Chimeric Bacteriophage fr Virus-Like Particles Harboring the Immunodominant C-Terminal Region of Hamster Polyomavirus VP1 Induce a Strong VP1-Specific Antibody Response in Rabbits

VIRAL IMMUNOLOGYVolume 15, Number 4, 2002© Mary Ann Liebert, Inc.Pp. 627–643

Chimeric Bacteriophage fr Virus-Like Particles Harboringthe Immunodominant C-Terminal Region of Hamster

Polyomavirus VP1 Induce a Strong VP1-SpecificAntibody Response in Rabbits and Mice

TATYANA VORONKOVA,1,2 ADRIAN GROSCH,2 ANDRIS KAZAKS,1 VELTA OSE,1

DACE SKRASTINA,1 KESTUTIS SASNAUSKAS,3 BURKHARD JANDRIG,4

WOLFGANG ARNOLD,5 SIEGFRIED SCHERNECK,4 PAUL PUMPENS,1

and RAINER ULRICH2

ABSTRACT

The late region of the hamster polyomavirus (HaPyV, former HaPV) genome encodes threestructural proteins VP1, VP2, and VP3, where VP1 represents the major capsid protein of384 amino acids. Screening of sera from HaPyV-infected papilloma-bearing and papilloma-free hamsters demonstrated the immunodominant features of all three capsid proteins. Forboth groups of hamsters in the C-terminal region of VP1 immunodominant B-cell epitopeswere identified in the regions between amino acids 305 and 351 and amino acids 351 and384. The high flexibility of the C-terminal region of VP1 was confirmed by the formation ofchimeric virus-like particles based on the coat protein of the RNA bacteriophage fr whichwas previously found to tolerate only very short-sized foreign insertions. Phage fr coat pro-tein-derived virus-like particles tolerated the N-terminal fusion of amino acids 333–384,351–384, 351–374, and 364–384, respectively, of VP1. The induction of VP1-specific anti-bodies in rabbits and mice by immunization with chimeric virus-like particles harboringamino acids 333–384, 351–384, and 364–384, respectively, of VP1 suggested the immun-odominant nature of the C-terminal region of VP1.

INTRODUCTION

MEMBERS OF THE FAMILY Polyomaviridae represented by only one genus Polyomavirus are able to in-duce tumors in various host species and to transform mammalian cells in vitro. The double stranded

circular DNA genome of all polyomaviruses demonstrates a similar organization: It contains an early ge-

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1Biomedical Research and Study Centre, Riga, Latvia.2Institute of Virology, Charité Medical School, Humboldt University, Berlin, Germany.3Institute of Biotechnology, Vilnius, Lithuania.4Max Delbrück Center for Molecular Medicine, Berlin, Germany.5Atugen AG, Berlin, Germany.

nomic region encoding the tumor (T) antigens and a late region coding for the capsid proteins. These re-gions are separated by the regulatory region containing the origin of replication and the transcriptional reg-ulatory signals for the early and late region. The virions are non-enveloped and approximately 40 nm in di-ameter. The icosahedral capsid consists of three virus-encoded structural proteins: 360 molecules of themajor capsid protein VP1 and 30–60 molecules of each of the minor capsid proteins VP2 and VP3. Thecapsid is built up by 72 pentameric capsomers in skewed (T 5 7) arrangement, each containing five mol-ecules of VP1. The C-terminal arm of VP1 provides interactions between the capsomers. This flexible geom-etry of the pentamer interactions allows the formation of an icosahedral capsid (6,52). The VP1 of variouspolyomaviruses expressed in heterologous hosts has been demonstrated to assemble spontaneously in vitroand in vivo to virus-like particles (VLPs) (5,25,30,33,34,36).

The hamster polyomavirus (HaPyV [52], former HaPV) was primarily discovered as the etiologic agentof hair follicle epitheliomas in spontaneously infected Syrian Z3/Bln hamsters (15), but induces lymphomasin newborn hamsters of another hamster colony bred in Potsdam (HaP [16]; for review, see [39]). Re-cently, HaPyV-like viruses have been identified as causative agents for lymphomas and trichoepitheliomasin pet Syrian hamsters in the United States and United Kingdom, respectively (9,40). HaPyV contains a5,366-bp long DNA genome, which demonstrates a highly similar organization as the murine polyomavirusMPyV (8,41). The early region encodes 3 T antigens, large, middle and small T, which are involved inthe immortalization of primary rodent cells (large T) and transformation of established rodent cells (mid-dle and small T [14]). The late region encodes three capsid proteins (Fig. 1A,B) where the VP1 ORF (384amino acids) overlaps that of VP2/VP3 (345/221 amino acids) but in different reading frames. As the VP2-and VP3-encoding sequences are in the same reading frame, VP2 represents an N-terminally extended VP3.The analysis of HaPyV virions revealed VP1 as the major capsid protein of approximately 42 kDa (41).The N-terminal region of VP1 representing a nuclear localization signal (3,27,53) and a DNA-bindingregion (4,26) is highly homologous between HaPyV and MPyV (41). If expressed in heterologous hosts,as yeast (37), bacteria, and insect cells (Voronkova et al., in preparation), the HaPyV-VP1 assembles toVLPs.

