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Definitions

• the present invention relates generally to avian vaccines against avian influenza (AI). More specifically, the present invention provides a recombinant turkey herpesvirus modified by the presence of the cDNA encoding the hemagglutinin (HA) protein of avian influenza virus under a promoter.
• AI avian influenza
• the present invention provides a recombinant turkey herpesvirus modified by the presence of the cDNA encoding the hemagglutinin (HA) protein of avian influenza virus under a promoter.
• Avian influenza is caused by avian influenza viruses that are classified in the family Orthomyxoviridae, genus Influenzavirus A.
• the genome of the avian influenza virus consists of eight segments of single-stranded, negative-sense RNA.
• the viral genome encodes ten proteins, of which eight proteins are structural proteins including HA and neuraminidase (NA), and two proteins are nonstructural.
• Influenza A viruses are divided into subtypes based on antigenicity of HA and NA proteins. There are 16 HA antigens and nine NA antigens recognized. HA is considered the major antigen that can elicit protective antibodies in birds.
• Influenza A viruses from poultry are categorized into two pathotypes based on their pathogenicity: highly pathogenic avian influenza (HPAI) viruses and low pathogenic avian influenza (LPAI) viruses.
• HPAI highly pathogenic avian influenza
• LPAI low pathogenic avian influenza
• Most avian influenza viruses are of low virulence, but a few viruses of H5 and H7 subtypes can cause severe systemic disease that results in high mortality.
• H5 and H7 avian influenza viruses are of high virulence, all H5 and H7 viruses are identified as notifiable avian influenza virus by World Organization for Animal Health (OIE) because of the risk of low virulent viruses increasing virulence by mutation.
• OEM World Organization for Animal Health
• the commercial fowlpox virus vectored recombinant AI vaccine contains the HA gene of the AI virus A/turkey/Ireland/1378/83 (H5N8) (J.R. Taylor et al., 1988, Vaccine, 6: 504- 508).
• H5N8 The commercial fowlpox virus vectored recombinant AI vaccine contains the HA gene of the AI virus A/turkey/Ireland/1378/83 (H5N8) (J.R. Taylor et al., 1988, Vaccine, 6: 504- 508).
• Several other experimental fowlpox vectored recombinant vaccines have been developed and shown to be efficacious against challenge with AI viruses in experimental conditions.
• Avian influenza virus genes contained in the fowlpox vectored recombinant vaccines include the HA gene from A/Chicken/Scotland/59 (H5N1) (CW.
• H5N1 A/Goose/Guangdond/3/96
• M. Mingxiao et al. fused the HA genes from H5N1 subtype and H7N1 subtype to form a single open frame and inserted into a recombinant fowlpox virus along with chicken Interleukin-18 (M. Mingxiao et al., 2006, Vaccine, 24: 4304-4311). Broad cross protection among the AI virus H5 subtypes has been observed.
• the fowlpox virus vectored recombinant AI vaccine and the inactivated whole AI vaccines for avian influenza H5 subtypes have been demonstrated to protect chickens against challenge with diverse H5 subtype AI viruses, of which deduced HA amino acid sequence similarities with the vaccines are as low as 87% (D.E. Swayne et al., 2000, Veterinary Microbiol., 74: 165-172).
• Next generation vaccines under development include recombinant Newcastle disease virus vaccines (D.E. Swayne et al., 2003, Avian Diseases, 47: 1047-1050; J. Veits et al., 2006, Proc. Natl. Acad. Sci. U.S.A., 103:8197-8202; M. Park et al., 2006, Proc. Natl. Acad. Sci. U.S.A., 103:8203-8208; and J. Ge et al., 2007, J. Virol. 81 : 150-158), recombinant infectious laryngotracheitis virus vaccines (D. Luschow et al., 2001, Vaccine 19: 4249-4259 and J.
• Newcastle disease virus vaccines D.E. Swayne et al., 2003, Avian Diseases, 47: 1047-1050
• J. Veits et al. 2006, Proc. Natl. Acad. Sci. U.S.A., 103
• the recombinant Newcastle disease virus vaccines were able to confer partial to semi-complete protection against AI challenge in specific pathogen free chickens, their efficacy in chickens with maternal antibodies to the vector viruses or AI, or in chickens with previous infection or vaccination with the vector viruses remains to be demonstrated.
