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Originally published In Press as doi:10.1074/jbc.M513571200 on April 18, 2006

J. Biol. Chem., Vol. 281, Issue 25, 17474-17481, June 23, 2006
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Hepatitis B Virus Capsid-like Particles Can Display the Complete, Dimeric Outer Surface Protein C and Stimulate Production of Protective Antibody Responses against Borrelia burgdorferi Infection*Formula

Claudia Skamel{ddagger}, Martin Ploss§, Bettina Böttcher§, Thomas Stehle, Reinhard Wallich||, Markus M. Simon, and Michael Nassal{ddagger}1

From the {ddagger}University Hospital Freiburg, Internal Medicine II/Molecular Biology, D-79106 Freiburg, Germany, §European Molecular Biology Laboratory, D-69117 Heidelberg, Germany, Max Planck Institute of Immunobiology, D-79108 Freiburg, Germany, and ||University Hospital Heidelberg, Institute of Immunology, D-61920 Heidelberg, Germany

Received for publication, December 21, 2005 , and in revised form, April 12, 2006.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Hepatitis B virus capsid-like particles (CLPs), icosahedral assemblies formed by 90 or 120 core protein dimers, hold promise as immune-enhancing vaccine carriers for heterologous antigens. Insertions into the immunodominant c/e1 B cell epitope, a surface-exposed loop, are especially immunogenic. However, display of whole proteins, desirable to induce multispecific and possibly neutralizing antibody responses, can be restrained by an unsuitable structure of the foreign protein and by its propensity to undergo homomeric interactions. Here we analyzed CLP formation by core fusions with two distinct variants of the dimeric outer surface lipoprotein C (OspC) of the Lyme disease agent Borrelia burgdorferi. Although the topology of the termini in the OspC dimer does not match that of the insertion sites in the carrier dimer, both fusions, coreOspCa and coreOspCb, efficiently formed stable CLPs. Electron cryomicroscopy clearly revealed the surface disposition of the OspC domains, possibly with OspC dimerization occurring across different core protein dimers. In mice, both CLP preparations induced high-titered antibody responses against the homologous OspC variant, but with substantial cross-reactivity against the other variant. Importantly, both conferred protection to mice challenged with B. burgdorferi. These data show the principal applicability of hepatitis B virus CLPs for the display of dimeric proteins, demonstrate the presence in OspC of hitherto uncharacterized epitopes, and suggest that OspC, despite its genetic variability, may be a valid vaccine candidate.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
The icosahedral nucleocapsid of hepatitis B virus (HBV)2 is formed by multiple dimeric copies of the 183-residue core protein. The particle, serologically termed hepatitis B core antigen (HBcAg), is exceptionally immunogenic and can act as a T cell-dependent and as a T cell-independent antigen (1), most likely due to the repetitive presentation of the epitopes on its surface (2). This property, shared by some other viral particles (3), has been exploited to design recombinant HBV capsid-like particles (CLPs) as an immune-enhancing carrier for heterologous peptides (reviewed in Refs. 4, 5) and recently for whole chain polypeptides (6-9). Fusions into the most exposed region on the capsid surface (10-12), encompassing the immunodominant c/e1 B cell epitope of HBcAg (13), are particularly immunogenic for B cells. However, the need to conserve the particulate structure for high immunogenicity together with the central location in the primary sequence of the c/e1 epitope imposes restraints on the nature of the heterologous sequence that can be inserted.

The core protein consists of an N-terminal assembly domain encompassing the first ~140 aa (14, 15) and an Arg-rich C-terminal nucleic acid binding domain (16, 17). The structure of HBV CLPs is known from electron cryomicroscopy (10, 18-21) and x-ray crystallography (22). The capsids occur in two size classes (triangulation numbers T 3 and T 4) consisting of 90 and 120, respectively, core protein dimers (Fig. 1A). Dimerization involves a four-helix bundle to which each monomer contributes two long antiparallel {alpha}-helices ({alpha}3 and {alpha}4) (22); these bundles protrude as spikes from the capsid surface, with the c/e1 epitope at their tips (11, 12, 23).

