 21, 28 post infection nasal swabs, temps and clinical scores Day 0, 7, 14, 21, 28 serum 28 dpi (vaccination) intranasal challenge with BHV-1 Cooper strain- 4 10.sup.7 PFUs (2 10.sup.7 PFUs/ml/nostril). Day 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 12, 14 post challenge nasal swabs, temps and clinical scores Day 0, 7, 14 post challenge - whole blood and serum Day 15 post challenge euthanize-necropsy

[0216] Clinical records, including rectal temperatures, nasal discharge and nasal lesions, were recorded during each visit and clinical scores were assigned for each parameter. Rectal temperatures were scored 0-4 (less than 39.0 C., 39.5 C., 40.0 C., greater than 40.9 C.), nasal discharges were scored 0-4 (normal, serous, mild and severe mucopurulent), and nasal lesions were scored 0-3 (normal, hyperemia, pustules, ulcers).

[0217] The clinical score data following primary infection/vaccination showed that like the gE-deleted BoHV-1, BoHV-1 tmv is highly attenuated in calves because the infected calves did not show any detectable clinical signs and their clinical scores were not significantly different from sham-infected controls.

[0218] Following virulent wild type BoHV-1 virus challenge, both the BoHV-1 tmv and gE-deleted virus-immunized groups showed no clinical signs. As expected, the control uninfected calves had high fever, nasal discharge and lesions in the nasal mucosa after challenge which is typical for wild type BoHV-1 infection. As a result, their clinical scores were significantly higher following challenge (FIG. 6A).

Example 4: Nasal Virus Shedding, Pathogenicity and Protective Vaccine Efficacy of BoHV-1 Tmv Against Virulent BoHV-1 wt Challenge in Calves Compared with that of BoHV-1 gEdel

[0219] This Example describes statistical analysis of the effects of BoHV-1 tmv treatment of calves followed by challenge with virulent BoHV-1 virus.

[0220] Calves were treated with BoHV-1 tmv and gE-deleted viruses as described in Example 3. Nasal virus shedding was recorded. To evaluate protective immune responses, blood was collected on 0, 7, 14 and 21 days post immunization/dpi and at day 0 (28 dpi), 7, 12 and 15 days post challenge (dpc) (see, Table 1).

[0221] Serum interferon levels were measured using high binding EIA plates (Costar, Corning, N.Y.) having wells coated with anti-bovine IFN-specific rabbit polyclonal antibodies (10 g/ml, Endogen, Rockford, Ill.) to capture IFN- from serially diluted calf sera (100 l) from different treatment groups. As a control, 100 l of the serial diluted recombinant bovine IFN (Thermo, Pierce, Ill.) were added instead of sample sera. After incubation (for 1 hour), the test wells were washed and incubated (1 hr) with biotinylated rabbit anti-bovine IFN polyclonal Ab (Endogen, Rockford, Ill.). After washing, the test wells were incubated (for 1 hour) further with avidin-HRP (eBioscience, San Diego, Calif.) and developed with substrate 2,2-azino-bis(3-ethylbenzothiazoline-6-sulphonic acid) (ABTS, Sigma). The test results were measured at OD.sub.405 by using an ELISA reader.

[0222] Data from the pathogenicity and vaccine efficacy studies, specifically the viral shedding titers, serum neutralization titers, and IFN- concentrations were analyzed using a repeated measures ANOVA to detect significant differences among treatments over the period of challenge. The SAS (Version 9.1.3) was used for statistical analysis. Post-hoc, pairwise comparisons were employed to estimate pairwise differences and conduct t tests. Significance was assessed at the P<0.05 level for all tests.

Results

[0223] Nasal virus shedding data showed that while both triple mutant BoHV-1 tmv viruses and gE-deleted viruses replicated with similar yield during primary infection/immunization, following challenge with virulent BoHV-1, shedding of the triple mutant BoHV-1 tmv virus from immunized calves was for only the first 7 days after challenge (FIG. 6B). In contrast, the gE-deleted virus immunized calves shed virus for about 10 days (3 days longer than BoHV-1 tmv-immunized calves: FIG. 6B). The sham-infected/vaccinated control calves shed virus for 11 days (4 days longer than the BoHV-1 tmv-immunized calves, FIG. 6B). Additionally, on 6 and 7 days post-challenge, nasal virus shedding in the BoHV-1 tmv vaccinated group was reduced significantly compared with the gE-deleted virus group (FIG. 6B).

[0224] As shown in FIG. 6C, virus neutralizing antibody titers following primary infection/immunization with BoHV-1 tmv were similar to those observed for BoHV-1 tmv vaccinated group. However, following challenge by virulent wild type BoHV-1, the BoHV-1 tmv vaccinated group had significantly higher (p<0.05) virus neutralizing antibody titer (650) than the gE-deleted virus immunized group (300).

[0225] Notably, significantly increased levels of interferon- were produced in the triple mutant BoHV-1 tmv virus-immunized group both at 7 days (p<0.01) and 15 days (p<0.05) post challenge when compared with the gE-deleted virus immunized group (FIG. 6D).

[0226] Taken together these results show that the triple mutant the BoHV-1 tmv virus can prime the humoral and cellular responses for a more rapid and exuberant neutralizing antibody and IFN- responses compared with the gE-deleted virus. The data indicated that the BoHV-1 tmv virus was better able to clear the challenge virus (FIG. 6B). These results show that the triple mutant BoHV-1 tmv virus lacking the MHC-I down regulation function is a more efficacious vaccine than the gE-deleted virus vaccine.

Example 5: Serological Properties of BoHV-1 Tmv

[0227] This Example illustrates some of the serological properties of the BoHV-1 tmv virus.

[0228] The sera of BoHV-1 tmv-infected calves and gE-deleted virally-infected calves were collected before virulent wild type BoHV-1 challenge, and tested for expression of gE antigens. In a first assay, the IDEXX gE antibody detection competitive ELISA test kit was used for detection of gE proteins. Based on the ELISA test results, BoHV-1 tmv-infected calves were slightly positive for gE protein expression (data not shown). However, the BoHV-1 tmv virus encodes and may express a portion of the gE protein (i.e., the cytoplasmic tail, CT). The part of glycoprotein E that is missing in the BoHV-1 tmv vaccine are amino acids 451-575, with the sequence shown below (SEQ ID NO:43).

