Physicochemical and immunological characterization of recombinant host-protective antigen (VP2) of infectious bursal disease virus A.A. Azad *++,N.M. McKern*, I.G. Macreadie*, P. Failla*, H.-G. Heine*, A. Chapman*, C.W. Ward* and K.J. Fahey* Small.fusions to the N-terminal end oJ' the host-protective antigen (VP2) of infectious bursal disease virus lead to stable expression of VP2 in Escherichia coli and ),east, and reduce the levels of inclusion body jbrmation in E. coli in comparison to VP2 constructs with larger N-terminal fusions. VP2 produced with small N-terminal fusions, like native viral VP2, can be .fi'aetionated into a hi qh molecular weight 'multimeric'./brm and a monomeric form. A virus-neutralizing monoclonal antibody that only recognizes undenatured VP2 preferentially reacts with multimeric jorms of recombinant VP2. Both native and recombinant monomeric jbrms of VP2 are non-immunogenic. The multimeric Jbrms of viral and yeast-derived VP2 are hi(4hh' immuno.qenic, while those produced in E. coli are not. Keywords: Infectious bursal disease virus; recombinant antigen;

E. coli: yeast; N-terminal fusions; immune response

INTRODUCTION Infectious bursal disease virus (IBDV), a member of the Birnaviridae family ~, is a pathogen of major economic importance to the world's poultry industries. It causes severe immunodepression in young chickens by destroying the developing B lymphocytes in the bursa of Fabricius 2'3. This leads to increased susceptibility to other avian pathogens'*. Young chickens can be protected by maternal antibodies transmitted through the egg yolk 5. Many of the currently used inactivated vaccines, employed for boosting anti-IBDV antibodies in laying hens 6, are produced from virus propagated in the bursae of specific pathogen free (SPF) chickens, greatly adding to the cost of production. A recombinant subunit vaccine against IBDV should overcome this problem. The bisegmented double-stranded RNA genome of IBDV strain 002-73 has been fully characterized, cloned and sequenced v 9. The smaller segment (~2900bp) encodes a 90 kDa polypeptide (VP1) that is believed to be a replicase 9'1°. The larger segment (~3200bp) encodes the structural proteins VP2 (41 kDa) and VP3 (32 kDa) and the non-structural protein VP4. These viral polypeptides are produced from a precursor polyprotein in which they are present in the order N-VP2-VP4-VP3-C *CSIRO Division of Biomolecular Engineering, 343 Royal Parade, Parkville, Victoria 3052, Australia. *CSIRO DMsion of Animal Health, Private Bag No. 1, Parkville, Victoria 3052, Australia. +*Towhom correspondence should be addressed: (Received 30 October 1990; revised 11 February 1991; accepted 27 February 1991) 0264-410X/91/100715-08 ~ 1991 Butterworth-HeinemannLtd

(ref. 8). The non-structural protein VP4 is involved in the processing of the precursor polyprotein of IBDV1 ~.12 similar to the processing function of VP4 of another birnavirus, infectious pancreatic necrosis virus ~3. The structural protein VP2 of IBDV strain 002-73 has a calculated molecular ratio of 52 kDa s, but migrates as two bands on SDS-polyacrylamide gels with apparent molecular ratios of 41kDa (VP2a) and 37kDa (VP2b)11.14. Peptide mapping studies have shown that VP2a is a precursor of VP2b (N. McKern, unpublished results). Deletion mapping studies of the large genomic segment have led to the identification of a discontinuous conformational epitope (145 amino acids) within VP2 that is recognized by virus neutralizing (VN) monoclonal antibodies (mAbs) 11. In another IBDV strain (Cul) it has been shown ~5 that VN mAbs can immunoprecipitate detergent-solubilized VP2. Isolated native VP2, but not denatured VP2, induced the production in chickens of VN antibodies that passively protected newly hatched chickens from IBDV infection 16 We have recently described the expression of IBDV VP2 in a highly immunogenic form in Saccharomyces cerevisiae 17"1s. In this report we describe the expression of IBDV VP2 in E. coli and Kluyveromyces lactis and compare the physicochemical and immunological properties of the recombinant antigens produced in yeast and