The c/e1 loop lends itself as an insertion site; however, both the N and C termini of an insert must fit to the geometry of the core acceptor sites (Fig. 1B), a requirement not easily be met by many natively folded proteins. In the green fluorescent protein (GFP), the first complete protein displayed on HBcAg CLPs (6), the N and C termini are closely juxtaposed (24). By contrast, the termini of the outer surface protein A (OspA) of the Lyme disease agent Borrelia burgdorferi (see below) are at opposite ends of the elongated structure (25), such that generation of a multimerization-competent fusion required very long connecting linkers (9). Less obvious but equally critical is the propensity of a candidate insert to undergo homomeric interactions. Dimerizing and tetramerizing color variants of GFP, though isostructural to GFP, failed to form CLPs; the key role of insert quaternary structure was demonstrated by successful CLP formation when engineered monomeric variants were used instead (8). However, monomerization may involve the exchange of many amino acids (e.g. 33 in the case of the monomeric red fluorescent protein) (26) and thus not be suited for a protein to be used as a vaccine. Here we explored the possibility of generating HBcAg CLPs displaying a naturally dimeric whole protein antigen.

The outer surface protein C (OspC) of B. burgdorferi appeared as an ideal model insert. OspCs are ~210 aa in length, including an 18-aa signal peptide, cleavage of which exposes an N-terminal Cys residue for subsequent lipidation. The solved structures of three OspC variants (27, 28) revealed, for the aa sequence encompassing ~positions 40-200, a common mushroom-shaped dimeric structure with each monomer contributing half the stalk and half the head (Fig. 1A) and with all four termini protruding from the foot of the stalk at the start and the end, respectively, of long helices ({alpha}1 and {alpha}5). Though reminiscent of the four acceptor sites in the core protein dimer, the topology of the termini in the two dimers is different (Fig. 1, B and C). Hence, within one fusion protein dimer either the core parts or the OspC parts, but not both, could interact as in the unfused proteins. Successful CLP formation would therefore require substantial flexibility in either the carrier or the insert or not be possible at all.


Figure 1
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  		FIGURE 1.
Structural aspects. A, three-dimensional structures of OspC and core protein. Both proteins dimerize through formation of four-helix bundles; the involved helices ({alpha}1 and {alpha}5 in OspC and {alpha}3 and {alpha}4 in the core protein) are shown as ribbons, the remaining sequences as a backbone trace. Upward pointing helices are drawn in green, downward pointing helices in red. Residues differing between OspCa and OspCb are highlighted in the OspC monomer (top left) by space-filled representations. B-D, schematic representation of the topology of the termini of the OspC dimer and the acceptor sites in the core protein dimer. Helix {alpha}3 of the core protein must be fused to {alpha}1 of OspC and {alpha}5 of OspC to {alpha}4 of the core protein. B, the helices of the four-helix bundles are represented by straight cylinders with their directions indicated by arrows. C and D, close-ups of the connection sites. If arranged as in panel C, the termini of OspC dimer subunits a and b do not match to those of core dimer subunits a' and b'. D, the ends of OspC subunit a fit to those of core subunit b', but OspC subunit b would be dislodged. Possible outcomes include that (i) OspC dimerization prevents core dimerization and particle assembly, (ii) core dimerizes and assembles but prevents OspC dimerization, (iii) both proteins dimerize. This would require massive distortion of the connecting sequences such that in panel D OspC subunit b would fit to core subunit a. Alternatively, OspC subunit b could be contributed by a neighboring fusion protein dimer in the particle.

 
Second, OspC is of medical interest as a major surface antigen of Lyme disease-causing Borreliae. Lyme borreliosis, the most common vector-borne disease in Europe and the United States (for reviews see Refs. 29-31), is a progressive inflammatory disorder with dermal, cardiac, musculoskeletal, and neurological manifestations. It is transmitted to humans by Ixodes ticks from animals, mostly rodents, infected with various strains of three species of Borrelia, in the U.S. mainly by B. burgdorferi sensu stricto and in Europe by B. afzelii and B. garinii as well. Of the various outer surface proteins, OspA probably mediates colonization of the tick gut (32), whereas OspC is crucial for infection of the mammalian host (33). Hence, despite the substantial inter-strain and intra-strain variability of OspC (34-36), which has questioned its usefulness as a vaccine, the different OspC variants might have common interaction partners, e.g. the immunosuppressive Salp15 protein from tick saliva (37, 38), and consequently shared epitopes. Importantly, mouse models of Lyme disease are established that allow us to assess the capacity of a given vaccine formulation to induce neutralizing antibodies, which play the major role in clearing infection (39). For instance, a recombinant glutathione S-transferase, OspC fusion protein (recGST-OspC), but not the same OspC protein after removal of the GST part (rec-OspC), induced antibodies with prophylactic, and upon passive transfer but not active immunization, also therapeutic potential (40, 41).

