AU596154B2 - Fusion proteins - Google Patents

Description

COMMONWEALTH OF AUSTRALIA.

\G'~9Q 3669A 596154 COMMONWEALTH OF AUSTRALIA PATENTS ACT 1952 COMPLETE SPECIFICATION FOR OFFICE USE Form Short Title: Int. Cl: j Application Number: 6 Y2?//7.

Lodged: Complete Specification-Lodged: Accepted: Lapsed: Published: Priority: tatnt C z ontains (be M Pade ut frie an Related Art: TO BE COMPLETED BY APPLICANT 00 t o a 0 00 0000 o0 0 00 a o 0 0 000 0990 0 0 0 00 0 0 0 01 0 00 Name of Applicant: Address of Applicant: Actual Inventor: Address for Service: THE WELLCOME FOUNDATION LIMITED 183-193 Euston Road, LONDON NW1 2BP,

ENGLAND

Peter Edmund Highfield Berwyn Ewart Clarke and Anthony Robin Carroll GRIFFITH HASSEL FRAZER 71 YORK STREET SYDNEY NSW 2000

AUSTRALIA

o0 0 Complete Specification for the invention entitled: o 00. FUSION PROTEINS o a The following statement is a full description of this o invention, including the best method of performing it known to me/us:- 4121A:rk ~i~ nr place ano a e or signatur. ecTarea at England ti'"s yGn uy U Signature of declarant() (no atlestation required) Note: Initial all alterations. Griffith, Hassel Frazer t S- 1/1- TITLE: FUSION PROTEINS This invention relates to the construction of fusion proteins.

Hepatitis B virus iS a DNA virus with a partly double stranded genome of 3200 nucleotides. The viral DNA is surrounded by the viral coded core antigen (HBcAg) which is enclosed by the similarly coded surface antigen (Robinson, 1977). Removal of the surface antigen by mild detergent treatment leaves a core particle 27nm in diameter composed of HBcAg and the viral DNA, HBcAg has been expressed in microbial cells as the native polypeptide and as a derivative fused to the terminal eight residues of beta-galactosidase (see Murray et al, 1984 for refs).

When synthesized in E. coli the core protein self assembles into 27 nm particles which can be visualized 15 under the electron microscope (Cohen and Richmond, 1982) 0 0 t and which are immunogenic in laboratory animals (Stahl et al, 1982). The amino acid sequence of the core antigen «o t .O shows a region towards the carboxy terminus which is homologous with that found in protamines (DNA binding proteins). By o o inference, it has been suggested that this part of the molecule interacts with DNA during assembly of core 0 particles (Pasek et al, 1979).

We have previously shown that it is possible to So'0. express immunogenic epitopes of foot-and-mouth disease @00

II

-2 virus (FMDV) as fusion proteins to beta-galactosidase in bacterial systems and also in cells infected with recombinant vaccinia virus (Winther et al, 1986; Newton et al, 1986). Since beta-galactosidase in its active form exists as a tetrameric structure, the nature of the fusion was such that only four copies of the FMDV sequence would be present on each complex but these only represent about 2% of the weight of the fusion protein. Further, animals vaccinated with the recombinant vaccinia virus failed to oeoo 10 produce neutralizing antibody (Newton et al, 1986).- The reason for this poor response may be because o.o. beta-galactosidase is expressed in the cytoplasm.

o o o o In order to improve the presentation of FMDV epitope to the immune system, we have fused the FMDV sequence to the HBcAg. DNA sequences were constructed, .oO each encoding a fusion protein comprising HBcAg to the 0040 ooo amino terminus of which was linked FMDV VP1 residues 141 to 160. The fusion gene sequence was incorporated into the S" vaccinia virus genome. Cells infected with the recombinant virus expressed the fusion protein strongly. The o o recombinant protein self-assembled into particulate structures. These have a high density of externally r located FMDV epitopes. By expressing the FMDV epitope in this way, it is possible to present it in a more virus-like form.

3 The FMDV-HBcAg fusion protein tan be expressed in yeast cells. Accordingly, the present invention provides a process for the' preparation of particles composed of a fusion protein comprising HBcAg and an antigenic epitope of foot-and-mouth disease virus (FMDV) linked to the amino terminus of the -HBcAg, the epitope being exposed on the outer surface of the particles, which process comprisises culturing such as to cause expression of the fusion protein yeast cells in which is provided a vector S incorporating a DNA sequence encoding the fusion protein and Sro capable, when provided in the said cells, of expressing the °oo° o fusion protein; and obtaining the fusion protein o o9 thus-produced as particles.

For expression of the fusion protein, the DNA sequence is incorporated in an expression vector. The DNA °o sequence is incorporated in a vector such that the vector, 0 o when provided in the yeast host, is capable of expressing a"0 the fusion protein. The vector may be a plasmid. The o o fusion protein is obtained in particulate form. The fusion protein is used as a vaccine. Vaccines may therefore comprise the fusion protein particles and a physiologically acceptable carrier or diluent.

