CA1252042A - Infectious bursal disease virus vaccine - Google Patents

Abstract

ABSTRACT OF THE DISCLOSURE

A non-infectious sub-unit vaccine for use against infectious bursal disease (IBD) virus comprises the structural polypeptide of approximate MW 32 Kd contained in the IBD virus, or an immunogenic peptide derived therefrom, together with, if desired, an adjuvant. A method of increasing the level of protective antibodies in poultry by administering the vaccine, and diagnostic methods are also disclosed.

Description

~sz~

This invention relates to the identification and char-acterisation of the major structural protein of infectious bursal ~ disease (IBD) virus of chickens (host-protective immunogen) which stimulates the production of antibody that neutralises the in-fectivity of IBD virus in vitro and which protects susceptible chickens against inEection with virulent IBD virus. The invention further relates to the production of an effective sub-unit vaccine against the virus utilising this major host-protective immunogen, as ~ell as to the use of this immunogen in diagnostic tests, assays and the like.
Infectious bursal disease virus is a pathogen of major economic importance throughout the world poultry industry and is a ubiquitous contaminant of commercial poultry environments. The virus causes a highly contagious immunodepressive disease of young chickens, and selectively proliferates in the bursa of Fabricius (one of the two major avian immunological organs) thereby des troying the precursors of the antibody producing plasma cells. In young chickens (day old to 4 weeks) it directly causes morbidity and mortality, while the capacity to produce antibody responses is inhibited or depressed in chickens which survive infection. Such 1~19-jms ~~

:LZ'jZ~

chickens respond poorly to vaccination programs aimed at other avian infections, remain highly susceptible to a variety of bac-terial, mycoplasmal and viral pathogens and exhibit very poor weight gain and food conversion ratios.
Protection against IBD virus infection is mediated by humoral antibody alone and does not require the presence oE cell-mediated immune effector systems. Thus chickens that receive an adequate amount of maternal antibody from an imMune breeder hen via the yolk sac are protected through the critical first ~ to 5 weeks after hatching.
Current vaccination strategies are aimed a-t achieving the deposition of high levels of maternal antibody in fertilized eggs to protect the chickens throughout these critical weeks after hatching. The presently used vaccination regimens to control IBDV
involve injecting breeder hens (previously exposed to live IBDV) with an inactivated oil-emulsion whole virus vaccine prior to the onset of their period of egg production around 22 weeks of age.
This inactivated vaccine provokes a major secondary antibody res-ponse -that is several orders of magnitude (> 100 fold) greater and persists for longer than the responses obtained by repeated vac-cination with live virus. This results in the transmission of high levels of protec-tive antibody to each egg throughout the next ~0 weeks or so of the egg production cycle. If the antibody levels can be boosted sufficiently it should be possible Eor dG)4~1 broiler chickens, that are slaughtered at 6 weeks of age, to go through their complete rearing period totally protected Erom IBD
virus by maternally derived antibody.
The presently available inactivated IBD virus vaccine is expensive and difficult to produce since the virus cannot be grown to sufficiently high titres in simple culture systems such as embryonated eggs or -tissue culture. Currently the viral material required for vaccine production is obtained by infecting six-week-old specified-pathogen-free (SPF) chickens and then harvesting the virus from the infected bursae 3 or 4 days after infection. This procedure for producing an IBD virus vaccine from infected SPF
chickens is both laborious and expensive. The identification and isolation of the major host-protective immunogen in accordance with the present invention opens the way for development of a safe and inexpensive sub-unit vaccine which is effective in stimulating prolonged high-titre antibody responses in hens to allow the transfer of sufficient maternal antibody to protect young chickens, at least during the critical first few weeks after hatching.
One object of the work leading to the present invention has been to identify the IBD virus-encoded protein(s~ that induce antibody in chickens following natural infection or the injection of a commercial inactivated oil-emulsion whole virus vaccine.

~ 2S ~

Tn this work, the purification of the Australian strain of IBD virus from chicken hursae by successive rate-zonal and density-equilibrium centrifugation using sucrose and caesium chloride respectively has been studied. The virus so purified has been analysed and found to be composed of two major structural protein or polypeptide components of molecular weights (MW~ approximately 37 kilodal-ton (Kd) and 32 Kd, and three other components of ~Ws approximately 91.5 Kd, 41.5 Kd and 29 Rd. Only the major polypeptides reacted strongly with serum from chickens naturally infected or hyperimmunized with IBD
virus, and of these the reaction of the 32 Kd polypeptide with antibody was iypically the most intense. The 3~ Kd polypeptide was a major component of all preparations (as revealed by Coomassie-blue staining of gels) of purified bursal grown ~ustralian IBD virus, with a buoyant density in CsCl of 1.33 g/ml. The relative amounts of the other polypeptides varied between preparations. It should be noted that from preliminary work in this re~ard, the 32 Kd polypeptide was estimated to have a molecular w~ight of approximately 31 Kd when compared with standard molecular wei~ht markers. As a result of further work, howeverj the molecular weight of approximately 32 Kd is considered to be more accurate.

The major 32Kd polypeptide of the Australian isolate is comparable in size to the VP-3 protein (M~l of 32 to 35 Kd) detected in studi~s overseas on the Cu-l isolate of IBD virus grown in vitro or in vivo - 5 - ~ ~5 Z~ ~

(Nick et al, 1976; Dobos 1979; Todd & McNulty, 1979; Muller &
Becht, 1982). The major 37 Kd polypeptide is, however, smaller than the VP-2 (MW of 40 to 41 Kd) of overseas isolates, while the VP-X protein (MW of 47 to 4~ Kd - Dobos, 1979; Muller & Becht 1982) was not detectecl in preparations of the intact Australian virus. ~he 41.5 Kd polypeptide of the Australian isolate is, however, the precursor of the 37 Kd polypeptide, and it is suggested that the 41.5 Kd polypep-tide is analagous to VP-X of cverseas isolates.
It has no~7 been demonstrated that serum from naturally infected or hyperimmunised chickens contains antibodies to all polypeptides of the Australian isolate other than the minor 91.5 Kd polypeptide. It has also been shown that antibodies specific for the 32 Kd polypeptide of IBD virus appear first during the primary immune response of SPF chickens to the infectious Virus, and these are also the predominant antibodies detected in the primary response of chickens to an inacti~ated oil-emulsion whole virus vaccine prepared from IBD virus propogated in the bursa. Only later in the response to live virus or following revaccination with an inactivated vaccine could antibodies to other structural polypeptides be readily detected.
The 32 Kd polypeptide thus appears to be a major, if not the major, immunogen of IBD virus.
The protective ability of chicken sera which contain only antibodies specific for the 32 Kd polypeptide, as assessed by Western blotting analysis on viral ~,S~ Z

polypeptides separated by SDS-PAGE, is attested to by the capacity oE such sera to neutralise the infectivity of IBD virus in vitro and to passively protect highly~susceptible 2-day-old SPF chickens.
Furthermore, chickens immunised with the purified 32 Kd polypeptide produced antibody detectable by ElISA and the virus neutralisation assay, while chickens ir~unised with the 37 Kd or ~1.5 Kd polypeptides produced antibody detectable by ELISA, but only low levels of virus neutralising ability.
Adsorption of the anti-32 Kd sera with the 37 Kd or 41.5 Kd polypeptides did not reduce the virus neutralising titre of the anti-32 ~d sera. ~hese results confirm that the 32 Kd polypeptide is a major protective immunogen of IBD virus.