• the DNA vaccines have also shown to provide protective immunity in chickens, but they require at least two vaccinations and individual administration to each chicken.
• the baculovirus-expressed subunit vaccines also require individual administration to each chicken.
• Turkey herpesvirus (HVT), Marek's disease virus (MDV) serotype-3 has been used as a vector to express antigens from avian pathogens.
• Wild type HVT or recombinant HVT can be administered to either the late developmental stage of embryos via the in ovo route or one- day-old chicks via the subcutaneous route at hatcheries.
• Recombinant, cell-associated HVT vaccines after inoculation into embryos or one-day-old chicks with maternal antibodies to inserted antigens, are demonstrated to be able to overcome influences of maternal antibodies and confer protective immunity to chickens as maternal antibodies wane (U.S. Pat. No. 6,764,684 and U.S. Pat. No. 6,866,852).
• HVT recombinant HVT
• HVT may be considered an excellent vector for avian pathogens.
• HVT may be considered an excellent vector for avian pathogens.
• Claim 15 of U.S. Pat. No. 5,853,733 describes the recombinant HVT comprising a polypeptide gene of AI virus inserted within a region which corresponds to an EcoRI #9 fragment of the HVT genome, there is no actual example of constructing recombinant HVT with avian influenza antigens.
• U.S. Pat. No. 6,225,111 describes construction of recombinant equine herpesviruses containing the HA gene of equine influenza virus, but there is no data about vaccine efficacy of these recombinants.
• the present invention provides a recombinant HVT modified by the presence of the cDNA encoding the HA protein of avian influenza virus under a promoter.
• the recombinant HVT is able to elicit a serological response that is easily detected by the HI assay but not by commercially available diagnostic ELISA kits. This feature of the recombinant virus enables easy differentiation between vaccination and field infection.
• a poultry vaccine comprising the recombinant HVT is also provided. The present invention is described below in more detail.
• the hemagglutinin gene may be obtained from any subtype or any strain of avian influenza virus.
• the HA gene is obtained from an avian influenza virus of the H5 subtype. More preferably, the HA gene is obtained from avian influenza virus of the H5N9 subtype. Most preferably, the HA gene is obtained from the avian influenza virus A/Turkey/Wisconsin/68 (H5N9) strain.
• a nucleotide sequence of the HA gene from the A/Turkey/Wisconsin/68 (H5N9) strain is shown in SEQ ID NO: 1.
• SEQ ID NO: 1 differs from the published nucleotide sequence of the HA gene of the A/Turkey/Wisconsin/68 (H5N9) strain (M. Garcia et al., 1997, Virus Res. 51 : 115-124, GenBank Accession# U79456) by several bases. These differences are probably due to the genetically unstable nature of avian influenza viruses, which have an RNA genome. Therefore, the sequence shown in SEQ ID NO: 1 is only an example and the present invention should not be restricted to the sequence.
• a regulatory DNA sequence which is referred to here as a promoter, is included in order to control transcription of the HA gene, and thereby to control expression of the HA gene (generation of the HA protein).
• a promoter When transcription and thereby expression of a gene is controlled by a promoter, the gene is considered under control of the promoter, hi the present invention, the HA gene is under control of the cytomegalovirus immediate early promoter (CMV promoter).
• CMV promoter cytomegalovirus immediate early promoter
• recombinant HVT with the HA gene in combination with the CMV promoter was capable of conferring higher and more uniform serology titers by HI in chickens than the recombinant HVT with other promoters such as the chicken beta-actin promoter (T.A. Kost et al., 1983, Nucleic Acids Res. 11 :8287-8301) and a modified chicken beta-actin promoter (U.S. Pat. No. 6,866,852).
• a nucleotide sequence of the CMV promoter is described in the literature (M. Boshart et al., 1985, Cell 41 : 521-530, GenBank Accession# K03104).
• the nucleotide sequence of a promoter does not have to be identical to the sequence in the literature.
• the CMV promoter the sequence of which is shown in SEQ ID NO: 3, is slightly modified from the original sequence by the inventors, but was demonstrated to express the HA gene effectively.
• Turkey herpesvirus is a double-stranded linear DNA virus in the Herpesviridae family and Alphaherpesvirinae subfamily.
• HVT is ubiquitous and non-oncogenic in domestic turkeys and it is classified as serotype 3 of Marek's disease virus.
