Expression in Escherichia coli of the major outer capsid protein of infectious pancreatic necrosis virus

369

Gene, 79 (1989) 369-374 Elsevier GEN 03020

Expression in Escherichia coli of the major outer capsid protein of infectious pancreatic necrosis virus (Recombinant DNA; lac promoter; hybrid plasmids; birnavirus)

fusion protein;

monoclonal

antibody;

veterinary

pathogen;

viral vaccines;

William R. Lawrence”, Eva Nagy b, Roy Duncan c, Peter Krell d and Peter DobosS a Department of Microbiology, College of Biological Sciences, University of Guelph, Gue[ph, Ontario (Canada Nl G 2 Wl); b Department of Veterinary Microbiology and Immunology. University of Guelph, Guelph. Ontario (Canada Nl G 2Wl) Tel. (519)824-4120, ext. 4783, and’ Department ofMicrobiology and Infectious Diseases, Universityof Calgary Health Sciences Centre, Calgary, Alberta (Canada T2N 4NI) Tel. (403)220-4222 Received

by D.T. Denhardt:

Accepted:

29 November

21 October

1988

1988

SUMMARY

The outer capsid polypeptide, VP2, represents the major neutralizing antigen of infectious pancreatic necrosis virus (IPNV). A 926-bp viral cDNA, encoding an N-terminal truncated VP2, was cloned into the pWR590 expression plasmid family resulting in a C-terminal extension of a truncated Escherichia coli /I-galactosidase @Gal) under the control of the lac promoter. When cells transformed by in-phase hybrid plasmids were induced by isopropylthiogalactoside, high levels of the lOO-kDa /?Gal-VP2 fusion protein accumulated within 4 h after induction. The fusion protein reacted in Western blots both with rabbit anti$Gal and with neutralizing mouse anti-VP2 monoclonal antibody. Sera of rabbits immunized with semipurilied fusion protein reacted with the VP2 polypeptide in Western blots and with intact purified virus in ELBA and also neutralized IPNV infectivity in a plaque-reduction assay. Out-of-phase hybrid plasmids did not produce the fusion protein but expressed a small amount of structurally discrete VP2-specific sequences probably by internal initiation of translation at an in-phase

AUG

codon near the 5’ end of the VP2 gene.

CorrespondenceCO:Dr. P. Dobos, Department College Ontario

of Biological (Canada

Sciences, NlG 2Wl)

University

of Microbiology, of Guelph,

Tel. (519)824-4120,

Guelph, ext. 2279;

bursal

disease

virus;

IPNV,

tide(s); Abbreviations: bp, base pair(s); salmon

A, absorbance; aa, amino acid(s); Ap, ampicillin; BGal, E. coli r?-galactosidase; CHSE, chinook

embryo

cells; ds, double

linked immunosorbent fer (see section

0378-l 119/89/$03.50

strand(ed);

ELISA,

assay; ESB, electrophoresis

a); FP, fusion

0 1989 Elsevier

polypeptide;

sample buf-

IBDV,

Science Publishers

enzyme-

phate-buffered sor polypeptide protein; VP2.

infectious

B.V. (Biomedical

NS, protease PAGE,

Division)

infectious

pancreatic

propyl-/I-D-thiogalactoside; antibody;

Fax (5 19) 836-9967.

virus; IHNV, infectious

hematopoietic

necrosis

LB, Luria broth; (non-structural

polyacrylamide-gel

polypeptide);

polypeptide;

iso-

nt, nucleoPBS, phos-

unit(s); pVP2, precur-

of VP2; SDS, sodium dodecyl capsid

necrosis IPTG,

mAb, monoclonal

electrophoresis;

saline; pfu, plaque-forming

VP2, outer

virus;

sulfate; VP, viral

VP2, gene coding

for

370

IPNV are VPZspecific (Nagy and Dobos, 1987). One such mAb, developed against the Jasper sero-

INTRODUCTION

Infectious

pancreatic

necrosis virus (IPNV) is the

causal agent of a contagious, high-mortality disease of young, hatchery-reared salmonids (Pilcher and Fryer, carriers