According to one aspect of the present invention, there is provided a non-infectious sub-unit vaccine for use against IBD virus which comprises the structural polypeptide of approximate .~ 32 Kd ~ contained in the IBD virus, or an immunogenic peptide derived therefrom, tog~ther with, if desired, an adjuvant.

- In a related aspect, this invention provides a method of increasing the level of protective antibodies against IBD virus in poultry, particularly breeding hens, which method comprises administering the aforesaid vaccine to said poultry.

- 7 ~

In yet another aspect, this invention provides a method of providing passive immunity to IBD virus in poultry, which method comprises administering to said poultry an antiserum con-taining antibodies specific for th~e 32 Kd structural polypeptide or an immunogenic peptide derived therefrom.
The 32 Kd polypeptide may be isolated from IBD Virus, for example IBD virus which has been grown in and purified from infected bursae of Fabricius in chickens.
As mentioned above, the vaccine according to this inven-tion may comprise an immunogenic peptide derived from the 3? Kdpolypeptide, for example~ by "genetic engineering" or chemical synthesis. A suitable immunogenic peptide may be derived so that it comprises all or at least the major immunogenic determinants of the 32 Kd polypeptide contained in the IBD virus and thus exhibits the same or similar immunogenicity to the 32 Kd polypeptide. If required the 32 Kd polypeptide may also be coupled to a carrier molecule to increase its immunogenicity and hence its efficacy as a vaccine.
Preferably, the non-infectious sub-unit vaccine of this inven~ion comprises an adjuvant. The vaccine may, for example, be delivered in an aqueous-mineral oil emulsion, such as an emulsion achieved by using an oil-phase emulsifier (e.g. Arlacel 80) and :`~
an aqueous-phase emulsifier (e.g. Tween 80) as described by -~D f' ~ ?~

~:~S2~
Stone _ al., 1978. Additional adjuvants may also be included if required, for example Al OH3 (Wells et al., 1979), saponin or a derivative of muramyl dipeptide (Wells et al., 1982).
In other aspects of the present invention there are provided methods for assaying both quantitatively and quali-ta-tively the levels of protec-tive antibodies in poultry, including breeding hens and their progeny, and methods for assaying the relative concentrations of protective immunogen in preparations of IBD virus produced for experimental and commercial inactivated vaccines, which methods are characterised by the use as an immuno gen of the polypeptide of approximate MW 32 Kd isolated from IBD
virus r or an immunogenic peptide derived therefrom. Further details of the methods by which these immunoassays can be carried out are well known in the art, and are accordingly not described in detail here. ~hese methods include the well known ELISA and radioimmunoassays.
In another aspect of the invention, there is provided a process for preparing a non-infectious sub-unit vaccine for use against IBD virus which comprises a structural polypeptide of approximate M~1 32 Kd contained in the IBD virus, or an immuno-genic peptide derived therefrom together with, if required, an adjuvant which process comprises separating said 32 Kd polypeptide or an immunogenic peptide derived therefrom from IBD virus and, if required, mixing said separated 32 Kd polypeptide or said immunogenic peptide with an adjuvant.

~5~ 23199-66 -8a-The following detailed description relates to the isola-tion and characterisation by elec-trophoresis and Western blotting of an Australian isolate o:E IBD virus. In the accompanying dia-grams:
Figure l shows the electrophoretic profile of the total RNA isolated from IBD virus which had been fractionated on a 25 to 50% sucrose gradient (lO ml) at 28,000 rpm for 90 mins. The white doublets towards the top of the gel in Eractions 3, 4 and

2~Z

9 from the gradient comprise the two seçments of ds-RNA present in IBD virus. Arrow indicates increasing concentration of sucrose.

Figure 2 shows (a) Polyacrylamide ge]
electrophoresis of purified IBD virus in a 12.5 gel using the discontinuous SDS-gel system described by Laemmli (1970). The gel was stained with Coomassie brilliant blue to reveal 2 major bands of approximate ~ 37 Xd and 32 Kd and 3 others (arrowed) of approximate M~l 91.5 Kd, 41.5 Kd and 29 Kd.
(b) Autoradiograph of a Western blot of the virus preparation from (a) after probing with serum fro~ a chicken experimentally infected with live IBD virus 2 months previously. ~olecular weight standards are on the left side of each gel [(a) Pharmacia standards and (b) Amersham 1 4c-standards]

~ Figure 3 shows the specificity of the serum antibody response of a 6 week-old chicken to live IBD
virus, as assessed by reacting 1:500 dilutions of serum collected 3,5,7,10 and 14 days after infection with Western blots of the viral polypeptides separated by SDS-PAGE. 14C-~markers (Amersham, U.X.) are on the left-hand side.

Figure 4 shows (a) the specificity of antibodies in serum collected from six 6-week-old chickens, 14 ~2~2~14~

days after they had been infected with IBD virus (tracks 1-6) or in the serum from a chicken tha~
had been infected 28 days previously (track 7).
Only one (track 1) of the 6 chickens had antibody with demonstrate specificity ~or other than the 32 Kd structures polypeptide by 14 days after infection. Autoradiograph exposed for 7 da~s.
Amersham 14C-~bl markers on left-hand side.

Figure 5 shows the specificity of the primary antibody response of 2 SPF chickens (l and 2) injected at 5 weeks of age with a commercial inactivated oil-emulsion vaccine. Serum obtained from the chickens 4 and 8 weeks (a and b) a~ter primary vaccination are compared with serum obtained 4 weeks (c) after a second injection of inactivated vaccine at 13 weeks of age. Amersham 14C-MW markers on left-hand side.

Figure 6 shows the specificity of antibodies in sera - from a chicken infected at 5 weeks of a~e with live virus when exam'ned 4 and 20 weeks post infection ~a and b). The chicken was then reimmunised with a commercial inactivated whole-virus vaccine at 25 weeks of age and the specificity of the response examined 4 and 8 weeks later (c) and d). Amersham 14C-~ markers on left-hand side.

MATERIALS AND METHODS

~L252(~

Growth and purification of IBD virus.

The isolate 002/73 of IBD virus was originally obtained in Australia by Firth (197~) from commercial poultry with varying degrees of bursitis and identified serologically as IBD virus at the Central Yeterinary Laboratory, ~eybridge, UK. Following propagation at a limiting a:ilution of infectivity, the virus was routinely propagated by intraocular inoculation of 4 to 6 week old specified pathogen free (SPF) white leghorn chickens (CSIRO SPF Poultry Unit -Maribyrnong, Victoria, Aust.). ~omogenates of infected bursae of Fabricius were prepared as 10~
(w/v) suspensions in phosphate buffered saline (PBS) and stored at -80C. The IBD virus stocks appeared to be free of contamination by other poultry viruses on electromicroscopic examination and did not cross react in the agar-gel precipitation test with antisera to avian reovirus.

