EP0948637A1 - Live recombinant avian vaccine, using the infectious laryngotracheitis avian virus as vector - Google Patents

Live recombinant avian vaccine, using the infectious laryngotracheitis avian virus as vector

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Publication number
EP0948637A1
EP0948637A1 EP97952066A EP97952066A EP0948637A1 EP 0948637 A1 EP0948637 A1 EP 0948637A1 EP 97952066 A EP97952066 A EP 97952066A EP 97952066 A EP97952066 A EP 97952066A EP 0948637 A1 EP0948637 A1 EP 0948637A1
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Prior art keywords
virus
recombinant
plasmid
nucleotide sequence
promoter
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German (de)
French (fr)
Inventor
Jean-Christophe Francis Audonnet
Michel Joseph Marie Bublot
Eliane Louise Françoise LAPLACE
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Boehringer Ingelheim Animal Health France SAS
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Merial SAS
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    • C12N15/09Recombinant DNA-technology
    • C12N15/63Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
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Definitions

  • the present invention relates to vaccines for avian use based on infectious laryngotracheitis virus (ILTV), into which has been inserted, by genetic recombination, at least one heterologous nucleotide sequence, in particular coding for and expressing, an antigenic polypeptide of an avian pathogen, under conditions ensuring immunization leading to effective protection of the animal vaccinated against said pathogen.
  • ILTV infectious laryngotracheitis virus
  • Infectious laryngotracheitis virus is an alphaherpesvirus (B. Roizman, Arch. Virol. 1992. 123. 425-449) which causes an important respiratory pathology (infectious laryngotracheitis or ILT) in chicken (LE Hanson and TJ Bagust, Diseases of Poultry 9th edn 1991. pp 485-495. Ames, lowa State University Press).
  • the vaccines currently available against this condition contain an attenuated strain which can be administered by different routes including the intranasal, conjunctival, cloacal in the drinking water and aerosol (LE and T Hanson. Bagust, Diseases of Poultry 9th Edition 1991. pp 485-495. Ames, Iowa State University Press).
  • ILTV Molecular biology studies ILTV have characterized the viral genome (MA Johnson et al., Arch. Vlrot. 1991. 119. 181-198) and iden t ify some virus genes (AM Griffin, J. Gen. Virol. 1989. 70. 3085-3089) including the genes coding for thy idine inase (UL23) (AM Griffin and MEG Boursnell, /. Gen. Virol. 1990. 71. 841-850; CL. Eeier et al ., Avion Dis. 1991. 35. 920-929), the glycoprotein gB (UL27) (AM Griffin, /. Gen. Virol. 1991. 72. 393-398;. Kongsuwan et al., Virology 1991. 184.
  • the aim of the present invendon is to develop an avian vaccine based on a recombinant ILTV virus expressing a heterologous gene, this virus being capable of replicating and of inducing immunity in the vaccinated host while maintaining good safety.
  • Another objective of the invention is to provide such a vaccine which is at the same time particularly effective against infectious laryngotracheitis (ILT).
  • ILT infectious laryngotracheitis
  • Another objective of the invention is to propose such a vaccine which can be used in mass vaccination by mucosal route, for example by aerosol route or in drinking water, in such a way that the replication of the virus at the mucosal level allows to induce mucosal and systemic immunity.
  • mucosal immunity will be particularly effective against respiratory diseases and con t re other diseases for which the input po ⁇ e pathogen is mucosal.
  • Another object of the invention is to provide such a vaccine which can be used both in adults and in young animals.
  • a specific objective is to propose such a vaccine usable in mass vaccination by mucosal route of very young animals such as day-old chicks.
  • Another objective of the invention is to provide a vaccine against the ILT which has an increased efficacy compared to the parental strain and which may even possibly allow the insertion and expression of a heterologous gene.
  • a genomic region which has proved quite suitable as a site for the insertion of heterologous genes.
  • NDV Newcastle disease virus
  • MDV Mare Disease Virus gB glycoprotein
  • IBDV Gumboro Disease Virus VP2 protein
  • IBV Infectious Bronchitis Virus S and M proteins
  • the present invention therefore relates to a live recombinant avian vaccine comprising, as a vector, the ILTV virus comprising at least one heterologous nucleotide sequence, in particular coding for and expressing, an antigenic polypeptide of an avian pathogenic agent, inserted into the locus d 'inse ⁇ ion formed of the intergene located between the stop codons of COL-B and COL-C of the ILTV virus and which, in a particular ILTV strain, is defined between nucleotides 908 and 994 with the sequence SEQ ID NO: l . If the specific sequence described in the request (SEQ ID NO: 1) comes from the ILTV vaccine strain T-20 12-8-66 from Select Laboratories (10026 Main
  • the COL-B and COL-C correspond respectively to the UL3.5 and UL4 genes described in the article by W. Fuchs and T.C. Mettreleiter (/. Gen. Virol. 1996. 77. 2221-
  • SEQ ID NO: 19 reproduces for this pathogenic strain, the sequence equivalent to SEQ ID NO: 1.
  • the intergene serving as insertion locus according to the invention is included in SEQ ID NO: 19 between nucleotides 908 and 994.
  • heterologous sequence is meant a sequence which does not originate from this insertion locus, c '' is to say both a sequence not originating from the ILTV virus, as well as a sequence originating from another genomic region of this virus, or also originating from another ILTV strain, in particular a virulent strain.
  • insertion into the insertion region is meant in particular simple insertion or after total or partial deletion of the insertion locus.
  • One or more expression cassettes can be inserted, each comprising at least one sequence to be expressed.
  • a strong eukaryotic promoter such as the immediate early CMV promoter (IE), the Rous sarcoma virus LTR (RSV), and the early promoter of the SV40 virus.
  • immediate early CMV promoter IE
  • the CMV IE promoter can be the human promoter (HCMV IE) or the murine promoter (MCMV IE), or else a CMV IE promoter from another origin, for example from monkeys, rats, guinea pigs or pigs.
  • promoters of viral or cellular origin can also be used.
  • promoters of viral origin mention may also be made of promoters of genes for ILTN virus (genes considered to be early-immediate (ICP4, ICP27, ...), early fthymidine kinase, D ⁇ A helicase, ribonucleotide reductase, ...), or late (gB, gD, gC, gK, ...)) .
  • Marek's disease virus (MDV) gB, gC, pp38, ppl4, ICP4, Meq, ... genes
  • h ⁇ ès turkey virus he ⁇ èsvirus of rurkey
  • the nucleotide sequence inserted into the ILTN vector to be expressed can be any sequence coding for an antigenic polypeptide, of an avian pathogenic agent, capable, once expressed under the favorable conditions provided by the invention, of ensuring immunization leading to effective protection of the animal vaccinated against the pathogen. It will therefore be possible to insert, under the conditions of the invention, the nucleotide sequences coding for the antigens of interest for a given disease.
  • This nucleotide sequence inserted into the ILTV vector can also code for an immunomodulatory polypeptide, and in particular a cytokine.
  • the vaccines according to the invention can be used for vaccination in ovo, day-old chicks or more and adults.
  • Different routes of administration could be used: the parenteral route, or the mucosa routes such as oronasale (drinking water, aerosol), conjunctival (drop in the eye) or cloacale, with a preference for the routes allowing a mucosal vaccination of mass (drinking water, aerosol).
  • the invention proves to be particularly useful both for protection against respiratory pathologies and against systemic pathologies by blocking the natural pathways of entry of the pathogenic agent.
  • the invention can in particular be used for the insertion of a nucleotide sequence suitably coding for an antigenic protein of the ⁇ DV virus and in particular, the glycoprotein H ⁇ or the glycoprotein F.
  • a nucleotide sequence suitably coding for an antigenic protein of the ⁇ DV virus and in particular, the glycoprotein H ⁇ or the glycoprotein F.
  • the recombinant ⁇ ewcastle disease vaccine will preferably contain 10 to 10 4 PFU / dose.
  • nucleotide sequences coding for antigens of other avian pathogens are inserted of nucleotide sequences coding for antigens of other avian pathogens, and in particular, but not limited to, antigens of the ⁇ arek disease virus, in particular genes gB, gC, gD, and gH + gL (WO-A-90 / 02803), the Gumboro disease virus, in particular the VP2 gene, the infectious bronchitis virus (IBV), in particular the S and M genes (M. Binns et al., J. Gen. Virol. 1985. 66. 719-726; M. Boursnell et al., Virus Research 1984. 1.
  • CAV chicken anemia virus
  • ILTN virus in particular the genes coding for gB (AM Griffin, /. Gen. Virol.
  • gp60 KK Kongsuwan et al., Genes Virus 1993. 7. 297-303
  • swelling head syndrome virus swollen head syndrome
  • chicken pneumovirus or turkey rhinotracheitis vires TRTV
  • pneumovirus in particular the fusion glycoprotein F (Q. Yu et al., J. Gen. Virol. 1991. 72. 75-81), or the attachment glycoprotein G (R. Ling et al., J. Gen. Virol.
  • the doses will preferably be the same as those for the castewcastie vaccine.
  • the promoter CMV IE is associated with another promoter so that their 5 'ends are adjacent (which implies transcriptions in opposite directions), which makes it possible to insert, in the insertion zone, two nucleotide sequences , one under the control of the CMV IE promoter, the other under that of the associated promoter.
  • the associated promoter may in particular be a promoter of a gene of the ILTN virus or of the MDV or HVT virus.
  • An interesting case of the invention is a vaccine comprising a nucleotide sequence coding for HN of NDV and a nucleotide sequence coding for F of
  • NDV or an antigen of another avian disease, in particular those mentioned above, one of the genes being under the control of the CMV IE promoter, and the other under the control of the associated promoter.
  • IVS Internai Ribosome Entry Site
  • SVDV swine vesicular disease virus
  • EMCV encephalomyocarditis virus
  • FMDV foot-and-mouth disease virus
  • the expression cassette for two genes would therefore have the following minimum structure: promoter - gene 1 - IRES - gene2 - polyadenylation signal.
  • the recombinant live vaccine according to the invention may therefore comprise, inserted into the insertion locus, an expression cassette successively comprising a promoter, two or more genes separated in pairs by an IRES, and a polyadenylation signal.
  • one or more other insertions, one or more mutations, or one or more deletions can be made elsewhere in the genome; if the parental strain is virulent, it is possible, for example, to inactivate (by deletion, insertion or mutation) of the genes involved in virulence such as the thymidine kinase gene, the ribonucleotide reductase gene, the gE gene, etc. In all cases, insertion into another locus than that described in the invention makes it possible to express other genes,
  • the subject of the present invention is also a vaccine against ILT comprising a recombinant ILTV virus in which genes coding for major immunogens of ILTV have been inserted upstream, preferably the genes coding for gB (AM Griffin, J. Gen. Virol. 1991. 72. 393-398), or for gD (MA Johnson et ai, DNA Séquence- Journal of Sequencing and Mapping 1995. Vol. 5. ppl91- 194. Harwood Académie Publishers GmbH), or for gp60 (KK Kongsuwan and ai , Virus Genes 1993. 7. 297-303), an exogenous promoter, in particular a strong promoter as described above. This increases the level of expression of one or more of these genes and thus leads to a vaccine with increased efficacy against ILT.
  • a recombinant ILTV virus in which genes coding for major immunogens of ILTV have been inserted upstream, preferably the genes coding for gB (AM Griffin,
  • the present invention also relates to a multivalent vaccine formula, comprising, as a mixture or to be mixed, a vaccine as defined above with another vaccine, and in particular another live recombinant avian vaccine as defined above, these vaccines comprising different inserted sequences, in particular of different pathogens.
  • the present invention also relates to a method for preparing the vaccines according to the invention, as it emerges from the description.
  • the present invention also relates to a method of avian vaccination comprising the administration of a recombinant live vaccine or of a multivalent vaccine formula as defined above. It relates in particular to such a method for vaccination in ovo, day-old chicks and adults.
  • Different routes of administration of the vaccine can be used (see above) with a preference for the routes allowing mass vaccination by mucosal route (aerosol, drinking water), the dose of vaccine being preferably chosen between 10 * and 10 * per animal.
  • the present invention also relates to an ILTV virus comprising at least one heterologous nucleotide sequence as described above inserted into the insertion locus as defined above.
  • the present invention also relates to all or part of the sequence S ⁇ Q ID NO: 1; by parts of this sequence is meant not only the characterized COLs taken individually or their fragments, but also the intergene located between the COL B and COL C and the fragments located on either side of this intergene, which may possibly include a part of this intergene, and which can serve as flanking arms for homologous recombination, a technique which is moreover well known to those skilled in the art.
  • the flanking arms can have from 100 to 800 base pairs.
  • Figure 2 Sequence of 4161 bp and translation of COLs A, B, C and D of the T-20 vaccine strain from Select Laboratories ( LT BLEN vaccine)
  • Figure 6 Diagram of the plasmid pEL158
  • Figure 9 Diagram of the plasmid pEL159
  • FIG. 12 Diagram of plasmid pEL160
  • FIG. 14 Diagram of the plasmid pEL106
  • FIG. 17 Diagram of plasmid pEL163
  • the virus used as parental virus can be chosen from the vaccine strains described in J.R. Andreasen et al. (Avion Diseases 1990. 34. 646-656) or the strain T-20 12-8-66 from Select laboratories 10026 Main Street P.O. Box 6 Berlin, Maryiand 21811, USA. It is also possible to use virulent strains such as the Lûtticken strain (see above), the N-71851 strain (ATCC VR-783) or the USDA strain 83-2, which can be attenuated by known techniques, for example that described in WO-A-95/08622.
  • Example 1 Culture of the ELTV virus:
  • the ILTV virus (strain T20 from Select Laboratories) is cultured on primary chicken kidney cells (CRP); these cells are cultured in MEM medium supplemented with 3% fetal calf serum (S VF) in culture flasks of 75 cm 2 (2 10 3 cells / cm 2 ) one or two days before inoculation.
  • CRP primary chicken kidney cells
  • S VF fetal calf serum
  • the culture of the ILTN virus can also be done on immortalized chicken liver cells, and in particular on the LMH line (.M. Schnitzlein et al. Avion Diseases 1994. 38. 211-217).
  • the culture of ILTV (2 flasks of 75 cm 2 ) is harvested and centrifuged at low speed (5000 rpm in a rotor 20, Beckman JA21 centrifuge, for 5 minutes) to remove large cellular debris. The supernatant is then ultracentrifuged (100,000 rpm TLA100.3 rotor, Beckman TL100 centrifuge, for 1 hour).
  • the pellet is then taken up in 1.6 ml of TE ⁇ -SDS (Tris pH 8.0 lOmM; EDTA ImM; ⁇ aCI 0.5M; sodium dodecyl sulfate 0.5%), and 35 ⁇ ⁇ of a proteinase K solution at 20 mg / mi are then added; the solution is incubated 3 to 4 hours in a water bath at 37 "C, and the AD ⁇ is then extracted 3 times with phenol / chloroform and 1 time with chloroform, then it is precipitated with emanol at -20 ⁇ C.
  • TE ⁇ -SDS Tris pH 8.0 lOmM; EDTA ImM; ⁇ aCI 0.5M; sodium dodecyl sulfate 0.5%
  • the pellet is rinsed with 70% ethanol, dried and resuspended in 200 ⁇ l TE (Tris pH8.0 10 mM; EDTA ImM).
  • the nucleic acid concentration is then determined using a spectrophotometer (DO 260 ). be directly digested with the appropriate restriction enzymes, and then be cloned into the plasmid pBlue Script II SIC; likewise, it can also be used in transfection experiments for obtaining a recombinant virus.
  • Example 3 Isolation and purification of recombinant ELTN virus
  • the donor plasmid composed of an expression cassette for a polypeptide inserted between two flanking regions of the insertion locus is digested with a restriction enzyme ⁇ ⁇ repet ⁇ , ⁇ O 98/27215
  • CRP cells are then transfected with the following mixture: 0.2 to 1 ⁇ g of linearized donor plasmid + 2 to 5 ⁇ g of ILTV viral DNA (prepared as in Example 2) in 300 ⁇ ⁇ of OptiMEM ' medium (Gibco BRL Cat # 041-01985H) and 100 ⁇ g of LipofectAMINE diluted in 300 ⁇ l of medium (final volume of the mixture ⁇ 600 ⁇ l). These 600 ⁇ l are then diluted in 3 ml (final volume) of medium and spread on 5.10 * CRP.
  • the mixture is left in contact with the cells for 5 hours, then eliminated and replaced with 5 ml of culture medium.
  • the cells are then left in culture for 3 to 8 days at + 37 ° C, and then, when cyropatho misleading effect appeared, they are frozen at -70 ° C.
  • the viral population is cloned dilution limit in microplates (96 wells) in order to isolate a homogeneous population of recombinant virus. These plates are left in culture for 1 to 3 days, then the supernatant is collected in an empty 96-well plate and the plate containing the supernatants is placed at 4 ⁇ C or at -70 ° C.
  • the cells remaining in the other plates are then fixed with 95% acetone for 20 to 30 minutes at -20 ° C., or for 5 minutes at room temperature.
  • An indirect immunofluoresce ⁇ ce (IFT) reaction is carried out with a monoclonal antibody directed against the expressed polypeptide to search for the ranges expressing this polypeptide.
  • a new cloning is then carried out in the same manner (in dilution limit in 96-well plates) from the supernatant present in the wells of the plates set at 4 e C or at -70 "C and corresponding to the wells having positive plaques in IFT
  • 4 successive isolation cycles are sufficient to obtain recombinant viruses whose entire progeny exhibit specific fluorescence.
  • the genomic DNA of these recombinant viruses is characterized at the molecular level by conventional PCR and Southern blot techniques using the appropriate oligonucleotides and DNA probes.
  • the isolation of recombinant virus can also be done by hybridization with a specific probe of the inserted expression cassette.
  • the viral population harvested after transfection is diluted and deposited on cells. s CRP (grown in Petri dish) so as to obtain isolated areas.
  • the medium of infection is removed and replaced with 5 ml of MEM medium containing 1% agarose, maintained molten at 42 ° C.
  • the dishes are incubated 48 to 72 hours at 37 ⁇ C in a CO 2 oven until the appearance of plaques, the agarose layer is then eliminated and a transfer of the viral plaques is carried out on a sterile membrane.
  • nitrocellulose of the same diameter as the Petri dish used for the culture This membrane is itself transferred to another nitrocellulose membrane so as to obtain an inverted "copy" of the first transfer.