Although the three-dimensional structure of SV40 and MPyV has been solved (17,22,44,45), littleis known about epitopes along the VP1 of polyomaviruses. Recently we have identified a major B-cell epitope region in HaPyV-VP1 at the C-terminal amino acids (aa) 320–384 (42). However, whenusing synthetic peptides differences in the epitope-specificity between serum pools of papilloma-bear-ing (Pap) and papilloma-free (PF) HaPyV-infected Z3 hamsters were observed: Whereas the serumpool of Pap hamsters reacted with VP1 peptides aa 79–97 and aa 353–367, the PF serum pool de-tected two other regions (aa 101–113, aa 165–179). According to structural predictions four regionslocated at aa 81–88 (site 1), aa 222/223 (site 2), aa 244–246 (site 3), and aa 289-294 (site 4) and theC-terminus of HaPyV-VP1 seem to be highly flexible and surface-exposed. A short-sized foreign pep-tide inserted into sites 1, 2, 3, and 4, respectively, has been exposed on chimeric HaPyV-VP1 VLPsand induced an insert-specific antibody response in mice (11). Similarly, Langner et al. (20) describedthe generation of chimeric B-lymphotropic papovavirus (LPyV) VP1-derived VLPs carrying a for-eign RGD motif at regions homologous to sites 1 and 4. In addition, a homologue of site 4 in MPyV-VP1 allowed the formation of VLPs harboring an enzymatically active dihydrofolate reductase (12).

In the past years VLPs became an interesting new strategy to generate highly efficient and safe an-tiviral vaccines (for review see 31,48). VLPs based on coat proteins (CP) of RNA bacteriophages havebeen attracted attention also for the presentation of foreign epitopes on their surface (chimeric VLPs).These phages belong to the genera Levivirus and Allolevivirus (50). In contrast to Levivirus represen-tatives, the Allolevivirus virions contain the essential protein A1 of unknown function within their cap-sids (18), which is a 329-aa-long read-through variant of CP consisting of the CP body and its C-ter-minal, 195-aa long extension separated from the CP body by an opal (UGA) stop codon. Both RNAphage CP body and its A1 extension represent possible targets for introduction of foreign epitopes, butthe A1 extension of RNA phage Qb was declared to be a preferential target for providing correct andefficient self-assembly of VLPs (51). The Levivirus representatives fr (2,19) and MS2 (24) were firstproposed as RNA-phage carriers for the presentation of foreign immunological epitopes on the CP body.

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The N- and C-termini of frCP supported inclusion of foreign epitopes, but not longer than 12-aa residues(19,32).

The major objective of the present study was to prove whether larger-sized protein segments derivedfrom a highly flexible sequence, that is, the C-terminus of HaPyV-VP1, can be accommodated by phagefrCP, when fused N-terminally, without disturbing the self-assembly of frCP-derived VLPs.

In this paper, we present evidence that the C-terminal region of HaPyV-VP1 bears immunodominant B-cell epitopes in the regions between aa 305–351 and aa 351–384 which are recognized both by sera of Papand PF hamsters. The high flexibility and immunogenicity of the C-terminal region of VP1 is demonstratedby the formation of chimeric VLPs based on the bacteriophage frCP and the induction of a strong anti-VP1antibody response in rabbits and mice. These data suggested the potential of the RNA phage fr as an effi-cient VLP carrier also for long foreign insertions, which could be added to its N-terminus.

CHIMERIC BACTERIOPHAGE fr VIRUS-LIKE PARTICLES

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FIG. 1. Immunoreactivity of sera from HaPyV-infected hamsters with recombinant HaPyV capsid proteins VP1,VP2 and VP3. (A) Restriction map of the late encoding region of HaPyV H, HindIII; B, BamHI; Pv, PvuII; Bc, BclI;E, EcoRI. (B) Coding sequences of the His6-tagged DHFR fusion proteins with entire HaPyV-VP1, -VP2 and -VP3,respectively. (C) Reactivity of sera from Pap (Pap 581, left) and PF (PF 598, right) hamsters with DHFR fusion pro-teins in the immunoblot. The full-sized DHFR fusion proteins with entire HaPyV-VP1, -VP2, and -VP3, respectively,are marked by arrows. Molecular weight marker proteins: 97.0 (phosphorylase b), 66.0 (albumin), 45.0 (ovalbumin),30.0 (carbonic anhydrase), 20.1 (trypsin inhibitor), and 14.4 (a-lactalbumin) kDa.

MATERIALS AND METHODS

Antisera. Hamster sera were collected from a colony of spontaneously HaPyV-infected Syrian Z3/Blnhamsters. This colony was bred since the end of the 1960s in Berlin-Buch and is now located at Schön-walde (Brandenburg, Germany). The HaPyV infection in this colony is transmitted horizontally. The breed-ing animals were selected for a high papilloma frequency and early occurrence of papillomas. A frequentpairing was found to favour tumor development. The serum panel from these hamsters includes samplesfrom HaPyV-infected papilloma-bearing (Pap) and papilloma-free (PF) hamsters. Pap animals were identi-fied by regular inspections of the hamster population. Papillomas are visible as dark or light nodules in thecutaneous region. Although they can be found on the whole body surface, predominant sites are eye lids,ears, jaw area and back. Progressive stages often exhibit extensive confluent structures.

A HaPyV-VP1-specific serum was generated by immunization of rabbits with HaPyV-VP1-VLPs ex-pressed in E. coli (rabbit no. 1564; our unpublished data). BKPyV- and JCPyV-VP1-specific antibodieswere generated by immunization of mice with yeast-expressed purified BKPyV- and JCPyV-VP1-VLPs,respectively [38]. A rabbit serum raised against JCPyV-VP1-derived VLPs was kindly provided by W. Lüke(Deutsches Primatenzentrum Göttingen). Rabbit anti-frCP antibodies were kindly provided by V. Bauma-nis (BMRC, Riga).

Generation of recombinant plasmids. The DNA cloning was performed according to standard proce-dures (35). For the generation of the VP1 expression library, the E. coli vector pFR36 was used (43). Itcontains the frCP gene downstream to the E. coli tryptophan operon promoter (Ptrp) and a polylinker in-serted at the original AsuII site at codon positions 1–3 of frCP.

