WO2001092486A1

WO2001092486A1 - Serotype chimeric ibdv strains

Info

Publication number: WO2001092486A1
Application number: PCT/EP2001/006005
Authority: WO
Inventors: Egbert Mundt; Adriaan Antonius Wilhelmus Maria Van Loon
Original assignee: Akzo Nobel NV
Current assignee: Akzo Nobel NV
Priority date: 2000-05-31
Filing date: 2001-05-25
Publication date: 2001-12-06
Anticipated expiration: 2002-11-30
Also published as: EP1290146A1; AU2001266025A1

Abstract

The present invention provides an IBDV mutant which is suited as a vaccine candidate in eradication control programmes. The IBDV mutant is a chimeric IBDV serotype 1 strain comprising the coding sequences of a serotype 2 VP3 or VP4 protein.

Description

Serotype chimeric IBDV strains

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.

Infectious bursal disease virus (IBDN) is a member of the 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) RΝA. 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,

1986). 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.

For IBDV, 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.

Furthermore, subtypes of serotype 1 have been isolated. These so-called "variant" viruses of serotype 1 can be identified by cross-neutralisation tests (Diseases of Poultry, 9th edition, 1991, Wolfe Publishing Ltd, ISBN 0 7234 1706 7, Chapter 28, P.D. Lukert and Y.M. Saif, 648- 663), a panel of monoclonal antibodies (Snyder, D.B. et al., Arch. Virol., 127, 89-101. 1992.) or RT-PCR (Jackwood, D.J., Proceedings of the International symposium on infectious bursal disease and chicken infectious anaemia, Rauischholzhausen, Germany, 155-161, 1994). Most of these subtypes of serotype 1 of IBDV have been described in literature, for example: Classical,

Variant-E, GLS, RS593 and DS326 strains (Van Loon, et al. Proceedings of the International symposium on infectious bursal disease and chicken infectious anaemia, Rauischholzhausen, Germany, 179-187, 1994).

Infectious Bursal disease (IBD), also called 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.

For many years 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.

Recently, very virulent strains of IBDV have caused outbreaks of disease with high mortality in Europe. The current vaccination programs failed to protect chicks sufficiently. Vaccination failures were mainly due to the inability of live vaccines to infect the birds before challenge with virulent field virus.

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 (Akzo Nobel NV) 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. 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. It is further known that 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. Despite this, it has been found that 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

VP4 protein while retaining its ability to infect and replicate in a host animal. Moreover, it is demonstrated that 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. In addition, the new IBDV mutant also displays specific VP3 and/or VP4 epitopes that are generally present on serotype 2 IBDV strains only. As a result of the absence of serotype 1 specific VP3 and/or VP4 epitopes or the presence of serotype 2 specific VP3 and/or VP4 epitopes on an IBDV mutant according to the invention, 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. In order to test the serological status of an animal in the field use is made of serotype specific monoclonal antibodies (Moabs) that react with the serotype specific VP3 or VP4 epitopes.

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.

In particular, use is made in this invention of 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

VP3 or VP4 epitope. In the latter case 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

D78, Cu-1, 52/70 and LZ228TC. 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,

395-404, 1980).

As described above, the genomic organisation of the IBDV is well established: the segment A of IBDV comprises a large open reading frame (ORF) encoding a polyprotein of about 110 kDa (VP2-NP4-VP3). 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. The 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. In a preferred embodiment of the invention 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)). To this end the complete region encoding for VP3 and or VP4 of a serotype 1 strain or part of it will be exchanged by site directed mutagenisis using oligionucleotides with substitution in the nucleotide sequence leading to amino acid sequence exchanges from the sequence of serotype 1 to sequences of serotype 2. 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.

In particular, the live, attenuated serotype 1 IBDV mutant according to the invention is a mild, intermediate or invasive IBDV.

Presently, live IBDV vaccine strains of these three types are commonly used in the field. These three types of vaccine strains are characterised as follows:

(i) 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 10⁵⁰ TCID₅₀/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 10⁵⁰ 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 EID₅₀/animal via the eye-drop route, within 14 days after vaccination. In this case, 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. Preferably, the IBDV mutant according to the invention is derived from a classical, Variant E or GLS strain, preferably from a classical strain. In a particular preferred embodiment of the invention the IBDV mutant is derived from strain D78

(commercially available from Intervet International BV).

Furthermore, an IBDV mutant according to the invention is provided that 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).