1980). Survivors shedding

of epizootics

may become

the virus in their feces and repro-

ductive fluids. Since the disease cannot the development

of a safe, efficacious,

vaccine against IPNV is highly desirable. immunized

against infectious

tion (adults) or by immersion

be treated,

cost-effective Fish can be

agents either by injecin water containing

vaccine (both adults and fry) (Paterson,

the

198 1; Tebbit

et al., 1981). Although vaccines are available for several bacterial fish diseases, none are available against viral diseases. One of the major reasons for this is the high cost of producing large quantities of virus for use as inactivated (killed) vaccine. The use of live, attenuated virus vaccine is not an acceptable alternative for it may revert to virulence or turn out to be pathogenic for other hosts. Furthermore, its presence would interfere with diagnostic tests used in fish-health certification. To circumvent these problems we propose the development of a cloned subunit vaccine as a safe and cost-effective alternative. Such a potential cloned vaccine has been produced recently against another fish virus, IHNV (Gilmore et al., 1988). IPNV is a naked icosahedron containing a bisegmented dsRNA genome. The genome segments have been cloned, sequenced and the polypeptides encoded by them mapped (Duncan and Dobos, 1986; Nagy et al., 1987). The smaller genome segment (2900 bp) encodes the putative viral polymerase VP1 (90 kDa), whereas the larger genome segment (3097 bp) encodes a 104-kDa polyprotein which is cleaved by rapid post-translational cleavage to produce two structural polypeptides, pVP2 (62 kDa) and VP3 (30 kDa), and one non-structural polypeptide, NS (27 kDa) (Nagy et al., 1987). The pVP2 polypeptide is further cleaved to generate VP2 (52 kDa). The order of these polypeptides within the polyprotein is NH,-pVP2-NS-VP3-COOH and the NS polypeptide has been identified as the viruscoded protease responsible for polyprotein processing (Duncan et al., 1987). VP3 is an internal protein, whereas VP2 constitutes the outer capsid of the virus and contains the major neutralizing epitopes since all neutralizing mAbs developed against

type of IPNV neutralized the infectivity of eleven out of twelve different virus types tested making VP2 an ideal antigen

to be cloned for vaccine

purposes.

This communication describes the construction of a hybrid plasmid which in E. coli can express, under the control of the Zuc promoter,

a major neutralizing

epitope of IPNV in the form of a fusion protein. The cloned virus antigen induces bodies in immunized

animals

didate as a potential

subunit

ous pancreatic

EXPERIMENTAL

necrosis

virus-neutralizing

anti-

making it a good canvaccine

against infecti-

of salmonids.

AND DISCUSSION

(a) Construction of expression tion of fusion polypeptide

plasmids and induc-

The 926-bp HincII fragment of IPNV cDNA (from bp 576-1501) was inserted by blunt-end ligation into the SmaI site of the polylinker region of each of the three pWR590 expression plasmids (pWR590, pWR590-1, pWR590-2). This region encodes a large portion of pVP2 but none of the NS protease (Fig. 1A). Plasmid DNA from 12-24 E. coli HBlOl transformants was purified using the smallscale alkaline lysis procedure (Maniatis et al., 1982). The DNA was screened for the presence of the 926-bp IPNV cDNA fragment by digesting a portion of each preparation with EcoRI + Hind111 followed by agarose-gel electrophoresis. Recombinant plasmids were physically mapped to ensure that the insert was in the correct orientation for gene expression. For induction of FP synthesis, single colonies were picked and inoculated into 5 ml of Ap-containing LB at 37°C. When the A,,, reached 0.2, the cultures were induced with IPTG (1 mM final concentration) and incubated for another 4 h. The cells were disrupted by sonication and centrifuged at 12000rev./min for 15 min. The fiGal-VP2 FP formed insoluble inclusion bodies and were quantitatively pelleted at this speed. They could be solubilized by boiling in ESB (0.0625 M Tris - HCl pH 6.8/l % SDS/2% 2-mercaptoethanol/lO% glycerol/a trace of bromphenol blue).

