WO2000012677A2 - Generation of recombinant infectious bursal disease viruses by reverse genetics technology and the use of the recombinant viruses as attenuated vaccines - Google Patents

Generation of recombinant infectious bursal disease viruses by reverse genetics technology and the use of the recombinant viruses as attenuated vaccines Download PDF

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WO2000012677A2
WO2000012677A2 PCT/IB1999/001604 IB9901604W WO0012677A2 WO 2000012677 A2 WO2000012677 A2 WO 2000012677A2 IB 9901604 W IB9901604 W IB 9901604W WO 0012677 A2 WO0012677 A2 WO 0012677A2
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recombinant
ibdv
seq
strains
ibdv mutant
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PCT/IB1999/001604
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WO2000012677A3 (en
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Boon-Leong Lim
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The University Of Hong Kong
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/005—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from viruses
    • 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
    • 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
    • C12N2720/10022—New viral proteins or individual genes, new structural or functional aspects of known viral proteins or genes

Definitions

  • This invention is in the field of molecular biology.
  • This invention relates to the generation of recombinant infectious bursal disease virus and the manipulation of the viral genome by the reverse genetics technology.
  • it relates to two DNA molecules comprising artificially constructed polynucleotide sequences substantially corresponding to all or a portion of infectious bursal disease d.s. RNA genome and the mutagenesis of certain amino acid residues of the virus so as to generate mutated attenuated vaccine strains against the virus.
  • IBD Infectious bursal disease
  • IBDV infectious bursal disease virus
  • serotype 1 and serotype 2 Two distinct serotypes of IBDV, designated as serotype 1 and serotype 2, have been identified.
  • serotype 1 strains are pathogenic to chicken and vary in their virulence
  • serotype 2 strains isolated from turkey are apathogenic for both turkey and chicken.
  • serotype 1 strains can be divided into several groups: classical virulent strains, attenuated strains, antigenic variant strains and very virulent (w) strains.
  • Classical virulent strains cause bursal inflammation and severe lymphoid necrosis in infected chicken, resulting in immunodeficiency and moderate mortality. Generally, mortality peaks by the third day post-infection but death may still occur over the next 5 to 7 days resulting in a mortality up to 20% ⁇ 30% in specific pathogen free (SPF) chickens.
  • Attenuated strains have been adapted to chick embryo fibroblast (CEF) cells or other cell lines. They do not cause diseases in chickens, and therefore some of them are being used as live vaccines.
  • Antigenic variant strains were recognized by their ability to escape cross- neutralization by the antisera against classical strains. Chickens affected by the variant strains are characterized by severe atrophy of the bursa, without showing the inflammation symptom associated with the infection of classical strains
  • IBDV is a member of the Birnaviridae family, as its genome consists of two segments of double-stranded RNA (dsRNA).
  • VP2 and VP3 are the major structural proteins of the virion, of which VP2 is the major host-protective immunogen of IBDV that contains the antigenic regions responsible for the induction of neutralizing antibodies (Brown, M. D. and Skinner, M. A. Virus Res,
  • 25 immunogen VP2 are highly conserved, except the central Accl-Spel restriction fragment which has been designated as the hypervariable region.
  • GZ902 has the highest identity to the American variant strains A, " E and GLS and they share unique amino acid residue at position 249K and 254S, which are not present in the other serotype 1 strains.
  • Attenuated strain GZ911 like other cell culture-adapted strains, has substitutions at positions 279(D to N) and 284(A to T) as well as in the serine-rich heptapeptide region. Hence, these substitutions may play important roles in the reduced virulence of these strains.
  • the four very virulent strains have the highest identity to the European very virulent strain UK661 and Japanese strain OKYM.
  • the principal methods of controlling IBD in young chickens are by vaccination with live attenuated strain of IBDV at the age of 0-5 weeks, or by in ovo vaccination, or by transferring high levels of maternal antibody induced by the administration of live and killed IBD vaccines to the breeder hens (Wyeth and Cullen, 1979). Both live attenuated virus vaccine and inactivated virus vaccine for this disease are available on the markets.
  • the live attenuated vaccine available on the markets are generally derived by serial passage of classical or variant strains in embryonated SPF eggs, or in cells derived from embryonated SPF eggs , such as primary or secondary chicken embryo fibroblasts (CEF).
  • Inactivated vaccine is prepared by culturing the virus itself, or by propagating the virus in SPF chicken, which are subsequently inactivated by heat, chemical treatment and the like.
  • the attenuated strains derived from the classical strains and the variant strains can usually protect the birds from the attacks of the classical strains and the variant strains, respectively.
  • these vaccine strains do not always offer 100% protection against the very virulent strains discovered in the recent years. Hence, it is important to develop an attenuated vaccine strains from the very virulent strains.
  • Attenuated live virus vaccines are preferred because they provoke a more long-lasting immune response (often both humoral and cellular), a good mucosal immunity and are easy to produce.
  • live infectious bursal disease vaccines are developed by multiple passages of the field virus in chicken embryonic fibroblasts or in embryonated eggs.
  • the method of producing live attenuated virus is a random, uncontrolled process and as a result, these vaccines suffer from a number of drawbacks.
  • a field virus may not be adapted to CEF after many attempts and hence no live attenuated virus could be derived.
  • the present invention involves the generation of full length cDNA from the double strand RNA genome of the a very virulent IBDV strain by reverse transcription.
  • the two cDNAs were individually cloned into eukaryotic expression vectors before carrying out site-directed mutagenesis.
  • the transfection of the mutated expression vectors into chicken embryo fibroblasts allowed the regeneration of a CEF adapted, live virus, whereas the transfection of the non- mutated expression vectors could not generate any live virus.
  • the present invention provide a novel technique that creates CEF-adapted IBDV strains by site-directed mutagenesis which can be used for the preparation of live and inactivated vaccines against IBDV infection.
  • the mutant viruses being attenuated in a controlled manner can elicits a strong immune response in host animals.
  • Fig. 1 shows an alignment of the hypervariable regions of various ISDV strains.
  • Fig. 2 shows the phylogenetic tree showing the relationship between various IBDV strains.
  • Fig. 3 shows construction of plasmid FA-pBssK containing full-length cDNA copy of segment
  • Fig. 4 shows construction of plasmid FB-pBssK containing full-length cDNA copy od segment B of IBDV HK46 strain.
  • Fig. 5 shows changes in Bursa Weight after virus inoculation.
  • the hypervariable region of the VP2 protein of seven Chinese IBDV strains were characterized.
  • sequences Fig.l
  • several amino acid residues on the VP2 protein were postulated to be responsible for the attenuation of the virus or the antigenicity of the virus.
  • this invention developed a molecular procedure that can be used to engineering IBDV genome in a controlled manner, which can change the property of the virus, such as generating a live attenuated strain of specific antigenicity.