E. coli. MATERIALS AND METHODS E. coil expression clones

In all the E. coli constructs, the 1BDV VP2 sequence,

Vaccine, Vol. 9, October 1991 715

Characterization of recombinant IBDV antigen. A.A. Azad et al. Table

1

Expression

of I B D V

VP2

i n E. coil w i t h

various

lengths

of N - t e r m i n a l

fusions So]Ublhly ',, present

N

terminal 12K Pellet

.

.

.

.

12K S u p e l n a t e

Clone

Vector

Promoter

h~sert

PO"

pEX a°

/ P~{

VP2-VP4-VP3'

.- 110 kDa fi-gal

10O

PO XhoI-Pstl

pEX

/ PH

VP2

.- 110 kDa fi-gal

100

0

pUR VP2 pRIT5 VP2 ~'

pUR ~" pRIT5 ( P h a r m a c m )

/ Pt SpA

VP2 VP2

." ? 10 kDa /f-gal Protein A

90 70

10 30

POt"

pPL (Pharmacia)

/ Pt

VP2-VP4-VP3 a

.- 7 kDa KN Protein

50

50

P O 1 VP2 pTTQ18 VP2

pPL pTTQ18 { A m e r s h a m )

4 Pl Ptac

VP2 VP2

-~ 7 kDa KN Protein --- 0 3 kDa (MCS) c

50 20

50 80

pJLA602 VP2

pJLA60232

/ Pt

VP2

-~ 0 7 kDa (MCS)

20

80

pCQV2 VP2

pCOV2 a~

/

VP2

Met (from vector)

10

go

pEV VP2

pEV ~

/ P[

VP2

Met (from vector)

10

90

PH

fusion

.

Yield reactloq w d h '

II1

.

.

.

rl/Ab 6, I

0

.

.

.

mAb 9/6 *

Reaction ~1111 VN mAb 39A ! u n d e n a t u r e d VP2)

?

*

i

"The expressed polyprotein was cleaved at the VP2 VP4 junction to produce a VP2 molecule with N-terminal fusion'" ~'The expression product was probed with Fab fragments of the mAbs because of the presence of Protein A ' M C S multiple cloning site 'lThe extent of reaction of recombinant VP2 with the morloclonal antibodies was monitored by serial dot-blot analyses: ~ minimum intensity on dot-blot

+ - m a x i m u m intensity on dot-blot The

specific reactivities of lhe mAbs are described in the lext

minus the live N-terminal amino acids, was cloned in-phase into vectors which contained ribosome binding sites tit optimal distances from the promoters and provided at least the initiating methionine. The properties of the E. coil expression clones are described in detail in TaMe I. The construction of clones P0 and P0.1, which encode the IBDV polyprotein (N-VP2-VP4-VP3-C), have been described previously ~. In these clones the expressed polyprotein is cleaved to produce VP2 with either 110 kDa fi-galactosidase or 7 kDa of ),-N protein, respectively, at the N-terminus. Clones P0 X h o l - P s t l ~ and P0.1.VP2 were constructed from clones P0 and P0.1, respectively, by deleting the VP4 and VP3 encoding regions. Clone pUR.VP2 produced a fusion protein with a near full-length fi-galactosidase at the N-terminus of VP2. In clone pRIT5.VP2, the VP2 molecule was cloned tit the C-terminus of Protein A. In clones pTTQ18.VP2 and pJLA602.VP2, very small fusions (less than I kDa) from the multiple cloning sites of the vectors were added to the N-terminus of the VP2 molecule. In clones pCQV2.VP2 and pEV.VP2, only the initiating methionine was provided by the vector so that VP2 was produced essentially as tin unfused protein.

