+

WO2001092486A1 - Souches de virus de bursite infectieuse chimeriques a serotype - Google Patents

Souches de virus de bursite infectieuse chimeriques a serotype Download PDF

Info

Publication number
WO2001092486A1
WO2001092486A1 PCT/EP2001/006005 EP0106005W WO0192486A1 WO 2001092486 A1 WO2001092486 A1 WO 2001092486A1 EP 0106005 W EP0106005 W EP 0106005W WO 0192486 A1 WO0192486 A1 WO 0192486A1
Authority
WO
WIPO (PCT)
Prior art keywords
ibdv
serotype
mutant
vaccine
protein
Prior art date
Application number
PCT/EP2001/006005
Other languages
English (en)
Inventor
Egbert Mundt
Adriaan Antonius Wilhelmus Maria Van Loon
Original Assignee
Akzo Nobel N.V.
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Akzo Nobel N.V. filed Critical Akzo Nobel N.V.
Priority to EP01943448A priority Critical patent/EP1290146A1/fr
Priority to AU2001266025A priority patent/AU2001266025A1/en
Publication of WO2001092486A1 publication Critical patent/WO2001092486A1/fr

Links

Classifications

    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N7/00—Viruses; Bacteriophages; Compositions thereof; Preparation or purification thereof
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/51—Medicinal preparations containing antigens or antibodies comprising whole cells, viruses or DNA/RNA
    • A61K2039/525—Virus
    • A61K2039/5256—Virus expressing foreign proteins
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2720/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA dsRNA viruses
    • C12N2720/00011—Details
    • C12N2720/10011—Birnaviridae
    • C12N2720/10021—Viruses as such, e.g. new isolates, mutants or their genomic sequences

