Cloning and expression of host-protective immunogens of IBDV

This invention relates to the cloning and characterization of the infectious bursal disease virus (IBDV) genome, to the identification of cloned genes for host-protective antigens of IBDV, to the expression of cDNA inserts encoding the whole or part of host-protective antigens of IBDV in E.coli or other host cells, and to the use of the expressed antigens in the production of virus neutralizing antibodies in chickens. The invention also relates to the production of an effective sub-unit vaccine against IBDV utilizing the expressed antigens, as well as to the use of the expressed antigens in diagnostic tests, assays and the like.

This invention relates to the cloning and characterisation of the 
infectious bursal disease virus (IBDV) genome, to the identification of 
cloned genes for host-protective antigens of IBDV, to the expression of 
cDNA inserts encoding the whole or part of host-protective antigens of 
IBDV in E.coli or other host cells, and to the use of the expressed 
antigens in the production of virus neutralizing antibodies in chickens. 
The invention further relates to the production of an effective sub-unit 
vaccine against IBDV utilising the expressed antigens, as well as to the 
use of the expressed antigens in diagnostic tests, assays and the like. 
In one particularly preferred aspect, this invention relates to a method 
for the use of recombinant DNA techniques in the production of "correctly" 
processed antigens of IBDV. The production of such "correctly" processed 
antigens is of particular importance in ensuring, for example, that these 
antigens may be effectively used as vaccine components for the production 
of neutralising and protective antibodies. 
The polypeptides of an Australian strain (002-73) of IBDV have recently 
been characterised. In prior International Patent Specification No. 
PCT/AU84/00256, it is disclosed that the 32 Kd structural protein is a 
major immunogen of IBDV, and produces antibodies in chickens that 
neutralize the virus in vitro and protect the chickens from IBDV 
infection. 
Further work has now led to the characterisation and molecular cloning of 
the genome of IBDV strain 002-73, and this genome has been shown to 
consist of two segments of double-stranded (ds) RNA which are 
approximately 3400 b.p. (MW 2.06.times.10.sup.6) and 2900 b.p. (MW 
1.76.times.10.sup.6) long, respectively. In vitro translation studies show 
that the large RNA segment codes for three major structural proteins, 
including the 32 Kd host-protective antigen previously identified. A novel 
method for the cloning of long double-stranded RNA molecules has been 
developed and used to clone the entire genome of IBDV. Molecular 
hybridization and expression studies involving cloned cDNA inserts have 
allowed the identification of the region of the IBDV genome that codes for 
the 32 Kd host-protective antigen. Cloned genes encoding the entire or 
part of this antigen have been sequenced and expressed in E.coli. In 
addition the immunogenicity in chickens of the expressed polypeptides has 
been tested, as well as their ability to produce virus neutralising 
antibodies. 
Initial work in this regard has lead to the production of the 32 Kd 
host-protective antigen of IBDV in the form of fusion proteins. The test 
results show that the fusion proteins are highly immunogenic and produce 
antibodies that recognise denatured 32 Kd protein. These antibodies, 
however, have weak ELISA and virus neutralising titres. The fusion 
proteins react strongly with MAb 17-80 (monoclonal antibody that 
recognises denatured 32 Kd viral protein), but weakly with the virus 
neutralising MAb 17-82. These results suggest that these genetically 
engineered fusion proteins may not have the correct three-dimensional 
structure necessary for the production of virus neutralising and 
protective antibodies, or that other viral proteins possess epitopes or 
are important in the formation of epitopes involved in the neutralisation 
of whole virus. 
Further work has shown that a monoclonal antibody (MAb 17-82), that 
neutralises the infecivity of IBDV, recognises an epitope encoded within 
the gene for the 52 Kd precursor protein; a protein processed into the 41 
Kd and 37 Kd structural proteins of IBDV. The expressed polypeptide from 
the 52 Kd region that reacts with MAb 17-82, does not contain epitopes 
recognised by the monoclonal antibody specific for the 32 Kd structural 
protein (MAb 17-80). 
According to one aspect of the present invention, there is provided a 
recombinant DNA molecule comprising a nucleotide sequence substantially 
corresponding to all or a portion of IBDV RNA, particularly the IBDV RNA 
segment of approximately 3400 b.p. Preferably, the nucleotide sequence 
codes for all or part of at least one structural protein of IBDV. In one 
particular aspect of the invention, the DNA molecule is capable of being 
expressed as a polypeptide displaying antigenicity substantially 
corresponding to the 32 Kd or 41/37 Kd structural protein of IBDV. 
By way of exemplification of this aspect of the invention, the nucleotide 
sequence may be characterised by at least a portion thereof having the 
base sequence substantially as shown in FIG. 10 hereinafter or one or more 
portions of said base sequence. 
The complete nucleotide sequence of the large segment of the IBDV genome 
and the amino acid sequence derived from it are shown in FIG. 10. 
Translation, in vitro, of the IBDV large segment genomic RNA in rabbit 
reticulocyte and wheat germ cell-free systems has led to the synthesis of 
discrete polypeptides identical in size to the viral proteins although 
there is only one stop codon at the 3' end of the large segment of the 
IBDV genome. While the rabbit reticulocyte and wheat germ cell-free 
systems may contain protease(s) which help to process viral polyproteins, 
it would appear more likely that one of the polypeptides encoded by the 
IBDV genome is a specific protease. Further work in this regard has 
enabled the production of correctly processed 32 Kd or 41/37 Kd protein of 
IBDV instead of the fused proteins described above. 
Accordingly, in a particularly preferred embodiment of this invention, 
there is provided a recombinant DNA molecule comprising a nucleotide 
sequence coding for all or part of the 32 Kd structural protein or the 52 
Kd precursor protein of IBDV, together with further portion(s) of the 3400 
b.p. segment coding for further polypeptides or proteins to correctly 
process said 32 Kd or 41/37 Kd structural protein. Expression of this 
molecule leads to the expression of the 32 Kd or 41/37 Kd structural 
protein as a correctly processed protein. Such a molecule may encode both 
the 32 Kd structural protein as well as additional polypeptides or 
proteins, including proteases, required to correctly process the 32 Kd 
structural protein. 
It will be appreciated that the nucleotide sequence of this aspect of the 
invention may be obtained from natural, synthetic or semi-synthetic 
sources, or by manipulation of the natural material; furthermore, this 
nucleotide sequence may be a naturally-occurring sequence, or it may be 
related by mutation, including single or multiple base substitutions, 
deletions, insertions and inversions, to such a naturally-occurring 
sequence, provided always that the DNA molecule comprising such a sequence 
is capable of being expressed as a polypeptide displaying the antigenicity 
of one or more structural proteins of IBDV. 
The nucleotide sequence may have expression control sequences positioned 
adjacent to it, such control sequences being derived either from IBDV 
nucleic acid or from a heterologous source. 
This invention also provides a recombinant DNA molecule comprising an 
expression control sequence having promoter sequences and initiator 
sequences, and a nucleotide sequence coding for all or part of at least 
one structural protein of IBDV. 
In yet another aspect, the invention provides a recombinant DNA cloning 
vehicle capable of expressing all or part of at least one structural 
protein of IBDV, comprising an expression control sequence having promotor 
sequences and initiator sequences, and a nucleotide sequence coding for 
all or part of at least one structural protein of IBDV. 
In a further aspect, there is provided a host cell containing a recombinant 
DNA cloning vehicle and/or a recombinant DNA molecule as described above. 
In yet further aspects, there are provided polypeptides displaying IBDV 
antigenicity which can be produced by a host cell transformed or infected 
with a recombinant DNA cloning vehicle as described above. Such expressed 
polypeptides may comprise all or part of at least one structural protein 
of IBDV as derived from the base sequence substantially as shown in FIG. 
10 or one or more portion(s) of the said sequence. Such polypeptides can 
be isolated from the host cell, and if necessary purified to provide the 
polypeptide substantially free of host cell or other proteins. Where the 
expressed polypeptides are in the form of a fused polypeptide, they may be 
cleaved to remove the "foreign" peptide portion. 
It will be appreciated that such expressed polypeptides as described above 
may be constructed by permutation and combinations of portions of the 
nucleotide sequence presented in FIG. 10. 
The present invention also extends to synthetic peptides or polypeptides 
displaying the antigenicity of all or a portion of at least one structural 
protein of IBDV, particularly the 32 Kd and/or 41/37 Kd structural 
proteins. 
As used herein, the term "synthetic" means that the peptides or 
polypeptides have been produced by chemical and/or molecular biological 
means, such as by means of chemical synthesis or by recombinant DNA 
techniques leading to biological synthesis. Such polypeptides can, of 
course, be obtained by direct expression by a host-cell of a correctly 
processed and folded protein, or by cleavage of a fused polypeptid (an 
IBDV polypeptide fused to a non-IBDV polypeptide) produced by a host cell 
and separation of the desired Polypeptide from additional polypeptide 
coded for by the DNA of the host cell or cloning vehicle by methods well 
known in the art. Alternatively, once the amino acid sequence of the 
desired polypeptide has been established, for example, by determination of 
the nucleotide sequence coding for the desired polypeptide, the 
polypeptide may be produced synthetically, for example by the well-known 
Merrifield solid-phase synthesis procedure [Marglin and Merrifield, 
(1970)]. 