In view of the apparent involvement of the carboxy terminus of the HBcAg in DNA interaction at the centre of the hepatitis B virus, the FMDV epitope is fused to the amino terminus of the HBCAg. The epitope may be fused directly to the HBcAg. Alternatively, the epitope may be 4 fused to the HBcAg via an intervening'linker. Such a linkter may be composed of one or more, for example up to ten, amino acid residues. The precore HBcAg signal amino acid sequence, which normally is located immediately before the amino terminus of the HBcAg, may therefore be absent from the fusion protein or part of it may comprise the linker. To the amino-terminus of the epitope may be located one or more amino acid residues prior to a Met residue o",o corresponding to a translational start codon.

o oo oo The major FMDV antigenic sites correspond to amino o acid residues 141 to 160 and 200 to 213 of the VP1 capsid protein. Either or both of these sequences of anino acid 0. 4 residues may therefore constitute the heterologous antigenic epitope(s). Alternatively, parts of these sequences may be provided e.g. residues 142 to 145, 146 to 151, 142 to 151 or o"O 142 to 160. A suitable DNA construct incorporating VP1 00 amino acid residues 142 to 160 of FMDV type 0 Kaufbeuren, 0o 0 and its corresponding amino acid sequence as denoted by the one-letter code, is shown below.

0 0i 5 TTTTTT T C TATG CTATAAATGAATTCAG CT Pllk Promoter M N S A C CGAACCTG C GTGGTGACCTGCAGGTTCT G P N L R G D L Q V L [VP1 G CT CAGAAAG T T G C T C GTA C C T G C C G G GA SA Q K V A R T L P G

S

1 0 VP1] o 0 0 0 G C T C C G G A T C C G C G C G C C C T T G G G T G G C T T oo A P D P R A L G W L LINKER o o 0 0 0 0 0o T G G G G CAT G GACAT TGAC C C T TATAAAGAA S W G M D I D P Y K E HEPATITIS B CORE ANTIGEN

TTT----

0

F

The DNA sequence encoding the fusion protein can be prepared starting from a DNA sequence encoding HBcAg, for example a plasmid incorporating the HBcAg gene. A DNA sequence encoding the FMDV epitope to be

-V-

OF 6 included in the fusion protein is ligated in the correct frame to the 5' end of the HBcAg gene. A vector capable of expressing the 'fusion protein may be prepared by incorporating a DNA sequence encoding the fusion protein between translational start and stop signals in a vector and providing a promoter for the sequence. By transforming suitable host cells with such an expression vector, the fusion protein can be produced.

°o °.The fusion protein incorporating the antigenic 0°o° S epitope is expressed in yeast cells, infected with the vector. The fusion protein is obtained from the cells in O conventional manner e.g. by lysing the cells followed by centrifugation. The fusion protein self-assembles within the cells to form particles. These particles closely resemble the 27 nm core particles composed of HBcAg and viral DNA which can be obtained by denaturing hepatitis B 0000 o virus. The FMDV epitope is exposed on the outer particle o surface.

Yeast cultures are used as host cells. Saccharomyces cerevisiae strains can therefore be employed. A:plasmid S. vector such as plasmid YRp7 is typically utilised to transform such hosts. Any plasmid vector containing a yeast-compatible promoter, origin of replication and termination sequences is suitable.

The fusion protein may be used as a vaccine for an animal. It may be administered in any appropriate fashion.

7 The choice of whether an oral route ot a parenteral route such as sub-cutaneous, intravenous or intramuscular administration 'is adopted and of the dose depends upon the purpose of the vaccination. Similar criteria govern the physiologically acceptable carrier or diluent employed in the vaccine preparation. Conventional formulations, carriers or diluents may be used. Typically, however, the fusion protein is administered in an amount of 10-1000 ug o°0o per dose, more preferably from 10-100 ug per dose, by either 0 00 o" oa the oral or the parenteral route.

O o aoo The following Example illustrates the present o° invention. A Reference Example is provided. In the o0 o accompanying drawings: Figure 1 shows the construction of plasmid pFOHc; Figure 2 shows the shuttle vector pvFOHC, the sequence of o0°O which contains two in-phase initiation codons separated by 0000 0 the FMDV VP1 1 4 2 -160 sequence and six amino acids of the 0 authentic HB "pre-core" sequence; Figure 3 presents the results of sandwich ELISA of cell.

lysates from wild-type (Wyeth) or recombinant (vFOHC) o0 o infected cells; Figure 4 shows the sucrose gradient profile of core reactive v) material obtained in the Reference Example; Figure 5 shows the results of labelling wild-type-vaccinia virus and recombinant (vFOHc) infected cells with 35 S-methionine and analysing the cellular supernatants by 8 immunoprecipitation with HBcAg reactive antisera and polyacrylamide gel electrophoresis (PAGE); Figure 6 shows 'the sequence of the synthetic GAL7 promoter used in pWYG7. The synthetic DNA extends from the left hand XhoI site to the right hand BamHI site; sequences to the right of BamHI are the natural GAL7 sequences. RNA start site indicated with an asterisk, initiation codon boxed. Sequences different to those in GAL7 are underlined.