TABLE-US-00039 1 ASQKRTYDILNPFGPVYTSLPTNEPLDVVVPVSDDEFSLD 41 EDSFADDDSDDDGPASNPPADAYDLAGAPEPTSGFARAPA 81 NGTRSSRSGFKVWFRDPLEDDAAPARTPAAPDYTVVAARL 121 KSILR

[0229] Therefore, the inventors have developed an ELISA test to detect the gE expressing wild type viruses, but not the gE-cytoplasmic tail expressed from the BoHV-1 tmv viral genome. ELISA plate test wells are coated with 120 amino acids of the gE SEQ ID NO:43 shown above that are missing from the BoHV-1 tmv viral genome. An HRP-conjugated indicator antibody was gE-CT specific was also used.

[0230] A protein containing BoHV-1 gE amino acid residues 451-564 (entire BoHV-1 gE CT coding region) was expressed as a GST fusion protein and purified GST fusion protein was purified and cleaved to release the gE CT protein. Monoclonal antibodies specific to the gE-CT antigen were generated. The gE CT-specific MAb specifically recognized the full length BoHV-1 gE but not the gE CT in immunoblotting (FIG. 7). An optimized gE-CT-based serological marker assay employs an affinity purified-HRP conjugated gE-CT-specific monoclonal antibody and gE-CT antigen coated ELISA plates.

Example 6: Construction of BVDV Type 1 E2 (E2.1) Specific Chimeric Gene and a BoHV-1 Tmv Expressing E2.1

[0231] This Example illustrates construction of BVDV E2-specific chimeric gene and a BoHV-1 vector that expresses the E2 protein.

Construction of a BVDV E2-Specific Chimeric Gene

[0232] A nucleic acid segment encoding a BVDV1 E2 open reading frame (GenBank accession# GI:7960753) was codon-optimized with a bias for expression in bovine cells and for commercial synthesis. Codon-optimized 1.18 Kb long synthetic oligonucleotides for BVDV E2.1 also incorporated a consensus Kozak sequence (GCCATGG) plus BoHV-1 gD signal sequence (57 nucleotide) (BoHV-1 gD amino terminal 19 amino acids; Genbank accession #GI:2653359) at the amino terminal end of the E2.1 sequence. The codon optimized synthetic BVDV1E2 gene (1.23 Kb), in a plasmid backbone, was amplified by PCR using Fail safe PCR kit and cloned into the TA cloning site of the eukaryotic expression vector plasmid pEF6/V5-His-TOPO (Invitrogen) (FIG. 8, panel A). The cloned fragment was verified by sequencing for the integrity of the E2.1 open reading frame, and for the in-frame fusion of the C-terminal end of the E2.1 open reading frame with the N-terminus (beginning) of the V5 epitope coding region. The PCR fragment containing the BVD E2.1 open reading frame was inserted in the correct orientation downstream of the strong EF-1 promoter and upstream of the V5 epitope. A Kozak sequence followed by gD signal sequence preceded the BVD E2.1 open reading frame, and a poly His tag with a bovine growth hormone (BGH) polyA signal followed the BVD E2.1 open reading frame (FIG. 8A). The in-frame fusion of the C-terminus of the E2.1 open reading frame with the vector sequence was designed to allow the expression of the V5 epitope and termination of the chimeric E2.1 transcript immediately downstream of the His tag within the pEF6/V5-His-TOPO vector. Therefore, the adjusted and predicted unprocessed molecular mass of the E2.1 fused to V5 epitope and His tag was approximately 48 kD, where the E2.1 portion was 43 kD, and the V5+His tag portion was 5 kD. Transient expression of V5 epitope by the chimeric E2.1/V5 gene was verified after transfection of the plasmid DNA into T293 cells (data not shown).

Construction of a BoHV-1 Vector that Expresses the E2 Protein

[0233] The BVDV type 1 E2 (E2.1) chimeric expression cassette containing the PEF-1 promoter at the upstream 5 end, and the bGH poly A sequence at the 3 end was amplified by using a p1F and p2R primer pair, where the p1F primer has GCggtaccTC-GTGAGGCTCCGGTGCCCGTCAGTG (SEQ ID NO:44), which is specific for PEF-la promoter sequences, and the p2R primer has GCggtaccCCATAGAGCCCACC-GCATCC-CCAGCATGCCTG (SEQ ID NO:45), which is specific for sequences immediately downstream of the bGH poly A (FIG. 8A). The p1F/p2R primer pair incorporated KpnI sites at each end of the amplified fragment. The PCR-amplified BVDV1 E2 expression cassette (approximately 2.9 kb) was digested with KpnI and cloned into the KpnI site of the insertion plasmid pBoHV-1gECT/Us9 (FIG. 8B), resulting in a pBoHV-1 BVDV E2.1 insertion vector (pBoHV-1 INS/E2.1) (FIG. 8C).

[0234] To generate a modified BoHV-1 tmv vector that expresses the chimeric BVDV E2.1 protein, linearized pBoHV-1 INS/E2.1 plasmid DNA (FIG. 8C) was co-transfected with the full-length BoHV-1 tmv DNA (FIG. 3D) into MDBK cells. Putative recombinant viral plaques expressing BVDV E2.1 were initially screened by PCR and verified by sequencing. Several recombinant BoHV-1 tmv containing BVDV E2.1 chimeric gene sequences were further analyzed by immunoblotting using goat anti-BVDV-specific polyclonal antibody (VMRD Cat #210-70-BVD) or an anti-V5 monoclonal antibody (Invitrogen Cat # R96025).

Construction of a Stable Cell Line for Expressing BVDV Type 1 and 2 E2:

[0235] In another series of experiments, nucleic acid segments encoding BVDVE2-1 and/or BVDVE2-2 along with Kozak, gD signal and V5 were amplified using the forward primer-TAATCAAAGCTTCGCCGCCACCATGCAGGGACC (SEQ ID NO:46) and reverse primer CTCCGGAATTCCGTAGAATCGAGACCGAGGAGAGG (SEQ ID NO:47) using a BVDV1-E2 expression cassette (e.g., SEQ ID NO:9) and/or a BVDV2-E2 expression cassette (SEQ ID NO: 14) as template. Forward and reverse primers incorporated HindIII and EcoRI restriction sites (shown in italics), respectively. The PCRgenerated fragment was digested with HindIII and EcoRI and cloned into the corresponding sites in the multiple cloning site of pcDNA3.1(+) (Invitrogen, cat# V790-20). PCDNA3.1-BVDV1-E2 or BVDV2-E2 constructs were used to transfect MDBK cells. Transfected MDBK cells were selected for resistance against gentamycin (1 mg/ml) containing 5% DMEM media. Stably transfected isolated colonies were screened for determining the expression of BVDV1-E2 and BVDV2-E2 protein by immunoblotting and indirect immunofluorescence using anti-BVDV1-E2 and BVDV2-E2 specific monoclonal antibody (VMRD, cat#157 and cat#BA-2, respectively). These cell lines can be used for the cellular immune response against BVDV types 1 and 2 by cytotoxicity and cell proliferation assays.