The virus was purified by a modification of the method of Todd ~ McNulty ~1979). An equal weight of chilled PBS was added to the freshly harvested bursae which were homogenised in an ice bath by 3 x 20s bursts of a Polytron (PT-10-OD, Kinematica, GM~H, Luzern, Schweiz) on setting 5. The homogenate was frozen to -80C and thawed rapidly before an equal volume of the fluorocarbon Arklone (Wertheim Labs., Melb. Australia) was added and the mixture rehomogenised. After centrifugation at 10,000 g for 30 min at 5C, the aqueous phase (ca 7ml) was prepared in 0.1 M NaCl, ~25~C3aL~

0.01 M Tris-HCl buffer, pH 7.6. Afte~ centrifugation at 28,000 rpm for 1.5 h in a Beckmann SW28 rotor at 5C, the gradients were harvested from the bottom in 1 ml fractions. These were then examined by gel electrophoresis, Western blGtting with hyperimmune sera, ELISA and by an assay for ds-RNA in order to determine the position of complete or incomplete particles of virus and soluble proteins of the virus (in later preparations the virus was collected from the interface of a stepwise gradient prepared by overlaying l0 ml of 40~ sucrose on 5 ml of 60%
sucrose). Fractions containing complete (intact) virus were pooled and layered onto chilled preformed 20 to ~0% (w/v) CsCl gradients (10 ml) which were centrifuged at 30,000 rpm for 18 h at 5C in a Beckmann ~0 Ti rotor and the band of IBD virus harvested through the side of the tube. The virus was dialysed against NaCl-Tris buffer to remove CsCl and then against MaCl-Tris buffer containing 0.05~ (w/v) sodium azide before being stored at 4C or else made 50% (v/vJ in glycerol and stored at -20C.

Chicken antisera to IBD virus SPF chickens were infected intraocularly with IBD
virus and bled 0,3,5,7,10 and 14 days later, then every two weeks. Chickens injected intramuscularly with 0.5 ml of commercial inactivated oil-emulsion vaccine (Arthur Webster P-ty.Ltd., Morthmead, NSW, Aust.) were bled fortnightly, as were chickens given a second intraocular infection with IBD virus or D f~ R ~

3~ 2 previously-infected chickens yiven an intramuscular injection of commercial inactivated vaccine. The sera were collected by centrifugation at 400 g for 15 min and stored at -20C.

Enzyme linked immunosorbent assav (ELLSA) The ELISA method used to assess the presence of I~D
viral antigen in various gradient fractions or chicken antibody to IBD virus was essentially that described by York et al., (1983), except that the microtiter trays (Nunc Immunoplate I) were coated with rabbit anti-IBD virus IgG prepared by hy~erimmunising rabbits with 002/73. To detect viral antigens in the gradient, serial dilutions of the fractions were added to the wells, which were then treated with a dilution of chicken anti-serum to IBD virus which produced a maximum OD450nm of 1Ø To titrate antibodies to IBD
virus, dilutions of chicken sera were added to the wells that had first been coated with rabbit ant~body followed by a standardised concentration of an extract of infected bursae. The amount of chicXen antibody binding to the viral antigen in each assay was then quantitated by adding sheep IgG-anti-chicken IgG-horseradish peroxidase conjugate, followed by 5-aminosalicylic acid. The trays were shaken for 30 min and the OD450nm was then read immediately on a Titertek Multiscan (Flow Lahoratories, Australia).

Polyacrylamide ~el electrophoresis ~PAGE) and Western blotting.

~.ZS2(~

la Aliquots (40~1) of each sucrose-gradient fraction were dried down under vacuum, resuspended in 20~1 of the sample buffer described by Laemmli (1970) which contained SDS and a trace of solid bromophenol-blue dye, then heated for 3 min in a boiling water bath.
These samples were examined by discontinuous P~GE
(Laemmi, 1970~ with Coomassie blue staining. The structural proteins of the virus, separated by SDS-PAGE, were also examined by the Western blotting procedure described by Burnette (1981). The viral proteins were transferred to nitrocellulose membrane-filter (Schleicher and Sch~ll BA83 0.2~m) and probed with chicken antisera diluted 1: 500 in 1%
(w/v) gelatin in NaC1-Tris buffer. The chicken antibodies binding to viral polypeptides were identified with rabbit IgG anti-chicken IgG (Cappel Labs, Cochranville, USA) diluted 1:1000 -n NaC1-Tris-gelatin buffer followed by I~Ci of 125I-Protein A (Amersham, U.K.) in the same buffer.
An autoradiograph of the nitrocellulose membrane was prepared using Fuji Rx Medical X-ray film and Ilford Fast Tungstate intensif~ing screens usually for 16-24 h at -70C.

Detection of_IBD virus by the RMA content of various fractions.

Sucrose-gradient fractions were diluted to 1:4 with 10 mM Tris-HCl, 50 mM NaCl, 0.2~ SDS buffer, pH 7.05 and treated with 0.5 mg/ml ribonuclease-free Pronase (Worthington, USA) for 1 h at 37C. The solutions ~25~ Z

were made 0.3 M with respect to NaCl and the nucleic acids extracted with 1 volume of phenol at 56C for 5 min. One volume of chloroform was added to the mixture which was then shaken at room temperature ror 10 min before being centrifuged at 12,0Q0 rpm for 2 min in an micro-centrifuge (Eppendorf, ~J.Germany~.
The nucleic acids in the upper aqueous phase were recovered by precipitation with 2 volumes of ethanol at -20C for 30 min followed by centrifugation. The R-I~A pellets were washed thoroughly with 67% tv/v) ethanol, dried and then dissolved in ~0~1 water.
Samples of RNA were electrophoresed under non-denaturing conditions in 1% (w/v) agarose slab gels in 20 mM phosphate buffer, pH 6.8, together with ~DNA standards (Boehringer, W.Germany). When the gels were stained with acridine orange and illuminated with W light, the ds-DNA or ~IA appeared as green bands while single-stranded (ss) R~A appeared as red bands (McMaster & Carmichael, 1977).

RESULTS

Purification of I~DV from infected bursae Following centrifugation of the clari~ied bursal homogenates in 25 to 50% continuous sucrose gradients, a major band of material was visible approximately 3/5 the way down the gradient. SDS-PAG~ analysis of the sucrose fractions indicated that the highest concent~ation of ~iral proteins was in the visible band, while fractions immediately above and below the 2,S2~2 major band contained lesser amounts of the viral proteins. Similarly, the ~LISA for viral antigen revealed peaks of IBD viral antigen throughout the gradient, although the visible band again contained the highest relative concentration.

The electrophoretic profiles of total RNA from different sucrose-gradient ~Eractions located 2 segments of viral RNA in fractions 3 and 4, corresponding to the major visible band and in fraction 9 near the top of the gradient (Fig.l).
Colour reaction with acridine orange showed that the two viral ~A segments were double-stranded. Fraction 10 contained only low ~^l RNAs (e.g. tRNA) and fractions 6-9 contained mainly ribosomal RNA (18S and 28S) and low ~l RNAs. The viral ds-RMA bands were much more resistant to ribonuclease digestion than the ribosomal ss-RNAs. When electrophoresed under non-denaturing conditions with ds-~DNA as standsrds, the two viral RNA segments appeared to have molecular weights of 2.5 x 106 and 2.2 x 106 respectively.

CsCl density-e~uilibrium centrifugation of complete virus from the continuous sucrose gradients revealed one major band which was visible under reflected light and had a mean buoyant density of 1.33 g/ml. When the crude virus from the interface of a 40-60% stepwise sucrose gradient was further purified on CsCl, a second, less dense, band was frequently seen which appeared by electron microscopy to contain a high proportion of "core" particles.

~ zs~2o~æ

The effect of the duration of infection on the yield of virus No band of IBD virus was visible in CsC1 gradients of virus from bursae harvested from chickens 2 days after infection. A distinct band of virus was visible in CsCl gradients of bursae from chickens infected for 3 daysy but was more diffuse when the virus was purified from bursae harvested 4 days after infection. An ELI5A for IBD viral antigen also found maximum titres of antigen in bursal homogenates obtained 3 or 4 days after infection. Consequently, virus was routinely prepared from bursae that were collected 3 days after infection.