• Vaccination of chickens with HVT has been extensively conducted to prevent Marek's disease in chickens.
• any HVT can be used in the present invention.
• the following HVT strains, FC126, PB-THVl, H-2, YT-7, WTHV-I , and HPRS-26 are suitable for the backbone virus.
• the FC 126 strain is favorable for use in the present invention.
• the HA gene and the CMV promoter are inserted into an HVT DNA genome.
• the HA gene and the CMV promoter are inserted into a region in the HVT genome that is not essential for virus growth, which is referred to here as a non-essential region.
• a non-essential region may be defined as a region where modification or insertion of a foreign gene does not prevent the virus from replicating successfully in vitro or in vivo.
• the HA gene and the CMV promoter can be inserted into, but not limited to UL43 (WO 89/01040), US2 (WO 93/25665) or inter-ORF region between UL44 and UL46 (WO 99/18215). Most preferably, the HA gene and the CMV promoter are inserted into the inter-ORF region between UL45 and UL46.
• a non-essential region may be newly identified by the following general procedure.
• HVT DNA fragments of appropriate lengths are cloned into an E. coli plasmid and physically mapped by restriction enzyme analysis.
• a gene cassette consisting of a promoter and a marker gene is inserted into an appropriate restriction site of the cloned DNA fragment resulting in a homology plasmid. If homologous recombination with the obtained homology plasmid results in a recombinant virus expressing the inserted marker gene and if the virus is stable in vitro and in vivo, the originally selected DNA fragment should be a non-essential region suitable for HA gene and CMV promoter insertion.
• any known method of generating recombinant HVT is applicable.
• a typical example is as follows. (1) First, as described above, a recombinant plasmid that contains a non-essential region of the HVT genome is constructed. Then, preferably with a promoter at the 5' terminus and a polyadenylation signal at the 3' terminus, the HA gene is inserted into the non-essential region to generate a homology plasmid. (2) The homology plasmid is transfected into chicken embryo fibroblast (CEF) cells infected with parent HVT or co-transfected into CEF cells with infectious HVT genomic DNA. Transfection can be performed by any known method.
• CEF chicken embryo fibroblast
• the transfected CEF cells are planted on tissue culture plates and incubated until virus plaques become visible.
• the identifiable plaques include recombinant virus as well as parent wild-type virus.
• the recombinant virus may be purified from wild type virus by any known method to detect expression of inserted foreign genes.
• the recombinant HVT containing the HA gene in the present invention may be used as a bivalent vaccine against AI and Marek's disease or as a monovalent vaccine against AI.
• the vaccine consisting mainly of the recombinant HVT in the present invention, may also include avian cells, ingredients of culture media, buffers such as a phosphate buffer and HEPES buffer, and/or adjuvants such as cytokines and CpG oligodeoxynucleotide. As long as not pharmacologically detrimental, the vaccine may contain any ingredients such as preservatives.
• the vaccine of the present invention can be used in a mixture with any recombinant or non-recombinant viruses such as the MDV serotype 1 or serotype 2 vaccine strains.
• the recombinant HVT may be inoculated into permissive culture cells such as CEF cells and grown to an appropriate titer. Then, the cells are removed from tissue culture plates or roller bottles with cell scrapers or by trypsin treatment and collected by centrifugation. The pelleted cells are then suspended in culture medium containing dimethyl sulfoxide, frozen slowly, and then stored in liquid nitrogen.
• the recombinant HVT may be released from the infected cells by disrupting the cells in diluents containing stabilizers such as sucrose and bovine albumin. These released HVT is called cell-free HVT. Cell-free HVT may be lyophilized and stored at 4 0 C.
• the bivalent recombinant HVT vaccine can be administered to chickens by any known method of inoculating Marek's disease vaccine.
• the vaccine of the present invention is diluted to give lO'-lO 5 , or more favorably 10 2 -10 4 plaque forming units (pfu) per dose with a diluent containing buffer components, sugars, and dye.
• the diluted vaccine may be inoculated subcutaneously behind the neck of one-day-old chicks or into embryonating eggs via the in ovo route with syringes or with any apparatus for injection.