Results

[0236] As depicted in FIG. 9B, the BVDV-specific polyclonal antibody detected two bands with molecular mass of 48 kD and 58/59 kD in the infected cell lysates of one representative BoHV-1 tmv/E2.1 recombinant virus. The 48 kD band is the unprocessed chimeric E2.1 expressed by BoHV-1 tmv/E2.1 recombinant virus which is predicted to be 5 kD larger (due to additional V5 epitope and His tag residues) than the 43 kD unprocessed BVDV E2.1 protein detected in the BVDV-infected cell lysates (FIG. 9B). The 58/59 kD protein is the processed chimeric E2.1 form, which in BVDV infected cells is the 53/54 kD band (FIG. 9B). These results indicate that BoHV-1 tmv vector encoding the E2.1 protein is processed appropriately in the infected MDBK cells.

[0237] Preliminary results also showed that in MDBK cells, the BoHV-1 tmv/E2.1 virus replicates with very similar kinetics and yield compared with the parent BoHV-1 tmv (data not shown).

Example 7: Immunoblotting and/or Immunoprecipitation Analysis

[0238] This Example describes radiolabeling infected cell proteins, immunoblotting, immunoprecipitation, and biochemical analysis of viral glycoproteins.

[0239] For immunoblotting and/or immunoprecipitation analysis, MDBK cells are mock-infected and infected with BoHV-1 tmv expression vector that encodes one or more antigenic proteins. The MDBK can be mock-infected or infected with BoHV-1 tmv expression vectors at a multiplicity of five.

[0240] Pulse labeling of cells with [.sup.35S] methionine and [.sup.35S] cysteine can be performed for 30 minutes starting at six hours post-infection as described (1, 2). Steady-state labeling is performed with [.sup.35S] methionine and [.sup.35S] cysteine for 10-12 hours starting at the same time as for pulse labeling. Processing of cell lysates, immunoprecipitation and SDS-PAGE are performed as described earlier (1).

[0241] For deglycosylation analysis of proteins, first [.sup.35S] methionine and [.sup.35S] cysteine labeled infected cell and purified virion proteins are immunoprecipitated. Then for N-glycosidase F and EndoH digestion, the immunoprecipitated viral proteins are incubated with 1,000 U of N-glycosidase F (New England Biolabs; NEB) or 100 U of EndoH (NEB) for 1 h at 37 C. as described earlier (1, 2). For neuraminidase and O-glycosidase digestion, immunoprecipitated proteins are digested overnight with 1 mU of neuraminidase (Boehringer) or 1 mU of neuraminidase and 1.5 mU of O-glycosidase (Boehringer). After the appropriate enzyme digestions above, the protein samples are subjected to SDS-PAGE and the results are visualized by autoradiography (1).

[0242] As described above a BoHV-1 vectored E2.1 protein has been generated and expression of antigenic protein therefrom has been characterized (FIG. 9). For BoHV-1 vectored E2.2, similar molecular weights of the ER processed precursor E2.2 (48 kD) and the Golgi processed mature E2.2 (58 kD/59 kD) are observed (FIG. 9). In addition for both E2.1 and E2.2, Golgi processed mature E2.1 and E 2.2 (58 kD/59 kD band) would be Endo H resistant. However, the ER processed precursor E2.2-specific 48 kD band would be Endo H sensitive and its molecular weight will be slightly reduced after Endo H treatment. In the case of glucosidase F treatment, both the 58 kD/59 kD and 48 kD bands would be deglycosylated and the digested bands will migrate faster than the respective untreated bands, as will the corresponding Endo H digested, ER processed 48 kD precursor-specific band above.

[0243] The BRSV F protein is synthesized as precursor 70 kD (F.sub.0), which is proteolytically processed to F1 (48 kD) and F2 (18 kD) bands. However, in the chimeric BRSV F ORF coding sequence, the two Furin cleavage sites FCS 1 and FCS 2 are mutated. Therefore, 70 kD F protein (F.sub.0) will not be cleaved to their F1 (48 kD) and F2 (18 kD) processed forms. To determine processing of the 70 kD chimeric F.sub.0 protein, we will chase the .sup.35S labeled 70 kD band for at least 120 minutes after pulse labeling. Pulse chase data analysis combined with Endo H and glycopeptidase F digestion data of pulse labeled chimeric F protein (expressed in BoHV-1 tmv/F- or BoHV-1 tmv/G-infected MDBK cells) will be critically evaluated and compared with the similarly treated pulse labeled BRSV F protein from BRSV-infected bovine turbinate (BT) cells. In BRSV-infected cells, F protein is glycosylated by N-linked oligosaccharides (41, 57). Therefore, the mature Golgi processed BoHV-1 tmv expressed 70 kD F-specific band is expected to be resistant to EndoH after 60-120 min. chase in the pulse-chase assay. Whereas a slightly smaller ER processed band containing high mannose glycans, immediately after pulse (0 min. chase) is expected to be Endo H sensitive and would be reduced to a slightly smaller size following Endo H treatment.

[0244] The BRSV G precursor is synthesized as a 43 kD protein which is further processed in the Golgi to 68-90 kD broad band form (51). The 43 kD band is the N-linked high mannose form and the 68-90 Kd band is both O-linked and N-linked complex carbohydrate modified forms. Since the chimeric G protein expressed by BoHV-1 tmv/G may lack residues 173-186 (14 amino acids), the corresponding band for the chimeric G precursor protein is expected to be approximately 38 kD. The mobility of the approximate 38 Kd band may migrate to a larger size if further processing of the protein in the Golgi has taken place. However, the nature of processing can only be determined based on the results from deglycosylation assays. The 38 kD chimeric G precursor band may also be sensitive to both glycopeptidase F and Endo H, in deglycosylation assays indicating that the 38 kD form is the N-linked precursor F protein synthesized in the ER, whereas the larger Golgi processed form (Approx. 70-76 kD) may be resistant to EndoH but partially sensitive to neuraminidase and O-glycosidase.