It will be appreciated that polypeptides displaying antigenicity 
characteristic of structural proteins of IBDV will have utility in 
serological diagnosis, and in the preparation of single or multivalent 
vaccines against IBDV by methods well known in the art of vaccine 
manufacture. Further details of such vaccines, and of methods of use 
thereof, as well as of quantitative and qualitative assays, are disclosed 
in International Patent Specification No. PCT/AU84/00256.

MATERIALS AND METHODS 
Materials and their sources are Klenow fragment of DNA polymerase 1, S1 
nuclease, DNase 1, and RNase A (Boehringer); rabbit reticulocyte lysate, 
[.alpha.-.sup.32 P]ATP, [.gamma.-.sup.32 P] ATP, [.sup.35 S]methionine, 
and PstI (Amerisham); RNase-free sucrose, DNA polymerase 1, and wheat germ 
lysate (Bethesda Research Laboratories); RNase-free Pronase (Calbiochem); 
agarose and lysozyme (Sigma); low melting point agarose and SDS (Bio-Rad), 
diethylpyrocarbonate and acridine orange (Merck); nitrocellulose filters 
and NA45 membrane filters (Schleicher and Schuell); reverse transcriptase 
(RTase) (Life Sciences Inc., St. Petersburg, Fla.); terminal transferase 
(Ratliffe, Los Alamos, N. Mex.); RNasin (Promega Biotech, Madison, Wis.). 
Random primers were prepared from sheep DNA by the method described by 
Taylor et al. (1976). UK bovine rotavirus ds RNA was prepared by Dr. M. 
Dyall-Smith. Virus: IBDV strain 002-73 was first reported by Firth (1974) 
in commercial chicken flocks in Australia and confirmed as IBDV at the 
Central Veterinary Laboratory, Weybridge, U.K. The virus was routinely 
passaged in 4- to 6-week old SPF White Leghorn chickens, isolated from 
bursas 3 days after infection, and purified by successive fractionations 
on sucrose and CsCl gradients. 
Isolation and Purification of IBDV RNA 
Homogenates of fresh infected bursas were spun at 17,000 g for 15 minutes 
at 0.degree.. The clear supernatant was layered on top of 2-ml sucrose 
cushions (40%) and the virus particles were pelleted through the cushions 
in a Beckman SW40 rotor at 22,000 rpm at 2.degree. for 2.5 hours. The 
pellets were suspended in 10 mM Tris. pH 7.5, 10 mM NaCl, 10 mM EDTA, 0.2% 
SDS, and 0.1% diethylpyrocarbonate and diaested with RNase-free Pronase (1 
mg/ml) for 1 hour at 37.degree., The solution was extracted with phenol 
and chloroform (1:1) and the RNA in the aqueous phase recovered by 
precipitation with ethanol. The ds viral RNA was purified from the chicken 
cellular RNA by differential salt precipitation (Diaz-Ruiz and Kaper, 
1978). 
Individual RNA or DNA segments were isolated from agarose gels by 
electrophoresis onto NA45 membrane filters followed by elution in 1M NaCl 
and 0.05 M arginine at 70.degree.. Alternatively, RNA bands were excised 
from low melting point agarose slab gel and melted (70.degree.) in 5 mol. 
of low salt buffer containing 0.5% SDS. The solution was extracted with 
phenol and the RNA in the aqueous phase precipitated with ethanol. 
Hybridization Probes 
IBDV RNA was labeled with [.gamma.-.sup.32 P] following mild alkaline 
digestion (Goldbach et al. 1978). cDNA probes were prepared from denatured 
ds RNA using random primers to initiate cDNA synthesis in the presence of 
RTase. The RNA template was then destroyed by digestion with NaOH. Nick 
translation of cloned DNA fragments was carried out essentially as 
described by Rigby et al. (1977). All radioactively labeled probes were 
purified from unreacted isotopes by precipitation (3.times.) from 2 M 
ammonium acetate and isopropanol at room temperature. 
Translation of IBDV RNA in vitro 
IBDV RNA (1-2 .mu.g) in 3 .mu.l of 10 mM phosphate, pH 6.8 was heated at 
100.degree. for 2 minutes and snap chilled in dry ice/ethanol. 
Methylmercuric hydroxide (1 .mu.l of 40 mM) was then added and the mixture 
left at room temperature for 10 minutes. .beta.-Mercaptoethanol (1 .mu.l 
of 700 mM) and 1 .mu.l of RNasin (25 units) were added and the solution 
was incubated for a further 5 minutes at room temperature. Aliquots (1 
.mu.l) were transferred to tubes containing 5 .mu.Ci of [.sup.35 
S]methionine (dried down) and 30 .mu.l of rabbit reticulocyte lysate and 
the solution was incubated at 30.degree. for 1 hour. The reaction mixture 
was reacted in succession with chicken antiserum, rabbit anti-chicken IgG, 
and protein A-Sepharose (Pharmacia). The protein 
A-Sepharose-antigen-antibody complex was washed extensively with 
phosphate-buffered saline containing 0.1% NP-40 and then boiled in buffer 
containing 2% SDS. The protein A-Sepharose was spun down and the 
translated proteins in the supernate were analysed by polyacrylamide gel 
electrophoresis (12.5% gel). The gel was then treated with AMPLIFY 
(Amersham), dried and exposed to Fuji RX film with intensifying screen 
(Dupont Cronex Lightening Plus AA). 
Synthesis of ds cDNA from ds RNA. 
IBDV RNA (5 .mu.g) in 9 .mu.; pf 5 mM phosphate buffer, pH 6.8, was heated 
at 1000 for 2 minutes and then snap frozen. After the RNA had thawed 1 
.mu.l of 100 mM methylmercuric hydroxide was added and the mixture left at 
room temperature for 10 minutes. Two microliters of RNasin (50 units) and 
4 .mu.l of 700 mM .beta.-mercaptoethanol were then added and the mixture 
was left at room temperature for a further 5 minutes. Ten microliters of 
random primers (50 .mu.g), which had been separately denatured by boiling 
and snap chilling, was then added to the mixture to prime cDNA synthesis. 
The mixture (100 .mu.l final volume) contained RTase (50 units) and other 
reactants required for cDNA synthesis. Following incubation at 42.degree. 
for 2 hours the RNA template was destroyed by digestion with NaOH, and the 
cDNA purified by gel filtration. Complementary cDNA fragments were 
annealed in 0.3 M NaCl at 65.degree. for 2 hours following initial heating 
at 90.degree. for 3 minutes. The solution was then allowed to cool 
gradually to room temperature over 1 hour. The annealed cDNA segments were 
repaired and chains. extended with DNA polymerase 1. The ds cDNA chains 
were further extended with RTase, treated with DNA ligase and S1 nuclease, 
and finally purified by gel filtration. 
Cloning of IBDV ds cDNA 
The ds cDNA was C tailed with terminal transferase, annealed to G-tailed 
Pst-cut pBR322 (New England Nuclear), and cloned in Escherichia coli RR1 
cells. The recombinant colonies were hybridized with radioactive probes 
made from IBDV RNA segments and autoradiographed. Biological containment 
levels specified by the Australian Recombinant DNA Monitoring Committee 
were used. 
Isolation of Plasmid DNA 
Plasmid DNA was isolated essentially by the Ish-Horowicz and Burke (1981) 
modification of the method described by Birnboim and Doly (1979) with the 
following further modifications. RNase digestion was carried out 
concommitantly with lysozyme treatment, and the plasmid DNA was purified 
from RNA breakdown products by precipitation from polyethylene glycol 
(6.5% PEG, 0.8 M NaCl, 0.degree., 1 hour). 
Colony Hybridization 
The recombinant colonies were hybridized with radioactive probes as 
described by Grunstein and Hogness (1975). The solution used for 
prehybridization and hybridization contained 5.times.Denhardt's solution, 
10 mM HEPES (pH 7.0), 0.1% SDS, 3.times.SSC, 10 .mu.g/ml E. coli tRNA, and 
18 .mu.g /ml sonicated and denatured herring sperm DNA. The filters were 
prehybridized at 65.degree. for 2 hours, and then hybridized with 
radioactive probes for 16-20 hours at 65.degree.. The filters were washed 
4.times.30 minutes with 0.5.times.SSC,0.1% SDS at 65.degree., and then 
autoradiographed using Fuji RX film and intensifying screen. 
Results 
1. Isolation and purification of RNA. The RNA isolation procedure described 
above is simple and rapid, and results in a high yield of good quality 
RNA. A low-speed spin of the bursal homogenate followed by sedimentation 
of the virus particles through a 40% sucrose cushion resulted in the 
removal of virtually all the cellular DNA and over 90% of the cellular 
RNA. Following digestion with Pronase and extraction with phenol and 
chloroform, the total RNA was fractionated by differential salt 
precipitation (Diaz-Ruiz and Kaper, 1978). The cellular ss RNA was 
precipitated from 2 M LiCl and the viral ds RNA in the supernatant could 
be further purified from low MW contaminants and any contaminating DNA by 
precipitation from 4M LiCl. 