• the present avian bivalent vaccine is able to confer serological titer by HI of more than 50 (geometric mean titer) in groups of vaccinated chickens by 5 weeks post inoculation, when using four hemagglutination units of an inactivated avian influenza virus homologous H5 subtype antigen for the HI tests. Also the bivalent vaccine in the present invention provided excellent protection against lethal challenge with highly pathogenic avian influenza virus (H5N1).
• Fig. 1 Construction of the plasmid pGICMVpA Fig. 2 Construction of the homology plasmid p45CMVH5Wis68 Fig. 3 Construction of the plasmid pGIBacpA2nd Fig. 4 Construction of the homology plasmid p45BacH5Wis68 Fig. 5 Construction of the homology plasmid p45PecH5Wis68 Fig.
• FIG. 9 ELISA titers in chickens vaccinated with the recombinant turkey herpesvirus with hemagglutinin gene using a commercial ELISA kit (SYNBIOTICS, PROFLOK AJV
• the avian influenza virus A/Turkey/Wisconsin/68 (H5N9) strain was propagated in the allantoic sac of specific pathogen free embryonating chicken eggs.
• Total genomic RNA from the A/Turkey/Wisconsin/68 virus was extracted using RNEASY MINI KIT (QIAGEN, Cat# 74104).
• First-strand cDNA was synthesized with SUPERSCRIPT FIRST-STRAND System for RT-PCR (Invitrogen, Cat# 11904-018).
• the HA gene was amplified by polymerase chain reaction (PCR) with PFUULTRA HIGH FIDELITY DNA Polymerase (STRATAGENE, Cat# 600380) and PCR primers.
• PCR primers BamHA-F primer (SEQ ID NO: 4) and SaIHA-R primer (SEQ ED NO: 5), anneal to the start and stop sequences of the HA gene and each primer contains a sequence at the 5' ends for a restriction enzyme, BamHI or Sail, respectively.
• Taq polymerase PROMEGA, Cat# M2665 was added to the PCR mixture to add 3' A-overhangs to the PCR products.
• BamHA-F primer (SEQ ID NO: 4) 5'-TGACGGATCCATGGAAAGAATAGTGATTG-S ' SaIHA-R primer (SEQ ED NO: 5) 5 '-CTGACAGTCGACCTAGATGCAAATTCTGC-S '
• the amplified 1.8 kilobase (kb) HA cDNA was inserted into PCR2.1-TOPO vector (ENVITROGEN, Cat# K4500-01), resulting in pCR2.1-H5Wis68.
• Nucleotide sequences of the HA genes in a few clones of the plasmid pCR2.1-H5Wis68 and the PCR product were determined using ABI PRISM 3730XL DNA Analyzer (APPLIED BIOSYSTEMS) with six primers; BamHA-F primer (SEQ ED NO: 4), SaIHA-R primer (SEQ ID NO: 5), Ml 3 Forward primer (SEQ ED NO: 6), Ml 3 Reverse primer (SEQ ID NO: 7), HA-F primer (SEQ ED NO: 8), and HA-R primer (SEQ ED NO: 9).
• the sequences in the clones of the plasmid pCR2.1-H5Wis68 were identical to each other and to the sequence of the PCR product.
• the deduced amino acid sequence was different from the reported sequence of A/Turkey/Wisconsin/68 (H5N9) (M. Garcia et al., 1997, Virus Res. 51 : 115-124, GenBank Accession# U79456) by four amino acids, the amino acids we obtained were the same as the amino acids of a majority of H5 subtype HA proteins.
• the nucleotide sequence and the deduced amino acid sequence of the HA gene obtained from A/Turkey/Wisconsin/68 (H5N9) are shown in SEQ ID NO: 1 and SEQ ID NO: 2.
• three promoters the CMV promoter, the chicken beta-actin promoter (Bac promoter), and a modified chicken beta-actin promoter (Pec promoter), were used to control expression of the HA gene of the AI virus A/Turkey/Wisconsin/68 (H5N9) strain.
• homology plasmids with the HA gene and one of the promoters were constructed and then recombinant turkey herpesviruses were generated using the homology plasmids.
• the recombinant turkey herpesviruses with different promoters were compared for capabilities of conferring serological titers against AI in chickens as shown in EXAMPLE 6.
• a recombinant HVT with the CMV promoter is presented here as an example and recombinant viruses with the Bac promoter or the Pec promoter are presented here as comparative examples.