Example 8: Growth Properties of BoHV-1 Tmv Expressing BVDV E2.1, BVDV E2.2, BRSV F or BRSV G

[0245] Characterization of the recombinant viruses with respect to their replication kinetics and plaque morphology can be performed to determine whether non-BoHV-1 proteins have effects on virus replication and/or plaque phenotype. One step growth curve studies can be performed as described earlier (90). In addition, incorporation of BVDV E2.1, BVDV E2.2, BRSV F and BRSV G chimeric proteins in the respective purified recombinant BoHV-1 vector viruses can be investigated by analyzing immunoblots containing purified virion lysates. Based on previous reports, both BVDV type 1 E2 and BRSV G proteins expressed by recombinant BoHV-1 were incorporated in the virus envelope (45, 77). Therefore, recombinant BoHV-1 expression of the BVDV E2.1 and BRSV G proteins may be incorporated in the virus envelope. The BRSV F chimeric protein may or may not be incorporated into the envelope.

Example 9: Vaccine Efficacy of Recombinant BoHV-1 Tmv Vectors

[0246] This Example describes procedures for evaluating the efficacy of individual BoHV-1 vector vaccine virus expressing BVDV E2.1 protein, BVDV E.2 protein, BRSV F protein. BRSV G protein, or a combination.

Determination of Sample Size for Animal Study

[0247] Sample size can be calculated following a power analysis of the primary variable of interest to maximize confidence in that metric. Data presented herein concerning viral titers and serum virus neutralizing antibody titers were used to estimate the expected differences (i.e. difference of 0.90 for viral titers) among treatment(s) at time points 6, 7 and 8 days post challenge. As these data indicate, days 6, 7 and 8 are most relevant for detecting differences in our variables of interest. Means and associated variances from our preliminary studies were then utilized in our sample size calculation, requesting a power of 90%. This resulted in a sufficient sample size of 6 calves per group.

Vaccination and Challenge of Calves:

[0248] For each of the three BoHV-1 vectored subunit vaccination challenge experiments, 18 calves (total 54 calves for the three vaccine efficacy study) free of BoHV-1, BVDV and BRSV antibodies or very low (<4 virus neutralization titers) will be purchased. For each vaccine efficacy study, they will be randomly divided into three groups of 6 calves each (BoHV-1 tmv vectored subunit vaccine group. Bovishield vaccinated and control unvaccinated) and housed in separate biocontainment rooms. Six calves in the control group will be sham-infected, six calves in the experimental vaccinated group will be infected with BoHV-1 vectored subunit vaccine virus and the remaining six calves will be vaccinated I/M with Bovishield following the manufacturers' recommendation. For vaccinating the calves with BoHV-1 vectored E2.1 or BoHV-1 vectored E2.2 vaccine, 110.sup.7 PFU/ml/nostril (total 210.sup.7 PFU/animal) of virus will be inoculated. For infecting the calves with BoHV-1 vectored BRSV F and G subunit vaccines, mixtures of each protein expressing BoHV-1 vector virus at IX 10.sup.7 PFU/ml/nostril for each virus (total 210.sup.7 PFU/nostril for F and G combined) will be inoculated. At 28 days post infection, all sixteen calves for each experiment will be challenged with virulent BVDV type 1 or BVDV type 2 or BRSV virus depending on their specific subunit vaccination protocol (see tables 2 and 3 for detail).

[0249] For BVDV challenge, 1 ml of virus suspension/nostril containing 610.sup.6.2 TCID.sub.50 type strain Singer or type 2 strain 125-C will be inoculated using a syringe. For BRSV challenge, the calves will be inoculated with 1 ml of viral suspension containing 610.sup.6.2 TCID.sub.50 strain 236-652 (41) in each nostril via aerosolization (61).

[0250] Aerosolization will be accomplished with a siphon-fed spray gun (Sears Model 491, Hoffman Estates, Ill.) fitted to a 10 cm long by 10 mm i.d. plastic tube, which is inserted into the nasal passages. The gun is powered by compressed nitrogen gas at 40 p.s.i.

TABLE-US-00040 TABLE 2 Vaccine efficacy study scheme for BoHV-1 tmv expressing BVDV type 1 or 2 specific E2.1 and E2.2 BHV-1 tmv/ Bovi-shield Control BVDV-E2.1 Gold 5 6 calves 2 or BVDV-E2.2 6 calves 2 6 calves 2 Day 0 Day 0 Day 0 1 10.sup.7 2 ml/IM Sham-infected with PFUs/ml/nostril 1 ml media/nostril Day 1, 2, 3, 4, 5, 6, 7, 8, 9, 12, 15, 21, 28 post infection nasal swabs, temps and clinical scores Day 0, 7, 14, 21, 28 serum, whole blood 28 dpi (vaccination) intranasal challenge with BVDV Type 1 strain Singer- 6 10.sup.6.2 TCID.sub.50 (18 calves) or with BVDV Type 2 strain 125 -C 6 10.sup.5.2 TCID.sub.50 (18 calves) corresponding to their vaccination group above. Day 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 12, 14 post challenge nasal swabs, temps and clinical scores Day 0, 7, 14 day post challenge - whole blood and serum Day 15 post challenge euthanize, necropsy and score lung lesions

TABLE-US-00041 TABLE 3 Vaccine efficacy study scheme for BoHV-1 tmv expressing chimeric BRSV F- and G-specific protective antigens. BHV-1 tmv/ Bovi-shield Control BRSV -F/ Gold 5 6 calves BRSV-G 6 calves 6 calves Day 0 Day 0 Day 0 1 10.sup.7 2 ml/IM Sham-infected with 1 PFUs/0.5 ml ml media/nostril each of BHV-1 triple/BRSV-F and BRSV-G/nostril Day 1, 2, 3, 4, 5, 6, 7, 8, 9, 12, 15, 21, 28 post infection nasal swabs, temps and clinical scores Day 0, 7, 14, 21, 28 serum, whole blood 28 dpi (vaccination) intranasal challenge with BRSV strain 236-652 6 10.sup.6.2 TCID.sub.50 Day 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 12, 14 post challenge deep naso- pharyngeal swabs, lung lavage, temps and clinical scores Day 0, 7, 14 day post challenge - whole blood and serum Day 15 post challenge euthanize, necropsy and score lung lesions

