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WO1992003554A1 - Vaccin contre le virus de la laryngotracheite infectieuse - Google Patents

Vaccin contre le virus de la laryngotracheite infectieuse Download PDF

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WO1992003554A1
WO1992003554A1 PCT/AU1991/000383 AU9100383W WO9203554A1 WO 1992003554 A1 WO1992003554 A1 WO 1992003554A1 AU 9100383 W AU9100383 W AU 9100383W WO 9203554 A1 WO9203554 A1 WO 9203554A1
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iltv
glycoprotein
virus
recombinant
glycoproteins
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PCT/AU1991/000383
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Michael George Sheppard
Christopher Prideaux
Michael Johnson
Kevin John Fahey
Jennifer Joy York
Kritaya Kongsuwan
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Arthur Webster Pty. Ltd.
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Publication of WO1992003554A1 publication Critical patent/WO1992003554A1/fr

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    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/85—Vectors or expression systems specially adapted for eukaryotic hosts for animal cells
    • C12N15/86—Viral vectors
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    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/005—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from viruses
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    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/85—Vectors or expression systems specially adapted for eukaryotic hosts for animal cells
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    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
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    • C12N2710/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA dsDNA viruses
    • C12N2710/00011—Details
    • C12N2710/16011—Herpesviridae
    • C12N2710/16041—Use of virus, viral particle or viral elements as a vector
    • C12N2710/16043—Use of virus, viral particle or viral elements as a vector viral genome or elements thereof as genetic vector
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    • C12N2830/00—Vector systems having a special element relevant for transcription
    • C12N2830/60—Vector systems having a special element relevant for transcription from viruses

Definitions

  • This invention relates to the use of specific glycoproteins of infectious laryngotracheitis virus (ILTV), which are major immunogens in chickens, in vaccines against infectious laryngotracheitis (ILT).
  • ILTV infectious laryngotracheitis virus
  • the invention also relates to the use of ILTV as a delivery vector for heterologous genes inserted into the glycoprotein or other region(s) of the ILTV genome where they are expressed by the homologous ILTV promoters or by heterologous promoters such as other herpesvirus promoters.
  • Infectious laryngotracheitis virus is a herpesvirus belonging to the alphaherpesvirinae subfamily (Gallid herpesvirus 1; Roizman et.al, 1981) which causes an acute upper respiratory tract infection in chickens.
  • the disease is found worldwide and sporadic outbreaks occur in which the severity of clinical symptoms may vary considerably. As outbreaks can result in mortalities of 10-40% and reduced egg production, the disease is of considerable importance to the intensive poultry industry. In recent years, a milder form of infectious laryngotracheitis has become widespread in England (Curtis and Wallis, 1983).
  • Naive chickens can be protected against challenge infection by the transfer of immune lymphoid cells (Fahey et.al, 1984), while bursectomised chickens that are unable to synthesise specific antibodies are protected against a challenge infection by vaccination (Fahey and York, 1990).
  • the viral glycoproteins produced in cells infected with either vaccine strain or virulent isolates of ILTV have been identified by in vitro labelling using [ 14 C] glucosamine and [ 14 C] mannose (York et.al, 1987).
  • Chicken antisera to the vaccine strain and to a virulent isolate, and rabbit antisera to the vaccine strain immunoprecipitated four major viral glycoproteins of 205, 115, 90 and 60K moLwt. Additional glycoprotein bands were recognised by immune chicken and rabbit sera in Western blotting using a glycoprotein fraction purified from extracts of virus infected cells.
  • Two antigenically distinct families of ILT viral glycoproteins have been defined by monoclonal antibodies; the 205 K complex of glycoproteins (205, 115 and 90K glycoproteins) and the 60K glycoproteins.
  • a non- infectious subunit vaccine against ILTV which comprises as active immunogen at least one glycoprotein of ILTV, or an immunogenic peptide derived therefrom, together with, if desired, an adjuvant.
  • die vaccine may comprise a non-infectious subunit vaccine containing one or more glycoproteins of ILTV which have been obtained by isolation from virus-infected cells, or by synthetic methods, particularly by recombinant DNA techniques, or from transformed cell cultures.
  • the vaccine may be in the form of a recombinant live virus vector having inserted therein a nucleotide sequence coding for at least one glycoprotein of ILTV or an immunogenic peptide derived therefrom.
  • the active immimogen in the vaccine is selected from die group consisting of the 205K complex of glycoproteins and the 60K glycoprotein of ILTV.
  • the invention provides a method for protecting chickens and other poultry against ILTV, which method comprises administering die vaccine described above to said chickens or other poultry
  • die vaccine described above to said chickens or other poultry
  • a subunit vaccine containing essentially the glycoproteins of the 205K complex protects 100% of the chickens.
  • the vaccine according to this invention may comprise an immunogenic peptide derived from a glycoprotein of ILTV, for example, by recombinant DNA techniques or chemical synthesis.
  • a suitable immunogenic peptide may be derived so tiiat it comprises all or at least the major immunogenic determinants of a glycoprotein of ILTV and dius exhibits the same or similar immunogenicity.
  • the glycoprotein(s) may also be coupled to a carrier molecule to increase immunogenicity and hence efficacy as a vaccine.
  • the non-infectious subunit vaccine of this invention comprises an adjuvant.
  • the vaccine may, for example, be delivered in an aqueous-mineral oil emulsion, such as an emulsion achieved by using an oil-phase emulsifier (e.g. Tween 80). Additional adjuvants may also be included if required, for example Al OH 3 , saponin or a derivative of muramyl dipeptide.
  • die vaccine of this invention may be in the form of a live recombinant viral vaccine which contains the nucleotide sequence (or sequences) coding for one or more of the immunogenic ILTV glycoproteins disclosed herein, or an immunogenic peptide derived therefrom.
  • such a live recombinant vaccine will induce protective immunity against ILT in avian species.
  • a live recombinant vaccine may comprise fowlpox virus, avian adenovirus or other avian virus expressing one or more nucleotide sequences for ILTV glycoproteins. Because of the protective response obtained when purified ILTV glycoproteins were formulated into subunit vaccines, the genes for these glycoproteins have now been cloned and characterised. In particular, restriction endonuclease maps of the ILTV genome have now been produced, and die locations of the two glycoprotein genes (gp60 and gp205) identified.
  • clones encoding gp60 and gp205 have been sequenced and relevant promoter regions identified.
  • production of die glycoproteins as recombinant products for use in subunit vaccines, as well as production of live recombinant viral vaccines expressing these glycoproteins maybe achieved using methods well known to persons skilled in die art.
  • a recombinant DNA molecule comprising a nucleotide sequence capable of being expressed as all or at least a substantial portion of the 205K complex of glycoproteins or the 60K glycoprotein of ILTV, or an immunogenic peptide derived therefrom.
  • the nucleotide sequence may have expression control sequences operatively linked tiiereto, such control sequences being derived from a homologous or heterologous source.
  • the invention also provides a recombinant DNA cloning vehicle (such as a plasmid or bacteriophage) comprising an expression control sequence operatively linked to a nucleotide sequence capable of being expressed as all or a substantial portion of the 205K complex of glycoproteins or the 60K glycoprotein of ILT, or an immunogenic peptide derived therefrom, as well as a host organism (such as a bacterium or yeast) containing such a cloning vehicle.
  • a recombinant DNA cloning vehicle such as a plasmid or bacteriophage
  • an expression control sequence operatively linked to a nucleotide sequence capable of being expressed as all or a substantial portion of the 205K complex of glycoproteins or the 60K glycoprotein of ILT, or an immunogenic peptide derived therefrom
  • a host organism such as a bacterium or yeast
  • the present invention also extends to syntiietic polypeptides displaying the antigenicity of the ILTV glycoproteins discussed above.
  • Such syntiietic polypeptides may comprise fusion polypeptides wherein die sequence displaying die desired antigenicity is fused to an additional heterologous polypeptide sequence.
  • die term "synthetic" means that the polypeptides have been produced by chemical or biological synthesis.
  • ILTV infectious laryngotracheitis virus
  • genes include (1) die ILTV glycoprotein gp60 gene which encodes a protein of 995 amino acids; (2) the Kprl/YL fragment ORF3 gene encoding a protein of 298 amino acids; and (3) die ILTV homologue of the HSV protein kinase gene.
  • herpesviruses can be administered in an aerosol form, tiius permitting an easy and inexpensive delivery system suitable for the highly intensive poultry industry.
  • herpesviruses used in such a way are known as “vectors” and genes, other than their own, expressed in such a way are referred to as “foreign genes”.
  • vectors genes, other than their own, expressed in such a way are referred to as "foreign genes”.
  • foreign genes In order to develop a vector system based on herpesviruses it is necessary to identify suitable promoters for die expression of foreign genes and "non-essential" regions of herpesviruses into which a foreign gene can be inserted without disrupting an essential function of the virus.
  • non-essential in this context means non-essential for growth under at least some conditions in which die virus can be grown in vitro and under at least some conditions in which it survives in vivo.
  • the present invention provides a recombinant ILT virus, characterised in that heterologous DNA is inserted into a non-essential region of the ILTV genome.
  • the region of the ILTV genome into which the heterologous DNA is inserted is the region corresponding to the gp60 gene.