The generation of the plasmids pQE41-VP1 (1–384; Fig.1B) and pFR36-VP1/2-12 has been describedrecently (41,42). The entire VP2-encoding sequence was isolated as a BglII fragment from plasmid pCR-R394 (41) and subcloned into BglII-linearized pQE40 (QIAGEN, Hilden, Germany; Fig. 1B). The VP3-en-coding sequence was PCR-amplified with the primers VP3XbaN-59-GGTCTAGACATGGCACTTATTC-CCTGGAGACCA-39 and VP3XbaC-59-GGTCTAGACTACTGGAAGCGCCGCTTTTTCTT-39, clonedinto pFR36 (pFR36-VP3-3) and subcloned as a BamHI-PstI fragment into BglII-PstI-digested pQE41 (Fig.1B).

Using pFR36-VP1/2-12 as a template PCR amplificates were generated encoding the following segmentsof VP1: aa 364–384, aa 351–374, aa 351–384, aa 333–384, aa 305–384, aa 291–384, aa 276–384, aa256–384, and aa 212–384. Except the forward PCR primers for segments encoding aa 364–384 and aa305–384, in all other forward primers a SmaI site was added to facilitate subcloning into pFR36; a uniquecleavage site for XbaI was included in all reverse primers (Table 1). The PCR amplification products werepurified by 1% agarose gel electrophoresis. After digestion with SmaI and XbaI or only XbaI the fragmentswere inserted into the SmaI/XbaI-cleaved vector pFR36. The authenticity of the nucleotide sequence wasverified by dideoxy chain termination DNA sequence analysis. For the screening of recombinant plasmidsthe E. coli K12 strains XL-1 Blue and RR1 were used.

Expression of recombinant HaPyV proteins. The pQE-derived expression plasmids were retransformedinto E.coli K12 M15pREP4 cells (QIAGEN). Expression of the recombinant dihydrofolate reductase(DHFR)/HaPyV fusion proteins was performed as described recently (42).

The pFR36-derived recombinant plasmids were retransformed into the E.coli K12 strain JM109. Re-combinant bacteria were screened for the expression level of the respective VP1 derivatives: Individualcolonies were inoculated to 10 mL of LB medium and incubated overnight without shaking at 37°C; forexpression, 0.5 mL of the stock was added to 5 mL of M9C minimal medium supplemented with 1%casamino acids (Difco, Detroit, MI)/0.2% glucose and grown overnight on a rotary shaker at 37°C to anoptical density of OD540 of 3–4 (usually 16–20 h). For preparative purposes, 0.5 mL of the stock was trans-ferred to 300 mL of M9C medium and incubated as described above.

Purification of E.coli-expressed authentic HaPyV-VP1-VLPs and frCP-derived VLPs. The initialsteps of VP1 protein purification, including the preparation and sonication of bacterial cell lysates, wereperformed according to Leavitt et al. (21) with a buffer composed of 50 mM Tris-HCl (pH 7.9), 2 mMEDTA, 5% glycerol, 15 mM 2-mercaptoethanol, and 250 mM NaCl. After centrifugation, proteins wereprecipitated from supernatant with ammonium sulphate at 35% saturation over 3 h at 4°C. The pellet was

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resuspended in 1 mL of a buffer containing 10 mM Tris-HCl (pH 7.5), 1 mM EDTA, 1 M NaCl, and loadedonto a Sephacryl S-200 (Sigma, Steinheim, Germany) column. After elution with a buffer containing 10mM Tris (pH 7.2), 1 mM EDTA, and 250 mM NaCl, fractions were collected and the presence of VLPswas tested by negative staining electron microscopy.

The purification of frCP-VLPs (without insertion) was performed as described previously (32). Briefly,cells were disrupted in lysis buffer (50 mM Tris-HCl, pH 8.0; 5 mM EDTA; 50 mg/mL phenylmethyl-sulphonylfluoride; 2 mg/mL lysozyme) by incubation on ice for 30 min, freeze-thawing (three times), andsonication. After centrifugation, proteins were precipitated from the supernatant with ammonium sulphateat 50% saturation over 12 h at 4°C. The ammonium sulphate pellet was resuspended in 1.5 mL of PBS andloaded onto a CL4B (Sigma) column. After elution with PBS, fractions were collected and tested for VLPformation by electron microscopy.

The expression level of the VP1/frCP fusion proteins was determined by double radial immunodiffusion.Their solubility was estimated by comparison of equal amounts of soluble and insoluble protein fractionsin the immunoblot. Briefly, 20 optical units of cells were harvested and lysed in 200 mL of lysis buffer.The soluble fraction was obtained after centrifugation for 15 min at 10,000 rpm; the insoluble fraction waswashed with PBS, centrifuged again and resuspended in PBS (the same amount as for the soluble fraction).To both fractions, Laemmli buffer was added 1:1.

Expression in yeast and purification of polyomavirus VP1-derived VLPs. The entire VP1 proteinsof HaPyV, JCPyV, BKPyV, and SV40 were expressed in yeast Saccharomyces cerevisiae and purified bycesium chloride density gradient centrifugation as described (37,38).

Double radial immunodiffusion. The double radial immunodiffusion was basically done according toOuchterlony (28). Serial dilutions of lysates from frCP and VP1/frCP-expressing cells (2 ODs in 200 mLof Laemmli buffer) were analyzed with polyclonal anti-frCP antibodies in 0.8% agarose M (PharmaciaBiotech AB, Uppsala, Sweden)/PBS.

SDS–polyacrylamide gel electrophoresis and immunoblot. For SDS–polyacrylamide gel elec-trophoresis (SDS-PAGE), bacteria were pelleted, resuspended in SDS-PAGE sample buffer containing 2%SDS and 2% 2-mercaptoethanol and lysed by heating at 100°C for 5 min. Protein samples were applied toa 12–18% gradient running gel and 4% stacking gel.