Further to the unexpected finding by the present inventors that a serotype 1 IBDV mutant according to the present invention is able to elicit an antibody response that can be distinguished from that elicited by naturally occurring IBDV strains, it has also been found that an IBDV mutant according to the invention is able to induce a protective immune response, i.e. animals immunised with a vaccine comprising the IBDV mutant are protected against virulent challenge.

Therefore, in another aspect of this invention a vaccine for use in the protection of animals against disease caused by IBDV infection is provided that 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. Usually, after inoculation of the cells, the virus is propagated for 3-10 days, after which the cell culture supernatant is harvested, and if desired filtered or centrifuged in order to remove cell debris.

Alternatively, the IBDV mutant is propagated in embryonated chicken eggs. In particular, 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 10² TCID₅₀ per egg, and subsequently incubated at 37 °C. After about 2-5 days 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 μm).

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.

Examples of suitable compounds and compositions with adjuvant activity are the same as mentioned below.

Although administration by injection, e.g. intramuscular, subcutaneous of the live vaccine according to the present invention is possible, the vaccine is preferably administered by the inexpensive mass application techniques commonly used for IBDV vaccination. For IBDV vaccination these techniques include drinking water and spray vaccination.

Alternative methods for the administration of the live vaccine include in ovo, eye drop and beak dipping administration. h another aspect of the present invention a vaccine is provided 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.

Preferably, 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.

Typically, the live vaccine can be administered in a dose of 10²⁰-10⁹⁰ TCID₅₀ infectious dose₅₀ (TCTD₅₀) per animal, preferably in a dose ranging from 10⁴⁰-10⁷⁰ TQD₅₀. Inactivated vaccines may contain the antigenic equivalent of 10⁵⁰-10⁹⁰ TCIDj₀ per animal.

Inactivated vaccines are usually administered parenterally, e.g. intramuscularly or subcutaneously.

Although, 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 age of the animals receiving a live or inactivated vaccine according to the invention is the same as that of the animals receiving the conventional live- or inactivated IBDV vaccines. For example, broilers (free of maternally derived antibodies-MDA) may be vaccinated at one- day-old, whereas broilers with high levels of MDA are preferably vaccinated at 2-3 weeks of age. Laying stock or reproduction stock with low levels of MDA may be vaccinated at 1-10 days of age followed by booster vaccinations with inactivated vaccine on 6-12 and 16-20 weeks of age. The invention also includes combination vaccines comprising, in addition to the IBDV mutant according to the invention, one or more immunogens derived from other pathogens infectious to poultry.

Preferably, the combination vaccine additionally comprises one or more vaccine strains of Mareks Disease virus (MDV), infectious bronchitis virus (IBN), Newcastle disease virus

(NDN), egg drop syndrome (EDS) virus, turkey rhinotracheitis virus (TRTN) or reovirus.

In addition to a marker vaccine, the availability of an appropriate diagnostic test is an important requirement for the application of an IBDN eradication control programme. Such a diagnostic test is provided herein and 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.

Therefore, 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.

Preferably, 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. In this method use is made of 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.

A preferred method for the detection of antibodies directed against a serotype 1 specific

VP3 or VP4 epitope comprises the steps of: (i) incubating a sample suspected of containing anti-IBDV antibodies with serotype 1 VP3 or

VP4 antigen material,

(ii) allowing the formation of antibody-antigen comple , and

(ii) detecting the presence of the antibody-antigen complex.

The design of this immunoassay may vary. For example, 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

Western blot analysis. hi an exemplifying ELISA, the wells of a polystyrene micro-titration plate are coated with VP3 or VP4 antigen. Next, the wells of the coated plates are filled with chicken test serum and serial dilutions are made. After incubation, the presence or absence of the specific chicken antibodies in the serum is determined by adding a (labelled) detecting monoclonal antibody reactive with the serotype 1 specific VP3 or VP4 epitope. The (labelled) Moab may bind to free antigens that have not been occupied by the specific VP3 or VP4 antibodies in the chicken serum. For example, 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. For example, the VP3 or VP4 antigen material may be serotype 1 IBDV infected cells or purified virus material.

Alternatively, 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).

In a further embodiment of the present invention a diagnostic test kit is provided which is suitable for performing the diagnostic test according to the invention as described above. In particular, 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. EXAMPLES

Example 1

Generation of a serotype 1/2 chimeric IBDV

Material and Methods Virus and Cells.

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. hnmunofluorescence assays were performed using quail muscle cells (QM-7, ATCC) grown in medium 199 supplemented with 10% FCS.

Construction of the chimeric segment A.