  • the plaques transferred to this last copy are then hybridized, according to the usual techniques known to those skilled in the art, with a DNA fragment of the expression cassette labeled with digoxigenin (DNA Labeling Kit, Boehringer Mannheim, CAT # 1175033). After hybridization, washing and contact with the development substrate, the nitrocellulose membrane is brought into contact with an autoradiographic film. The positive hybridization images on this membrane indicate which plaques contain recombinant ILTV viruses which have inserted the expression cassette.
  • plaques corresponding to these positive plaques are cut sterile on the first nitrocellulose membrane, placed in an Eppendorf tube containing 0.5 ml of MEM medium and sonicated to release the virions from the membrane.
  • the medium contained in the Eppendorf tube is then diluted in MEM medium and the dilutions thus obtained are used to infect new cultures of CRP cells.
  • the AD ⁇ extracted from the ILTN virus was digested with the restriction enzyme Kpn1 for 2 hours at 37 ° C.
  • the restriction enzyme was then removed by extraction with phenol / chloroform, followed by precipitation with ethanol.
  • the fragments resulting from this digestion were then ligated (overnight at 14 ⁇ C) with the plasmid pBlueScriptlI SK + (pBS SK +; Stratagene) digested with Kpnl and treated with alkaline phosphatase; analysis of the clones obtained after transformation of bacteria E coli DH5 ⁇ and culture on dishes of medium supplemented with ampicilin made it possible to identify Kpnl-Kpnl inserts of different sizes, including a fragment of approximately 4.2 kb (plasmid pEL112). 15
  • the sequence between the COL B and C STOP codons can be used to insert polypeptide expression cassettes into the ILTV genome. This sequence is called the insertion locus.
  • the insertion can be done with or without deletion in the intergenic region (see example 5).
  • Example 5 Construction of the donor plasmid pEL157 for insertion into the intergenic region between COLs B and C
  • the plasmid pEL112 (7116 bp), was digested with the enzymes Not1 and Spel to isolate the fragment Notl-Spel of 4.5 kb.
  • the fragment thus digested was then treated with DNA poiymerase (Klenow fragment) in the presence of dNTP to make the ends blunt; after ligament and transformation of the E. coli bacteria, the clone pEL156 (4503 bp) was obtained.
  • the oligonucleotides EL001 (SEQ ID No: 2) and EL002 (SEQ ID No: 3) served as a primer for a first chain amplification by Taq poymerase (PCR).
  • EL002 (SEQ ID No: 3): 5 'ACGC ⁇ AATTCAAATACGAGCATTTAATTATTGCG 3'
  • EL003 (SEQ ID No: 4): 5 'TCTCCAGAATCGCTGGAGTGTCC 3'
  • PCRs were carried out in the presence of PCR buffer, of dNTP, of DNA of the plasmid pEL156, of Taq poymerase, and for the first PCR, of oligonucleotides EL001 and EL002, and for the second PCR, of oligonucleotides EL003 and EL004.
  • RI for 2 h at 37 ⁇ C to give a fragment p7i ⁇ I- £ ccRI DNA of 85 bp which was eluted after agarose gel electrophoresis.
  • the plasmid pEL156 was digested with the enzymes Xbal and Xhol.
  • the two fragments of PCR-XE ⁇ 1-EcoI (120 bp) and ⁇ 7 ⁇ oI -E> RI (85 bp) were ligated overnight at 14 ° C. with the plasmid pEL156 digested with Xbal and Xhol. mation of E.
  • the clone pEL157 (4531 bp), comprising an EcoRl - Hindlll - EcoRV - Sali polylinker was obtained (see diagram for obtaining pEL157 in FIG. 3 ).
  • VP2 gene from the Gumboro disease virus (IBDV) and construction of a VP2 expression cassette under the control of the HCMV IE promoter
  • the plasmid pEL004 (see FIG. 4; ⁇ plasmid pGH004 described in the patent application French 92.13109) containing the IBDV VP2 gene in the form of a BamHI-HindIII cassette was digested with BamHI and Xbal to isolate the BamHI-Xbal fragment (truncated VP2 gene) of 1104 bp.
  • This fragment was cloned into the vector pBS SK +, previously digested with Xbal and BamHI to give the plasmid pEL022 of 4052 bp (FIG. 4.
  • the vector pBS-SK-f- was digested with £ c ⁇ RV and Xbal, then ligated on itself to give pBS-SK * (modified)
  • the plasmid pEL004 was digested with Kpnl and Hindlll to isolate the Kpnl-Hindlll fragment of 1387 bp containing the complete IBDV VP2 gene.
  • Plasmid pEL022 was digested with BamKL and NotI to isolate the BamHI-NotI fragment of 1122 bp (fragment A). Plasmid pEL023 was digested with BamHI and NotI to isolate the fragment BamHI-NotI of 333 bp (fragment B).
  • Plasmid pEL024 was digested with NotI to isolate the f ragment Notl-Notl of 1445 bp. This fragment was ligated with the plasmid pCMV ⁇ (Clontech Ca 6177-1, FIG. 5), previously digested with Notl, to give the plasmid pEL026 of 5095 bp (FIG. 5).
  • the plasmid pEL026 was digested with JS ⁇ RI, Sali and Xmnl to isolate the EcoKl-SalI fragment of 2428 bp. This fragment was ligated with the vector pBS-SK +, previously digested with ⁇ c ⁇ RI and SalI, to give the plasmid pEL027 of 5379 bp (FIG. 5).
  • the plasmid pEL027 was digested with £ c ⁇ RI, S ⁇ and Xmnl to isolate the £? RI-Sall fragment of 2428 bp. This fragment was ligated into the plasmid pEL157 (see example 5 and FIG. 3), previously digested with £ c ⁇ RI and SalI, to give the plasmid pEL158 of 6950 bp (FIG. 6).
  • vILTV8 virus was isolated and purified after cotransfection of the DNA of the plasmid pEL158 previously linearized with the enzyme Kpnl and of the viral DNA, as described in Example 3.
  • This recombinant contains an HCMV-IE / IBDV VP2 cassette in the intergenic site between the COLs B and C of the ILTV virus (see example 5).
  • Example 7 Construction of the donor plasmid pEL159 for the insertion of an expression cassette for the VP2 gene of EBDV under the control of the MCMN EE promoter in the intergenic site between the COLs B and C and isolation of vELTN9:
  • the plasmid pCMV ⁇ (Clontech Catf 6177-1, FIG. 7) was digested with Sali and Smal to isolate the Sall-Smal ragment of 3679 bp containing the lacZ gene as well as the poiy-adenylation signal of the late gene of the SV40 virus. This fragment was inserted into the vector pBS-SK +, previously digested with SalI and Ec ⁇ RV, to give the plasmid pCD002 of 6625 bp (FIG. 7). This plasmid contains the lacZ reporter gene but no promoter is located upstream of this gene.
  • the MCMI virus Smiui strain was obtained from the American Type Culture Collection, Rockville, Maryland, USA (ATCC ⁇ "VR-194). This virus was cultured on Balb / C mouse embryo cells and viral AD of this virus was prepared as described by Ebeling A. et al. (J. Virol. 1983. 47. 421-433) This viral genomic DNA was digested with Pstl to isolate the Pstl-Pstl fragment of 2285 bp. fragment was cloned into the vector pBS-SK, previously digested with Pstl and treated with alkaline phosphatase, to give the plasmid pCD004 (FIG. 7).
  • the plasmid pCD004 was digested with Hpal and Pstl to isolate the Hpal-Pstl fragment from 1389 bp which contains the promoter / activator region of the Immediate-Early gene of the murine cytomegalovirus (Murine CytoMegalo Virus ⁇ MCMV) (Dorsch-Hâsler K. et al. Proc. Natl. Acad. Sci. 1985. 82. 8325-8329, and request WO-A-87/03905).
  • This fragment was cloned into the plasmid pCD002, previously digested with Pstl and Smal, p or give the plasmid pCD009 of 8007 bp ( Figure 7).
  • a double-stranded oligonucleotide was obtained by hybridization of the following two oligonucleotides:
  • the plasmid pEL024 (see example 6, paragraph 6.1 and FIG. 5) was digested with HindIII and NotI to isolate the HindIII-NotI fragment of 1390 bp (fragment A).
  • the plasmid pEL027 (see example 6, paragraph 6.1 and FIG. 5) was digested with HindIII and SalI to isolate the HindIII-SalI fragment of 235 bp (fragment B). Fragments A and B were ligated together with the plasmid pEL068, previously digested with NotI and SalI, to give the plasmid pEL070 of 5908 bp (FIG. 8).
  • This plasmid therefore contains a casse ⁇ e expression consisting of the IE promoter of MCMV, the VP2 gene and the polyA signal of SV40.
  • the plasmid pEL070 was digested with JE oRI, Sali and Xmnl to isolate the EcoTU.- Sali fragment of 3035 bp. This fragment was ligated into the plasmid pEL157 (see example 5 and FIG. 3), previously digested with EcoRL and SalI, to give the plasmid pEL159 of 7545 bp (FIG. 9). This plasmid allows the insertion of the MCMV-IE / IBDV-VP2 expression cassette into the intergenic site between the COLs B and C of the ILTV virus.
  • vILTN9 virus was isolated and purified after cotransfection of the AD ⁇ of the plasmid pEL159 previously linearized with the enzyme BgR and the viral DNA, as described in Example 3.
  • This recombinant contains an MCMV-IE / IBDV VP2 cassette in the intergenic site between the COLs B and C of the ILTN virus (see example 5).
  • Example 8 Construction of the donor plasmid pEL160 for the insertion of an expression cassette for the NDV HN gene into the intergenic site between COLs B and C and isolation of vELTN10:
  • NDV Newcastle disease virus
  • a pBR322 clone containing the end of the fusion (F) gene, the entire hemagglutinin- neuraminida.se (HN) gene and the start of the poiymerase gene was identified pHNOl.
  • the sequence of the NDV HN gene contained in this clone is presented in FIG. 10 (SEQ ID NO: 8).
  • the plasmid pHNOl was digested with Sphl and Xbal to isolate the Sphl-Xbal fragment of 2520 bp. This fragment was ligated with the vector pUC19, previously digested with Sphl and Xbal, to give the plasmid pHN02 of 5192 bp.
  • the plasmid pHN02 was digested with ClaI and Pstl to isolate the Cl ⁇ l-Pstl fragment of 700 bp (fragment A).
  • a PCR was carried out with the following oligonucleotides:
  • EL073 (SEQ ID NO: 10) 5 'GTATTCGGGACAATGC 3' and the pHN02 matrix to produce a PCR fragment of 270 bp.
  • This fragment was digested with HindIII and Pst1 to isolate a HindIII-Pst1 fragment of 220 bp (fragment B).
  • Fragments A and B were ligated together with the vector pBS-SK- previously digested with ClaI and HindIII, to give the plasmid pEL028 of 3872 bp (FIG. 11).
  • the plasmid pHN02 was digested with Bsphl and ClaI to isolate the Bsp l-ClaI fragment of 425 bp (fragment C).
  • a PCR was carried out with the following oligonucleotides: EL074 (SEQ ID NO: 11) 5 'GTGACATCACTAGCGTCATCC 3' EL075 (SEQ ID NO: 12)
  • fragment E 5 'CCGCATCATCAGCGGCCGCGATCGGTCATGGACAGT 3' and the pHN02 matrix to produce a PCR fragment of 465 bp.
  • This fragment was digested with Bsphl and Notl to isolate the Bsphl-Notl fragment of 390 bp (fragment D).
  • Fragments C and D were ligated together with the vector pBS-SK +, previously digested with ClaI and NotI, to give the plasmid pEL029bis of 3727 bp (FIG. 11).
  • the plasmid pEL028 was digested with ClaI and SacIl to isolate the ClaI-SacII fragment of 960 bp (fragment E).
  • the plasmid pEL029bis was digested with ClaI and NotI to isolate the ClaI-NotI fragment of 820 bp (fragment F). Fragments E and F were ligated together with the vector pBS-SK-, previously digested with NotI and Sacll, to give the plasmid pEL030 of 4745 bp (FIG. 11).
  • the plasmid pEL030 was digested with NotI to isolate the NotI-NotI fragment of 1780 bp (whole NDV HN gene) . This fragment was inserted into the NotI sites of the plasmid pEL159 (Example 7, FIG. 9) in place of the NotI-NotI fragment of 1405 bp containing the gene coding for the protein VP2 of IBDV; this cloning made it possible to isolate the plasmid pEL160 of 7921 bp (FIG. 12). This plasmid allows the insertion of the MCMV-IE / NDV-HN expression cassette into the intergenic site between the COLs B and C of the ILTN virus.
  • the vILTVIO virus was isolated and purified after cotransfection of the AD ⁇ of the plasmid pEL160 previously linearized with the enzyme BgH and of the AD virai, as described in Example 3.
  • This recombinant contains an MCMV-IE / ⁇ DV H ⁇ cassette in the intergenic site between COLs B and C of the ILTV virus (see example 5).
  • Example 9 Construction of the donor plasmid pEL161 for the insertion of an expression cassette for the ⁇ DV F gene into the intergenic site between COLs B and C and isolation of ylLTVll:
  • NDV Newcastle disease virus
  • pNDV81 A clone originating from the DNA bank complementary to the genome of the Newcastle disease virus (see example 8, paragraph 8.1) and containing the fusion gene (F ) in its entirety was called pNDV81.
  • This plasmid has been described previously and the sequence of the NDV F gene present on this clone has been published (Taylor J. et al. J. Virol., 1990, 64, 1441-1450).
  • the plasmid pNDV81 was digested with Narl and Pstl to isolate the Narl-Pstl fragment of 1870 bp (fragment A).
  • a PCR was carried out with the following oligonucleotides:
  • Plasmid pEL033 was digested with NotI to isolate the 1935 bp NotI-NotI fragment (whole F gene). This fragment was inserted into the NotI sites of the plasmid pEL159 (Example 7, FIG. 9) in place of the NotI-NotI fragment of 1405 bp containing the gene coding for the protein VP2 of ITBDV; this cloning made it possible to isolate the plasmid pEL161 from 8074 bp (FIG. 14). This plasmid allows the insertion of the MCMV-IE / NDV-F expression cassette into the intergenic site between the COLs B and C of the ILTN virus.
  • vILTVll 9.3 - Isolation and purification of the recombinant virus vILTVll
  • the vILT ll virus was isolated and purified after cotransfection of the AD ⁇ of the plasmid pEL161 previously linearized with the enzyme Bg ⁇ and of the viral AD ⁇ , as described in Example 3.
  • This recombinant contains an MCMV-IE / ⁇ DV F cassette in the intergenic site between the COLs B and C of the ILTV virus (see example 5).
  • Example 10 Construction of a donor plasmid for the insertion of a double cassette for expression of the H ⁇ and F genes of ⁇ DV in the intergenic site between the COLs B and C and isolation of a recombinant ELTN virus:
  • a double expression cassette for two genes for example the H ⁇ and F genes of the ⁇ DV virus, can be constructed.
  • Such a construction is shown schematically in Figure 15.
  • the 5 ′ end of the two promoters are adjacent so that the transcription of the two genes takes place in opposite directions.
  • One of the two promoters is the MCMV IE promoter and the other promoter (called associated promoter) is the SV40 promoter (present in the plasmid pSVbeta, Clontech Laboratories, Palo Alto, California 94303-4607, USA).
  • the associated promoter is activated by the activating region of the CMV IE promoter.
  • This double expression cassette can then be inserted into the donor plasmid described above (pEL157 described in Example 5 and represented in FIG. 3).
  • the isolation of the recombinant viruses is done in the same way as above (see example 3).
  • Example 11 Construction of the donor plasmid pEL163 for the insertion of an MDV gB gene expression cassette into the intergenic site between COLs B and C and isolation of vILTV12:
  • CD002 (SEQ ID NO: 16) 5 'T ⁇ CGGGACATTTTCGCGG 3' and the matrix pCD007 to produce a PCR fragment of 222 bp.
  • Another PCR was carried out with the following oligonucleotides:
  • CD003 (SEQ ID NO: 17) 5 'TATATGGCGTTAGTCTCC 3'
  • CD004 (SEQ ID NO: 18)
  • vILTV11 recombinant virus 11.3 - Isolation and purification of the vILTV11 recombinant virus
  • the vILTV12 virus was isolated and purified after cotransfection of the AD ⁇ of the plasmid pELl ⁇ l previously linearized with the enzyme BgH and of the viral AD ⁇ , as described in Example 3.
  • This recombinant contains an MCMV-IE / MDV gB cassette in the intergenic site between the COLs B and C of the ILTV virus (see example 5).
  • Example 13 Construction of Donor Plasmids for Insertion of Cassette Expression of Gene (s) of Other Avian Pathogens or Immunomodulatory Peptide into the Site Described and Isolation of Recombinant ELTV Viruses: According to the Same Strategy as That described above for the insertion of single cassettes (examples 6, 7, 8, 9 and 11) for the insertion of double cassettes (example 10), in the site described above (example 5), it is possible to make recombinant ILTV viruses expressing at a high level CAV immunogens (and in particular a double expression cassette for genes coding for VP1 and for VP2), chicken pneumovirus virus, or other avian pathogens, or still immunomodulatory peptides and in particular cytokines.
  • the recombinant viruses obtained according to the invention are produced on embryonated eggs.
  • the harvested viral solution is then diluted in a stabilizing solution for lyophilization, distributed at the rate of 1000 vaccine doses per vial, and finally lyophilized.

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Abstract

The invention concerns a live recombinant avian vaccine containing, as vector, an ILTV virus comprising and expressing at least a heterologous nucleotide sequence, said sequence being inserted in the insertion locus formed by the IRES (internal ribosome entry site) located between the stop coding units of COL B and COL C of the ILTV and which, in a particular ILTV strain, is defined between nucleotides 908 and 994 at the SEQ ID NO: 1.

Description

Vaccin vivant recombinant aviaire, utilisant comme vecteur le virus de la laryngotrachéite infectieuse aviaire.Avian recombinant live vaccine, using avian infectious laryngotracheitis virus as a vector.
La présente invention a trait à des vaccins à usage aviaire à base de virus de la laryngotrachéite infectieuse (ILTV), dans lequei a été insérée, par recombinaison génétique, au moins une séquence nucléotidique hétέrologue, notamment codant pour, et exprimant, un polypeptide antigénique d'un agent pathogène aviaire, dans des conditions assurant une immunisation conduisant à une protection efficace de l'animal vacciné contre ledit agent pathogène.The present invention relates to vaccines for avian use based on infectious laryngotracheitis virus (ILTV), into which has been inserted, by genetic recombination, at least one heterologous nucleotide sequence, in particular coding for and expressing, an antigenic polypeptide of an avian pathogen, under conditions ensuring immunization leading to effective protection of the animal vaccinated against said pathogen.