  • the invention comprises of the RNA genomes derived from a very virulent IBDV strain HK46, including a biological pure RNA encoding SEQ ID NO:l, which encodes a 1012 amino acids long polypeptide, and a biological pure RNA encoding SEQ ID NO:2, which encode a polypeptide of 879 amino acids long.
  • the RNA genomes were reverse transcripted into cDNAs which were then subcloned into various vectors, of which some of them containing an eukaryotic promoter, such as CMV promoter or RSV promoters.
  • the transfection of two of these vectors, which individually carries the segment A cDNA and segment B cDNA of the very virulent strain HK46, into CEF together, can not yield a viable virus.
  • a preferred IBDV mutant according to the invention can propagate in CEF or cell lines as a result of point mutation and/or a deletion and/or insertion of an
  • the mutation can be introduced in the IBDV segment A genomic region encoding the VP2 having an amino acid sequence (SEQ ID NO:32) shown in SEQ ID NO:l.
  • the nucleic acid sequence of the whole segment A genome was determined and is shown in SEQ ID NO:l.
  • the mutation can be introduced in the IBDV segment B genomic region encoding the VP1 having an amino acid sequence (SEQ ID NO:33) shown in SEQ ID NO:2.
  • the nucleic acid sequence of the whole segment B genome was determined and is shown in SEQ ID NO:2.
  • 15 into the vectors can be mutated by site-directed mutagenesis or PCR at one or more amino acid residues at positions 222, 249, 254, 256, 279, 284, 294, 299, 326 to 332 and combinations thereof.
  • the transfection of certain mutated vectors together with the vector carrying the segment B cDNA insert into CEF can yield viable viruses, which are in the embodiment of this invention.
  • the viable mutant virus consists of substitutions at the amino acids residues 249(Q-> K) and 254(G- >S) of segment A protein, which can be passaged in CEF.
  • the viable mutant virus consists of substitutions at the amino acids residues 279(D->N),
  • segment A protein which can be passaged in CEF.
  • mutant IBDVs are generated in a controlled, non-random manner.
  • the mutation introduced into the viruses are site- directed and the mutations can result in the change of the virulence and antigenicity of the virus.
  • the IBDV mutant according to the invention is derived from strain
  • any IBDV strain can be used to prepare the IBDV mutant, e.g. the very virulent strains G9201, G9303, F9502, the classical CJ801 and the variant strain
  • the Ndel-Spel restriction fragment (nucleotides 652-1192) can be subcloned into the vectors containing SEQ ID NO:l.
  • the vectors generated thereof can be used for mutagenesis as described above to generate cell culture-adapted, attenuated virus.
  • IBDV mutants according to the invention can also be obtained by inserting a nucleic acid sequence into the VP2 coding region.
  • a nucleic acid sequence can inter alia be an oligonucleotide, for example of about 10-90 bp, or a gene encoding a polypeptide.
  • Said nucleic acid sequence can be derived from any source, e.g. synthetic, viral, prokaryotic or eukaryotic.
  • the live recombinant vaccine generated according to this invention is administered in amounts sufficient to stimulate the immune system to confer resistance to IBD.
  • the vaccine according to the invention is administered in such amount that will induce immunity in a chicken against challenge by a virulent IBDV.
  • Immunity is defined as the induction of a significant level of protection in a population of chickens after vaccination compared to an unvaccinated group. A dose of IO 2 to IO 5 EID 50 of the IBDV mutant per chicken is recommended in general.
  • the vaccine according to the invention may also be in a lyophilized form.
  • a vaccine according to the invention also comprises a pharmaceutically acceptable carrier compatible with the IBDV recombinant vaccine.
  • a sterilized isotonic solution such as a physiological saline and a phosphate buffer may be added to the IBDV recombinant vaccine.
  • suitable excipients are skimmed milk, glycerol, dextrose, sorbitol, starch, mannitol and the like.
  • the vaccine can contain amounts of auxiliary substances which enhance the effectiveness of the vaccine, such as emulsifiers and adjuvants.
  • Adjuvants such as aluminum hydroxide, aluminum phosphate, plant and mineral oil and the like, are administrated with the vaccine in amounts adequate to enhance the immune response to the IBDV. The amount of adjuvant added to the vaccine will vary dependent on the nature of the adjuvant.
  • the live IBDV recombinant vaccine can be given to the animal orally, nasally, ophthalmically, intradermally, subcutaneously, intramuscularly or by aerosol, spray and drinking water. It can also be given to fertilized eggs by in ovo injection. It is preferred to vaccinate the chickens by mass administration techniques such as by placing the vaccine in drinking water or by spraying the vaccine.
  • An live IBDV recombinant vaccine according to the invention can also be used to prepare an inactivated vaccine, which can be administered by injection.
  • Parenteral administration of the inactivated vaccine can be carried out intradermally, subcutaneously, intramuscularly.
  • the recombinant vaccine generated by this invention is administered to poultry, including breeders, broilers, chickens, hens, layers, roasters, roosters and turkeys.
  • the vaccine is administrated to the poultry to prevent IBD before birth, and after hatching.
  • the bursa were removed and homogenized in TNE buffer (10 mM Tris/HCl, 100 mM NaCl, 1 mM EDTA, pH8.0). After freezing and thawing for three times, the homogenates were centrifuged at 14 000 rpm for 15 min at 4°C, and the supematants were collected for virus purification.
  • bursal homogenate supematants were loaded onto 4 ml of 40% sucrose cushion and ultracentrifuged at 55,000 g (Beckman sw55Ti rotor, 22,000 rpm) for 2.5 hr at 4°C. After removing the cushion, the virus particle pellets at the bottom were stored at -20°C.
  • Purified IBDV particles were resuspended in proteinase buffer (TNE + 0.5%SDS), digested with 1 mg/ml proteinase K for 1 hr at 37°C, and extracted with phenol-chloroform-isoamylalcohol (25:24:1, V/V) twice. Finally the viral dsRNA were precipitated from the upper aqueous-phase by ethanol.
  • the amplifications were performed using Robocyclera Gradient 96 (Stratagene) following the program of 94°C for 3 min; 30 cycles of 94°C for 40 sec, 60°C (for FA5 and FA3 amplification) or 52°C (for FB5 and FB3 amplification) for 40 sec, 72°C for 2 min 30 sec; and finally 72°C for 10 min.
  • Robocyclera Gradient 96 (Stratagene) following the program of 94°C for 3 min; 30 cycles of 94°C for 40 sec, 60°C (for FA5 and FA3 amplification) or 52°C (for FB5 and FB3 amplification) for 40 sec, 72°C for 2 min 30 sec; and finally 72°C for 10 min.
  • the insert (FA3) and the vector (FA5- pBssk) were ligated with T4 DNA ligase.
  • T4 DNA ligase After transforming the ligated DNA into E.coli XLl-Blue strain, positive colonies were screened by PCR. Plasmid DNAs are extracted from positive colonies by a mini-prep kit (Wizard Plus Minipreps DNA purification system, Promega) and their identities were confirmed by digestion with EcoR I and Kpn I.
  • the size of the plasmid FA-pBssK is about 6.2kb. This plasmid contains a full-length cDNA copy of segment A of IBDV HK46 (Fig.3).
  • pBssK vector lacks Bgl II site
  • another plasmid pBssK-Bgl in which an unrelated cDNA containing Bgl II sites was cloned into the EcoR I site of pBssK, was employed to construct full-length cDNA clones of fragment B.
  • Fragment FB3 digested with Bgl II and ' Xba I was cloned into the Bgl II/Xba I site of plasmid pBssK-Bgl to obtain plasmid FB3-pBssK.
  • the insert (FB5) and the vector (FB3-pBssk) were ligated with T4 DNA ligase. After transforming the ligated DNA into E.coli XLl-Blue strain, positive colonies were screened by PCR. Plasmid DNAs were extracted from positive colonies by a mini-prep kit (Wizard Plus Minipreps DNA purification system, Promega) and their identities were confirmed by digestion with EcoRI and Xbal. The size of the plasmid FA-pBssK is about 5.7 kb.