Definitions

  • the present invention is concerned with a serotype 1 IBDN mutant comprising a coding region of a NP3 and NP4 protein in segment A of the viral genome, a vaccine comprising this mutant, a method for determining IBDN infection in an animal, as well as with a test kit for carrying out this method.
  • IBDN Infectious bursal disease virus
  • Birnaviridae family Viruses in this family have a very similar genomic organisation and a similar replication cycle.
  • the genomes of these viruses consist of 2 segments (A and B) of double-stranded (ds) R ⁇ A.
  • the larger segment A encodes a polyprotein which is cleaved by autoproteolysis to form mature viral proteins NP2, NP3 and VP4 (Hudson, P.J. et al, Nucleic Acids Res., 14, 5001-50012,
  • VP2 and VP3 are the major structural proteins of the virion.
  • VP2 is the major host- protective immunogen of bimaviruses, and contains the antigenic regions responsible for the induction of neutralising antibodies.
  • the VP4 protein appears to be a virus-coded protease that is involved in the processing of a precursor polyprotein of the VP2, VP3 and VP4 proteins.
  • the larger segment A possesses also a second open reading frame (ORF), preceding and partially overlapping the polyprotein gene. This second open reading frame encodes a protein VP5 of unknown function that is present in IBDV infected cells.
  • the smaller segment B encodes VP1, a 90 kDa multifunctional protein with polymerase and capping enzyme activities.
  • serotype 1 two serotypes exist, serotype 1 and 2.
  • Serotype 1 viruses are pathogenic to chickens, whereas serotype 2 viruses infect chickens and turkeys but do not induce lesions in these animals.
  • the two serotypes are differentiated by virus neutralisation tests, but they are not distinguishable by fluorescent antibody tests or ELIS A using polyclonal antiserum.
  • IBD Infectious Bursal disease
  • Gumboro disease is an acute, highly- contagious viral infection in chickens that has lymphoid tissue as its primary target with a selective tropism for cells of the bursa of Fabricius.
  • the morbidity rate in susceptible flocks is high, with rapid weight loss and moderate mortality rates.
  • Chicks that recover from the disease may have immune deficiencies because of the destruction of the bursa of Fabricius which is essential to the defence mechanism of the chicken.
  • the IBD virus causes severe immunosuppression in chickens younger than 3 weeks of age and induces bursal lesions in chicks up to 3 months old.
  • the disease could be prevented by inducing high levels of antibodies in breeder flocks by the application of an inactivated vaccine to chickens that had been primed with an attenuated live IBDV vaccine. This has kept economic losses caused by IBD to a minimum. Maternal antibodies in chickens derived from vaccinated breeders prevents early infection with IBDV and diminishes problems associated with immunosuppression. In addition, attenuated live vaccines have also been used successfully in commercial chicken flocks after maternal antibodies had declined.
  • a further cause of acute disease in vaccinated flocks is the emerging of antigenic variants in the mid-1980s, in particular in the USA.
  • the most important new antigenic subtypes of serotype 1 IBDV strains are the Delaware Variant E and GLS strains. Eradication of the disease by other preventative measures than vaccination has not been feasible, because the virus is widely spread and because with currently used live attenuated or inactivated IBDV vaccines it is not possible to determine whether a specific animal is infected with an IBDV field virus or whether the animal was vaccinated with an IBDV vaccine strain. In order to be able to start an eradication control programme for IBDV it is highly desirable to have a marker vaccine available.
  • the principle feature of a marker vaccine application is the differentiation of the antibody response.
  • the introduction of, for example, a serologically identifiable marker in a vaccine strain can be achieved by introducing a mutation in a gene encoding a non-essential protein of the IBDV which still give rise to the production of antibodies in an infected host animal.
  • the great advantage of a marker vaccine is that serological detection of infected animals in the field is still possible despite the concurrent vaccination programme.
  • a combined vaccination/eradication programme may lead to a decrease of the prevalence of the virus in the field while remaining cases of field infection are eradicated. Such a programme may finally lead to a total elimination of the virus from the field.
  • the prerequisites for the development of an effective IBDV marker vaccine is the identification of a position or region in the genomic IBDV sequence that (i) can be used for the incorporation of a mutation without disrupting essential functions of IBDV, such as those necessary for infection and replication, (ii) encodes (an) immunogenic epitope(s) that is able to induce a solid antibody response in vivo in the infected animal, and that (iii) does not encode an antigenic determinant involved in the induction of protection against virus infection.
  • European patent application no. 98201704.8 discloses a serotype 1 IBDN mutant that can be used as a marker vaccine strain.
  • This IBDN mutant lacks the ability to express the VP5 protein as a result of a mutation in the VP5 gene of the viral genome.
  • the present inventors have unexpectedly identified a further IBDV mutant that can be used as a marker vaccine.
  • the new IBDV mutant allows a serological distinction to be made between animals infected with wild-type IBDV and animals immunised with a vaccine based on this IBDV mutant.
  • the present invention provides a serotype 1 IBDV mutant comprising a coding region of a VP3 and VP4 protein in segment A of the viral genome, characterised in that the mutant comprises a coding region encoding the VP3 and/or VP4 protein of a serotype 2 IBDV.