Le virus de la laryngotrachéite infectieuse (ILTV) est un alphaherpèsvirus (B. Roizman, Arch. Virol. 1992. 123. 425-449) qui provoque une pathologie respiratoire importante (la laryngotrachéite infectieuse ou ILT) chez le poulet (L.E. Hanson et TJ. Bagust, Diseases of Poultry 9th edn 1991. pp 485-495. Ames, lowa State University Press). Les vaccins actuellement disponibles contre cette affection contiennent une souche atténuée administrable par différentes voies dont les voies intranasales, conjonctivales, cloacales, dans l'eau de boisson et par aérosol (L.E. Hanson et T . Bagust, Diseases of Poultry 9th Edition 1991. pp 485-495. Ames, lowa State University Press).Infectious laryngotracheitis virus (ILTV) is an alphaherpesvirus (B. Roizman, Arch. Virol. 1992. 123. 425-449) which causes an important respiratory pathology (infectious laryngotracheitis or ILT) in chicken (LE Hanson and TJ Bagust, Diseases of Poultry 9th edn 1991. pp 485-495. Ames, lowa State University Press). The vaccines currently available against this condition contain an attenuated strain which can be administered by different routes including the intranasal, conjunctival, cloacal in the drinking water and aerosol (LE and T Hanson. Bagust, Diseases of Poultry 9th Edition 1991. pp 485-495. Ames, Iowa State University Press).
Les études de biologie moléculaire du virus ILTV ont permis de caractériser le génome viral (M. A. Johnson et al. , Arch. Vlrot. 1991. 119. 181-198) et d'identifier quelques gènes du virus (A.M. Griffin, J. Gen. Virol. 1989. 70. 3085-3089) dont les gènes codant pour la thy idine inase (UL23) (A.M. Griffin et M.E.G. Boursnell, /. Gen. Virol. 1990. 71. 841-850; CL. eeier ét al., Avion Dis. 1991. 35. 920-929), la glycoprotéine gB (UL27) (A.M. Griffin, /. Gen. Virol. 1991. 72. 393-398; . Kongsuwan et al., Virology 1991. 184. 404-410; D.J. Poulsen ét al.. Virus G nes 1991. 5. 335-347), la glycoprotéine gC (UL44) (D.H. Kingsley ét al., Virology 1994. 203. 336-343), la protéine de capside p40 (UL26) (A.M. Griffin, Nucl. Acids Res. 1990. 18. 3664), la protéine homologue de la protéine ICP4 de l'herpès simplex (HSV-l) (M. A. Johnson et al., Virus Research 1995. 35. 193-204), les protéines homologues aux protéines ICP27 (UL54), glycoprotéine gK (UL53) et DNA hélicase (UL52) de l' HSV-l (M. A. Johnson et ai, Arch. Virol. 1995. 140. 623-634), la ribonuciéotide réductase (A.M. Griffin, /. Gen. Virol. 1989. 70. 3085-3089, O-A-90/02802), les gène UL1 à UL5 (W. Fuchs et T.C. Mettentleiter, /. Gen. Virol. 1996. 77. 2221-2229), les gènes présents dans la séquence unique courte du génome (U,) (M. A. Johnson et ai, DNA S quence- The Journal of Sequencing and Mapping 1995. Vol. 5. pp 191-194; K. Kongsuwan et al., Arch. Virol. 1995. 140. 27-39; K. Kongsu an et ai. Virus Research 1993. 29. 125-140; K. Kongsuwan et ai. Virus G ne 1993. 7. 297-303; M.A. Wild et ai. Virus Gènes 1996. 12. 107-116; WO-A-92/03554; WO-A-95/08622).Molecular biology studies ILTV have characterized the viral genome (MA Johnson et al., Arch. Vlrot. 1991. 119. 181-198) and iden t ify some virus genes (AM Griffin, J. Gen. Virol. 1989. 70. 3085-3089) including the genes coding for thy idine inase (UL23) (AM Griffin and MEG Boursnell, /. Gen. Virol. 1990. 71. 841-850; CL. Eeier et al ., Avion Dis. 1991. 35. 920-929), the glycoprotein gB (UL27) (AM Griffin, /. Gen. Virol. 1991. 72. 393-398;. Kongsuwan et al., Virology 1991. 184. 404 -410; DJ Poulsen et al. Virus G nes 1991. 5. 335-347), the glycoprotein gC (UL44) (DH Kingsley et al., Virology 1994. 203. 336-343), the capsid protein p40 ( UL26) (AM Griffin, Nucl. Acids Res. 1990. 18. 3664), the protein homologous to the ICP4 protein of herpes simplex (HSV-1) (MA Johnson et al., Virus Research 1995. 35. 193- 204), proteins homologous to proteins ICP27 (UL54), glycoprotein gK (UL53) and DNA helicase (UL52) of HSV-1 (MA Johns on and ai, Arch Virol. 1995. 140. 623-634), ribonuciéotide reductase (AM Griffin, /. Gen. Virol. 1989. 70. 3085-3089, OA-90/02802), the UL1 to UL5 genes (W. Fuchs and TC Mettreleiter, /. Gen. Virol. 1996. 77. 2221-2229), the genes present in the unique short genome sequence (U,) (MA Johnson et ai, DNA S quence- The Journal of Sequencing and Mapping 1995. Vol. 5 . pp 191-194; K. Kongsuwan et al., Arch. Virol. 1995. 140. 27-39; K. Kongsu an et al. Virus Research 1993. 29. 125-140; K. Kongsuwan et al. Virus G ne 1993. 7. 297-303; MA Wild et al. Genes Virus 1996. 12. 107-116; WO-A-92/03554; WO-A-95/08622).
La présente invendon a pour objectif de mettre au point un vaccin aviaire à base de virus ILTV recombinant exprimant un gène hétérologue, ce virus étant capable de se répliquer et d'induire une immunité chez l'hôte vacciné tout en conservant une bonne innocuité.The aim of the present invendon is to develop an avian vaccine based on a recombinant ILTV virus expressing a heterologous gene, this virus being capable of replicating and of inducing immunity in the vaccinated host while maintaining good safety.
Un autre objectif de l'invention tst de proposer un tel vaccin qui soit en même temps particulièrement efficace contre la laryngotrachéite infectieuse (ILT).Another objective of the invention is to provide such a vaccine which is at the same time particularly effective against infectious laryngotracheitis (ILT).
Un autre objectif de l'invention est de proposer un tel vaccin qui soit utilisable dans la vaccination de masse par voie mucosale, par exemple par voie aérosol ou dans l'eau de boisson, de telle manière que la réplication du virus au niveau mucosal permette d' induire une immunité mucosale et systémique. Une telle immunité mucosale sera particulièrement efficace pour lutter contre les maladies respiratoires, ainsi que contre les autres maladies pour lesquelles la poπe d'entrée de l'agent pathogène est mucosale.Another objective of the invention is to propose such a vaccine which can be used in mass vaccination by mucosal route, for example by aerosol route or in drinking water, in such a way that the replication of the virus at the mucosal level allows to induce mucosal and systemic immunity. Such mucosal immunity will be particularly effective against respiratory diseases and con t re other diseases for which the input poπe pathogen is mucosal.
Un autre objectif de l'invention est de proposer un tel vaccin qui soit utilisable aussi bien chez les adultes que chez les jeunes animaux.Another object of the invention is to provide such a vaccine which can be used both in adults and in young animals.
Un objectif spécifique est de proposer un tel vaccin utilisable dans la vaccination de masse par voie mucosale des tout jeunes animaux tels que les poussins d'un jour. Un autre objectif de l'invention est de proposer un vaccin contre l'ILT qui ait une efficacité accrue par rapport à la souche parentale et qui puisse même éventuellement permettre l' insertion et l'expression d'un gène hétérologue.A specific objective is to propose such a vaccine usable in mass vaccination by mucosal route of very young animals such as day-old chicks. Another objective of the invention is to provide a vaccine against the ILT which has an increased efficacy compared to the parental strain and which may even possibly allow the insertion and expression of a heterologous gene.
Au cours de leurs travaux sur le virus ILTV, les inventeurs ont trouvé une région génomique qui s'est révélée tout à fait appropriée comme site d'inseπion de gènes hétérologues. Cela a permis de mettre au point un vaccin vivant recombinant à base d'un vecteur ILTV dans lequel est insérée au moins une séquence codant pour un immunogène aviaire, en particulier les protéines HN et F du virus de la maladie de Newcastle (NDV), et/ou la glycoprotéine gB du virus de la maladie de Mare (MDV), et/ou la protéine VP2 du virus de la maladie de Gumboro (IBDV), et/ou les protéines S et M du virus de la bronchite infectieuse (IBV). Un tel vaccin incorporant une séquence codant pour des protéines du NDV, du MDV et/ou de l'IBV assure une protection satisfaisante des animaux contre la maladie de Newcastle, contre la maladie de Marek, contre la maladie de Gumboro, et contre la bronchite infectieuse respectivement.In the course of their work on the ILTV virus, the inventors have found a genomic region which has proved quite suitable as a site for the insertion of heterologous genes. This has made it possible to develop a recombinant live vaccine based on an ILTV vector into which is inserted at least one sequence coding for an avian immunogen, in particular the HN and F proteins of Newcastle disease virus (NDV), and / or the Mare Disease Virus gB glycoprotein (MDV), and / or the Gumboro Disease Virus VP2 protein (IBDV), and / or the Infectious Bronchitis Virus S and M proteins (IBV) ). Such a vaccine incorporating a sequence coding for NDV, MDV and / or IBV proteins provides satisfactory protection of animals against Newcastle disease, against Marek's disease, against Gumboro disease, and against bronchitis infectious respectively.
La présente invention a donc pour objet un vaccin vivant recombinant aviaire comprenant, comme vecteur, le virus ILTV comprenant au moins une séquence nucléotidique hétérologue, notamment codant pour, et exprimant, un polypeptide antigénique d'un agent pathogène aviaire, insérée dans le locus d' inseπion formé de l' intergène situé entre les codons "stop" des COL-B et COL-C du virus ILTV et qui, dans une souche ILTV paπiculière, est défini entre les nucleotides 908 et 994 à la séquence SEQ ID NO: l. Si la séquence paπiculière décrite dans la demande (SEQ ID NO: 1) provient de la souche vaccinale d'ILTV T-20 12-8-66 provenant de Select Laboratories (10026 MainThe present invention therefore relates to a live recombinant avian vaccine comprising, as a vector, the ILTV virus comprising at least one heterologous nucleotide sequence, in particular coding for and expressing, an antigenic polypeptide of an avian pathogenic agent, inserted into the locus d 'inseπion formed of the intergene located between the stop codons of COL-B and COL-C of the ILTV virus and which, in a particular ILTV strain, is defined between nucleotides 908 and 994 with the sequence SEQ ID NO: l . If the specific sequence described in the request (SEQ ID NO: 1) comes from the ILTV vaccine strain T-20 12-8-66 from Select Laboratories (10026 Main
Street P.O. Box 6 Berlin, Maryland 21811, USA), il est bien évident que l'homme du métier pourra utiliser les autres souches d'ILTV, compte-tenu des informations données dans la présente sur la souche vaccinale.Street P.O. Box 6 Berlin, Maryland 21811, USA), it is obvious that a person skilled in the art will be able to use the other strains of ILTV, taking into account the information given herein on the vaccine strain.
Les COL-B et COL-C correspondent respectivement aux gènes UL3.5 et UL4 décrits dans l'aπicle de W. Fuchs et T.C. Mettentleiter (/. Gen. Virol. 1996. 77. 2221-The COL-B and COL-C correspond respectively to the UL3.5 and UL4 genes described in the article by W. Fuchs and T.C. Mettreleiter (/. Gen. Virol. 1996. 77. 2221-
2229) d'une souche pathogène provenant de D. Lutticken, Boxmeer, Pays-Bas. Cet aπicle ne suggère en aucune manière que cet intergène puisse être utilisé comme locus d'inseπion.2229) of a pathogenic strain from D. Lutticken, Boxmeer, Netherlands. This article in no way suggests that this intergene can be used as a locus of inclusion.
La séquence référencée SEQ ID NO: 19 reproduit pour cette souche pathogène, la séquence équivalente à SEQ ID NO: l. L' intergène servant de locus d'insertion conformément à l'invention est compris à la SEQ ID NO: 19 entre les nucleotides 908 et 994. Par séquence hétérologue, on entend une séquence qui ne provient pas de ce locus d'inseπion, c'est-à-dire aussi bien une séquence n'ayant pas pour origine le virus ILTV, qu'une séquence provenant d'une autre région génomique de ce virus, ou encore provenant d'une autre souche ILTV, notamment une souche virulente.The sequence referenced SEQ ID NO: 19 reproduces for this pathogenic strain, the sequence equivalent to SEQ ID NO: 1. The intergene serving as insertion locus according to the invention is included in SEQ ID NO: 19 between nucleotides 908 and 994. By heterologous sequence is meant a sequence which does not originate from this insertion locus, c '' is to say both a sequence not originating from the ILTV virus, as well as a sequence originating from another genomic region of this virus, or also originating from another ILTV strain, in particular a virulent strain.
Par insertion dans la région d'inseπion, on entend notamment inseπion simple ou après délétion totale ou partielle du locus d'inseπion.By insertion into the insertion region is meant in particular simple insertion or after total or partial deletion of the insertion locus.
On peut insérer une ou plusieurs cassettes d'expression chacune comprenant au moins une séquence à exprimer.One or more expression cassettes can be inserted, each comprising at least one sequence to be expressed.
Pour exprimer la séquence insérée, on préfère utiliser un promoteur eucaryote fort tel que le promoteur CMV immédiate early (IE), le LTR du virus du sarcome de Rous (RSV), et le promoteur précoce du virus SV40. Par promoteur CMV immédiate early (IE), on entend notamment le fragment donné dans les exemples ainsi que ses sous-fragments conservant la même activité promotrice. Le promoteur CMV IE peut être le promoteur humain (HCMV IE) ou le promoteur murin (MCMV IE), ou encore un promoteur CMV IE d'une autre origine, par exemple du singe, du rat, du cobaye ou du porc.To express the inserted sequence, it is preferred to use a strong eukaryotic promoter such as the immediate early CMV promoter (IE), the Rous sarcoma virus LTR (RSV), and the early promoter of the SV40 virus. By immediate early CMV promoter (IE) is meant in particular the fragment given in the examples as well as its subfragments retaining the same promoter activity. The CMV IE promoter can be the human promoter (HCMV IE) or the murine promoter (MCMV IE), or else a CMV IE promoter from another origin, for example from monkeys, rats, guinea pigs or pigs.
D'autres promoteurs d'origine virale ou cellulaire peuvent également être utilisés.Other promoters of viral or cellular origin can also be used.
Parmi les promoteurs d'origine virale, on peut encore citer les promoteurs de gènes du virus ILTN (gènes considérés comme précoce-immédiats (ICP4, ICP27, ...), précoces fthymidine kinase, DΝA helicase, ribonucléotide réductase, ...), ou tardifs (gB, gD, gC, gK, ...)). du virus de la maladie de Marek (MDV) (gènes gB, gC, pp38, ppl4, ICP4, Meq, ...) ou du virus de l'hβ ès de la dinde (heφèsvirus of rurkey) (gènes gB, gC, ICP4, ...).Among the promoters of viral origin, mention may also be made of promoters of genes for ILTN virus (genes considered to be early-immediate (ICP4, ICP27, ...), early fthymidine kinase, DΝA helicase, ribonucleotide reductase, ...), or late (gB, gD, gC, gK, ...)) . Marek's disease virus (MDV) (gB, gC, pp38, ppl4, ICP4, Meq, ... genes) or hβ ès turkey virus (heφèsvirus of rurkey) (gB, gC genes, ICP4, ...).
La séquence nucléotidique insérée dans le vecteur ILTN pour être exprimée peut être toute séquence codant pour un polypeptide antigénique, d'un agent pathogène aviaire, capable, une fois exprimé dans les conditions favorables procurées par l'invention, d'assurer une immunisation conduisant à une protection efficace de l'animal vacciné contre l'agent pathogène. On pourra donc insérer, dans les conditions de l' invention, les séquences nucléotidiques codant pour les antigènes d'intérêt pour une maladie donnée.The nucleotide sequence inserted into the ILTN vector to be expressed can be any sequence coding for an antigenic polypeptide, of an avian pathogenic agent, capable, once expressed under the favorable conditions provided by the invention, of ensuring immunization leading to effective protection of the animal vaccinated against the pathogen. It will therefore be possible to insert, under the conditions of the invention, the nucleotide sequences coding for the antigens of interest for a given disease.
Cette séquence nucléotidique insérée dans le vecteur ILTV peut également coder pour un polypeptide immunomodulateur, et notamment une cytokine. De manière remarquable, les vaccins selon l'invention pourront être utilisés pour la vaccination in ovo, des poussins d'un jour ou plus et des adultes. Différentes voies d'administration pourront être utilisées: la voie parentérale, ou les voies mucosaies telles que oronasale (eau de boisson, aérosol), conjonctivale (goutte dans l'oeil) ou cloacale, avec une préférence pour les voies permettant une vaccination mucosale de masse (eau de boisson, aérosol).This nucleotide sequence inserted into the ILTV vector can also code for an immunomodulatory polypeptide, and in particular a cytokine. Remarkably, the vaccines according to the invention can be used for vaccination in ovo, day-old chicks or more and adults. Different routes of administration could be used: the parenteral route, or the mucosa routes such as oronasale (drinking water, aerosol), conjunctival (drop in the eye) or cloacale, with a preference for the routes allowing a mucosal vaccination of mass (drinking water, aerosol).
L'invention se révèle paπiculièrement utile aussi bien pour la protection contre les pathologies respiratoires que contre les pathologies systέmiques en bloquant les voies d'entrée naturelles de l'agent pathogène.The invention proves to be particularly useful both for protection against respiratory pathologies and against systemic pathologies by blocking the natural pathways of entry of the pathogenic agent.
L' invention peut notamment être utilisée pour l'inseπion d'une séquence nucléotidique codant convenablement pour une protéine antigénique du virus ΝDV et en paπiculier, la glycoprotéine HΝ ou la glycoprotéine F. On obtient ainsi un vaccin vivant recombinant assurant, en plus d'une protection contre la laryngotrachéite infectieuse, une protection satisfaisante contre la maladie de Νewcastle.The invention can in particular be used for the insertion of a nucleotide sequence suitably coding for an antigenic protein of the ΝDV virus and in particular, the glycoprotein HΝ or the glycoprotein F. This gives a recombinant live vaccine ensuring, in addition to protection against infectious laryngotracheitis, satisfactory protection against castleewcastle disease.