Here we generated fusions of the HBV core protein with two variants of OspC, termed OspCa and OspCb, that according to molecular modeling form the typical mushroom-like dimer structures but differ at 15 surface-exposed aa positions (Figs. 1A and 2B). We addressed their ability to form CLPs, looked into the structural consequences of the nonmatching dimeric insert structure by electron microscopy and image reconstruction, and monitored their immunogenicity and protective potential in the mouse model.


   EXPERIMENTAL PROCEDURES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
RESULTS
 DISCUSSION
 REFERENCES
 
Plasmids—The parental expression vector pET28a2-HBc149_H6 encodes the HBV core protein from aa 1 to 149 with a C-terminal His6 tag; in the context of similar fusions with color variants of GFP the tag increased the assembly efficiency (8). The genes for OspCa and OspCb were isolated from a single B. burgdorferi ZS7-infected tick and cloned into the pGEX vector.3 The GST fusion protein encoded by pGEX-OspCb, recGST-OspCb, served as control antigen in the immunization experiments; recombinant OspCb without the GST part was obtained by thrombin cleavage (41). Using PCR primers OspC(+) GGAGCTCGAG.GAT.gga.ggg.ggt.ggt.acc.GGA.AAA.GAT.GGG.AAT.GCA and OspC(-), complementary to GTG.GCA.GAA.AGT.CCA.AAA.AAA.CCT.ggt.ggc.TCT.AGA.GTGTG (codons separated by dots, XhoI and XbaI cloning sites in underlined italics; linker codons Gly4-Thr, and Gly2 in lowercase), the genes for OspCa and OspCb, starting with codon Gly-23 and ending with the authentic C-terminal codon for Pro-211 (Gly-5 to Pro-193 in the mature protein) were amplified from the pGEX vectors and cloned into the parental HBcAg vector such that Pro-79 and Ala-80 were replaced (Fig. 2A). The new vectors were termed pET28a2-c149_OspCa_H6 and pET28a2-c149_OspCb_H6.

Fusion Protein Expression and Purification—The pET vectors were transformed into Escherichia coli BL21<DE> Codonplus RIL cells (Stratagene). Fusion protein expression was induced by isopropyl-1-thio-beta-D-galactopyranoside addition, and after shaking for 10 h at room temperature the cultures were harvested and cleared lysates were prepared as previously described (8). To enrich for particles, the lysates were sedimented through 10-60% sucrose step gradients (8). Aliquots from the gradient fractions were analyzed by SDS-PAGE using the Laemmli buffer system. Proteins were stained by Coomassie Blue, or the fusion proteins were detected by Western blotting using a monoclonal antibody (mAb) against HBcAg (mAb158, recognizing a linear epitope around aa 130 of the core protein) (42) or a mAb against OspC (LA97.1) (43). According to densitometric scanning of the Coomassie Blue-stained gel lanes, the fusion proteins in the center gradient fractions amounted to more than 90% of the total protein present, which was sufficiently pure for electron microscopy. For immunizations, the preparations were dialyzed (Slide-a-lyzer cassettes, 30-kDa molecular mass cutoff; Pierce), concentrated (Amicon Ultra devices, 30-kDa cutoff; Millipore), and subjected to a second round of sucrose gradient sedimentation. The center gradient fractions were pooled, dialyzed against phosphate-buffered saline (PBS), and concentrated to ~0.75 mg/ml; purity as estimated by densitometric scanning was ~94%.