Amino acid analysis of an acid hydrolysate, assuming a mean amino acid-residue weight of 110, showed that up to 250~g of viral protein could be obtained from a single bursa.

Coomassie-blue staining of SDS-PAGE of purified virus As shown in Fig.2, purified preparations of intact virus contained 2 major polypeptides with approximate MW of 37 ~d and 32 Kd, and 3 other components (arrows in Fig.2) with approximate M~ of 91.5 Kd, 41.5 Kd and 2q Kd. Although the polypeptide of ~ 32 Kd was a major component of all preparations of virus, densitometer tracings from polyacrylamide gels of different preparations of virus revealed that the ~L'2,5~2 relative amounts of the polypeptides varied between preparations.

The kinetics and specificity of the primary antibody res~onse of chickens infectled with IBD virus.
L

The appearance of serum antibody -to IBD virus, as determined by ELISA, was followed in 6 SPF chickens infected intraocularly at 6 weeks of age with isolate 002/73. Antibody was first detected on day 5 (mean titre 250), rising quickly to a mean titre of 10,000 on day 10 and 17,~00 on day 14. The analyses by Western blotting of the sera ob-tained from one of these chickens is shown in Fig.3. Antikody binding to the 32 Kd polypeptide of IBD virus was detected on day 5, with the intensity of binding increasing with time after infection. The antibodies present in the circulation of this chicken remained relatively specific ~or the 32 Kd polypeptide, at least until day 14 of the response. ~estern blots of the antibody present in sera obtained 14 days after infection from all 6 chickens are shown in Fig.~ (tracks 1-6). The sera from 5 of these 6 chickens demonstrated specificity for the 32 Kd polypeptide, even when the autoradiographs were exposed for 7 days. By 28 days after a primary infection the chickens had produced antibodies to all I~D virus polypeptides, except the 91.5 Kd polypeptide ~Fig.4, track 7~.

Response of SPF chickens to vaccination with an inactivated oil-emulsion IBD vaccine Six chickens, when 5 ~eeks of age, were injected intramuscularly with 0.5 ml of a commercial inactivated whole virus vaccine. The sera from the 2 chickens with the highest ELISA titres at 8 weeks post-vaccination (titres of 25, 600 and 51, 200 respectively) were analysed by Western blotting. ~t 4 and 8 weeks after vaccination the primary antibody response of both chickens to the inactivated vaccine was relatively specific for the 32 Kd polypeptide of IBD
virus (Fig.5, tracks a and b). By 4 weeks after a second intra-muscular injection of inactivated vaccine at 13 weeks of age, however, both chickens had produced serum antibodies which reacted with the 3 most abundant structural proteins (Fig.5, tracks c)~
Response of sensitised chickens to an inactivated oil-emulsion IBD vaccine Chickens that had been given live IBD virus at 5 weeks of age were injected at 25 weeks of age with a commercial inactivated vaccine~
Sera were obtained 4 weeks and 20 weeks after the primary infec-tion and then 4 and ~ weeks following revaccination. Analysis by Western blotting showed that initially there had been a response to 4 of the structural proteins, which had waned by 20 weeks post-infection (Fig.6). An injection of inactivated vaccine at thattime resulted in a heightened response to all the IBD viral poly-peptides, including the 91.5 Kd polypeptide (Fig.6, track d).

~L~5~

The following detailed description describes experiments detected towards defining the major immunogen of IBD virus and assessing the protective efficacy o antibodies to that immunogen in vitro and in vivo. In the accompanying drawings:
Figure 7 shows Western-blots of immune serum collected from three 10-week-old chickens (tracks 1, 2 and 3), 1~ days after in-fection with IBD virus (002/73). Nitrocellulose strips reacted with 25~1 of serum diluted 1:100. Hyperimmune serum (track 4) included as a positive control.
Figure 8 shows (a) Protein profile (OD 280nm) of an S200 column fractionation of day 10 immune serum from a 10-week-old chicken infected with IBD virus (002/73).
(b) Antibody activity de~ectable by ELISA (OD 450nm) in a 1:10 dilution of each fraction.
Figure 9 shows Western-blots of whole virus with pools of the (a) IgM, and (b) IgG fractions of 10 day immune sera obtained from two 10-week-old chickens (1 and 2) infected with IBD virus (002/73), 0 Figure 10 shows ~estern-blots of sera from adult chickens obtained 3 weeks after immunisation with approximately 50~g of puri-fied structural polypeptides oE IBD virus (002/73). Chickens A, B and C immunised with 32 Kd polypeptide: D, E and F

~L2~2C3~2 immunised with 37 Kd polypeptide; G, H and I immunised with 41.5 Kd polypeptide. Track K reacted with hyperimmwne serum.
Amersham C14 MW markers on left-hand side.
Figure 11 shows Western-blots of 2 anti-32 Kd polypeptide sera (A
and B) before (a) and after adsorption with (b) the 37 Kd polypeptide or (c) the 41.5 Kd polypeptide and of 2 anti-37 Kd polypeptide sera (E and F) before (a) and after adsorption with (d) the 32 Kd polypepticle. The extraneous antibody activity removed by adsorption is arrowed on each original serum (a). Refer to Table 5.
Figure 12 shows Western-blots of day 10 immune serum from a chicken infected with 002/73 (L) and day 28 immune serum from a chicken immunised with inactivated vaccine (K) before (a) and after adsorption with the (b) 32 Xd, (c) 37 Kd or (d) 41.5 Xd polypeptides. Refer to Table 5.

MATERIALS AND METHODS
Animals White Leghorn chickens of the "CSIRO-W" line were supplied by the CSIRO Specified Pathogen Free (SPF) Poultry Unit, Maribyrnong, Victoria and transferred into flexible-film plastic poultry iso-lators, Dennett and Bagust (1979) at one-day-old.

A~L

Virus The Australian isolate of IBD virus (002/73) used in these studies was originally described by Firth (1974) and identified sero-logically as IBD virus at the Central Veterinary Laboratory, Weybridge, U.K. Following one passage at limiting dilution of infectivity, the virus was routinely passaged by intraocular (i~o.) infection of ~ to 6 week old SPF chickens. Virus infect-ivity was titrated by inoculating 3-day-old SPF chickens i.o. with 25~1 of log1o dilutions of a 10% (w/v) homogenate of infected bursae. The bursae were harvested from 'hese chickens 72h later, homogenised and IBD viral antigen detected by an enzyme-linked immunosorbent assay (ELISA). The titre of virus was expressed as the reciprocal of the dilution of virus that infected 50~ of the chickens inoculated (CID50).
IBD virus adapted to propagate in chick embryo fihroblast (CEF) culture was initially made available by A. Webster Pty. Ltd., Sydney, Australia. The virus has been designated TC-IBD virus (GT101) and was routinely passaged in CEF cultures. GT101 virus is neutralised in vitro by chicken antisera to type-1 IBD, virus, but not type-2 IBD virus (unpublished data - Central Veterinary Laboratory, Weybridge, UK).
Purification of virus IBD virus (002/73 was grown in bursae then purified as described above. Briefly, a 50~ homogenate of the infected bursae in O.OlM
Tris-HCl, 0.15M NaCl, pH 7.6 (TBS) was frozen/thawed and homogenised with an equal IL25j2~

volume of the fluorocarbon Arklone (Wertheim Labs., Melbourne, Australla). The clarified aclueous phase was centrifuged on s-tepwise gradients of ~0% and 60 (w/v) sucrose. The sucrose interface was collected and centrifuged on preformed 25~ to 50gO (w/v) CsCl gradients. The purified intact virus banded at a density on CsCl of 1.33g/ml.