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 pD78ABgiπ. The sequence of the inserted fragment was controlled by sequencing. A plasmid containing the mutation only at positions

1420 (C to A) and 1423 (C to T) was used for construction of the chimeric segment A. To this end pD78ABglII was cleaved with Bgl II and EcoRI and the appropriate f agment containing the T7 promoter sequence and the sequence of segment A of strain D78 up to the nucleotide 1421 was eluted. This fragment was ligated into the appropriate cleaved plasmid containing the full length cDNA clone of segment A of strain 23/82 (US patent 5,871,744) to obtain the chimeric plasmid pD78-23A (SEQ ID No. 1). Table 1. Oligonucleotides used for the construction of full length chimeric cDNA clones of IBDV segment A containing

Designation Nucleotide sequence Orientation Nucleotide no.

FKA5' EcøRI sense 1-18 AGAGAATTCr^lMCG^CT Cr^r^GGATACGATCGGTC TGA

Bgl ϊl antisense 1437-1427

D7823BglII TGAGaTCtGCCACCTCCATGAAGTATTCACG

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)

Virus recovery from cRNA in tissue culture.

For in vitro transcription of 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. Two days after transfection cells were freeze/thawed, centrifuged at 700 x g to eliminate cellular debris, and the resulting supematants were further clarified by filtration through 0.45 μm filters and stored at -70°C. For immunofluorescence studies cells were grown on sterile cover slips .

Results

Construction of chimeric plasmid. To construct a chimeric full length cDNA clone of segment A containing sequences of serotype I strain D78 and serotype II strain 23/82 mutagenisis was necessary in the sequence of segment A of strain D78. The nucleotide substitutions at position 1420 (C to A) and 1423 (C to T) resulted in the restriction enzyme cleavage site Bgl II. Both mutations did not affect amino acid substitution of the sequence of segment A of strain D78. By creation of this restriction enzyme cleavage site it was possible to use the single Bgl II cleavage site located in the full length cDNA clone ρUC18FLA23 located at position 1424. 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.

Transfection experiments with chimeric cRNA.

For transfection experiments full length cDNA clone of chimeric segments pD78-23A was transcribed into synthetic cRNA and cotransfected with segment B (pD78B) full length cRNA into CEC. Two days after transfection cells were freeze/ thawed and the resulting supematants were passaged once on CEC. After freeze/ thawing transfection and passage supematants were tested for IBDV antigen by IFA using QM-7 cells. Virus was generated after transfection of cRNA from plasmid pD78B in combination with pD78-23A leading to the mutant virus D78- 23. SEQ ID No. 1 and 2 show the nucleotide sequence corresponding to the complete segment

A of the chimeric vims and the amino acid sequence of the polyprotein expressed by this virus.

Example 2

Preparation and properties of IBDV marker vaccine

Materials and Methods

Chimeric serotype 1/serotype 2 IBDV marker vaccine.

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 10⁶ CEF/ml and 5% FCS. After incubation for 3-6 days, the supernatant (passage level 4) had an infectious titer of 10^7,3 TCID₅₀/ml and an antigenic mass of approximately 2100 EU/ml as determined by the R63 antigenic mass ELISA.

To carry out animal experiment 1 the supernatant was diluted to result in a vaccine dose of 10⁴² or l0⁵² TCID₅₀/animal. For the second animal experiment, the IBDV-strain D78-23 was cultured in the same was as described above. Next the live virus (at passage level 4) was inactivated by means of 0.05% formaldehyde for 48h. The inactivated virus was mixed with mineral oil to prepare a water in oil emulsion (45:55 w/o emulsion). The inactivated vaccine used in the animal experiment contained 500 EU/animal dose.

Serotype 1 IBDV vaccine.

In the second experiment for comparison reasons an 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. Lϋtticken 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.

Experiment 1 hi the potency test the effect of the vaccine is assessed by measurement of the serological response and resistance to challenge obtained from administering a challenge vims 14 days after administering the Gumboro vaccine marker vaccine strain D78-23. Fourteen days old chickens were divided in 2 groups of 30 chickens each. The groups were vaccinated with different doses of marker vaccine strain D78-23 via the intramuscular route. At 3, 7, 10 and 14 days after the vaccination bursae were isolated from 3 chickens per group. The bursae were examined for lesions and Hie presence of viral antigen. Fourteen days after the vaccination five non- vaccinated SPF chickens of the same age and source were added to each group, blood was taken from all animals and all animals were challenged with vimlent IBDV strain F52/70 via the eye drop route. For a period of 10 days after challenge clinical examination took place. At three days after challenge, bursae were isolated of 3 chickens per group. The bursae were examined for lesions and the presence of viral antigen. At ten days after challenge, bursae from the surviving animals were examined for the presence of microscopic lesions. Microscopic lesions in the bursae were scored as follows: the bursae were fixed in 10% formalin immediately after killing of the birds. After dehydration and embedding in Paraplast

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:

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.