371

A

PVPP H

5L-y

I

575

301

H

X

I

H

1501

s55

mid pWI926-0

2280

should

the two out-of-phase pWI926-2

1791

PO

due to the presence

:

frames

Sm ori 1

(four and

tions for pWR590

p”I926-0

DC0

AGC

TCG

AAT

TCO

ADC

TCG

ccc

pv1926-1

DGC

GAG

CTC

GAA

TTC

GAO

CTC

DCC

I

GCT

CGA

ATT

CGA

fusion

GCT

pWI926-1

cot

and

only the truncated

of stop codons 18 codons,

/IGal

in both reading

respectively)

down-

cDNA junction.

in Fig. 2a confirm these predic-

and pWI926-0

(lanes A and B).

au

Asn AAC

Asn Am

CAA

-

-

-

CAA

CM

TCA

-

-

-

CCA ACA ATC -

-

-

I, CGA

recombinants

encode

The data presented

1838

GO0

a fiGal-VP2

stream from the polylinker-IPNV

5

w

should

s

PWR590

p.W1926-2

sequence, the hybrid plasencode

polypeptide of approx. 100 kDa (66 kDa truncated fiGal and 34 kDa pVP2). The pWR590 plasmid and

2’

I /4616

Based on the nucleotide

P _--_-3'

2045

Q-q

B

(b) Characterization of expressed gene products

NS

A

926%-

Fig. 1. Partial map of the IPNV and map of the pWR590 vector plasmids. (A) Physical map of the relevant portion of the IPNV cDNA. The location of pVP2 and NS gene products is shown above the map, with the dashed line representing the proposed area of proteolytic cleavage. The exact locations of restriction sites are indicated on the map below, together with the number of bp, counting from the 5’ end of viral cDNA. The replicative form of recombinant phage M 13mp 19 containing a subclone of segment A cDNA from BarnHI site (301 bp) to the first PstI site (2280 bp) prepared as described previously (Duncan et al., 1987) was the source of IPNV cDNA. The dashed line at the 5’ and 3’ ends represents M13mp19 DNA sequences. Abbreviations: B, EarnHI; E, EcoRI; H, HincII; P, PstI; S, SacI; Sm, SmaI; X, XhoI. (B) Map of pWR590 expression vector family redrafted from the original

publication

by Dr. D. Thomas (Biotechnology

Institute,

Canada).

luc promoter

590 aa N-terminal

indicated cDNA the

parental

in the pWR590

codons

Recombinant

plasmids

vector

plasmid

respectively),

Below the

arrow-

(lane A) and pWI926-0

expression

vector

uninfected

represent

IPNV

0.1%

site at 576 bp. The first 3 aa are

the first three

followed

of the in-phase are

IPNV

designated

as

the first two letters indicate (pWR590,

pWR590-1

by the letter I indicating

0, 1 or 2.

indicates

the reading

frame

SDS-7.5%

blotted

(lane B), and IPNV-infected

(lane D) CHSE

cell lysates

polyacrylamide

onto nitrocellulose.

gel (Laemmli,

1970)

One half of the nitrocellulose

in and was

stained with amido black (panel a); the other half (panel b) was with mouse anti VP2 mAb. In panel a, the arrowhead

probed

lane A indicates

the position

of the truncated

in lane B indicates

the

tions of the FP and that of VP2, detected

of the

(lane C) and

were electrophoresed

and

of IPNV cDNA insert, the size of the insert in numbers

of bp and the last number vector,

cDNA

reading frame. The upward

pWI926- 1 and pWI926-2;

pWR590-2, presence

at the polylinker-IPNV

Fig. 2. Western-blot analysis of the /IGal-VP2 fusion protein probed with anti-VP2 mouse mAb. Duplicate samples of wholecell lysates of E. coli HBlOl cells carrying plasmids pWR590

from the H&II

insert.

pWI926-0,

contains

expres-

of replication.

on the right of the arrowheads

above

the

of the recombinant

sequences

SmaI cleavages

cDNA starting

D

1791 and 1838 bp. Abbreviations:

family are shown

family; sequences

C

for approx.

of BGal (Z). The plasmid

for each translational

heads indicate

AB

b.