2. Physico-chemical characterization of the viral genome. To determine 
whether the RNA of the Australian isolate IBDV 002-73 is double stranded, 
viral RNA which had not been totally purified from single-stranded 
cellular RNA was electrophoresed under nondenaturing conditions, stained 
with acridine orange, and the nucleic acid bands were visualized on a uv 
transilluminator. The DNA standards, ds UK bovine rotavirus RNA segments, 
and the two segments of IBDV RNA in the upper part of the gel appeared as 
bright green bands as expected of ds nucleic acids (Lerman, 1963), while 
the single-stranded cellular RNA near the bottom of the gel appeared 
bright red (Blake and Peacocke, 1968). Moreover, under RNase A-digestion 
conditions that completely destroy 28 S and 18 S rRNA the two segments of 
IBDV RNA remained intact when electrophoresed under nondenaturing 
conditions. Thus, the genome of IBDV strain 002-73 consists of two 
segments of ds RNA as has been shown to be the case for strain Cu-1 
(Muller et al, 1979) and a strain isolated at the Central Veterinary 
Laboratories, Weybridge, U.K. (Todd and McNulty, 1979). 
When electrophoresed under nondenaturing conditions the two segments of 
IBDV RNA appear to be 3825 and 3400 bp, respectively, when compared with 
DNA standards. These values correspond to MW of 2.52.times.10.sup.6 and 
2.2.times.10.sup.6, respectively, for the two segments. When compared 
under nondenaturing conditions with ds RNA segments of UK bovine 
rotavirus, the sizes of which were obtained by electron microscopy (Rixon 
et al, 1984), the two segments of IBDV ds RNA appear to be about 3400 bp 
(MW 2.06.times.10.sup.6) and 2900 bp (MW 1.76.times.10.sup.6), 
respectively. 
3. Translation, in vitro, of IBDV RNA. The ds RNA has to be extensively 
denatured for any in vitro protein synthesis to take place. Heating at 
100.degree. followed by snap chilling in dry ice/ethanol was not 
sufficient, and heating the RNA in 90% dimethylsulfoxide gave inconsistent 
results. The best results were obtained when the heat-denatured RNA was 
further denatured in 10 mM methylmercuric hydroxide. Even after these 
treatments the amount of radioactivity incorporated into TCA-precipitable 
material was only between 10 and 20% of that obtained when translating 
similar amounts of rotovirus ss RNA or globin mRNA. 
Immunoprecipitation of the translation product shows that total IBDV RNA 
codes for six polypeptides of Ca. MW 90 Kd, 52 Kd, 41 Kd, 32 Kd, 18 Kd, 
and 16 Kd (FIG. 1 (ii). The larger RNA segment, purified by gel 
electrophoresis, produces all the translation products except the 90-Kd 
polypeptide (FIG. 1 (iii). When the smaller RNA segment, which we have not 
been able to completely purify by gel electrophoresis, is translated in 
vitro traces of all the translation products are seen but the 90-Kd 
protein is consistently the most prominent one (FIG. 1 (iv). Since this 
90-Kd protein is consistently absent among the translation products of the 
larger RNA segment it would appear that all the IBDV proteins except the 
90-Kd protein are encoded by the larger RNA segment. 
4. Molecular cloning of IBDV ds DNA. To overcome problems encountered in 
the synthesis of cDNA covering the entire IBDV genome an alternative 
method was developed for the cloning of long ds RNA molecules. The ds RNA 
was denatured in methylmercuric hydroxide and random primers were used to 
initiate cDNA synthesis on both strands of the RNA simultaneously in the 
presence of RTase. The RNA was then destroyed and complementary cDNA 
strands were allowed to reanneal. DNA polymerase 1 was used to repair and 
extend the cDNA chains, which were then extended further with RTase. The 
ds cDNA molecules were then treated with DNA ligase followed by S1 
nuclease. The ds cDNA molecules were C tailed and annealed to G-tailed 
pBR322, and used to transform E.coli RR1 cells. 
Recombinant colonies were hybridized with radioactive probes made from the 
large or small segment of IBDV RNA, and 200 colonies positive to each of 
the probes were randomly selected for further characterization. The 
positive colonies were screened a for plasmid size by electrophoresing 
colony lysates on agarose mini-gels. A few of these colonies, positive to 
the large segment probes, were grown up in 5 ml L broth for plasmid DNA 
isolation. The plasmids were digested with PstI and the sizes of the 
inserts determined by electrophoresis. These inserts of defined size were 
"nick translated" and used separately to probe identical sets of positive 
colonies. Inserts from clones D6 (1100 bp), L6 (1900 bp), and M7 (450 bp) 
hybridized with three basically different sets of colonies. Inserts from 
clone G2 (1600 bp) hybridized to colonies which previously hybridized 
either with D6 or L6 probe but not with both. Similarly, a N9 insert (950 
bp) hybridized with colonies which were positive either to the L6 or M7 
probes but not to both. From the sizes of the insert and the extent and 
ability to cross-hybridize with colonies positive to the large RNA segment 
it was possible to construct a tentative map to show that overlapping cDNA 
fragments covering the entire large RNA segment had been cloned (FIG. 2), 
and the relative positions of all the positive colonies could be 
determined on this map. 
The following detailed description relates to the expression in E.coli of 
cDNA fragments encoding the gene for the host-protective antigen of IBDV. 
In the accompanying diagrams: 
FIGS. 3A-3C show some E.coli colonies expressing proteins positive to a 
monoclonal antibody (Mab 17-80) that reacts with denatured 32 Kd protein 
of IBDV. 
FIGS. 4A and 2B show proteins from E.coli colonies subjected to 
electrophoresis and (a) stained with Coomassie Blue, or (b) Western 
blotted and reacted with MAb 17-80. Arrows 1 and 2 indicate the positions 
of fusion protein and .beta.-galactosidase, respectively. Samples are (i) 
HB 101 cells, (ii) HB 101 with pUR 290, (iii)-(viii) some recombinant 
clones that were identified as possible positives by reaction with MAb 
17-80 (FIG. 3). 
FIG. 5 shows the position of the insert from clone D6 on the large segment 
of IBDV genome; the restriction map of the inserts from clones D6 and D1. 
FIG. 6 shows the determination of optimum conditions for expression of 
fusion proteins. Cells were grown to an O.D..sub.660 of 0.2, (i) then 
grown further with or without induction with 1.5 mM IPTG, (ii) 1.5 hr, 
(iii) 1.5 hr+IPTG, (iv) 3.0 hr, (v) 3.0 hr+IPTG, (vi) 4.0 hr, (vii) 4.0 
hr+IPTG. Samples were electrophoresed and the gel was stained with 
Coomassie blue. The arrow indicates the position of the fusion protein. 
FIG. 7 shows affinity purification of fusion protein from clone D1. (i) 
Total E.coli protein; (ii)-(vii) fractions eluted from column. 
FIGS. 8A-C show affinity purified proteins from clones D1 and D6, subjected 
to electrophoresis and stained with Coomassie blue (a), reacted with 
anti-.beta.-galactosidase (b), and reacted with anti-32 Kd-monoclonal 
antibody (c). 
FIG. 9 shows Western blot analysis of sera from unprimed (1.degree.) or 
primed (2.degree.) chickens injected with fusion proteins from clones D1 
or D6 in Freund's adjuvant. Sera obtained prior to vaccination (0), 3 
weeks after injection of fusion proteins (3), or 4 weeks after a second 
injection of fusion proteins (7). 
Materials and Methods 
The materials and their sources are: DNase 1, lysozyme, agarose, BSA, 
isopropyl .beta.-D-thiogalactoside (1 PTG) and 1-ethyl-3 
(3-dimethylaminopropyl) carbodtmide (Sigma): goat anti-mouse IgG horse 
radish peroxidase conjugate (GAM HRP), goat anti-rabbit IgG horse radish 
peroxidate conjugate (GAR HRP), and HRP colour developing reagent 
(BioRad): .alpha.[.sup.32 P] dATP, [.sup.125 I] Protein A and Pst 1 
(amersham); nitrocellulose filters and MA45 membrane filters (Schleicher 
and Schuell); CH-Sepharose 4B (Pharmacia): DNA polymerase (Boehringer); 
rabbit anti-mouse IgG (Dako immunoglobulins (Denmark). Monoclonal 
antibodies against IBDV were produced and characterized as described 
below. 
IBDV strain 002-73 was grown and isolated as described earlier. 
Colony and Southern blot hybridization, isolation of plasmid DNA, 
production of hybridization probes, agarose gel electrophoresis, 
polyacrylamide gel electrophoresis (Laemli) and autoradiography were 
performed as described earlier. 