Polyacryl mide gel electrophoresis ~PAGE~ and immunoblotting of viral proteins.

Viral polypeptides were analysed in 12.5% (w/v) polyacrylamide slab gels using the discontinuous SDS
gel system of Lael~mli ~1970), then transferred from the gel onto nitrocellulose paper by the Western blotting technique described by Burnette (1981~ and reacted with chicken antibodies as described above.
Brieflyr the nitrocellulose membrane filter was blocked with a 5% ~w/v) solution of dried skim milk powder (blotto) in TBS (Johnson, et al, 1984), cut into 5mm strips then reacted with a 1:100 dilution of chick antisera in blotto, followed by a 1:1000 dilution of rabbit anti-chicken IgG (Cappel Labs., Cochranville, USA) in blotto and finally 1 ~Ci of 125I-Protein A (Amersham, U.K.). Autoradiographs were prepared using Fugi Medical X-ray film and Ilford Fast Tungstate intensifying screens.

Purification of the structural pol~peptides OL IBD
virus.

~L~52~

Purified virus was boiled with SDS for 2 min in the absence of reducing agents and the peptiAes separated by SDS-PAG~. The gels were lightly stained with Coomassie-blue, destained and the 29 Kd, 32 Kd, 37 Kd, 41.5 Kd and 91.5 Kd bands of protein cut from the gels. The polypeptides were eluted from the gel strips into 0.05M Tris-acetic acid buffer (pH8.0) containing 0.1% (w/v) SDS at 50 volts for 40h. The eluted polypeptides were d:ialysed against multiple changes of distilled H2O for 48h and the approximate concentration of the polypeptides assessed by SDS-PAGE
against known concentrations of MW markers (Pharmacia, Sweden). The purity of the polypeptides was assessed by Western-blotting with hyperimmune chicken serum.

Chicken antisera to whole virus or the structural polypeptides of IBD vir Six to ten-week-old SPF chickenc were inoculated i.o.
with infectious virus (002/73) and bled 10 and 14 days later. The serum was collected by centrifugation and stored at -20C. SPF chickens were also injected intramuscularly (i.m.) with 0.5ml of a commercial inactivated IBD vaccine (Arthur Webster, Pty.Ltd.), and sera collected 28 days later, a-t the peak of the primary antibody response.

Adult SPF chickens were injected i.m. with approximately ~O~g of either the 32 Kd, 37 Kd or 41.5 Kd polypeptides of IBD virus emulsified in 2 ii20~

volumes of Freund's complete adjuvant (FCA) (Difco, U.S.A.). Three weeks later the chickens were reinjected i.m. with the same amount of the respective polypeptides in Freund's incomplete adjuvant (FIA) (Difco). Approximately 40ml bleeds were collected at approximately weekly intervals into preservative free heparin (a final concentration of lOU/ml~ and the plasma collected and fro2en at -20C. When the plasma was thawed, the fibrin clot was removed by centrifugation. Other chickens were immunised with the 29 Kd and 91.5 Kd polypeptides, except that the amounts of polypeptide injected were very much less and the antibody responses were correspondingly weak.

ELISA for anti-IBD virus antibody and IBD vlral anti~en.

Anti-IBD virus antibody in chicken sera and IBD viral antigen in bursal homogenates were both quantitated using the ELISA as described above.
-Plaque-reduction serum neutralising (SN) assay Serial dilutions of heat inactivated 56C/30 min) serum in biocarbona~e buffered medium l99 (Gibco, USA) were added to an equal volume of TC-IBD virus, which had been diluted to approximately ~00 plaque-forming units (pfu)/ml. Virus-serum mi~tures were incubated at room temperature for 60 min with occasional shaking and then 0.lml of each mixture inoculated into 3 secondary CEF cultures (35mm diam.plastic petri ~L25;2(~2 dishes, Kayline, Sth.Australia) to test for residual viral infectivity. The virus was allowed to adsorb to monolayers for 60 min at 37C before each dish was overlayed with 2ml of 0.7~O (w/v~ agar (Baco-Difco, USA) in HEPES (0.015 M) buffered medium 199 containing 5% (w/v) calf serum~ The cultures were incubated at 37C for a further 6 days and stained by the addition of 0.15~o (w/v) neutral red -in a lgo agar overlay. The end-point of the neutralisation assay was the dilution of serum which caused a 50~ reduction in the number of IBD virus plaques.

The Micro-SN assay Serial dilutions of inactivated serum were prepared in a volume of 25~1 of 199 containing 10% (w/v) TPB
(Difco) and 2~o heat inactivated fetal calf serum in flat-bottomed microtiter trays (Linbro, USA) before an equal volume of TC-IBD virus (500 pfu/ml) was added and incubated at 37~ for lh. Following incuba-tion, 50~1 of a 7.5 105 cell/ml suspension of secondary CEF
cells were added and the trays incubated at 37 DC for 5 days. The trays were then stained with 1~ (w/v) crystal violet in methanol and the end point read as the last dilution to completely inhibit virus replication.
assive protection of chickens with specific antibody r Chickens were injectcd intraperitoneally (i.p.) at 2 days of age with various immune serum or control serum 2s~

free of antibody to IBD virus. One day later the chickens were challenged i.o. with 25~l of bursal homogenate, usually containing a minimum of 1000 CID50 of IBD virus 002/73. Three days after infection, the chickens were exsanguinated and their bursae re-moved, weighed and made into a 5% homogenate in saline. Both the levels of antibody in the sera and the presence of viral antigen in the bursae were quantitated by ELISA.
Column chromatography Five ml of immune serum was separated on a 2.5cm x 100cm S200 (Pharmacia) column by elution with 0.01M phosphate, 0.15M NaCl, pH
7.6 (PBS). The fractions were assayed for antibody activity by ELISA before the IgM region and IgG regions were pooled, deleting one 6ml fraction overlapping the two regions. The pools were concentrated to 4ml using an ~M100A membrane (Amicon, USA) and sterile filtered.
Affinity chromatography Only the 32 Kd, 37 Kd and 41.5 Kd polypep~ides were obtained in sufficient quantity to prepare absorption columns. The polypep-tides were obtained from unstained gels, guided by Coomassie stained strips taken from each margin of the gel. The purity of the eluted polypeptides was assessed by Western blotting with hyperimmune serum. The polypeptides were quantitated by a Lowry protein assay (Hartree, 1972) and between 150 and 300 ~g of the respective polypeptides were reacted with lml of ~25~

Affigel ~0 (Biorad, USA) according to the manufacturers instructions.

One ml of each antipolypeptide sera was run slowly through the adsorption columns and eluted with 3 volumes of PBS. The columns were reactivated by passing 5ml of lM proprionic acid through the columns followed by 20ml of PBS. sera from chickens immunised with whole virus or inactivated vaccine were adsorbed by running 0O25ml of serum onto the columns and eluting with 1.75ml of PBS. The effectiveness of the adsorption was assessed by immunoblotting.