Research

(4.6 kb) contains

(O), and codons

gene; ori, origin

the nucleotide

sion plasmid junction

a-portion

region between

ApR, Ap-resistance diagram,

The vector

(P), and operator

a polylinker

30-

of Guo et al. (1984). The plasmids

were kindly provided Montreal,

45-

panel

the position

of the BGal-VP2

b. The sixes of the marker

indicated

in kDa on the left margin

identical in the two panels.

in

BGal’ while that FP. The posi-

by mAb are shown in

proteins

(in lane MW) are

of panel a. Lanes A-D are

372

The polypeptide patterns of E. coli carrying the outof-phase recombinant plasmids were similar to that of lane A (not shown). Densitomet~ of Coomassie blue-stained gels showed that the fusion protein represents approx. 15y0 of total bacterial cell protein 4 h after induction. Rabbit anti$?Gal serum reacted in Western blots both with the truncated /?Gal’ and with the FP (not shown). In contrast, mouse mAb did not react with the truncated BGal’ or any of the E. cob HBlOl polypeptides but reacted strongly with the FP (Fig. 2b, lanes A and B). A few smaller polypeptides were also detected (in lane B) and were considered to represent partially degraded FP. The only other polypeptide that reacted strongly with the mAb was the authentic pVP2 polypeptide present in virusinfected CHSE214 cell lysates (lane C) (Dobos and Rowe, 1977). Thus it may be concluded that the 926 bp viral cDNA insert encodes a major neutralizing epitope of the virus which can be expressed at high levels in E. coli carrying the expression plasmid pWI926-0. The out-of-phase hybrid plasmid (pWI926-1) containing cell lysate was also subjected to Western blotting and probing with mouse mAb. The results presented in Fig. 3 show that mAb reacted strongly with pVP2 from infected CHSE214 cell lysates (lane C) as well as with a 34-kDa polypeptide from E. co& cells carrying the out-of-phase hybrid plasmid (lane B). This polypeptide was undete~tabie in Coomassie blue-stained gels (not shown). The size of this polypeptide suggests that an in-phase internal initiation of translation occurred at the first or second ATG near the 5’ end of the insert (at 681 or 696 bp) which would theoretic~ly generate a 28-kDa pol~eptide 575

681

*

‘-31

Fig. 3. Western-blot analysis, of cell lysates of E. cob HBlOl transformed with pWI926-1 (out-of-phase recombinant plasmid) and probed with anti-VP2 mAb. Lanes: A, lysate of E. cobcarrying the vector pWRS90; B, out-of-phase recombinant plasmid pWI926-1; C, IPNV-infected CHSE cell lysate; D, uninfected CHSE cell lysate. The samples were electrophoresed in 10% polyacrylamide gels containing 0.1% SDS (Laemmli, 1970). The arrowhead indicates the position of the 62-kDa pVP2 polypeptide. Molecular sizes of marker proteins (in kDa) are indicated on the right margin.

which may correspond to the 34-kDa polypeptide detected in Fig. 3. Similar results were obtained with the other out-of-phase hybrid plasmid pWI926-2 (not shown). Analysis of the 926-bp cDNA sequence around the first two in-phase ATGs (at nt 681 and 696) indicate that the second of these initiation sites is preceded by a better Shine-Dalgamo sequence than the first one (Fig. 4) and is most likely the start codon used. The next two internal in-phase start 696

1502

nt

nt 5'....C

A

C C C A G T C C oe

16s rRNA

Fig. 4. Nucleotide sequence in the region of the fast two in-phase start codons of the 926-bp IPNV cDNA insert and its complementa~ty with the nucleotide sequence of the 3’ OH terminus of l&S rRNA. The numbers of nt from the 5’ end of the virat genome segment A cDNA are shown above the sequence. The first two in-phase ATGs at nt 681 and 696 are underlined. Heavy black dots 5’ proximal to the ATGs indicate complementarity to the nt ofthe 3’ OH terminus of 16s rRNA (Shine and Dalgarno, 1975). The degree ofhomology is greater (see boxed areas) for the sequence preceding the second ATG at nt 696.