  • IBDV it is known for IBDV that only one or a few mutations in the genome of an IBDV can lead to a virus with a different phenotype or even to a non-viable virus.
  • the VP3 protein of an IBDV interacts with the VP1 polymerase protein that has an important function in the replication of the virus and that the VP4 is a protease essential for the processing of the polyprotein.
  • the coding region of the VP3 and/or VP4 protein of a serotype 1 virus can be replaced by the coding region of a serotype 2 VP3 and/or
  • the new chimeric serotype 1/serotype 2 IBDV mutant is able to induce a protective immune response in chickens, and that the new IBDV mutant is antigenically distinct from the parent, serotype 1 virus as a result of wliich the IBDV mutant can be used as a marker vaccine virus.
  • a serotype 1 IBDV mutant as defined above can be used as marker vaccine strain, because the antigenic make-up of the serotype 1 virus is modified.
  • the new IBDV mutant lacks specific VP3 and or VP4 epitopes that are generally present on serotype 1 IBDV strains.
  • the new IBDV mutant also displays specific VP3 and/or VP4 epitopes that are generally present on serotype 2 IBDV strains only.
  • the presence or absence of antibodies directed against these specific epitopes in a sample of an animal indicate whether an animal in the field is vaccinated with a marker vaccine strain as described herein or infected with a naturally occurring IBDV serotype 1 strain.
  • serotype specific monoclonal antibodies Moabs
  • IBDV serotype specific VP3 Moabs The generation of such IBDV serotype specific VP3 Moabs is described in Mahardika et al., (Arch. Virol. 140, 765-774, 1995) and Oppling et al. (J. Gen. Virol. 72, 2275-2278, 1991). Granzow et al. (J. Virology 71, 8879-8885, 1997) disclose the generation of VP3 and VP4 specific Moabs.
  • Moab TBDV-2 cited in Granzow et al. (1997, supra).
  • This Moab specifically reacts with an epitope on the VP3 protein of serotype 1 IBDVs.
  • the coding region of the serotype 2 VP3 or VP4 protein includes the complete coding region of these proteins, or a fragment thereof that encodes part of the serotype 2 VP3 or VP4 protem that lacks a serotype 1 specific VP3 or VP4 epitope, or expresses a serotype 2 specific
  • the VP3 or VP4 coding region comprises a hybrid serotype 1/serotype 2 region.
  • the new IBDV mutant defined herein is a serotype 1 IBDV based on the presence of a serotype 1 VP2 protein which is the major antigen of IBDV that is capable of inducing virus neutralising (VN) antibodies in a host animal, which afford protection against pathogenic IBDV strains.
  • the IBDV mutant according to the invention is a serotype 1 virus, as the mutant is able to induce VN antibodies that are able to neutralise well known serotype 1 IBDV strains, such as
  • Serotype 1 IBDV strains can be distinguished from serotype 2 IBDV strains by standard VN assays, as demonstrated by McFerran et al. (Avian Pathology 9,
  • the segment A of IBDV comprises a large open reading frame (ORF) encoding a polyprotein of about 110 kDa (VP2-NP4-VP3).
  • ORF open reading frame
  • the coding regions of the VP3 and VP4 protein of serotype 1 and 2 viruses are disclosed in the prior art.
  • the VP3 coding region comprises the nucleotide sequence encoding the third, terminal protein of the polyprotein, whereas the VP4 coding region comprises the nucleotide sequence encoding the protein following VPl and preceding VP3 on the polyprotein (see Figure 1).
  • the VP3 coding region encodes a protein of 257 ainino acids resulting in a molecular mass of about 32 kDa.
  • the VP4 coding region encodes a protein of 233 amino acids resulting in a molecular mass of about 28 kDa.
  • the VP3 or VP4 coding region can be derived from any serotype 2 IBDV strain, such as the 23/82, TY89 and OH strain.
  • nucleotide sequences of the VP3 and VP4 coding regions of a serotype 2 IBDV that replace the VP3 or VP4 coding region of the serotype 1 IBDV are known from the prior art (Kibenge et al, Virology 184, 437-440, 1991). US patent no. 5,871,744 also discloses the nucleotide sequence and amino acid sequence of the complete serotype 2 genomic segment A and polyprotein, respectively. Furthermore, the nucleotide sequence of the serotype 2 VP3 and VP4 coding regions and the amino acid sequences of the corresponding VP3 and VP4 proteins are shown in SEQ ID No. 1 and 2, respectively.
  • the VP2-VP4 and VP4-VP3 post-ttanslational cleavage of the polyprotein takes place at the junction between amino acids 512/513 and 755/756 of the polyprotein, respectively.
  • the VP3 coding region in the segment A extends until the stop codon at position 3167-3169.
  • the serotype 1 IBDV mutant comprises the serotype 2 VP3 and/or VP4 coding region encoding a VP3 and/or VP4 protein having an amino acid sequence shown in SEQ ID. No. 2.
  • the construction of the IBDV mutants can be achieved by means of the recently established infectious cRNA system for IBDV (Mundt and Vakharia, Proc. Natl. Acad. Sci. USA 93, 11131-11136, 1996).
  • This reverse genetics system opens the possibility to introduce mutations in the RNA genome of the IBDV.
  • the most important step in this reverse genetics system is to provide full length cDNA clones of the segments A and B of IBD virus.
  • cDNA constructs comprising the segment A or B, including the nucleotides of the 5'- and 3'- ends of both these segments can be generated according to the method described by Mundt and Vakharia (1996, supra). Additionally, these constructs comprise a RNA polymerase promoter operably linked to either of the segments.
  • the promoter can be the promoter for the T7, SP6 or T3 polymerase, the T7 promoter being preferred.
  • Methods for the exchange of the VP3 or VP4 coding region between serotype 1 and serotype 2 IBDVs are well known in the art. For example, the exchange of the coding sequence of the complete VP3, VP4 or part of these coding regions of serotype 1 IBDV with those of serotype 2 viruses can be performed by applying the classic approach of using restriction enzyme cleavage sites originally located in the cDNA in both genomes of segments A of both serotypes.