P~ESULTS

Passive protection with monospecific anti-32 Kd sera When antisera obtained from 6-week-old chickens, 14 days after the had been infected with IBD virus, were analysed by ~estern-blotting~ 5 were found to contain only antibodies specific for the 32 Kd structural polypeptide. The sera had E~ISA titers between 6,620 and 51,200 and virus neutralisation titers of 6,250 or greater (~able 1~. The ability of these sera to passively protect chickens was assessed by injecting lml of each serum into each o four 2-day-old chickens, which were challenged 1 day later with 1000 CID50 of 002/73. All the chickens that received immune serum resisted infection and had circulating antibody when exsanguinated 3 days after challenge, while those that received antibody negative serum had ~f~Pkfi~ /S

~ 29 -no antibody and were susceptible to infection (Table 1).
The experiment was repeated with day 10 and day 14 immune serum from three 10-week-old chickens infected with IBD virus. Again these sera were monospecific by Western-blotting (Fig.7) and had ELISA titers between 57,000 and 77,000 at day 10 and 70,000 and 166,000 at day 14. Two ml of each serum was injected into each of 3 chickens. In addition to the groups of chickens that were challenged with 1000 CIDso of 002/~3, small groups oE chickens that received either pooled immune serum or control serum remained unchallenged and served as in-contact controls. Neither the chickens that received immune serum and virus (Table 2) nor the in-contact chickens were infected. The chickens that were given normal chicken serum and virus were all susceptible to infection.
Characterisation of the protective antibodies in anti-32 Kd serum When sera from two 10-week-old chickens, obtained 10 days after infection with IBD virus, were fractionated b~ S200 column chroma-tography and assayed for antibody detectable by the ELISA, all activity was confined to the IgG region of the column profile (Fig.8). The IgM region and ~he IgE regions were pooled and titrated by the micro-SN assay. Both the IgM and IgG pools of these sera were found to have virus neutralising activity; The IgG pool having t~ice the SN-titer of the IgM pool (~able 3,.
Immunoblotting whole virus with these antibody pools showed that this assay also primarily detected IgG antibodies which were specific for the 32 Kd polypeptide (Fig.9).
One ml oE the IgM and IgG pools from each serum were injected into groups of four 2-day-old chickens, which were then challenged 1 day later with 10 CIDso f virus. The IgG antibody pools were found to confer protection while chickens injected with the IgM
pools of antibody were all susceptible to infection (Table 3).
Antibody responses to purified viral polypeptides Adult SPF chickens did not produce substantial titers of antibody that could be detected by either the ELISA or micro-SN assay during the early stages of the response to approximately 50~9 of either the 32 Kd, 37 Kd or 41.5 Kd purified viral polypeptides in FCA. Western-blotting with serum obtained 3 weeks after immuni-sation, however, showed that the chickens had synthesised anti-bodies to their respective po~ypeptides (Fig.10). Chicken B, which produced the strongest response to the 32 Kd polypeptide, also produced antibodies to the 37 Kd and 41.5 Kd polypeptides (Fig.10, track B) and the sera from the 3 chickens (~, E and F) injected with the 37 Kd polypeptide were almost indistinguishable from sera from the 3 chickens (G, H and I) injected with the 41.5 Kd polypeptide ~2S2~

(Fig.10, tracks D to I). Tt was noted that all of the chickens (D to I~ injected with either the 37 Kd or 41.5 Kd polypeptides also produced antibodies that reacted with low MW material on the blots, material that was not recognised by hyperimmune serum from vaccinated chickens ~Fig.10, track K).

One week after a second injection of viral polypeptides in FIA, 1 of the 3 chickens injected with the 32 Kd polypeptide had a micro-SM titre of 256 (Table 4). Further bleedings at 3, 4 and 6 weeks after the second immunisation showed that 2 of the chickens injected with the 32 Kd polypeptide had neutralising titers for IBD virus of between 160 and 1280, while those injected with the 37 Kd or 41.5 Kd polypeptides had neutralising titers of 40 or less.
One o:F the chickens injected with the 41.5 Kd polypeptides had the highest ELISA titers followed the second injection of polypeptides (Table 4).

Affinity chromatography of an-ti-pol~peptide sera Probing ~estern blots of the virus with sera o~tained 3 to 6 weeks after the second injection of the purified polypeptides showed that they all contained antibodies to the other polypeptides. Monospecific antisera were prepared by passing antipolypeptide sera through adsorption columns prepared by binding the various viral polypeptides to Affigel-10.

~,~52~2 Passing the anti-32 Kd sera through a 37 Kd column removed all antibody acti~ity to the 37 Kd and 41~5 ~d polypeptides (Fig.ll, A and B), but did not reduce the micro-SN titre (Table 5). The 41.5 Kd column was less efficient, but gave similar results in that it did not reduce the SN-titer of the anti-32 Kd sera. Passing the 2 anti-37 Kd sera with the highest S~ activity, through the 32 Kd adsorpticn column removed all anti-32 Kd antibody (Fig.Ll, E and F) r but did not reduce the low levels of SN antibodies present in these sera (Table 5). The mcre discriminating plaque-reduction assay confirmed that passing the anti-3~ Kd sera through the 37 Kd column, did not reduce the activity in the sera ~Table 6). Passing the antipolypeptide sera through the homologous adsorption column either had no detectable effect on the micro-SN titre or reduced it by no more than 50~0 (Table 5).

Day ln serum from an infected chicken and day 28 serum froma chicken injected with an inactivated I~D
vaccine, were also passed through the absorption columns. In neither case did the 37 Kd and 41 5 Kd columns affect the Western-blotting patterns (Fig.12) or the micro-SN titers of the serum (Table 5). Passing the serum through the 32 Rd col~mn, however, mar~edly reduced the intensity of the Western-blotting pattern (Fig.12) and reduced the micro-SN titre of the sera by half (Table 5~. In an attempt to improve the efficacy of absorption, day 10 serum was diluted 1:100 prior to being passed through the 32 ~d column. In this case, ;20~2 the plaque-reduction assay tTable 6) showed that the virus neu-tralising activity in a 1:20,000 dilution of serum was reduced by almost 50%.
Synergism between anti-polypeptide sera Mixing equal volumes of an anti-32 Rd serum, which did not have a detectable micro-SN titre, with 3 different anti-37 Kd sera, did not enhance the SN titre of the mixtures more than could be accounted for by the activity of the anti-37 Kd sera alone (Table 7). Similarly, mixing an anti-32 Kd serum which had a micro-SN titre of 320 with the 3 anti-37 Kd sera, resulted in mixtures with SN titers which could be completely accounted for by the activity of the anti-32 Kd serum (Table 7).
Passive protection with anti-32 Kd polypePtide serum The concentration of antibody in 1 to 2ml of anti-32 Kd poly peptide serum was found to be too low to produce detectable levels of circulating antibody when injected i.p. into young chickens.
An (NH~)2SO4 precipitate of 20ml of anti-32 Kd polypeptide serum (Chicken B) was prepared, redissolved in ~ml of PBS, sterile filtered and lml injected each of 3 chickens of 2 days of age.
These chickens, together with 3 control chickens, were challenged one date later with 10 CIDso f 002~73, exsanguinated 3 days after challenge and their bursae and serum assessed by the ELISA. The 3 chickens that received the precipitated antibody had residual - 33a -ELISA titres between 160 and 320 and 2 of them had no detectable viral antigen in their bursae. The 3 control chickens had no detectable antibody and all had ELISA titres of viral antigen ~128.