After separation, proteins were electro-transferred onto nitrocellulose sheets (Schleicher & Schuell, Das-sel, Germany) and immunoblot was conducted according to a standard semi-dry blotting procedure. Nitro-


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TABLE 1. PRIMERS USED FOR PCR AMPLIFICATION OF THE DIFFERENT VP1-ENCODING FRAGMENTS

Codon position

Forward primeraa 364* 59-GTGAATAGGTTCATTGACAAG-3 9

aa 351 59-ACCCGGGATTTATGAGGGTACAGAGGCT-3 9

aa 333 59-ACCCGGGACAATTGAAGGCCAGCCCATG-3 9

aa 305 (2384)* 59-AATGTGACCTTGAGAAAAAGA-3 9

aa 305 (2351) 59-ACCCGGGAATGTGACCTTGAGAAAAAGA-3 9

aa 291 59-GCACCCGGGAACAGTGCAGGCTGGCATTGG-3 9

aa 276 59-GCACCCGGGCTTTATCTCAGTGCAGCAGAT-3 9

aa 256 59-GCACCCGGGACCTTGACTACTGTGCTTCTG-3 9

aa 212 59-GCACCCGGGGGAAAATTGGACAAGGATGGT-3 9

Reverse primeraa 384 59-CGATCTAGAGTGTTTGCTGGTTTTGCA-3 9

aa 374 59-CGATCTAGAGTCTGTTGGCCATACTTGTCAAT-3 9

aa 351 59-CGATCTAGAGTAATCCTGACTTCTTCTACC-3 9

Except forward primers marked by *, all primers contain unique cleavage sites for SmaI or XbaI tofacilitate subcloning into SmaI/XbaI-cleaved pFR36.

cellulose sheets were incubated with polyclonal rabbit anti-frCP antibodies (dilution 1:1,000), rabbit anti-HaPyV-VP1 (aa 1–384) antibodies (dilution 1:100), and sera of HaPyV-infected hamsters (dilution 1:200)at room temperature overnight, washed three times with PBS/Tween-20, and then incubated for 1–2 h atroom temperature with peroxidase-labeled anti-rabbit or anti-hamster IgG antibodies. The immune reactionwas detected by addition of 3,39-diaminobenzidine.

Electron microscopy. The protein samples were adsorbed on carbon-formvar coated grids and stainedwith 2% phosphotungstic acid (pH 6.8). The grids were examined with a JEM 100C electron microscope(JEOL Ltd., Tokyo, Japan) at an accelerating voltage of 80 kV.

Direct and competitive ELISA. For the direct ELISA, 96-well polysorb microtiter plates (Nunc,Roskilde, Denmark) were coated with VP1/frCP-, frCP-, and VP1-derived VLPs (10 mg/mL per well), re-spectively, and incubated for 12–20 h at 4°C. After removal of the liquid and postcoating with 1% BSA(in PBS/0.05% Tween-20; 200 mL per well) for 1 h at 37°C, serial dilutions of the various antibodies wereadded: rabbit anti-HaPyV-VP1 serum (aa 1–384; no. 1564), sera from HaPyV-infected Pap and PF ham-sters, JCPyV- and BKPyV-VP1-specific sera, and sera from mice and rabbits immunized with HaPyV-VP1/frCP-VLPs. After an incubation for 1 h at 37°C, the appropriate secondary antibodies (Sigma, St. Louis,MO) conjugated to horse radish peroxidase were added. IgG subtype analysis of sera from mice immunizedwith VP1/frCP-VLPs was done with a mouse monoclonal antibody isotyping reagent kit according to theprotocol of the manufacturer (Sigma). Between all incubation steps, the plates were washed three times with0.05% Tween-20 in PBS. The immune reaction was detected by adding of o-phenylendiamine/H2O2 sub-strate (Sigma) for 20 min at room temperature in dark phase. Optical densities (ODs) were measured at 492nm using an automatic Immunoscan MS reader (Labsystems, Helsinki, Finland).

For the competitive ELISA, purified HaPyV-VP1-VLPs were adsorbed on polysorb microtiter plates. Be-fore adding, increasing amounts of VP1/frCP-VLPs as competitor were preincubated with a standard dilu-tion (1:25,000) of the rabbit anti-HaPyV-VP1 serum (aa 1–384; no. 1564) for 20 min at 37°C, and ELISAreaction was continued as described above. According to a calibration curve of serial dilutions of antibody,the standard antibody dilution (1:25,000) yielding 50% of the maximal OD492 value was chosen. As con-

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TABLE 2. EXPRESSION LEVEL, SOLUBILITY, AND ASSEMBLY COMPETENCE

OF VP1/frCP FUSION PROTEINS IN COMPARISON WITH frCP

frCP fusionconstruct: aapositions of Predicted Expression ParticleVP1 MW, kDa levela Solubilityb formationc

364–384 21.0 111 1 111

351–374 21.4 111 1 111

351–384 22.6 111 1 11

333–384 24.9 111 1 11

305–384 28.7 11 1 1

305–351 24.6 1 ND ND291–384 30.7 1 ND —276–384 32.7 11 ND ND256–384 35.1 111 1 ND212–384 40.7 11 1 —

aDetermined by double radial immunodiffusion test of E. coli lysates according to Ouchterlony (28): 111, 75–100%;11, 50–75%; 1, 25–50% from frCP production.

bDetermined by immunoblotting comparing soluble and insoluble fractions: 1, more than 50% of protein in solublefraction; ND, not determined.

cDetermined by electron microscopy: 111, stable particles in amounts similar to frCP; 11, stable particles, loweramount compared with frCP; 1, particles formed (detected in crude lysate) but destroyed during chromatography; —, noparticles detected.

trols frCP-VLPs and VP1-VLPs were used as competitors when VP1- and frCP-VLPs, respectively, havebeen adsorbed to the solid phase.