Immunoassay of Expressed Proteins in Recombinant Colonies 
Recombinant colonies were grown (37.degree.) on nitrocellulose filters on 
LB plates containing 30 .mu.g/ml ampicillin. All subsequent steps were 
carried out at room temperature. The nitrocellulose filters were placed in 
a chloroform atmosphere on Whatman No. 3 paper saturated with 1% SDS for 
30 minutes to 1 hour. The filters were rinsed with 50 mM Tris-HCl (pH 
7.5), 150 mM NaCl (TBS) to remove cell debris, and then incubated for 1 
hour with shaking in TBS containing 3% BSA, 5 mM MgCl.sub.2, 1 .mu.g/ml 
Dnase and 40 .mu.g/ml lysozyme. This was followed by incubation for 1 hour 
in supernatant from monoclonal antibodies. The filters were then washed 
for 10 minutes in TBS, 10 minutes in TBS-0.1% NP40 and finally for 10 
minutes in TBS. Sometimes the filters were reacted with a second antibody 
(rabbit anti-mouse IgG) in TBS containing 3% BSA, and washed as described 
earlier. Initial experiments the recombinant colonies expressing the 
desired protein were identified by using [.sup.125 I] Protein A. After 
reactions with the antibodies the filters were incubated with [.sup.125 I] 
Protein A in TBS containing 3% BSA. The filters were then washed for 90 
minutes in 50 mM Tris-Hcl (pH 7.5), 1M NaCl, 5 mM EDTA, 0.25% gelatin, 
0.4% Sarkosyl, and autoradiographed as described earlier. Tn later 
experiments the filters after reaction with monoclonal antibody and 
washing were reacted with goat anti-mouse IgG horse radish peroxidase 
conjugate or with goat anti-rabbit IgG horse radish peroxidase conjugate 
(when amplified with a second antibody) in TBS-3% BSA for 1 hour. The 
filters were then washed for 20 minutes in phosphate buffered saline, 
followed by colour development using the HRP colour developing reagent as 
described by BioRad. 
Assay of Small Amounts of Proteins Isolated from E.coli Cells 
E.coli cultures (0.8 ml in Eppendorf tubes) were grown in L broth 
containing ampicillin for 1-2 hours, induced with IPTG, if required, and 
the cells collected by centrifugation. 
If the proteins were to be analyzed by polyacrylamide gel electrophoresis, 
the cell pellet was suspended directly in the loading buffer containing 60 
mM Tris-Hcl (pH 7.5), 2% SDS, 10% glycerol, 5% .beta.-mercaptoethanol, 
0.001% bromophenol blue, and boiled for two minutes. 50 .mu.l aliquots 
were loaded in duplicate on two gels. Proteins on one gel were stained 
with Coomassie blue, and proteins on the duplicate gel transferred to 
nitrocellulose filter. 
For quick immunoassays of the isolated protein, the cell pellets were 
suspended in 300.mu.l TBS buffer containing 40 .mu.g/ml lysozyme 
(0.degree., 15 minutes) and then SDS was added to 1% and the solution left 
at room temperature for 30 minutes. Alternatively, the cell pellet was 
suspended in 300 .mu.l TBS buffer and sonicated. In either case, cell 
debris were removed by centifigation, and 100 .mu.l of the supernatant 
blotted onto nitrocellulose filter using Schleicher and Schuell Manifold 
apparatus. The filter was then immunoassayed as described earlier for 
recombinant colonies. 
Western Blotting 
Proteins electrophoresed on acrylamide gels were transferred to NC filters 
with Bio-Rad Transblot apparatus using buffers and protocol described by 
Bio-Rad. Proteins of interest were detected by immunoassaying the filter 
as described earlier. 
Purification of the Expressed Fusion Protein 
The fusion protein was purified by affinity chromatography (Ullmann, 1984). 
Vaccination of Chicken with Fusion Proteins 
Preparations of affinity purified fusion proteins D1 and D6 were emulsified 
in an equal volume of Freund's complete adjuvant and 1 ml injected 
intramuscularly into a series of adult White Leghorn chickens. The 
vaccines were injected into both specific pathogen free (SPF) chickens and 
chickens that had previously (&gt;8 weeks) been sensitized by inoculation 
with live IBDV. The chickens were revaccinated three weeks later with the 
respective fusion proteins emulsified in Freund's incomplete adjuvant and 
bled at weekly intervals throughout. 
Results and Discussion 
1. Subcloning of cDNA Inserts into pUR Vectors: 
The large segment of IBDV RNA encodes three major structural proteins 
including the 32 Kd host-protective antigen. cDNA inserts hybridizable to 
the large segment of IBDV RNA were recovered from the cDNA library by 
digestion of the "mixed" plasmids with Pst 1, and the "mixed" inserts were 
subcloned into the Pst 1 site of pUR expression vectors 290, 291 and 292, 
(Ruther and Muller-Hill, 1983) and these were used to transform E.coli 
HB101 cells. These three vectors together contain restriction sites in all 
three frames at the 3' end of the lacZ gene. Insertion of cDNA in the 
proper cloning site leads to a fusion protein of active 
.beta.-galactosidase and the peptide encoded by the forein cDNA. 
2. Identification of Colonies Expressing the 32 Kd Polypeptide or Parts of 
It 
Recombinant colonies containing cDNA inserts hybridizable to the large 
segment of IBDV RNA were grown on nitrocellulose filters on LB plates 
containing ampicillin (30 .mu.g/ml). The colonies were induced with 
isopropyl .beta.-D-thiogalactopyranoside (IPTG) and then lysed by placing 
the filters on Whatman No. 3 paper soaked in 1% SDS in a 
chloroform-saturated atmosphere. After blocking with BSA, the filters were 
reacted with monoclonal antibodies that recognize the 32 Kd polypeptide on 
Western blots (MAbs17-80). The filters were then reacted with rabbit anti 
mouse IgG followed by [.sup.125 I] Protein A and autoradiographed. A 
number of possible positive clones expressing proteins that react with 
monoclonal antibody specific to the 32 Kd structural proteins could be 
seen on the autoradiograph (FIGS. 3A-3C). The protocol was modified for 
later experiments. After incubation with the monoclonal antibody the 
filter was reacted with goat anti-Mouse IgG Horse Radish Peroxidase 
(BioRad) and subjected to colour development. 
A total of 20 possible positives were selected for further 
characterization. These colonies were spread on LB plates and resultant 
individual colonies were reprobed with monoclonal antibodies specific for 
the denatured 32 Kd protein. Only three of the original possible positives 
expressed polypeptides that reacted with the monoclonal antibody. 
3. Characterization of the Expressed Proteins 
The expressed proteins were characterized by polyacrylamide gel 
electrophoresis and Western blotting. The cells grown in Eppendorf tubes 
in L broth were spun down and boiled in 2% SDS for 2 minutes and loaded in 
duplicate on two separate gels. After electrophoresis one gel was stained 
with Coomassie blue, and proteins from the other gel were electroblotted 
onto nitrocellulose filter and probed with monoclonal antibody specific 
for the 32 Kd polypeptide. 
Examination of the stained gel showed no prominent polypeptide band larger 
than .beta.-galactosidase (FIG. 4a), but the Western blot of the duplicate 
samples showed very prominant polypeptide bands larger than 
.beta.-galactosidase (FIG. 4b). The expressed fusion proteins from all the 
positive clones were of the same size, but some clones produced more of 
the expressed proteins than others, and this allowed us to identify clones 
that grew faster and expressed more of the fusion protein. 
Identification of the Region of the IBDV Genome that Codes for the 32 Kd 
Host-protective Antigen 
The cDNA inserts, obtained by digestion with Pst 1, from all the positive 
clones were of identical size of about 450 b.p. These inserts were 
"nick-translated" and hybridized with a series of cDNA clones that contain 
a network of overlapping fragments covering the entire large segment of 
the IBDV genome. The inserts from the expressing clones, in every case, 
hybridized specifically with clone D6 which spans the 3' end of the large 
segment of IBDV RNA (see FIG. 5a) and other cDNA clones containing inserts 
of varying sizes from the same region of the IBDV genome. 
The inserts from the expressing clones had identical restriction maps and 
were of identical size. Therefore one clone, D1, that grew well and 
expressed the fusion protein to a high level, was selected for further 
studies. The insert of D1 is present in the vector pUR 290. Comparison of 
the restriction maps (FIG. 5) of inserts from clones D1 (450 b.p.) and D6 
(1100 b.p.) show that the D1 insert is situated towards the 3' end of the 
D6 insert. Sequencing studies (see later) confirm the location of the D1 
insert and show that it lacks the initiation and termination codons, and 
constitutes about 50% of the 32 Kd host-protective antigen. The insert of 
clone D6 on the other hand is large enough to encode the entire 32 Kd 
polypeptide. Therefore, the insert from clone D6 has been subcloned in the 
pUR vectors and clones expressing fusion proteins larger than that from 
clone D1 have been obtained. 
A clone containing the D6 insert in pUR vector 291 which grows well and 
expresses the fusion protein to a high level was selected for further 
studies. Clone D1 (450 b.p. insert) and clone D6 (1100 b.p. insert) both 
produce fusion proteins in which the C-terminal polypeptides fused to 
.beta.-galactosidase react strongly with monoclonal antibodies specific 
for the 32 Kd host-protective antigen. Clones D1 and D6 have been used for 
all subsequent studies. 
Optimum Conditions for Expression 
The optimum conditions for the expression of the fused proteins were as 
follows (FIG. 6). Cells were grown in L Broth in presence of ampicillin 
(30 .mu.g/ml) to an O.D. 660 of 0.2 and then induced with 1.5 mM 1PTC for 
4 hours. There was no significant synthesis of the fused proteins at 3 
hours after start of induction, and there was a dramatic increase in 
synthesis of the fused proteins after four hours of induction. Induction 
for longer periods or at higher cell concentrations did not result in 
higher yields of the fused protein. 
Purification of the Fusion Proteins 
The fusion proteins from clones D1 and D6 were affinity purified as 
described by Ullmann (1984). When pUR vectors are used for expression the 
.beta.-galactosidase moiety of the fusion protein is enzymatically active 
and will bind to a substrate for .beta.-galactosidase (Ullmann (1984)). 