[0251] For BoHV-1 tmv vectored BVDV type 1- or type 2-specific subunit vaccine efficacy studies, the schemes listed in Table 2 will be followed for the collection and evaluation of nasal swabs. Nasal swabs will be processed as described before (90) and MDBK cells will be used for plaque assay. For BoHV-1 triple mutant vectored BRSV subunit vaccine efficacy study, the scheme listed in Table 3 is similar up to 28 dpi as above in Table 2. However, after challenge with virulent BRSV strain 236-652 (41), deep nasopharyngeal swabs will be collected and twice during the first week of post challenge (3 pc and 6 dpc) lung lavage will be collected for virus isolation and/or for preparing formalin fixed slides containing cells pelleted from lung lavage (Table 3). Bovine turbinate cells will be used for BRSV isolation from lung lavage or nasopharyngeal swabs. In addition, formalin fixed slides containing smears prepared with cells pelleted from lung lavage will be tested for virus-specific antigens by FA. Alternatively, RNA isolated from the lung lavage/nasopharyngeal swab samples will be subjected to BRSV-specific semi quantitative RT-PCR as described earlier (55). In the case of calves challenged with BVDV, virus isolation from buffy coat cells and virus-specific RT-PCR will also be performed.

Determining Effects of BoHV-1 Tmv Vectored Subunit Vaccination Relative to Bovishield Vaccination on Challenge and Virus Shedding in the Nose.

[0252] For this study, challenge virus replication in the nasal epithelium and/or in the lung tissues and clinical scores in infected verses uninfected control animals will be determined. Based on viral plaque assays on nasal swab samples (BVDV) or on deep nasopharyngeal swabs and lung lavage samples (BRSV), the level of protection conferred by recombinant viruses against challenge by wild type virus can be evaluated. Following challenge, a significant reduction in the duration and amount of virus in the nasal swabs (BVDV) or nasopharyngeal swabs (BRSV) or lung lavage (BRSV) of vaccinated animals compared with the similarly challenged sham-infected control animals will indicate efficacy.

[0253] We expect that like their parental virus (BoHV-1 tmv), the BoHV-1 tmv vector virus expressing BVDV E2.1 or E2.2 or BRSV-F/BRSV-G will replicate efficiently in the nasal epithelium of calves. As illustrated in FIG. 6A, the recombinant viruses may cause very mild or no respiratory disease. Although highly unlikely, increased clinical scores following primary infection with a recombinant virus would indicate that expression of the chimeric protein has affected the pathogenicity of the virus.

Determine Respective Protein-Specific Antibody Responses in Infected Calves.

[0254] Antigen-specific antibody (generated against subunit proteins BVDV E2.1, BVDV E2.2, BRSV F, BRSV G, or a combination thereof) can be detected by Cell ELISA test using respective virus-infected cells. Alternatively, ELISA plates can be coated with solubilized subunit protein-specific purified virion lysates (BVDV types 1 or 2 or BRSV) and tested by standard ELISA test (14). In addition, for the BRSV subunit vaccine group, sera from animals after primary infection/vaccination can be tested using a commercially available BRSV ELISA kit specific for F protein-specific antibodies (Cat # BIO K 061; Bio-X diagnostics, Belgium). Further, Western blots containing each wild-type BVDV types 1 and 2 and BRSV purified virion lysates, purified virion lysates of BoHV-1 triple mutant vector expressing BVDV E2.1/E2.2, BRSV-F/G will be tested to detect the subunit protein-specific as well as their corresponding band in their respective wt virion lysates.

Determine Virus Neutralizing Antibody and Cellular Immune Responses in BoHV-1 Triple Mutant Virus Vectored Subunit Vaccinated Relative to Bovishield Vaccinated and Uninfected Control Calves Before and after Challenge.

[0255] Virus neutralizing serum antibody titers against BoHV-1 can be determined by a plaque reduction assay described earlier (90, 91). For BVDV and BRSV, an immunofluorescence (IFA) based plaque reduction assay can be used in which after fixing the plates with formaldehyde, FA staining with virus-specific antibodies can be performed and fluorescent plaques will be counted. Compared with the uninfected control animals, the vaccinated animals can have higher serum neutralization (SN) titers against the BoHV-1 as well as against the respective subunit vaccine-specific wild-type virus at 21 dpi-28 dpi.

[0256] For assessing BoHV-1 specific cellular immune response, serum IFN- levels can be determined as described for FIG. 6D. In addition, cell proliferation assays and ELISPOT assays for interferon-, using PBMCs collected during primary infections from different vaccinated and control groups will be performed as described earlier (91). For assessing BVDV E2.1-, BVDV E2.2- and BRSV F/G-specific cell proliferation assay, PBMCs collected from vaccinated (subunit and Bovishield) and control group before and after specific challenge can be sensitized with the corresponding UV-inactivated purified virions or for BVDV additionally with commercially available BVDV E2-specific codon optimized proteins (Bioclone Inc. Cat#PN0146, Cat # PN0143).

[0257] Following challenge, a rapid spike in virus-specific SN antibody and cell mediated immunity (CMI) responses in the vaccinated animals is expected. This spike will indicate that protective subunit vaccine antigen primed the vaccinated animal for a rapid memory response and a rapid rise in neutralizing antibody titers. For specific subunit antigen-specific cellular immune response in BoHV-1 triple mutant-infected calves expressing BVDV E2.1 or E2.2 or BRSV F/G, there can be increased proliferation of CD8 positive T cells and increased secretion of IFN- when PBMCs collected after primary vaccination as well as after challenge, are specifically stimulated with respective UV-inactivated virus or with specific BVDV codon optimized antigen. Following challenge, a rapid spike in virus-specific SN antibody and CMI responses in the vaccinated animals should also correlate with virus shedding/clearance data.

Determine Respiratory Tissue Lesions in BoHV-1 Triple Mutant Subunit Vaccinated Calves Relative to Bovishield Vaccinated and Uninfected Control Calves

[0258] Comparison of lung lesions of vaccinated animals with similarly challenged control animals using the subunit vaccine-specific wild type virus will show the protective effects in lungs following challenge. Relative to the similarly challenged control calves, a significant reduction in the lung lesions (>70%) or no lesions in the vaccinated animals challenged with either subunit-specific wild type virus will indicate protective efficacy of the vaccine. Lung lesions will be scored as follows: 0=normal, 1-=mild; 2=moderate; and 3=severe. The changes evaluated with the above scores will include bronchial epithelial necrosis; bronchial inflammation: peribronchial/perivascular inflammation: alveolar inflammation/necrosis; and syncytial cell formation (BRSV only).