Immunization of mice and rabbits. Female 6–8-week-old BALB/c mice (5 per group) were immunizedat day 0 with 0.02 mg (full dose: 0.01 mg intraperitoneally, i.p., and 0.01 mg subcutaneously, s.c.) ofchimeric VP1/frCP-VLPs in Complete Freund’s adjuvant (CFA, Sigma), followed by two booster immu-nizations in incomplete Freund’s adjuvant (IFA, Sigma) given at days 10 (half of full dose: 0.005 mg i.p.and 0.005 mg s.c.) and 24 (full dose, at the same way). Sera obtained on day 32 were analyzed by ELISAfor production of frCP- and VP1-specific antibodies.


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FIG. 2. Detection of frCP fusion proteins in immunoblot by rabbit sera raised against frCP (A), HaPyV-VP1 (aa1–384) (B), and JCPyV-VP1 (C). Crude lysates of JM109 cells expressing frCP fusion proteins carrying aa 364–384(lane 3), 351–374 (4), 351–384 (5), 305–351 (6), 333–384 (7), 305–384 (8), 291–384 (9), 276–384 (10), 256–384 (11),and 212–384 (12), respectively, were applied to the gel. As controls purified E.coli–expressed VP1-derived VLPs (en-tire VP1, aa 1–384; lane 1) and frCP (lane 2) were used. The full-sized VP1/frCP fusion proteins are marked by ar-rows. Molecular weight marker proteins: 97.0 (phosphorylase b), 66.0 (albumin), 45.0 (ovalbumin), 30.0 (carbonic an-hydrase), 20.1 (trypsin inhibitor), and 14.4 (a-lactalbumin) kDa.

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Immunization of rabbits with frCP-derived VLPs harboring aa 333–384 and aa 351–384, respectively, ofHaPyV-VP1 was performed by BioGenes (Berlin, Germany).

RESULTS

The highly flexible C-terminal region of VP1 bears immunodominant B-cell epitopes. Hamster seracollected from a spontaneously HaPyV-infected hamster colony, including papilloma-bearing (Pap) and pa-pilloma-free (PF) animals, were screened for the presence of HaPyV-VP1-, VP2-, and VP3-specific anti-bodies using dihydrofolate reductase (DHFR) fusion proteins with VP1, VP2, and VP3, respectively, ex-pressed in E.coli (Fig. 1B,C). All 18 and 16 sera from Pap and PF animals, respectively, contained antibodiesdirected against VP1 and VP2. In only one out of 34 animals, no anti-VP3 antibodies were detected (datanot shown).

Recently, we have observed differences in the epitope specificity of antibodies of Pap and PF hamsterswhen using linear synthetic peptides (42). Here, we used a C-terminal expression library of HaPyV-VP1based on N-terminal fusions of VP1 segments to frCP. These gene fusions were generated by the means ofPCR amplification of segments encoding selected portions of VP1. The different VP1/frCP fusion proteinswere detected in crude lysates of E.coli cells by immunoprecipitation with rabbit anti-frCP polyclonal an-tibodies that recognize denaturated frCP (data not shown). The expression level of frCP fusions with aa364–384, aa 351–374, aa 351–384, aa 333–384, and aa 256–384, respectively, correspond to that observedfor frCP alone, whereas the other derivatives were found in lower quantities (Table 2). The expression of

TABLE 3. REACTIVITY OF SERA OF HaPyV-INFECTED Z3 HAMSTERS

WITH VP1/frCP FUSION PROTEINS IN THE IMMUNOBLOT

Entire VP1,Serum frCP aa 1–384 aa 305–351 aa 333–384 aa 351–374 aa 364–384

Pap a4 — 1 1 1 1 —Pap a8 — 1 1 1 1 —Pap 33 — 1 1 1 (1) —Pap 581 — 1 (1) (1) (1) (1)Pap 608 — 1 2 1 1 —Pap M1091 — 1 1 1 1 —Pap M1142 — 1 1 1 1 1

Pap M1286 — 1 1 1 (1) —Pap a2 — 1 1 1 1 1

PF a1 — 1 1 1 1 1

PF 556 — 1 1 1 1 —PF 598 — 1 1 1 1 1

PF 607 — 1 1 1 1 1

PF F1151 — 1 1 1 1 1

PF F1208 — 1 1 1 1 1

PF 523 — 1 1 1 1 —PF 602 — 1 1 1 1 —

NK — — — — — —

1, strong reactivity; (1), weak reactivity; —, no reactivity; Pap, papilloma-bearing; PF, papilloma-free HaPyV-infected hamsters; NK, serum pool from noninfected HaP hamsters.

frCP fusions with HaPyV-VP1 segments

the HaPyV-VP1 derivatives of the predicted size was confirmed by immunoblot analysis using the frCP-specific rabbit serum (Fig. 2A, lanes 3–12) and a HaPyV-VP1 (aa 1–384) specific rabbit serum (Fig. 2B,lanes 3–12). In addition to the predicted full-sized VP1/frCP fusion proteins, shorter immunoreactive pro-tein bands were observed, which may result from proteolytic degradation. The control proteins, entireHaPyV-VP1 and frCP (without insertion), demonstrated the expected reactivities with the correspondingantisera (lanes 1 and 2 in Fig. 2A,B).

When analysing crude lysates from E.coli cells of the C-terminal VP1 expression library with hamstersera in the immunoblot (Table 3), all 17 hamster sera tested (nine Pap and eight PF hamster sera) reactedwith the entire VP1 (aa 1–384), aa 333–384 (Table 3) and all larger VP1 segments (data not shown). Six-teen out of 17 and all 17 sera reacted also with aa 305–351 and aa 351–374, respectively. In contrast, thesegment aa 364-384 was recognized by only eight out of 17 sera, including three out of nine Pap and fiveout of eight PF sera.

In addition, the HaPyV-VP1 expression library was tested in the immunoblot with a rabbit anti-JCPyV-VP1 serum (Fig. 2C). As found already earlier with the DHFR-VP1 fusion protein (42), the serum reactedwith the entire VP1 (lane 1). In addition, the anti-JCPyV-VP1 serum reacted with all C-terminal segmentsof VP1, including the shortest segments aa 305–351, aa 351–384, aa 351–374, and aa 364–384 (lanes 3–6).