E.coli cell lysate, in buffer containing 1.6M NaCl, was passed through an 
affinity column containing CH Sepharose coupled to 
p-aminophenyl-.beta.-D-thiodalactoside and equilibrated with the same 
buffer. Only .beta.-galactosidase or proteins fused to it will bind to the 
affinity column under these conditions. The bound protein was 
quantitatively eluted with 100 mM borate, pH10. The purification of fusion 
proteins from clone D1 is shown in FIG. 7. 
The highly purified fusion proteins (D1 and D6) and free 
.beta.-galactosidase (FIG. 8a) were recovered at a fairly high 
concentration of ca. 1-2 mg/ml, and yielded up to 20 mg of affinity 
purified protein per liter of culture. However, the fusion protein was 
subject to proteolytic degradation as evidenced by the presence of 
substantial amounts of polypeptides having electrophoretic mobilities 
similar to or faster than .beta.-galactosidase. Three bands are seen in 
affinity purified proteins from clones D6 and D1. All of the bands react 
with anti-.beta.-galactosidase IgG (FIG. 8b), while only bands 1a and 1b, 
from D6 and D1 respectively, react with the anti-32 Kd monoclonal (FIG. 
8c). However, it is mainly the C-terminal IBDV protein that is 
substantially degraded. This degradation of the IBDV expressed protein 
does not seem to be caused by the isolation procedure since cells which 
were directly boiled in SDS prior to electrophoresis also contain 
substantial amounts of free .beta.-galactosidase in addition to the intact 
fusion product. 
Reaction of the Expressed Proteins with Monoclonal Antibodies Specific for 
the 32 Kd Host-protective Antigen 
A number of monoclonal antibodies (Mab)that recognise the 32 Kd structural 
protein of IBDV and/or neutralize the virus have been produced (see 
later). These fall into two classes. One class of Mabs (e.g.17-80) reacts 
with the 32 Kd proteins on Western blots but do not neutralize the virus, 
while the other class of Mabs (e.g.17-82) neutralize the virus but do not 
significantly react with the 32 Kd protein on Western blots. This suggests 
that the virus neutralizing monoclonal antibodies recognize a 
conformational epitope. 
The fusion proteins expressed in clones D1 and D6 when boiled in SDS, react 
very strongly with monoclonal antibodies that recognize the 32 Kd 
structural proteins on Western blots. Both the expressed fusion proteins, 
when not treated with SDS, also react weakly but specifically with 
monoclonal antibodies that neutralize the virus. What is significant is 
that the IBDV polypeptide expressed in clone D1 is only 150 amino acid 
residues long and constitutes about half of the 32 Kd protein but contains 
the epitope that is recognized by the MAb that is specific for the 32 Kd 
protein on Western Blots (17-80), and at least a part of the epitope 
recognised by the MAb that neutralizes the virus (17-82). 
Immunogenicity of the Expressed Proteins 
Fusion Proteins from Clones D1 and D6 were injected into both SPF chickens 
and chickens previously sensitised with live IBDV as described under 
Materials and Methods. The specificity of the antibodies in the sera 
obtained from both groups of chickens was analysed by Western blotting of 
whole IBDV particles boiled in SDS prior to electrophoresis (FIG. 9). 
Previously sensitized chickens had antibodies to the 32 Kd, 37 Kd and 42 Kd 
structural polypeptides of IBDV at relatively low levels prior to 
vaccination with the fusion proteins. Fusion proteins from clones D6 and 
D1 recalled a specific anti-32 Kd antibody response in all these chickens, 
while the intensity of binding to the other structural proteins remained 
unchanged. 
In unprimed SPF chickens the fusion proteins induced the synthesis of 
antibodies in only some of the chickens. When antibodies were detected, 
however, they were specific by Western blotting for the 32 Kd structural 
polypeptide of IBDV. Thus the fusion proteins expressed in clones D6 and 
D1 induce antibodies specific for the 32 Kd polypeptide in both primed and 
unprimed chickens. 
The sera obtained from the sensitized and SPF chickens vaccinated with the 
fusion proteins were assessed by the ELISA and micro-virus neutralization 
assays which were designed to recognize the protective immunogen in its 
native conformation. The levels of antibody detectable by ELISA did not 
increase by more than 2-4 fold above pre-existing levels in sensitized 
chickens or above base-line levels (&lt;1:100) in SPF chickens, even though 
they reacted very strongly with Western blotted viral proteins. 
The virus neutralization assay also showed no dramatic increase in the 
levels of antibody in previously sensitized chickens, but detected a titre 
of 1:320 to 1:160 in one of two SPF chickens vaccinated with affinity 
purified protein from clone D1. The antibody titre peaked 3 to 4 weeks 
after the second injection of protein from clone D1 and persisted for more 
than 6 weeks. By Western blotting, the polyclonal response of this chicken 
to D1 protein was specific for the 32 Kd polypeptide of IBDV. 
Thus the antibodies produced against the fusion proteins react very 
specifically and strongly with Western blotted 32 Kd host-protective 
antiaen of IBDV, but have relatively weak ELISA titres, and virus 
neutralization activity in only 1 out of 4 chickens. In addition, the 
expressed proteins react very weakly with monoclonal antibody that 
neutralizes the virus. These results strongly suggest that the expressed 
IBDV proteins fused to .beta.-galactosidase, though immunogenic, do not 
have the right conformation necessary for the consistent induction of 
virus neutralizing or protective antibodies. The expression of unfused 
proteins with the right conformation will probably be required to produce 
a more effective subunit vaccine against IBDV. 
In this context, it should be reiterated that the serum of one chicken, 
injected with fusion protein from clone D1, had significant virus 
neutralization activity. In this instance, the IBDV protein could have 
been proteolytically cleaved off the .beta.-galactosidase and assumed the 
conformation required for inducing virus neutralizing antibody response. 
In subsequent experiments, unfused 32 Kd protein has been produced by 
expressing the gene for the 32 Kd protein in vectors that produce unfused 
proteins. This unfused protein reacted with the virus neutralizing MAb 
17-82, though to a lesser extent than with MAb 17-80 that preferentially 
reacts with denatured 32 Kd protein. Thus, one avenue for producing the 32 
Kd antigen with the correct three-dimensional structure is to cleave off 
the IBDV antigen from affinity purified fusion protein by chemical or 
enzymatic cleavage at the junction of the two proteins. Although this 
method may require a further refolding step, the level of expression of 
fusion protein, in comparison to unfused protein, is very high and the 
fusion protein can be readily purified by affinity chromatography. 
The following detailed description relates to the determination of the 
nucleotide sequence of the large segment of IBDV RNA and the amino acid 
sequence of cDNA clones that encode the 32 Kd host-protective antigen of 
IBDV. In the accompanying diagrams: 
FIGS. 10A-10F show sequence analysis of the large RNA segment of IBDV. The 
predicted amino acid sequence is presented in single letter code above the 
nucleotide sequence derived from cDNA clones. There are no other extensive 
open reading frames. The amino acid sequences are numbered sequentially 
from the N-terminus of the 37 Kd protein as position 1. The region 
encompassed by cDNA clones M7, G6, L6, D6 and D1 are indicated. Dibasic 
residues are boxed and the repeat unit A-X-A-A-S is similarly highlighted. 
N-terminal sequences derived from tryptic peptides are shown overlined as 
(- - - -&gt;) for the 37 Kd, (....&gt;) for 28 Kd and (----&gt;) for the 32 Kd 
protein. Only the N-terminus of the 37 Kd protein could be obtained by 
direct sequencing on intact proteins and this is shown from residue 1. 
Results and Discussion 
Random Nucleotide Sequencing 
A mixed population of cDNA inserts (350-2000 bp) spanning the entire large 
RNA segment of IBDV was recovered on DEAE-cellulose from a 1% agarose gel 
after Pst 1 digestion of the selected cDNA library. After purification on 
a NACS column (Schleicher & Schull) the homopolymeric tails were removed 
using Bal 31 exonuclease in a controlled reaction (2 units, 20.degree. C., 
10 minutes) designed to digest no more than 50 nucleotides from either 
end. The fragments were then blunt-ended with DNA polymerase (Klenow 
fragment) and ligated into a Sma I restricted M13mp10 vector followed by 
transformation of E.coli JM101 (Sanger et.al., 1980). Single-stranded 
templates were sequenced by the primed synthesis method using an 
M13-specific primer (Sanger et.al., 1980) but with modificatons that 
improved transcription fidelity over regions of secondary structure in the 
template. These included removal of NaCl from the buffer, using reverse 
transcriptase and optimized ratios of dideoxy:deoxy nucleotides (1:30A; 
2:15C; 1:15G; 2:3T) and performing the reaction at 30.degree. C. or 
greater. Sequences were compiled using a VAX/UMS computer system using the 
programmes of Staden (1982) with modifications by Dr. T. Kyne. 
Directed Chemical Sequencing 
Specific cDNA fragments in either pBR322 or pUR expression vectors were 
sequenced by the Maxam and Gilbert (1977) procedure after first 
identifying a restriction site which could be end labelled with reverse 
transcriptase and either .alpha.-.sup.32 P-dATP or .alpha.-.sup.32 P-dCTP 
at 37.degree. C. for one hour. This method often required a second 
restriction digest after the reverse transcriptase step to generate a 
molecule with a radiolabel at only one end. The fragments were then 
purified by electroelution from an 8% polyacrylamide gel. After chemical 
degradation the sequencing samples were loaded on denaturing 
polyacrylamide gels (Sanger and Coulson, 1978) which contained 90% 
formamide. Under these conditions when 20 cm.times.40 cm gels were run at 
25 W on an apparatus that maintained the temperature above 50.degree. C. 
the secondary structure was completely disrupted. 