Experiment 2 hi a second potency test the effect of the inactivated marker vaccine was assessed by measurement of the serological response 4 and 6 weeks after administering the Gumboro vaccine strain D78-23 via the intramuscular route.

Four weeks old SPF chickens were divided in 2 groups. One group was vaccinated with inactivated maker vaccine D78-23 (Passage level 4; 500 EU/animal) the other group with classical Gumboro vims strain D78, which is also present in our commercial vaccines (500 EU/animal dose).

Results

Identification of IBDV vaccines by means of panel test.

As can be seen in table 2 and 3, 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. * Specific MCA directed against a VP2 epitope present on serotype 1 viruses. ** Specific MCA directed against a VP3 epitope present on serotype 1 viruses. ' Specific MCA directed against a VP3 epitope present on serotype 1 and 2 viruses.

Table 3: Reactivity of a rabbit polyclonal anti-IBDV serum and a Moab with different IBDV strains

Experiment 1

1. Average microscopic lesion score in the bursa.

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).

Table 4: Average bursal lesion score of D78-23; (IM) = intramuscular route; A = acute lesions; * 1 out of 3 animals had a score of 5.0A the other 2 animals had a score of 0.

2. Serological response against IBDV

It can be seen in table 5, that D78-23 induced a good serological response against IBDV.

Table 5: Serological response 14 days after vaccination (VN-titre is expressed as log2 of the dilution). IM = intramuscular route.

3. Detection of IBD-viral-antigen in the bursa.

As can be seen from table 6, no viral antigen could be detected from the bursa before challenge. In one out of 3 animal, viral antigen could be detected, 3 days after challenge indicating that this animal was not protected against challenge.

Table 6: Presence of IBDN in the bursa of Fabricius. 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.

Because no viral antigen could be detected after vaccination it was decided to try to re-isolate the vaccine vims by means of serial blind passages on CEF. Results are depicted in table 7. It was possible to re-isolate the vaccine vims 3 and 7 days after vaccination. The isolated vims had an identical reaction pattern with the different Moab as the marker vaccine strain D78-23 (for reaction pattern see table 2), indicating that the animal passage had no influence on the epitopes present on the vims.

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.

Experiment 2

1. Serological response against IBDV

It can be seen in table 8, that the inactivated marker vaccine D78-23 induced a good serological response against IBDV, 4 and 6 weeks after vaccination. Similar results were obtained in the group vaccinated with an identical dose of D78 inac.

Table 8: Serological response 4 and 6 weeks after vaccination (VN-tihe is expressed as log2 of the dilution).

Claims

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.

A mutant according to claim 1 characterised in that the serotype 2 VP3 and VP4 protein have an a ino acid sequence as shown in SEQ ID No. 2

A mutant according to claims 1 or 2, characterised in that the mutant is a mild, intermediate or invasive virus.

A mutant according to claims 1-3, characterised in that the mutant is of the classical, variant E or GLS subtype of serotype 1 IBDVs.

A mutant according to claim 4, characterised in that the coding region for the serotype 2 VP3 and/or VP4 protein is introduced in IBDV strain D78.

A mutant according to claims 1-5, characterised in that the mutant is in an inactivated form.

A vaccine for use in the protection of animals against disease caused by IBDV infection, characterised in that it comprises a serotype 1 IBDV mutant according to claims 1-6, together with a pharmaceutical acceptable carrier or diluent.

A method for the detection of an IBDV infection in an animal comprising the step of examining a sample of the animal for the presence or absence of antibodies directed against a serotype specific IBDV VP3 or VP4 epitope. _ A method according to claim 8, characterised in that the method comprises the steps of:

(i) incubating a sample suspected of containing anti-IBDV antibodies with serotype 1 VP3 or VP4 antigen material,

(ii) allowing the formation of antibody-antigen complex , and (iii) detecting the presence of the antibody-antigen complex.

10 A method according to claim 9, characterised in that in step (iii) a serotype 1 specific VP3 or VP4 Moab is used. 1 A diagnostic kit suitable for carrying out a method according to claims 8-10.