Electron Cryomicroscopy and Image Reconstruction—Samples were frozen as described (44). Micrographs were taken under low dose conditions at a nominal magnification of 52,000 at a Philips CM 120 Biotwin operating at 100 kV and equipped with a LaB6 filament. For cryomicroscopy a Gatan cryoholder maintaining a temperature of 94 K was used. Micrographs were recorded on Kodak SO 163 film and scanned with a Zeiss Scai scanner with a step size of 21 µm/pixel corresponding to 4 Å at specimen level. From 5 micrographs a total of 407 small particle images were selected. Image processing was done with the IMAGIC 5 software package (45, 46). For two-dimensional reconstructions, single particle images were normalized in their gray value distribution, inverted in contrast, and centered by translational alignment to a rotationally averaged sum of all particles. Each particle was rotated by increments of 45° (0-315°), and the rotated particles were added to the data set enabling comparison. For accessing the local organization of OspC a segment of the outer rim of the particles (140 by 150 Å, including the edge of the HBc core shell and the peripheral density of the OspC layer) was analyzed where OspC is seen in side view projection and, due to the curvature, overlaps only a little with other features. The variation in density distribution in this segment was analyzed by multivariate statistical analysis, and particles were grouped according to their similarity. Particles in the same group were averaged, showing side view projections of OspC bound to the spikes. A three-dimensional icosahedral reconstruction approach is described in the supplemental material.

Immunization of Mice—For generation of immune sera (IS), four adult female BALB/c mice (H-2d; bred under specific pathogen-free conditions at the Max-Planck-Institut für Immunbiologie, Freiburg, Germany) per construct were repeatedly immunized into the base of the tail with initially 30 µg and subsequently 10 µg of the respective recombinant proteins (coreOspCa, coreOspb, and recGST-OspCb) in the presence of 100 µl of MPL-TDM adjuvant (composed of monophosphoryl Lipid A and synthetic trehalose dicorynomycolate; Sigma) and bled on the indicated days post-immunization. As control, IS was generated against wild-type core protein 1-149 following the same protocol. Prior to further analyses, the IS from each group of animals immunized with the same construct were pooled. OspC- and core protein-specific Ab in IS were quantified by a solid-phase ELISA as described (43) using 1 µg/ml of either recombinant OspCb or wild-type core protein 1-149 CLPs as substrate and mAb 97.1 or the core-specific mAb 312 (42) as reference antibodies. Western blot analysis using recGST-OspCb as antigen (250 ng/lane) was done as described (47). Pooled preimmune sera from the same animals served as negative controls.

Disease Model and Protection Experiments—Pools of IS to be used in protection experiments were collected from immunized BALB/c mice (groups of 3-4 mice) every 7-10 days for up to 93 days post-immunization starting after the third challenge. For passive immunization, three C.B-17 SCID mice (bred under specific pathogen-free conditions) were injected intraperitoneally with IS containing the equivalent of 10 µg of antibody specific for the individual recombinant protein or with the OspA-specific mAb LA-2 (5 µg/mouse) (43) and challenged experimentally (subcutaneously) with 103 spirochetes (strain ZS7)/mouse 2 h later. Control mice received either anti-wild-type core IS (30 µg of specific antibody), normal mouse serum, or PBS (300 µl each). Development of clinical arthritis in the tibiotarsal joints was monitored in the right and left tibiotarsal joint and scored under double-blind conditions (40). Testing for the outgrowth of viable spirochetes from cultured ear biopsies was done on day 42 post inoculation as described (41).


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
DISCUSSION
 REFERENCES
 
Generation of and Efficient CLP Formation by CoreOspCa and CoreOspCb Fusion Proteins—As carrier we used a HBV core protein truncated after aa 149 with a C-terminal His6 tag that efficiently forms CLPs in E. coli (48). The inserts (Fig. 2) encompassed the nearly full-length mature proteins from aa residues 23 to 211 of OspCa and OspCb. In the OspC crystal structures, residues preceding aa 40 and following aa 204 were not resolved (27, 28), suggesting these sequences are flexible; therefore, only short flanking linkers were added (G4T and G2).

Both the OspCa and the OspCb fusions were well expressed and largely soluble. Upon sedimentation in sucrose gradients of the cleared bacterial lysates, both proteins formed a sharp peak in the center of the gradient (shown in Fig. 3A for coreOspCa), as is typical for ordered HBcAg CLPs (8). A band of identical mobility was detected by immunoblotting using an anti-HBcAg mAb (Fig. 3A) as well as an anti-OspC mAb (not shown). Direct confirmation of efficient CLP formation was obtained by negative staining electron microscopy (EM) that revealed abundant particles exhibiting, compared with wild-type CLPs, a rough surface that on some particles appeared to consist of distinct spikes (Fig. 3B).