Figure 7 is a map of pWYG7; o Figure 8 shows expression cassettes for HBcAg and the oa FMDV-HBcAg fusion protein expressed in the Example; 0oo no Figure 9 shows the peptide extension of the FMDV-HBcAg Sfusion protein and the DNA encoding it; and Figure 10 shows the sucrose gradient profile of core reactive material obtained in the Example.

44 REFERENCE EXAMPLE Two clones were used to construct the fusion protein described in this study. One clone representing hepatitis B core antigen (HBcAg) was obtained from Dr. P. High-field o (pWRL 3123). This clone had been modified at the NH 2 terminus such that it could be expressed in bacteria as a fusion protein to the E. coli protein TRP E. coli HB101 harbouring pWRL 3123 were deposited at the National Collection of Industrial Bacteria, Aberdeen, GB on 6 March 1987 under accession number NCIB 12423. A second clone fi9 to-the amino te'rminus of p-q9.lactosidase was obtained from Dr. M1. Winther (pWRL 201) (Winther et al, 1986).

Restriction maps of each clone are shown in Figure '00- 1 10 1. As can be seen in Figure 1, the junction between the FMDV sequence and the 0-galactosidase comprises a Bam HI restriction site. The strategy undertaken therefore involved the fusion of the FMDV sequence and the HBcAg sequence through this Bam HI site.

The initial stage in the construction therefore involved insertion of a synthetic oligonucleotide linker for Bam HI at the 5' end of the HBcAg gene of pWRL 3123.

The site used for insertion of the linker was the Nar I site at position 290. However a second Nar I site at position 1230 was also present in this plasmid. The plasmid was therefore partially digested with Nar I so that So. a population of plasmid molecules which had been cut at only one Nar I site could be observed by agarose gel electrophoresis and purified. After flush ending the Nar I sites using the Klenow fragment of DNA polymerase I, a Dol o. synthetic oligonucleotide linker representing a Bam HI site was ligated into the partial Nar I digest and the resulting plasmids were used to transform E. coli. Clones were then analysed for the presence of a Bam HI linker in the correct Nar I site by restriction mapping.

SOne such clone, designated pEB208, was isolated and DNA prepared. The length of the Bam HI linker had been specifically chosen so that, when ligated to the FMDV portion of pWRL201 (Fig the translational reading frame ~I44 C.,p~o +a 1.

11 would be continuous and a fusion protein could be produced.

Concomitant with the insertion of the Bam HI linker, the Nar I site into which it had been inserted, was destroyed.

It was therefore possible to remove the HBcAg sequence from pEB208 by Bam HI Nar I digestion whereupon a DNA fragment of 940 bases was produced. Similarly a Bam HI Nar I fragment from pWRL201 of approximately 3.5 kilobases was purified. These two fragments were ligated together and the correct clone (pFOHc) was identified by restriction 0 0 10 mapping.

c As can be seen from Figure 1, pFOHc can be oo 0 0 expressed in bacterial cells under the control of the tac C0 oo 0 promoter. In order to facilitate the transfer of the 0 0 hybrid gene to a vaccinia virus (VV) shuttle vector, however, plasmid pFOHc was cut at the single Nar I site and a second EcoRI site was introduced as a synthetic linker.

o .00 This enabled the complete hybrid gene to be isolated as an n EcoRI fragment.

o o0 The VV shuttle vector was pVpllk which was 0 0 derived from the vector pH3JAR1A (Newton et al, 1986) by ,o deletion of extraneous W sequences. This shuttle vector o 0 has a W promoter (in this case pllK) inserted into VV "i/ thymidine kinase (TK) gene. This vector has a unique EcoRI site immediately following the W pllk promoter and AUG (Bertholet et al, 1985). The EcoRI site and AUG are in the same translational reading frame as the amino terminal 1 12 EcoRI site of the hybrid gene in pFOHc. The FMDV-HBcAg gene was therefore inserted as the EcoRI fragment into EcoRI cut dephosphorylated pVpllk. Clones with the hybrid gene in the correct orientation relative to the pllk promoter were identified by restriction mapping. This clone was designated pvFOHc (Figure 2).

This shuttle plasmid was then inserted into the genome of the Wyeth (US vaccine) strain of VV, under the control of the pllk promoter, by homologous recombination o0 0 10 using the flanking TK sequences (Mackett et al, 1985 a and 0 0 o. Individual progeny plaques with a TK phenotype were 0 o S'O screened for the presence of FMD-HBcAg DNA by dot blot O 00 o hybridisation.

o CV-1 Cell lysates from wild-type (Wyeth) and recombinant (vFOHc) infected cells were screened for the presence of core antigen and for FMDV sequences by sandwich o o o. ELISA. Antigen from infected cells was bound to ELISA plates using either FMD virus particle (146S) or FMD VP1 0 141-160 antisera raised in rabbits. Each trapped antigen was then assessed for the presence of either HBc, FMD 146S o or FMD VP1 142-160 epitopes by binding with the respective guinea pig antisera and development with anti guinea pig peroxidase conjugate. The results are shown in Figure 3.