  • tiiat the promoter regions in the ILTV genome particularly those for the glycoprotein genes gp60, gp205 (gpB) and ORF3, are major promoter regions. Accordingly, knowledge of the sequences of these regions, and particularly the gp60 promoter region, enables these promoter regions to be used for the expression of heterologous genes, either in ILTV or in a foreign host cell or organism.
  • the invention further provides a recombinant virus, particularly ILTV, characterised in that heterologous DNA is inserted into a non-essential region of the host virus genome.
  • a recombinant virus particularly ILTV
  • expression of said heterologous DNA is controlled by an ILTV promoter region br by a heterologous promoter.
  • This example demonstrates die specificity of the serum antibody response of chickens to glycoprotein antigens of ILTV as determined by Western blotting, and the ability of ILTV glycoproteins to elicit a cell-mediated immune response.
  • SA-2 the vaccine strain of ILTV used in Australia, was propagated and assayed in monolayer cultures of primary chicken kidney (CK) cells (Fahey et.al, 1983).
  • Detergent extracts of virus-infected cells were prepared at 18-20 h post infection using 1% (v/v) Nonidet P40 and 1% (w/v) sodium deoxycholate (York et.al., 1987).
  • the glycoprotein fraction of die detergent extract was obtained by affinity chromatography on a lentil lectin Sepharose 4B column (York et.al, 1987).
  • Immune chicken serum was collected 8 to 12 weeks after eyedrop vaccination of 6-week-old specified patiiogen-free (SPF) White Leghorn chickens (CSIRO SPF Poultry Unit, Maribyrnong) with approximately 10 5 PFU of SA-2 ILT vaccine (Arthur Webster Pty.Ltd., Castle Hill).
  • SPF patiiogen-free
  • CSIRO SPF Poultry Unit Maribyrnong
  • SA-2 ILT vaccine Arthur Webster Pty.Ltd., Castle Hill
  • ILTV antigens obtained by immunoprecipitation of detergent extracts with monoclonal antibodies (Mabs) to ILTV were tested for their ability to elicit a delayed-type hypersensitivity (DTH) reaction in the wattle of cockerels vaccinated witii SA-2 by eyedrop 4 weeks earlier.
  • DTH delayed-type hypersensitivity
  • the ILTV-specific Mabs have been described previously (York et.al, 1987).
  • Mab 39-2 (Group I) recognises a single glycoprotein of 60K molecular weight in Western blotting
  • Mabs 22- 37, 131-6 and 12-1 (Group II) recognise a complex of glycoproteins of 205, 160, 115, 90 and 85K molecular weight.
  • Immunoprecipitations were carried out using Protein A-Sepharose beads as described previously.
  • the antigen-antibody complexes were dissociated by incubation with 1% SDS for 10 min at room temperature as preliminary experiments had shown tiiat the recovery of antigens in a form able to elicit DTH reactions was maximal when 1% SDS was used to dissociate the antigen-antibody complexes, ratiier than 1 M propionic acid, 3 M potassium thiocyanate or 8 M urea (data not shown).
  • the thickness of each wattle was measured at time zero (Ag 0 , C 0 ) and a 0.1 ml volume of antigen (Ag) was injected subcutaneously into the right wattle.
  • a control antigen (C) was prepared by an identical treatment of uninfected cells and 0.1 ml of the control preparation was injected into the left wattle.
  • the thickness of both wattles was measured (Ag 24 , C 24 ) and die DTH index was calculated as the difference between the increase in thickness of the test wattle and die increase in the control wattie, i.e. (Ag 24 -Ag 0 )-(C 24 -C 0 ).
  • a DTH index of greater than 0.4 was considered positive.
  • the differences between group mean indices were analysed by die non-parametric Kruskal-Wallis Q test as the variance of treatment groups was strongly heterogenous.
  • glycoproteins precipitated by Mabs 39-2, 22-37 and 131-6 also elicited positive DTH reactions in most chickens showing that both tiiese groups of glycoproteins were able to elicit cell-mediated immune responses to ILTV.
  • the response to the antigens precipitated by tiiese Mabs was significantly different (p ⁇ 0.05) to the response to antigens precipitated by normal mouse serum (Table 2).
  • none of the chickens injected witii glycoproteins precipitated by Mab 12-1 (Group II) showed a positive DTH reaction.
  • This example demonstrates the vaccination of chickens with ILTV glycoproteins purified by lentil lectin affinity chromatography. Immune responses to the vaccines were measured by a virus neutralisation assay and a delayed-type hypersensitivity (DTH) assay and cprrelated with protection. The efficacy of the glycoprotein vaccines in providing protection against a challenge infection was assessed by both clinical signs and by detecting viral antigen in tracheal scrapings by ELISA
  • Figure 1 is a Western blot of glycoprotein vaccines used in Experiment 1.
  • the glycoprotein preparations used for the primary (lanes 1,3) and secondary (lanes 2, 4) vaccination were reacted in Western blotting with chicken antiserum to ILTV (lanes 1,2) or normal chicken serum (NCS; lanes 3, 4) diluted 1:400.
  • the molecular weights (kilodaltons) of the glycoproteins are indicated on the left hand side.
  • Figure 2 is a Western blot of glycoprotein preparations for Experiment 2.
  • the glycoprotein fractions use- to prepare the primary vaccines were probed with chicken antiserum to ILTV diluted 1:400.
  • Lane 1 shows the original glycoprotein fraction, and lanes 2 and 3 the glycoprotein fraction after treatment once (lane 2) or twice (lane 3) with Mab 10-2.
  • the material that bound to the antibody- coated Protein A-Sepharose beads after one (lane 4) or two (lane 5) treatments with Mab 10-2 is shown in lanes 4 and 5.
  • the molecular weights of the glycoproteins are indicated on die left hand side.
  • Figure 3 is a Western blot of sera from chickens in Experiment 2 that were protected against challenge. Sera were diluted 1:50 and reacted in Western blotting with a detergent extract of virus-infected cells.
  • Panels a and b show 5 representative sera from chickens that were protected following vaccination with total glycoprotein vaccine (a, lanes 1-5) and with depleted glycoprotein vaccine (b, lanes 1-5), respectively. The molecular weights of the glycoproteins are indicated at the centre.
  • ILTV strains were propagated in monolayer cultures of chicken kidney (CK) cells which were grown in Eagle's basal medium supplemented with 10% tryptose phosphate broth, 5% newborn calf serum, Hepes buffer (0.015 M), araphotericin B (2.5 ⁇ g/ml), penicillin (0.06 mg/ml) and streptomycin (0. 1 rag/ml).
  • CK chicken kidney
  • araphotericin B 2.5 ⁇ g/ml
  • penicillin 0.06 mg/ml
  • streptomycin 0. 1 rag/ml
  • Extracts were prepared at 18-20 hr post infection, using 1% Nonidct P40 and 1% sodium deoxycholate (York et al, 1987), from cells that had been infected at a multiplicity of approximately 5 plaque-forming units (PFU) per cell.
  • PFU plaque-forming units
  • the glycoprotein fraction of the detergent extract was obtained by affinity chromatography on a lentil lectin Sepharose 4B column (Pharmacia (Australia) Pty Ltd). The extract was allowed to bind to the column for 1 hr, then the column was washed with 5 column volumes of equilibration buffer (0.05 M Tris-HCl, pH 8.0, 0.15 M Nad, 0.1% Nonidet P40). The bound glycoproteiis were eluted with 0.2 M methyl glucoside in equilibration buffer. Fractions were concentrated to the original volume by membrane filtration using a YM10 membrane (Amicon Corporation, Danvers, MA.) and phenylmethylsulfonylfluoride was added to a final concentration of 1 mM.
  • the glycoprotein fraction was depleted of the 60K glycoprotein by reacting it with monoclonal antibody (Mab) 10-2 (York et al., 1987) bound to protein A-Sepharose beads coated with rabbit anti-mouse lg, using the immunoprecipitation protocol described previously (York et al., 1987).
  • the depleted preparation was treated a second time with the monoclonal antibody to ensure that all material reacting with the antibody was removed.
  • the 60K glycoprotein (and also the antibody) was removed from the beads by treatment with 1% sodium dodecyl sulphate (SDS) for 15 min at room temperature.
  • SDS sodium dodecyl sulphate
  • glycoprotein preparations were inactivated by treatment with ⁇ -propiolactone.
  • the glycoprotein preparation (18 parts) was mixed with 0.5 M Na 2 HPO 4 pH 8.0 (1 part) and the mixture was added to 2% (v/v) ⁇ -propiolactone. After thorough mixing, the tube was incubaied for 1 hr at 37o. The preparation was transferred to a fresh glass tube, incubated as before and then incubated at 4o overnight. Infectious virus was not detected in any inactivated glycoprotein preparation after three passages in CK cells. Protein concentrations were determined using the bicincboninic acid (Pierce Chemical Co., Rockford, IL, USA) assay (Smith et.al., 1985; Redinbaugh and Turley, 1986).