  • this plasmid contains a full-length cDNA copy of segment B of IBDV HK46 strain, but does not contain the unrelated cDNA fragment (Fig.4) .
  • Example 6 DNA Sequencing PCR sequencing reactions were carried out using an Autocycle DNA Sequencing Kit (Pharmacia) following the manufacturer's recommended conditions. Specific primers with fluorescein at 5' end, as listed in table 2, were used for the sequencing. Sequence determination was performed using Automated Laser Fluorescein (ALF) nucleotide sequencer (Pharmacia). Sequences data were assembled and analyzed using the GCG package (Genetics Computer Group, Madison, Wisconsin, USA). Phylogenetic trees were prepared by the UPGMA method (Unweighted Pair Group with Arithmetic Mean) of the GeneWorks program (IntelliGenetics Inc, Mountain View, CA, USA).
  • amino acid substitutions at position 279 (D®N) and 284 (A®T), as well as in the serine- rich heptapeptide region (330 S®R), may result in the reduced virulence of classical IBDV strains.
  • two other specific amino acid residues, 249K and 254S may be the signature residues of the variant IBDV strains.
  • plasmids NT- FA-pBssK and KS-FA-pBssK were constructed by oligo-nucleotide directed mutagenesis, or by using specific primer pairs and FA-pBssK as template in PCR reactions.
  • A5 SEQ ID NO: 3
  • NTA SEQ ID NO: 11
  • NTS SEQ ID NO: 12
  • A3AP SEQ ID NO: 7
  • the PCR product of 1756 bp were then digested with EcoR I and Sal I.
  • the resulting fragment of 1726 bp was cloned into the EcoR I / Sal I site of FA-pBssK to substitute the EcoR I/Sal I insert of the plasmid FA-pBssK to obtain the plasmid NT-FA-pBssK.
  • the mutated sequences were confirmed by cycling PCR sequencing.
  • the plasmid NT-FA-pBssK contains full-length cDNA of segment A of HK46 strain but has substitutions at nucleotide positions 966 (G®A) and 981 (G®A).
  • KS-FA-pBssK Two primer pairs, A5 (SEQ ID NO: 3) and KSA (SEQ ID NO: 13), KSS (SEQ ID NO: 14) and A3AP(SEQ ID NO: 7), were used to amplify the DNA fragments of 902 and 874 bp respectively, using FA-pBssK as template.
  • a PCR product of 1756 bp was then amplified from the two PCR products as described above, which was subsequently cloned into the EcoRI/Sall site of the plasmid FA-pBssK.
  • the mutated sequences were confirmed by cycling PCR sequencing using sequencing primers.
  • the new plasmid KS-FA-pBssK contains full- length cDNA of segment A of HK46 but has substitutions at nucleotide positions 876 (C®A) and 891 (C®A).
  • the pALTER-MAX vector may be used for mutagenesis and expression of genes in eukaryotic cells (Promega) .
  • segment A cDNA (SEQ ID NO:l) of strain HK46 was subcloned into the pALTER-MAX vector.
  • the plasmid FA-pBssK was digested with EcoR I, Kpn I and Bgl I.
  • the fragment FA and pBssK can easily be separated by 1% agarose gel after the plasmids were digested with Bgl I. After digestion, the FA fragment (3269 bp) was purified and cloned into the EcoR I/Kpn I site of pALTER-MAX vector to create plasmid FA- pALTER.
  • the nucleotide T at position 1127 was changed to A using Altered sites II mammalian mutagenesis system (Promega) .
  • the single strand (ss) DNA of plasmids FA-pALTER was produced using the methods recommended by supplier.
  • a primer designated as 330R (5'ACTGCTAGGCTCCCTCTTGCGGA CCA3') that was phosphorylated at the 5'-end was synthesized and used for producing a mutation on plasmid FA-pALTER in which the nucleotide 1127(T) was substituted with A.
  • annealing reaction mixture containing 0.05 pmole of ssDNA of FA-pALTER, 1.25 pmole of primer 330R, 0.25 pmole of ampicillin repair oligonucleotide, 20mM Tris- HC1 ( ⁇ H7.5), 10 mM MgCl 2 and 50 mM NaCl was heated to 75°C for 5 min, and then cooled slowly down to room temperature.
  • synthesis buffer containing 100 mM Tris-HCl (pH7.5), 5 mM dNTPs, 10 mMATP and 20 mM DTT, ten units of T 4 DNA polymerase and 3 units of T4 DNA ligase were mixed with annealing reaction mixture, and the total volume of DNA synthesis reaction was adjusted to 30 ul with deionized water. Subsequently, synthesis mixture was incubated at 37°C for 2 hr to perform mutant strand synthesis and ligation. After incubation, the synthesis mixture was transformed into E coli strain ES1301 mutS competent cells by electroporation.
  • the transformed ES1301 mutS cells were resuspended in 1 ml of LB broth without any antibiotics, and incubated at 37°C for 3 hr. By adding 0.5 ml of transformed cell culture to 4.5 ml of selective medium (LB broth with 100 ug/ml ampicillin), the transformed ES1301 mutS cells were cultured at 37°C with shaking at 250rpm overnight. Plasmid DNA was then extracted by the Wizard 5 Plus Minipreps DNA purification system and then transformed into Ecoli strain JM109 competent cells. Ten colonies were screened by direct sequencing with primer A768S (SEQ ID NO: 20) using an automatic sequencer. The resulting mutant plasmid designated as R-FA-pALTER was obtained.
  • segment A cDNAs from plasmids FA-FA-pBssK, NT-FA- pBssK, KS-FA- pBssK and R-FA-pALTER were released by EcoR I, Kpn I and Bgl I digestion and then subcloned into a plasmid at a site downstream to a CMV promoter.
  • the plasmid FB-pBssK was digested with three restriction enzymes EcoR I, Xba I and Pvu II, where Pvu II can only digest pBssK vector but can not digest fragment FB.
  • fragment FB was purified and inserted into the EcoR 1/ Xba I site of the plamid carrying a CMV promoter.
  • This plasmid contained the full-length cDNA copy of segment B downstream to a CMV promoter.
  • the CEFs were grown to 80% confluence in a 12-well tissue culture plate (Falcon) and washed once with phosphate-buffered-saline (PBS) .
  • PBS phosphate-buffered-saline
  • One ml of serum free EME medium (SF-EMEM) (Life Technologies) were added to monolayers of each well, and the cells were incubated at 37°C for 1 hr in 5% CO 2 incubator. 5ul (0.2ug/ul) of both plasmids, CMV-FA and CMV-FB were then transfected to the CEF by Lipofectamine (Life Technologies), according to the manufacturer's procedures.
  • the transfection mixture was replaced with EMEM containing 10% FBS, and the cells were further incubated at 37°C in 5% CO 2 incubator for desired time intervals.
  • the plasmids containing mutants of segment A cDNAs (KS-FA or NT-FA or R-FA) and/or CMV-FB were also transfected into CEF using the same procedure described above. After 96 hours, cytopathic effect was observed. The ability of the recombinant viruses in propagating in CEF was further tested by adding 0.1 ml vims suspension collected 96 hr posttransfection into a well of a 12 well plate containing 80% confluent CEF with 1 ml cell culture medium.
  • RI, R2 Transfected with two independent CMV-R plasmids and CMV-FB plasmid.
  • NT Transfected with CMV-NT-FA plasmid and CMV-FB plasmid.
  • KS Transfeced with CMV-KS-FA plasmid and CMV-FB plasmid.
  • Example 11 After virus propagation in CEF, cell debris was removed from the supernatant by centrifugation at 14,000rpm for 20min at 4°C. One ml of supernatant was loaded onto 4 ml of 40%(v/v) sucrose cushion and ultracentrifuged at 55,000g (Beckman sw55Ti rotor, 22,000rpm) for 2.5 hr at 4°C. The vims at the pellet was processed as stated in Examples 3&4 to produce first stranded cDNA. PCR reaction as stated in Example 5 was carried out to amplify the 5'cDNA segment of genome A (equivalent to FA5), which was then sequenced to confirm the mutation (Table 2). Example 11
  • the recombinant virus (HK46-NT) generated from transfection was passaged and titred in CEF.
  • 3.2xl05TCID50 HK46-NT and 1.6xl05EID50 HK46 were administrated to 4 weeks old SPF birds. No birds vaccinated with HK46-NT showed any symptomes nor mortality, whereas all birds challenged by the parent strain HK46 were sick and the mortality reached 60%.
  • the weights of the bursa after chanllenge were recorded in figure 5.
  • HK46-NT To test the immunologicity of HK46-NT, it was administrated to SPF chickens at 25 days old (106TCID50/bird) and antibody titre at 43 days old was measured by a commercial ELISA kit (Idexx Inc.) .
  • the signal-to-noise (S/N) ratios were 3.41 +/- 0.68 and 1.37+/-0.13, respectively.