~eutralisation of ty~e-1 IBD viruses by chicken anti-serum specific for the 32 Kd polypeptide Five S~F chickens of 7 weeks of age were infected with IBD virus (002/73) and bled on days 10, 11 and 12 post-infection. The 15 individual sera were assessed by Western-blotting, and all con-tained antibodies specific for the 32 Kd polypeptide of IBD virus.
The sera were pooled and sent to the Central Veterinary Labora-tory, Weybridge, UK. When assessed by the serum neutralisation assay, the pooled serum was found to neutralise the type-1 strains PGB-98, Cu-1 and G-13 in addition to GT-101, but not the type-2 IBD virus strain Ty-89.

Table 1. Passive pro~ection of chickens wiLh serum conLaining anLibodies specific for Lhe 32k polypep~ide of IBD v;rus (002/73) Group Antibody titers of Mean ELI~SA titers of t No. donor serum*
__ _ _ .
viral antigen in residual antibody _ ELISA S~ bursae in circulation 1 <20 <?0 >256 <20 2 6,620 6,250 <1~477 317,000 >6,250 <11,830 422,300 >6,250 <11,740 525,600 >6,250 <11,930 6>25,600>6,250 <13,340 * Each group of 3 chickens of 2 days of age was injected i.p. ~i~h 1 ml of serum from 5 different donor chickens obtained 14 days after they had been infected with IBD virus (Groups 2-6) or antibody negative serum from an SPF chicken (Group 1). Western blots of immune serum - shown in Fig.4.
t The chickens were challenged i.o. with 1000 CIDso of IBD virus 1 day after receiving the serum and then exsanguinated 3 days later. The bursae were examined by ELISA for the presence of IBD viral antigen and the corresponding sera assayed for residual specific antibody.
<1 = undiluted bursal homogenate.

2~Z

Table 2. Passive protection of chickells wi~h serum containin~ antibodies specific for the 32k polypep~ide of I~D virus*

. ._ Mean El ,ISA titers of Donor Serum ELISA titer No of Virus viral anLigen residual antibody Type-Number of Donor Serum Chickens Inoculated in bursae in circulation . __ _ .
SPF <5 3 _ <1 <5 SPF <5 3 + >12$ <5 Day 10 Immuue 6357 77,000 3 + <1 3,900 6362 58,000 3 + <1 1,900 6-366 76,000 2 + <1 3,000 Pool N.T. 2 _ <1 2,200 Day 14 Immune . ..__ 6357 166,000 3 + <1 5~800 6362 78,000 3 + <1 6,600 6366 70,000 2 + <1 3,800 Pool N.T. 2 _ <1 8,000 * Experimental design as per Table 1, excepc that groups of 3 chickens received 2ml of serum i.p., which was lethal for some chickens and some of the groups remained unchallenged, but in contact with the challenged chickens. Western-blots of sera shown in Fig. 7.
N.T. Not tested.

~2szl~4~ .

Table 3. Passive protcction of chickens* with IgG and IgM anLlbodies specific for the 32k polypeptide of IBD virus .
Donor Titer of antibody t Mean ELISA titer of Antibody in donol serum viral antigen residual antibody - Serum No.ELISA Micro-SN in bursal in circulation .. . , .. __ IgM-1 <40 3,200 >128 N.A.
IgM-2 <40 12,800 >128 N.A.
IgG-1 12,800 6,400 <8 515 IgG-2 25,600 25,600 <8 800 . _ , `

* Groups of 3 chickens of 2 days of age were injected I.P. with IgM or IgG antibody obtained from 2 donor chickens, 10 days after being infected with IBD virus. The antibodies, particularly the IgG antibodies, were specific for the 32k polypeptide of IBD virus (Fig.9 ).
t The chickens wer-e challenged at 3 days of age with 10 I~so of 002/73 and exsanguinated 3 days later. The bursae were homogenised and assessed by ELISA
for the presence of antigen and the sera assayed for residual IgG antibody.
N.A. Not applicable.
-~ 2 5~7 Table 4. Antibody responses of SPF chickens immunised wiLh Lhe 32k, 37k or 41.5k structural polypepLides of IBD virus Tlme after ~InmunlsaLion* (weeks~
Antigen Antibody Preparation Assay . _ 32k polypeptide Chicken A ELISA <100 <100<100100 300 300 Micro-SN <4 4 4 320 160 320 Chicken B ELISA <100 <1003006001,200 800 Micro-SN <4 4 2561,2801,280 640 . _ .
37k polypeptide Chicken E ELISA <100 <1008004001,200 1,200 Micro-SN <4 4 ~, 40 40 20 Chicken F ELISA <100 <1003001,2001,6001,600 Micro-SN <4 <4 8 20 40 40 . _ .
41.5k polypeptide Chicken G ELISA <100 <100674006,40012,8009,600 _ Micro-SN <4 <4 4 40 10 20 Chicken H ELISA <100 <1004001,6001,600 800 Micro-SN <4 <4 <4 20 <10 <20 * Three adult SPF chickens injected i.m. with approximately 50~ of the respective polypeptides in FCA. Three weeks after the first immunisation they were reinjected with the polypeptides in FIA. Only 2 of each group of 3 chickens synthesised antibody detectable by the micro-neutralisation assay.

~5j2C~Z
38 ~
Table 5. The effect of adsorption with the viral polypepLi _s on the micro-SN titer of antisera from chickens immunised with . _ _ _ _ _ IBD viral polypeptides, inactivaLed whole virlls vaccine or live virus.

_ . . _ SN-titer post-adsorption with viral polypeptides of mol wt Antisera Original SN-titer _ .
32k 37k 41.5k Anti-32k _ _ _ _ __ .
1,280t 1,280 1,280t 1,280t B 2,560t 1,280 2,560t 2,560t - . . _ Anti-37k _ 32t 32 64t N.T.
F 16t 16 32t N.T.
_ _ . __ Inactivated* 12,800 6,400 12,800 25,600 Vaccine _ . . _ Live** 51,200 25,600 102,400 102,400 Vaccine _ * Sera obtained 28 days* or 10 days** post-vaccinaLion and were specific for the 32k ** polypeptide by Western-blotting as shown in Fig. 12.

t Western-blots of sera pre and post-adsorption shown in Fig.ll.
N.T. NoL tested.

~252(11~
39 ~
Table 6. The effect of adsorption wlth the viral po]ypeptldes on Lhe percenta~e plaque-reduction of dilutions of antisera from chickens immunised with IBD viral polypeptides, inactivated whole virus vaccine or live virus % p1aque-reduction following Dilution % plaque- adsorption with viral Antisera of serum reduction polypeptides of mol w~

_ 32k37k 41.5k Anti-32k At 1:2G0 61.5 61.5 92.3 N.T, Bt 1.200 92.3 88.5 80.8 N.T.
_ Inactivated 1:16,000 69.2 42.3 80.8 61.5 Vaccinet Live 1:20,000 80.8 80.8 84.6 N.D.
Vaccinet 42.3 .

t Antisera as per Table 5.
* Antiserum diluted 1:100 prior to adsorption.
N.T. Not tested.

,~ZS;2~

--'10 --Table 7. Micro-SN antibody tlL~rs of 1:1 mixtures of anti-32k and anti-371c sera -Anti-37k sera .__ . .
Anti-~2k Original SN-titers 40 40 20 . . - . .. _ C <20 20* 80* 20*
A 320 320* 160* 160*
.
* Micro-SN titer of 1:1 mixtures have been doubled for direct comparison with titer of original sera.