• a list of the homology plasmids and the recombinant turkey herpesviruses constructed in the present invention is shown in TABLE 1. Table 1. A list of homology plasmids and recombinant turkey herpesviruses
• the CMV promoter was obtained from pBK-CMV (STRATAGENE, Cat. #212209). Three BgII restriction enzyme sites in the CMV promoter were disrupted for ease of the plasmid construction process by PCR in vitro mutagenesis using four pairs of primers.
• the primer pairs were PrCMVl (SEQ ID NO: 10) and PrCMV3 (SEQ ID NO: 12), PrCMV4 (SEQ ID NO: 13) and PrCMV5 (SEQ ID NO: 14), PrCMV6 (SEQ ID NO: 15) and PrCMV2' (SEQ ID NO: 11), and PrCMVoI (SEQ ID NO: 16) and PrCMVRl (SEQ ID NO: 17).
• PCR reactions were conducted separately using each pair of primers and pBK-CMV as a template. Then four PCR products were combined and used as a template for the secondary PCR with primers PrCMVl and PrCMVRl, yielding the 604 bp fragment with a modified CMV promoter sequence.
• the nucleotide sequence of the CMV promoter used to express HA gene is provided in SEQ ID. NO. 3.
• the CMV promoter fragment was digested with Pstl and Xbal and inserted into Pstl and Xbal digested pUCl ⁇ polyASfi (U.S. Pat. No. 6,866,852), resulting in pGICMV(-).
• the SV40 polyA signal was obtained from pBK-CMV by PCR using primers PolyA-SalKpn (SEQ ID NO: 18) and PolyA-SfiF2 (SEQ ID NO: 19).
• the PCR fragment containing SV40 polyA signal was digested with Sail and Sfil and ligated to pGICMV(-) digested with Sail and Sfil resulting in pGICMVpA (FIGURE 1).
• PrCMVl SEQ ID NO: 10 5 '-GGGCTGCAGAGTTATTAATAGTAATCAATT-S '
• PrCMV3 (SEQ ID NO: 12) 5 '-GGGTCGTTGGGCGGTCAGCCGGCGG-S '
• PrCMV4 (SEQ ID NO: 13) 5 '-CTTACGGTAAATGGCCCGCCGGCTG-S '
• PrCMV ⁇ (SEQ ID NO: 15) 5 '-TATTTACGGTAAACTGCCCATTGGC-S '
• PrCMVoI (SEQ ID NO: 16) 5 '-ACGTCAATGACGGTAAATGGCGCGCCTGGC-S '
• PrCMVRl (SEQ ID NO: 17) 5 '-CGTCTAGAGGATCTGACGGTTCACTAAACC-S '
• PolyA-SalKpn (SEQ ID NO: 18) 5 '-TGTGGTACCGTCGACGATTCACAGTCCCAAGGC-
• PolyA-SfiF2 (SEQ ID NO: 19) 5'- CTTGGCCTTATTGGCCTAAGATACATTGATGAG -3'
• the CMV promoter and the SV40 polyA signal (940 bp) were excised from pGICMVpA by BgII and ligated into SfII digested p45/46Sfi (US 6,866,852), resulting in p45/46CMVpA.
• the HA gene from A/Turkey/Wisconsin/68 H5N9 was excised from pCR2.1-H5Wis68 using Sail and BamHI.
• the 1701 bp HA gene was inserted into p45/46CMVpA digested with Sail and BamHI, resulting in P 45CMVH5Wis68 (FIGURE 2).
• the plasmid p45CMVH5Wis68 was used as a homology plasmid to generate recombinant turkey herpesvirus.
• the Bac promoter was obtained by PCR using cellular DNA of CEF cells as a template. PrBacl (SEQ ID NO: 18) and PrBac2' (SEQ ID NO: 19) were the primer set used for PCR. An obtained 1.5-kilobase DNA fragment was digested with Pstl and Xbal and inserted into Pstl and Xbal digested pUCl ⁇ polyASfi, resulting in pGIBac2.
• the SV40 polyA signal obtained PCR using primers PolyA-SalKpn (SEQ ID NO: 18) and PolyA-SfiF2 (SEQ ID NO: 19) was digested with Sail and Sfil and ligated to pGDBac2 digested with Sail and Sfil resulting in pGIBacpA2nd (FIGURE 3).