Analysis of Statistical Significance

[0259] Nasal virus shedding, clinical scores and specific virus neutralizing titers will be analyzed as described herein, for example, utilizing repeated measures ANOVA and post-hoc pairwise comparisons. However, as explained in response to previous reviews, lung lesion scores will be analyzed using a nonparametric Kruskal-Wallis t-test.

[0260] Based on data provided herein, BoHV-1 triple mutant virus-infected calves will have BoHV-1-specific SN antibody titers and cellular immune responses. After vaccination with the novel BoHV-1 vectored subunit vaccines, similar BoHV-1-specific SN titers and cellular immune responses can be as illustrated in the foregoing Examples. Although unlikely, reduced levels of SN titers and/or weaker cellular immune responses against BoHV-1 vector would indicate that the specific subunit proteins have inhibitory effects on the host response to the vector virus. Based on the results from vaccine efficacy of vaccinia virus and adenovirus vectored subunit vaccines against BRSV (2) and the DNA subunit vaccine against BVDV (52, 53), SN antibody titers and cellular immune responses against the subunit vaccine-specific wild type viruses will be induced in the vaccinated calves, and the calves will be protected against the respective wild type viral challenges (reduced and shorter virus shedding, spike in SN antibody titers, increased level of IFN- production by PBMCs at 7 days post vaccination and increased CD8+ T cell proliferation following challenge with respective wt viruses). In the case of BVDV, an ideal protection would be when there is no viremia following challenge as judged by RT-PCR.

Potential Problems

[0261] One potential problem could be that in the case of BVDV and BRSV challenges, calves may come down with secondary bacterial infections. To avoid such problems following challenge infections, injection of Micotil 300 (8 mg/lbs.) on the day of challenge can be used.

[0262] BRSV is a highly sensitive to freezing and thawing. Therefore, another potential problem could be that virus in the nasopharyngeal swabs or in lung lavage samples may be inactivated due to freezing and thawing. To avoid such problems, the viral plaque assay can be performed on the day of sample collection without freezing the samples. Alternatively, FA staining can be performed on slides containing smears of nasopharyngeal swabs and/or lung lavage cells.

Example 10: Construction of Novel Bovine Herpesvirus Type 1 (BoHV-1) Vectored Vaccine Against Viral Infections Associated with Bovine Respiratory Disease Complex (BRDC)

[0263] The Example describes experiments relating to the following.

[0264] 1. Construction of BoHV-1 tmv expressing BVDV types 1 and 2 envelope proteins E2.1 and E2.2, respectively.

[0265] 2. Construction of BoHV-1 tmv expressing BRSV envelope proteins F and G.

[0266] 3. Test the vaccine efficacy of the above constructs against the BVDV and BRSV infections compared with a commercial multivalent viral BRD vaccine Bovishield).

[0267] The construction and testing of these recombinant viruses can provide a better and safer strategy to vaccinate beef and dairy cattle against BoHV-1, BVDV and BRSV, the three most significant bovine respiratory viruses that predispose to fatal secondary bacterial pneumonia. Ultimately, this vaccine will reduce the mortality and morbidity associated with BRDC.

[0268] FIG. 3A shows the structure of the BoHV-1 tmv recombinant virus previously developed by the inventors.

[0269] To construct BoHV-1 recombinants expressing BVDV E2-1 or 2, first the BVDV E2-1 or 2 insertion vectors were generated (FIG. 11). Second, linearized pBoHV-1 gECTUs9/BVDV E2-1 or 2 insertion vector DNA was used for transfection with full-length BoHV-1 tmv DNA (FIG. 3A). Putative recombinant plaques were verified initially by PCR for the incorporation of BVDV type 1- or type 2-specific E2 (E2-1 or E2-2) sequence. Subsequently, selected plaques were further characterized for the expression of E2-1 or E2-2 (FIG. 12)

[0270] FIG. 12A-12B illustrate expression of BVDV E2 antigens from a recombinant BoHV-1 tmv vector. FIG. 12A shows an immunoblot of MDBK cell lysates that were either mock-transfected or transfected with BoHV-1tmv recombinant vectors expressing either BVDV type 1 (BoHV-1 tmv E2-1 clone 8 and clone 2) or type 2 E2 proteins (BoHV-1 tmv E2-2) as detected by a BVDV type 1 E2-specific monoclonal antibody (VMRD #157). FIG. 12B shows an immunoblot of MDBK cell lysates that were either mock-transfected or transfected with BoHV-1tmv recombinant vectors expressing either BVDV type 1 (BoHV-1 tmv E2-1 clone 8 and clone 2) or type 2 E2 proteins (BoHV-1 tmv E2-2) as detected by a BVDV type 2 E2-specific monoclonal antibody (VMRD # BA-2). As control, the reactivity of the monoclonal antibody Ab 157 and the monoclonal antibody mAb BA-2 is illustrated for mock and BVDV-1 type 1 (strain sanger) and BVDV type 2 (strain 125) infected MDBK cell lysates.

[0271] FIG. 13 graphically illustrates single step growth kinetics of BoHV-1tmv, BoHV-1tmv/E2-1 and BoHV-1tmv/E2-2. Samples were collected at 0 h, 3 h, 7 h, 11 h, 17 h, 23 h, 29 h, 35 h, 41 h and 47 hours post infection and titrated by plaque assay.

[0272] FIG. 14 graphically illustrates the plaque morphology of BoHV-1 tmv/E2-1 and BoHV-1tmv/E2. Plaque sizes produced by parental BoHV-1tmv, BoHV-1 tmv/E2-1 and BoHV-1tmv/E2 viruses were measured at 48 hours post-infection. Average plaque diameters of 50 randomly selected plaques are shown as meanstandard deviation.

[0273] Thus, two BoHV-1tmv recombinants expressing BVDV type 1 or 2 E2 proteins separately have successfully been constructed.

Example 11: Recombinant BHV-1 Tmv that Express BRSV F and G Proteins

[0274] Recombinant BHV-1 tmv that express BRSV F and G were generated as illustrated in FIG. 3E-3J. First BRSV F and G insertion vectors were generated (FIG. 3F, 3I, 3J). Second, linearized pBHV-1 gECTUs9/BRSV F or G insertion vector DNA was transfected into host cells (Botur cells) with full-length BHV-1 tmv DNA (FIG. 3E).