Nucleotide Sequence Analysis of the large RNA Segment of IBDV 
Since the cDNA library was constructed with G/C homopolymeric tails of 
average length 20-30 nucleotides we were unable to obtain clear sequences 
directly over these tails by the simple subcloning of PstI fragments into 
M13 ventors. Instead we adopted the strategy of using Bal 31 exonuclease 
to remove the tails and then subdlone random cDNA fragments of the IBDV 
genome by blunt-end ligation into Sma I digested M13mp10. The cDNA 
fragments were initially selected as having originated from the large RNA 
segment by colony hybridization with a specific probe. Random nucleotide 
sequences were rapidly sorted and overlapped into a consensus sequence 
with the aid of computer programmes. The final alignment comprised 2950 bp 
and was constructed from over 60 overlapping sequences. No point mutations 
or rearrangements were found in the overlapping sequences which confirms 
that the original library construction by randomly primed transcripts 
followed by reannealing and polymerase elongation was remarkably 
error-free. 
However, two problems emerged with this approach. Firstly the subcloning 
was definitely not random; some regions were sequenced many times whereas 
cDNA fragments containing the region from nucleotides 2250-2600 could not 
be subcloned into M13. Secondly the general quality of nucleotide sequence 
obtained by the chain termination method of Sanger et.al. (1980) was poor 
due to multiple regions of secondary structure causing premature 
terminations in the transcriptase reaction. This latter problem was partly 
overcome by the use of reverse transcriptase under optimized conditions 
rather than the standard DNA polymerase (Klenow fragment). These secondary 
structure problems appear to be particularly severe for this double 
stranded RNA virus since other genes being sequenced at the same time 
(Hudson et.al., 1984; McIntyre et.al., 1985). To overcome these problems 
we resorted to the chemical degradation technique of Maxam and Gilbert 
(1977) which is less affected by secondary structure and the use of 
denaturing formamide gels to resolve the sequencing ladder. Interestingly, 
the region with the most severe secondary structure problems (nucleotides 
2540-2565) was contained within the fragment which could not be subcloned 
into M13. The significance of this structure which is lethal to M13 has 
not been further characterized; it is contained within the coding region 
of the 32 Kd protein product. 
Identification of the Gene Encoding the 32 Kd Host-protective Immunogen 
Two methods were attempted in parallel; protein sequencing of tryptic 
peptides derived from purified 32 Kd protein and identification by 
immunoblot assay of cDNA clones expressing fragments of the 32 Kd antigen 
as a fusion protein. For the expression studies vectors have recently been 
described in which cDNA fragments can be ligated into the 3'end of the 
.beta.-galactosidase gene (Ruther and Muller-Hill, 1983; Stanley and Luzio 
1984). The fusion proteins produced by these constructions appear to be 
particularly stable and has lead to claims of hybrid-protein synthesis up 
to 30% of the host cell proteins. With suitable inducible promoters 
sufficient protein is produced to form amorphous aggregates appearing as 
inclusion bodies. Plasmid vectors which have been designed to express only 
the gene encoded by the cDNA (pUC, pCQV) do not appear to produce such 
high levels of expressed protein. For these reaons we chose to subclone a 
mixed population of cDNA fragments spanning the entire IBDV genome, still 
containing homopolymeric tails, into the Pst I site of vectors pUR290, 
pUR291, pUR292 to ensure translation in all three reading frames. 
Recombinant colonies were screened by an immunoblot assay using a 
monoclonal antisera raised against denatured 32 Kd protein (see later) 
followed either by autoradiography using .sup.125 I-protein A or visible 
detection by a peroxidase conjugated second antisera. Two colonies 
expressed the epitope recognised by the anti-32 Kd antisera; one was a 
direct subclone of the 1100 bp fragment D6 described previously, and the 
other a shorter 450 bp cDNA fragment D1 entirely contained within D6 (FIG. 
10A-10F). 
Directed nucleotide sequencing over the homopolymeric tails from the EcoRI 
site in the pUR vector readily identified both the cloning vector (pUR290 
for D1; pUR291 for D6 and the translation phase of the recombinant 
product. The entire nucleotide sequence of D6 and D1 was obtained by the 
Maxam and Gilbert technique on suitable end-labelled fragments. This 
sequence overlapped the consensus generated by the random sequencing 
approach, thus spanning the region which could not be subcloned into M13 
and completing the 3129 bp genome presented in FIG. 10. 
With the exception of the D6 region the rest of the genomic sequence had 
been compiled from multiple independent cDNA clones. Although this random 
approach showed that the original construction of the cDNA library was 
remarkably error-free we were concerned that regions with secondary 
structure in D6 could have been transcribed incorrectly. To settle this 
point two further clones (G2 and N1) spanning residues 1250-2750 and 
2210-3150 respectively were sequenced completely by the direct chemical 
method. No ambiguities were found between D6 and these clones indicating 
that the transcription of the IBDV genome was correct. The only 
differences observed between any cDNA clones were always located in the 
last ten nucleotides adjacent to the homopolymeric tails. These sequences 
are known to contain potential errors generated by the DNA polymerase 
fill-in reaction (Hudson et.al., 1984) and were therefore not included in 
the consensus. Any areas of potential ambiguity due to secondary structure 
affecting the random sequencing approach were resolved by direct chemical 
sequencing from a suitable restriction site within the cDNA inserts of M7, 
A3, L6, G2 or D6 which together span the entire sequence presented in FIG. 
10. 
The 5' and 3' terminal sequences of the consensus are defined by the ends 
of M7 and D6 respectively. 
Structure of the 32 Kd Antigen 
On FIGS. 10A-10F arrows indicate the translational phase in the pUR 
subclones of D6 and D1 bearing in mind that the initial residue adjacent 
to the homopolymeric tails has not been included. Although the termination 
codon at residue 3065 is unambiguous, the N-terminal residue of the 32 K 
antigen is not clear. If the protein is generated from a polycistronic RNA 
template as implicated for the related IPNV or Drosphila.times.viruses we 
would expect initiation at MET 2287 giving rise to a product of 29 Kd 
which is consistent from size estimates. However if the 32 Kd protein is 
generated by processing of a precursor we might expect proteolytic 
cleavage somewhere before the MET residues, assuming the C-terminus is 
intact. 
Peptide sequencing of tryptic fragments has confirmed both the reading 
phase predicted from the D6 and D1 expression vectors and that the 32 Kd 
protein spans residues 2372-3008. All nine peptides sequenced to date are 
located from the region 3' to MET 2287. However, the intact 32 Kd antigen 
has a blocked N-terminus which perhaps suggests Gln 2274 as the N-terminal 
residue after proteolysis. 
The amino acid sequence derived from the complete nucleotide sequence of 
the large segment of the IBDV genome is shown in FIG. 10A-10F. Partial 
peptide sequencing of purified viral proteins has confirmed these 
sequences and allowed the positioning of the coding regions of the viral 
proteins on the large segment of the genome. There is only one translation 
termination codon at the 3' end of the genomic RNA, and it would appear 
that the entire genome is expressed as a single polyprotein in which the 
viral proteins are arranged in the following order: N-41/37 Kd -28 Kd -32 
Kd-C. The exact processing mechanism of this large precursor to the viral 
proteins has not yet been defined. Dibasic residues, which are frequent 
targets for eukaryotic precursor proteins, are conveniently situated at 
residues 451-452 and 721-722 and cleavage at these sites would excise a 
predicted 28.2 Kd protein. The cleavage sites are consistent with peptide 
sequencing data which confirms that the 37 Kd protein spans at least base 
residues 32-1310, the 28 Kd protein at least base residues 1660-1870, and 
the 32 Kd protein at least base residues 2310-3030. The region encoding 
the 37 Kd protein is also expected to encode the larger 52 Kd and 41 Kd 
precursors of the 37 Kd protein. An alternative cleavage site could be the 
peptide sequence A-X-A-A-S which is repeated three times between residues 
483-503 and also appears at residues 752-756. 
The following detailed description relates to the production of monoclonal 
antibodies (MAbs) to IBDV and to the identification of a neutralising 
epitope on IBDV using these monoclonal antibodies. In the accompanying 
diagrams: 
FIG. 11 shows Western blot analysis of anti-IBDV MAbs against whole virus 
following SDS-PAGE. 
FIGS. 12A and 2B show competitive ELISA between anti-IBDV MAbs and a 
chicken anti-32 Kd specific antisera to IBDV. 
Results and Discussion 
Mouse monoclonal antibodies (Mabs) to IBD virus were prepared by 
hyper-immunising Balb/C mice with purified virus and fusing the immune 
spleen cells with SP2/0 myeloma cells according to the method of Hewish 
et.al (1984). Antibody secreting colonies were detected by an Immunodot 
assay (Bio Rad) on whole virus and by the IBD virus ELISA described in 
International Patent Specification No. PCT/AU84/00256, modified to detect 
mouse antibodies by using goat anti-mouse Ig-HRP (Bio Rad). The positive 
colonies were cloned by limiting dilution on at least 3 occasions, 
selecting positive colonies by the above assays at each cloning. 