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Abstract

The invention relates to the generation and mutagenesis of recombinant infectious bursal disease virus by reverse genetics technology. Site-directed mutagenesis of certain amino acid residues on the Non-CEF adapted, very virulent strains of IBDV transforms the viruses into attenuated, CEF-adapted strains. The attenuated, CEF-adapted strains can be used as live vaccines against the very virulent strains. Curently available live attenuated vaccines were generated by serial passage through cell culture and the like, whereas the present invention provides a novel molecular mechanism in manipulation of the viral genome and the generation of attenuated vaccines by site-directed mutagenesis. The same approach can be used to produce vaccine strains from newly evolved IBDV viruses or to manipulate the antigenicity and pathogenicity of the virus.

Description

GENERATION OF RECOMBINANT INFECTIOUS BURSAL DISEASE
VIRUSES BY REVERSE GENETICS TECHNOLOGY AND THE USE OF
THE RECOMBINANT VIRUSES AS ATTENUATED VACCINES
RELATED APPLICATION
This application claims the priority of provisional patent application no. 60/098,684, filed on September 1, 1998.
FIELD OF THE INVENTION This invention is in the field of molecular biology. This invention relates to the generation of recombinant infectious bursal disease virus and the manipulation of the viral genome by the reverse genetics technology. In particular, it relates to two DNA molecules comprising artificially constructed polynucleotide sequences substantially corresponding to all or a portion of infectious bursal disease d.s. RNA genome and the mutagenesis of certain amino acid residues of the virus so as to generate mutated attenuated vaccine strains against the virus.
BACKGROUND OF INVENTION Infectious bursal disease (IBD), caused by infectious bursal disease virus (IBDV), is a highly contagious disease of young chickens, which results in significant losses to the poultry industry. After infection, IBDV multiplies rapidly in the developing B lymphocytes of the bursa of Fabricius, leading to immunosuppression and increased susceptibility to other diseases. Two distinct serotypes of IBDV, designated as serotype 1 and serotype 2, have been identified. The serotype 1 strains are pathogenic to chicken and vary in their virulence, whereas serotype 2 strains isolated from turkey, are apathogenic for both turkey and chicken. According to antigenic variation and virulence, serotype 1 strains can be divided into several groups: classical virulent strains, attenuated strains, antigenic variant strains and very virulent (w) strains. Classical virulent strains cause bursal inflammation and severe lymphoid necrosis in infected chicken, resulting in immunodeficiency and moderate mortality. Generally, mortality peaks by the third day post-infection but death may still occur over the next 5 to 7 days resulting in a mortality up to 20%~30% in specific pathogen free (SPF) chickens. Attenuated strains have been adapted to chick embryo fibroblast (CEF) cells or other cell lines. They do not cause diseases in chickens, and therefore some of them are being used as live vaccines. Antigenic variant strains were recognized by their ability to escape cross- neutralization by the antisera against classical strains. Chickens affected by the variant strains are characterized by severe atrophy of the bursa, without showing the inflammation symptom associated with the infection of classical strains
5 (Vakharia et al. Virus Res 31:265-273, (1994)). Since the late eighties, the outbreaks of newly evolved, very virulent (w) strains in Europe, Japan and China cause significant economical losses to the poultry industry. The w strains can cause up to 60-100% mortality in SPF birds. These w strains cause typical lesions of IBDV and are antigenically similar to the classical strains (Cao, Y. et al, Avian Dis. 42,
10 340-351, (1998)).
IBDV is a member of the Birnaviridae family, as its genome consists of two segments of double-stranded RNA (dsRNA). The smaller RNA segment (segment B) with a length of about 2.8kb, encodes VP1, a 90 kDa multifunctional protein with polymerase and capping enzyme activities. The larger RNA segment (segment A),
15 with a length of about 3.2kb, encodes a polyprotein that is cleaved by autoproteolysis to form mature viral proteins VP2, VP3 and VP4. VP2 and VP3 are the major structural proteins of the virion, of which VP2 is the major host-protective immunogen of IBDV that contains the antigenic regions responsible for the induction of neutralizing antibodies (Brown, M. D. and Skinner, M. A. Virus Res,
20 40:1-15, (1996)). A second open reading frame (ORF), preceding and partially overlapping the polyprotein gene, encodes VP5, a 17kD polypeptide present in IBDV-infected cells. However, the function of the polypeptide is still unknown. The nucleotide sequences of genome segments A and B of various IBDV strains have been published, which conclude that the sequences of the major host-protective
25 immunogen VP2 are highly conserved, except the central Accl-Spel restriction fragment which has been designated as the hypervariable region.
Seven infectious bursal disease virus strains isolated from China have recently been characterized (Cao, Y. et al, Avian Dis. 42, 340-351, (1998)), including a classical strain CJ801, an attenuated strain GZ911, a variant strain
30 GZ902, and four very virulent strains G9201, G9302, F9502 and HK46. Using reverse transcription-polymerase chain reaction (RT-PCR), the full length VP2 genes were amplified and the hypervariable regions were sequenced. Protein sequences of the hypervariable region (a.a. 143-382) of the field isolates confirmed their identities (Figure 1). CJ801 has the highest identity to the classical strains STC and
35 52/70. GZ902 has the highest identity to the American variant strains A," E and GLS and they share unique amino acid residue at position 249K and 254S, which are not present in the other serotype 1 strains. Attenuated strain GZ911, like other cell culture-adapted strains, has substitutions at positions 279(D to N) and 284(A to T) as well as in the serine-rich heptapeptide region. Hence, these substitutions may play important roles in the reduced virulence of these strains. The four very virulent strains have the highest identity to the European very virulent strain UK661 and Japanese strain OKYM. They share unique amino acid residues at positions 222A, 2561, 2941, and 299S which are not present in other less virulent strains. The very virulent strains isolated in Guangdong (G9201, G9303) and Fujian (F9502) provinces have 1 to 5 amino acid substitutions at the two hydrophilic domains of VP2 comparing to UK661 and OKYM, indicating that new very virulent strains are evolving. Phylogenetic analysis suggests that Chinese very virulent IBDVs and European very virulent strains are derived from similar origin (Figure 2). The principal methods of controlling IBD in young chickens are by vaccination with live attenuated strain of IBDV at the age of 0-5 weeks, or by in ovo vaccination, or by transferring high levels of maternal antibody induced by the administration of live and killed IBD vaccines to the breeder hens (Wyeth and Cullen, 1979). Both live attenuated virus vaccine and inactivated virus vaccine for this disease are available on the markets. The live attenuated vaccine available on the markets are generally derived by serial passage of classical or variant strains in embryonated SPF eggs, or in cells derived from embryonated SPF eggs , such as primary or secondary chicken embryo fibroblasts (CEF). Only the strains with their virulence reduced or eliminated by these methods can be used as live attenuated vaccines. Inactivated vaccine is prepared by culturing the virus itself, or by propagating the virus in SPF chicken, which are subsequently inactivated by heat, chemical treatment and the like. The attenuated strains derived from the classical strains and the variant strains can usually protect the birds from the attacks of the classical strains and the variant strains, respectively. However, these vaccine strains do not always offer 100% protection against the very virulent strains discovered in the recent years. Hence, it is important to develop an attenuated vaccine strains from the very virulent strains.
SUMMARY OF INVENTION In general, attenuated live virus vaccines are preferred because they provoke a more long-lasting immune response (often both humoral and cellular), a good mucosal immunity and are easy to produce. Presently live infectious bursal disease vaccines are developed by multiple passages of the field virus in chicken embryonic fibroblasts or in embryonated eggs. Hence, the method of producing live attenuated virus is a random, uncontrolled process and as a result, these vaccines suffer from a number of drawbacks. Firstly, a field virus may not be adapted to CEF after many attempts and hence no live attenuated virus could be derived. Secondly, even some of the field viruses can be adapted in CEF, a population of viruses of different characteristics and different degree of virulence may be generated. Because uncontrolled mutations are introduced into the viral genome during serial passages, a population of virus particles heterogeneous in their virulence and immunizing properties are generated. Single virus has to be cloned and the pathogenicity of each cloned virus has to be tested. Thirdly, it is possible that such traditional attenuated live virus vaccines can revert to virulence, resulting in disease of the inoculated animals and the possible spread of the pathogen to other animals. In this invention, the development of techniques for controlled manipulation of genetic material has allowed the creation of CEF-adapted virus vaccines which avoids the disadvantages of the attenuated virus vaccines generated by conventional techniques.