• PrBac2' (SEQ ID NO: 21) 5'- GCTCTAGAGGCGTGGAGCTTGGGGGCTGCGGAGGAACAGAGAAGGGAAG-S'
• the Bac promoter and the SV40 polyA signal (1866 bp) were excised from pGIBacpA2nd by BgII and ligated into Sfil digested p45/46Sf ⁇ , resulting in p45/46BacpA2nd.
• the HA gene of A/Turkey/Wisconsin/68 (H5N9) excised from pCR2.1-H5Wis68 using Sail and BamHI was inserted into p45/46BacpA2nd digested with Sail and BamHI, resulting in P 45BacH5Wis68 (FIGURE 4).
• Pec promoter Construction of the Pec promoter is described in U.S. Pat. No. 6,866,852.
• the Pec promoter was synthesized by fusing a part of the chicken beta-actin promoter with the enhancer region of the CMV promoter.
• the Pec promoter was excise from pGIPec (U.S. Pat. No. 6,866,852) with Pstl and BamHI and inserted into Pstl and BamHI digested p45/46BacpA2nd described in EXAMPLE 2-4, resulting in p45/46PecpA2nd.
• H5N9 A/Turkey/Wisconsin/68 excised from pCR2.1-H5Wis68 using Sail and BamHI was inserted into p45/46PecpA2nd digested with Sail and BamHI, resulting in p45PecH5Wis68 (FIGURE 5).
• Viral DNA of the HVT FC 126 strain was prepared as described by Morgan et al. (Avian Diseases, 1990, 34:345-351).
• 10 7 secondary chicken embryo fibroblast (CEF) cells were suspended in Saline G (0.14 M NaCl, 0.5 mM KCl, 1.1 mM Na 2 HPO 4 , 1.5 mM NaH 2 PO 4 , 0.5 mM MgCl 2 , and 0.011% glucose) and co-transfected with HVT viral DNA and 5 to 25 ⁇ g of the homology plasmid, p45CMVH5Wis68, p45BacH5Wis68, or p45PecH5Wis68 by electroporation. Electroporation was performed using BIO-RAD GENE PULSER. Transfected cells were incubated for 10 minutes at room temperature and transferred to wells of 96-well plates.
• the cells were detached from the plates by trypsinization, transferred equally to two 96-well plates with secondary CEF and incubated for 3 to 4 days until plaques were observed. Screening was conducted by the black plaque assay, staining only plaques expressing HA protein. Briefly, one of the two plates was fixed with methanol: acetone mixture (1 :2) and incubated with chicken anti-HA antiserum.
• rHVT/CMVH5Wis68 Purified recombinant virus with the HA gene under the CMV promoter was designated as rHVT/CMVH5Wis68 (the present invention).
• Recombinant viruses with the Bac promoter or the Pec promoter were designated as rHVT/BacH5Wis68 and rHVT/PecH5Wis68, respectively (comparative examples).
• Chicken embryo fibroblast cells in a 100-mm dish that were infected with the recombinant virus, rHVT/CMVH5Wis68 or the HVT FC 126 parent strain were used in the Southern blot analysis to confirm the insertion of the HA gene in the desired insertion site.
• the cells were collected by a cell scraper and by centrifugation at 913 x g for 5 minutes.
• the harvested cells were washed with phosphate buffered saline (PBS) and resuspended in 1.0 milliliter (ml) of lysis buffer (0.5% TRITON X-100, 100 mM 2-mercaptethanol, and 20 mM EDTA in PBS).
• the cell suspension was vortexed for a total of 30 seconds and incubated for 15 minutes at room temperature.
• Cell nucleus and cell debris were removed by centrifuging at 2,060 x g for 5 minutes and the supernatant was transferred to a 1.5-ml tube.
• Viruses were collected by centrifugation at 20,800 x g for 20 minutes at 4 0 C.
• the pellet was suspended in 0.33 ml of a nuclease solution (12.5 mM Tris-Cl (pH7.5), 1 ⁇ g/ml DNase I and 1 ⁇ g/ml RNase A) and incubated at 37 0 C for 30 minutes.
• SDS-protease solution 50 mM EDTA, 5% SDS, 0.5mg/ml protease K, and 25 mM 2-mercaptoethanol
• 50 mM EDTA, 5% SDS, 0.5mg/ml protease K, and 25 mM 2-mercaptoethanol was added to the virus suspension and incubated at 55 0 C for 30 minutes to disrupt virus envelopes.