As can be seen in Fig 3, a protein recombinant from (vFOHc) infected cell lysates was trapped with anti-FMDV 141-160 antiserum and this protein could then react with anti HBc, -iod I I I r -,L 13 anti-FMDV 141-160 and FMDV antivirion serum in a sandwich

ELISA.

Furthermore, this protein could be purified by ultracentrifugation suggesting that it was particulate in nature. This was illustrated more clearly when the products of centrifugation were sedimented on a sucrose density gradient and fractions were re-assayed for the presence of core antinen by ELISA. Cell lysates from recombinant (vFOHc) vaccinia virus infected cells or o oo 10 bacteria expressing native core antigen were fractionated 0 00 oo on 15-45% sucrose gradients. Fractions were assayed for 00 Soo the presence of core reactive material by indirect sandwich o o 0 0 ELISA using human anticore antiserum as trapping antibody o and guinea pig HBc antigen antiserum for detection. The results are shown in Figure 4. The position at which FMD virus sediments is also indicated. Fig 4 shows that a peak 0 1 of HBcAg reactive material was observed in a position S' similar to that observed when core particles expressed in o4 bacteria wre centrifuged in parallel. Thus it appears that the presence of the FMDV VP1 142 -160 sequence does not interfere with the particulate nature of the core 0O S particles.

SThe ability of the fusion protein to self assemble into regular, 27nm core like particles was confirmed by electron microsocopic examination of imnune complexes formed with sucrose gradient purified material.

4121A:rk 14 The complexes were formed by reacting the FMDV-HBcAg particles with antiserum raised to intact foot and mouth disease virus. The complexes were adsorbed to form over coated grids and negatively stained with phosphotungstic acid. As was to be expected from the ELISA data shown in Fig 3, immune complexes were also seen after reacting the particles with antisera to HBcAg or to synthetic FMDV peptide 141-160.

Finally, the nature of the polypeptides 0o o 10 synthesized in CV-1 cells infected with wild-type vaccinia coo0 "o o virus or vFOHc by labelling the cells with aS-methionine aso °and analysing the cellular supernatants by o S immunprecipitation with HBcAg reactive antisera and PAGE.

oo oSO,. Infected cells were pulse labelled with 5 S-methionine for 1 hour and total cell lysates prepared.

Immunoprecipitation of labelled proteins was carried out with vaccinia virus antiserum human HepB antiserum or guinea pig HBc antigen antiserum F).

i Precipitated proteins were then analysed by PAGE and fluorography. The results are shown in Figure 5. The 0 position of the FMDV VP1 1 42 -_16 -HBcAg fusion protein is arrowed. As shown in Fig 5, several proteins are specifically precipitated from extracts of recombinant (vHOFc) infected cells by HBcAg antiserum. Two of these proteins (Mol. Wt. 25KD and 20 KD) represent the complete fusion protein and a derivative having lost a 5Kd fragment 15 from its carboxy terminus by proteolytic digestion which would correspond to FMDV VP1 142-_I-HBeAg (Mackay et al, 1981).

EXAMPLE: Expression of FMDV-HBcAg fusion protein in yeast 1. Construction of yeast expression vector pWYG7 The vector pWYG7 is derived from the 2/ vector pJDB219 (Beggs, 1978) modified to contain a kanamycin- Sresistance marker (kanr) and the yeast galactose-regulated GAL 7 promoter. The construction of pWYG7 is as follows.

First the kanr marker (HincII fragment from pUC4K; Vieira a .and Messing, 1982) was ligated into the unique Smal site of 0 1 pJDB219 to give the kanr tetr vector pJDB219K. Second, a synthetic GAL7 promoter fragment (XhoI-BamHI fragment, sequence shown in Fig. 6) was cloned into the unique SalI n and BamHI sites of pJDB219K. The resulting vector, pWYG7, has the GAL7 promoter with unique BamHI and BclI sites o upstream of the yeast 2p plasmid FLP gene transcriptional terminator (Sutton and Broach, 1985). Foreign genes to be oo~a expressed from pWYG7 are inserted between the BamHI and BclI sites.

SThe smallest fragment of DNA upstream.of the GAL7 gene which exhibits full promoter activity has been defined by deletion mapping (Tajima et al., 1985). Based on this information a 260bp GAL7'promoter fragment was synthesised (Fig. 6 for sequence). The 260bp promoter was synthesised I II r Lr -16as four overlapping oligonucleotides using a Pharmacia Gene Assembler (protocol supplied by Pharmacia). These oligonucleotide's were phosphorylated and annealed using standard techniques, then ligated into XhoI-BamHI cut (Marsh et al., 1984). Positive clones were identified and their DNA sequenced using the double-stranded DNA sequencing method with universal and reversed sequencing primers (Hong, 1982). The sequence of the GAL7 inserts was confirmed, and then the XhoI-BamHI GAL7 insert was excised t and cloned into pJDB219K as described above.