  • Experiment 1 Three groups of 4-week-old chickens were used. One group of 16 chickens was vaccinated by eyedrop with approximately 10 5 PFU of commercial live ILT vaccine (Arthur Webster Pty Ltd, Sydney) and a second group of 17 chickens was vaccinated intraperitoneally with 70 ⁇ % of affinity purified glycoproteins emulsified in an equal volume of Freund's complete adjuvant (FCA), in a total volume of 1 ml. A third group of 16 chickens were held as unvaccinated controls.
  • FCA Freund's complete adjuvant
  • Groups 2 and 3 were vaccinated twice intraperitoneally with different glycoprotein vaccines which were emulsified in FCA for the primary vaccination and in F1A for the secondary vaccination.
  • Group 2 received doses of 350 ⁇ g of the complete glycoprotein vaccine in the primary vaccination and 160 ⁇ g in the secondary vaccination.
  • Group 3 were vaccinated with 260 ⁇ g of the depleted glycoprotein preparation in the primary vaccination and 56 ⁇ g in the secondary vaccination.
  • Group 4 were vaccinated with the immunoprecipitated 60K glycoprotein. A fifth group was held as unvaccinated controls.
  • Groups 2, 3 and 4 were revacdnated intrapcritoneally at 4 weeks after primary vaccination with the appropriate antigen in FIA
  • the DTH reaction of 6 cockerels from each group was tested 6 weeks after primary vaccination. All chickens were bled and then challenged intratracheally with 2.5 ⁇ 10 5 PFU CSW-1 in a 200 ⁇ l volume at 8 weeks after primary vaccination. At day 3 after challenge the chickens were eutbanased and tracbeal scrapings were collected for examination for ELTV antigen by ELISA.
  • a detergent extract was prepared from virus-infected and uninfected CK cells and dialysed overnight against phosphate-buffered saline (PBS). The thickness of each wattle was measured at time zero and a 0.1 ml volume of the antigen extracted from infected cells was injected subcutaneously into the right wattle. The same volume of the control antigen prepared from uninfected cells was injected into the left wattle. At 24 hr after injection the thickness of both wattles was measured and the DTH index was calculated as the difference between the increase in thickness of the test wattle and the increase in thickness of the control wattle. A DTH index of greater than 0.5 was considered positive. The differences between group mean indices were analysed by the non-parametric Kruskal-Wallis Q test as the variance of treatment groups was strongly heterogeneous. Virus neutralization
  • Neutralizing activity in serum was assayedby a plaque reduction test asdescribed by York et al.
  • the neutralizing titre was taken as the dilution which produced a 70% reduction in the number of plaques compared to controls.
  • Viral antigen present in tracheal scrapings was assayed by ELISA as described previously (York and Fahey, 1988). In brief, an equal volume of PBS containing 1% Nonidet P40 was added to the scrapings and the sample was vortexed for 30 sec. Debris was removed by centrifugation in a microfuge at 12.000 rpm for 1 min. The supernatant fluid was added to polyvinyl chloride microtitre Plates coated with rabbit IgG antibody to ILTV.
  • Antiserum to the SA-2 strain of ILTV was a pool of serum collected from 5 chickens 12 weeks after eyedrop ino ⁇ lalion of approximately 10 5 PFU of commercial ILT vaccine.
  • glycoprotein preparations used for the primary and secondary immunizations in Experiment 1 are shown in Fig. 1 after Western bloning with chicken antiserum.
  • Major glycoprotein bands of 205, 115, 90. 85, 74, 60 and 50K, plus a minor band of 160K were present in both preparations ie. glycoproteins of the two major families of ILTV glycoproteins previously defined by monocional antibodies, the 205K complex and the MK glycoprotein, plus glycoproteins of 74 and 50K molecular weight.
  • Figures 3a and 3b show the reactivity in Western biorting of 5 representative sera obtained immediately before challenge from chickens receiving the glycoprotein vaccine and 5 representative sera from the group that received the depleted glycoprotein vaccine.
  • Sera from both groups reacted with bands of 205, 160, 135, 115, 90, 85, 67, 60 and 50K. Only sera from some of the birds from each group recognised the 135K band: 8 of 12 from the total glycoprotein vaccine group and 5 of 12 of the depleted glycoprotein vaccine group.
  • Sera from the chickens that were immunized with the depleted glycoprotein preparation showed Western blotting reactivity to bands in the 60K region.
  • the 2 chickens in the total glycoprotein vaccine group that were not protected had virus neutralizing antibody titres of 1/40 and 1/160 respectively, and also produced antibody detectable by Western blotting.
  • the one chicken immunized with the glycoprotein vaccine that was negative for DTH 2 weeks prior to challenge was protected against infection.
  • glycoproteins were indeed protective immunogens of ILTV they were purified by lectin affinity chromatography and formulated into glycoprotein subunit vaccines. Vaccination with various preparations of affinity purified ELTV glycoproteins protected up to 100% of chickens. They were protected not only against clinical disease, but also against replication of the challenge virus in the trachea. As far as we are aware, this is the only reported instance of this degree of protection against a herpesvirus infection following vaccination with a subunit glycoprotein vaccine.
  • Vaccination with HSV glycoproteins has also been shown to reduce the incidence of recurrent (Stanberry et al ., 1987; Wachsman et.al, 1987) and latcnt infections (Cremer et.al., 1985) in mice and guinea pigs. It is possible therefore that vaccination with ILTV glycoproteins could decrease the occurrence of latent virus, but this question was not addressed in the present study.
  • This may be a co-migrating antigen unrelated to the 60K glycoprotein recognised by Mab 10-2, or the antibodies may be to 60K glycoprotein in which the epitope recognised by Mab 10-2 was destroyed or denatured.
  • mre could be a number of reasons for the lack of efficacy of the 60K glycoprotein as a vaccine. Firstly, the dose of the glycoprotein may have been insufficient Secondly, the presence of 1% SDS in the vaccine preparation may have affected the conformation and hence the immunogenicity of if 60K glycopro.ein, panicularly as no antibody responses were detected Alternatively, the glycoprotein may have remained bound to the Mab used for depletion which may have interfered with its recognition by the immune system.
  • Experiment 2 bad serum neutralizing antibody, while the one chicken in Experiment 1 without detectable neutralizing antibody was protected. Immunization studies with glycoproteins of other herpesviruses have also shown that protection does not always correlate with the presence of neutralizing antibody responses in serum. Animals can be protected in the absence of neutralizing antibody (Zweerink et.al, 1981; Schrier et.al., 1983; Chan et al, 1985; Wacbsman et al., 1987) while the presence of neutralizing antibody does not ensure protection (Israel et al.,
  • 160 and 135K glycoproteins were absent or poorly represented in the vaccines, sera from many vaccinated chickens recognised these bands in Western blotting, particularly the 160K glycoprotein, suggesting that either these two antigens are related to one or more of the other glycoproteins present in tbe detergent extract, or they are very strong immunogens.
  • T-belper cells and cytotoxic T-cells have been shown to play an important role in recovery from HSV infections in experimental animals (Nash et al., 1981; Larsen et al, 1983; Sethi et al, 1983). It may be that measures of cell-mediated immune responses other than DTH, such as the cytotoxic T-cell response or tbe generation of T-helper cells, or alternatively, tbe magnitude of local immune responses would provide a better correlation of protection against ILT than either serum neutralizing antibody or DTH responses.
  • This example details the analysis of the genome of ILTV and construction of a restriction enzyme map thereof.
  • SA-2 the vaccine strain of ILTV used in Australia, was propagated in monolayer cultures of chicken kidney (CK) cells.
  • CK cells Confluent monolayers of CK cells were infected with virus at a MOI of 1:100 and incubated at 37 oC until more than 90% of the cells showed CPE.
  • Virus was isolated from both cytoplasmic and cell free fractions and DNA extracted essentially according to the method of Wilkie (1973) by treatment with 2% SDS,
  • NTE-saturated phenol 10mM Tris-HCl, pH 8.0, 100 mM NaCl, 1 mM
  • a cosmid library was prepared by ligating partially digested Sau3A ILTV DNA with BamHI digested pHC79 (Collins and Hohns, 1978; Hohns and Collins, 1980) and packaged by standard techniques (Sambrook et.al, 1989).
  • a set of EcoRI clones was prepared in a similar way using sonicated ILTV treated with T4 polymerase, EcoRI linkers attached and finally digested with EcoRI before ligation with EcoRI digested pHC79 and packaged.
  • the transforming strain was E.coli MB406 and transformants were screened using colony blot hybridisation.
  • ILTV DNA was digested with SmaI, KpnI or EcoRI and the fragments separated through 0.5% agarose gels, stained with ethidium bromide and photographed. After photography, DNA was transferred and fixed to nylon membranes (Southern, 1975). The relative amount of DNA in each band was estimated from photographic negatives of gels scanned with a laser microdensitometer. SmaI, KpnI, NotI or EcoRI ILTV fragments cut from low melting point agarose gels, or plasmids containing ILTV fragments, were radiolabelled using a random hexamer priming kit (BRESATEC) (Feinberg and Vogelstein, 1983).
  • BRESATEC random hexamer priming kit
  • ILTV genome The terminii of ILTV genome were tentatively identified by end-labelling of viral DNA with dATP ( 32 P), then confirmed by Exonuclease III digestion. Labelled DNA was then digested with NoS or SmaI and hybridised to corresponding digests of ILTV DNA.