Le vaccin recombinant contre la maladie de Νewcastle contiendra de préférence de 10 à 104 PFU/dose.The recombinant Νewcastle disease vaccine will preferably contain 10 to 10 4 PFU / dose.
D'autres cas préférés de l'invention sont l' inseπion de séquences nucléotidiques codant pour des antigènes d'autres agents pathogènes aviaires, et notamment, mais de manière non limitative, des antigènes du virus de la maladie de ïarek, en paπiculier gènes gB, gC, gD, et gH + gL (WO-A-90/02803), du virus de la maladie de Gumboro, en particulier gène VP2, du virus de la bronchite infectieuse (IBV), en paπiculier gènes S et M (M. Binns ét al., J. Gen. Virol. 1985. 66. 719-726 ; M. Boursnell ét al., Virus Research 1984. 1. 303-313), du virus de l'anémie du poulet (CAV), en paπiculier VP1 (52 kDa) + VP2 (24 Da) (N.H.M. Notebom ét al., J. Virol. 1991. 65. 3131-3139), du virus ILTN, en particulier les gènes codant pour gB (A.M. Griffin, /. Gen. Virol.Other preferred cases of the invention are the insertion of nucleotide sequences coding for antigens of other avian pathogens, and in particular, but not limited to, antigens of the ïarek disease virus, in particular genes gB, gC, gD, and gH + gL (WO-A-90 / 02803), the Gumboro disease virus, in particular the VP2 gene, the infectious bronchitis virus (IBV), in particular the S and M genes (M. Binns et al., J. Gen. Virol. 1985. 66. 719-726; M. Boursnell et al., Virus Research 1984. 1. 303-313), chicken anemia virus (CAV), in particular VP1 (52 kDa) + VP2 (24 Da) (NHM Notebom et al., J. Virol. 1991. 65. 3131-3139), of the ILTN virus, in particular the genes coding for gB (AM Griffin, /. Gen. Virol.
1991. 72. 393-398), ou pour gD (M.A. Johnson et al., DNA Sequence-The Journal of Sequencing and Mapping 1995. Vol. 5. ppl91-194. Harwood Académie Publishers1991. 72. 393-398), or for gD (M.A. Johnson et al., DNA Sequence-The Journal of Sequencing and Mapping 1995. Vol. 5. ppl91-194. Harwood Académie Publishers
GmbH), ou pour gp60 (K.K. Kongsuwan ét al., Virus Gènes 1993. 7. 297-303), et du virus du syndrome infectieux du gonflement de la tête ("swollen head syndrome" ou pneumovirose du poulet ou turkey rhinotracheitis vires (TRTV) de la dinde; pneumovirus), en particulier la glycoprotéine de fusion F (Q. Yu ét al., J. Gen. Virol. 1991. 72. 75-81), ou la glycoprotéine d'attachement G (R. Ling et al., J. Gen. Virol.GmbH), or for gp60 (KK Kongsuwan et al., Genes Virus 1993. 7. 297-303), and the swelling head syndrome virus (swollen head syndrome) or chicken pneumovirus or turkey rhinotracheitis vires ( TRTV) of turkey; pneumovirus), in particular the fusion glycoprotein F (Q. Yu et al., J. Gen. Virol. 1991. 72. 75-81), or the attachment glycoprotein G (R. Ling et al., J. Gen. Virol.
1992. 73. 1709-1715; K. Juhasz et J. Easton, J. Gen. Virol. 1994. 75. 2873-2880). Les doses seront de préférence les mêmes que celles pour le vaccin de Νewcastie.1992. 73. 1709-1715; K. Juhasz and J. Easton, J. Gen. Virol. 1994. 75. 2873-2880). The doses will preferably be the same as those for the castewcastie vaccine.
Dans le cadre de la présente invention, on peut bien entendu insérer plus d'une séquence hétérologue dans le même virus ILTV, notamment dans ce locus. On peut notamment y insérer des séquences provenant d'un même virus ou de virus différents, ce qui comprend également l'insertion de séquences d'ILTV et d'un autre virus aviaire. On peut également y associer des séquences codant pour des immunomodulateurs, et en paπicuiier des cytokines.In the context of the present invention, it is of course possible to insert more than one heterologous sequence into the same ILTV virus, in particular in this locus. In particular, it is possible to insert sequences therefrom from the same virus or from different viruses, which also includes the insertion of sequences from ILTV and from another avian virus. It is also possible to associate therewith sequences coding for immunomodulators, and in particular cytokines.
Par exemple, on associe au promoteur CMV IE un autre promoteur de façon que leurs extrémités 5' soient adjacentes (ce qui implique des transcriptions dans des sens opposés), ce qui permet d' insérer, dans la zone d'inseπion, deux séquences nucléotidiques, l'une sous la dépendance du promoteur CMV IE, l'autre sous celle du promoteur associé. Cette construction est remarquable par le fait que la présence du promoteur CMV IE, et notamment de sa paπie activatrice (enhancer), active la transcription induite par le promoteur associé. Le promoteur associé peut être en paπicuiier un promoteur d'un gène du virus ILTN ou du virus MDV ou HVT. Un cas intéressant de l'invention est un vaccin comprenant une séquence nucléotidique codant pour HN de NDV et une séquence nucléotidique codant pour F deFor example, the promoter CMV IE is associated with another promoter so that their 5 'ends are adjacent (which implies transcriptions in opposite directions), which makes it possible to insert, in the insertion zone, two nucleotide sequences , one under the control of the CMV IE promoter, the other under that of the associated promoter. This construction is remarkable by the fact that the presence of the CMV IE promoter, and in particular of its activating part (enhancer), activates the transcription induced by the associated promoter. The associated promoter may in particular be a promoter of a gene of the ILTN virus or of the MDV or HVT virus. An interesting case of the invention is a vaccine comprising a nucleotide sequence coding for HN of NDV and a nucleotide sequence coding for F of
NDV ou un antigène d'une autre maladie aviaire, notamment celles citées plus haut, l'un des gènes étant sous le contrôle du promoteur CMV IE, et l'autre sous le contrôle du promoteur associé.NDV or an antigen of another avian disease, in particular those mentioned above, one of the genes being under the control of the CMV IE promoter, and the other under the control of the associated promoter.
On peut aussi monter deux promoteurs CMV IE d'origines différentes avec leurs extrémités 5' adjacentes.It is also possible to mount two CMV IE promoters of different origins with their adjacent 5 ′ ends.
L'expression de plusieurs gènes hétérologues insérés dans le locus d'insertion peut également être rendu possible par insertion entre les cadres ouverts de lecture 'de ces gènes d'une séquence appelée "1RES" (Internai Ribosome Entry Site) provenant notamment d'un picomavirus tel le virus de la maladie vésiculaire du porc (swine vesicular disease virus, SVDV; B.-F. Chen ét al., J. Virology, 1993, 67, 2142-2148), le virus de i'encéphalomyocardite (EMCV; R.J. Kauftnan étal., NucleicAcids Research, 1991, 19, 4485-4490), le virus de la fièvre aphteuse (FMDV; N. Luz et E. Beck, J. Virology, 1991, 65, 6486-6494), ou encore d'une autre origine. Le contenu des 3 articles cités est incoφonî par référence. La cassette d'expression de deux gènes aurait donc la structure minimale suivante: promoteur - gène 1 - IRES - gène2 - signal de polyadénylation. Le vaccin vivant recombinant selon l'invention pourra donc comprendre, insérée dans le locus d'insertion,' une cassette d'expression comprenant successivement un promoteur, deux ou plusieurs gènes séparés deux à deux par un IRES, et un signal de polyadénylation.The expression of several heterologous genes inserted into the insertion locus can also be made possible by insertion between the open reading frames of these genes of a sequence called "IRES" (Internai Ribosome Entry Site) originating in particular from a picomavirus such as swine vesicular disease virus (SVDV; B.-F. Chen et al., J. Virology, 1993, 67, 2142-2148), encephalomyocarditis virus (EMCV; RJ Kauftnan et al., NucleicAcids Research, 1991, 19, 4485-4490), the foot-and-mouth disease virus (FMDV; N. Luz and E. Beck, J. Virology, 1991, 65, 6486-6494), or d 'another origin. The content of the 3 cited articles is incoφonî by reference. The expression cassette for two genes would therefore have the following minimum structure: promoter - gene 1 - IRES - gene2 - polyadenylation signal. The recombinant live vaccine according to the invention may therefore comprise, inserted into the insertion locus, an expression cassette successively comprising a promoter, two or more genes separated in pairs by an IRES, and a polyadenylation signal.
En plus de l'insertion dans le locus selon l'invention, on peut réaliser une ou plusieurs autres insertions, une ou plusieurs mutations, ou une ou plusieurs délétions ailleurs dans le génome; si la souche parentale est virulente, on peut par exemple inactiver (par délétion, insertion ou mutation) des gènes impliqués dans la virulence tels que le gène thymidine kinase, le gène ribonucléotide réductase, le gène gE,... Dans tous les cas, l'insertion dans un autre locus que celui décrit dans l'invention, permet d'exprimer d'autres gènes,In addition to insertion into the locus according to the invention, one or more other insertions, one or more mutations, or one or more deletions can be made elsewhere in the genome; if the parental strain is virulent, it is possible, for example, to inactivate (by deletion, insertion or mutation) of the genes involved in virulence such as the thymidine kinase gene, the ribonucleotide reductase gene, the gE gene, etc. In all cases, insertion into another locus than that described in the invention makes it possible to express other genes,
La présente invention a aussi pour objet un vaccin contre l'ILT comprenant un virus ILTV recombinant dans lequel on a inséré en amont des gènes codant pour des immunogènes majeurs de l'ILTV, de préférence les gènes codant pour gB (A.M. Griffin, J. Gen. Virol. 1991. 72. 393-398), ou pour gD (M. A. Johnson et ai , DNA Séquence- Journal of Sequencing and Mapping 1995. Vol. 5. ppl91- 194. Harwood Académie Publishers GmbH), ou pour gp60 (K.K. Kongsuwan et ai , Virus Gènes 1993. 7. 297- 303), un promoteur exogène, en paπiculier un promoteur fort tel que décrit plus haut. Cela permet d'augmenter le niveau d'expression de l'un ou plusieurs de ces gènes et ainsi conduire à un vaccin à efficacité accrue contre l'ILT. On peut bien sûr combiner cela avec une construction telle que décrite plus haut comprenant l'insertion d'une séquence hétérologue dans le locus d'insertion.The subject of the present invention is also a vaccine against ILT comprising a recombinant ILTV virus in which genes coding for major immunogens of ILTV have been inserted upstream, preferably the genes coding for gB (AM Griffin, J. Gen. Virol. 1991. 72. 393-398), or for gD (MA Johnson et ai, DNA Séquence- Journal of Sequencing and Mapping 1995. Vol. 5. ppl91- 194. Harwood Académie Publishers GmbH), or for gp60 (KK Kongsuwan and ai , Virus Genes 1993. 7. 297-303), an exogenous promoter, in particular a strong promoter as described above. This increases the level of expression of one or more of these genes and thus leads to a vaccine with increased efficacy against ILT. One can of course combine this with a construction as described above comprising the insertion of a heterologous sequence into the insertion locus.
La présente invention a aussi pour objet une formule de vaccin multivalent, comprenant, en mélange ou à mélanger, un vaccin tel que défini plus haut avec un autre vaccin, et notamment un autre vaccin vivant recombinant aviaire tel que défini plus haut, ces vaccins comprenant des séquences insérées différentes, notamment de pathogènes différents.The present invention also relates to a multivalent vaccine formula, comprising, as a mixture or to be mixed, a vaccine as defined above with another vaccine, and in particular another live recombinant avian vaccine as defined above, these vaccines comprising different inserted sequences, in particular of different pathogens.
La présente invention a aussi pour objet une méthode de préparation des vaccins selon l'invention, telle qu'elle ressort de la description.The present invention also relates to a method for preparing the vaccines according to the invention, as it emerges from the description.
La présente invention a aussi pour objet une méthode de vaccination aviaire comprenant l'administration d'un vaccin vivant recombinant ou d'une formule de vaccin multivalent tel que défini plus haut Elle a notamment pour objet une telle méthode pour la vaccination in ovo, des poussins d'un jour ou plus et des adultes. Différentes voies d'administration du vaccin peuvent être utilisées (voir plus haut) avec une préférence pour les voies permettant une vaccination de masse par voie mucosale (aérosol, eau de boisson), la dose de vaccin étant choisie de préférence entre 10* et 10* par animal.The present invention also relates to a method of avian vaccination comprising the administration of a recombinant live vaccine or of a multivalent vaccine formula as defined above. It relates in particular to such a method for vaccination in ovo, day-old chicks and adults. Different routes of administration of the vaccine can be used (see above) with a preference for the routes allowing mass vaccination by mucosal route (aerosol, drinking water), the dose of vaccine being preferably chosen between 10 * and 10 * per animal.
La présente invention a aussi pour objet un virus ILTV comprenant au moins une séquence nucléotidique hétérologue telle que décrite ci-dessus insérée dans le locus d'insertion tel que défini plus haut. La présente invention a aussi pour objet tout ou partie de la séquence SΞQ ID NO : 1 ; par parties de cette séquence, on entend non seulement les COL caractérisés pris isolément ou leurs fragments, mais aussi l' intergène situé entre les COL B et COL C et les fragments situés de part et d'autre de cet intergène, pouvant éventuellement inclure une partie de cet intergène, et qui pourront servir de bras flanquants pour une recombinaison homologue, technique par ailleurs parfaitement connue de l'homme du métier. De manière générale, mais sans que cela soit limitatif, les bras flanquants peuvent avoir de 100 à 800 paires de bases.The present invention also relates to an ILTV virus comprising at least one heterologous nucleotide sequence as described above inserted into the insertion locus as defined above. The present invention also relates to all or part of the sequence SΞQ ID NO: 1; by parts of this sequence is meant not only the characterized COLs taken individually or their fragments, but also the intergene located between the COL B and COL C and the fragments located on either side of this intergene, which may possibly include a part of this intergene, and which can serve as flanking arms for homologous recombination, a technique which is moreover well known to those skilled in the art. In general, but without being limiting, the flanking arms can have from 100 to 800 base pairs.
L'invention va être maintenant décrite plus en détail à. l'ai¬ de d'exemples de réalisation non limitatifs, pris en référen¬ ce au dessin, dans lequel :. The invention will now be described in more detail at. ¬ have examples of non-limiting embodiments taken referen ¬ this drawing, in which:.
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Figure 1 Cane de restriction du fragment clone et position des COLsFigure 1 Restriction cane of the cloned fragment and position of the COLs
Figure 2 : Séquence de 4161 pb et traduction des COLs A, B, C et D de la souche vaccinale T-20 de Select Laboratories (Vaccin LT BLEN)Figure 2: Sequence of 4161 bp and translation of COLs A, B, C and D of the T-20 vaccine strain from Select Laboratories ( LT BLEN vaccine)
Figure 3 : Schéma d'obtention du plasmide pEL157Figure 3: Scheme for obtaining the plasmid pEL157
Figure 4 : Schéma d'obtention du plasmide pEL024Figure 4: Scheme for obtaining the plasmid pEL024
Figure 5 : Schéma d'obtention du plasmide pEL027Figure 5: Scheme for obtaining the plasmid pEL027
Figure 6 : Schéma du plasmide pEL158Figure 6: Diagram of the plasmid pEL158
Figure 7 : Schéma d'obtention du plasmide pCD009Figure 7: Scheme for obtaining the plasmid pCD009
Figure 8 : Schéma d'obtention du plasmide pEL070Figure 8: Scheme for obtaining the plasmid pEL070
Figure 9 : Schéma du plasmide pEL159Figure 9: Diagram of the plasmid pEL159
Figure 10 séquence du gène HN du NDVFigure 10 NDV HN gene sequence
Figure 11 Schéma d'obtention du plasmide pEL030Figure 11 Scheme for obtaining the plasmid pEL030
Figure 12 Schéma du plasmide pEL160Figure 12 Diagram of plasmid pEL160
Figure 13 Schéma du plasmide pEL033Figure 13 Diagram of plasmid pEL033
Figure 14 Schéma du plasmide pELlôlFigure 14 Diagram of the plasmid pEL106
Figure 15 Schéma de double cassette d'expressionFigure 15 Diagram of double expression cassette
Figure 16 Schéma du plasmide pCDOllFigure 16 Diagram of the plasmid pCDOll
Figure 17 Schéma du plasmide pEL163Figure 17 Diagram of plasmid pEL163
Figure 18 Séquence de 4161 pb et traduction des UL3, 3.5, 4 et 5 de la souche pathogène de LϋttickenFigure 18 Sequence of 4161 bp and translation of UL3, 3.5, 4 and 5 from the pathogenic strain of Lϋtticken
Liste des séquencesList of sequences
SEQ ID NO:l Séquence du fragment Kpnl-Kpnl (4161 pb, voir figure 2) SEQ ID NO:2 OHgonuciéotide EL001 SEQ ID NO:3 Oligonucléotide EL002 SEQ ID NO:4 Oligonuciéotide EL003 SEQ ID NO:5 Oligonucléotide EL004 SEQ ID NO:6 Oligonucléotide MB070 SEQ D NO:7 Oligonuciéotide MB071 215SEQ ID NO: l Sequence of the Kpnl-Kpnl fragment (4161 bp, see FIG. 2) SEQ ID NO: 2 OHgonuciéotide EL001 SEQ ID NO: 3 Oligonucleotide EL002 SEQ ID NO: 4 Oligonuciéotide EL003 SEQ ID NO: 5 Oligonucleotide EL004 SEQ ID NO : 6 Oligonucleotide MB070 SEQ D NO: 7 Oligonucleotide MB071 215
- 11-- 11-
SEQ ED NO:8 Séquence du gène HN du NDV (voir figure 10) SEQ ED NO:9 Oligonuciéotide EL071 SEQ ED NO:10 Oligonucléotide EL073 SEQ ED NO: 11 Oligonucléotide EL074 SEQ ED NO: 12 Oligonucléotide EL075 SEQ ED NO:13 Oligonucléotide EL076 SEQ ED NO: 14 Oligonucléotide EL077 SEQ ED NO: 15 Oligonucléotide CD001 SEQ ED NO: 16 Oligonucléotide CD002 SEQ ED NO: 17 Oligonucléotide CD003 SEQ ED NO:18 Oligonucléotide CD004 SEQ ED NO: 19 Séquence du fragment Kpnl-Kpnl (4161 pb, voir figure 18)SEQ ED NO: 8 Sequence of the HN gene of NDV (see Figure 10) SEQ ED NO: 9 Oligonucleotide EL071 SEQ ED NO: 10 Oligonucleotide EL073 SEQ ED NO: 11 Oligonucleotide EL074 SEQ ED NO: 12 Oligonucleotide EL075 SEQ ED NO: 13 Oligonucleotide EL076 SEQ ED NO: 14 Oligonucleotide EL077 SEQ ED NO: 15 Oligonucleotide CD001 SEQ ED NO: 16 Oligonucleotide CD002 SEQ ED NO: 17 Oligonucleotide CD003 SEQ ED NO: 18 Oligonucleotide CD004 SEQ ED NO: 19 Sequence of the fragment Kpnl-Kpnl (41 , see figure 18)
EXEMPLESEXAMPLES
Toutes les constructions de plasmides ont été réalisées en utilisant les techniques standards de biologie moléculaire décrites par Sambrook J. et al. (Molecular Cloning:All the plasmid constructions were carried out using the standard molecular biology techniques described by Sambrook J. et al. (Molecular Cloning:
A Laboratory Manual. 2n Edition. Cold Spring Harbor Laboratory. Cold Spring Harbor.A Laboratory Manual. 2 n Edition. Cold Spring Harbor Laboratory. Cold Spring Harbor.