Figure 7 is a map of pWYG7. The design of the GAL7 4o4. promoter fragment in pWYG7 is such that the natural GAL7 DNA 04 sequence has been slightly modified (2bp changed) in .order to make the BamHI cloning site upstream of the GAL7 mRNA start sites. The foreign gene to be expressed is then linked with synthetic DNA to the BamHI site, such that the GAL7 mRNA start sites are introduced, along with the GAL7 upstream untranslated sequences. Thus the first non-yeast DNA downstream of the promoter is the initiating ATG codon of the foreign gene, and the transcript produced:will have a yeast GAL7 leader rather than a foreign leader which could reduce efficiency of translation.

2. Construction of the FMDV-HBcAg expression vector, pWYG7-HBF In order to express the FMDV-HBcAg fusion protein in r 17 yeast, the coding sequence had to be adapted at both the and 3' ends for cloning into pWYG7. The expression cassettes for HBcAg and the FMDV-HBcAg fusion protein are shown in Figure 8. The polypeptide extension for the FMDV-HBcAg fusion protein and the DNA encoding it are shown in Figure 9.

Intermediate vector pKGF.

The vaccinia expression vector pvFOHc of the Reference Example was used as a source of DNA. Its conversion to an 0 expression cassette that could be introduced into pWYG7 is aa as follows. First, a SalI/EcoRI synthetic linker, containing 3' terminal sequences (Aval site to stop codon) from the HBcAg gene, was cloned into the SalI/EcoRI sites of pUCl8 (Vieira and Messing, 1982). Second, the resulting vector was digested with HindIII/Aval, and the 2.7kb fragment was isolated. This was used in a 3-way ligation with the 640bp EcoRI/Aval fragment from pvFOHc and a HindIII/EcoRI synthetic linker containing the 5' end of the FMDV-HBcAg gene in a form which can be transferred, to the j o ,yeast vector pWYG7. The sequence of the linker is: a..

SalI Aval "stop"

TCGACTCGGGAATCTAATGT...

GAGCCCTTAGATTACA...

BamHI

TAAGGATCCTCTAG

ATTCCTAGGAGATCTTAA

EcoRI a4 The sequences of the synthetic regions of the plasmid were confirmed by double-stranded DNA sequencing using universal and reverse sequencing primers (Hong, 1982),.

(ii) Construction of pWYG7HBF The BclI/BamHI expression cassette was isolated from pKGF.

This was carried out by partially digesting pKGF with BamHI, then digesting with BclI, and isolating the 707bp BclI/BamHI fragment. Since BclI does not cut dam methylated DNA, pKGF for this purpose was purified from a dam- host strain. The fragment was ligated with pWYG7 (dam-) that had been digested with BamHI and BclI and treated with calf-intestinal alkaline phosphatase. After.transformation, kanr colonies were tested for inserts of the correct orientation and pWYG7HBF was isolated.

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1 Y Hi As A o 4r o ai to 01 19 3. Transformation of yeast with pWYG7HBF The vector pWYG7HBF was introduced into the Saccharomyces c'erevisiae strain S150-2B leu2, his3, ura3, trpl, McCleod et al., 1984) using the lithium transformation procedure of Ito et al., (1983). Transformed yeast cells were incubated in YPD broth (Sherman et al., 1983) at 30 0 C overnight prior to plating out on selective medium (YPD plus 500pg/ml G418). This allows expression of G418-resistance and increases transformation frequency.

Colonies that came up as G418r were checked on minimal medium lacking leucine (YNB+glucose+histidine+uracil +tryptophan, Sherman et al., 1983) to check for the Leu phenotype also conferred by pWYG7HBF. Positive transformants (G418r Leu were used for expression analysis.

4. Galactose induction of expression of FMDV-HBcAg Transformants were grown to the mid-logarithmic stage (10 7 cells/ml) in YP broth containing 2% raffinose and 500pg/ml G418 at 30 0 C in an orbital shaker. An aliquot of 40% galactose was then added to a final concentration of 2%, and the culture was incubated for a further 48h. The cells were then harvested by low speed centrifugation, washed once in distilled water, and resuspended in ice-cold break buffer sodium phosphate pH 7.0, 0.1% triton X-100, 4mM phenylmethanesulphonyl fluoride, 4mM EGTA, and 2pg/ml each of pepstatin, antipain, leupeptin, and chymostatin; 5ml for

I,

0 4 20 cells from a 250 ml culture). Acid washed glass beads (0.45mm) were added and the cells were broken by vigorous vortexing. The crude cell lysate was cleared by centrifugation for 15 min at 10,000g. The protein concentration of the cleared supernatant was determined using the BioRad protein assay (BioRad, according to the manufacturer's instructions), and the material was stored at 0 C in aliquots.

