VIROLOGY

190,

840-844

(1992)

Presentation

of Hepatitis

B Virus preS, A. S.

*Laboratory

Epitope

BELYAEV,*

AND

on Bluetongue POLLY

Virus Core-like

Roy*+’

of Molecular Biophyscis, University of Oxford and NfRC Institute of Virology and Environmental Mansfield Road, Oxford, OX1 3SR, tuniversity of Alabama at Birmingham, Birmingham, Alabama Received

February

4, 1992;

accepted

Particles

Microbiology, 35294

June 3, 1992

A chimeric protein containing most of the hepatitis B virus preS, region (amino acid residues l-48) upstream to, and colinear with the amino-terminus of bluetongue virus VP7 protein (preS,-VP7) was expressed by a recombinant Autographa californica nuclear polyhedrosis virus (AcNPV). The chimeric protein formed BTV core-like particles (CLPs) in Spodoptera frugiperda cells only when the cells were coinfected with this recombinant virus and a recombinant baculovirus that expresses unmodified VP7 and VP3 of BTV. The ratio of preS,-VP7 incorporated into CLPs was influenced by the relative multiplicities of infection of the two viruses. lmmunoelectron microscopy of the chimeric particles indicated that the preS, epitope was exposed on the surface of the CLPs. When insect cells were coinfected with the preS,-VP7 recombinant virus and a baculovirus vector that synthesized only the VP3 protein, no CLPs were identified. 0 1992 Academic Press, Inc.

The development of particulate vector systems for the presentation of immunogenic epitopes provides a powerful approach for the delivery of antigens. Various types of particles have been used to present foreign epitopes, including particles formed from the hepatitis B virus (HBV) surface or core antigens, polioviruses, and yeast Ty-particles (I-5). We have recently reported that when the two major inner capsid proteins (VP3 and VP7) of bluetongue virus (BTV, Orbivirus genus, Reoviridae) are synthesized in insect cells by a dual recombinant baculovirus, viral core-like particles (CLPs) are formed (6). These particles can be isolated by one-step sucrose gradient centrifugation. The formation of CLPs has lead us to investigate whether CLPs can be used to present foreign epitopes. The VP7 protein is located on the outer surface of the CLPs with VP3 forming an inner icosahedral subcore (7). The introduction of a lo-aminoacid sequence representing an immunogenic region of rabies G protein to the amino terminus of VP3 led to the expression of the expected chimeric protein but did not result in its inclusion into CLPs when coexpressed with VP7 (unpublished data). However, when the rabies 1Oamino-acid residues were incorporated into the amino terminus of VP7, not only was the chimeric protein expressed in infected cells but also it was incorporated into CLPs on coexpression with VP3 (unpublished data). In this investigation, we report the expression of a chimeric VP7 protein containing 48 amino acid residues of the hepatitis B virus (HBV) pre-S, region (8, 9). The chimeric protein, although not included into CLPs ’ To whom 0042.6822/92

reprint

requests

should

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when expressed with VP3, was included into CLPs when coexpressed with VP3 and VP7, indicating that the particles can accomodate various forms of VP7. The biological and immunological characteristics of these particles have been analyzed. The HBV epitopes were localized to the surface of the CLPs as demonstrated using immunogold electron microscopy. A plasmid containing a chimeric preS,-VP7 gene was generated (Fig. 1) by manipulating an /%oRI-Xhol fragment derived from the ayw strain of HBV (8, 9) into the amino terminus of BTV-10 VP7 DNA in a pAcYM lbased transfer vector (pAcBTV10.7) so that it was under the control of the polyhedrin piomoter (10, 11). The orientation of the chimeric gene in the transfer vector was confirmed by sequence analysis (12). To generate a recombinant virus, monolayers of S. frugiper& cells were cotransfected with the recombinant transfer vector and AcRP23-IacZ DNA in the presence of lipofectin (13, 14). Progeny viruses with a IacZ-negative phenotype were plaque purified and a recombinant AcBTV7-preS, virus recovered and a high-titered virus stock prepared. The synthesis of proteins by AcBTV7-preS, virus was investigated by infecting monolayers of S. frugiper& cells with five plaque forming units (PFU) of AcBTV7-preS, per cell. Cells were harvested at 48 hr postinfection, lysed as described previously (6), and extracts analyzed by 10% SDS-polyacrylamide gel electrophoresis (SDS-PAGE). By comparison with wild-type AcNPV or AcBTV10.7 that expressed only the 38-kDa VP7 protein (15), a protein of the size expected for the HBV-VP7 chimera (preS,-VP7, 43 kDa) was identified (Fig. 2A). Confirmation that the expressed protein represented the preS, region of HBV