  • Figure 4 shows restriction maps of SA-2 ILTV DNA for the enzymes EccRI, KpnI and SmaI.
  • the filled rectangles above the maps indicate the gp205 (gp200) gene and the gp60 gene, respectively.
  • the 155 kpb of ILTV genome is represented as two unique sequences (U L , U S ) and 2 large inverted repeat sequences (IR s , TR s ) flanking U s .
  • the scale below shows fractional genome length.
  • This example shows the cloning and sequencing of the ILTV gp60, Kpn K/ORF3 and PK genes.
  • SA-2 vaccine strain of ILTV was used to obtain DNA for cloning, SA-2 is used commercially as a vaccine throughout Australia and was obtained from the National Biological Standards Laboratory, Parkville, Australia and used at passage levels 5 and 20.
  • ILTV was propagated in monolayer cultures of primary chicken kidney (CK) cells.
  • CK cells were grown in Eagle's basal medium (Commonwealth Ser ⁇ m Laboratories) supplemented with 5% newborn calf serum (Flow Laboratories Australasia Pty Ltd), 2 ⁇ g/ml fungizone, 100 ⁇ g/ml streptomycin and 100 IU/ml penicillin.
  • SA-2 ILT virus was inoculated on to confluent monolayers of CK cells at a multiplicity of infection of approximately 0.5 plaque forming unit (pfu) per cell.
  • Cells were pre-washed in PBS (phosphate saline buffer), and the virus inoculum was added to the cells in 1ml of serum-free medium per 90 mm x 14 mm petri dish. After 1 incubation at 37°C to allow the virus to adsorb to the cells, 5 mis of medium containing 5% newborn calf serum was added. After 24 b when 80-100% of the cell monolayers showed cytopathic effe ⁇ , the cell monolayers were harvested.
  • PBS phosphate saline buffer
  • ILTV DNA was sheared by sonication; made flush-ended with T4 DNA polymerase; methylated by EcoRI methyltransferase; ligated to EcoRI linkers (Biolabs); and digested with EcoRI fragments. Fragments of 0.5-2 kilobases were isolated by agarose gel electrophoresis and ligated to ⁇ gt11 arms previously digested with EcoRI and dephosphatased with calf intestine alkaline phosphatase (Amersham). After ligation, the lambda DNA was packaged using Packagene extract (Promega). The result was a library of 100,000 phages. The library was plated on lawn of E.
  • ILTV DNA 1 ⁇ g was cut with the restriction enzyme BamHl and Kpnl (Pharmacia) and the resulting DNA fragments separated by agarose gel electrophoreses and transferred to Hybond membranes (Amersham). The filters were then probed with EcoRI inserts from ⁇ gt11-ILTV recombinant phages (X24-4, X27-1) which were positive with gp60 monoclonal antibodies. The DNA fragments from these two phages were labelled with deoxyadenosine [ ⁇ 32 P] triphosphate by using a random hexamer priming method.
  • Hybridization was in 5xSSC, 0.5% sodium dedocyl sulphate (SDS), 5xDenhardt's solution and 100 ⁇ g/ml denatured herring sperm DNA at 65°C. Washes were 0.5 ⁇ SSC, 0.1% SDS at 65oC
  • DNA sequencing was carried out by the dideoxynucleotide termination method with [ 35 S]dATP.
  • the DNA synthesis reaction was primed with either a 17-mer residue that hybridized to pUC or M13 sequences adjacent to the insert DNA or with custom 20-mer oligonucleotides complementary to a specific region of the insert DNA.
  • Specific oligonucleotides were synthesized using Pharmacia LKB Gene Assembler Plus.
  • Figure 5 shows the genome structure of ILTV showing the KpnI site distribution and the location of the KpnI/K fragment that has been sequenced. The latter has been expanded in the lower part of the figure to show the overlapping BamHI 4.5 Kbp fragment
  • Open reading frames ORFs
  • ORFs Open reading frames
  • Figure 6 shows the nucleotide and predicted amino acid sequence of the ILTV gp60 gene with the numbering referring to the nucleotides and amino acids.
  • the EcoRI inserts of ⁇ 27 and ⁇ 24-4 were labelled with 32 P dATP, and used to probe the KpnI and BamHl digests of ILTV DNA. Hybridization resuhs showed that the DNA cloned into the two recombinant phages mapped to the small 678 bp Kpnl fragment (cross-hatched), the KpnI/K fragment and to the 4.5 Kbp BamHI fragment ( Figures). Nucleotide analysis of the 5.3 Kbp Kpnl/K and the 4,557
  • Kbp BamHI fragment revealed several potential open reading frames (data not shown).
  • One ORF encodes the C-terminal part of the gene referred to as the KpnK/ORF3 and this ORF is described in Figure7 .
  • a second ORF specifying the FLTV gp60 gene product and has a size of 2985 bp.
  • the predicted translated protein is 995 amino acids.
  • the gp60 coding region contains an average base composition of 24%A, 22%T, 26%G and 28%C. with the G+C content of 54%, which is higher than the estimate of 45% for total ILTV DNA as determined from buoyant density measurements (Plummer, G.
  • the deduced amino acid sequence of the gp60 gene has several features of a glycoprolein. These include 19 hydrophobic amino acid residues at the N- terminus which may commpond to the signal sequence. A second region of hydrophobic amino acids (positions 960 to 989) at the C-terminus could function as a transmembrane anchor sequence. There are nine potential N-linked glycosylation sites on the ILTV gp60 protein (underlined). One of these sites at residue 677, may not be active due to the presence of a proline residue within the N-X-S/T signal.
  • TAATAAA at positions 294-301 could be a potential TATA boo element for this gene. There is no obvious polyadcnylation signal downstream for the stop codon.
  • the predicted molecular weight of the coded amino acid sequence is 107,503 daltons which is significantly larger than the apparent mass of the 63Kd polypeptide detected by monoclonal antibodies on Western bot. It is noted that there is a potential internal proteolytic cleavage for the gp60 precursor polypeptide. A potential cleavage site (R-R-S) is present at residues 679 to 681. The predicted (cleaved) polypeptide product of this gene would have unglycosylated M r value of approximately 70Kd which is close to the apparent mass of 63 Kd.
  • F ⁇ gure 7 shows the nucleotide and predicted amino acid sequence of the SA-2 strain of ILTV KpnK/ORF3 gene.
  • KpnK/ORF3 is 298 amino acids long and is located 5' of the gp60 gene. It has a predicted M r of 32309 daltons and contains four potential glycosylation sites (underlined). The protein has an N-terminal hydrophobic region, which might be a signal sequence for translation on membrane-bound ribosomes.
  • the DNA sequences upstream and downstream of this open reading frame have common features in eukaryotic transcription initiation and termination signals.
  • a consensus "TATA” box (5'-TATAAA-3'), characteristic of many eukaryotic and also herpesviral promoters is present 81 nucleotides upstream from the proposed start codon.
  • the sequences 5'-GGCTCCATA-3' which resides 25 nucleotides upstream from the "TATA" box exhibits similarities to the "CAT” box consensus sequence (5'-GGOTCAATCT-3').
  • two potential polyadcnylatio ⁇ signals 5'-AATAAA-3' are found at the 3' end of the gene.
  • FIG. 8 shows the DNA sequence of part of the protein kinase (PK) gene of ILTV. The sequence starts from the left end of KpnI/K fragment and ends 656 bp from the KpnI.
  • PK protein kinase
  • FIG. 9 shows mapping of recombinant phage ⁇ 26-2 DNA on the ILTV genome.
  • ELTV DNA was digested with EcoRI, resulting DNA fragments were separated by agarose gel electrophoresis and transferred to Hybond membrane.
  • One set of fragments was hybridised with 32 P-labelled EcoRI insert from A26-2 (lane 2), while the other set was hybridised with 32P-labelled ILTV DNA (lane 1).
  • the letters denote the assignments for each hybridising ILTV- EcoRI restriction enzyme fragment based on the previous restriction endonuclease cleavage maps of ILTV genomic DNA.
  • Figure 10 she a physical map of EcoRI restriction sites for ILTV DNA and an expanded restriction map of the EcoRI "U” fragment.
  • the EcoRI restriction cleavage map is that described above.
  • U L and U s long and short unique regions of the genome, respectively; TR and IR terminal and inverted repeat regions of the genome, respectively.
  • the position and direction of the ILTV "gB" transcript is shown below the expanded map of the EcoRI "U”.
  • Nucleotide sequence strategy is indicated by arrows.
  • Figure 11 shows the nucleotide sequence of the EcoRI “U” fragment encoding the "gB" homologue of ILTV and the deduced amino acid sequence.
  • the putative TATA box of the promoter is boxed.
  • the polyadenylation site, AATAAA is double underlined.
  • Broken lines indicate GC-rich regions.
  • the presumed signal sequence at the N-terminus and the membrane-spanning region at the C-terminus are indicated by italics and bracketed ([]).
  • the putative N-linked glycosylation sites of the consensus N-X-S/T are underlined. Brackets ( ⁇ ) indicate the beginning and end of the insert present in the recombinant ⁇ 26-2.
  • Figure 12 shows expression of ILTV "gB" gene.