Analysis of cell lysates for expression The proteins in the induced yeast cell lysates were o0°o analysed by separation in SDS-polyacylamide gels (Laemmli, So 1970). 50g of soluble protein were loaded per track, and o i as a negative control an extract of induced S150-2B was o 0 loaded. A new protein band was detected by staining with 0 0o °ooo° Coomassie blue in the pWYG7HBF-transformed cell extracts, migrating at about 24,000KDa. It was confirmed that this polypeptide is FMDV-HBcAg by using Western blot analysis 0oo with either HBcAg or FMDV-peptide antiserum.

s Coomassie-stained gels were scanned in order to es'timate oo FMDV-HBcAg as a proportion of the total soluble yeast 0 00 protein. Gel scans have given figures of 2-3% and these agree with ELISA quantitation data.

o0 6. FMDV-HBcAg fusion protein expressed in yeast associates to form core particles In order to test whether or not the FMDV-HBcAg

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21 protein made in yeast was present as core particles, the induced cell extract was layered over a 15%-45% w/v sucrose density gradient (in phosphate-buffered saline) and centrifuged in a Beckman SW28 rotor (28,000 rpm, 4h). The gradient was fractionated and the fractions analysed for the presence of HBcAg- or FMDV-reacting material. Using ELISA with HBcAg antiserum, a peak of reacting material was detected in the middle fractions of the gradient (Fig. and none was detected at the top of the gradient. This indicates that all the FMDV-HBcAg protein made in yeast associated to form core particles which sedimented in a o",o sucrose nradient. For confirmation, an aliquot from a peak gradient fraction was sent for electron microscopy SRo (phosphotugstic acid stain). A large number of viral core oo o particles were clearly seen.

0 00 7. In vitro analysis of FMDV-HBcAg particles and immune response of guinea pigs to the particles o o 0o° Methods o Enzyme linked immunosorbent assay (ELISA) Modifications of the indirect and double antibody sandwich ELISA techniques described by Voller et al (1976)' 0oo 0 o were used for antigen and antibody assays.

Antigen assay. Microplates were coated, overnight at +4 0

C,

with rabbit anti-HBcAg IgG. After washing, 0.5 log 1 0 dilutions of FMDV peptid/HBcAg fusion protein were added

N

22 and the plates incubated for 1hr at 37 0 C. Plates were then washed and guinea pig anti-HBcAg or guinea pig anti-FMDV peptide 142-160' or mouse monoclonal antibody (MAb) against the FMDV peptide at predetermined fixed dilutions was added.

After a further incubation for 1 hr at 37 0 C, plates were washed and anti-guinea-pig or anti-mouse IgG-peroxidase conjugate was added. After a final incubation for lhr at 37 0 C the plates were washed and an enzyme substrate (0.04% o-phenylenediamine 0.004% hydrogen peroxide in phosphate/citrate buffer) was added. The resulting colour development was stopped with 12.5% sulphuric acid after a n 0 few minutes and the absorbance at 492nm was measured in a o Titertek Multiskan (Flow Laboratories, Irvine, Ayrshire).

o 0 0o The A492 values obtained were plotted against the logo o ooo 0 0 0 o""o reciprocal fusion protein dilution and a 50% endpoint titre 0o 0 o° was calculated by reference to minimum and maximum A492 values.

Antibody assay. Microplates were coated overnight at +4 0

C

o oo oo with bacterially expressed HBcAg or synthetic FMDV peptide 141-160. Plates were washed and test guinea pig serum 0 0 0 0 samples at 0.5 logo dilutions from 1:10 were added. After incubation for lhr at 37 0 C, plates were washed and 0o 0 anti-guinea pig IgG-peroxidase conjugate was added. After a further hour at 37 0 C plates were developed with substrate solution as described above. The A492 values obtained were

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23 plotted against the log,, reciprocal antiserum dilution and the antibody endpoint titres were calculated by reference to a negative standard (a 1:10 dilution of pre-inoculation guinea pig serum).

Neutralisation assay. The FMDV neutralizing activity of serum samples against 100 TCIDs 0 of virus was determined using a micro neutralization test with IBRS-2 cells (Francis Black, 1983). Each test was performed in duplicate and the results were recorded as the mean log,, reciprocal of the serum dilution that gave confluent cell sheets in 50% of the microplate wells (SNs 0 Animals. Four female Dunkin-Hartley guinea pigs o a 41 weighing approximately 450g were inoculated intramuscularly with a 6.5ug dose of HBcAg fusion protein, containing 0.65ug of FMDV peptide sequence, formulated in incomplete Freund's adjuvant (IFA). All animals were boosted with a similar inoculum at 56 days. Serum samples, collected at 7 u0 to 14 day intervals were stored at -200C prior to ,analysis 0 0 6 for antibody activity.

(ii) Results In vitro analysis of FMDV-core preparations The FMDV-cores were tested to determine whether the VPl 142-160 peptide was intact. Dilutions of N V 24 two FMDV-core preparations (Al A2) were analysed for HBcAg and FMDV peptide using a double antibody sandwich ELISA.

Results showed '(Table 1 below) that the preparations contained equivalent activity against HBcAg and FMDV 142-160 peptide, suggesting that the peptide was present on all HBcAg particles. Furthermore, an anti-peptide MAb, produced to an FMDV 137-162 peptide 8-galactosidase fusion protein (Broekhuijsen et al, 1987) also reacted to a similar degree with the FMDV-core preparations. Since this MAb recognises a 142-160 synthetic peptide but not 143-160 or other synthetic peptides shortened at their amino termini (Parry et al, 1989), it appears that the FMDV peptide epitope on FMDV-cores is intact.