New York. 1989). Tous les fragments de restriction utilisés pour la présente invention ont été isolés en utilisant le kit "Geneciean" (BIO101 Inc. La Jolla, CA).New York. 1989). All the restriction fragments used for the present invention were isolated using the "Geneciean" kit (BIO101 Inc. La Jolla, CA).
Le virus utilisé comme virus parental peut être choisi parmi les souches vaccinales décrites dans J.R. Andreasen et al. (Avion Diseases 1990. 34. 646-656) ou la souche T- 20 12-8-66 provenant de Select laboratories 10026 Main Street P.O. Box 6 Berlin, Maryiand 21811, USA. On peut également utiliser des souches virulentes telles que la souche de Lûtticken (voir ci-dessus), la souche N-71851 (ATCC VR-783) ou la souche 83-2 de l' USDA, que l'on peut atténuer par les techniques connues, par exemple celle décrite dans WO-A-95/08622.The virus used as parental virus can be chosen from the vaccine strains described in J.R. Andreasen et al. (Avion Diseases 1990. 34. 646-656) or the strain T-20 12-8-66 from Select laboratories 10026 Main Street P.O. Box 6 Berlin, Maryiand 21811, USA. It is also possible to use virulent strains such as the Lûtticken strain (see above), the N-71851 strain (ATCC VR-783) or the USDA strain 83-2, which can be attenuated by known techniques, for example that described in WO-A-95/08622.
Exemple 1: Culture du virus ELTV:Example 1: Culture of the ELTV virus:
Le virus ILTV (souche T20 de Select Laboratories) est cultivé sur des cellules primaires de reins de poulets (CRP); ces cellules sont mises en culture en milieu MEM complémenté avec 3 % de sérum de veau foetal (S VF) dans des flacons de culture de 75 cm2 (2 103 cellules/cm2) un ou deux jours avant inoculation.The ILTV virus (strain T20 from Select Laboratories) is cultured on primary chicken kidney cells (CRP); these cells are cultured in MEM medium supplemented with 3% fetal calf serum (S VF) in culture flasks of 75 cm 2 (2 10 3 cells / cm 2 ) one or two days before inoculation.
Le jour de l'inoculation, un flacon de 1000 doses de vaccin lyophilisé est resuspendu dans 10 ml de milieu MEM complémenté avec 1 % de SVF; environ 0,5 ml de cette solution est ensuite déposé sur la culture de CRP. Le lendemain, le milieu est changé, et le surlendemain, lorsque l'effet cytopathogène (ECP) se généralise, les flacons de culture sont congelés à -70" C.The day of inoculation, one vial of lyophilized 1000 doses of vaccine is resuspended in 10 ml of MEM medium supplemented with 1% FCS; about 0.5 ml of this solution tt e is then deposited on the culture of CRP. The next day, the medium is changed, and the day after, when the cytopathogenic effect (ECP) becomes generalized, the culture flasks are frozen at -70 "C.
La culture du virus ILTN peut également être faite sur des cellules immortalisées de foie de poulet, et notamment sur la lignée LMH ( .M. Schnitzlein ét al.. Avion Diseases 1994. 38. 211-217).The culture of the ILTN virus can also be done on immortalized chicken liver cells, and in particular on the LMH line (.M. Schnitzlein et al. Avion Diseases 1994. 38. 211-217).
Exemple 2: Préparation de l'ADΝ génomique de l'ILTV:Example 2: Preparation of the genomic ADΝ of ILTV:
Après 2 cycles de congélation/décongélation, la culture d'ILTV (2 flacons de 75 cm2) est récoltée et centrifugée à basse vitesse (5000 tr/min dans un rotor 20, centrifugeuse Beckman JA21, pendant 5 minutes) pour éliminer les gros débris cellulaires. Le surnageant sst ensuite ultracentrifugé (100000 tr/min rotor TLA100.3, centrifugeuse Beckman TL100, pendant 1 heure). Le culot est alors repris dans 1,6 ml de TEΝ-SDS (Tris pH 8,0 lOmM; EDTA ImM; ΝaCI 0,5M; sodium dodecyl sulfate 0,5 %), et 35 μ\ d'une solution de protéinase K à 20 mg/mi sont ensuite ajoutés; la solution est incubée 3 à 4 heures au bain marie à 37"C, et l'ADΝ est ensuite extrait 3 fois au phénol/chloroforme et 1 fois au chloroforme, puis il est précipité à l'émanol à -20βC. Après centrifugation, le culot est rincé i l'éthanol 70%, séché et resuspendu dans 200 μl TE (Tris pH8.0 lOmM; EDTA ImM). La concentration en acide nucléique est ensuite dosée au spectrophotomètre (DO260). L'ADΝ peut être directement digéré par les enzymes de restriction appropriées, pour être ensuite clone dans le plasmide pBlue Script II SIC ; de même, il pourra également être utilisé dans les expériences de transfection pour l'obtention d'un virus recombinant.After 2 freeze / thaw cycles, the culture of ILTV (2 flasks of 75 cm 2 ) is harvested and centrifuged at low speed (5000 rpm in a rotor 20, Beckman JA21 centrifuge, for 5 minutes) to remove large cellular debris. The supernatant is then ultracentrifuged (100,000 rpm TLA100.3 rotor, Beckman TL100 centrifuge, for 1 hour). The pellet is then taken up in 1.6 ml of TEΝ-SDS (Tris pH 8.0 lOmM; EDTA ImM; ΝaCI 0.5M; sodium dodecyl sulfate 0.5%), and 35 μ \ of a proteinase K solution at 20 mg / mi are then added; the solution is incubated 3 to 4 hours in a water bath at 37 "C, and the ADΝ is then extracted 3 times with phenol / chloroform and 1 time with chloroform, then it is precipitated with emanol at -20 β C. After centrifugation, the pellet is rinsed with 70% ethanol, dried and resuspended in 200 μl TE (Tris pH8.0 10 mM; EDTA ImM). The nucleic acid concentration is then determined using a spectrophotometer (DO 260 ). be directly digested with the appropriate restriction enzymes, and then be cloned into the plasmid pBlue Script II SIC; likewise, it can also be used in transfection experiments for obtaining a recombinant virus.
Exemple 3: Isolement et purification de virus recombinant ELTNExample 3: Isolation and purification of recombinant ELTN virus
Le plasmide donneur composé d'une cassette d'expression d'un polypeptide inséré entre deux régions flanquantes du locus d'insertion est digéré par une enzyme de restriction Λ Λ^ ,^ O 98/27215The donor plasmid composed of an expression cassette for a polypeptide inserted between two flanking regions of the insertion locus is digested with a restriction enzyme Λ Λ^ , ^ O 98/27215
1 31 3
permettant la linéarisation du plasmide, puis il est extrait avec un mélange phénol/chloroforme, précipité avec de l'éthanol absolu, et repris dans de l'eau stérile. Des cellules CRP primaires de 24 heures sont ensuite transfectées avec le mélange suivant: 0,2 à 1 μg de plasmide donneur linéarisé + 2 à 5 μg d'ADN viral d'ILTV (préparé comme dans l'exemple 2) dans 300 μ\ de milieu OptiMEM' (Gibco BRL Cat# 041-01985H) et 100 μg de LipofectAMINE dilués dans 300 μl de milieu (volume final du mélange ≈ 600 μï). Ces 600μl sont ensuite dilués dans 3 ml (volume final) de milieu et étalés sur 5.10* CRP. Le mélange est laissé en contact avec les cellules pendant 5 heures, puis éliminé et remplacé par 5 mi de milieu de culture. Les cellules sont alors laissées en culture pendant 3 à 8 jours à + 37 *C, puis, lorsque l'effet cyropathogène est apparu, elles sont congelées à -70*C. Après décongélation et éventuellement sonication, cette population virale est clonée en dilution limite en microplaques (96 puits) afin d' isoler une population homogène de virus recombinant. Ces plaques sont laissées en culture pendant 1 à 3 jours, puis le surnageant est récolté dans une plaque 96 puits vide et la plaque contenant les surnageants est placée à 4βC ou à -70°C. Les cellules restant dans les autres plaques sont ensuite fixées à l'acétone 95% pendant 20 à 30 minutes à - 20°C, ou pendant 5 minutes à température ambiante. Une réaction d' immunofluoresceπce indirecte (IFT) est réalisée avec un anticorps monoclonal dirigé contre le polypeptide exprimé pour rechercher les plages exprimant ce polypeptide. Un nouveau clonage est ensuite effectué de la même manière (en dilution limite en plaques 96 puits) à partir du surnageant présent dans les cupules des plaques mises à 4e C ou à -70" C et correspondant aux cupules présentant des plages positives en IFT. En générai, 4 cycles d'isolement successifs (dilution limite, récoite du surnageant, contrôle des cellules par IFT, dilution limite à partir du surnageant...) suffisent pour obtenir des virus recombinants dont la totalité de la progénie présente une fluorescence spécifique. L' ADN génomique de ces virus recombinants est caractérisé au niveau moléculaire par des techniques classiques de PCR et de Southern blot en utilisant les oligonucléotides et les sondes d'ADN appropriés. L'isolement de virus recombinant peut également se faire par hybridation avec une sonde spécifique de la cassette d'expression insérée. Pour cela, la population virale récoltée après transfection est diluée et déposée sur des cellules CRP (cultivées en boîte de Pétri) de manière à obtenir des plages isolées. Après un contact d' 1 heure à 37βC, le milieu d'infection est éliminé et remplacé par 5 ml de milieu MEM à 1 % d'agarose, maintenu en surfusion à 42 °C. Lorsque l'agarose est solidifié, les boîtes sont incubées 48 à 72 heures à 37βC en étuve CO2 jusqu'à apparition de plages, la couche d'agarose est alors éliminée et un transfert des plages virales est réalisé sur une membrane stérile de nitrocellulose de même diamètre que la boîte de Pétri ayant servi à la culture. Cette membrane est elle-même transférée sur une autre membrane de nitrocellulose de manière à obtenir une "copie" inversée du premier transfert. Les plages transférées sur ceπe dernière copie sont alors hybridées, selon les techniques usuelles connues de l'homme de l'art, avec un fragment d'ADN de la cassette d'expression marqué à la digoxigénine (DNA Labelling Kit, Boehringer Mannheim, CAT # 1175033). Après hybridation, lavages et mise en contact avec le substrat de révélation, la membrane de nitrocellulose st mise en contact avec un film autoradiographique. Les images d'hybridation positive sur cette membrane indiquent quelles sont les plages qui contiennent des virus ILTV recombinants ayant inséré la cassette d'expression. Les plages correspondant à ces plages positives sont découpées stérilement sur la première membrane de nitrocellulose, placées dans un tube Eppendorf contenant 0,5 ml de milieu MEM et soniquées pour libérer les virions de la membrane. Le milieu contenu dans le tube Eppendorf est ensuite dilué en milieu MEM et les dilutions ainsi obtenues servent à infecter de nouvelles cultures de cellules CRP.allowing the linearization of the plasmid, then it is extracted with a phenol / chloroform mixture, precipitated with absolute ethanol, and taken up in sterile water. Primary 24-hour CRP cells are then transfected with the following mixture: 0.2 to 1 μg of linearized donor plasmid + 2 to 5 μg of ILTV viral DNA (prepared as in Example 2) in 300 μ \ of OptiMEM ' medium (Gibco BRL Cat # 041-01985H) and 100 μg of LipofectAMINE diluted in 300 μl of medium (final volume of the mixture ≈ 600 μl). These 600 μl are then diluted in 3 ml (final volume) of medium and spread on 5.10 * CRP. The mixture is left in contact with the cells for 5 hours, then eliminated and replaced with 5 ml of culture medium. The cells are then left in culture for 3 to 8 days at + 37 ° C, and then, when cyropathogène effect appeared, they are frozen at -70 ° C. After thawing and possibly sonication, the viral population is cloned dilution limit in microplates (96 wells) in order to isolate a homogeneous population of recombinant virus. These plates are left in culture for 1 to 3 days, then the supernatant is collected in an empty 96-well plate and the plate containing the supernatants is placed at 4 β C or at -70 ° C. The cells remaining in the other plates are then fixed with 95% acetone for 20 to 30 minutes at -20 ° C., or for 5 minutes at room temperature. An indirect immunofluoresceπce (IFT) reaction is carried out with a monoclonal antibody directed against the expressed polypeptide to search for the ranges expressing this polypeptide. A new cloning is then carried out in the same manner (in dilution limit in 96-well plates) from the supernatant present in the wells of the plates set at 4 e C or at -70 "C and corresponding to the wells having positive plaques in IFT In general, 4 successive isolation cycles (limiting dilution, receiving the supernatant, cell control by IFT, limiting dilution from the supernatant, etc.) are sufficient to obtain recombinant viruses whose entire progeny exhibit specific fluorescence. The genomic DNA of these recombinant viruses is characterized at the molecular level by conventional PCR and Southern blot techniques using the appropriate oligonucleotides and DNA probes. The isolation of recombinant virus can also be done by hybridization with a specific probe of the inserted expression cassette. To do this, the viral population harvested after transfection is diluted and deposited on cells. s CRP (grown in Petri dish) so as to obtain isolated areas. After contact for 1 hour at 37 β C, the medium of infection is removed and replaced with 5 ml of MEM medium containing 1% agarose, maintained molten at 42 ° C. When the agarose is solidified, the dishes are incubated 48 to 72 hours at 37 β C in a CO 2 oven until the appearance of plaques, the agarose layer is then eliminated and a transfer of the viral plaques is carried out on a sterile membrane. nitrocellulose of the same diameter as the Petri dish used for the culture. This membrane is itself transferred to another nitrocellulose membrane so as to obtain an inverted "copy" of the first transfer. The plaques transferred to this last copy are then hybridized, according to the usual techniques known to those skilled in the art, with a DNA fragment of the expression cassette labeled with digoxigenin (DNA Labeling Kit, Boehringer Mannheim, CAT # 1175033). After hybridization, washing and contact with the development substrate, the nitrocellulose membrane is brought into contact with an autoradiographic film. The positive hybridization images on this membrane indicate which plaques contain recombinant ILTV viruses which have inserted the expression cassette. The plaques corresponding to these positive plaques are cut sterile on the first nitrocellulose membrane, placed in an Eppendorf tube containing 0.5 ml of MEM medium and sonicated to release the virions from the membrane. The medium contained in the Eppendorf tube is then diluted in MEM medium and the dilutions thus obtained are used to infect new cultures of CRP cells.
Exemple 4: Clonage et caractérisation d'une région génomique de l'ELTNExample 4 Cloning and Characterization of an ELTN Genomic Region
L'ADΝ extrait du virus ILTN a été digéré par l'enzyme de restriction Kpnl pendant 2 heures à 37"C. L' enzyme de restriction a ensuite été éliminé par une extraction au phénol/chloroforme, suivie d'une précipitation à Péthanol. Les fragments résultant de ceπe digestion ont ensuite été ligaturés (une nuit à 14βC) avec le plasmide pBlueScriptlI SK+ (pBS SK+ ; Stratagene) digéré par Kpnl et traité à la phosphatase alcaline ; l'analyse des clones obtenus après transformation de bactéries E. coli DH5α et culture sur boîtes de milieu complémenté en ampiciline a permis d'identifier des inserts Kpnl- Kpnl de tailles différentes, dont un fragment d'environ 4,2 kb (plasmide pEL112). 15The ADΝ extracted from the ILTN virus was digested with the restriction enzyme Kpn1 for 2 hours at 37 ° C. The restriction enzyme was then removed by extraction with phenol / chloroform, followed by precipitation with ethanol. The fragments resulting from this digestion were then ligated (overnight at 14 β C) with the plasmid pBlueScriptlI SK + (pBS SK +; Stratagene) digested with Kpnl and treated with alkaline phosphatase; analysis of the clones obtained after transformation of bacteria E coli DH5α and culture on dishes of medium supplemented with ampicilin made it possible to identify Kpnl-Kpnl inserts of different sizes, including a fragment of approximately 4.2 kb (plasmid pEL112). 15
Le séquençage complet de l' insert présent dans pEL112 (voir figure 1) a permis de meπre en évidence deux cadres ouverts de lecture (COLs) complets (COL B et COL C), et une grande partie de deux autres COLs (COL A et COL D). La carte de restriction de cette région génomique cionée et séquencée, est montrée à la figure 1; la séquence de 4161 pb (SEQ ID NO: 1) est montrée à la figure 2. La position et la séquence en acides aminés des COLs A, B, C et D sont également montrées sur les figures 1 et 2 respectivement.The complete sequencing of the insert present in pEL112 (see Figure 1) made it possible to highlight two complete open reading frames (COLs) (COL B and COL C), and a large part of two other COLs (COL A and COL D). The restriction map of this cionated and sequenced genomic region is shown in Figure 1; the sequence of 4161 bp (SEQ ID NO: 1) is shown in FIG. 2. The position and the amino acid sequence of COLs A, B, C and D are also shown in FIGS. 1 and 2 respectively.