Yeast expression clones IBDV sequences, minus the first five N-terminal amino acids of VP2, were cloned into pYELC5 ~'), a vector designed for the copper-inducible expression of foreign proteins in yeast. The construction of the expression clones pYELC5.P0, p Y E L C 5 . P O A X h o l , and pYELC5. VP2T have been described in detail earlier ~8. Clone pYELC5.P0 encodes the IBDV polyprotein N-VP2-VP4VP3-C s. In clone pYELC5.POAXhol, the coding sequences for VP4 and VP3 have been deleted, but because of the absence of a cleavage site or a translation stop codon at the C-terminal end of VP2, about 12 kDa of irrelevant protein sequence derived from the E. coil sequences in the vector are added on the C-terminus of the VP2 molecule ~s. In clone pYELC5.VP2T, a translation stop codon has been introduced near the C-terminus of VP2 ~. In all the above constructs, the first live N-terminal amino acids of VP2 have been replaced by eight amino acids of CUP1 in pYELC5 ~s The growth and induction conditions for these clones have been described earlier 18.1,) Clone p E I . c u p l B . V P 2 T was constructed by cloning

716

Vaccine, Vol. 9, October 1991

the cupl-VP2 resort from the S. cererisiae clone pYELC5.VP2T 1~ into the K. lactis expression vector pE12°. K. lactis cells were transformed with this vector essentially as described by Bianchi et al. 2~, except that protoplasts were prepared by treatment with 1 mg ml Novozyme 234 (Novo Biolabs, Denmark) for 10 min tit 3 0 C instead of Cytohelicase. K. lactis strain MW98-9C was obtained from Dr H. Fukuhara (Institut Curie, Orsay, France). Media and growth conditions were as described previously -'° .

Preparation of E. coil and yeast lysates For preparation of tin E. coil lysate an overnight culture was used to inoculate 11 of Leuria Broth containing 501~gml ] ampicillin, and grown to tin absorbance at 620nm (At,2o) of 0.4. Clones PO, POXhoI-Psti, P0. I, P0.1.VP2, pJLA602.VP2, pCQV2.VP2 and PEV.VP2 were induced by elevating the temperature from 32 to 42 C. Clones pUR.VP2 and p T T Q I S . V P 2 were induced by the addition of 1.5 mM isopropyl fl-D-thiogalactopyranoside (IPTG). In clone pRIT5.VP2, the VP2 molecule was produced without induction. The cultures were then grown for a further 2 h tit the required temperature, the cells collected by centrifugation, and resuspended in 10ml of lysis buffer (PBS containing 40 fig ml i lysozyme, 2.5/~g ml E RNaseA, 1 fig ml t DNase, and 20 t*g ml ] phenylmethyl-sulphonylfluoride (PMSFI). After 30min, the cells were disrupted by sonication (4 x 30 s bursts on ice), and incubated in ice for a further 30 rain after addition of fresh PMSF. The lysate was then spun at 3000 rev. min ~ in a Beckman JA20 rotor for 10 rain at 2 C, and the 3K supernatant was used for further studies. The preparation of lysates for yeast cells has been described earlier ~s. Briefly, yeast cells, recovered by centrifugation, from an overnight culture were transferred to 1 1 of fresh yeast medium and allowed to grow at 30 ( ' for 2 h. CuSO,~ was then added to a concentration of 0.5 mM (only to S. cererisiae) and the culture grown for a further 2 h. The cells were collected by centrifugation and resuspended in 50ml of 1.2M sorbitol containing 1 mg ml 1 zymolyase and P M S F and incubated with shakmg at 30 C for 1 h. The spheroplasts were recovered by centrifugation and resuspended in lysis buffer as used for 15. coil cells. After a brief sonication, the 3K supernatant was prepared as described for E. coll.

Characterization of recombinant IBDV antigen: A.A. Azad et al.

Gel filtration of E. coil and yeast lysates The 3K supernatant from E. coli or yeast (~