  • Total cytoplasmic RNAs from ILTV infected (lane 1) and mock infected (lane 2) were fractionated on an agarose /formaldehyde gel and transferred onto Hybond-N membrane. The membrane was hybridised to the BglII- PstI fragment (Fig.10).
  • Arrowheads indicate the location of chicken ribosomal RNAs 28S and 18S which were used as size standard and were estimated to be 4.2kb and 1.6kb respectively.
  • Figure 13 is a hydropathy plot of the predicted ILTV "gB" amino acid sequence. The plot was based on the algorithm of Kyte and Doolittle (1982) by using a 11-amino acid window. The two most hydrophobic regions in the N- and C-termini are predicted to represent the signal sequence and the transmembrane anchor region, respectively, of the glycoprotein molecule.
  • Figure 14 shows homology of ILTV "gB” with those of other herpesviruses. Multiple alignment of the amino acid sequences predicted for the "gB"-like proteins of 10 different herpesviruses. Sequences were aligned using CLUSTAL program (Higgins and Sharp, 1988). Asterisks indicate identical amino acids and dots represent conserved amino acid substitutions. Putative N-linked glycosylation sites are shown in bold and underlined. The signal sequences are double underlined and the triple transmembrane domains are boxed. conserveed cysteine and proline residues are shown by ⁇ and ⁇ respectively.
  • the strain used in the present study is the SA-2 vaccine strain of ILTV used in Australia.
  • ILTV was propagated in monolayer cultures of primary chicken kidney (CK) cells grown in Eagle's basal medium (Commonwealth Serum Laboratories) supplemented with 5% newborn calf serum (Flow Laboratories Australasia Pty.Ltd.).
  • Cell associated viral DNA was prepared using the slightly modified method of Whalley et.al. (1981) from infected cell cultures at 24hr post infection. Infected cells were harvested by scraping the cells from tissue culture dishes and centrifuged for 10 min at 7000 rpm.
  • the cell pellet was then resuspended in CAV buffer (10 mM KCl, 15 mM MgCl 2 and 10 mM Tris-HCl pH7.5) and disrupted by repeated freeze-thawing in a solution of 0.5% NP40.
  • CAV buffer 10 mM KCl, 15 mM MgCl 2 and 10 mM Tris-HCl pH7.5
  • the supernatant was pooled and extracted with phenolchloroform and incubated with RNase A (10 ug/ml). DNA was precipitated by addition of 50% volume of isopropanol.
  • Enzymes were purchased from Pharmacia, or Promega and used as specified by the manufacturers.
  • ILTV DNA was sheared by sonication, made flush-ended with T4 DNA polymerase, methylated by EcoRI methyltransferase, ligated to ⁇ c ⁇ RI linkers (Biolabs) and digested with EcoRI endonuclease. Fragments of 0.5-2 kb were isolated by agarose gel electrophoresis and ligated to ⁇ gt11 arms previously digested with EcoRI and dephosphorylated with calf intestine alkaline phosphatase (Amersham). After ligation, the DNA was packaged using Packagene extract (Promega), transfected and resulted in a library of 100,000 phages. The library was plated on a lawn of E.
  • ILTV or plasmid DNAs were digested with appropriate restriction enzymes and the resulting DNA fragments separated by agarose gel electrophoresis and transferred to Hybond membranes (Amersham). The filters were then probed with DNA fragments which were labeled with deoxyadenosine [ ⁇ 32 P] triphosphate using a random hexamer priming kit (Bresatec, South Australia). Hybridization was in 5 X SSC, 0.5% sodium dedocyl sulphate (SDS), 5 X Denhardt's solution and 100 ug/ml denatured herring sperm DNA at 65°C. Washes were performed using 0.5 X SSC, 0.1% SDS at 65°C.
  • DNA sequencing was carried out by the dideoxynucleotide chain termination method using the Seque ⁇ ase sequencing kit (United States Biochemical).
  • the DNA synthesis reactions were primed with either a 17-mer oligonucleotide residue that hybridized to pUC or M13 sequences adjacent to the insert DNA or with custom 20-mer oligonucleotides complementary to a specific region of the insert DNA.
  • Specific oligonucleotides were synthesized using Pharmacia LKB Gene Assembler Plus.
  • DNA sequence reading and analyses were done using a HIBIO DNAsis software package (Hitachi America, Ltd.) and with the programs available through the Australian National Sequence Analysis Facility (ANSAF). Searches of protein databases and comparison of homologous sequences were performed with the FASTN/P program of Lipman and Pearson (1985).
  • guanidium thiocyanate technique was used to isolate cytoplasmic RNA from ILTV-infected or uninfected control CK cells (Maniatis et.al., 1982). About 15 ug of total RNA was denatured and separated on 1% agarose/formaldehyde gels and transferred onto Hybond-N membrane. Blots were probed with a 32 P-labcled 632 base pairs (bp) PstI-BglII fragment (see Fig. 10).
  • a ⁇ gt11 library of randomly generated 500 bp to 2 kb ILTV DNA fragments inserted into the EcoRI site of the lacZ gene was constructed.
  • the translational products of inserted open reading frames are expected to be part of a ⁇ -galactosidasc fusion protein (Young and Davis, 1983).
  • the library was grown on E. coli Y1090; fusion proteins were induced by isopropyl-) ⁇ -D-thiogalactopyranoside (IPTG) and screened with a mixture of appropriately diluted monoclonal antibodies (kindly provided by Dr Jenny York).
  • Monoclonal antibodies (MAbs) used were 10-1, 10-2, 39-2 (group 1) which immunoprecipitated the 60K glycoprotein and 12-1, 22-7, 23-1, 131-6 (group 2) which reacted with the 205K glycoprotein complex in Western blot (York and Fahey, 1988). From among 50,000 plaques screened, thirteen positive clones were identified. Ten clones reacted with group 1 MAbs and the other three reacted with group 2 MAbs. Of the three recombinant phages which were positive with group 2 MAbs, one phage designated 26-2, was plaque-purified and DNA prepared for further studies.
  • FIG. 10 A restriction map for EcoRI sites in the ILTV genome and a more detailed map for the region spanning 0.23-0.25 map units are shown in Fig. 10.
  • the complete nucleotide sequence of the region containing the putative gp205K coding sequence is shown in Fig 11.
  • Fig.11 There is a single large open reading frame within this region extending from the ATG codon beginning 185 bp 3' of the EcoRI site (Fig.11 to a TAA termination codon, starting at nucleotide 2804, (Fig. 11). Translation of this 2619 bp would produce a polypeptide of 873 amino acids.
  • initiation codon ATG at 185 bp resides within the sequence GACATGG which conforms well to the consensus sequence (A/G)CCATGG (Kozak, 1984). It has a purine (G) at position -3, C at-1, and a G at +4 which is considered to be the most strongly conserved features of the flanking sequence of the initiation codon of eukaryotic mRNAs.
  • TATA box position at bp 38-41 is the functional TATA box of this gene for two reasons: (i) its local sequence TATATTT has some features proposed for the consensus TATA box sequence TATA (A/T)A(A/T) (Corden et.al., 1980), and (ii) SI nuclease mapping indicated that the potential RNA polymerase initiation site of this gene mapped at about 144 nucleotides upstream of the ATG (C. T. Prideaux, unpublished data).
  • Other putative cis-regulatory elements found are the GC-rich regions (Fig .11 indicated by broken lines) which are potential binding sites for the promoter-specific transcription factor Spl (Briggs et al, 1986).
  • a potential polyadenylation signal AATAAA was found 20 bp downstream from the termination codon (Fig. 1 1).
  • the G+C content of the sequence is 44.4%, which is close to the estimate of 45% for total ILTV DNA as determined from buoyant density measurements (Plummet et al, 1969).
  • the deduced amino acid sequence for the polypeptide encoded by the 2619 bp open reading frame is shown above the DNA sequence in Fig. 11.
  • the molecular mass of the 873 amino acids primary translation product is 98,895 daltons.
  • the predicted protein has features common to other membrane-spanning glycoproteins.
  • a hydrophobicity plot (Fig. 13)identified a sequence of 16 hydrophobic amino acids at the extreme NH 2 end (Figs. 1 land 13 which may function as the signal peptide. Applying the weight-matrices criteria of von Heijne (1986) for the prediction of the cleavage site, the cleavage might occur at the isoleucine residues 14.
  • a broad hydrophobic domain at amino acids 690 to 761 near the C-terminus represents a membrane anchor sequence.
  • a targe extracellular domain (amino acid. 17 to 689) contains nine potential N-linked glycosylation sites (underlined in Fig. 11).
  • C-terminal amino acids 762 to 873 have net positive charge and may function as the cytoplasmic domain.
  • gp205 shares significant amino acid bomology with gB-like glycoproteins of other rerpesviruses
  • Fig. 14 Muitiple alignments of ten herpesvirus gBs (Fig. 14)have highlighted several charaaeristics of conserved sequence.
  • the common structural features of the gB-like proteins shown in Fig. 14 are: (i) the conservation of tea cysteine (C) residues which were perfectly aligned in gB of ail ten viruses. This accounts for all cysteines of ILTV protein except the two which occur in srgnal sequence. This observation indicates that the proteins are conserved in their secondary and tertiary structures since C-C disulfide bonds are important determinants of the tertiary structure of the protein, (ii) Six sites of prolines occur at conserved positions (Fig.