La séquence entre les codons STOP des COL B et C (position de 908 à 994 sur SEQ ID NO: 1), est utilisable pour insérer des cassettes d'expression de polypeptides dans le génome de i'ILTV. Cette séquence est appelée locus d'insertion. L'insertion peut se faire avec ou sans délétion dans la région intergénique (voir exemple 5).The sequence between the COL B and C STOP codons (position 908 to 994 on SEQ ID NO: 1) can be used to insert polypeptide expression cassettes into the ILTV genome. This sequence is called the insertion locus. The insertion can be done with or without deletion in the intergenic region (see example 5).
Exemple 5: Construction du plasmide donneur pEL157 pour l'insertion dans la région intergénique entre les COLs B et CExample 5: Construction of the donor plasmid pEL157 for insertion into the intergenic region between COLs B and C
Le plasmide pEL112 (7116 pb), a été digéré par les enzyme Notl et Spel pour isoler le fragment Notl-Spel de 4,5 kb. Le fragment ainsi digéré a ensuite été traité à l'ADN poiymérase (fragment de Klenow) en présence de dNTP pour rendre les bouts francs ; après ligamre et transformation des bactéries E. coli, le clone pEL156 (4503 pb) a été obtenu.The plasmid pEL112 (7116 bp), was digested with the enzymes Not1 and Spel to isolate the fragment Notl-Spel of 4.5 kb. The fragment thus digested was then treated with DNA poiymerase (Klenow fragment) in the presence of dNTP to make the ends blunt; after ligament and transformation of the E. coli bacteria, the clone pEL156 (4503 bp) was obtained.
Les oligonucléotides EL001 (SEQ ID No:2) et EL002 (SEQ ID No:3) ont servi d'amorce pour une première amplification en chaîne par la Taq poiymérase (PCR). Les oligonucléotides EL003 (SEQ ID No:4) et EL004 (SEQ ID No:5) ont servi d'amorce pour une deuxième amplification en chaîne par la Taq poiymérase (PCR).The oligonucleotides EL001 (SEQ ID No: 2) and EL002 (SEQ ID No: 3) served as a primer for a first chain amplification by Taq poymerase (PCR). The oligonucleotides EL003 (SEQ ID No: 4) and EL004 (SEQ ID No: 5) served as a primer for a second chain amplification by Taq poymerase (PCR).
EL001 (SEQ ID No:2) : 5' TATTσCTTTCTACCσAAGTCGG 3'EL001 (SEQ ID No: 2): 5 'TATTσCTTTCTACCσσAAGTCGG 3'
EL002 (SEQ ID No:3) : 5' ACGCσAATTCAAATACGAGCATTTAATTATTGCG 3'EL002 (SEQ ID No: 3): 5 'ACGCσAATTCAAATACGAGCATTTAATTATTGCG 3'
EL003 (SEQ ID No: 4) : 5' TCTCCAGAATCGCTGGAGTGTCC 3'EL003 (SEQ ID No: 4): 5 'TCTCCAGAATCGCTGGAGTGTCC 3'
EL004 (SEQ IDNσ:5) : 5' TGCGCGAATCGTAAGCTTTGATATCCAGTCGACA ~ „ .,f.~ e O 98/27215EL004 (SEQ IDNσ: 5): 5 'TGCGCGAATCGTAAGCTTTGATATCCAGTCGACA ~ „., F. ~ E O 98/27215
1616
TAAτTTσGτσ TTATTACTTTTA 3'TAAτTTσGτσ TTATTACTTTTA 3 '
Les PCR ont été effectuées en présence de tampon PCR, de dNTP, d'ADN du plasmide pEL156, de Taq poiymérase, et pour la première PCR, des oligonucléotides EL001 et EL002, et pour la deuxième PCR, des oligonucléotides EL003 et EL004.The PCRs were carried out in the presence of PCR buffer, of dNTP, of DNA of the plasmid pEL156, of Taq poymerase, and for the first PCR, of oligonucleotides EL001 and EL002, and for the second PCR, of oligonucleotides EL003 and EL004.
Pour les deux PCR, 25 cycles ont été effectués (30 secondes à 94"C ; 30 secondes à 60 °C et 30 secondes à 72°C). Les produits des deux PCR ont été purifiés par une extraction au phénol/chloroforme, suivie d'une purification par l'éthaπol. Le produit de la première PCR (EL001/EL002) a ensuite été digéré par les enzymes de restriction Xbal et EcoRI pendant 2 h à 37βC pour donner un fragement d'ADN Xbal-EcoRl de 120 pb qui a été élue après électrophorèse en gel d'agarose. Le produit de la deuxième PCR (EL003/EL004) a ensuite été digéré par les enzymes de restriction Xhol et £ ?RI pendant 2 h à 37βC pour donner un fragment d'ADN Λ7iσI-£ccRI de 85 pb qui a été élue après électrophorèse en gel d'agarose. Le plasmide pEL156 a été digéré par les enzymes Xbal et Xhol. Les deux fragements de PCR-XEβl-EcoI (120 pb) et Λ7ιoI-.E >RI (85 pb) ont été ligaturés une nuit à 14°C avec le plasmide pEL156 digéré par Xbal et Xhol. Après transformation des bactéries E. coli, et culture sur boites de milieu complémenté en ampicilline, le clone pEL157 (4531 pb), comprenant un polylinker EcoRl - Hindlll - EcoRV - Sali a été obtenu (voir schéma d'obtention de pEL157 à la figure 3).For the two PCRs, 25 cycles were performed (30 seconds at 94 "C; 30 seconds at 60 ° C and 30 seconds at 72 ° C). The products of the two PCRs were purified by phenol / chloroform extraction, followed purification by ethaπol. The product of the first PCR (EL001 / EL002) was then digested with the restriction enzymes Xbal and EcoRI for 2 h at 37 β C to give a DNA fragment of Xbal-EcoRl of 120 bp which was eluted after agarose gel electrophoresis The product of the second PCR (EL003 / EL004) was then digested with the restriction enzymes Xhol and £? RI for 2 h at 37 β C to give a fragment p7iσI- £ ccRI DNA of 85 bp which was eluted after agarose gel electrophoresis. The plasmid pEL156 was digested with the enzymes Xbal and Xhol. The two fragments of PCR-XEβ1-EcoI (120 bp) and Λ7ιoI -E> RI (85 bp) were ligated overnight at 14 ° C. with the plasmid pEL156 digested with Xbal and Xhol. mation of E. coli bacteria, and culture on boxes of medium supplemented with ampicillin, the clone pEL157 (4531 bp), comprising an EcoRl - Hindlll - EcoRV - Sali polylinker was obtained (see diagram for obtaining pEL157 in FIG. 3 ).
Exemple 6: Construction du plasmide donneur pEL158 pour l'insertion d'une cassette d'expression du gène VP2 de l'EBDV sous contrôle du promoteur HCMV EE dans le site intergénique entre les COLs B et C, et isolement de vELTV8:Example 6 Construction of the Donor Plasmid pEL158 for the Insertion of an EBDV VP2 Gene Expression Cassette Under the Control of the HCMV EE Promoter in the Intergenic Site Between COLs B and C, and Isolation of vELTV8:
6.1 - Clonage du gène VP2 du virus de la maladie de Gumboro (IBDV) et construction d'une cassette d'expression de VP2 sous contrôle du promoteur HCMV IE Le plasmide pEL004 (voir figure 4; ≈ plasmide pGH004 décrit dans la demande de brevet français 92.13109) contenant le gène IBDV VP2 sous forme d'une cassette BamHl- Hindlll a été digéré par Bamϋl et Xbal pour isoler le fragment BamHl-Xbal (gène VP2 tronqué) de 1104 pb. Ce fragment a été clone dans le vecteur pBS SK+ , préalablement digéré avec Xbal et BamHl pour donner le plasmide pEL022 de 4052 pb (figure 4. Le vecteur pBS-SK-f- a été digéré par £cσRV et Xbal, puis ligaturé sur lui- même pour donner pBS-SK* (modifié). Le plasmide pEL004 a été digéré par Kpnl et Hindlll pour isoler le fragment Kpnl-Hindlll de 1387 pb contenant le gène IBDV VP2 complet. Ce fragment a été clone dans le vecteur pBS-SK*, préalablement digéré par Kpnl et Hindlll, pour donner le plasmide pEL023 de 4292 pb (figure 4). Le plasmide pEL022 a été digéré par BamKL et Notl pour isoler le fragment BamHl-Notl de 1122 pb (fragment A). Le plasmide pEL023 a été digéré par BamHl et Notl pour isoler le fragment BamHl-Notl de 333 pb (fragment B). Les fragments A et B ont été ligaturés ensemble avec le vecteur pBS-SK+, préalablement digéré par Notl et traité avec la phosphatase alcaline, pour donner le plasmide pEL024 de 4369 pb (figure 4). Le plasmide pEL024 a été digéré par Notl pour isoler le fragment Notl-Notl de 1445 pb. Ce fragment a été ligaturé avec le plasmide pCMVβ (Clontech Ca 6177-1, figure 5), préalablement digéré par Notl, pour donner le plasmide pEL026 de 5095 pb (figure 5).6.1 - Cloning of the VP2 gene from the Gumboro disease virus (IBDV) and construction of a VP2 expression cassette under the control of the HCMV IE promoter The plasmid pEL004 (see FIG. 4; ≈ plasmid pGH004 described in the patent application French 92.13109) containing the IBDV VP2 gene in the form of a BamHI-HindIII cassette was digested with BamHI and Xbal to isolate the BamHI-Xbal fragment (truncated VP2 gene) of 1104 bp. This fragment was cloned into the vector pBS SK +, previously digested with Xbal and BamHI to give the plasmid pEL022 of 4052 bp (FIG. 4. The vector pBS-SK-f- was digested with £ cσRV and Xbal, then ligated on itself to give pBS-SK * (modified) The plasmid pEL004 was digested with Kpnl and Hindlll to isolate the Kpnl-Hindlll fragment of 1387 bp containing the complete IBDV VP2 gene. This fragment was cloned in the vector pBS-SK *, previously digested with Kpnl and Hindlll, to give plasmid pEL023 of 4292 bp (FIG. 4). Plasmid pEL022 was digested with BamKL and NotI to isolate the BamHI-NotI fragment of 1122 bp (fragment A). Plasmid pEL023 was digested with BamHI and NotI to isolate the fragment BamHI-NotI of 333 bp (fragment B). The fragments A and B were ligated together with the vector pBS-SK +, previously digested with NotI and treated with alkaline phosphatase, to give the plasmid pEL024 of 4369 bp (FIG. 4) Plasmid pEL024 was digested with NotI to isolate the f ragment Notl-Notl of 1445 bp. This fragment was ligated with the plasmid pCMVβ (Clontech Ca 6177-1, FIG. 5), previously digested with Notl, to give the plasmid pEL026 of 5095 bp (FIG. 5).
Le plasmide pEL026 a été digéré par JS σRI, Sali et Xmnl pour isoler le fragment EcoKl-Sall de 2428 pb. Ce fragment a été ligaturé avec le vecteur pBS-SK+, préalablement digéré par ϋcσRI et Sali, pour donner le plasmide pEL027 de 5379 pb (figure 5).The plasmid pEL026 was digested with JS σRI, Sali and Xmnl to isolate the EcoKl-SalI fragment of 2428 bp. This fragment was ligated with the vector pBS-SK +, previously digested with ϋcσRI and SalI, to give the plasmid pEL027 of 5379 bp (FIG. 5).
6.1 - Construction du plasmide donneur p ELI 58 Le plasmide pEL027 a été digéré par £cσRI, S Λ et Xmnl pour isoler le fragment £ ?RI- Sall de 2428 pb. Ce fragment a été ligaturé dans le plasmide pEL157 (voir exemple 5 et figure 3), préalablement digéré par £cσRI et Sali, pour donner le plasmide pEL158 de 6950 pb (figure 6).6.1 - Construction of the donor plasmid p ELI 58 The plasmid pEL027 was digested with £ cσRI, S Λ and Xmnl to isolate the £? RI-Sall fragment of 2428 bp. This fragment was ligated into the plasmid pEL157 (see example 5 and FIG. 3), previously digested with £ cσRI and SalI, to give the plasmid pEL158 of 6950 bp (FIG. 6).
6.3 - Isolement et purification du virus recombinant vILTVS Le virus vILTV8 a été isolé et purifié après cotransfection de l'ADN du plasmide pEL158 préalablement linéarisé par l'enzyme Kpnl et de l'ADN viral, comme décrit dans l'exemple 3. Ce recombinant contient une cassette HCMV-IE/IBDV VP2 dans le site intergénique entre les COLs B et C du virus ILTV (voir exemple 5). Exemple 7: Construction du plasmide donneur pEL159 pour l'insertion d'une cassette d'expression du gène VP2 de l'EBDV sous contrôle du promoteur MCMN EE dans le site intergénique entre les COLs B et C et isolement de vELTN9:6.3 - Isolation and purification of the vILTVS recombinant virus The vILTV8 virus was isolated and purified after cotransfection of the DNA of the plasmid pEL158 previously linearized with the enzyme Kpnl and of the viral DNA, as described in Example 3. This recombinant contains an HCMV-IE / IBDV VP2 cassette in the intergenic site between the COLs B and C of the ILTV virus (see example 5). Example 7: Construction of the donor plasmid pEL159 for the insertion of an expression cassette for the VP2 gene of EBDV under the control of the MCMN EE promoter in the intergenic site between the COLs B and C and isolation of vELTN9:
7.1 • Construction de pEUOJO contenant une cassette d'expression du gène VP2 de l'IBDV sous contrôle du promoteur immédiate early (IE) du MCMV (Mouse CytoMegalo Virus)7.1 • Construction of pEUOJO containing an expression cassette for the IBDV VP2 gene under the control of the immediate early (IE) promoter of MCMV (Mouse CytoMegalo Virus)
Le plasmide pCMVβ (Clontech Catf 6177-1, figure 7) a été digéré par Sali et Smal pour isoler le ragment Sall-Smal de 3679 pb contenant le gène lacZ ainsi que le signal de poiy-adénylation du gène tardif du virus SV40. Ce fragment a été inséré dans le vecteur pBS-SK+, préalablement digéré par Salï et EcσRV, pour donner le plasmide pCD002 de 6625 pb (figure 7). Ce plasmide contient le gène reporter lacZ mais aucun promoteur n'est situé en amont de ce gène.The plasmid pCMVβ (Clontech Catf 6177-1, FIG. 7) was digested with Sali and Smal to isolate the Sall-Smal ragment of 3679 bp containing the lacZ gene as well as the poiy-adenylation signal of the late gene of the SV40 virus. This fragment was inserted into the vector pBS-SK +, previously digested with SalI and EcσRV, to give the plasmid pCD002 of 6625 bp (FIG. 7). This plasmid contains the lacZ reporter gene but no promoter is located upstream of this gene.
Le virus MCMV souche Smiui a été obtenu de l'American Type Culture Collection, Rockville, Maryland, USA (ATCC Ν" VR-194). Ce virus a été cultivé sur cellules d'embryon de souris Balb/C et l'AD viral de ce virus a été préparé comme décrit par Ebeling A. ét al. (J. Virol. 1983. 47. 421-433). Cet ADN génomique viral a été digéré par Pstl pour isoler le fragment Pstl-Pstl de 2285 pb. Ce fragment a été clone dans le vecteur pBS-SK , préalablement digéré par Pstl et traité avec la phosphatase alcaline, pour donner le plasmide pCD004 (figure 7). Le plasmide pCD004 a été digéré par Hpal et Pstl pour isoler le fragment Hpal-Pstl de 1389 pb qui contient la région promotrice/activatrice du gène Immediate-Early du cytomégalovirus murin (Murine CytoMegalo Virus ≈ MCMV) (Dorsch-Hâsler K. étal. Proc. Natl. Acad. Sci. 1985. 82. 8325-8329, et demande de brevet WO-A-87/03905). Ce fragment a été clone dans le plasmide pCD002, préalablement digéré par Pstl et Smal, pour donner le plasmide pCD009 de 8007 pb (figure 7).The MCMI virus Smiui strain was obtained from the American Type Culture Collection, Rockville, Maryland, USA (ATCC Ν "VR-194). This virus was cultured on Balb / C mouse embryo cells and viral AD of this virus was prepared as described by Ebeling A. et al. (J. Virol. 1983. 47. 421-433) This viral genomic DNA was digested with Pstl to isolate the Pstl-Pstl fragment of 2285 bp. fragment was cloned into the vector pBS-SK, previously digested with Pstl and treated with alkaline phosphatase, to give the plasmid pCD004 (FIG. 7). The plasmid pCD004 was digested with Hpal and Pstl to isolate the Hpal-Pstl fragment from 1389 bp which contains the promoter / activator region of the Immediate-Early gene of the murine cytomegalovirus (Murine CytoMegalo Virus ≈ MCMV) (Dorsch-Hâsler K. et al. Proc. Natl. Acad. Sci. 1985. 82. 8325-8329, and request WO-A-87/03905). This fragment was cloned into the plasmid pCD002, previously digested with Pstl and Smal, p or give the plasmid pCD009 of 8007 bp (Figure 7).
Un oligonucléotide double brin a été obtenu par hybridation des deux oligonucléotides suivants :A double-stranded oligonucleotide was obtained by hybridization of the following two oligonucleotides:
MB070 (SEQ ID NO:6)MB070 (SEQ ID NO: 6)
5 ' CG AATTCACTAGτGTGTGTCTGCAGGCGGCCσCGTGTGτσTCσ ACGGTAC 3'5 'CG AATTCACTAGτGTGTGTCTGCAGGCGGCCσCGTGTGτσTCσ ACGGTAC 3 '
MB071 (SEQ ID NO:7)MB071 (SEQ ID NO: 7)
5 ' CGTCGACACACACGCGGCCGCCTGCAGAC ACAC ACTAGTGAATTCG AGCT 3' Cet oligonucléotide double brin a été ligaturé avec le vecteur pBS-SK-H, préalablement digéré par Kpnl et Sacl, pour donner le plasmide pEL067 (figure 8). Le plasmide pCD009 a été digéré par Pstl et Spel pour isoler le fragment Pstl-Spel de 1396 pb. Ce fragment a été ligaturé avec le plasmide pEL067, préalablement digéré par Pstl et Spel, pour donner le plasmide pEL068 de 4297 pb (figure 8). Le plasmide pEL024 (voir exemple 6, paragraphe 6.1 et figure 5) a été digéré par Hindlll et Notl pour isoler le fragment Hindlll-Notl de 1390 pb (fragment A). Le plasmide pEL027 (voir exemple 6, paragraphe 6.1 et figure 5) a été digéré par Hindlll et Sali pour isoler le fragment Hindlïl-Sall de 235 pb (fragment B). Les fragments A et B ont été ligaturés ensemble avec le plasmide pEL068, préalablement digéré par Notl et Sali, pour donner le plasmide pEL070 de 5908 pb (figure 8). Ce plasmide contient donc une casseπe d'expression constituée du promoteur IE du MCMV, du gène VP2 et du signal polyA de SV40.5 'CGTCGACACACACGCGGCCGCCTGCAGAC ACAC ACTAGTGAATTCG AGCT 3' This double-stranded oligonucleotide was ligated with the vector pBS-SK-H, previously digested with Kpn1 and Sac1, to give the plasmid pEL067 (Figure 8). Plasmid pCD009 was digested with Pstl and Spel to isolate the Pstl-Spel fragment of 1396 bp. This fragment was ligated with the plasmid pEL067, previously digested with Pstl and Spel, to give the plasmid pEL068 of 4297 bp (FIG. 8). The plasmid pEL024 (see example 6, paragraph 6.1 and FIG. 5) was digested with HindIII and NotI to isolate the HindIII-NotI fragment of 1390 bp (fragment A). The plasmid pEL027 (see example 6, paragraph 6.1 and FIG. 5) was digested with HindIII and SalI to isolate the HindIII-SalI fragment of 235 bp (fragment B). Fragments A and B were ligated together with the plasmid pEL068, previously digested with NotI and SalI, to give the plasmid pEL070 of 5908 bp (FIG. 8). This plasmid therefore contains a casseπe expression consisting of the IE promoter of MCMV, the VP2 gene and the polyA signal of SV40.