The specificity of the MAbs was assessed by Western blotting whole virus 
(Patent Specification No. PCT/AU84/00256) again modified to detect mouse 
antibodies by using rabbit anti-mouse Ig (Sera-lab). The majority of MAbs 
were specific for the 32 Kd structural polypeptides of IBD virus, as 
exemplified by the series 1 and 17 MAbs shown in FIG. 11. Only one series 
of MAbs, series 6, recognised the 42 Kd polypeptide (FIG. 11) and none 
have yet been obtained that specifically react with the 37 Kd polypeptide. 
A subclone of the series 17 MAbs, designated 17-82, did not bind to SDS 
denatured IBD viral polypeptides (FIG. 11). All the MAbs that were 
positive for viral polypeptides on Western blots also bound to material on 
the blots that were of lower mol. wt. than any of the known structural 
proteins of IBD virus (Dobos, 1979; Patent Specification No. 
PCT/AU84/00256) and may therefore represent degraded viral proteins. The 
anti-32 Kd monoclones, particularly of the 17-80 and 17-83 lineage bound 
to a large molecule which had an approximate mol. wt. of 55 Kd and may 
represent the unprocessed precursor molecule described earlier. 
The relative antibody activity of the MAbs was assessed by the ELISA and 
the Immunodot assay; the latter on both denatured and nondenatured virus 
(Table 1). Ascites fluid from mice inoculated with myeloma cells of the 
series 1 and 17 all had high titres of antibodies by the ELISA and 
Immunodot assays (2.sub.--.sup.14 2.sup.19) performed on native virus, 
although the Immunodot reactivity of the 17-82 lines was abolished by 
treatment of the virus with SDS and boiling. The series 6 MAbs reacted 
weakly in both assays (Table 1), although this time the reactivity of the 
MAbs in the Immunodot assay was enhanced by treating the virus with SDS 
and boiling. 
TABLE 1 
______________________________________ 
Summary of the specificity, relative activity 
and isotypes of the MAbs to IBD virus. 
AB Activity 
AB Specificity Virus AB 
Western Competi- Immunodot 
Neutral- 
Iso- 
MAb blot ive ELISA 
ELISA -SDS +SDS isation 
type 
______________________________________ 
1 32 Kd ? ++++ +++ +++ - G.sub.1 
6 42 Kd ? + + +++ - G.sub.1 
17-80 32 Kd ? +++ +++ ++++ - G.sub.1 
17-82 ? 32 Kd ++++ ++++ - ++++ G.sub.2b 
______________________________________ 
When the virus neutralizing activity of the MAbs was assessed in the 
micro-virus neutralization assay (Patent Specification No. PCT/AU84/00256) 
only the MAbs of the 17-82 lineage neutralized the infectivity of the 
virus; the ascites fluid having a titre of 2.sup.14. MAbs of the series 1 
and 6 and of the 17-80 lineage were all negative (&lt;2.sup.4). 
The specificity of the 17-82 MAbs was investigated in a competitive 
inhibition ELISA against chicken antisera specific for the 32 Kd 
polypeptide of IBD virus by Western blotting. The 17-82 MAbs effectively 
competed out the chicken anti-32 Kd antibodies (FIG. 12), while the 17-80 
and series 6 MAbs were much less effective (FIG. 12). The 17-82 MAbs also 
completed out a polyspecific chicken antisera, which recognized the 32 Kd, 
37 Kd and 42 Kd viral polypeptides on Western blots (data not shown), 
indicating that the 17-82 MAbs were against a dominant immunogen on the 
virus. 
The isotype of the anti-IBD virus MAbs was determined by an ELISA utilising 
either anti-mouse lambda chain, IgM, IgG1, IgG2a+2b, IgG2b or IgG3 as the 
second step reagent. All blAbs were of the mouse IgG1 class except the 
17-82 line MAbs, which were of the IgG2b class. 
The series 6 MAbs were of particular interest as we believe from HPLC 
analysis of a tryptic and chrymotryptic digest of the 37 Kd and 42 Kd 
polypeptides that the latter is the precursor of the former. It would seem 
likely therefore that the series 6 MAbs recognise the peptide sequence 
cleaved off during the formation of the major 37 Kd structural polypeptide 
of the Australian type-1 IBD virus. 
Because of their ability to recognise SDS denatured 32 Kd polypeptide of 
IBD virus, MAbs of the 17-80 lineage were used to select recombinant 
bacterial colonies expressing part or all of the 32 Kd polypeptides as 
described above. 
The following detailed description relates to the production of the 32 Kd 
structural protein in its unfused form. In the accompanying diagrams: 
FIG. 13 shows proteins expressed in clones D6, D1 and P1, and IBDV 
proteins, which were Western blotted and reacted with MAb 17-80. The 
insert of clone P1 was constructed by ligating the L6 and D6 inserts via 
the Apa I restriction site to retain the exact genomic sequence of native 
IBDV over this region. 
FIG. 14 shows clones D1, D6 and P1 which were lysed by various treatments 
and the proteins blotted onto nitrocellulose filters, then reacted with 
either MAb 17-80 or MAb 17-82. The expressed proteins were visualised by 
reaction with [.sup.125 I] Protein A followed by autoradiography. 
FIG. 15 indicates the minimum size of precursor polypeptide that has to be 
expressed for the correct processing of the 32 Kd antigen. The insert of 
clone PO, that contains the entire coding region of the large segment of 
IBDV genome, was progressively shortened at the 5' end at specific 
restriction sites and the resultant fragments were expressed in pPL vector 
in E.coli. Expressed gene products were Western blotted and reacted with 
MAb 17-80. 
FIG. 16 indicates the regions of the precursor polypeptide that may 
contribute to the antigenic determinant recognised by the virus 
neutralising monoclonal MAb 17-82. Undenatured proteins from clones 
containing precursors of varying sizes were blotted onto nitrocellulose 
filter and reacted with MAb 17-80 or MAb 17-82. 
A large recombinant molecule spanning bases 425-3145 was constructed by 
joining the inserts of clone D6 (which encodes the 32 Kd protein) and 
clone L6 (which encodes the 28 Kd protein and the major part of the 41/37 
Kd protein)--full details of both these clones are set out above. L6 and 
D6 inserts were ligated via the Apa I restriction site to retain the exact 
genomic sequence of native IBDV over this region. This large recombinant 
molecule (PI) was expressed in pUR plasmids in E.coli and the expressed 
protein analysed by Western blotting and reaction with MAb 17-80 (FIG. 
13). The large insert was expected to express a viral polyprotein of M&gt;80 
Kd (or .about.190 Kd as a fusion protein) but instead produced a discrete 
32 Kd protein that specifically reacted with MAb 17-80. 
To see if the correct processing of the expressed polypeptides leads to 
their being correctly folded the proteins expressed in clone P1 were 
analysed by an immunoblot assay (FIG. 14) using a monoclonal antibody (Mab 
17-82) that neutralises the virus but does not react with denatured 32 Kd 
viral protein. The expressed proteins reacted quite strongly with MAb 
17-82, but this reaction was completely abolished when the expressed 
protein is first denatured in SDS. After denaturation in SDS, the 
expressed protein reacted strongly with MAb 17-80 which recognises 
denatured 32 Kd protein. Thus, the genetically engineered polypeptides 
mimic the immune response of the whole virus particle towards MAb 17-82 
and MAb 17-80. 
These results clearly demonstrate that the expression of a large cDNA 
fragment encoding the 32 Kd protein, the 28 Kd protein and a major portion 
of the 41/37 Kd protein results in the synthesis of an unfused 32 Kd 
protein that is recognised by a monoclonal antibody (Mab 17-80) which 
reacts with denatured 32 Kd host-protective antigens of IBDV. Tn the 
"native" form, the genetically engineered polypeptides react specifically 
with the virus neutralising monoclonal antibody (Mab 17-82) suggesting 
that they may be folded in the same conformation as the native viral 
antigen. 
A larger recombinant molecule (P0) containing the entire coding region of 
the large RNA segment of IBDV was constructed by ligating the insert of 
clone P1 to the insert of another clone G6 through a common Nde1 
restriction site. P0 was expressed in pEX vector (Stanley and Luzio, 
(1984)), in E.coli. As in the case of P1, this resulted in the production 
of a correctly processed 32 Kd polypeptide that reacted with MAb 17-80. 
The 32 Kd protein produced in clones P1 and P0 might be processed by a 
virus-specified protease. Alternatively, a translation initiation site 
recognised by E.coli ribosomes may be present within or just before the 
gene for the 32 Kd protein. If this is the case then the introduction of 
frame shifts within the 28 Kd protein should not affect the production of 
the 32 Kd polypeptide in clone P0. Frame shifts were introduced by 
inserting the 1.3 Kb Km.sup.R fragment (Vieira and Messing (1982)) into 
the EcoR1 or Bam H1 sites within the gene for the 28 Kd protein, or by 
deleting the EcoR1--Bam H1 fragment. In none of these instances was a 32 
Kd or higher MW protein produced that reacted with MAb 17-80 on Western 
blots. This ruled out the possibility that the 32 Kd protein is expressed 
from an independent translation initiation site. 