be addressed. 840

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COMMUNICATIONS

841

pre S2 I MDPNSTTFHQTLQDPRVRGLYFPAGGSSSG GGATCCTCTAGAAAATGGATCCGAATTCCACAACCTTCCAGATCCCAGAGTGAGAGGCCTGTATTTCCCTGCTGGTGGCTCCAGTTCAGGA VP7 b 4 T V N P V P T T V S P I S S I F S R I G D G D ACAGT~CCCTGTTCCGACTACTGTCTCTCCCATATCGTC~TCTTCTCGAGGATTGGGGATGGGGACACT.................

1 T..................

FIG. 1. Construction of a recombinant transfervector containing an HBV preS, sequence upstream and colinearwith BTVVP7. An oligonucleotide (5’ GCGGGATCCCCTCAGACCCGGGGACACTATCGCCGCA) was employed using the polymerase chain reaction to mutate the 5’ coding region of a BTV VP7 transfer vector (15) in order to introduce upstream BarnHI, Xbal, and Smal sites. Since the BlV VP7 transfer vector also contains a downstream BarnHI site, the modified vector was digested with 5amHl and inserted into pAcYM1 (71). The product was then digested with Xbal and Smal and ligated to an EcoRI-Xhol fragment derived from the ayw strain of HBV (8) modified at both ends with adapters (underlined) to provide a 5’ overhang complimentary to a cut Xbal sequence and a 3’ blunt end. The sequence of the amino terminus of the derived chimeric gene is shown.

was provided by Western blot analyses (Fig. 2B) using a rabbit antiserum that had been raised to an HBV preS, peptide (amino acid residues 14-32). Due to the strong titer of the pre-S2 antisera, several low-level (not detached by staining) degraded products were also reacted. Both the modified and unmodified VP7 proteins reacted with antisera raised to BTVlO virus partcles (Fig. 2C). To investigate whether the recombinant protein would assemble with BTV-VP3 protein to form CLPs

% z3E i

preS,-Vp7,

12

3

4

(6), a suspension culture of S. frugiperda cells was coinfected with the AcBTV-preS, recombinant virus and AcBTV17.3, a recombinant baculovirus that expresses only VP3 protein (16). The infected cells were harvested, lysed, and analyzed by gradient centrifugation as described previously (6). Despite the high level of expression of both proteins, no morphological structures were recovered (data not shown). As a control, cells coinfected with AcBTV10.7 and AcBTV17.3 yielded CLPs (6; data not shown).

12

preS,-Vp7

VP7 b

3

4

1234

ves, -VP7, vp7,

36.5 26.5

4 Polyhedrin

FIG. 2. Expression of preS,-VP7 protein. S. frugiperda cells were infected at a m.o.i. of 5 PFU per cell with AcBTV7-preS, (lane l), AcBTV10.7 (lane 2), AcNPV (lane 3) or mock-infected (lane 4). Cell lysates were processed as described in the text. Proteins were separated by 10% SDS-PAGE and (A) stained with Coomassie blue, (B) electroblotted onto an lmmobilon membrane and reacted with rabbit anti-preS,, or (C) blotted and reacted with rabbit BTV-10 antiserum. Bound antibody was detected using an alkaline phosphatase conjugate. Protein molecular weight markers (kDa) are shown in the left-hand lane of (A). The positions of the AcNPV polyhedrin (P), preS,-VP7, and VP7 proteins are indicated.