373

codons are located at 10 18 bp and 1191 bp and their usage would generate much smaller polypeptides. Similar internal initiation of translation of eukaryotic genes in prokaryotic cells has been reported for Herpes simplex virus thymidine kinase (Garapin et al. 1981), porcine parvovirus DNA (Halling and Smith, 1985), and tetrahydrofolate dehydrogenase DNA (Chang et al., 1980). (c) Antigenicity

TABLE

I

Virus neutralization IPNV rabbit

by plaque reduction

Serum a

Dilution

Decrease

of

in

plaque number b

serum used a

(%) Control

serum

1: 10

0

Anti-FP

serum

1:40

86

of the fusion protein Anti-IPNV

Sera of rabbits immunized with FP, reacted in Western blots with both pVP2 from lysates of infected CHSE-214 cells as well as with FP from lysates of E. coli carrying the pWI926-0 recombinant vector (not shown). Furthermore, a 1: 10 000 dilution of anti FP serum still gave a positive ELISA result (vs. 1: 100000 dilution of anti IPNV serum) indicating that the fusion protein elicited antibodies that were able to react with complete virus particles. Although the antiviral titer of the anti-FP serum as measured in plaque reduction assays was not very high compared to the antiviral serum, it did specifically and reproducibly neutralize virus infectivity causing a 52% plaque reduction when diluted 1: 100 (Table I). Recent reports indicate that viral antigens produced in E. coli as fusion proteins exhibit different levels of immunogenicity when injected into experimental animals. For example, Azad et al. (1987) produced in E. coli the outer capsid polypeptide of another bimavirus (IBDV) in the form of a 150-kDa fiGal-VP2 fusion protein. Reporting preliminary results, the authors state that the recombinant IBDV VP2 elicited the production of high levels of neutralizing and protective antibodies when injected into chickens. Another recent report described the production of the major neutralizing antigen (VP7c) of bovine rotavirus in E. coli as a C-terminal extension of bGa.l (McCrae and McCorquodale, 1987). Similar to IPNV most of the synthesized FP was present in the cells as insoluble inclusions so preparative SDS-PAGE was used to purify it for immunization purposes. The authors reported that a 1: 300 dilution of the rabbit anti-FP serum reduced the number of plaques by 60% in a neutralization assay, a value which is higher than the one we obtained with antiBGal-VP2 FP (Table I).

using anti FP and anti

sera

serum

a To produce

anti-FP

were collected section

1: 100

52

1: 2000

78

serum, the insoluble

a. The preparation

was dissolved

followed by dialysis at room temperature 0.1%

SDS.

adjuvant

After

an equal

was added,

into rabbits

volume

the mixture

(three injections

FP inclusion

E. coli lysates,

from sonicated

by boiling

in

in ESB

against PBS containing of Freund’s

was injected

at 1Cday

bodies

as described

incomplete

intramuscularly

intervals).

Immune

sera

were collected two weeks after the last injection. A similar preparation

of E. coli that

injected

into rabbits

serum. To prepare

carried

the pWR590

in an identical anti-IPNV

manner

vector

intramuscularly

intervals).

Immune

was

serum, purified virus in PBS was

mixed with an equal volume of Freund’s incomplete injected

alone

to serve as control

into rabbits

adjuvant

(three injections

serum was collected

and

at 1Cday

two weeks after the last

injection. b Virus dilutions

containing

equal volume of diluted for 1 h. Residual

100-200

antiviral

pfu were mixed with an

serum and incubated

virus was then titrated

described

by

McCrae

CHSE-214

cell monolayers

and

by plaque

McCorquodale

and the assay end-point

the highest serum dilution giving a 50% reduction tivity. The represent

% reduction averages

values

(decrease

at 20°C assay

(1987)

as

using

was taken as of virus infec-

in plaque

number)

of five replicates.

Another report by Gilmore et al. (1988) suggests that fusion protein that gives rise to a low neutralizing immune response can still elicit a good protective response. In this work, Suu3AI fragments of cDNA encoding the glycoprotein of IHNV (a fish rhabdovirus) were expressed as a fusion protein with the TrpE protein of E. coli. The fusion protein was purified by preparative SDS-PAGE and used to innnunize rabbits. The resulting anti-FP serum reacted with IHNV in ELISA and also detected the viral glycoprotein in Western immunoblots but only weakly neutralized IHNV in a plaque-reduction assay. Nevertheless, immersion immunization trials in fish with crude bacterial lysates containing the FP induced good protection against virulent virus challenge (20% mortality in immunized fish vs. 92% mortality in unvaccinated controls).

374

These data indicate that the relatively low neutralizing titer of our rabbit anti-FP serum does not rule out this protein as a potential subunit vaccine against IPNV in salmonid fish populations.

This work was supported by grants from the Natural Sciences and Engineering Research Council of Canada and the Department of Fisheries and Oceans of Canada.