  • a second approach is to create an additional restriction enzyme cleavage site by PCR (as described in Example 1) or by the method using single stranded viral cDNA following the method of Kunkel et al. (Methods in Enzymology; 204, 125-139, 1991). Following this the mutated viral cDNA can be exchanged as described above.
  • a third approach is the exchange of the complete coding region of VP3, VP4 or parts of it by using the site directed mutagenisis as described by Kunkel et al. (1991, supra)).
  • the serotype 1 IBDV mutant according to the invention is provided in a live or inactivated form.
  • An inactivated serotype 1 IBDV mutant can be used as the basis of an inactivated IBDV vaccine.
  • a live IBDV mutant can be either a pathogenic virus or an attenuated IBDV.
  • a serotype 1 IBDV mutant according to the invention that is still pathogenic for chickens can be used for further processing into an inactivated vaccine. Such a pathogenic
  • IBDV mutant can de derived from IBDV strains 52/70, UK661 and virulent Variant E strains.
  • a live, attenuated serotype 1 IBDV mutant according to the invention can be used as a basis for a live vaccine.
  • the live, attenuated serotype 1 IBDV mutant according to the invention is a mild, intermediate or invasive IBDV.
  • a mild IBDV strain is a strain that is able to induce no or only very mild lesions (lymphocytic depletion ⁇ 20%) to the bursa of Fabricius, when applied at day old to SPF white leghorns with a dose of 10 50 TCID 50 /animal via the eye-drop route, for a period of 24 days after vaccination.
  • Typical mild, serotype 1 IBDV strains are the Gumboro vaccine Nobilis strain PBG98 and strain 8903 (Intervet International BV, Boxmeer, the Netherlands), (ii) An intermediate IBDV strain is able to induce mild to moderate lesions (20% ⁇ lymphocytic depletion ⁇ 80%) to the bursa of Fabricius , when applied at day old to SPF white leghorns with a dose of 10 50 TCID 5r /animal via the eye-drop route, within a period of 14 days after vaccination. In this case, re-population of the bursa of Fabricius may occur leading to a lymphocytic depletion ⁇ 40%, 21 days after vaccination.
  • Typical intermediate serotype 1 IBDV-strains are Gumboro vaccine Nobilis strain D78 and strain LZ228TC (Intervet International B V) (iii)
  • An invasive IBDV strain is able to induce severe lesions (lymphocytic depletion 80- 100%) to the bursa of Fabricius, when applied at day old to SPF white leghorns with a dose of 100 EID 50 /animal via the eye-drop route, within 14 days after vaccination.
  • re- population of the bursa of Fabricius may occur within 3 weeks after vaccination leading to a lymphocytic depletion ⁇ 60%, 4 weeks after vaccination.
  • Typical invasive serotype 1 IBDV strains are Gumboro vaccine Nobilis strain 228E (Intervet International BV) and Bursa plus strain V877 (Fort Dodge, USA).
  • the serotype 1 IBDV mutant according to the invention can de derived from any of the serotype 1 subtype strains.
  • the IBDV mutant according to the invention is derived from a classical, Variant E or GLS strain, preferably from a classical strain.
  • the IBDV mutant is derived from strain D78
  • an IBDV mutant according to the invention comprises in addition to the coding region of the serotype 2 VP3 and or VP4 protein, a mutation in the VP2 gene, as a result of which this gene expresses a chimeric VP2 protein comprising neutralising epitopes of more than one antigenic subtype of IBDV (e.g. classic, Variant-E and or GLS).
  • a mutation in the VP2 gene as a result of which this gene expresses a chimeric VP2 protein comprising neutralising epitopes of more than one antigenic subtype of IBDV (e.g. classic, Variant-E and or GLS).
  • a vaccine for use in the protection of animals against disease caused by IBDV infection comprises the serotype 1 IBDV mutant as characterised above, together with a pharmaceutical acceptable carrier or diluent.
  • the IBDV mutant according to the present invention can be incorporated into the vaccine as live or inactivated virus.
  • a vaccine according to the invention can be prepared by conventional methods such as for example commonly used for the commercially available live- and inactivated IBDV vaccines. Briefly, a susceptible substrate is inoculated with an IBDV mutant according to the invention and propagated until the virus replicated to a desired infectious titre after which
  • IBDV containing material is harvested.
  • Every substrate which is able to support the replication of IBD viruses can be used in the present invention, including primary (avian) cell cultures, such as chicken embryo fibroblast cells (CEF) or chicken kidney cells (CK), mammalian cell lines such as the VERO cell line or the BGM-70 cell line, or avian cell lines such as QT-35, QM-7 or LMH.
  • primary (avian) cell cultures such as chicken embryo fibroblast cells (CEF) or chicken kidney cells (CK)
  • mammalian cell lines such as the VERO cell line or the BGM-70 cell line
  • avian cell lines such as QT-35, QM-7 or LMH.
  • the IBDV mutant is propagated in embryonated chicken eggs.
  • the substrate on which these IBD viruses are propagated are SPF embryonated eggs.
  • Embryonated eggs can be inoculated with, for example 0.2 ml IBDV mutant containing suspension or homogenate comprising at least 10 2 TCID 50 per egg, and subsequently incubated at 37 °C.
  • the IBD virus product can be harvested by collecting the embryo's and or the membranes and/or the allantoic fluid followed by appropriate homogenising of this material.
  • the homogenate can be centrifuged thereafter for 10 min at 2500 x g followed by filtering the supernatant through a filter (100 ⁇ m).
  • the vaccine according to the invention containing the live virus can be prepared and marketed in the form of a suspension or in a lyophilised form and additionally contains a pharmaceutically acceptable carrier or diluent customary used for such compositions.
  • Carriers include stabilisers, preservatives and buffers.