The present invention involves the generation of full length cDNA from the double strand RNA genome of the a very virulent IBDV strain by reverse transcription. The two cDNAs were individually cloned into eukaryotic expression vectors before carrying out site-directed mutagenesis. The transfection of the mutated expression vectors into chicken embryo fibroblasts allowed the regeneration of a CEF adapted, live virus, whereas the transfection of the non- mutated expression vectors could not generate any live virus.
The present invention provide a novel technique that creates CEF-adapted IBDV strains by site-directed mutagenesis which can be used for the preparation of live and inactivated vaccines against IBDV infection. The mutant viruses being attenuated in a controlled manner can elicits a strong immune response in host animals.
It is another object of the present invention to create new live vaccine strains from classical, variant, very virulent or any other newly evolved IBDV strains.
It is another object of the present invention to provide a mutant IBDV which can be used not only for the preparation of a vaccine against IBDV infection but also against other infectious diseases. It is a further object of the present invention to produce subunit vaccines, pharmaceutical and diagnostic preparations comprising a heterologous polypeptide expressed by an IBDV mutant according to the invention.
BRIEF DESCRIPTION OF THE DRAWINGS
These and other objects, features and elements of the present invention will be better understood from the following detailed description of preferred embodiments of the invention in which:
Fig. 1 shows an alignment of the hypervariable regions of various ISDV strains.
Fig. 2 shows the phylogenetic tree showing the relationship between various IBDV strains.
Fig. 3 shows construction of plasmid FA-pBssK containing full-length cDNA copy of segment Fig. 4 shows construction of plasmid FB-pBssK containing full-length cDNA copy od segment B of IBDV HK46 strain.
Fig. 5 shows changes in Bursa Weight after virus inoculation.
DETAILED DESCRIPTION From a previous publication of our group (Cao, Y. et al, Avian Dis. 42, 340-
351, (1998)), the hypervariable region of the VP2 protein of seven Chinese IBDV strains were characterized. By comparing the sequences (Fig.l), several amino acid residues on the VP2 protein were postulated to be responsible for the attenuation of the virus or the antigenicity of the virus. Based on these data, this invention developed a molecular procedure that can be used to engineering IBDV genome in a controlled manner, which can change the property of the virus, such as generating a live attenuated strain of specific antigenicity.
The invention comprises of the RNA genomes derived from a very virulent IBDV strain HK46, including a biological pure RNA encoding SEQ ID NO:l, which encodes a 1012 amino acids long polypeptide, and a biological pure RNA encoding SEQ ID NO:2, which encode a polypeptide of 879 amino acids long. The RNA genomes were reverse transcripted into cDNAs which were then subcloned into various vectors, of which some of them containing an eukaryotic promoter, such as CMV promoter or RSV promoters. The transfection of two of these vectors, which individually carries the segment A cDNA and segment B cDNA of the very virulent strain HK46, into CEF together, can not yield a viable virus.
A preferred IBDV mutant according to the invention can propagate in CEF or cell lines as a result of point mutation and/or a deletion and/or insertion of an
5 oligo- or polynucleotide sequence in the gene encoding VP2.
Preferably, the mutation can be introduced in the IBDV segment A genomic region encoding the VP2 having an amino acid sequence (SEQ ID NO:32) shown in SEQ ID NO:l. The nucleic acid sequence of the whole segment A genome was determined and is shown in SEQ ID NO:l.
10 In addition, the mutation can be introduced in the IBDV segment B genomic region encoding the VP1 having an amino acid sequence (SEQ ID NO:33) shown in SEQ ID NO:2. The nucleic acid sequence of the whole segment B genome was determined and is shown in SEQ ID NO:2.
Certain amino acid residues encoded by the segment A cDNA inserts cloned
15 into the vectors can be mutated by site-directed mutagenesis or PCR at one or more amino acid residues at positions 222, 249, 254, 256, 279, 284, 294, 299, 326 to 332 and combinations thereof. The transfection of certain mutated vectors together with the vector carrying the segment B cDNA insert into CEF can yield viable viruses, which are in the embodiment of this invention.
20 In a particular preferred embodiment of the invention, the viable mutant virus consists of substitutions at the amino acids residues 249(Q-> K) and 254(G- >S) of segment A protein, which can be passaged in CEF.
In another particular preferred embodiment of the invention, the viable mutant virus consists of substitutions at the amino acids residues 279(D->N),
25 254(A~>T) of segment A protein, which can be passaged in CEF.
According to the present invention such mutant IBDVs are generated in a controlled, non-random manner. The mutation introduced into the viruses are site- directed and the mutations can result in the change of the virulence and antigenicity of the virus.
30 Although, the IBDV mutant according to the invention is derived from strain
HK46, an isolate recovered from infected tissues of 6 week old broilers by the Agricultural and Fishery Department of the Hong Kong Special Administration Region, any IBDV strain can be used to prepare the IBDV mutant, e.g. the very virulent strains G9201, G9303, F9502, the classical CJ801 and the variant strain
35 GZ902 described in a publication (Cao, Y. et al, Avian Dis. 42, 340-351, (1998)), or any newly evolved virulent strains.
From the cDNA derived from the genome segment A of any other IBDV, the Ndel-Spel restriction fragment (nucleotides 652-1192) can be subcloned into the vectors containing SEQ ID NO:l. The vectors generated thereof can be used for mutagenesis as described above to generate cell culture-adapted, attenuated virus. IBDV mutants according to the invention can also be obtained by inserting a nucleic acid sequence into the VP2 coding region. Such a nucleic acid sequence can inter alia be an oligonucleotide, for example of about 10-90 bp, or a gene encoding a polypeptide. Said nucleic acid sequence can be derived from any source, e.g. synthetic, viral, prokaryotic or eukaryotic.
The live recombinant vaccine generated according to this invention is administered in amounts sufficient to stimulate the immune system to confer resistance to IBD. The vaccine according to the invention is administered in such amount that will induce immunity in a chicken against challenge by a virulent IBDV. Immunity is defined as the induction of a significant level of protection in a population of chickens after vaccination compared to an unvaccinated group. A dose of IO2 to IO5 EID50 of the IBDV mutant per chicken is recommended in general. The vaccine according to the invention may also be in a lyophilized form. In addition to the IBDV recombinant vaccine described above, a vaccine according to the invention also comprises a pharmaceutically acceptable carrier compatible with the IBDV recombinant vaccine. As a pharmaceutically acceptable carrier a sterilized isotonic solution such as a physiological saline and a phosphate buffer may be added to the IBDV recombinant vaccine. Further suitable excipients are skimmed milk, glycerol, dextrose, sorbitol, starch, mannitol and the like. In addition, if desired, the vaccine can contain amounts of auxiliary substances which enhance the effectiveness of the vaccine, such as emulsifiers and adjuvants. Adjuvants such as aluminum hydroxide, aluminum phosphate, plant and mineral oil and the like, are administrated with the vaccine in amounts adequate to enhance the immune response to the IBDV. The amount of adjuvant added to the vaccine will vary dependent on the nature of the adjuvant.
For administration to animals, the live IBDV recombinant vaccine can be given to the animal orally, nasally, ophthalmically, intradermally, subcutaneously, intramuscularly or by aerosol, spray and drinking water. It can also be given to fertilized eggs by in ovo injection. It is preferred to vaccinate the chickens by mass administration techniques such as by placing the vaccine in drinking water or by spraying the vaccine.
An live IBDV recombinant vaccine according to the invention can also be used to prepare an inactivated vaccine, which can be administered by injection. Parenteral administration of the inactivated vaccine can be carried out intradermally, subcutaneously, intramuscularly.