[0275] Expression of BRSV F protein from recombinant BHV-1 tmv was determined by preparing an immunoblot of infected Botur cell lysates and staining the blot with a rabbit anti-BRSV F specific antibody (GenScript). Lysate from Botur cells infected with an unmodified BRSV Nebraska 236-652 strain served as positive control.

[0276] Expression of BRSV G protein from recombinant BHV-1 tmv was determined by preparing an immunoblot of infected MDBK cell lysates and staining the blot with anti-V5 specific monoclonal antibodies (Invitrogen, #R960-25). In a separate experiment, expression of BRSV G protein from recombinant BHV-1 tmv was determined by an immunoblot of infected Botur cell lysates using a rabbit anti-BRSV G specific antibody (GenScript).

[0277] Expression of expression of BRSV F or G proteins from the recombinant BHV-1 tmv vectors is illustrated in FIG. 24A-24C.

[0278] BRSV F protein is typically synthesized as 67 kDa precursor protein that is proteolytically cleaved at two separate furin cleavage sites, to yield a peptide of 48 kDa and peptide of 18 kDa. However, the coding region of the F protein inserted into the recombinant BHV-1 tmv vector had mutated Furin cleavage sites to prevent such proteolytic cleavage. In particular, modified BRSV F1 was generated by mutating some arginines in furin cleavage sites to alanines, while a modified BRSV F2 is generated by mutating all arginines and lysines in furin cleavage sites to alanines. Hence, the uncleaved F protein is visible in FIG. 24A.

[0279] BRSV-G is synthesized as 43 kDa precursor protein which is further processed in the Golgi to a 68-90 kDa mature protein G. The recombinant BRSV-G1 expressed from the recombinant BHV-1 tmv vector has modifications that are not present in the wild type G protein. In particular, the recombinant BRSV-G1 expressed from the recombinant BHV-1 tmv vector has all four cysteines in the cysteine noose replaced by serines (amino acids C173S, C176S, C182S and C186S; .sub.173SST.sub.176SEGNLA.sub.182SLSL.sub.186S; GenBank accession #AL49398.1). The recombinant BRSV-G2 expressed from the recombinant BHV-1 tmv vector has a complete deletion of the cysteine noose (i.e., a deletion of CSTCEGNLACLSLC (SEQ ID NO:48), GenBank accession #AL49398.1, amino acids 173 . . . 186).

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[0379] All patents and publications referenced or mentioned herein are indicative of the levels of skill of those skilled in the art to which the invention pertains, and each such referenced patent or publication is hereby specifically incorporated by reference to the same extent as if it had been incorporated by reference in its entirety individually or set forth herein in its entirety. Applicants reserve the right to physically incorporate into this specification any and all materials and information from any such cited patents or publications.

[0380] The following statements are intended to describe and summarize various embodiments of the invention according to the foregoing description in the specification.

Statements:

[0381] 1. A BoHV-1 recombinant vector comprising BoHV-1 tmv with at least one heterologous antigen from a respiratory RNA virus. [0382] 2. The BoHV-1 recombinant vector of statement 1, wherein the at least one antigen is from BoHV, BVDV, BRSV, or a combination thereof. [0383] 3. The BoHV-1 recombinant vector of statement 1 or 2, wherein the at least one antigen is BoHV-1, BVDV type 1, BVDV type 2, BRSV, or a combination thereof. [0384] 4. The BoHV-1 recombinant vector of statement 1, 2, or 3, wherein the at least one antigen is an E2 protein, BVDV Ems protein, BRSV F protein, BRSV G protein, or a combination thereof. [0385] 5. The BoHV-1 recombinant vector of statement 1-3 or 4, wherein at least heterologous antigen is inserted into a BoHV-1 tmv vector. [0386] 6. The BoHV-1 recombinant vector of statement 1-4 or 5, wherein at least heterologous antigen is inserted into a BoHV-1 tmv vector, where the BoHV-1 tmv vector has a deletion of a cytoplasmic tail of envelope glycoprotein gE (gE-CT), a BoHV-1 tmv deletion of an entire envelope protein, a BoHV-1 tmv deletion of envelope protein UL49.5 residues 30-32, a BoHV-1 tmv deletion of UL49.5 cytoplasmic tail residues 80-96, or a combination thereof. [0387] 7. The BoHV-1 recombinant vector of statement 1-5 or 6, wherein at least one heterologous antigen is a BVDV E2 antigen having at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 99.5% amino acid sequence identity to SEQ ID NO:2, 3, 4, 5, 7, 10, 12, 55, or 57. [0388] 8. The BoHV-1 recombinant vector of statement 1-6 or 7, wherein at least one heterologous antigen is a BVDV E2 antigen encoded by a nucleic acid segment with at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 99.5% nucleotide sequence identity to SEQ ID NO:6, 8, 9, 11, 13, 14, 56, or 58. [0389] 9. The BoHV-1 recombinant vector of statement 1-7 or 8, wherein at least one heterologous antigen is a BVDV Ems antigen having at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 99.5% amino acid sequence identity to SEQ ID NO:39 or 42. [0390] 10. The BoHV-1 recombinant vector of statement 1-8 or 9, wherein at least one heterologous antigen is a BVDV Ems antigen encoded by a nucleic acid segment with at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 99.5% nucleotide sequence identity to SEQ ID NO:40 or 42. [0391] 11. The BoHV-1 recombinant vector of statement 1-9 or 10, wherein at least one heterologous antigen is a BRSV F protein antigen having at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 99.5% amino acid sequence identity to SEQ ID NO:15, 16, 17, 20, 22, or 24. [0392] 12. The BoHV-1 recombinant vector of statement 1-10 or 11, wherein at least one heterologous antigen is a BRSV F protein antigen encoded by a nucleic acid segment with at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 99.5% nucleotide sequence identity to SEQ ID NO: 18, 19, 21, 23, or 25. [0393] 13. The BoHV-1 recombinant vector of statement 1-11 or 12, wherein at least one heterologous antigen is a BRSV G protein antigen having at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 99.5% amino acid sequence identity to SEQ ID NO:26, 28, 29, 31, 34, or 36. [0394] 14. The BoHV-1 recombinant vector of statement 1-12 or 13, wherein at least one heterologous antigen is a BRSV G protein antigen encoded by a nucleic acid segment with at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 99.5% nucleotide sequence identity to SEQ ID NO:30, 32, 33, 35, 37, or 38. [0395] 15. The BoHV-1 recombinant vector of statement 1-13 or 14, wherein at least one heterologous antigen is expressed from a heterologous promoter. [0396] 16. The BoHV-1 recombinant vector of statement 1-14 or 15, wherein at least one heterologous antigen is expressed from a heterologous promoter selected from a viral promoter, a bacterial promoter or a mammalian promoter. [0397] 17. The BoHV-1 recombinant vector of statement 1-15 or 16, wherein at least one heterologous antigen is expressed from a heterologous promoter selected from a strong, weak, or inducible promoter. [0398] 18. The BoHV-1 recombinant vector of statement 1-16 or 17, wherein at least one heterologous antigen is expressed from a heterologous promoter selected from a human elongation factor 1 promoter, a T7 promoter (e.g., optionally with the lac operator), a CaMV 35S promoter, a CaMV 19S promoter, a nos promoter, Adh1 promoter, sucrose synthase promoter, -tubulin promoter, ubiquitin promoter, actin promoter, cab, PEPCase promoter, the CCR promoter (cinnamoyl CoA:NADP oxidoreductase, EC 1.2.1.44, e.g., isolated from Lollium perenne, a baculovirus derived promoter, a p10 promoter, or a combination thereof. [0399] 19. The BoHV-1 recombinant vector of statement 1-17 or 18, wherein at least one heterologous antigen is expressed from a heterologous promoter that include one or more enhancer elements. [0400] 20. The BoHV-1 recombinant vector of statement 1-18 or 19, wherein at least one heterologous antigen is expressed from an elongation factor 1 promoter [0401] 21. The BoHV-1 recombinant vector of statement 1-19 or 20, wherein at least one heterologous antigen is expressed as a fusion protein. [0402] 22. The BoHV-1 recombinant vector of statement 1-20 or 21, wherein at least one heterologous antigen is expressed as a fusion protein with a fusion partner selected from a signal sequence, an epitope, a histidine tail, GM-CSF, or any combination thereof. [0403] 23. The BoHV-1 recombinant vector of statement 1-20 or 21, wherein at least one heterologous antigen is expressed as a fusion protein with a fusion partner selected from a gD signal sequence, a V5 epitope, a histidine tail comprising 2-10 histidine residues, GM-CSF, or any combination thereof. [0404] 24. The BoHV-1 recombinant vector of statement 1-22 or 23, wherein at least one heterologous antigen is encoded by a nucleic acid segment that includes a 3 polyA tail. [0405] 25. A composition comprising at least one BoHV-1 recombinant vector of statement 1-23 or 24. [0406] 26. A composition comprising at least two, or at least three, or at least four, or at least five of the BoHV-1 recombinant vectors of statement 1-23 or 24. [0407] 27. The composition of statement 25 or 26, further comprising a carrier. [0408] 28. The composition of statement 25, 26, or 27, further comprising a polypeptide or peptide. [0409] 29. The composition of statement 25-27 or 28, further comprising at least one antigenic polypeptide or peptide. [0410] 30. The composition of statement 25-28 or 29, further comprising at least one antigenic polypeptide or peptide selected from a M. hemolytica, Pasteurella maltocida, Histophilus somni or Mycoplasma bovis antigenic polypeptide or peptide. [0411] 31. The composition of statement 25-29 or 30, further comprising a GM-CSF polypeptide. [0412] 32. The composition of statement 25-30 or 31, further comprising an adjuvant. [0413] 33. The composition of statement 25-31 or 32, formulated for administration by an intranasal, subcutaneous, intravenous, intradermal, intramuscular, intraperitoneal, oral, nasal, or transdermal route. [0414] 34. The composition of statement 25-32 or 33, formulated for administration by an intranasal route. [0415] 35. A method comprising administering at least one BoHV-1 recombinant vector of statement 1-23 or 24 to a mammal. [0416] 36. The method of statement 35, which reduces the incidence or severity of respiratory symptoms in the mammal. [0417] 37. The method of statement 35 or 36, which reduces the incidence or severity of respiratory symptoms in the mammal. [0418] 38. The method of statement 35, 36, or 37, wherein the mammal is a bovine animal or an experimental animal. [0419] 39. The method of statement 35-37 or 38, which reduces the incidence or severity of respiratory symptoms of bovine respiratory disease complex (BRDC) in a bovine mammal to which the composition is administered. [0420] 40. The method of statement 35-38 or 39, which protects the mammal against respiratory disease. [0421] 41. The method of statement 35-39 or 40, which protects the mammal against bovine respiratory disease complex (BRDC). [0422] 42. A BoHV-1 recombinant vector comprising BoHV-1 gECTUs9/BVDV E2-1. [0423] 43. A BoHV-1 recombinant vector comprising BoHV-1 gECTUs9/BVDV E2-2.

[0424] The specific constructs, compositions, and methods described herein are representative of preferred embodiments and are exemplary and not intended as limitations on the scope of the invention. Other objects, aspects, and embodiments will occur to those skilled in the art upon consideration of this specification, and are encompassed within the spirit of the invention as defined by the scope of the claims. It will be readily apparent to one skilled in the art that varying substitutions and modifications may be made to the invention disclosed herein without departing from the scope and spirit of the invention. The invention illustratively described herein suitably may be practiced in the absence of any element or elements, or limitation or limitations, which is not specifically disclosed herein as essential. The methods and processes illustratively described herein suitably may be practiced in differing orders of steps, and the methods and processes are not necessarily restricted to the orders of steps indicated herein or in the claims.

[0425] The terms and expressions that have been employed are used as terms of description and not of limitation, and there is no intent in the use of such terms and expressions to exclude any equivalent of the features shown and described or portions thereof, but it is recognized that various modifications are possible within the scope of the invention as claimed. Thus, it will be understood that although the present invention has been specifically disclosed by preferred embodiments and optional features, modification and variation of the concepts herein disclosed may be resorted to by those skilled in the art, and that such modifications and variations are considered to be within the scope of this invention as defined by the appended claims and statements of the invention. Under no circumstances may the patent be interpreted to be limited to the specific examples or embodiments or methods specifically disclosed herein. Under no circumstances may the patent be interpreted to be limited by any statement made by any Examiner or any other official or employee of the Patent and Trademark Office unless such statement is specifically and without qualification or reservation expressly adopted in a responsive writing by Applicants.