  • Suitable stabilisers are, for example SPGA, carbohydrates (such as sorbitol, mannitol, starch, sucrose, dextran, glutamate or glucose), proteins (such as dried milk serum, albumin or casein) or degradation products thereof.
  • Suitable buffers are for example alkali metal phosphates.
  • Suitable preservatives are thimerosal, merthiolate and gentamicin.
  • Diluents include water, aqueous buffer (such as buffered saline), alcohols and polyols (such as glycerol). If desired, tlie live vaccines according to the invention may contain an adjuvant.
  • the vaccine is preferably administered by the inexpensive mass application techniques commonly used for IBDV vaccination.
  • these techniques include drinking water and spray vaccination.
  • a vaccine comprising the IBDV mutant in an inactivated form.
  • the major advantage of an inactivated vaccine is the extremely high levels of protective antibodies of long duration that can be achieved.
  • the aim of inactivation of the viruses harvested after the propagation step is to eliminate reproduction of the viruses. In general, this can be achieved by chemical or physical means.
  • a vaccine containing the inactivated IBDV mutant according to the invention can, for example, comprise one or more of tlie above-mentioned pharmaceutically acceptable carriers or diluents suited for this purpose.
  • an inactivated vaccine according to the invention comprises one or more compounds with adjuvant activity.
  • Suitable compounds or compositions for this purpose nclude aluminium hydroxide, -phosphate or -oxide, oil-in- water or water-in-oil emulsion based on, for example a mineral oil, such as Bayol F® or Marcol 52® or a vegetable oil such as vitamin E acetate, and saponins.
  • the vaccine according to the invention comprises an effective dosage of the IBDV mutant as the active component, i.e. an amount of immunising IBDV material that will induce immunity in the vaccinated birds against challenge by a virulent virus.
  • Immunity is defined herein as the induction of a significant higher level of protection in a population of birds after vaccination compared to an unvaccinated group.
  • the live vaccine can be administered in a dose of 10 20 -10 90 TCID 50 infectious dose 50 (TCTD 50 ) per animal, preferably in a dose ranging from 10 40 -10 70 TQD 50 .
  • Inactivated vaccines may contain the antigenic equivalent of 10 50 -10 90 TCIDj 0 per animal.
  • Inactivated vaccines are usually administered parenterally, e.g. intramuscularly or subcutaneously.
  • the IBDV vaccine according to the present invention may be used effectively in chickens, also other poultry such as turkeys, guinea fowl and partridges may be successfully vaccinated with the vaccine.
  • Chickens include broilers, reproduction stock and laying stock.
  • the combination vaccine additionally comprises one or more vaccine strains of Mareks Disease virus (MDV), infectious bronchitis virus (IBN), Newcastle disease virus
  • MDV Mareks Disease virus
  • IBN infectious bronchitis virus
  • Newcastle disease virus Newcastle disease virus
  • NTN egg drop syndrome
  • EDS egg drop syndrome
  • TRTN turkey rhinotracheitis virus
  • diagnostic test comprises a method for determining IBDN infection in poultry.
  • a method is provided for distinguishing an animal in the field vaccinated with a vaccine as described above from an animal infected with a naturally-occurring IBDN serotype 1 strains.
  • the present invention provides a method for the detection of an IBDN infection in an animal comprising the step of examining a sample of the animal for the presence or absence of antibodies directed against serotype specific IBDN NP3 or VP4 epitopes.
  • the method comprises the step of examining a sample of the animal for the presence or absence of antibodies directed against a serotype 1 specific VP3 or VP4 epitope.
  • a serotype 1 specific Moab that specifically recognises a VP3 or VP4 epitope present on serotype 1 IBDV strains.
  • the animal sample used in this method may be any sample in which IBDV antibodies are present, e.g. a blood, serum or tissue sample, the serum sample being preferred.
  • VP3 or VP4 epitope comprises the steps of: (i) incubating a sample suspected of containing anti-IBDV antibodies with serotype 1 VP3 or
  • this immunoassay may vary.
  • the immunoassay may be based upon competition or direct reaction.
  • the detection of the antibody-antigen complex may involve the use of labelled antibodies; the labels may be, for example, enzymes, fluorescent-, chemiluminescent-, radio-active- or dye molecules.
  • Suitable methods for the detection of the specific VP3 or VP4 antibodies in the sample include the enzyme-linked immunosorbent assay (ELISA), immunofluorescent test (IFT) and
  • horse radish peroxidase coupled to avidin may be added and the amount of peroxidase is measured by an enzymatic reaction. If no specific antibodies against the serotype 1 specific VP3 or VP4 epitope are present in the chicken serum sample then a maximum binding of the Moab is obtained. If the serum contains many antibodies against the serotype 1 specific VP3 or VP4 epitope, the added Moab will not bind to the epitope.
  • the VP3 or VP4 antigen material to be used in the assay may be any serotype 1 IBDV antigen material comprising the serotype 1 specific epitope that binds with the Moab used in the assay.
  • the VP3 or VP4 antigen material may be serotype 1 IBDV infected cells or purified virus material.
  • the VP3or VP4 antigen material is the expression product of a recombinant host cell or virus, e.g. such as E.coli or baculovirus expressed VP3 or VP4 (Vakharia et al., Vaccine 12, 452-456, 1994; Vakharia et al., J. Gen Virol. 74, 1201-1206, 1993; Pitcovski et al. Avian Dis. 43, 8-515, 1999 andLejal et al., J. Gen. Virol. 81, 983-992, 2000).
  • a recombinant host cell or virus e.g. such as E.coli or baculovirus expressed VP3 or VP4 (Vakharia et al., Vaccine 12, 452-456, 1994; Vakharia et al., J. Gen Virol. 74, 1201-1206, 1993; Pitcovski et al. Avian Dis. 43, 8-515, 1999 andLejal et al.,