The recombinant vaccine generated by this invention is administered to poultry, including breeders, broilers, chickens, hens, layers, roasters, roosters and turkeys. Preferably, the vaccine is administrated to the poultry to prevent IBD before birth, and after hatching.
EXAMPLES Example 1 Propagation and Purification of IBDV from Bursae The very virulent strains of infectious bursal disease virus (IBDV), HK46,
G9303, G9201, and F9502, which were isolated in the southern part of P R China, were propagated in SPF chickens at 5 weeks of age. At 72 hr post-infection, the bursa were removed and homogenized in TNE buffer (10 mM Tris/HCl, 100 mM NaCl, 1 mM EDTA, pH8.0). After freezing and thawing for three times, the homogenates were centrifuged at 14 000 rpm for 15 min at 4°C, and the supematants were collected for virus purification. One ml of bursal homogenate supematants were loaded onto 4 ml of 40% sucrose cushion and ultracentrifuged at 55,000 g (Beckman sw55Ti rotor, 22,000 rpm) for 2.5 hr at 4°C. After removing the cushion, the virus particle pellets at the bottom were stored at -20°C.
Example 2
Extraction of Viral RNA
Purified IBDV particles were resuspended in proteinase buffer (TNE + 0.5%SDS), digested with 1 mg/ml proteinase K for 1 hr at 37°C, and extracted with phenol-chloroform-isoamylalcohol (25:24:1, V/V) twice. Finally the viral dsRNA were precipitated from the upper aqueous-phase by ethanol.
Example 3
Synthesis of cDNA by Reverse Transcriptase The cDNA of IBDV segments A and B of the very virulent strain, HK46, were synthesized independently. Two primer pairs, designated as A5 (SEQ ID NO: 3) with EcoR I site and A3 (SEQ ID NO: 4) with Kpn I site, B5 (SEQ ID NO: 5) with EcoR I site and B3 (SEQ ID NO: 6) with Xba I site, were used for synthesizing cDNA of IBDV segments A and B. Viral ds RNA mixed with primers A5 and A3 (for segment A) or B5 and B3 (for segment B) were denatured by boiling for 5 min and cooled on ice for 2 min. After adding equal volume of 2X PCR buffer containing 10 mmole of dNTPs, 50 mmole of MgCl2, 200 mmole of DTT and 200 U of Superscript II RT (life Technologies), a total volume of 50 ul of reaction mixture was incubated at 50°C for 1 hr to synthesize the first strand cDNA. The reaction was terminated at 70°C for 15 min and the ds RNA was then digested by 1 U of RNase H at 37°C for 20 min.
Example 4 PCR Amplification of the Whole IBDV Genome
All primers used for PCR and mutagenesis are listed in Table 1. Four fragments, designated as FA5 (5' end fragment of segment A), FA3 (3' end fragment of segment A), FB5 (5' end fragment of segment B), FB3 (3' end fragment of segment B), were independently amplified by Expand™ High Fidelity PCR System (Boehringer Mannheim), using the first strand cDNA as templates. The amplifications were performed using Robocyclera Gradient 96 (Stratagene) following the program of 94°C for 3 min; 30 cycles of 94°C for 40 sec, 60°C (for FA5 and FA3 amplification) or 52°C (for FB5 and FB3 amplification) for 40 sec, 72°C for 2 min 30 sec; and finally 72°C for 10 min. Four primer pairs 'were used for amplifications of the corresponding fragments, in which A5 (SEQ ID NO:3) and A3AP (SEQ ID NO:7) were used for amplifications of fragment FA5, A5SP (SEQ ID NO:8) and A3 (SEQ ID NO:4) for fragment FA3, B5 (SEQ ID NO:5) and B3AP (SEQ ID NO:9) for fragment FB5, B5SP (SEQ ID NO:10) and B3 (SEQ ID NO:6) for fragment FB3. TABLE 1. Primers for genome cloning and mutagenesis of IBDV
SEQ Na Size RE direc locatio sequence
IDNO: me (bp) site tion n
3 A5 40 Eco + A,5' end 5ATGAATTCAGGATACGATCGGTCTGAC R I CCCGGGGGAGTCA3'
4 A3 33 Kpn - A,3' end 5'TAGGTACCAAGGGACCCGCGAACGGAT I CCAATT3'
5 B5 29 Eco + B,5' end 5'TTAGAATTCTAGGATACGATGGGTCTGA R I C3'
6 B3 29 Xba - B,3' end 5ΑTTTCTAGATGGGGGCCCCCGCAGGCG I AA3'
7 A3A 27 Sal . A, 5'CAGGTGAAGCARAGAATCCCGTCGACT
P I 1729- A3' 1755
8 A5S 28 Sal + A, 5*CCAGAATCCYGTAGTCGACGGGATTCTT
P I 1718- 3' 1745
9 B3A 21 Bgl - B, S'GATCCCRAGATCTTTGCTGTAS' P II 1848-
1868
10 B5S 24 — + B, 5'CCTTGCACAACCAGGGTACCTGAG3' P 1757-
1780
11 NT 34 A, 956- 5'GGTCGTTAGCCCATTGTTTCGGGCCACA A 989 GCTCTG3'
12 NTS 38 — + A, 969- 5'GCAAACAATGGGCGAACGACCGGCACT 1006 GACAACCTTAT3'
13 KSA 35 A, 866- 5'GGCTTTGGAeGCTTGrrπAAACACGAG 900 CTCTCCC3'
14 KSS 33 — + A, 881- 5'TAAAACAAGCGTCCAAAGCCTTATACTT 913 GGGTGC3'
15 330 27 — - A, 5'pACTGCTAGGCTCCCTCTTGCTGACCAT R 1115- 3'
1141
16 mu 35 — - A, 873- 5'pAGTATAAGGCTTTGGACGCTTGTTTTA t-KS 907 AACACGAG3'
17 mu 36 — - A, 963- 5'pGTCAGTGCCGGTCGTTAGCCCATTGTT t- 998 TGCGGCCAC3' NT
R = A or G; Y= C or T Example 5
Construction of Full Length cDNA Clones of IBDV Genome
Full-length cDNA clones of IBDV segments A and B genome were independently prepared. To obtain cDNA clones of segment A of HK46 strain, two cDNA fragments FA5 and FA3 were amplified by PCR using two primer pairs, A5 (SEQ ID NO:3) and A3AP(SEQ ID NO:7), A5SP(SEQ ID NO:8) and A3(SEQ ID NO: 4) and using first strand cDNA of segment A as template. Fragment FA5 digested with EcoR I and Sal I was cloned into the EcoR I/Sal I site of pBssK vector (Strategene) to obtain plasmid FA5-pBssK. After digesting fragment FA3 and plasmid FA5-pBssK with Sal I and Kpn I, the insert (FA3) and the vector (FA5- pBssk) were ligated with T4 DNA ligase. After transforming the ligated DNA into E.coli XLl-Blue strain, positive colonies were screened by PCR. Plasmid DNAs are extracted from positive colonies by a mini-prep kit (Wizard Plus Minipreps DNA purification system, Promega) and their identities were confirmed by digestion with EcoR I and Kpn I. The size of the plasmid FA-pBssK is about 6.2kb. This plasmid contains a full-length cDNA copy of segment A of IBDV HK46 (Fig.3).
To obtain cDNA clones of segment B of HK46, two fragments FB5 and FB3 were amplified by PCR using two primer pairs, B5 (SEQ ID NO:5) and B3AP(SEQ ID NO:9), B5SP(SEQ ID NO:10) and B3(SEQ ID NO:6), respectively, and using first strand cDNA of segment B as template. There is an unique Bgl II site in the overlapping region of fragments FB5 and FB3. Because pBssK vector lacks Bgl II site, another plasmid pBssK-Bgl, in which an unrelated cDNA containing Bgl II sites was cloned into the EcoR I site of pBssK, was employed to construct full-length cDNA clones of fragment B. Fragment FB3 digested with Bgl II and'Xba I was cloned into the Bgl II/Xba I site of plasmid pBssK-Bgl to obtain plasmid FB3-pBssK. After digesting fragment FB5 and FB3- pBssK with EcoR I and Bgl II, the insert (FB5) and the vector (FB3-pBssk) were ligated with T4 DNA ligase. After transforming the ligated DNA into E.coli XLl-Blue strain, positive colonies were screened by PCR. Plasmid DNAs were extracted from positive colonies by a mini-prep kit (Wizard Plus Minipreps DNA purification system, Promega) and their identities were confirmed by digestion with EcoRI and Xbal. The size of the plasmid FA-pBssK is about 5.7 kb. Therefore this plasmid contains a full-length cDNA copy of segment B of IBDV HK46 strain, but does not contain the unrelated cDNA fragment (Fig.4) . Example 6 DNA Sequencing PCR sequencing reactions were carried out using an Autocycle DNA Sequencing Kit (Pharmacia) following the manufacturer's recommended conditions. Specific primers with fluorescein at 5' end, as listed in table 2, were used for the sequencing. Sequence determination was performed using Automated Laser Fluorescein (ALF) nucleotide sequencer (Pharmacia). Sequences data were assembled and analyzed using the GCG package (Genetics Computer Group, Madison, Wisconsin, USA). Phylogenetic trees were prepared by the UPGMA method (Unweighted Pair Group with Arithmetic Mean) of the GeneWorks program (IntelliGenetics Inc, Mountain View, CA, USA).
TABLE 2. Primers for DNA sequencing
SEQ Name Size direct locati Sequence
IDNO: (bp) ion on
18 A639S 19 + 639- 5ΑGCTTACCCACATCATATG3' 657
19 A723A 17 - 723- 5'TCACTGCTGTCACATGT3' 739
20 A768S 20 + 768- 5'GCCGATGATTACCAATTCTC3'
787
21 A1041 17 + 1041- 5ΑCCCAGCCAATCACATC3' S 1057
22 A1405 16 + 1405- 5'CGGAGTACTTCATGGA3'
S 1420
23 A1835 16 + 1835- 5*GGAAGACGCCATGACA3' S 1850
24 A2174 18 + 2174- S'CTATGGCGAGATTGAGAAS' S 2191
25 A2538 17 + 2538- 5ΑTGGCCAACTTCGCACT3' S 2554
26 B296S 17 + 296- 5'CAAGATTCTGCAGCCAC3' 312
27 B429S 17 + 429- 5'CAGGAGTACTTCCCAAA3' 446
28 B876S 19 + 876- 5ΑACCTCAAGTCATCAAGTG3' 894
29 B1029 17 - 1029- S'TCACTTAGCATGCTGAGS'
A 1045
30 B1229 19 + 1229- 5'GTCACTCTACAAGTTCAAC3' S 1247
31 B2568 17 - 2568- 5'GTCTGTGGGTTCTTAAC3' A 2584 Example 7
Site-directed Mutagenesis by PCR
According to the results of sequence analysis of VP2 cDNA of IBDV, amino acid substitutions at position 279 (D®N) and 284 (A®T), as well as in the serine- rich heptapeptide region (330 S®R), may result in the reduced virulence of classical IBDV strains. In addition, two other specific amino acid residues, 249K and 254S, may be the signature residues of the variant IBDV strains. So three combinations of mutations were introduced to the segment A cDNA of strain HK46: (a) Mutant NT: substitutions at nucleotide positions 966 (G®A) and 981 (G®A) can result in amino acid substitutions at residues 279 (D®N) and 284(A®T); (b) Mutant KS: nucleotide substitutions at positions 876 (C®A) and 891 (G®A) can result in amino acid substitutions at residues 249 (Q®K) and 254 (G®S); (c) Mutant R: nucleotide substitution at position 1121 (T®A) can result in amino acid substitution at position 330 (S®R).