842

SHORT

1

2

COMMUNICATIONS

3

180 118 84 58 48.5

pre.s2-Vp7 VP7

38.5 28.5

A

munoelectron microscopy using gold-labeled anti-HBV preS, serum. As shown in Fig. 4A the gold particles were located on the outer surface of the CLPs indicating that the preS, sequences were exposed on the surface of the particles. In a control experiment using CLPs derived from AcBTVl7.3-10.7 no gold particles were detected (Fig. 4B). When a cell lysate infected with the AcBTVl7.3-10.7 dual recombinant virus was mixed with a cell lysate recovered from an AcBTV7preS, infection, CLPs were recovered and similarly analyzed by immunoelectron microscopy using gold-labeled preS antiserum. No gold particles were detected on the surface of the CLPs (data not shown). To demonstrate whether the chimeric CLPs were indeed biologically active, the immunogenicity of these particles was assessed. Antisera against the purified chimeric CLPs was raised in mice as described previously (17) and the reactivities of the resulting antisera

B

FIG. 3. Ten percent SDS-PAGE and Western immunoblot analysis of CLPs. CLPs composed of VP3 and VP7 (lane 1) or VP3, VP7, and preS,-VP7 (lanes 2, 3) were synthesized in insect cells as described in the text. Purified CLPs were purified by discontinuous sucrose gradient centrifugation. Proteins were separated by 10% SDSPAGE and stained with Coomassie blue (A), or electroblotted onto an lmmobilon membrane and reacted with rabbit anti-preS, serum (B). In lanes 2 and 3 the CLPs came from coinfections of AcBTVl7.310.7 (m.o.i. of 2) and AcBTV7-preS, (m.o.i. = 8, lane 2) or AcBTV17.3-10.7 (m.o.i. = 2) and AcBTV7-preS, (m.o.i. = 2, lane 3).

In order to determine whether there was any condition in which the chimeric VP7 could be incorporated into particles, S. frugiperda cells were infected with a dual recombinant baculovirus (A&TV1 7.3-l 0.7) that expresses VP3 and VP7 (6) at a multiplicity of infection (m.o.i.) of 2 PFU per cell and the AcBTV7-preS, virus at a m.o.i. of either 2 or 8 PFU per cell. Infected cells were harvested at 3 days p.i., lysed, and CLPs purified by centrifugation as previously described (6). The protein profiles of the CLPs indicated that the two forms of VP7 were present in the both CLP preparations (Fig. 3). However, the amount of the chimeric VP7 in the CLPs was dependent on the m.o.i. of the AcBTV7-preS, recombinant virus used in the coinfection. At the m.o.i. of 2 the preS,-VP7 protein content relative to VP7 was significantly less than when AcBTV7-preS, was employed at a m.o.i. of 8 (Fig. 3A). At the high multiplicity the amount of the chimeric VP7 was estimated from the stained gel to be ca. 20% the amount of authentic VP7. The presence of the chimeric protein in the particles was confirmed by Western analysis using the HBV preS, peptide antiserum (Fig. 36). The location of preS,-VP7 protein on the particles was analyzed by im-

FIG. 4. Immunogold-electron microscopy. (A) Purified tained from coinfection of insect cells with AcVP7-preS,. purified from cells infected only with AcBTV17.3-10.7. nm.

CLPs ob(B) CLPs Bar = 100

SHORT COMMUNICATIONS A 12

3

0 4MW

1234

C 12

pres2-vp7*

4L MM PSp-GST, GST)

FIG. 5. Demonstration of immunogenicity of the chimeric CLPs by Western analysis using antisera raised against purified chimeric CLPs in mice. SDS 10% PAGE (A) and Western immunoblot (B) of 1, Spodopfera frugiperda cells infected with recombinant baculovirus expression preS,-VP7; 2, E. colicells expressing preS, epitopefused with C terminus of glutathione S-transferase (GST-preS,) in pGEX-1 vector induced with IPTG (18); 3, same as lane 2, without IPTG induction; 4, f. co/i cells expressing glutathione S-transferase (GST). (C) 1: Western immunoblot with 1 HBVsAg-positive patient serum; L, Large (preS,-preS,-S) HBV surface antigen, M, medium (preS,-S) HBV surface antigen. 2: HBVsAg-negative human serum. Antibody dilution 1:4000.