  • This example shows the identification of various ILTV promoters and use of the ILTV glycoprotein B (GbP) gene promoter in the regulation and expression of foreign vaccine antigens in ILTV viral vectors.
  • GbP ILTV glycoprotein B
  • Figure 15 shows the nucleotide sequence of the ILTV glycoprotein B promoter.
  • Binding positions of the oligonucleotides used to isolate the fragment by the polymerase chain reaction are underlined.
  • the ATG translation initiation codon is in bold, and possible TATA and CAAT consensus signal sequences are boxed.
  • Figure 16 shows the construction of pPgB-CAT.
  • the ILTV glycoprotein B promoter was isolated using the polymerase chain reaction. Restriction enzyme sites, Pstl and Xbal, engineered into the oligonucleotides used for the polymerase chain reaction were digested, facilitating the cloning of the fragment into pUC and subcloning into pCAT-BASIC
  • Figure 17 shows the nucleotide sequence of die 5' non-coding regions of the ILTV ORF3 and gp60 genes isolated by polymerase chain reaction. Binding positions of the oligonucleotides used to isolate the fragments by use of the polymerase chain reaction are underlined. The ATG translation initiation codon is in bold, and possible TATA and CAAT consensus signals are boxed. MATERIALS AND METHODS
  • CK cells Primary chicken kidney (CK ) cells were prepared by trypsinization of kidneys isolated from two to four week old specific pathogen free (CSIRO, SPF Poultry Unit, Maribyrnong, Victoria ) chickens as described by Fahey et al. ( 1983 ).
  • ILTV SA2 vaccine strain was obtained from Arthur Websters Pty. Ltd (Northmead, 2152, Australia ).
  • ILTV was grown on CK cells in Eagle's Basal medium ( Gibco Laboratories ) supplemented with 5% bovine calf serum ( BCS) and lOmM Hepes. Virus stocks were frozen ( -70 °C ) and thawed three times prior to infection to release ILTV from cells.
  • Monolayers of primary CK cells were prepared in 50 mm petri-dishes ( IxlO 6 cells) and infected with ILTV at a multiplicity of 2-5 plaque forming units (p.f.u.) per cell. Prior to addition of ILTV, the growth medium was removed from cells and the monolayer washed twice with phosphate buffered saline ( PBS ). After 2 hr of absorption at 39 °C, the monolayers were washed and growth medium added.
  • PBS phosphate buffered saline
  • Restriction enzymes and other DNA modifying enzymes were obtained from various sources and used according to the manufacturers' instructions or as oudinedbyManiatis et al. ( 1982 ).
  • Cellphect transfection and dideoxy sequencing kits were from Pharmacia.
  • Chloramphenicol acetyl transferase (CAT ) assay system was from Promega. Isolation of ILTV Genomic DNA
  • CK cells Confluent monolayers of CK cells were infected with ILTV at a level of 0.1 plaque forming units per cell and incubated at 39 °C until cellular cytopathic effects were greater than 90%.
  • Virus was isolated from both cytoplasmic and cell free fractions and DNA extracted essentially according to the method of Wilkie ( 1973 ) by treatment with 2% SDS, NTE-saturated phenol ( NTE: 10 mM Tris- HCl, pH8.0, 100 mM NaCl, 1 mM EDTA ) and chlorophorm/isoamylalcohol (24:1 ).
  • the DNA was precipitated in 70% ethanol and resuspended in TE buffer ( 10 mM Tris-HCl, pH7.5, 1 mM EDTA ).
  • Amplification of the 5' non-coding region from the ILTV gpB gene was performed using the polymerase chain reaction (PCR ). Reactions were performed in 50 ul volumes comprising 50 ng of ILTV DNA, 3 ng of each oligonucleotide primer, 50 mM KC1, 10 mM Tris-HCl, pH8.4, 2.5 mM MgCl 2 , 200 ug/ml BSA, 200 uM dATP, 200 uM dCTP, 200 uM dGTP, 200 uM dTTP and 1 unit of Taq polymerase ( Cetus ).
  • PCR polymerase chain reaction
  • Reaction mixtures were overlayed with an equal volume of paraffin oil and heated to 94 oC for 1 min, cooled to 55 °C for 2 min and heated to 72 °C for 5 min. This cycle was repeated 35 times using a Perkin Elmer Cetus DNA Thermal Cycler.
  • Monolayers of CK cells prepared in 50mm petri-dishes were transfected with CsCl 2 purified promoter-CAT plasmid constructs using a Pharmacia Cellphect transfection kit as detailed by the manufacturer. Briefly 1-10ug of plasmid DNA in 1ul of isotonic Tris-HCl, pH7.5, was mixed with an equal volume of DEAE-Dextran solution ( end cone. 0.5mg/ml ). The DNA/dextran solution was added drop-wise to monolayers of CK cells which had been washed twice with isotonic Tris-HCl, pH7.5, and incubated at room temperature. After 15mins the DNA/dextran solution was carefully removed, the monolayers washed with isotonic Tris-HCl, and complete growth medium added.
  • CAT activity in CK monolayers were made using a Promega CAT enzyme assay system. Briefly monolayers of transfected CK cells were scraped into the medium witii the use of a rubber policeman, pelleted at 1,000 ⁇ m for 5mins at 4oC, and washed once with PBS and once with TEN buffer ( 40mM Tris-HCl pH7.5, ImM EDTA, 15mM Nacl ). The pelleted cells were resuspended in 100ul of 0.25M Tris-HCl pH8.0, and subjected to three cycles of freeze thawing ( -70 °C to 37 °C ), with vortexing after each cycle.
  • Standard CAT assays contained 55ul of cell extract, 5ul of 14C-chloramphenicol ( Amersham Int. ) and 2.5ul n- butryl CoenzymeA ( 5mg/ml; Promega ). Reactions were carried out at 37 °C for 12hrs, and terminated by extraction with 150ul of mixed xylenes (Aldrich Chemical Co. ). The xylene phase containing the 14C n-butyryl chloramphenicol reaction products was isolated by microfugation for 3mins ( upper phase ) and purified by back extraction with 150ul 0.25M Tris-HCl pH8.0. Fifty microlitre aliquots were added to 1ml Econofluor scintillation fluid ( NEN Research Products ) and counted in a LKB Wallac 1209 Rackbeta scintillation counter.
  • oligonucleotides were designed to isolate the 5' noncoding region from the ILTV gpB gene ( PgB ), by use of the polymerase chain reaction ( Figure 15 ). To facilitate the cloning of the PCR products, the oligonucleotides were designed to contain unique restriction enzyme sites; the oligonucleotide furthest 5' to the open reading frame contained an internal Pstl site, while the oligonucleotide immediately 5' to the open reading frame contained an Xbal site. Oligonucleotide priming positions are given in figure 15.
  • the promoter fragment was sub-cloned from the pUC-PgB construct into the pCAT-Basic plasmid ( Promega ), to form pPgB-CAT ( Figure 16 ).
  • the pCAT-Basic plasmid contains the chloramphenicol acetlytransferase ( CAT ) gene without any transcription regulation signals, and was designed for the specific purpose of assaying DNA fragments for promoter activity.
  • the CAT gene product is readily assayed, allowing accurate qualification of promoter activity.
  • the orientation, and correct cloning of the promoter fragment adjacent to the CAT gene was confirmed by double stranded sequencing ( results not shown ) using a 15mer oligonucleotide which bound specifically to a region of the CAT gene 25bp 3' to the translation initiation signal.
  • the promoter fragment was cloned adjacent to the CAT gene in the same orientation as it was with respect to the ILTV gpB open reading frame.
  • Monolayers of CK cells were transfected with 1-10ug of pPgB-CAT.
  • transfected cells were infected with ILTV, or mock-infected with PBS 14 hr post-transfection, and assayed for CAT activity 48hrs post-infection ( Table 5 ). From Table 5 it can be seen that pPgB-CAT expresses CAT activity at levels substantially higher than back-ground CK cell levels. The level of CAT activity observed was seen to be affected by the level of input plasmid DNA, but not in a linear ratio.
  • the polymerase chain reaction was used to isolate a 573bp fragment of ILTV DNA extending upstream from the first nucleotide 5' to the open reading frame of the ILTV gpB gene.
  • the ATG translation initiation codon of the gpB gene was not included in the promoter fragment, thus eliminating the production of fusion proteins and the need to align open reading frames, when expressing foreign genes.
  • the ILTV gpB promter was aligned adjacent to the marker gene CAT, and transfected into CK cells, levels of CAT activity observed were significantly above background. The activity observed was shown to be dependent on the level of pPgB-CAT transfected into cells.
  • the infection of cells containing the pPgB-CAT construct resulted in an increased level of CAT expression.
  • This increased level of CAT activity is typical of herpesvirus promoters where trans-activating factors encoded by the virus serve to increase promter activity ( reviewed Roizman and Sears, 1990 ).
  • FIG. 18 shows the construction of a fowlpox virus (FPV) recombinant expressing the ELTV glycoprotein B gene, and its ability to protect chickens from infection with viruuent ILTV.
  • Figure 18 shows the structure of recombinant plasmid for insertion of the LacZ, Ecogpt and ILTV glycoprotein B gene into the FPV TK gene.