To introduce the mutation sequences into segment A of IBDV, plasmids NT- FA-pBssK and KS-FA-pBssK were constructed by oligo-nucleotide directed mutagenesis, or by using specific primer pairs and FA-pBssK as template in PCR reactions. To construct plasmid NT-FA-pBssK, two primer pairs, designated as A5 (SEQ ID NO: 3) and NTA (SEQ ID NO: 11), NTS (SEQ ID NO: 12) and A3AP (SEQ ID NO: 7), were used to amplify two DNA fragments of 991 and 786 bp, respectively. These two DNA fragments were purified, combined and reamplified by PCR using the primers A5 and A3AP. Pfu polymerase (Strategene) was used in the PCR reaction to increase the fidelity. To increase the yield of the combined fragment, five cycles of 94°C for 2 min, 50°C for 1 min and 72°C for 2 min were run without adding any primers in the PCR cocktails. Subsequently, the primer pair A5 and A3AP was added into the PCR mixtures, and the amplification was performed following the program of 94°C for 3 min, 30 cycles of 94°C for 40 sec, 60°C for 40 sec, 72°C for 2 min 30 sec, and finally 72°C for 10 min. After amplification, the PCR product of 1756 bp were then digested with EcoR I and Sal I. The resulting fragment of 1726 bp was cloned into the EcoR I / Sal I site of FA-pBssK to substitute the EcoR I/Sal I insert of the plasmid FA-pBssK to obtain the plasmid NT-FA-pBssK. The mutated sequences were confirmed by cycling PCR sequencing. The plasmid NT-FA-pBssK contains full-length cDNA of segment A of HK46 strain but has substitutions at nucleotide positions 966 (G®A) and 981 (G®A). To construct plasmid KS-FA-pBssK, two primer pairs, A5 (SEQ ID NO: 3) and KSA (SEQ ID NO: 13), KSS (SEQ ID NO: 14) and A3AP(SEQ ID NO: 7), were used to amplify the DNA fragments of 902 and 874 bp respectively, using FA-pBssK as template. A PCR product of 1756 bp was then amplified from the two PCR products as described above, which was subsequently cloned into the EcoRI/Sall site of the plasmid FA-pBssK. The mutated sequences were confirmed by cycling PCR sequencing using sequencing primers. The new plasmid KS-FA-pBssK contains full- length cDNA of segment A of HK46 but has substitutions at nucleotide positions 876 (C®A) and 891 (C®A).
Example 8
Mutation of the Serine-rich Heptapeptide Region on VP2 Using Altered Sites II Mammalian Mutagenesis System
Carrying the genes for ampicillin / chloramphenicol resistance and CMV I.E. enhancer / promotor, the pALTER-MAX vector may be used for mutagenesis and expression of genes in eukaryotic cells (Promega) . In this study, the segment A cDNA (SEQ ID NO:l) of strain HK46 was subcloned into the pALTER-MAX vector. The plasmid FA-pBssK was digested with EcoR I, Kpn I and Bgl I. Because there is an unique Bgl I site in the pBssK vector but not in the sequence of fragment FA, the fragment FA and pBssK can easily be separated by 1% agarose gel after the plasmids were digested with Bgl I. After digestion, the FA fragment (3269 bp) was purified and cloned into the EcoR I/Kpn I site of pALTER-MAX vector to create plasmid FA- pALTER.
To substitute serine at amino acid residue 330 with arginine, the nucleotide T at position 1127 was changed to A using Altered sites II mammalian mutagenesis system (Promega) . The single strand (ss) DNA of plasmids FA-pALTER and was produced using the methods recommended by supplier. A primer designated as 330R (5'ACTGCTAGGCTCCCTCTTGCGGA CCA3') that was phosphorylated at the 5'-end was synthesized and used for producing a mutation on plasmid FA-pALTER in which the nucleotide 1127(T) was substituted with A. Twenty microliters of annealing reaction mixture containing 0.05 pmole of ssDNA of FA-pALTER, 1.25 pmole of primer 330R, 0.25 pmole of ampicillin repair oligonucleotide, 20mM Tris- HC1 (ρH7.5), 10 mM MgCl2and 50 mM NaCl was heated to 75°C for 5 min, and then cooled slowly down to room temperature. After adding 3 ul of synthesis buffer containing 100 mM Tris-HCl (pH7.5), 5 mM dNTPs, 10 mMATP and 20 mM DTT, ten units of T4 DNA polymerase and 3 units of T4 DNA ligase were mixed with annealing reaction mixture, and the total volume of DNA synthesis reaction was adjusted to 30 ul with deionized water. Subsequently, synthesis mixture was incubated at 37°C for 2 hr to perform mutant strand synthesis and ligation. After incubation, the synthesis mixture was transformed into E coli strain ES1301 mutS competent cells by electroporation. The transformed ES1301 mutS cells were resuspended in 1 ml of LB broth without any antibiotics, and incubated at 37°C for 3 hr. By adding 0.5 ml of transformed cell culture to 4.5 ml of selective medium (LB broth with 100 ug/ml ampicillin), the transformed ES1301 mutS cells were cultured at 37°C with shaking at 250rpm overnight. Plasmid DNA was then extracted by the Wizard5 Plus Minipreps DNA purification system and then transformed into Ecoli strain JM109 competent cells. Ten colonies were screened by direct sequencing with primer A768S (SEQ ID NO: 20) using an automatic sequencer. The resulting mutant plasmid designated as R-FA-pALTER was obtained.
Example 9
Generation of Recombinant viruses by Transfection
The segment A cDNAs from plasmids FA-FA-pBssK, NT-FA- pBssK, KS-FA- pBssK and R-FA-pALTER were released by EcoR I, Kpn I and Bgl I digestion and then subcloned into a plasmid at a site downstream to a CMV promoter. To clone the fragment FB into this plasmid, the plasmid FB-pBssK was digested with three restriction enzymes EcoR I, Xba I and Pvu II, where Pvu II can only digest pBssK vector but can not digest fragment FB. Subsequently the fragment FB was purified and inserted into the EcoR 1/ Xba I site of the plamid carrying a CMV promoter. This plasmid contained the full-length cDNA copy of segment B downstream to a CMV promoter.
The CEFs were grown to 80% confluence in a 12-well tissue culture plate (Falcon) and washed once with phosphate-buffered-saline (PBS) . One ml of serum free EME medium (SF-EMEM) (Life Technologies) were added to monolayers of each well, and the cells were incubated at 37°C for 1 hr in 5% CO2 incubator. 5ul (0.2ug/ul) of both plasmids, CMV-FA and CMV-FB were then transfected to the CEF by Lipofectamine (Life Technologies), according to the manufacturer's procedures. After 5 hr of incubation at 37°C, the transfection mixture was replaced with EMEM containing 10% FBS, and the cells were further incubated at 37°C in 5% CO2 incubator for desired time intervals. The plasmids containing mutants of segment A cDNAs (KS-FA or NT-FA or R-FA) and/or CMV-FB were also transfected into CEF using the same procedure described above. After 96 hours, cytopathic effect was observed. The ability of the recombinant viruses in propagating in CEF was further tested by adding 0.1 ml vims suspension collected 96 hr posttransfection into a well of a 12 well plate containing 80% confluent CEF with 1 ml cell culture medium. The results on transfection and virus propagation are summarized in Table 3. TABLE 3. CPE on CEF momolayers transfeced by plasmids containing full-length cDNA of both segments A and B of HK46 strain or its mutants. The presence of virus was tested by passages in CEF cells.
RI R2 NT KS FA
Transfection I: + + + +
(96 hrs after transfection)
Transfection II + + + +
(96hrs after transfection)
Passage one in CEF + +
(with transfection I)
Passage two in CEF + +
RI, R2: Transfected with two independent CMV-R plasmids and CMV-FB plasmid.
NT: Transfected with CMV-NT-FA plasmid and CMV-FB plasmid.
KS: Transfeced with CMV-KS-FA plasmid and CMV-FB plasmid.
FA: Transfected with wild-type CMV-FA plasmid and CMV-FB plasmid.
Example 10
Confirmation of antigenic sequence of the recombinant virus by DNA sequencing
After virus propagation in CEF, cell debris was removed from the supernatant by centrifugation at 14,000rpm for 20min at 4°C. One ml of supernatant was loaded onto 4 ml of 40%(v/v) sucrose cushion and ultracentrifuged at 55,000g (Beckman sw55Ti rotor, 22,000rpm) for 2.5 hr at 4°C. The vims at the pellet was processed as stated in Examples 3&4 to produce first stranded cDNA. PCR reaction as stated in Example 5 was carried out to amplify the 5'cDNA segment of genome A (equivalent to FA5), which was then sequenced to confirm the mutation (Table 2). Example 11
Animal tests
The recombinant virus (HK46-NT) generated from transfection was passaged and titred in CEF. To compare its pathogenicity with its parent strains, 3.2xl05TCID50 HK46-NT and 1.6xl05EID50 HK46 were administrated to 4 weeks old SPF birds. No birds vaccinated with HK46-NT showed any symptomes nor mortality, whereas all birds challenged by the parent strain HK46 were sick and the mortality reached 60%. The weights of the bursa after chanllenge were recorded in figure 5. To test the immunologicity of HK46-NT, it was administrated to SPF chickens at 25 days old (106TCID50/bird) and antibody titre at 43 days old was measured by a commercial ELISA kit (Idexx Inc.) . The signal-to-noise (S/N) ratios were 3.41 +/- 0.68 and 1.37+/-0.13, respectively.
While the present invention has been described in a number of different exemplary embodiments, it will be understood that the principles of the invention can be extended to still further embodiments and that the embodiments illustrated here are not intended to limit the scope of the invention as set forth in the appended claims.