• Phenol chloroform extraction was conducted twice and DNA was precipitated by adding 2.5 volume of cold 100% ethanol and NaCl at a final concentration of 0.16 M. After centrifuging at 20,800 x g for 30 mininutes at 4 0 C, the pellet was washed with 70% ethanol and air-dried. The pellet was dissolved in TE buffer (10 mM Tris-Cl (pH8.0), and 1 mM EDTA).
• the viral DNA in TE buffer and the homology plasmid were digested with Xhol, BamHI and Spel and separated by agarose gel electrophoresis using 0.6% agarose gel. DNA fragments on the gel were transferred to a BIODYNE A nylon membrane (PALL, Cat# BNXF3R). The membrane was hybridized with either Digoxigenin (DIG)- labeled HA probe or DIG-labeled IS45/46 probe.
• DIG Digoxigenin
• the DIG-labeled HA probe and the IS45/46 probe were prepared with PCR DIG Probe Synthesis Kit (ROCHE APPLIED SCIENCE, Cat# 11636090910) using primers HAl-P-F (SEQ ID NO: 22) and HAl-P-R (SEQ ID NO: 23) and primers 45/46-F (SEQ ID NO: 24) and 45/46-R (SEQ ID NO: 25), respectively.
• HAl-P-F (SEQ ID NO: 22) 5 '-GGGGGTGGCAAGGAATG-S ' HAl-P-R (SEQ ID NO: 23) 5'-GCTAGGGAACTCGCCACTGT-S ' 45/46-F-B (SEQ ID NO: 24) 5 '-TAGCGGCACGGAAACAGATAGAGA-S ' 45/46-R-B (SEQ ID NO: 25) 5 '-TGGCGATACGGTTCCTGGTTTGAC ⁇ '
• the membrane was washed with 2 X SSC solution at room temperature and then with 0.5 X SSC solution at 68 0 C.
• the membrane was blocked and incubated with anti- Digoxigenin-AP, Fab fragments (ROCHE APPLIED SCIENCE, Cat# 11093274910) for 30 minutes at room temperature.
• maleic acid washing buffer 0.1 M maleic acid, 0.15 M NaCl, and 0.3% Tween20, pH 7.5
• DNA bands that were hybridized with the probes were visualized by incubating the membrane with BCIP/NBT solution.
• the HA probe hybridized with 3.6 kb bands in the recombinant virus DNA and the homology plasmid, while no bands were detected with the HVT parent.
• the IS45/46 probe hybridized with 4.9 kb and 0.8 kb bands in the recombinant DNA and the homology plasmid, and with 2.3 kb band in the HVT parent.
• the HA probe hybridized with 3.6 kb bands in the recombinant virus DNA and the homology plasmid, while no bands were detected with the HVT parent.
• the IS45/46 probe hybridized with 3.6 kb and 1.2 kb bands in the recombinant DNA and the homology plasmid, and with 2.3 kb band in the HVT parent.
• the recombinant viruses were passed 20 times blindly in CEF cells. After the 20 passages, the viruses were analyzed by the Southern blot analysis as described in EXAMPLE 4.1. Bands detected in DNA isolated from the virus after 20 passages were identical to the bands described in EXAMPLE 4.1, demonstrating that the recombinant viruses were stable even after 20 passages.
• EXAMPLE 5 HA Protein Expression by Recombinant HVT
• HA protein by the recombinant viruses, rHVT/CMVH5Wis68, rHVT/BacH5Wis68, and rHVT/PecH5Wis68, was confirmed by the black plaque assay and the Western blot assay. Procedures for the black plaque assay are described in EXAMPLE 3. The western blot was conducted using CEF cells infected with the recombinant viruses and chicken anti-HA antiserum. Briefly, CEF cells in 100-mm dishes were infected with one of the recombinant viruses or the parent HVT FC 126 strain at a multiplicity of infection of approximately 0.01.
• the membrane was incubated with alkaline phosphatase-conjugated anti-chicken IgG Fc antibody (BETHYL, Cat# A30-104AP). Protein bound with chicken anti-HA antiserum was visualized by adding BCIP/NBT solution. As shown in FIGURE 3, a protein band of 74 kilodaltons (kDa) was observed only in the lane with the recombinant virus infected cells, which was the expected size of the non-processed HA protein.