The coat protein of bacteriophage fr allow the formation of chimeric particles harboring the im-munodominant region of HaPyV-VP1. The panel of VP1/frCP fusion proteins was analysed for its ca-pacity to form chimeric VLPs. In lysates of all constructs tested, about 50% or more of the VP1/frCP fu-


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FIG. 3. Formation of frCP-derived VLPs harboring aa 333–384 (A), aa 351–384 (B), and aa 364–384 (C) of HaPyV-VP1, respectively, demonstrated by negative staining electron microscopy. frCP particles are shown as a control (D).Bar 5 50 nm.

sion proteins were found to be localized in the soluble fraction, but only frCP fusions harboring aa 364–384,aa 351–374, aa 351–384, and aa 333–384 of VP1, respectively, allowed the formation of VLPs which canbe purified to high yields by column chromatography. In the case of the construct carrying aa 305–384 ofVP1, particle formation was observed in total cell lysate, but particles were not obtained after the standardpurification procedure. This is probably due to destruction of the preformed VLPs during the following pu-rification steps. In contrast, no particles were detected in the case of constructs carrying VP1 aa 291–384and aa 212–384, respectively. Negative staining electron microscopy confirmed the formation of chimericVP1/frCP-VLPs (Fig. 3A–C) corresponding in their morphology and size to frCP-VLPs (Fig. 3D). How-ever, when lysing cells under the same conditions and analyse VLPs by electron microscopy at differentstages of purification (total cell lysate, ammonium sulphate precipitation, fractions collected after columnchromatography) chimeric VP1/frCP-VLPs seem to be not of the same stability as frCP VLPs.

The antigenicity of chimeric frCP-VLPs harboring aa 333–384, aa 351–384, and aa 364–384, respectively,was investigated by direct and competitive ELISA. In the direct ELISA, all three antigens reacted with the rab-bit anti-HaPyV-VP1 (aa 1–384) serum (no. 1564) at very high titers (Table 4). For ELISA, sera from HaPyV-infected Pap and PF hamsters and, as control antigens, frCP- and HaPyV-VP1-VLPs expressed in E.coli wereused (Table 4). In general, all sera reacted with the highest titers with the entire VP1 (aa 1–384); except fortwo sera (one Pap and one PF serum) the reciprocal titers were $51,200. Furthermore, all sera reacted with theC-terminal portions of VP1 (aa 333–384, aa 351–384, aa 364–384). When comparing the reactivity of the Papand PF sera with the entire VP1 and aa 333–384, equal or similar reciprocal titers were observed. When ana-lyzing the shortest VP1 derivative (aa 364–384) in comparison to the entire VP1, the reciprocal titers of fiveout of eight and five out of six sera from Pap and PF hamsters, respectively, were reduced.

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TABLE 4. REACTIVITY OF E. coli–EXPRESSED CHIMERIC HaPyV-VP1/frCP-VLPS

WITH SERA OF HaPyV-INFECTED HAMSTERS IN THE ELISA

HaPyV-VP1

Segments fused N-terminally to frCP

Serum frCP aa 1–384 aa 333–384 aa 351–384 aa 364–384

anti-VP1 ,400a $51,200 $51,200 $51,200 $51,200

Pap a2 ,400 $51,200 .25,600 .56,400 3,600Pap a4 ,400 .51,800 .51,800 .51,600 800Pap a36 ,400 $51,200 $51,200 .12,800 12,800Pap a46 ,400 $51,200 $51,200 .12,800 12,800Pap a33 ,400 $51,200 $51,200 .25,600 $51,200Pap 533 ,400 $51,200 $51,200 $51,200 12,800Pap 559 ,400 $51,200 .51,200 .25,600 12,800Pap 581 ,400 $51,200 $51,200 $51,200 $51,200

PF a1 ,400 .51,600 .51,600 .51,600 3,200PF a25 ,400 $51,200 .25,600 .53,200 6,400PF 555 ,400 $51,200 $51,200 $51,200 1,600PF 556 ,400 $51,200 .25,600 .53,200 6,400PF 582 ,400 $51,200 $51,200 $51,200 800PF 607 ,400 $51,200 $51,200 .12,800 12,800

NK ,400 ,400 ,400 ,400 ,400

aGiven are the highest reciprocal titers where the OD value was .0.4.Pap, papilloma-bearing; PF, papilloma-free HaPyV-infected hamsters; anti-VP1, rabbit serum (no. 1564) raised against

E. coli–expressed HaPyV-VP1 (aa 1–384) VLPs; NK, serum pool from noninfected HaP hamsters.

In a competitive ELISA the frCP-VLPs harboring VP1 aa 351–384, aa 364–384, and aa 333–384, re-spectively, competed to similar levels with the entire VP1 (adsorbed to the solid phase) for binding toHaPyV-VP1-specific rabbit antibodies (Fig. 4). As expected, the frCP alone did not show any competition(control 2) and, when frCP was adsorbed to solid phase, no binding of the VP1-specific antibodies was ob-served (control 1).

Chimeric VP1/frCP-VLPs induce a strong VP1-specific antibody response in rabbits and mice. Tostudy the humoral immunogenicity of chimeric VP1/frCP-VLPs, rabbits were immunized with VLPs har-boring aa 333–384 and aa 351–384 of HaPyV-VP1, respectively. The antibody response of rabbits wasanalysed in the ELISA using yeast-expressed purified VLPs derived from VP1 proteins of HaPyV, BKPyV,JCPyV, and SV40 (Table 5). In all four rabbits immunized with VLPs harboring aa 333–384 and aa 351–384,respectively, a high-titered antibody response against HaPyV-VP1 was induced. In addition, these sera werealso highly cross-reactive with VP1-derived VLPs from primate polyomaviruses BKPyV, JCPyV, and SV40.In general, the titers of the VP1-specific antibodies in animals immunized with VP1/frCP fusions are sim-ilar to those of the rabbit immunized with HaPyV-VP1-VLPs (rabbit no. 1564). Interestingly, when ana-lyzing mice sera raised against JCPyV- and BKPyV-VP1 only these antigens were recognized but notHaPyV- and SV40-VP1.