Claims

WHAT IS CLAIMED IS:
1. A biological pure RNA encoding SEQ ID NO:l or a fragment thereof of from 1 to 1012 amino acids long; or a biological DNA encoding to said biological pure RNA. (v. v. IBD segment A)
2. A biological pure RNA encoding SEQ ID NO: 2 or a fragment thereof of from 1 to 879 amino acids long; or a biological DNA encoding to said biological pure RNA. (v. v. IBD segment B)
3. The biological pure DNA of claim 1, consisting of SEQ ID NO:l.
4. The biological pure DNA of claim 2, consisting of SEQ ID NO:2.
5. The biological pure DNA of claim 3, consisting of one or more nucleotide substitutions generated by mutagenesis using molecular approaches.
6. The biological pure DNA of claim 4, consisting of one or more nucleotide substitutions generated by mutagenesis using molecular approaches.
7. The biological pure DNA of claim 3, consisting of one or more nucleotide substitutions resulting in point mutations at one or more amino acid residues at positions 222, 249, 254, 256, 279, 284, 294, 299 and 326 to 332.
8. A recombinant vector consisting of the biological pure DNA of claim 5.
9. A recombinant vector consisting of the biological pure DNA of claim 6.
10. A recombinant vector consisting of the biological pure DNA of claim 7.
11. The generation of recombinant infectious bursal disease virus by the co-transfection of the recombinant vectors of claims 8 and 9.
12. The generation of recombinant infectious bursal disease virus by the co-transfection of the recombinant vectors of claims 9 and 10.
13. An recombinant infectious bursal disease virus generated by the procedures described in claim 11, comprising mutations at one or more of the amino acid residues, wherein the IBDV mutant acquire the ability of propagation in chicken embryo fibroblasts, or other primary tissue culture or cell lines.
14. An recombinant infectious bursal disease vims generated by the procedures described in claim 12, comprising mutations at one or more of the amino acid residues, wherein the IBDV mutant acquire the ability of propagation in chicken embryo fibroblasts, or other primary tissue culture or cell lines.
15. An recombinant infectious bursal disease vims generated by the procedures described in claim 11, comprising mutations at one or more of the amino acid residues at positions 249, 254, 279, 284, 326 to 332 of the SEQ ID NO:l, wherein the IBDV mutant acquires the ability of propagation in chicken embryo fibroblasts, or other primary tissue culture or cell lines.
16. An recombinant infectious bursal disease virus generated by the procedures described in claim 12, comprising mutations at one or more of the amino acid residues at positions 249, 254, 279, 284, 326 to 332 of the SEQ ID NO:l, wherein the IBDV mutant acquires the ability of propagation in chicken embryo fibroblasts, or other primary tissue culture or cell lines.
17. An IBDV mutant according to claim 13, wherein the mutation is selected from the group consisting of a deletion, an insertion, and a deletion and insertion and point mutations.
18. An IBDV mutant according to claim 15, wherein the mutation is selected from the group consisting of a deletion, an insertion, and a deletion and insertion and point mutations.
19. An IBDV mutant according to claim 13, wherein at least one heterologous nucleic acid fragment from a virus or a pathogen encoding a polypeptide is inserted into the VP2 locus.
20. An IBDV mutant according to claim 15, wherein at least one heterologous nucleic acid fragment from a vims or a pathogen encoding a polypeptide is inserted into the VP2 locus.
5 21. A cell infected with an recombinant IBDV mutant according to claim
13.
22. A cell infected with an recombinant IBDV mutant according to claim 15.
10
23. A cell infected with an recombinant IBDV mutant according to claim 17.
24. A cell infected with an recombinant IBDV mutant according to claim 15 19.
20
25
30
35
PCT/IB1999/001604 1998-09-01 1999-09-01 Generation of recombinant infectious bursal disease viruses by reverse genetics technology and the use of the recombinant viruses as attenuated vaccines WO2000012677A2 (en)

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EP1170302A1 (en) * 2000-07-07 2002-01-09 Akzo Nobel N.V. An infectious bursal disease virus mutant and vaccines
US6485940B2 (en) 2000-07-07 2002-11-26 Akzo Nobel N.V. Broad spectrum infectious bursal disease virus vaccine
EP1689858A2 (en) * 2003-11-13 2006-08-16 University Of Georgia Research Foundation, Inc. Methods of characterizing infectious bursal disease virus
JP2007526215A (en) * 2003-05-05 2007-09-13 ダウ アグロサイエンス リミテッド ライアビリティー カンパニー Stable immunoprophylactic and therapeutic compositions derived from transgenic plant cells and methods for their production
CN102258777A (en) * 2011-06-30 2011-11-30 河南农业大学禽病研究所 Method for preparing inactivated vaccine and combined vaccine by breeding infectious bursal disease virus (IBDV) by chicken embryo source cell line
EP3620504A1 (en) 2018-09-07 2020-03-11 Henkel AG & Co. KGaA Platinum colloids as bleach boosting agents
EP3620505A1 (en) 2018-09-07 2020-03-11 Henkel AG & Co. KGaA Mixed metal colloids as bleach boosting agents

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WO1991016925A1 (en) * 1990-05-04 1991-11-14 University Of Maryland At College Park Specific dna sequences related to an ibdv protein including vectors, hosts and vaccines
WO1995026196A1 (en) * 1994-03-29 1995-10-05 The University Of Maryland College Park CHIMERIC INFECTIOUS BURSAL DISEASE VIRUS cDNA CLONES, EXPRESSION PRODUCTS AND VACCINES BASED THEREON
WO1998009646A1 (en) * 1996-09-05 1998-03-12 University Of Maryland - Biotechnology Institute A method for generating birnavirus from synthetic rna transcripts

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WO1991016925A1 (en) * 1990-05-04 1991-11-14 University Of Maryland At College Park Specific dna sequences related to an ibdv protein including vectors, hosts and vaccines
WO1995026196A1 (en) * 1994-03-29 1995-10-05 The University Of Maryland College Park CHIMERIC INFECTIOUS BURSAL DISEASE VIRUS cDNA CLONES, EXPRESSION PRODUCTS AND VACCINES BASED THEREON
WO1998009646A1 (en) * 1996-09-05 1998-03-12 University Of Maryland - Biotechnology Institute A method for generating birnavirus from synthetic rna transcripts

Cited By (8)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP1170302A1 (en) * 2000-07-07 2002-01-09 Akzo Nobel N.V. An infectious bursal disease virus mutant and vaccines
US6485940B2 (en) 2000-07-07 2002-11-26 Akzo Nobel N.V. Broad spectrum infectious bursal disease virus vaccine
JP2007526215A (en) * 2003-05-05 2007-09-13 ダウ アグロサイエンス リミテッド ライアビリティー カンパニー Stable immunoprophylactic and therapeutic compositions derived from transgenic plant cells and methods for their production
EP1689858A2 (en) * 2003-11-13 2006-08-16 University Of Georgia Research Foundation, Inc. Methods of characterizing infectious bursal disease virus
EP1689858A4 (en) * 2003-11-13 2007-07-11 Univ Georgia Res Found Methods of characterizing infectious bursal disease virus
CN102258777A (en) * 2011-06-30 2011-11-30 河南农业大学禽病研究所 Method for preparing inactivated vaccine and combined vaccine by breeding infectious bursal disease virus (IBDV) by chicken embryo source cell line
EP3620504A1 (en) 2018-09-07 2020-03-11 Henkel AG & Co. KGaA Platinum colloids as bleach boosting agents
EP3620505A1 (en) 2018-09-07 2020-03-11 Henkel AG & Co. KGaA Mixed metal colloids as bleach boosting agents

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