was examined against an expressed Escherichia co/i fusion protein (18) containing the preS, region (GSTpreS, unpublished data). Shown in Fig. 5A are the Coomassie-blue-stained gel of insect cells infected with recombinant-baculovirus-expressing chimeric preS,VP7 protein (lane l), E. colicells expressing GST-preS,, induced (lane 2) and noninduced (lane 3), and the GST of the control E. co/i culture. In Western analyses both expressed proteins reacted strongly with the antibody raised against chimeric particles. The high immunogenicity was further evidenced by the reactivity of the apparently unstainable fusion protein band of the noninduced GST-preS, with the antibody (in a 1 to 4000 titer). To investigate whether the antibody can indeed recognize HBV antigen, similar Western analyses were performed using surface-antigen-positive HBV patient serum (Fig. 5C). The positive signals against large (preS,-preS,) and medium (preS,-S) surface antigen of HBV confirmed the high immunogenic capability of the chimeric particles. In summary, the data reported here indicate that when a large epitope of 48 amino acids of HBV preS, was incorporated into the amino terminus of BTV VP7, it blocked the formation of CLPs on coexpression with BTV VP3. However, when coexpressed with a dual gene vector that made VP7 and VP3, the chimeric protein was incorporated. By contrast when a 1O-aminoacid sequence representing a rabies G sequence was incorporated into the VP7 amino terminus, CLPs were formed on coexpression with VP3 alone. The VP7 of

843

BTV exists on the surface of CLPs as trimers (19). Recent analyses (20) have shown that when insufficient VP7 is made in coinfections with vectors that synthesize large quantities of VP3, incomplete CLPs are produced, CLPs that lack some of the surface arrangement of VP7. It is possible that VP7 is added sequentially to the VP3 subcore, first to positions that stabilize the structure and second to positions that complete the CLP surface structure (19). By this model when a chimeric VP7 is unable to produce a CLP with VP3 alone, supplementation with unmodified VP7 protein allows incorporation of the chimeric protein. A similar situation exists in the case HBV. The 226amino-acid surface antigen (HBsAg) is essential for particle formation. Addition of a 55-amino-acid preS, region to the amino terminal region of HBVsAg (preS,S) allows self-assembly of particles, although the addition of 163 amino acids of preS, and preS, (preS,preS,-S) abolishes self assembly (21-23). Nevet-theless, the preS,-preS,-S protein is incorporated into particles when coexpressed with S and preS,-S proteins (24). The synthesis of high levels of BTV chimeric CLPs offers a novel approach for presenting large epitopes. In addition, these chimeric CLPs are highly immunogenic, implicating its potential use for delivery of foreign immunogens.

ACKNOWLEDGMENTS We thank Dr. T. F. Booth for the EM work and Mr. Christopher Hatton for photographic work, V. Samukov (Vector, Novosibirsk, Russia) for the generous gift of HBV preS, antisera and E. Gren (Institute of Molecular Biology, Riga, Latvia) for donating the plasmid DNA containing the HBV (ayw strain) sequence. We are also grateful to Dr. Chong-gee Teo of the Public Health Laboratories, Collindale, for providing us with the HBV patient sera. The work was partly funded by MRC Grant SPG8915519 and NIH Grant Al26829 whose support are gratefully acknowledged.

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8. GANEM, D., and VARMUS, H. E. (1987). H. E. Annu. Rev. Biochem. 56, 651-633. 9. GALIBERT, F., MANDART, E., FITOUSSI, R., TIOLLAIS, P., and CHARNAY, P. (1979). Nature 281, 646-650. 10. MANIATIS, T., FRITSCH, E. F.. and SAMBROOK, J. (1982). “Molecular Cloning: A Laboratory Manual.” Cold Spring Harbor Laboratory, Cold Spring Harbor, NY. 11. MATSUURA, Y., POSSEE, R. D., OVERTON, H. A., and BISHOP, D. H. L. (1987). /. Gen. viral. 68, 1233-l 250. 12. SANGER, D. V., NICKLEN, F. S., and COULSON, A. R. (1987). Proc. Nat/. Acad. Sci. USA 74, 5963-5467. 13. KITTS, P. A., AYRES, M. D., and POSSEE, R. D. (1990). Nucleic. Acids Res. 18119, 5667. 14. FELGNER, P. L., GADEK, T. R., HOLM, M., ROMAN, R., CHAN, H. W., WENZ, M., NORTHROP, J. P., RINGOLD, G. M., and DANIELSEN, M. (1987). Proc. Natl. Acad. Sci. USA 84, 7413-7417. 15. OLDFIELD, S., ADACHI, A., URAKAWA, T., HIRASAWA, T., and ROY, P. (1990). I. Gen. viral. 71, 2649-2656.

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Presentation of hepatitis B virus preS2 epitope on bluetongue virus core-like particles.

A chimeric protein containing most of the hepatitis B virus preS2 region (amino acid residues 1-48) upstream to, and colinear with the amino-terminus ...
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