To study the humoral immune response more precisely, BALB/c mice were immunized with VLPs har-boring aa 351–384 and aa 364–384 of HaPyV-VP1, respectively. All 10 animals developed specific IgG an-tibodies against frCP and VP1 (Fig. 5A). As expected, no specific IgM antibodies were detected (data notshown). In four out of five mice immunized with aa 351–384 (nos. 1, 2, 3, and 5), the anti-VP1 responsewas IgG1 dominated, whereas one mice showed a IgG2a-dominated response (Fig. 5B). In four out of fiveanimals (nos. 1, 2, 4 and 5), the frCP-specific antibody response was also mainly of IG1 isotype; in animal3, a low response by IgG1, IgG2a, and IgG2b was observed. Similarly, in four out of five mice (nos. 6, 7,8, and 10) immunized with aa 364–384, the antibody response against VP1 and frCP was IgG1 dominated(Fig. 5C).

DISCUSSION

The structural investigation of SV40 and MPyV capsids by x-ray analysis showed that the individualVP1 pentamers are hold together by the C-terminal arms of the VP1 monomers. The proximal 63 aa of VP1


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FIG. 4. Demonstration of VP1 antigenicity of chimeric VP1/frCP-VLPs by competitive ELISA using a rabbit an-tiserum raised against HaPyV-VP1 (aa 1–384). For competitive ELISA, plates were coated with E.coli–expressedHaPyV-VP1 (aa 1–384) VLPs and incubated with rabbit anti-HaPyV-VP1 (aa 1–384) antibodies (diluted 1:25,000).Before adding, the antiserum was preincubated with frCP VLPs (control 2) and frCP-derived VLPs containing aa333–384, aa 351–384, and aa 364–384 of HaPyV-VP1, respectively. As an additional control, frCP was adsorbed tothe solid phase and VP1-VLPs were used as competitor (control 1).

emerge from each monomer and invade another pentamer (22,45). The deletion of the C-terminal regionwas demonstrated to prevent assembly of VP1 VLPs (10). The prediction of the three-dimensional struc-ture of the HaPyV-VP1 showed the C-terminal part as highly flexible suggesting its role as potential epi-tope region (11). In fact, in the C-terminal region of SV40-VP1 a linear epitope was mapped (1). Epitopemapping studies using sera from HaPyV-infected hamsters proved the C-terminal region aa 320–384 ofHaPyV-VP1 to be an immunodominant, cross-reactive B-cell epitope region. However, when using linearsynthetic peptides spanning the HaPyV-VP1 differences in the epitope specificity of antibodies from Papand PF hamsters were observed (42).

Therefore the reactivity of sera from HaPyV-infected Pap and PF hamsters with the entire HaPyV-VP1,-VP2, and -VP3 and a C-terminal VP1 expression library was investigated. Our data confirmed previousdata that the major capsid protein VP1 as well as the minor capsid proteins VP2 and VP3 are immuno-

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TABLE 5. REACTIVITY OF RABBIT SERA RAISED AGAINST HaPyV-VP1/frCP-VLPS AND VP1-VLPS WITH YEAST-EXPRESSED VP1-DERIVED VLPS FROM DIFFERENT POLYOMAVIRUSES

Antigens

Sera raised against BKPyV-VP1a JCPyV-VP1a SV40-VP1a HaPyV-VP1a DOBV-N b BSAc

HaPyV-VP1/frCP $6 5 5 5 1 1(aa 333–384)(rabbit 3498)

HaPyV-VP1/frCP 5 5 2 5 2 1(aa 333–384)(rabbit 3497)

Rabbit 3497 1 1 ,1 ,1 1 1(preimmune)



Rabbit 3499 2 2 ,1 ,1 1 1(preimmune)

HaPyV-VP1 5 $6 5 $6 2 1(aa 1–384)(rabbit 1564)

JCPyV-VP1 $6 5 1 ,1 1 ,1(mouse)

BKPyV-VP1 $6 5 1 ,1 1 ,1(mouse)

aVLPs formed by yeast-expressed VP1 proteins of HaPyV, BKPyV, JCPyV and SV40, respectively.bDOBV-N, Dobrava hantavirus nucleocapsid protein expressed in yeast (negative control) kindly provided by A.

Dargeviciute (Vilnius).cBSA, bovine serum albumin (negative control).Given are the log10 of the highest reciprocal titers where the OD value was . 0.4.

FIG. 5. Antibody response of mice immunized with chimeric VP1/frCP-VLPs as shown by ELISA. (A) Total IgGantibody response to HaPyV-VP1 and frCP of mice 1–5 immunized with VP1 (aa 351–384)/frCP-VLPs and mice 6–10immunized with VP1 (aa 364–384)/frCP-VLPs. (B,C) Determination of the IgG isotypes of antibodies directed againstVP1 and frCP in mice immunized with VP1 (aa 351–384)/frCP-VLPs (B) and VP1 (aa 364–384)/ frCP-VLPs (C).

dominant inducing strong antibody responses in HaPyV-infected hamsters (9,41,42,47). In addition, no dif-ferences in the reactivity of Pap and PF sera to entire VP1, VP2, and VP3 as well as to C-terminal VP1segments were observed.