  • a diagnostic test kit which is suitable for performing the diagnostic test according to the invention as described above.
  • a diagnostic test kit is provided which comprises in addition to the components usually present, the VP3 or VP4 antigen material (if desired coated onto a solid phase).
  • Other components usually present in such a test kit include, biotin or horseradish peroxidase conjugated Moab, enzyme substrate, washing buffer etc.
  • CEF derived from embryonated SPF eggs were grown in Dulbeccos minimal essential medium (DMEM) supplemented with 10% fetal calf serum (FCS) and were used for rransfection experiments, propagation of recovered virus, and passaging of transfection supernatants.
  • DMEM Dulbeccos minimal essential medium
  • FCS fetal calf serum
  • hnmunofluorescence assays were performed using quail muscle cells (QM-7, ATCC) grown in medium 199 supplemented with 10% FCS.
  • PCR were performed for the construction of the chimeric segment A consisting of genomic sequences of segment A of serotype I strain D78 and serotype II strain 23/82, using a primer pair (FKA5'; D7823BglII, Table 1; SEQ ID No. 3 and 4) and the full length cDNA clone pD78A EK (Mundt et al., J. Virol. 71, 5647-5651, 1997).
  • the resulting PCR fragment encompassing the sequence of segment A of strain D78 up to the nucleotide 1427, in accordance to the sequence of segment A of strain D78 (US patent no. 5,871,744), were cloned blunt ended into the plasmid pUC18 to obtain pD78ABgi ⁇ .
  • the sequence of the inserted fragment was controlled by sequencing.
  • composition and location of the oligonucleoti.de primers used for site directed mutagenesis Changed nucleotides for mutagenesis are in small letter code and the used restriction enzyme sites are highlighted in boldface type. The positions where the primers bind (nucleotide no.) and are in accordance to the published sequence of strain P2 (Mundt & Muller, Virology 209, 209-218, 1995)
  • RNA of plasmid pD78-23 A containing chimeric segment A and pD78B (Mundt, J. Gen. Virol. 80, 2067-2076, 1999) were linearized by cleavage with either BsrGI or Pst I. Further treatment of linearized DNA and transcription were carried out as described by Mundt & Vakharia (1996, supra), but with the exceptions that the transcription mixtures were not purified by phenol/ chloroform extraction, and CEC were used for transfection experiments.
  • the constructed plasmid pD78-23A contains sequences of segment A of serotype I strain D78 (up to nucleotide 1419) and of segment A of serotype II strain 23/82 (starting with nucleotide 1420).
  • the construction of the chimeric plasmid is shown in Figure 1.
  • CEF cells Primary chicken embryo fibroblasts (CEF) cells were prepared at a final concentration of 1 x 107ml. The cells were cultured in Eagles minimum essential medium containing 5% fetal calf serum (FCS) at 37 °C. One ml of supernatant (passage level 3) was added to 150 ml medium containing 2 x 10 6 CEF/ml and 5% FCS. After incubation for 3-6 days, the supernatant (passage level 4) had an infectious titer of 10 7,3 TCID 50 /ml and an antigenic mass of approximately 2100 EU/ml as determined by the R63 antigenic mass ELISA.
  • FCS fetal calf serum
  • inactivated classical virus vaccine was used.
  • This vaccine also was an oil-emulsion vaccine as described above and contained 500 EU/animal dose.
  • the vims strain used to prepare the inactivated classical vaccine was D78. Identification of IBDV vaccines by means of IFT.
  • IBDV-strains were identified by means of IFT using different monoclonal antibodies.
  • the monoclonal antibodies used for identification have been described by Van Loon et al. (Van Loon, A.A.W.M., D. L ⁇ tticken and D.B. Snyder. Rapid quantification of infectious bursal disease (TBD) challenge, field or vaccine vims strains. International symposium on infectious bursal disease and chicken infectious anaemia, Rauischhilzhausen, Germany, 179-187, 1994).
  • An additional monoclonal antibody was used (IBDN-2) which is specific for serotype 1 vimses and recognises an epitope on VP3 of serotype 1 only.
  • He-stained sections of each chicken was prepared. These HE-stained sections were microscopically examined. Bursal lesions or lymphocyte depletion in the follicles were scored as follows:
  • lymphocyte depletion 0 no lesions; 1 0-20% lymphocyte depletion; 2 20%-40% lymphocyte depletion; 3 40% > -60% lymphocyte depletion; 4 60%-80% lymphocyte depletion and severe lesions, 80-100% lymphocyte depletion.
  • IBDV-strain D78-23 contains VP2-specific- epitopes that are present on a classical serotype 1 IBDV-virus (strain D78). Furthermore, the chimeric D78-23 marker vims does not posses the VP3-specific-serotype 1 epitope, recognised by monoclonal antibody IBDV-2. This indicates that the chimeric vims for the VP3-epitope reacts identical as the VP3 epitope of serotype 2.
  • Table 2 Panel pattern of different IBDV viruses with different Moab. + epitope present on virus, - epitope not present on vims.
  • Results are depicted in table 4.
  • the marker vaccine (D78-23) did not induce lesions after vaccination. Ten days after challenge the average lesions score in both vaccinated groups was 0.8. Based on the individual microscopic lesion scores the marker vaccine induced 82%-83% protection (15 out of 18 or 14 out of 17 animals). In contrast, the none-vaccinated control animals were not protected and the challenge vims induced complete lymphocytic depletion (score 5.0), 3 and 10 days after challenge (acute lesions are lesions induced by the challenge vims).
  • IM intramuscular route; * Number of positive animals with viral antigen present per total number investigated. Viral antigen was detected using a specific ELISA with monoclonal antibodies directed against IBDV.
  • Table 7 Re-isolation of marker vaccine D78-23 from the bursa. * Number of positive animals with viral antigen present per total number investigated. Identification of the isolated vims was done by IFT using different Moab.
  • Table 8 Serological response 4 and 6 weeks after vaccination (VN-tihe is expressed as log2 of the dilution).