7.2 - Construction du plasmide donneur pELl 597.2 - Construction of the donor plasmid pEL1 59
Le plasmide pEL070 a été digéré par JE oRI, Sali et Xmnl pour isoler le fragment EcoTU.- Sali de 3035 pb. Ce fragment a été ligaturé dans le plasmide pEL157 (voir exemple 5 et figure 3), préalablement digéré par EcoRL et Sali, pour donner le plasmide pEL159 de 7545 pb (figure 9). Ce plasmide permet l'insertion de la cassette d'expression MCMV-IE/IBDV-VP2 dans le site intergénique entre les COLs B et C du virus ILTV.The plasmid pEL070 was digested with JE oRI, Sali and Xmnl to isolate the EcoTU.- Sali fragment of 3035 bp. This fragment was ligated into the plasmid pEL157 (see example 5 and FIG. 3), previously digested with EcoRL and SalI, to give the plasmid pEL159 of 7545 bp (FIG. 9). This plasmid allows the insertion of the MCMV-IE / IBDV-VP2 expression cassette into the intergenic site between the COLs B and C of the ILTV virus.
7.3 - Isolement et purification du virus recombinant vILTV9 Le virus vILTN9 a été isolé et purifié après cotransfection de l'ADΝ du plasmide pEL159 préalablement linéarisé par l'enzyme BgR et de l' ADN viral, comme décrit dans l'exemple 3. Ce recombinant contient une cassette MCMV-IE/IBDV VP2 dans le site intergénique entre les COLs B et C du virus ILTN (voir exemple 5). Exemple 8: Construction du plasmide donneur pEL160 pour l'insertion d'une cassette d'expression du gène HN du NDV dans le site intergénique entre les COLs B et C et isolement de vELTNlO:7.3 - Isolation and purification of the recombinant virus vILTV9 The vILTN9 virus was isolated and purified after cotransfection of the ADΝ of the plasmid pEL159 previously linearized with the enzyme BgR and the viral DNA, as described in Example 3. This recombinant contains an MCMV-IE / IBDV VP2 cassette in the intergenic site between the COLs B and C of the ILTN virus (see example 5). Example 8: Construction of the donor plasmid pEL160 for the insertion of an expression cassette for the NDV HN gene into the intergenic site between COLs B and C and isolation of vELTN10:
8.1 - Clonage du gène HN du virus de la maladie de Newcastle (NDV) La constitution d'une banque d'ADN complémentaire du génome du virus de la maladie de Newcastle (NDV), souche Texas, a été réalisée comme décrit par Taylor J. et al. (J. Virol. 1990. 64. 1441-1450). Un clone pBR322 contenant la fin du gène fusion (F), la totalité du gène hemagglutinine-neuraminida.se (HN) et le début du gène de la poiymérase a été identifié pHNOl. La séquence du gène NDV HN contenue sur ce clone est présentée sur la figure 10 (SEQ ID NO:8). Le plasmide pHNOl a été digéré par Sphl et Xbal pour isoier le fragment Sphl-Xbal de 2520 pb. Ce fragment a été ligaturé avec le vecteur pUC19, préalablement digéré par Sphl et Xbal, pour donner le plasmide pHN02 de 5192 pb. Le plasmide pHN02 a été digéré par Clal et Pstl pour isoler le fragment Clάl-Pstl de 700 pb (fragment A). Une PCR a été réalisée avec les oligonucléotides suivants:8.1 - Cloning of the HN gene of the Newcastle disease virus (NDV) The constitution of a DNA library complementary to the genome of the Newcastle disease virus (NDV), Texas strain, was carried out as described by Taylor J . et al. (J. Virol. 1990. 64. 1441-1450). A pBR322 clone containing the end of the fusion (F) gene, the entire hemagglutinin- neuraminida.se (HN) gene and the start of the poiymerase gene was identified pHNOl. The sequence of the NDV HN gene contained in this clone is presented in FIG. 10 (SEQ ID NO: 8). The plasmid pHNOl was digested with Sphl and Xbal to isolate the Sphl-Xbal fragment of 2520 bp. This fragment was ligated with the vector pUC19, previously digested with Sphl and Xbal, to give the plasmid pHN02 of 5192 bp. The plasmid pHN02 was digested with ClaI and Pstl to isolate the Clάl-Pstl fragment of 700 bp (fragment A). A PCR was carried out with the following oligonucleotides:
EL071 (SEQ ID NO:9) 5' CAGACCAAGCTTCTTAAATCCC 3'EL071 (SEQ ID NO: 9) 5 'CAGACCAAGCTTCTTAAATCCC 3'
EL073 (SEQ ID NO: 10) 5' GTATTCGGGACAATGC 3' et la matrice pHN02 pour prod -.-e un fragment PCR de 270 pb. Ce fragment a été digéré par Hindlll et Pstl pour isoler un fragment Hindïïl-Pstl de 220 pb (fragment B). Les fragments A et B ont été ligaturés ensemble avec le vecteur pBS-SK- préalablement digéré par Clal et Hindlll, pour donner le plasmide pEL028 de 3872 pb (figure 11). Le plasmide pHN02 a été digéré par Bsphl et Clal pour isoler le fragment Bsp l-Clal de 425 pb (fragment C). Une PCR a été réalisée avec les oligonucléotides suivants: EL074 (SEQ ID NO: 11) 5' GTGACATCACTAGCGTCATCC 3' EL075 (SEQ ID NO: 12)EL073 (SEQ ID NO: 10) 5 'GTATTCGGGACAATGC 3' and the pHN02 matrix to produce a PCR fragment of 270 bp. This fragment was digested with HindIII and Pst1 to isolate a HindIII-Pst1 fragment of 220 bp (fragment B). Fragments A and B were ligated together with the vector pBS-SK- previously digested with ClaI and HindIII, to give the plasmid pEL028 of 3872 bp (FIG. 11). The plasmid pHN02 was digested with Bsphl and ClaI to isolate the Bsp l-ClaI fragment of 425 bp (fragment C). A PCR was carried out with the following oligonucleotides: EL074 (SEQ ID NO: 11) 5 'GTGACATCACTAGCGTCATCC 3' EL075 (SEQ ID NO: 12)
5' CCGCATCATCAGCGGCCGCGATCGGTCATGGACAGT 3' et la matrice pHN02 pour produire un fragment PCR de 465 pb. Ce fragment a été digéré par Bsphl et Notl pour isoler le fragment Bsphl-Notl de 390 pb (fragment D). Les fragments C et D ont été ligaturés ensemble avec le vecteur pBS-SK+, préalablement digéré par Clal et Notl, pour donner le plasmide pEL029bis de 3727 pb (figure 11). Le plasmide pEL028 a été digéré par Clal et Sacïl pour isoler le fragment Clal-Sacll de 960 pb (fragment E). Le plasmide pELÛ29bis a été digéré par Clal et Notl pour isoler le fragment Clal-Notl de 820 pb (fragment F). Les fragments E et F ont été ligaturés ensemble avec le vecteur pBS-SK- , préalablement digéré par Notl et Sacll, pour donner le plasmide pEL030 de 4745 pb (figure 11).5 'CCGCATCATCAGCGGCCGCGATCGGTCATGGACAGT 3' and the pHN02 matrix to produce a PCR fragment of 465 bp. This fragment was digested with Bsphl and Notl to isolate the Bsphl-Notl fragment of 390 bp (fragment D). Fragments C and D were ligated together with the vector pBS-SK +, previously digested with ClaI and NotI, to give the plasmid pEL029bis of 3727 bp (FIG. 11). The plasmid pEL028 was digested with ClaI and SacIl to isolate the ClaI-SacII fragment of 960 bp (fragment E). The plasmid pEL029bis was digested with ClaI and NotI to isolate the ClaI-NotI fragment of 820 bp (fragment F). Fragments E and F were ligated together with the vector pBS-SK-, previously digested with NotI and Sacll, to give the plasmid pEL030 of 4745 bp (FIG. 11).
8.2 - Construction du plasmide pEL160 contenant une cassette d'expression de HN du NDV dans le site intergénique entre les COLs B et C Le plasmide pEL030 a été digéré par Notl pour isoler le fragment Notl-Notl de 1780 pb (gène NDV HN entier). Ce fragment a été inséré dans les sites Notl du plasmide pEL159 (exemple 7, figure 9) à la place du fragment Notl-Notl de 1405 pb contenant le gène codant pour la protéine VP2 de l'IBDV; ce clonage a permis d'isoier le plasmide pEL160 de 7921 pb (figure 12). Ce plasmide permet l'insertion de la cassette d'expression MCMV-IE/NDV-HN dans le site intergénique entre les COLs B et C du virus ILTN.8.2 - Construction of the plasmid pEL160 containing an NDV HN expression cassette in the intergenic site between COLs B and C The plasmid pEL030 was digested with NotI to isolate the NotI-NotI fragment of 1780 bp (whole NDV HN gene) . This fragment was inserted into the NotI sites of the plasmid pEL159 (Example 7, FIG. 9) in place of the NotI-NotI fragment of 1405 bp containing the gene coding for the protein VP2 of IBDV; this cloning made it possible to isolate the plasmid pEL160 of 7921 bp (FIG. 12). This plasmid allows the insertion of the MCMV-IE / NDV-HN expression cassette into the intergenic site between the COLs B and C of the ILTN virus.
8.3 • Isolement et purification du virus recombinant vILTVIO8.3 • Isolation and purification of the recombinant virus vILTVIO
Le virus vILTVIO a été isolé et purifié après cotransfection de l'ADΝ du plasmide pEL160 préalablement linéarisé par l'enzyme BgH et de l'AD virai, comme décrit dans l'exemple 3. Ce recombinant contient une cassette MCMV-IE/ΝDV HΝ dans le site intergénique entre les COLs B et C du virus ILTV (voir exemple 5).The vILTVIO virus was isolated and purified after cotransfection of the ADΝ of the plasmid pEL160 previously linearized with the enzyme BgH and of the AD virai, as described in Example 3. This recombinant contains an MCMV-IE / ΝDV HΝ cassette in the intergenic site between COLs B and C of the ILTV virus (see example 5).
Exemple 9: Construction du plasmide donneur pEL161 pour l'insertion d'une cassette d'expression du gène F du ΝDV dans le site intergénique entre les COLs B et C et isolement de ylLTVll:Example 9: Construction of the donor plasmid pEL161 for the insertion of an expression cassette for the ΝDV F gene into the intergenic site between COLs B and C and isolation of ylLTVll:
9.1 - Clonage du gène F du virus de la maladie de Newcastle (NDV) Un clone provenant de la banque d'ADN complémentaire du génome du virus de la maladie de Newcastle (voir exemple 8, paragraphe 8.1) et contenant le gène fusion (F) en entier a été appelé pNDV81. Ce plasmide a été décrit précédemment et la séquence du gène NDV F présent sur ce clone a été publiée (Taylor J. et al. J. Virol., 1990, 64, 1441- 1450). Le plasmide pNDV81 a été digéré par Narl et Pstl pour isoler le fragment Narl-Pstl de 1870 pb (fragment A). Une PCR a été réalisée avec les oligonucléotides suivants:9.1 - Cloning of the Newcastle disease virus (NDV) F gene A clone originating from the DNA bank complementary to the genome of the Newcastle disease virus (see example 8, paragraph 8.1) and containing the fusion gene (F ) in its entirety was called pNDV81. This plasmid has been described previously and the sequence of the NDV F gene present on this clone has been published (Taylor J. et al. J. Virol., 1990, 64, 1441-1450). The plasmid pNDV81 was digested with Narl and Pstl to isolate the Narl-Pstl fragment of 1870 bp (fragment A). A PCR was carried out with the following oligonucleotides:
EL076 (SEQ ID N° 13) 5' TGACCCTGTCTGGGATGA 3' EL077 (SEQ ID N" 14)EL076 (SEQ ID N ° 13) 5 'TGACCCTGTCTGGGATGA 3' EL077 (SEQ ID N "14)
5' GGATCCCGGTCGACACATTGCGGCCGCAAGATGGGC 3' et la matrice pNDVδl pour produire un fragment de 160 pb. Ce fragment a été digéré par Pstl et Sali pour isoler le fragment Pstl-Sall de 130 pb (fragment B). Les fragments A et B ont été ligaturés ensemble avec le vecteur pBS-SK+, préalablement digéré par Clal et Sali, pour donner le plasmide pEL033 de 4846 pb (figure 13).5 'GGATCCCGGTCGACACATTGCGGCCGCAAGATGGGC 3' and the matrix pNDVδl to produce a 160 bp fragment. This fragment was digested with Pstl and SalI to isolate the Pstl-SalI fragment of 130 bp (fragment B). Fragments A and B were ligated together with the vector pBS-SK +, previously digested with ClaI and SalI, to give the plasmid pEL033 of 4846 bp (FIG. 13).
9.2 - Construction du plasmide pELl 61 contenant une cassette d'expression du gène F du NDV dans le site intergénique entre les COLs B et C9.2 - Construction of the plasmid pEL161 containing an expression cassette for the NDV F gene in the intergenic site between COLs B and C
Le plasmide pEL033 a été digéré par Notl pour isoler le fragment Notl-Notl de 1935 pb (gène F entier). Ce fragment a été inséré dans les sites Notl du plasmide pEL159 (exemple 7, figure 9) i la place du fragment Notl-Notl de 1405 pb contenant le gène codant pour la protéine VP2 de ITBDV; ce clonage a permis d'isoler le plasmide pEL161 de 8074 pb (figure 14). Ce plasmide permet l'insertion de la cassette d'expression MCMV-IE/NDV-F dans le site intergénique entre les COLs B et C du virus ILTN.Plasmid pEL033 was digested with NotI to isolate the 1935 bp NotI-NotI fragment (whole F gene). This fragment was inserted into the NotI sites of the plasmid pEL159 (Example 7, FIG. 9) in place of the NotI-NotI fragment of 1405 bp containing the gene coding for the protein VP2 of ITBDV; this cloning made it possible to isolate the plasmid pEL161 from 8074 bp (FIG. 14). This plasmid allows the insertion of the MCMV-IE / NDV-F expression cassette into the intergenic site between the COLs B and C of the ILTN virus.
9.3 - Isolement et purification du virus recombinant vILTVll Le virus vILT ll a été isolé et purifié après cotransfection de l'ADΝ du plasmide pEL161 préalablement linéarisé par l'enzyme BgΛ et de l'ADΝ viral, comme décrit dans l'exemple 3. Ce recombinant contient une cassette MCMV-IE/ΝDV F dans le site intergénique entre les COLs B et C du virus ILTV (voir exemple 5).9.3 - Isolation and purification of the recombinant virus vILTVll The vILT ll virus was isolated and purified after cotransfection of the ADΝ of the plasmid pEL161 previously linearized with the enzyme BgΛ and of the viral ADΝ, as described in Example 3. This recombinant contains an MCMV-IE / ΝDV F cassette in the intergenic site between the COLs B and C of the ILTV virus (see example 5).
Exemple 10: Construction d'un plasmide donneur pour l'insertion d'une double cassette d'expression des gènes HΝ et F du ΝDV dans le site intergénique entre les COLs B et C et isolement d'un virus recombinant ELTN:Example 10: Construction of a donor plasmid for the insertion of a double cassette for expression of the HΝ and F genes of ΝDV in the intergenic site between the COLs B and C and isolation of a recombinant ELTN virus:
Une double cassette d'expression de deux gènes, par exemple les gènes HΝ et F du virus ΝDV, peut être construite. Une telle construction est schématisée à la figure 15. Dans cette construction, l'extrémité 5' des deux promoteurs sont adjacentes de manière que la transcription des deux gènes se fasse en sens opposés. Un des deux promoteurs est le promoteur MCMV IE et l'autre promoteur (appelé promoteur associé) est le promoteur SV40 (présent dans le plasmide pSVbeta, Clontech Laboratories, Palo Alto, California 94303-4607, USA). Dans cette configuration, le promoteur associé est activé par la région activatrice du promoteur CMV IE.A double expression cassette for two genes, for example the HΝ and F genes of the ΝDV virus, can be constructed. Such a construction is shown schematically in Figure 15. In this construction, the 5 ′ end of the two promoters are adjacent so that the transcription of the two genes takes place in opposite directions. One of the two promoters is the MCMV IE promoter and the other promoter (called associated promoter) is the SV40 promoter (present in the plasmid pSVbeta, Clontech Laboratories, Palo Alto, California 94303-4607, USA). In this configuration, the associated promoter is activated by the activating region of the CMV IE promoter.
Ceπe double cassette d'expression peut ensuite être insérée dans le plasmide donneur décrit ci-dessus (pEL157 décrit dans l' exemple 5 et représenté dans la figure 3). L' isolement des virus recombinants se fait de la même manière que ci-dessus (voir exemple 3).This double expression cassette can then be inserted into the donor plasmid described above (pEL157 described in Example 5 and represented in FIG. 3). The isolation of the recombinant viruses is done in the same way as above (see example 3).