In line with our previous data (42), the C-terminal region of VP1 again was confirmed to be immuno-dominant. Here, we were able to define new B-cell epitopes at the C-terminus of HaPyV-VP1 in the re-gions between aa 305–351, aa 351–374, and aa 364–384, which were found to be cross-reactive also witha JCPyV-VP1-specific rabbit serum. The C-terminal portion of VP1 overlaps a region mapped by a pep-scan analysis (aa 353–367) exclusively in a serum pool of Pap hamsters (42). This difference in the reac-tivity of Pap and PF animals could not be confirmed: Both groups of sera, from Pap and PF animals, re-acted with the frCP fusion harboring aa 351–374 of VP1. Likely, the prolongation of the peptide by additionof some aa increased the reactivity with sera from PF hamsters. In addition, the reactivity of this segmentconfirmed the previously observed strong reactivity of the peptide aa 355–367 with a rabbit anti-JCPyV-VP1 serum. The other previously described highly cross-reactive peptide (aa 340–352) was also confirmedby the reactivity of the anti-JCPyV-VP1 serum with frCP fusion with aa 305–351 of VP1. Moreover, thesegments aa 333–384, aa 351–384, and aa 364–384, were also found to induce a strong antibody responsein mice and rabbits. Interestingly, the VP1-specific antibody response in four out of five mice immunizedwith VLPs harboring aa 351–384 and aa 364–384, respectively, was IgG1 dominated. Only one out of fiveanimals in both groups were found to be IgG2a dominated or to have an identical endpoint titer for IgG1and IgG2a. Although these data suggested a Th2-like dominated response, additional investigations to provethis by analysis of the cytokine profile induced by immunization are required. In contrast, only in two outof five mice immunized with yeast-expressed, adjuvant-complexed HaPyV-VP1-VLPs VP1-specific anti-bodies showed the highest reciprocal endpoint titer for IgG1 isotype, whereas in the remaining two and onemice for IgG1/IgG2a and IgG2a isotypes, respectively (our unpublished data).

RNA bacteriophages contributed markedly to the resolution of three-dimensional structure of virions andVLPs, including the construction of chimeric VLPs. Capsids of RNA phages R17 and f2 were among thefirst virions with clearly resolved symmetry (7). X-ray crystallography of the RNA phage fr virions andVLPs (23) showed their close similarity to the most popular representatives of the groups I–MS2 (49),II–GA (46), and III–Qb (13). According to the structure, the 180 CP subunits are arranged in dimers as ini-tial building blocks and form a lattice with the triangulation number T 5 3 (49). The CP subunit consistsof a five-stranded b-sheet facing the inside of the particle, and a hairpin and two helices on the outside.

Physical proximity of the N- and C-termini of two monomers from the same dimer and their close tosurface location put these protein regions in the forefront of candidate insertion sites. However, low toler-ance of RNA phage capsids to foreign insertions strongly limited their use as a basis for the generation ofchimeric VLPs in vaccine development. Although the N- and C-termini seemed to be more prospective thanother positions of the frCP, tested at aa residues 10, 51, 63, 69, 96, 114 (19,32; Kozlovskaya and Pushko,unpublished data), their capacity to foreign insertions also remained relatively low, not more than 12-aaresidues (19,32). Nevertheless, special vectors have been constructed for insertions at the N-terminus offrCP in all possible reading frames (2), and an universal marker, the preS1 epitope DPAFR (43), was suc-cessfully inserted and mapped as exposed on the particle surface (Pushko, unpublished data). Furthermore,attempts to introduce the 40-aa long V3 loop of the HIV-1 gp120 into the N-terminus of frCP failed andled to assembly-deficient products (Tars, unpublished data). Moreover, N-terminal insertion of a short Flagoctapeptide into the related MS2 CP prevented its self-assembly, which could be restored only by geneticfusion of the duplicated CP-encoding sequence (29). It resulted in the synthesis of covalently-linked MS2CP dimers considerably more tolerant to structural perturbations and able to overcome structural defectsaccompanying Flag peptide insertion.

Our results clearly show that frCP is able to provide unusually high vector capacity for N-terminal in-sertions, when addition of C-terminal segments of HaPyV-VP1 of 21, 34, and 52 aa to the N-terminus offrCP was found dispensable for frCP-VLP self-assembly. The decreased ability or failure of frCP-VLP for-mation for larger segments of VP1 (i.e., aa 305–384, aa 291–384, aa 212–384) is probably due to an in-creasing hydrophobicity, an increasing tendency of b-sheet formation and potential association of VP1 seg-ments with b-sheet regions of the phage frCP itself.

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For the first time the length of insertions into RNA phage coats was comparable to the length of the car-rier protein. These findings strongly suggest the importance of the primary and spatial structure of foreigninsertions over their length in terms of fitting to the VLP carrier. In addition, our data confirm the flexiblefeatures of the C-terminal part of HaPyV-VP1.

We plan to concentrate our further studies on the prediction of protein domains, which could be addedto the N-terminus without damage of self-assembly competence of RNA phage VLPs. Successful N-termi-nal insertions of relatively long, immunologically important HaPyV sequences will restore the interest tothe RNA phage coats as potent VLP carriers and vaccine candidates.

ACKNOWLEDGMENTS

We acknowledge very much the support of Drs. Detlev H. Krüger, Muhsin Özel, and Cornelius Fröm-mel, the technical assistance of Karin Dauer and Juris Ozols in SDS-PAGE analysis, and Eva Stankevicaand her group for the synthesis of oligonucleotides. The work was supported by Charité Medical School,Bundesministerium für Forschung und Technologie/Internationales Büro-DLR (grants no. LVA 00/001 andLTU 00/001) and by grant from the Latvian Council of Science (no. 96.0736)

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Address reprint requests to:Dr. Rainer Ulrich

Institute of VirologyCharité Medical School

Humboldt UniversityD-10098 Berlin, Germany

E-mail: [email protected]


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Chimeric Bacteriophage fr Virus-Like Particles Harboring the Immunodominant C-Terminal Region of Hamster Polyomavirus VP1 Induce a Strong VP1-Specific Antibody Response in Rabbits