In order to localize the putative virus-specified protease the insert from 
clone P0 was progressively shortened from the 5' end at specific 
restriction sites (FIG. 15) and resultant fragments of different sizes 
were inserted into vectors that produced either fusion proteins or unfused 
proteins, and the same results were obtained irrespective of the type of 
vector used. The proteins expressed in E.coli were Western blotted and 
reacted with MAb 17-80 (FIG. 15) to see how much of the coding sequence 
besides the 32 Kd polypeptide has to be expressed in order to produce the 
correctly processed 32 Kd protein. FIG. 15 shows the results obtained by 
expression in a fusion vector (pPL) that adds on about 7 Kd of the XN gene 
product to the expressed protein. 
The deletion of part or whole of the gene coding for the 52 Kd precursor of 
the 37 Kd protein or the N-terminal portion of the 28 Kd protein did not 
in any way interfere with the production of the 32 Kd polypeptide. 
However, the removal of further portions of the gene encoding the 28 Kd 
protein (Bam H1 and Hind III restriction sites) inhibited the processing 
of the 32 Kd protein even though the dibasic residues at the approximate 
junction between the 32 Kd and 28 Kd proteins were still present. Similar 
results were obtained by site-specific insertional mutagenesis studies 
using Km cassettes (Vieria and Messing, (1982)). Insertion of 10 codons 
`in-phase` into the EcoR1 site near the 5' end of the 28 Kd protein does 
not affect the production of the correctly processed 32 Kd protein, 
whereas the insertion `in-phase` of 4 codons into the Bam H1 site in the 
middle of the 28 Kd protein inhibits the processing of the 32 Kd protein 
and a much large precursor molecule is produced. 
These results together with the fact that the 28 Kd protein is present in 
very minute and variable quantities in the mature virus particle would 
suggest that the 28 Kd protein is an IBDV specific protease involved in 
the processing of the large precursor polypeptide. 
The proteins expressed in clones P1 and P0 react strongly with the virus 
neutralizing MAb 17-82. Since clones P1 and P0 produce correctly processed 
32 Kd protein and other proteins encoded by the large segment it was 
important to know whether the correct processing of the proteins resulting 
from the expression of large precursor molecules leads to the expressed 
polypeptides assuming the correct 3-dimensional structure that is 
recognised by the virus neutralizing MAb 17-82. Recombinant molecules of 
various sizes containing the gene for the 32 Kd protein and part or whole 
of the gene(s) for the 28 Kd and 52 Kd proteins, were expressed in E.coli. 
The undenatured expressed proteins were blotted onto nitrocellulose filter 
and reacted with MAb 17-80 or MAb 17-82 (FIG. 16). While MAb 17-80 reacted 
with proteins expressed in all the constructs, the virus neutralizing MAb 
17-82 only reacted with proteins expressed in clones in which the 
substantial portion of the 52 Kd precursor of the 37 Kd protein is 
retained (FIG. 16). On the other hand, FIG. 15 clearly shows that the 
correct processing of the 32 Kd protein does not require any portion of 
the 52 Kd protein or even the extreme N-terminal portion of the 28 Kd 
protein. Thus the correct processing of the 32 Kd protein alone does not 
ensure recognition by MAb 17-82, and a portion of the 52 Kd precursor 
protein may be directly or indirectly involved in the process. 
The antigenic determinant recognised by the virus neutralizing MAb 17-82 
may consist of a discontinuous epitope made up of contributing regions 
from both the 32 Kd and 41/37 Kd proteins. Fusion proteins from clones D6 
and D1 in the undenatured state, react weakly, but quite specifically with 
virus neutralising MAb 17-82. Unfused protein produced by the expression 
of the Aha II-Pst 1 fragment of the 32 Kd gene, also react with MAb 17-82. 
Thus the 32 Kd protein or part thereof, is recognised albeit weakly by MAb 
17-82. In order to see if MAb 17-82 also reacted with the 52 Kd precursor 
protein of the 41 and 37 Kd structural proteins, the gene encoding this 
region, without those genes coding for the 28 Kd and 32 Kd structural 
proteins, was expressed in pEX vector in E.coli. The undenatured expressed 
protein reacted strongly with MAb 17-82, indicating that the 52 Kd 
precursor also contained an epitope(s) recognised by the virus 
neutralising MAb. It is possible that an interaction between the 32 Kd and 
the 41/37 Kd structural proteins may be involved in formation of 
epitope(s) that induce virus neutralising and/or protective antibody. 
Thus one viable approach to producing the correctly processed and folded 
antigens is to express the entire coding region or precursors retaining 
the 32 Kd, 28 Kd and a substantial portion of the 52 Kd precursor 
proteins. The antigens produced by this method can be readily purified by 
affinity chromatography using monoclonal antibodies, or by engineering 
specific sequences at the termini of the expressed antigens. 
Another approach is to express the complete gene or fragments thereof for 
the 32 Kd and/or the 52 Kd protein. A susbsequent refolding step may or 
may not be required. This approach is quite feasible since we have 
previously demonstrated (International Patent Application PCT/AU84/00256) 
that the viral 32 Kd protein isolated from SDS-polyacrylamide gel can 
refold and when injected into chickens produce virus neutralizing and 
protective antibodies. Moreover, an unfused protein of 30 Kd produced by 
the expression of the AhaII-Pst 1 fragment of the 32 Kd gene in pCAV2 
vector reacts with the virus neutralizing MAb 17-82. The protein expressed 
from the gene for the 52 Kd precursor of the 41 Kd and 37 Kd structural 
proteins also reacts with the virus neutralising MAb 17-82. 
A third approach to producing the viral antigen in E.coli is to produce 
fusion proteins in which an enzymic or chemical cleavage site has been 
engineered at the junction between the IBDV and host proteins. The levels 
of expression of fusion proteins are very high and the expressed protein 
can be readily purified by affinity chromatography. The IBDV protein can 
be recovered by enzymic or chemical cleavage of the purified fusion 
protein. 
REFERENCES 
1. BIRNBOIM, H. D., and DOLY, J. (1979) Nucl. Acids Res. 7, 1513-1523. 
2. BLAKE, A. and PEACOCKE, A. R. (1968). Biopolymers 6, 1225-1253. 
3. DIAZ-RUIZ, J. R., and KAPER, J. M. (1978). Prep. Biochem. 8, 1-17. 
4. DOBOS, P. (1979). J. Virology, 32, 1046-1050. 
5. FIRTH, G. A., (1974). Aust. Vet. J. 50. 128-130. 
6. GOLDBACH, R. W., BORST, P., BOLLEN-DE BOER, J. E., and VAN BRUGGEN, E. 
F. J. (1978). Biochem, Biophys. Acta 521, 169-186. 
7. GRUNSTEIN, M. and HOGNESS, D. S. (1975). Proc. Natl. Acad. Sci. USA 72, 
3961-3965. 
8. HEWISH, D. R., ROBINSON, C. P. and SROW, L. G. (1984). 
Aust.J.Biol.Sci., 37, 17-23. 
9. HUDSON, P., JOHN, M., CRAWFORD, R., HARALAMBIDIS, J., SCANLON, D., 
GORMAN, J., TREGEAR, G., SHINE, J., and NIALL, H. (1984) EMBO J. 3, 
2333-2339. 
10. ISH-HOROWICZ, D., and BURKE, J. F. (1981). Nucl. Acids Res. 9, 
2989-2998. 
11. LERMAN, L. S. (1963). Proc. Natl. Acad. Sci. USA 49, 94-102. 
12. MARGLIN, A., and MERRIFIELD, R. B. (1970) Auno. Rev. Biochem., 39, 841. 
13. MAXAM, A. M. and GILBERT, W. (1977) Proc. Natl. Acad. Sci. 74, 
5463-5467. 
14. McINTYRE, P., GRAF, L., MERCER, J., WAKE, S., HUDSON, P. and 
HOOGENRAAD, N. (1985) DNA 4, in press. 
15. MULLER, H., SCHOLTISSEK, C. and BECHT, H. (1979). J. Virol. 31, 
584-589. 
16. RIGBY, P. W. J., DIECKMANN, M., RHODES, C., and BERG, P. (1977). J. 
Mol. Biol. 113, 237-251. 
17. RIXON, F., TAYLOR, P., and DESSELBERGER, U. (1984). J. Gen. Virol. 65, 
233-239. 
18. RUTHER, U. and MULLER-HILL, B. (1983) EMBO J. 2, 1791-1794. 
19. SANGER, F., COULSON, A. R., BARRELL, B. G., SMITH, A. J. H. and ROE, B. 
A. (1980) J. Molec. Biol. 143, 161-178. 
20. SANGER, F. and COULSON, A. R. (1978) FEBS Lett. 87, 107-110. 
21. STADEN, R. Nucleic Acids Res. (1982) 10, 4731-4751. 
22. STANLEY, K. K. and LUZIO, J. P. (1984) EMBO J. 3, 1429-1434. 
23. TAYLOR, J. M., ILLMENSEE, R., and SUMMERS, J. (1976). Biochem. Biophys. 
Acta 442, 324-330. 
24. TODD, D., and McNULTY, M. S. (1979). Arch. Virol. 60, 265-277. 
25. ULLMANN, A. (1984). Gene. 29, 27-31. 
26. VIEIRA, J., and MESSING, J. (1982). Gene. 19, 259-268.