~2~

REFERENCES
Burnette, W.N. 1981. "Western Blotting": electro-phoretic transfer of proteins from sodium dodecyl sulfate-polyacrylamide gels to unmodified nitrocellulose and radiographic detection with antibody and radioiodinated Protein A. Analytical Biochemistry, 112: 195-203.
Dennett, D.P. and Bagust, T.J. 1979. Application of a flexible-film isolator for rearing specific pathogen-free chickens and investigating poultry pathogens. Avian Pathology, 8:
289-300.
Dobos, P. 1979. Peptide map comparison of the proteins of infectious bursal disease virus. J. Virol., 32: 1046-1050.
Firth, G.A. 1974. Occurrence of an infectious bursal syndrome within an Australian poultry flock. Aust. Vet. J.,. 50:
128-130.
Hartree, E.F. 1972. Determination of protein: a modi-fication of the Lowry method that gives a linear photometric res-ponse. Analytical Biochem. 48: 422-427.
Johnson, D.A., Gautsch, J.~., Sportsman, J.R., and Elder, J.H. 19~4. Improved technique utili~ing nonfat dry milk for analysis of proteins and nucleic ~5~1~42 acids transferred to nitrocellulose. Gene Anal.Techn., 1 : 3-8.

~ aemmli, U.X. 1970. Clevage of structural proteins during assembly of the head of the bacteriophage T4. Nature (Lond.), 227 : 680-685.

McMaster, G~K., and Carmichael, G.G. 1977.
Analysis of single- and double-stranded nucleic acids on polyacrylamide and agarose gels by using glyoxal and ocridine orange. Proc.Natt.Acad.Sci., U.S.A., 74 : 4835-4838.

Muller, H. and Becht, H. 1982. Biosynthesis of virus-specific proteins in cells infected with infectious bursal disease virus and their significance as structural elements of infectious virus and incomplete particles. Journal of Virology, 44 :
384-392.

~ Nick, H., Cursiefen, D. and Becht, H. 1976.
Structural and growth characteristics of infectious bursal disease virus. J.Virol., 18 : 227 234.

Stone, H.D., Brugh, M., Hopkins, S.R., Yoder, H.W., and Beard, C.W. 1978. Preparation of inactivated oil-emulsion vaccines with avian viral or mycoplasma antigens. Avian Diseases, 22 : 666.

Todd, D. and McNulty, M.S. 1979. Biochemical studies with infectious bursal disease virus ~25~2 comparison of some of its properties with infectious pancreatic ne~rosis virus. Archives Virol., 60 :
265-277.

Wells, P.~., Gilmour, N.J.L., Burrells, C. and Thompson, D.A. 1979. A seralogical comparison of Pasteurella haemolytica vaccines containing dif erent adjuvants. Res.Vet.Sci., 27 : 248-250.

Wells, P.W., Emery, D.L., Hinson, C.A., Morrison, W.I. and Murray, M. 1982. Immunisation of cattle with a variant-specific surface antigen of Trypanosoma brucei : influence of different adjuvants.
Infect. Immunity, 36, 1-10.

York, J.J., Fahey, ~.J. and Bagust, T.J. 1983.
Development and evaluation of an ELISA for the detection of antibody to infectious laryn~otracheitis virus in chickens. Avian Diseases, 27 : 409-421.

~ It will be appreciated that many modifications and variations may be made to the particular methods described above by way of illustration of the present invention, and that the present invention includes all such modifications which fall within the scope of the invention as broadly described above.

Claims (15)

THE EMBODIMENTS OF THE INVENTION IN WHICH AN EXCLUSIVE
PROPERTY OR PRIVILEGE IS CLAIMED ARE DEFINED AS FOLLOWS-

1. A non-infectious sub-unit vaccine for use against IBD
virus which comprises the structural polypeptide of approximate MW
32 Kd contained in the IBD virus, or an immunogenic peptide derived therefrom, together with, if required, an adjuvant.

2. A vaccine according to claim 1, comprising said 32 Kd polypeptide isolated from IBD virus.

3. A vaccine according to claim 1, comprising an immunogenic peptide comprising all or at least the major immunogenic determin-ants of said 32 Kd polypeptide.

4. A vaccine according to claim 1, wherein said 32 Kd poly-peptide or said immunogenic peptide is coupled to a carrier molecule to increase its immunogenicity.

5. A vaccine according to claim 1, 2 or 3, wherein said adjuvant is an aqueous-mineral oil emulsion.

6. A method for assaying the levels of protective antibodies against IBD virus in poultry, characterized in that the structural polypeptide of approximate MW 32 Kd contained in the IBD virus, or an immunogenic peptide derived therefrom, is used as an immunogen in the said assay.

7. A method for assaying the levels of protective immunogen in preparations of IBD virus produced for use as vaccines, charact-erized in that the structural polypeptide of approximate MW 32 Kd contained in the IBD virus, or an immunogenic peptide derived therefrom, is used as an immunogen in the said assay.

8. A method of assaying a protective antibody against IBD
virus in poultry, which method comprises treating a sample obtained from poultry with a structural polypeptide of approximate MW 32 Kd contained in an IBD virus, or an immunogenic peptide derived there-from, as immunogen, and observing if any interaction between said sample and said immunogen occurs, an interaction therebetween indicating the presence of said antibody.

9. A method according to claim 8 wherein the interaction is measured by ELISA or RIA.

10. A method of assaying a protective immunogen in a pre-paration of an IBD virus, which method comprises treating a sample suspected of containing said protective immunogen with a structural polypeptide of approximate MW 32 Kd contained in an IBD virus, or an immunogenic peptide derived therefrom, and an antibody to said polypeptide and observing if any interaction between said sample and said polypeptide or said immunogenic peptide occurs, an inter-action therebetween indicating the presence of said immunogen.

11. A method according to claim 10 wherein the interaction is measured by competition RIA.

12. A process for preparing a non-infectious sub-unit vaccine for use against IBD virus which comprises a structural polypeptide of approximate MW 32 Kd contained in the IBD virus, or an immunogenic peptide derived therefrom together with, if required, an adjuvant, which process comprises:

(i) (a) separating said 32 Kd polypeptide, or said immunogenic peptide, from IBD virus; or, (b) synthesizing said 32 Kd polypeptide or said immunogenic peptide with a genetic template coding for said 32 Kd polypeptide or said immunogenic peptide said template being foreign to an organism in which it is placed for expression; or, (c) condensing a reagent A which comprises a first peptide fragment of said 32 Kd polypeptide or said immunogenic peptide with a reagent B which comprises the balance of the 32 Kd polypeptide or the immunogenic peptide, reagents A and B being optionally protected and, if required, removing any protecting group to yield the 32 Kd polypeptide or the immunogenic peptide;
and, if required, (ii) mixing said 32 Kd polypeptide or said immunogenic peptide with an adjuvant.

13. A process for preparing a non-infectious sub-unit vaccine for use against IBD virus which comprises a structural polypeptide of approximate MW 32 Kd contained in the IBD virus, or an immunogenic peptide derived therefrom together with, if required, an adjuvant which process comprises separating said 32 Kd polypeptide or an immunogenic peptide derived therefrom from IBD virus and, if required, mixing said separated 32 Kd polypep-tide or said immunogenic peptide with an adjuvant.

14. The process of claim 12 or 13 wherein separation of said 32 Kd polypeptide or the immunogenic peptide derived there-from comprises electrophoretic separation.

15. The process of claim 12 or 13 wherein separation of said 32 Kd polypeptide or the immunogenic peptide derived there-from comprises sodium dodecyl sulphate-polyacrylamide gel electro-phoretic separation.