Landscapes

  • Life Sciences & Earth Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Health & Medical Sciences (AREA)
  • Genetics & Genomics (AREA)
  • Organic Chemistry (AREA)
  • Zoology (AREA)
  • Engineering & Computer Science (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Wood Science & Technology (AREA)
  • Medicinal Chemistry (AREA)
  • Biotechnology (AREA)
  • Microbiology (AREA)
  • Biomedical Technology (AREA)
  • Virology (AREA)
  • Biochemistry (AREA)
  • General Engineering & Computer Science (AREA)
  • General Health & Medical Sciences (AREA)
  • Immunology (AREA)
  • Medicines Containing Antibodies Or Antigens For Use As Internal Diagnostic Agents (AREA)

Abstract

La présente invention concerne un mutant du virus de bursite infectieuse, qui convient en tant que vaccin candidat dans des programmes de contrôle d'éradication. Ledit mutant du virus de bursite infectieuse est une souche du virus de bursite infectieuse chimérique de sérotype 1, qui comprend les séquences de codage d'une protéine virale VP3 ou VP4 de sérotype 2.
PCT/EP2001/006005 2000-05-31 2001-05-25 Souches de virus de bursite infectieuse chimeriques a serotype WO2001092486A1 (fr)

Priority Applications (2)

Application Number Priority Date Filing Date Title
EP01943448A EP1290146A1 (fr) 2000-05-31 2001-05-25 Souches de virus de bursite infectieuse chimeriques a serotype
AU2001266025A AU2001266025A1 (en) 2000-05-31 2001-05-25 Serotype chimeric ibdv strains

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
EP00201933 2000-05-31
NL00201933.9 2000-05-31

Publications (1)

Publication Number Publication Date
WO2001092486A1 true WO2001092486A1 (fr) 2001-12-06

Family

ID=8171584

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/EP2001/006005 WO2001092486A1 (fr) 2000-05-31 2001-05-25 Souches de virus de bursite infectieuse chimeriques a serotype

Country Status (3)

Country Link
EP (1) EP1290146A1 (fr)
AU (1) AU2001266025A1 (fr)
WO (1) WO2001092486A1 (fr)

Cited By (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN100341889C (zh) * 2005-03-29 2007-10-10 河南省动物免疫学重点实验室 鸡ibdv vp3蛋白b细胞抗原表位ⅱ
FR2966605A1 (fr) * 2010-10-20 2012-04-27 Id Vet Utilisation d'anticorps anti-vp2 du btv-8 dans une methode diva appliquee aux virus btv-8
FR2966460A1 (fr) * 2010-10-20 2012-04-27 Id Vet Utilisation d'anticorps anti-ns1 du btv dans une methode diva appliquee aux virus btv
FR2966461A1 (fr) * 2010-10-20 2012-04-27 Id Vet Utilisation d'anticorps anti-vp2 du btv-8 pour le serotypage et le diagnostic des infections a btv

Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1990015140A1 (fr) * 1989-05-30 1990-12-13 Commonwealth Scientific And Industrial Research Organisation Production de vp2 d'ibdv sous forme hautement immunogene
WO1993025665A1 (fr) * 1992-06-12 1993-12-23 Syntro Corporation Virus de l'herpes des gallinaces type 2 recombine et ses utilisations
WO1994000586A2 (fr) * 1992-06-26 1994-01-06 Rhône Merieux Mutants et vaccins du virus de la rhinotracheite infectieuse bovine
WO1995026196A1 (fr) * 1994-03-29 1995-10-05 The University Of Maryland College Park CLONES D'ADNc CHIMERES DU VIRUS DE LA BURSITE INFECTIEUSE, PRODUITS D'EXPRESSION ET VACCINS A BASE DESDITS CLONES
EP1069187A1 (fr) * 1999-07-14 2001-01-17 Stichting Dienst Landbouwkundig Onderzoek Vaccin contenent un virus mosaique de la maladie infectieuse des bourses

Patent Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1990015140A1 (fr) * 1989-05-30 1990-12-13 Commonwealth Scientific And Industrial Research Organisation Production de vp2 d'ibdv sous forme hautement immunogene
WO1993025665A1 (fr) * 1992-06-12 1993-12-23 Syntro Corporation Virus de l'herpes des gallinaces type 2 recombine et ses utilisations
WO1994000586A2 (fr) * 1992-06-26 1994-01-06 Rhône Merieux Mutants et vaccins du virus de la rhinotracheite infectieuse bovine
WO1995026196A1 (fr) * 1994-03-29 1995-10-05 The University Of Maryland College Park CLONES D'ADNc CHIMERES DU VIRUS DE LA BURSITE INFECTIEUSE, PRODUITS D'EXPRESSION ET VACCINS A BASE DESDITS CLONES
EP1069187A1 (fr) * 1999-07-14 2001-01-17 Stichting Dienst Landbouwkundig Onderzoek Vaccin contenent un virus mosaique de la maladie infectieuse des bourses