• alkaline phosphatase-conjugated anti-chicken IgG Fc antibody BETHYL, Cat# A30-104AP
• EXAMPLE 6 Serological Evaluation of Chickens Inoculated with Recombinant HVT Serological responses against AI in chickens that were vaccinated with the recombinant viruses, rHVT/CMVH5Wis68, rHVT/PecH5Wis68, and rHVT/BacH5Wis68, were evaluated.
• One-day-old specific pathogen free chicks (SPAFAS, Flock T-IO) were vaccinated subcutaneously with one of the recombinant viruses.
• Groups 1 and 2 were inoculated with 1638 pfu per dose (0.2 ml) and 375 pfu per dose of rHVT/CMVH5Wis68, respectively (TABLE 2).
• Groups 3 and 4 contained chickens vaccinated with 2800 pfu (Group 3) or 550 pfu (Group4) of rHVT/PecH5Wis68. Groups 5 and 6 were inoculated with 4350 pfu and 720 pfu per dose of rHVT/BacH5Wis68, respectively. A group of chickens (Group 8) were held as non-inoculated negative controls. Another group of chickens (Group 7) was vaccinated subcutaneously with inactivated A/Turkey/Wisconsin/68 (H5N9) vaccine at three weeks old as an inactivated vaccine control.
• Chickens were bled between 3 to 7 weeks old and obtained sera were evaluated by the AI HI tests and AIV ELISA tests.
• the AI HI tests were conducted using four hemagglutination units of an inactivated avian influenza virus homologous antigen of the A/Turkey/Wisconsin/68 (H5N9) strain, the HA gene of which was used in the recombinant viruses, as described by D. E. Swayne et al (D.E. Swayne et al., 1998, Avian Influenza. In: A Laboratory Manual for the Isolation and Identification of Avian Pathogens, 150-155).
• the number of the hemagglutination units in the inactivated A/Turkey/Wisconsin/68 (H5N9) antigen was determined as the highest dilution of the antigen giving complete agglutination, and the antigen was diluted to contain four hemagglutination units in 25 ⁇ l.
• the sera were initially diluted 1 :5 and then serially diluted by two fold across the plates with phosphate buffered saline (PBS) to contain 25 ⁇ l per well.
• PBS phosphate buffered saline
• HI titers are the highest dilution of the sera exhibiting inhibition of hemagglutination. HI titers of equal to or more than 10 were considered positive.
• the ELISA tests were conducted using two commercial AJV ELISA kits (IDEXX Laboratories, FLOCKCHEK AIV and SYNBIOTICS, PROFLOK ATV Ab test kit) that are available in the United States.
• the recombinant HVT with the HA gene in combination with the CMV promoter provided vaccinated chickens with higher and more uniform serology titers by HI than the recombinant HVT with the Bac promoter (rHVT/BacH5Wis68) and the recombinant HVT with the Pec promoter (rHVT/PecH5Wis68), which are presented here as comparative examples.
• the sera collected from chickens vaccinated from rHVT/CMVH5Wis68 were negative by commercially available AIV ELISA kits although the sera were highly positive by the AI HI tests, thus enabling easy differentiation between reaction from vaccination and field infection.
• a group of chickens (Group 4) were held as non- inoculated negative controls. Chickens were bled between 2 to 7 weeks old and obtained sera were evaluated by the AI HI tests and AIV ELISA tests. The AI HI tests were conducted using four HA units of an inactivated A/Turkey/Wisconsin/68 (H5N9) antigen as described by D. E. Swayne et al (D. E. Swayne et al., 1998, Avian Influenza. In: A Laboratory Manual for the Isolation and Identification of Avian Pathogens, 150-155).
• rHVT/CMVH5Wis68 by subcutaneous administration (Group 2) again showed excellent potency as in the first trial.
• HI titers in this group reached 100 (geometric mean titer) by three weeks post vaccination and high levels of HI titers were maintained through seven weeks post vaccination.
• rHVT/CMVH5Wis68 (Group 1) is as potent as subcutaneous administration.
• HI titers in Group 1 were very similar to those in Group 2.
• Lys lie GIn lie He Pro Arg Asn Ser Trp Ser Asn His Asp Ala Ser 130 135 140

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