Exemple 11: Construction du plasmide donneur pEL163 pour l'insertion d'une cassette d'expression du gène gB du MDV dans le site intergénique entre les COLs B et C et isolement de vILTV12:Example 11: Construction of the donor plasmid pEL163 for the insertion of an MDV gB gene expression cassette into the intergenic site between COLs B and C and isolation of vILTV12:
11.1 • Clonage du gène gB du virus de la maladie de Marek Le fragment EcoRl-Scuï de 3,9 kpb de l'ADN génomique du virus MDV souche RB1B contenant le gène MDV gB (séquence publiée par Ross N. et al. J. Gen. Virol. 1989. 70, 1789-1804) a été ligaturé avec le vecteur pUC13, préalablement digéré par £ ?RI et Sali, pour donner le plasmide pCD007 de 6543 pb (figure 16). Ce plasmide a été digéré par Sacl et Xbal pour isoler le fragment Sacl-Xbal de 2260 pb (partie centrale du gène gB ≈ fragment A). Une PCR a été réalisée avec les oligonucléotides suivants: CD001 (SEQ ID O:15)11.1 • Cloning of the Marek disease virus gB gene The 3.9 kbp EcoRI-Scui fragment of the genomic DNA of the MDV virus strain RB1B containing the MDV gB gene (sequence published by Ross N. et al. J. Gen. Virol. 1989. 70, 1789-1804) was ligated with the vector pUC13, previously digested with RI and SalI, to give the plasmid pCD007 of 6543 bp (FIG. 16). This plasmid was digested with SacI and Xbal to isolate the SacI-Xbal fragment of 2260 bp (central part of the gB gene ≈ fragment A). A PCR was carried out with the following oligonucleotides: CD001 (SEQ ID O: 15)
5' GACΓGGTACCGCGGCCGCATGCACΠTΠΆGGCGGAATTG y5 'GACΓGGTACCGCGGCCGCATGCACΠTΠΆGGCGGAATTG y
CD002 (SEQ ID NO: 16) 5' TτCGGGACATTTTCGCGG 3' et la matrice pCD007 pour produire un fragment PCR de 222 pb. Ce fragment a été digéré par Kpnl et Xbal pour isoler un fragment Kpnl-Xbal de 190 pb (extrémité 5' du gène gB = fragment B). Une autre PCR a été réalisée avec les oligonucléotides suivants:CD002 (SEQ ID NO: 16) 5 'TτCGGGACATTTTCGCGG 3' and the matrix pCD007 to produce a PCR fragment of 222 bp. This fragment was digested with Kpnl and Xbal to isolate a Kpnl-Xbal fragment of 190 bp (5 'end of the gB gene = fragment B). Another PCR was carried out with the following oligonucleotides:
CD003 (SEQ ID NO: 17) 5' TATATGGCGTTAGTCTCC 3'CD003 (SEQ ID NO: 17) 5 'TATATGGCGTTAGTCTCC 3'
CD004 (SEQ ID NO: 18)CD004 (SEQ ID NO: 18)
5' TTσcσAGCTCGCGGCCGCTTATTACACAGCATCATCTTCτG 3' et la matrice pCD007 pour produire un fragment PCR de 195 pb. Ce fragment a été digéré par Sacl et Sacll pour isoler le fragment Sacl-Sacll de 162 pb (extrémité 3' du gέne gB ≈ fragment C). Les fragments A, B et C ont été ligaturés ensemble avec le vecteur pBS-SK+, préalablement digéré par Kpnl et Sacl. pour donner le plasmide pCDOi l de 5485 pb (figure 16).5 'TTσcσAGCTCGCGGCCGCTTATTACACAGCATCATCTTCτG 3' and the matrix pCD007 to produce a PCR fragment of 195 bp. This fragment was digested with Sacl and Sacll to isolate the Sacl-Sacll fragment of 162 bp (3 'end of the gene gB ≈ fragment C). Fragments A, B and C were ligated together with the vector pBS-SK +, previously digested with Kpnl and Sacl. to give the plasmid pCDOi l of 5485 bp (FIG. 16).
11.2 • Construction du plasmide pEÎΛ 63 contenant une cassette d'expression du gène gB du MDV dans le site intergénique entre les COLs B et C du virus ILTV Le plasmide pCDOil a été digéré par Notl pour isoler le fragment Notl-Notl de 2608 pb (gène gB MDV entier). Ce fragment a été inséré dans les sites Notl du piasmide pEL159 (exemple 7, figure 9) à la place du fragment Notl-Notl de 1405 pb contenant le gène codant pour la protéine VP2 de l'EBDV; ce clonage a permis d'isoler le plasmide pEL163 de 8749 pb (figure 17). Ce plasmide permet l'insertion de la cassette d'expression MCMV-IE/MDV-gB dans le site intergénique entre les COLs B et C du virus ILTN.11.2 • Construction of the plasmid pEÎΛ 63 containing an expression cassette for the MDV gB gene in the intergenic site between the COLs B and C of the ILTV virus The plasmid pCDOil was digested with NotI to isolate the NotI-NotI fragment of 2608 bp ( whole MDV gB gene). This fragment was inserted into the NotI sites of piasmide pEL159 (Example 7, FIG. 9) in place of the NotI-NotI fragment of 1405 bp containing the gene coding for the protein VP2 of EBDV; this cloning made it possible to isolate the plasmid pEL163 from 8749 bp (FIG. 17). This plasmid allows the insertion of the MCMV-IE / MDV-gB expression cassette into the intergenic site between the COLs B and C of the ILTN virus.
11.3 - Isolement et purification du virus recombinant vILTVll Le virus vILTV12 a été isolé et purifié après cotransfection de l'ADΝ du plasmide pELlόl préalablement linéarisé par l'enzyme BgH et de l'ADΝ viral, comme décrit dans l'exemple 3. Ce recombinant contient une cassette MCMV-IE/MDV gB dans le site intergénique entre les COLs B et C du virus ILTV (voir exemple 5).11.3 - Isolation and purification of the vILTV11 recombinant virus The vILTV12 virus was isolated and purified after cotransfection of the ADΝ of the plasmid pELlόl previously linearized with the enzyme BgH and of the viral ADΝ, as described in Example 3. This recombinant contains an MCMV-IE / MDV gB cassette in the intergenic site between the COLs B and C of the ILTV virus (see example 5).
Exemple 12: Construction d'un plasmide donneur pour l'Insertion d'une cassette d'expression de gène(s) de l'EBV dans le site Intergénique entre les COLs B et C et isolement de virus recombinant ILTV:Example 12 Construction of a Donor Plasmid for the Insertion of an EBV Gene Expression Cassette into the Intergenic Site Between COLs B and C and Isolation of Recombinant ILTV Virus:
Selon la même stratégie que celle décrite plus haut pour l'insertion de simples cassenes (exemples 6, 7, 8, 9 et 11) ou pour l'insertion de doubles cassettes (exemple 10), dans le site décrit ci-dessus (exemple 5), il est possible de réaliser des virus ILTV recombinants exprimant à un niveau élevé les protéines Membrane (M) ou Spike (S), ou partie de Spike (SI ou S2), ou Nucléocapside (N) du virus de la bronchite infectieuse aviaire (IBV). On réalise notamment une double cassette d'expression avec le gène S sous contrôle du promoteur CMV IETet le gène M sous contrôle du promoteur associé. 215According to the same strategy as that described above for the insertion of simple cassenes (examples 6, 7, 8, 9 and 11) or for the insertion of double cassettes (example 10), in the site described above (example 5), it is possible to make recombinant ILTV viruses expressing at a high level the Membrane (M) or Spike (S) proteins, or part of Spike (SI or S2), or Nucleocapsid (N) of the infectious bronchitis virus avian (IBV). A double expression cassette is produced in particular with the S gene under the control of the CMV IET promoter and the M gene under the control of the associated promoter. 215
2525
Exemple 13: Construction de plasmides donneurs pour l'insertion de cassettes d' expression de gène(s) d'autres agents pathogènes aviaires ou de peptide immunomoduiateur dans le site , décrit, at isolement de virus recombinants ELTV: Selon la même stratégie que celle décrite plus haut pour l'insertion de simples cassettes (exemples 6, 7, 8, 9 et 11) pour l'insertion de doubles cassettes (exemple 10), dans le site décrit ci-dessus (exemple 5), il est possible de réaliser des virus ILTV recombinants exprimant à un niveau élevé des immunogènes du CAV (et notamment une double cassette d'expression des gènes codant pour VPl et pour VP2), du virus de la pneumovirose du poulet, ou d'autres agents pathogènes aviaires, ou encore des peptides immunomodulateurs et notamment des cytokines.Example 13 Construction of Donor Plasmids for Insertion of Cassette Expression of Gene (s) of Other Avian Pathogens or Immunomodulatory Peptide into the Site Described and Isolation of Recombinant ELTV Viruses: According to the Same Strategy as That described above for the insertion of single cassettes (examples 6, 7, 8, 9 and 11) for the insertion of double cassettes (example 10), in the site described above (example 5), it is possible to make recombinant ILTV viruses expressing at a high level CAV immunogens (and in particular a double expression cassette for genes coding for VP1 and for VP2), chicken pneumovirus virus, or other avian pathogens, or still immunomodulatory peptides and in particular cytokines.
Exemple 1 : Production de vaccins:Example 1: Production of vaccines:
Les virus recombinants obtenus selon l'invention sont produits sur oeufs embryonnés.The recombinant viruses obtained according to the invention are produced on embryonated eggs.
La solution virale récoltée est ensuite diluée dans une solution stabilisatrice pour la lyophilisation, répartie à raison de 1000 doses vaccinales par flacon, et enfin lyophilisée. The harvested viral solution is then diluted in a stabilizing solution for lyophilization, distributed at the rate of 1000 vaccine doses per vial, and finally lyophilized.

Claims

REVENDICATIONS
1 - Vaccin vivant recombinant aviaire comprenant, comme vecteur, un virus ILTN comprenant et exprimant au moins une séquence nucléotidique hétérologue, cette séquence nucléotidique étant insérée dans le locus d'insertion formé par l' intergène situé entre les codons stop des COL B et COL C d'ILTV et qui, dans une souche d'ILTN particulière, est défini entre les nucleotides 908 et 994 à la SEQ ID ΝO: l.1 - Live recombinant avian vaccine comprising, as vector, an ILTN virus comprising and expressing at least one heterologous nucleotide sequence, this nucleotide sequence being inserted into the insertion locus formed by the intergene located between the stop codons of COL B and COL C of ILTV and which, in a particular ILTN strain, is defined between nucleotides 908 and 994 at SEQ ID ΝO: l.
2 - Vaccin vivant recombinant selon la revendication 1, caractérisé en ce que la ou les séquences nucléotidiques sont insérées par insertion simple, ou après délé ion totale ou partielle du locus d'insertion.2 - Recombinant live vaccine according to claim 1, characterized in that the nucleotide sequence or sequences are inserted by simple insertion, or after total or partial deletion of the insertion locus.
3 - Vaccin vivant recombinant selon l'une quelconque des revendications 1 à 2, caractérisé en ce que, pour exprimer la séquence nucléotidique insérée, le vecteur comprend un promoteur eucaryote fort.3 - Recombinant live vaccine according to any one of claims 1 to 2, characterized in that, to express the inserted nucleotide sequence, the vector comprises a strong eukaryotic promoter.
4 - Vaccin vivant recombinant selon la revendication 3, caractérisé en ce que le promoteur fort est choisi parmi le groupe consistant en: promoteur CMV immediate- early, de préférence le promoteur CMV immediate-early murin ou humain, promoteur LTR du virus du Sarcome de Rous (RSV), promoteur précoce du virus SV40.4 - Recombinant live vaccine according to claim 3, characterized in that the strong promoter is chosen from the group consisting of: immediate-early CMV promoter, preferably murine or human immediate-early CMV promoter, LTR promoter of the Sarcoma virus Rous (RSV), early promoter of the SV40 virus.
5 - Vaccin vivant recombinant selon l'une quelconque des revendications 1 à 4, caractérisé en ce qu'il comprend au moins deux séquences nucléotidiques insérées dans le locus d'insertion sous le contrôle de promoteurs eucaryotes différents.5 - Recombinant live vaccine according to any one of claims 1 to 4, characterized in that it comprises at least two nucleotide sequences inserted into the insertion locus under the control of different eukaryotic promoters.
6 - Vaccin vivant recombinant selon la revendication 5, caractérisé en ce que les promoteurs eucaryotes sont des promoteurs CMV immediate-early d'origines animales différentes.6 - Recombinant live vaccine according to claim 5, characterized in that the eukaryotic promoters are immediate-early CMV promoters of different animal origins.
7 - Vaccin vivant recombinant selon la revendication 5, caractérisé en ce qu' il comprend une première séquence nucléotidique associée au promoteur CMV immédiate early et un autre promoteur sous la dépendance duquel se trouve une autre séquence nucléotidique, ces deux promoteurs étant disposés de manière que leurs extrémités 5' soient adjacentes.7 - A live recombinant vaccine according to claim 5, characterized in that it comprises a first nucleotide sequence associated with the CMV immediate promoter early and another promoter under the control of which is another nucleotide sequence, these two promoters being arranged so that their 5 'ends are adjacent.
8 - Vaccin vivant recombinant selon l'une quelconque des revendications 1 à 7, caractérisé en ce qu'il comprend, insérée dans le locus d'insertion, une cassette d'expression comprenant successivement un promoteur, deux ou plusieurs gènes séparés deux à deux par un IRES, et un signal de polyadénylation.8 - Recombinant live vaccine according to any one of claims 1 to 7, characterized in that it comprises, inserted into the insertion locus, an expression cassette successively comprising a promoter, two or more genes separated in pairs by an IRES, and a polyadenylation signal.
9 - Vaccin vivant recombinant selon l'une quelconque des revendications 1 à 8, caractérisé en ce qu'il comprend une séquence nucléotidique codant pour un polypeptide antigénique d'un agent pathogène aviaire, cette séquence étant insérée dans le locus d'insertion.9 - Recombinant live vaccine according to any one of claims 1 to 8, characterized in that it comprises a nucleotide sequence coding for an antigenic polypeptide of an avian pathogenic agent, this sequence being inserted into the insertion locus.
10 - Vaccin vivant recombinant selon la revendication 9, caractérisé en ce qu'il comprend une séquence codant pour un antigène d'un agent pathogène aviaire choisi parmi le groupe consistant en le virus de la maladie de Newcastle (NDV), le virus de la maladie de Gumboro (IBDV), le virus de la maladie de Marek (MDV), le virus de la bronchite infectieuse (EBV), le virus de l'anémie du poulet (CAV), le virus de la pneumovirose du poulet10 - Recombinant live vaccine according to claim 9, characterized in that it comprises a sequence coding for an antigen of an avian pathogenic agent chosen from the group consisting of Newcastle disease virus (NDV), the virus of Gumboro disease (IBDV), Marek's disease virus (MDV), infectious bronchitis virus (EBV), chicken anemia virus (CAV), chicken pneumovirus virus
11 - Vaccin vivant recombinant selon la revendication 10, caractérisé en ce qu'il comprend une séquence nucléotidique, choisie parmi les séquences nucléotidiques codant pour les polypeptides F et HN du virus NDV.11 - Recombinant live vaccine according to claim 10, characterized in that it comprises a nucleotide sequence, chosen from the nucleotide sequences coding for the F and HN polypeptides of the NDV virus.
12 • Vaccin vivant recombinant selon la revendication 10, caractérisé en ce qu'il comprend une séquence nucléotidique, choisie parmi les séquences nucléotidiques codant pour les polypeptides gB, gC, gD, gH+gL du virus MDV.12 • Recombinant live vaccine according to claim 10, characterized in that it comprises a nucleotide sequence, chosen from the nucleotide sequences coding for the gB, gC, gD, gH + gL polypeptides of the MDV virus.
13 - Vaccin vivant recombinant selon la revendication 10, caractérisé en ce qu' il comprend au moins une séquence nucléotidique choisie parmi le groupe des séquences correspondant aux antigènes VP2 de l'IBDV, aux antigènes S, ou partie de S, M et N du virus IBV, aux antigènes VPl et VP2 du CAV, aux antigènes G et F du virus de la pneumovirose du poulet.13 - A live recombinant vaccine according to claim 10, characterized in that it comprises at least one nucleotide sequence selected from the sequences of the group corresponding to the IBDV VP2 antigens, to the S antigens, or part of S, M and N of the IBV virus, to the VP1 and VP2 antigens of the CAV, to the G and F antigens of the chicken pneumovirus virus.
14 - Vaccin vivant recombinant seion l'une quelconque des revendications 1 à 13, caractérisé en ce qu'il comprend une séquence nucléotidique codant pour un polypeptide immunomodulateur, cette séquence étant insérée dans le locus d'insertion.14 - Recombinant live vaccine according to any one of claims 1 to 13, characterized in that it comprises a nucleotide sequence coding for an immunomodulatory polypeptide, this sequence being inserted into the insertion locus.
15 - Vaccin vivant recombinant seion la revendication 14, caractérisé en ce que cette séquence nucléotidique est choisie parmi le groupe des séquences codant pour des cytokines.15 - Live recombinant vaccine according to claim 14, characterized in that this nucleotide sequence is chosen from the group of sequences coding for cytokines.
16 - Formule de vaccin multivalent comprenant, en mélange ou à mélanger, au moins deux vaccins vivants recombinants tels que définis dans l'une quelconque des revendications 1 à 15, ces vaccins comprenant des séquences insérées différentes.16 - Multivalent vaccine formula comprising, as a mixture or as a mixture, at least two recombinant live vaccines as defined in any one of claims 1 to 15, these vaccines comprising different inserted sequences.
17 - Un virus ELTV comprenant au moins un séquence nucléotidique hétérologue insérée dans le locus d'insertion formé par l' intergène situé entre les codons stop des COL B et COL C d'ILTV et qui, dans une souche d'ILTV particulière, est défini entre les nucleotides 908 et 994 à la SEQ ID NO:l. 17 - An ELTV virus comprising at least one heterologous nucleotide sequence inserted into the insertion locus formed by the intergene located between the stop codons of COL B and COL C of ILTV and which, in a particular ILTV strain, is defined between nucleotides 908 and 994 at SEQ ID NO: 1.
EP97952066A 1996-12-16 1997-12-15 Live recombinant avian vaccine, using the infectious laryngotracheitis avian virus as vector Withdrawn EP0948637A1 (en)

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AU5562798A (en) 1998-07-15
US6033670A (en) 2000-03-07
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AR010086A1 (en) 2000-05-17
FR2757061B1 (en) 1999-03-26
JP2001510338A (en) 2001-07-31
WO1998027215A1 (en) 1998-06-25
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CO4650231A1 (en) 1998-09-03
ZA9711247B (en) 1999-06-17

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