  • Figure 18A is a schematic representation (not drawn to scale) of the genes inserted into the FPV recombinant.
  • pAF09-gpB also contains the ampicillin resistance gene and an E.coli origin of replication.
  • Figure 18B shows the junction region between the FPV E/L promoter and the ILTV gpB gene. The ATG of the E/L promoter is in phase with two ATG codons of the gpB gene.
  • FPV chicken embryo skin
  • CES chicken embryo skin
  • Primary CES cells were prepared as described by Silim et al. (1982) with the modification the collagenase at 100 ⁇ g/ml (Sigma C2139) was used to digest the skin of 13-day-old specific pathogen free embryos (CSIRO, SPF Poultry Isolation Unit, Maribyrnong, Victoria, Australia) in place of trypsin.
  • CK cells Primary chicken kidney (CK) cells were prepared by trypsinisation of kidneys isolated from two to four week old specific pathogen free chickens as described by Fahey et.al. (1983).
  • ILTV SA2 vaccine strain was obtained from Arthur Websters Pty. Ltd.
  • ILTV was grown on CK cells in Eagle's Basal Medium (Gibco Laboratories) supplemented with 5% bovine calf serum and 10 mM Hepes. Virus stocks were frozen (-70 °C) and thawed three times prior to infection to release ILTV from cells.
  • Enzymes and Chemicals are prepared by trypsinisation of kidneys isolated from two to four week old specific pathogen free chickens as described by Fahey et.al. (1983).
  • ILTV SA2 vaccine strain was obtained from Arthur Websters Pty. Ltd.
  • ILTV was grown on CK cells in Eagle's Basal Medium (Gibco Laboratories) supplemented with 5% bovine cal
  • CK cells Confluent monolayers of CK cells were infected with ILTV at a level of 0.1 plaque forming units (p.f.u.) per cell and incubated at 39 °C until cellular cytopathic effects were greater than 90%.
  • Virus was isolated from both cytoplasmic and cell free fractions. DNA was extracted essentially according to the method of Wilkie (1973) by treatment with 2% SDS, NTE-saturated phenol (NTE: lOmM Tris-HCl. pH 8.0, 100mM NaCl, ImM EDTA) and chloroform/isoamylalcohol (24:1). The DNA was precipitated in 70% ethanol and resuspended in TE buffer (lOmM Tris-HCl, pH7.5, ImM EDTA). Isolation of the ILTV Glycoprotein B Gene:
  • Isolation of the ILTV glycoprotein B (gpB) gene was performed using the polymerase chain reaction (PCR). Reactions were carried out in 50 ⁇ l volumes comprising 50ng of ILTV genomic DNA, 3ng of each oligonucleotide primer, 50mM KCl, 10mM Tris- HCl, pH8.4, 2.5mM MgCl 2 , 200 ⁇ g/ml BSA, 200mM dATP, 200 ⁇ M dCTP, 200 ⁇ M dGTP, 200 ⁇ M dTTP and 1 unit of Taq polymerase (Cetus).
  • PCR polymerase chain reaction
  • Reaction mbctures were overlayed with an equal volume of paraffin oil and heated to 94 °C for 1 min, cooled to 55 °C for 2 min and heated to 72 °C for 5 min. This cycle was repeated 35 times using a Perkin Elmer Cetus DNA Thermal Cycler.
  • oligonucleotides were designed to isolate the gpB gene by use of the PCR, based on sequence data presented elsewhere in this patent.
  • the oligonucleotides bound to the ILTV gpB gene at nucleotides 140-172 and 3000-3030 as presented in this patent.
  • BamHl restriction enzyme sites were engineered into the oligonucleotides.
  • the isolated gpB gene was inserted into FPV using the plasmid vehicle pAF09 (provided by Dr. David Boyle, CSIR0, Australian Animal Health Lab., Geelong, Australia).
  • This plasmid vehicle is suitable for the insertion of foreign genes into the FPV thymidine kinase (TK) gene, and contains the E. coli xanthine-guanine phosphoribosyl transferase (Ecogpt) gene under the transcriptional control of the W P7.5 promoter, acting as a co-expressed selectable marker as previously described by Boyle and Coupar (1988a).
  • pAF09 also contains the E.coli LacZ gene under the transcription control of the FPV late promoter (Kumar and Boyle, 1990) allowing rapid identification of FPV recombinants (Prideaux et.al. 1990).
  • the ILTV gpB gene was introduced into the BamHI site of pAF09 ( Figure 18A; pAF09-gpB), 3' to the FPV E/L promoter (Kumar and Boyle, 1990).
  • the gpB gene and FPV promoter were aligned in such away that the gpB gene translation initiation codon was in frame with initiation codon of the E/L promoter, producing a fusion gene with 4 FPV amino acids upstream of the ILTV gpB gene.
  • the FPV recombinant (FPV-gpB) was constructed using the protocol described previously by Boyle and Coupar (1988b) utilising the co-expressed Ecogpt gene for recombinant virus selection.
  • the co-expression of the Ecogpt gene enables recombinant virus to replicate in medium containing MXHAT (MXHAT: 2 ⁇ g/ml mycophenolic acid, 250 ⁇ g/ml hypoxanthine, 0.4 ⁇ M aminopterin and 320 ⁇ M thymidine) selective conditions (Boyle and Coupar, 1988a).
  • Recombinant virus expressing ⁇ -galactosidase were selected by plaquing under non-selective conditions and staining with X-Gal (200 ⁇ /ml) in growth medium containing 1% agar. Recombinants expressing ⁇ -galactostdase produced characteristic b.ue plaques (Chakrabarti et.al., 1985; Panicali et.al, 1986). Recombinant virus was plaque purified three times prior to the production of working stocks.
  • Results of ILTV antigen ELISA's from tracheal exudates of all birds are given in Table 7 as percentage of birds protected against challenge with ILTV CSW-1. From Table 7 it can be seen that all birds vaccinated with the commercial ILTV SA2 vaccine were protected from challenge with ILTV CSW-1. The unvaccinated, and FPV M vaccinated birds both showed a protection level of 17%. This protection may be the result of insufficient challenge, or failure of virus to enter the trachea. The FPV-gpB recombinant protected 58% of birds challenged, significantly (Fisher's exact test) higher than the non-ILTV vaccinated birds. Table 1 ILTV proteir recognised by Immune chicken sera using Western blotting of a detergent extract of SA-2-lnfected cells separated by SDS-PAGE
  • glycoprotein 43 1.61 ⁇ 0.24 f 6/6 0 71
  • Live virus 153 2.49 ⁇ 0.429 6/6 0 100 a Geometric mean virus neutralization titre (reciprocal) of groups of 16-17 chickens.
  • e Protection was assessed 4 weeks after secondary vaccination by the absence of viral antigen in the trachea at day 5 after intratracheal challenge.
  • ILTV glycoprotein B promoter expression of the marker gene CAT was linked to the CAT gene and 1 or 10ug of plasmid DNA transfected into CK cells. Enzymes activity was determined for a minimum of two plates for each assay, following infection with ILTV (+ ILTV), or mock infection (-ILTV). Results are expressed as counts per minute.
  • ILTV SA2, FPV M, PBS or FPV-gpB are examples of ILTV SA2, FPV M, PBS or FPV-gpB.
  • Groups C and D revaccinated, in opposite wing web, as for Day O.

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Abstract

Vaccin à sous-unités non infectieux destiné à combattre le virus de la laryngotrachéite infectieuse (ILTV) comprenant en tant qu'immunogène actif au moins une glycoprotéine de ILTV, ou un peptide immunogène dérivé de ladite glycoprotéine, et éventuellement un adjuvant. La production de polypeptides présentant l'antigénicité de glycoprotéines ILTV au moyen d'ADN recombiné est décrite. On décrit également des vaccins à virus vivant recombiné contenant des séquences de gène pour des glycoprotéines ILTV, un virus ITL recombiné ayant de l'ADN hétérologue inséré dans une région non essentielle et l'utilisation de régions stimulatrices du ILTV, dans le virus recombiné.