Non-Patent Citations (4)

* Cited by examiner, † Cited by third party
Title
GRANZOW, H. ET AL: "A second form of infectious bursal disease virus-associated tubule contains VP4.", JOURNAL OF VIROLOGY, vol. 71, no. 11, 1997, pages 8879 - 8885, XP002150789, ISSN: 0022-538X *
MAHARDIKA G N K ET AL: "Mapping of cross-reacting and serotype-specific epitopes on the VP3 structural protein of the infectious bursal disease virus (IBDV).", ARCHIVES OF VIROLOGY, vol. 140, no. 4, 1995, pages 765 - 774, XP000953014, ISSN: 0304-8608 *
ÖPPLING V ET AL: "THE STRUCTURAL POLYPEPTIDE VP3 OF INFECTIOUS BURSAL DISEASE VIRUS CARRIES GROUP-SPECIFIC AND SEROTYPE-SPECIFIC EPITOPES", JOURNAL OF GENERAL VIROLOGY, vol. 72, no. 9, 1991, pages 2275 - 2278, XP000953048, ISSN: 0022-1317 *
SCHRÖDER, A. ET AL: "Chimeras in noncoding regions between serotypes I and II of segment A of infectious bursal disease virus are viable and show pathogenic phenotype in chickens.", JOURNAL OF GENERAL VIROLOGY., vol. 81, no. 2, February 2000 (2000-02-01), pages 533 - 540, XP002150788, ISSN: 0022-1317 *

Cited By (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN100341889C (zh) * 2005-03-29 2007-10-10 河南省动物免疫学重点实验室 鸡ibdv vp3蛋白b细胞抗原表位ⅱ
FR2966605A1 (fr) * 2010-10-20 2012-04-27 Id Vet Utilisation d'anticorps anti-vp2 du btv-8 dans une methode diva appliquee aux virus btv-8
FR2966460A1 (fr) * 2010-10-20 2012-04-27 Id Vet Utilisation d'anticorps anti-ns1 du btv dans une methode diva appliquee aux virus btv
FR2966461A1 (fr) * 2010-10-20 2012-04-27 Id Vet Utilisation d'anticorps anti-vp2 du btv-8 pour le serotypage et le diagnostic des infections a btv

Also Published As

Publication number Publication date
EP1290146A1 (fr) 2003-03-12
AU2001266025A1 (en) 2001-12-11

Similar Documents

Publication Publication Date Title
EP1035203B1 (fr) Mutants du virus de la maladie infectieuse des bourses (IBDV) adaptés par génie génétique pour la culture cellulaire
WO2009158614A1 (fr) Nouvel astrovirus
US9273287B2 (en) Avian reoviridae and vaccines thereof
US7022327B1 (en) Recombinant birnavirus vaccine
EP0887412B1 (fr) Vaccin recombinant du birnavirus
JP4646903B2 (ja) 伝染性ファブリキウス嚢病ウイルス（ｉｂｄｖ）の変異株およびワクチン
CA2349960A1 (fr) Souche du virus de la bursite infectieuse pour l'administration in ovo
WO2001092486A1 (fr) Souches de virus de bursite infectieuse chimeriques a serotype
WO2003074552A1 (fr) Nouvelle variante du virus de la bursite infectieuse
CA2351010C (fr) Vaccin a large spectre d'efficacite contre le virus de la bursite infectieuse
EP1170302B1 (fr) Vaccin a base des mutants du virus de la bursite infectieuse
US7431930B2 (en) Infectious bursal disease virus (IBDV) mutant expressing virus neutralising epitopes specific for classic-and variant IBDV strains
MXPA00002278A (en) Genetically engineered cell culture adapted infectious bursal diseases virus (ibdv) mutants

Legal Events

Date Code Title Description
AK Designated states

Kind code of ref document: A1

Designated state(s): AE AG AL AU BA BB BG BR BZ CA CN CO CR CU CZ DM DZ EC EE GD GE HR HU ID IL IN IS JP KP KR LC LK LR LT LV MA MG MK MN MX MZ NO NZ PL RO RU SG SI SK SL TR TT UA US UZ VN YU ZA

AL Designated countries for regional patents

Kind code of ref document: A1

Designated state(s): GH GM KE LS MW MZ SD SL SZ TZ UG ZW AM AZ BY KG KZ MD RU TJ TM AT BE CH CY DE DK ES FI FR GB GR IE IT LU MC NL PT SE TR BF BJ CF CG CI CM GA GN GW ML MR NE SN TD TG

121 Ep: the epo has been informed by wipo that ep was designated in this application
DFPE Request for preliminary examination filed prior to expiration of 19th month from priority date (pct application filed before 20040101)
WWE Wipo information: entry into national phase

Ref document number: 2001943448

Country of ref document: EP

WWP Wipo information: published in national office

Ref document number: 2001943448

Country of ref document: EP

WWW Wipo information: withdrawn in national office

Ref document number: 2001943448

Country of ref document: EP

NENP Non-entry into the national phase

Ref country code: JP

点击 这是indexloc提供的php浏览器服务，不要输入任何密码和下载