Immune Response of guinea-pigs to FMDV-cores Anti-HBcAg, anti-FMDV 142-160 peptide and FMDV neutralizing antibody titres elicited in guinea pigs following inoculation with FMDV-cores are given in Table 2.

High levels of anti-HBcAg (3.7 log 10 and anti-FMDV peptide (2.4 logo) antibody appeared within 14 days and:these continued to rise up to 56 days, when a second inoculation °o was given. In the presence of very high anti-HBcAg antibody levels (greater than 5.0 log 0 o) the second inoculation had' o little or no booster effect. However, antibody levels did persist at high levels for the following 28 days, at which time the experiment was terminated. The significant levels 00 4) o 0 0 00 00 00 U 00 0 00 0'!00 O 0 0o 6 0000 0 00 0 0 I 00 130 "0 25 of anti-FMDV peptide antibody were reflected in virus neutralizing activity which appeared at 14 days and increased to protective levels in all four animals (2.4-2.7 logo SN 5 0 at 56 days (Francis et al, 1988). These results with an inoculum containing only 0.65 ug of FMDV peptide of FMDV-cores) -confirm that the yeast-derived material has the same marked immunogenicity as the material made using a vaccinia virus expression system.

REFERENCES

Beggs (1978) Nature 275 104-109 Berthelot et al (1985) PNAS 82 2096 Broekhuijsen et al (1987) J. gen. Virol. 68 3137-3143 Cohen and Richmond (1982) Nature 296 677-678 Francis and Black (1983) J. Hygiene 91 329-334 Francis et al (1988) J. gen. Virol. 69 2483-2491 Hong (1982) Biosci. Report 2 907 Ito et al (1983) J. Bact. 153 163-168 Laemmli (1970) Nature 227 680-685 McCleod et al (1984) Cold Spring Harbor Symp. Quan't. Biol.

49 779-787 Mackay et al (1981) J. Med. Virol 8 237 Mackett et al (1985a) DNA Cloning, A Practical Approach (ed.

D.M. Glover) 2 191 IRL Press Oxford Mackett et al (1985b) Techniques in Gene Cloning Vol. 2 Marsh et al (1984) Gene 32 481-485 i

LA.

A- 0 Dh 4 ri'

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-26- Murray et al (1984) EMBO Journal 3 645-650 Newton et al (1986) Vaccines 86: New Approaches to Immunizatiu., Cald Spring Harbor Laboratory, 303-309 Parry et al (1989) J. gen. Virol. Pasek et al (1979) Nature 282 575-579 Robinson (1977) Ann. Rev. Microbiol 31 357-377 Sherman et al (1983) Methods in Yeast Genetics, Cold Spring Harbor, New York Stahl et al (1982) Proc. Natl. Acad. Sci. USA 79 1606-1610 Sutton and Broach (1985) Mol. Cell Biol. 5 2770-2780 Tajima et al (1985) Yeast 1 67-77 Vieira and Messing (1982) Gene 19 259 o Voller et al (1976) J. gen. Virol. 33 165-167 Winther et al (1986). J. Immunol. 136 1835 0 0 0 04 0 4 .0 4 0 0oo4 o0 4 o o o I 0 0

A

27 TABLE I ELISA analysis of FMDV-core samples Test antiserum Fusion pro tein sample Al A2 Anti-l-BcAg 1.77 2.24 Anti -FMDV 142-160 peptide 1.97 2.26 Anti-FMDV/B gal fusion protein mAb 1.85 2.26 lIogi 50% ELISA 0 0 4 04 00 04 0 4441 4 4 0O 4 0 44 04 4 444' 0414 4 4 44444 0 04 ,0 4 40 44 0 00 0 TABLE 2 Immune response of guinea pigs to FMDV-cores Days post primary inoculation 14 28 56 63 Antibody activity Anti -HBcAg Anti-FMDV peptide FMDV neutralization 0 4 40 ~4 0 -second inoculation *-mean log 1 0 ELISA endpoint titre **-mean log 1 0

SN

50 vs IOOTC1D 50 of virus

Claims (3)

1. A process for the preparation of particles composed of a fusion protein comprising HBcAg and an antigenic epitope of foot-and-mouth disease virus (FMDV) linked to the amino terminus of the HBcAg, the epitope being exposed on the outer surface of the particles, which process comprises culturing such as to cause expression of the fusion protein yeast cells in which is provided a vector incorporating a DNA sequence encoding the fusion protein and capable, when provided in the said cells, of expressing the fusion protein; and obtaining the fusion protein thus-produced as particles. o 2. A process according to claim 1, wherein the FMDV epitope is fused directly to the HBcAg. .o 3. A process according to claim 1, wherein the 0 d C Io FMDV epitope is fused to the HBcAg via a linker of from one to ten amino acid residues.