PCT/AU1991/000383 1990-08-24 1991-08-23 Vaccin contre le virus de la laryngotracheite infectieuse WO1992003554A1 (fr)

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Publication number Priority date Publication date Assignee Title
WO1995008622A1 (fr) * 1993-09-24 1995-03-30 Syntro Corporation Virus de recombinaison de la laryngotracheite infectieuse et utilisations desdits virus
US5443831A (en) * 1991-10-29 1995-08-22 University Of Delaware Gene encoding glycoprotein B of Infectious Laryngotracheitis Virus
WO1996000791A1 (fr) * 1994-06-30 1996-01-11 The Board Of Trustees Of The University Of Illinois Virus de recombinaison de la laryngo-tracheite infectieuse et vaccin
WO1996021034A1 (fr) * 1994-12-30 1996-07-11 Rhone Merieux Vaccin vivant recombinant aviaire
WO1996029396A1 (fr) * 1995-03-23 1996-09-26 Syntro Corporation Virus de la laryngotracheite infectieuse recombine et ses utilisations
EP0719864A3 (fr) * 1994-12-30 1996-12-04 Rhone Merieux Vaccin vivant recombinant aviaire, utilisant comme vecteur un virus herpès aviaire
WO1997049826A1 (fr) * 1996-06-27 1997-12-31 Merial Vaccin vivant recombinant aviaire, utilisant comme vecteur le virus de la laryngotracheite infectieuse aviaire
WO1998033928A1 (fr) * 1997-01-31 1998-08-06 Merial Vaccin vivant recombinant aviaire, utilisant comme vecteur le virus de la laryngotracheite infectieuse aviaire
US5853733A (en) * 1993-02-26 1998-12-29 Syntro Corporation Recombinant herpesvirus of turkeys and uses thereof
US5928648A (en) * 1985-09-06 1999-07-27 Syntro Corporation Recombinant herpesvirus of turkeys and uses thereof
US5965138A (en) * 1985-09-06 1999-10-12 Syntro Corporation Recombinant chimeric virus and uses thereof
US6033670A (en) * 1996-12-16 2000-03-07 Merial Recombinant live avian vaccine, using as vector the avian infectious laryngotracheitis virus
US6875856B2 (en) 1993-09-24 2005-04-05 Syntro Corporation Recombinant infectious laryngotracheitis virus and uses thereof
WO2013057236A1 (fr) 2011-10-21 2013-04-25 Intervet International B.V. Produits de recombinaison d'un virus non pathogène de la maladie de marek qui codent des antigènes du virus infectieux de la laryngotrachéite et du virus de la maladie de newcastle
US9096869B2 (en) 2011-10-21 2015-08-04 Intervet, Inc. Recombinant nonpathogenic MDV vector providing multivalent immunity
WO2017216287A1 (fr) 2016-06-17 2017-12-21 Intervet International B.V. Constructions de recombinaison d'un virus non pathogène de la maladie de marek codant pour des antigènes du virus infectieux de la laryngotrachéite et du virus de la maladie infectieuse de la bourse de fabricius
WO2019072964A1 (fr) 2017-10-12 2019-04-18 Intervet International B.V. Constructions de virus de la maladie de marek non pathogènes recombinantes codant de multiples antigènes hétérologues

Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1990002802A2 (fr) * 1988-09-13 1990-03-22 Institute For Animal Health Limited Sequences de nucleotides virales

Patent Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1990002802A2 (fr) * 1988-09-13 1990-03-22 Institute For Animal Health Limited Sequences de nucleotides virales

Non-Patent Citations (4)

* Cited by examiner, † Cited by third party
Title
ARCHIVES OF VIROLOGY, Volume 115, issued December 1990, (SPRINGER-VERLAG), J.J. YORK and K.J. FAHEY, "Humoraland cell-mediated immune responses to the glycoproteins of infectious laryngotracheitis herpesvirus", (see pages 289-297). *
ARCHIVES OF VIROLOGY, Volume 115, issued December 1990, (SPRINGER-VERLAG), J.J. YORK, S. SONZA et al., "Antigens of infectious laryngotracheitis herpesvirus defined by monoclonal antibodies", (see pages 147-162). *
TRENDS IN BIOTECHNOLOGY, Volume 7, No. 10, issued October 1989, (ELSEVIER SCIENCE PUBLISHERS LTD, U.K.), FINKELSTEIN and R.F. SILVA, "Live recombinant vaccines for poultry", (see whole document). *
VIROLOGY, Volume 161, No. 2, issued 1987, (ACADEMIC PRESS INC.,), J.J. YORK, S. SONZA et al., "Immunogenic Glycoproteins of Infectious Laryngotracheitis Herpesvirus", (see pages 340-347). *

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US5965138A (en) * 1985-09-06 1999-10-12 Syntro Corporation Recombinant chimeric virus and uses thereof
US5928648A (en) * 1985-09-06 1999-07-27 Syntro Corporation Recombinant herpesvirus of turkeys and uses thereof
US5443831A (en) * 1991-10-29 1995-08-22 University Of Delaware Gene encoding glycoprotein B of Infectious Laryngotracheitis Virus
US5853733A (en) * 1993-02-26 1998-12-29 Syntro Corporation Recombinant herpesvirus of turkeys and uses thereof
US6984728B2 (en) 1993-09-24 2006-01-10 Schering Corporation Recombinant infectious laryngotracheitis virus and uses thereof
US6875856B2 (en) 1993-09-24 2005-04-05 Syntro Corporation Recombinant infectious laryngotracheitis virus and uses thereof
US7045598B2 (en) 1993-09-24 2006-05-16 Schering-Plough Animal Health Recombinant infectious laryngotracheitis virus and uses thereof
US7364893B2 (en) 1993-09-24 2008-04-29 Schering-Plough Animal Health Corp. Recombinant infectious laryngotracheitis virus and uses thereof
WO1995008622A1 (fr) * 1993-09-24 1995-03-30 Syntro Corporation Virus de recombinaison de la laryngotracheite infectieuse et utilisations desdits virus
US7892564B2 (en) 1993-09-24 2011-02-22 Schering-Plough Animal Health Corp. Recombinant infectious laryngotracheitis virus and uses thereof
US7501491B2 (en) 1993-09-24 2009-03-10 Schering-Plough Animal Health Corp. Recombinant infectious laryngotracheitis virus and uses thereof
WO1996000791A1 (fr) * 1994-06-30 1996-01-11 The Board Of Trustees Of The University Of Illinois Virus de recombinaison de la laryngo-tracheite infectieuse et vaccin
EP0719864A3 (fr) * 1994-12-30 1996-12-04 Rhone Merieux Vaccin vivant recombinant aviaire, utilisant comme vecteur un virus herpès aviaire
US6045803A (en) * 1994-12-30 2000-04-04 Merial Live recombinant avian vaccine using an avian herpesvirus as vector
WO1996021034A1 (fr) * 1994-12-30 1996-07-11 Rhone Merieux Vaccin vivant recombinant aviaire
WO1996029396A1 (fr) * 1995-03-23 1996-09-26 Syntro Corporation Virus de la laryngotracheite infectieuse recombine et ses utilisations
US6153199A (en) * 1996-06-27 2000-11-28 Merial Avian recombinant live vaccine using, as vector, the avian infectious laryngotracheitis virus
FR2750866A1 (fr) * 1996-06-27 1998-01-16 Rhone Merieux Vaccin vivant recombinant aviaire, utilisant comme vecteur le virus de la laryngotracheite infectieuse aviaire
WO1997049826A1 (fr) * 1996-06-27 1997-12-31 Merial Vaccin vivant recombinant aviaire, utilisant comme vecteur le virus de la laryngotracheite infectieuse aviaire
US6033670A (en) * 1996-12-16 2000-03-07 Merial Recombinant live avian vaccine, using as vector the avian infectious laryngotracheitis virus
AU735265B2 (en) * 1997-01-31 2001-07-05 Merial Avian recombinant live vaccine using, as vector, the avian infectious laryngotracheitis virus
US6306400B1 (en) 1997-01-31 2001-10-23 Merial Avian recombinant live vaccine using, as vector, the avian infectious laryngotracheitis
FR2758986A1 (fr) * 1997-01-31 1998-08-07 Rhone Merieux Vaccin vivant recombinant aviaire, utilisant comme vecteur le virus de la laryngotracheite infectieuse aviaire
WO1998033928A1 (fr) * 1997-01-31 1998-08-06 Merial Vaccin vivant recombinant aviaire, utilisant comme vecteur le virus de la laryngotracheite infectieuse aviaire
US9409954B2 (en) 2011-10-21 2016-08-09 Intervet Inc. Recombinant non-pathogenic marek's disease virus constructs encoding infectious laryngotracheitis virus and newcastle disease virus antigens
US8932604B2 (en) 2011-10-21 2015-01-13 Intervet Inc. Recombinant non-pathogenic marek's disease virus constructs encoding infectious laryngotracheitis virus and newcastle disease virus antigens
US9096869B2 (en) 2011-10-21 2015-08-04 Intervet, Inc. Recombinant nonpathogenic MDV vector providing multivalent immunity
WO2013057236A1 (fr) 2011-10-21 2013-04-25 Intervet International B.V. Produits de recombinaison d'un virus non pathogène de la maladie de marek qui codent des antigènes du virus infectieux de la laryngotrachéite et du virus de la maladie de newcastle
WO2017216287A1 (fr) 2016-06-17 2017-12-21 Intervet International B.V. Constructions de recombinaison d'un virus non pathogène de la maladie de marek codant pour des antigènes du virus infectieux de la laryngotrachéite et du virus de la maladie infectieuse de la bourse de fabricius
US11596687B2 (en) 2016-06-17 2023-03-07 Intervet Inc. Recombinant non-pathogenic Marek's disease virus constructs encoding infectious laryngotracheitis virus and infectious bursal disease virus antigens
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WO2019072964A1 (fr) 2017-10-12 2019-04-18 Intervet International B.V. Constructions de virus de la maladie de marek non pathogènes recombinantes codant de multiples antigènes hétérologues
US11229698B2 (en) 2017-10-12 2022-01-25 Intervet Inc. Recombinant non-pathogenic Marek's Disease virus constructs encoding multiple heterologous antigens
US12239704B2 (en) 2017-10-12 2025-03-04 Intervet Inc. Recombinant non-pathogenic Marek's disease virus constructs encoding multiple heterologous antigens

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