Structural Analysis of CoreOspCa CLPs—For a more detailed analysis, the coreOspCa sample was subjected to electron cryomicroscopy (cryo-EM) and image processing. Cryoelectron micrographs confirmed the presence of abundant spherical, though sometimes distorted, shells of different size classes (Fig. 4A). Compared with CLPs from unmodified His-tagged core protein 1-149, the fraction of small particles was substantially increased, as was also found for other, including smaller, c/e1 insertions (49). All showed distinct, large, outward protruding spikes, indicating that the OspC domains are indeed surface exposed.

For an approximate comparison of the size of the outer spikes with the expected dimensions of OspC, a two-dimensional image-processing approach was used. Particle images were centered, and then segments of the outer rim showing the outer densities in side view were compared. Particles with similar peripheral density distributions were grouped, and those within one group were averaged. The corresponding projections from several such groups are shown in Fig. 4B. All displayed regularly arranged densities at the positions expected for the core dimers constituting the inner shell, plus elongated and slightly kinked outward reaching protrusions. The largest of these protrusions were on average ~6.5 nm long and ~4 nm wide, in good agreement with the dimensions of an OspC dimer (~6.2 by 4.2 nm when viewed from the broadside); others had a similar length but appeared narrower. This would be compatible with edgewise-viewed OspC dimers or with monomers (Fig. 4C).


Figure 2
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  		FIGURE 2.
Scheme of the constructs used. A, arrangement of the core protein and OspC parts in the coreOspC fusion proteins. The carrier part, HBc1-149_H6, encompasses the assembly domain of the core protein, with the C-terminal basic domain (clusters of Arg residues symbolized by + signs) replaced by a His6 tag. Amino acids Pro-79 and Ala-80 were replaced by OspCa or OspCb starting with aa Gly-23 and ending with Pro-211 (Gly-5 and Pro-193 in the mature protein), flanked by G4T and G2 linkers. B, sequence comparison between OspCa and OspCb. The 18-aa signal peptide is omitted. Numbering starts with the lipidated cysteine Cys-19. The complete sequence is that of OspCa; residues exchanged in OspCb are indicated in the line below. The known three-dimensional structures (27, 28) encompass residues Pro-40 to Pro-202. Helices{alpha}1 to {alpha}5 are indicated by bars; the bracketed region denoted loop 5 contains recently identified type-specific linear epitopes (36).

 


Figure 3
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  		FIGURE 3.
Expression of and CLP formation by the coreOspC fusion proteins. A, sucrose gradient sedimentation. Aliquots from fractions (top, fraction 1; bottom, fraction 14) of a sucrose gradient loaded with cleared bacterial lysate containing the coreOspCa fusion protein were analyzed by SDS-PAGE. The fusion protein (arrow) accumulated in fractions 7-10; a separate gel was used for immunoblotting with a mAb against the core protein (bottom panel). For immunization, the pooled fractions 7 to 10 were concentrated and resubjected to gradient sedimentation. 10 and 3 µl of this material were analyzed by SDS-PAGE (right panel). B, electron microscopy. Negative staining EM of coreOspCa (top) and coreOspCb (bottom) as obtained from the first sucrose gradient revealed abundant particles with a much more elaborate surface structure than seen with CLPs from the unmodified carrier protein HBc1-149H6 (middle). Arrows point to CLPs with well visible surface protrusions.

 
Obtaining three-dimensional icosahedral reconstructions was hampered by the variability in the arrangement of the OspC layer between individual particles and by difficulty in accurately assigning particle orientations. Nonetheless, a plausible model could be derived by combining a reconstruction based on a known reference map of wild-type T 3 particles that emphasizes the inner core shell with a single particle-based reconstruction emphasizing the outer OspC shell (see supplemental Fig. S1 and legend). Although little detail of the core dimer spikes was visible, indicating that the model is of limited accuracy, it strongly suggests that the packing of the core dimers and the location of the spikes in the inner shell are comparable with wild-type CLPs. Furthermore, because the OspC densities were not centered above the core dimer spikes (supplemental Fig. S2), OspC dimerization is unlikely to occur between the two OspC moieties present on one core dimer. The data are, however, compatible with dimerization between OspCs present on neighboring core dimers. Irrespective of how the OspC moieties are arranged in detail, these data provide strong evidence for the ability of the HBcAg carrier to accommodate the insertion of a dimerization-competent whole protein antigen and to present it on an intact CLP surface.