4. A process according to any one of the preceding oa claims, wherein the epitope has the sequence of amino acid o residues 141 to 160, 142 to 145, 146 to 151, 142 to 151 or O 0 142 to 160 of the FMDV VP1 capsid protein. I 5. A process according to claim 4,.wherein the epitope has the sequence of amino acid residues 142 to 160 a of FMDV type 01 Kaufbeuren. L EcoRi Fig -1. INor I partial Lklenow+BomHl linker EcoRi BamH I(Nor I0 P~q HBcAg pEB 208 3.7Kb Nor I Barn HI 1-golacto- sidase P. 0 01 0 04 0o 00 0 0 00 000 a 00 00 0 0 0P 0 006 0 0 0 Am Isolate ligate Barn HI Nor I f ragments EcoRI Barn HI pTac~ f DV HBCAg AmpRt VP'142-160 ~mp 4 pFOHc t or I ft Hind III Fig.2 6 amilno acids of 'pra~ore" TK -PIIK o. ATG i I cre TAA T K FMDV VP'1 42 16 0 0 0 0 ~0 00 0 0 0 I a-peptide o a-peptide o cr-peptide m a- 146S 1.5 1- p 00 00 0 0 0 0 0 vFOHc a-core* (anti HEP 8 core) Wyeth &--core* (wild type vaccinia control) vFOHc a-ieptide* (anti VP! 141-160) vFOHc a-146S' (anti FMD virus) Fig.3. I- 2 4 6 Log Antigen Dilution Fig. 4. FMDV 0.6* 000 o 03 S0 0 vFOHc 000 0 ~p312 3 (bacterIaHlY expressd native core antgn 0 04 1 00 4 8121 Fractionl no. 0 00 1/ 0 0 a 0 00 lh p.- wl VV vFOHc E v 7C 120k S24k 00 0 00 0000 o 0 00 0 00 00 0 0000 00 0 0 0 000 0O 00 000 0v or 0 0 0 0

18.4k .4 0 0 0000 ig 0 Fig.6. XholI AdtIT CTGGAGAGGT CTATACTTCG GAGCTCTGCA GATATGAAGC GAGCACTCTT GAGCGAAGGC TCATTAGATA TATTTTGTGT CTCGTGACAA CTCGCTTCCG AGTAATCTAT ATAAAAGAGA 80 90 100 110 120 00 0 0 0 p 0 *0 00 0 0 00 000 00 0 000 0 0 0 0000 CATTTTCCTT AACCCAAAAA TAAGGGAGAG GGTCGAAAAA GCGGTCGGAG AACTGTTGAG GTAAAAGGAA TTGGGTTTTT ATTGCCTGTC CCAGGTTTTT GGCGAGGCTG TTGACAACTG 130 140 150 160 170 180 CGTGATGCGA AGGACTGGCT ATACAGTGTT CAGAAAATAG CCAAGCTGAA AATAATGTGT GCACTAGGCT TCCTGAGCGA TATGTCACAA GTGTTTTATC GGTTGGACTT TTATTAGACA 0 000 0000 0000 cc 00 00 0 00 0 0 00 190 AGCCTTTAGG TCGGAAATCG 200 210 220 230 240 TATGTTGAGT TAGTTTGGCT AGCAAAGATA TAAAAGCAGG TCGGAAATAT ATACAAGTCA ATGAAACCGA TCGTTTCTAT ATTTTCGTGC AGCGTTTATA 0000 0 00 00 0 Ban-I 250 260 270 280 290 300 TTATGGGGAT TATTATGAG AGGATCGACA TGATAAAAAA AACAGTTGAA TATTCCCTGA AATACCCGTA ATAATACGTC TGCTAGGTGT AGTATTTTTT TTGTCAACTT ATAAGGGAGT 310 AAAgqACTG TTTTACTGAC IL- Fig. 7 EcoR I LP -EcoR I EcoR I 00 0 0 0 0000 0 00 0 00 000 0 0 0 0 0 00 00.0 BcI I Xho I ATG Barn HI RNA I GAL7 promoter Iforeign gene 0000 0000 0000 a 0 00 0 -300 -40 -22 +1 synthetic l inker 00 a Fig. 8. B N M 00 a HBcAg M Bc E BA I I I FM DV-HBcAgImi l A Aval B Barnl Bc Bcl E EcoRi M MaeIl N Ncol DGAL7 leader *DNA encoding FMDV peptide SHBcAg gene Synthetic DNA 100bp M, Fig. 9. FMDV fusion EcoRI ATO AATT CAGC TC C AAC C TGGTGGTGAG CTGCAGGTTC TGCTCAGAAAC TTGCTC GTAC CCTGCCC M N SAP NL RG DL QV LA QK VA R TL P -0FMDV peptide Aw B amP I CGAGCTCGCATCCGCCCCCCTTGCCTGCCTTTCGCCATG G A PDPR A L GWL WG M M.spacer pre-core Fig. HBcAg ELISA A 492 0 A 26 0 SUCROSE CONG