REFERENCES Azad, A.A., Jagadish, M.N., Brown, M.A. and Hudson, P.: Deletion mapping and expression in Escherichia coli of the large genomic segment of a bimavirus. Virology 161 (1987) 145-152. Chang, A.C.Y., Relich, H.A., Gunsalus, R.P., Hungerg, J.H., Daaufman, R.J., Schimke, R.T. and Cohen, S.N.: Initiation of protein synthesis in bacteria at a translational start codon of mammalian cDNA: effects of the preceding nucleotide sequence. Proc. Natl. Acad. Sci. USA 77 (1980) 1442-1446. Dobos, P. and Rowe, D.: Peptide map comparison of infectious pancreatic necrosis virus-specific polypeptides. J. Virol. 24 (1977) 805-820. Duncan, R. and Dobos, P.: The nucleotide sequence of infectious pancreatic necrosis virus (IPNV) dsRNA segment A reveals one large ORF encoding a precursor polyprotein. Nucleic Acids Res. 14 (1986) 5934. Duncan, R., Nagy, E., Krell, P.J. and Dobos, P.: Synthesis of the infectious pancreatic necrosis virus polyprotein, detection of a viral coded protease, and fine structure mapping of the genome segment A coding regions. J. Virol. 61 (1987) 3655-3664. Garapin, A.C., Colbere-Garapin, F., Cohen-Solal, M. and Horodniceanu, F.: Expression of herpes simplex virus type I

thymidine kinase gene in Escherichia COILProc. Natl. Acad. Sci. USA 78 (1981) 815-819. Gilmore Jr., R.D., Engelking, H.M., Manning, D.S. and Leong, J.C.: Expression in ~~che~ch~a eoli of an epitope of the glycoprotein of infectious hematopoietie necrosis virus protects against viral challenge. Bio/Technology 6 (1988) 295-300. Guo, L.-H., S&pie& P.P., Tso, J.Y., Brousseau, R., Narang, S., Thomas, D.Y. and Wu, R.: Synthesis of human insulin gene, VIII. Construction of expression vectors for fused proinsulin production in E~cke~c~~~ coii. Gene 29 (1984) 251-254. Halling, S.M. and Smith, S.: Expression in Escherichiu coli of multiple products from a chimaeric gene fusion: evidence for the presence of procaryotic translational control regions within eucaryotic genes. Bio/Technology 3 (1985) 715-720. Laemmli, U.K.: Cleavage ‘of structural proteins during the assembly of the head of bacteriophage T4. Nature 227 (1970) 680-685. Maniatis, T., Fritsch, E.F. and Sambrook, J.: Molecular Cloning. A Laboratory Manual. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY, 1982. McCrae, M.A. and McCorquodale, J.G.: Expression of a major bovine rotavirus neutr~isation antigen (VP7c) in Escherichiu coli. Gene 55 (1987) 9-18. Nagy, E. and Dobos, P.: Epitope mapping of infectious pancreatic necrosis virus (IPNV) polypeptides using monoclonal antibodies. VILInternational Congress of Virology, Edmonton, Canada. Abstracts, 1987, OP25.2, p. 311. Nagy, E., Duncan, R., Krell, P.J. and Dobos, P.: Mapping of the large RNA genome segment of infectious pancreatic necrosis virus by hybrid arrested translation. Virology 58 (1987) 211-217. Paterson, W.D.: Aeromonas salmonicida as an immunogen. In Anderson, D.P. and Hennessen, W. (Eds.), Developments in Biological Standardization, Vol. 49. Karger, Basel, 1981, pp. 375-386. Pilcher, D.S. and Fryer, J.L.: The viral diseases of fish: a review through 1978. Part I: diseases of proven viral etiology. CRC Crit. Rev. Microbial. 7 (1980) 297-363. Shine, J. and Dalgarno, L.: Determinant of cistron specificity in bacterial ribosomes. Nature 254 (1975) 34-38. Tebbit, G.L., Erickson, J.D. and Vande Water, R.B.: Development and use of Yersinia ruckeri bacterins to controt enteric redmouth disease. In Anderson, D.P. and Hennessen, W. (Eds.), Developments in Biological Standardization, Vol. 49. Karger, Basel, 1981, pp. 395-402.