Figure 4
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  		FIGURE 4.
Cryo-EM analysis of coreOspCa CLPs. A, cryoelectron micrographs. A sample of the gradient-enriched coreOspCa CLPs was visualized under near-native conditions in vitrified ice. Note the corona of extended spikes around each particle that is absent from the wild-type HBc1-149H6 CLPs shown below. B, two-dimensional image reconstructions. Images from individual CLPs were centered and grouped according to congruent peripheral density features. Averaged projections from representative groups are shown. Light shading corresponds to high electron density. C, correlation of density features with atomic models of the OspC and core dimer. In the projection from the lower right section in panel B, distinct density features in the inner and outer shell are outlined. The models in the upper panel are depicted at approximately the same scale. The dimers are shown once viewed versus their narrow and once versus their broad side together with their approximate dimensions.

 
Immunological Characterization of CoreOspCa and CoreOspCb CLPs— Next, coreOspCa and coreOspCbb were compared with recGST-OspCb in their potential to induce specific antibodies (Ab) in BALB/c mice. When tested at day 7 after the first immunization, IS from recipients immunized with coreOspCa and coreOspCb, but not with recGST-OspCb, contained specific Ab as revealed by Western blot analysis using recGST-OspCb as target (Fig. 5A). At day 15 following the second challenge with antigen, all three IS contained specific Ab (Fig. 5A). For semiquantitation of OspC- and core-specific Ab, IS were taken at different time points following repeated immunization and analyzed by ELISA using either rec-OspCb (lacking the GST part) or wild-type HBcAg as target protein (Fig. 5B). ELISA readings were related to those obtained with known amounts of mAbs against OspC and core. Assuming the same average affinity for the polyclonal antibody pools in the IS and the mAbs, IS to coreOspCb contained high titers (up to 450 µg/ml) of OspC-specific antibodies, which peaked after three immunizations and then declined but again increased after a further antigen challenge. Similar kinetics of OspC Ab were seen with IS to coreOspCa and recGST-OspCb, though at ~5-fold lower levels. Yet the Ab levels in IS to coreOspCa were still as high as those in IS to recGST-OspCb, although the test antigen was OspCb. As expected, Ab to HBcAg were seen only in IS of mice immunized with the core fusions, however at titers much lower compared with OspC Ab (Fig. 5B).

For a more detailed analysis of the respective IS, 2-fold serial dilutions obtained after the fourth immunization were tested by ELISA using either coreOspCa, coreOspCb, or rec-OspCb as targets (Fig. 5C). All three antigen preparations were recognized by the OspC-specific mAb LA97.1 (43), confirming the expression of authentic epitopes in the two coreOspC fusion proteins. When comparing IS to coreOspCa and coreOspCb, higher Ab titers (~4-15-fold) were always found when tested on the homologous OspC antigen, yet the response to the other OspC type was still substantial, indicating the existence of both distinct and common epitopes. In addition, titers of OspC Ab in IS to coreOspCb exceeded those in IS to recGST-OspCb by up to 8-fold when tested on either of the three antigen preparations, demonstrating a prominent adjuvant activity of the CLP carrier.

Passive Immunization with CoreOspCa and CoreOspCb Immune Sera Protects against Challenge with B. burgdorferi—In light of the previous findings that recGST-OspCb can confer active and passive protection against the homologous B. burgdorferi strain in both immunocompetent and immunodeficient mice (40, 41) IS to coreOspCa, coreOspCb, and recGST-OspCb were analyzed for neutralizing Ab in vivo. Accordingly, IS were passively transferred to C.B-17 SCID mice, known to develop chronic B. burgdorferi infection and disease, including arthritis and carditis (50), and the course of infection and disease (clinical arthritis) was monitored following challenge with the pathogen. The established neutralizing anti-OspA mAb LA-2 served as positive control (51), while IS to the core protein only, normal mouse serum, and PBS were used as negative controls. As shown in Table 1, transfer of IS to either coreOspCa, coreOspCa, or recGST-OspCb into C.B-17 SCID mice (3 mice/group) 2 h prior to challenge led to complete protection against infection, as monitored by reculturing of spirochetes from ear biopsies, and disease (development of clinical arthritis) except in one coreOspCb IS-treated animal. In comparison, 3/3 mice pretreated with LA-2 but none of the mice pretreated with either IS to the core protein only, normal mouse serum, or PBS was protected against infection and disease.


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  		TABLE 1
Prophylactic effect of different immune sera specific for OspC on B.burgdorferi ZS7 infection in C.B-17 SCID mice

 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
REFERENCES
 
The immune-enhancing property of HBV CLPs presenting short heterologous peptides or other small molecules is well established (4, 5, 52). Surface display of whole protein antigens, desirable for a vaccine addressing multiple epitopes and therefore less vulnerable to genetic variability, is structurally much more demanding, and as yet only few protein-chemical and immunological data are available (6-9, 48). The present study demonstrates that two distinct variants of a complex dimeric lipoprotein of B. burgdorferi, OspCa and OspCb, can successfully be presented on such CLPs; that the OspC polypeptide chains are exposed on the particle surface, possibly in a patchwork of OspC dimers across different carrier dimers; that these CLPs are highly immunogenic in mice, inducing IS that are specific for their cognate OspC but cross-reactive with the other; and that both IS are able to passively protect mice from experimental challenge with B. burgdorferi.


Figure 5
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  		FIGURE 5.
Immunological characterization of immune sera induced by coreOspCa and coreOspCb CLPs. A, immunoblot. The ability of IS induced by the coreOspC preparations, or the recGST-OspCb fusion, to recognize recGST-OspCb was tested on day 7 after the first and on day 4 after the second immunization; the OspC-specific mAb LA 97.1 served as positive control. No signal was observed with preimmune sera (strip labeled pre-immune). B, kinetics of OspC- and core-specific antibody induction. ELISA plates were coated with either rec-OspCb (top) or wild-type core protein (bottom) and incubated with the indicated IS. ELISA readings were normalized using known amounts of OspC-(mAb LA97.1) or core-specific (mAb 312) antibody. Assuming comparable affinities, values from the IS samples are given as µg/ml of OspC- or core-specific antibody. Note the different scales for the OspC-versus the core-specific response. C, specificity and cross-reactivity of IS against the homologous and heterologous OspC antigens. ELISA plates were coated with the indicated antigen, and binding of the different IS and mAb LA97.1 was monitored using 2-fold dilution series; the first dilution was 10-fold for the IS and 70-fold for LA97.1 (100 ng/well). The y-axis shows the absorbance at 405 nm. In panels A and C, OspCa and OspCb are abbreviated as OCa and OCb.

 
Structural Implications—Both the HBV core protein dimer (22) and the OspC dimer (27, 28, 53) are stabilized through large, mostly hydrophobic interfaces, keeping the constituent monomers in a defined arrangement. Because of the non-matching topology of the termini to be joined in the fusion protein (Fig. 1, B and C), it was possible that the dimerization tendency of one of the partners overrides that of the other, such that no regular particles would form. However, both coreOspC fusions efficiently formed particles, as demonstrated by the absence of non-assembled fusion protein in the gradients and by the abundant presence of particles upon EM inspection (Fig. 3). Furthermore, the solubility of the fusion proteins argues for proper OspC folding and dimerization because solvent exposure of the hydrophobic dimer interface would probably lead to aggregation. It is likely therefore that both partners, despite the non-matching topology, were able to dimerize.

Surface exposure of the OspC chains was clearly corroborated by cryoelectron microscopy (Fig. 4A) showing the CLPs as spheres of wild-type-like appearance but surrounded by a corona of prominent outward-pointing spikes. Two-dimensional image reconstructions (Fig. 4B) showed that the protrusions had approximately the length of 6.5 nm expected for OspC; the width varied between ~3 and 4 nm, with the latter corresponding to that of an OspC dimer viewed toward its broadside. The smaller values are compatible with both an edgewise-viewed dimer or a monomer. The original micrographs and the two-dimensional reconstructions indicated that the OspC domains are not rigidly fixed at topologically equivalent positions, similar to what was seen with CLPs displaying GFP flanked by long connecting linkers (6) or a 120-aa segment of a hantavirus nucleocapsid protein (7). The terminal OspC sequences flanking the structured part would provide for sufficient flexibility such that each OspC domain can occupy alternative positions at the CLP surface. The partial disorder in the OspC layer and possible deviations from strict icosahedral symmetry plus the difficult assignment of the exact particle orientations complicated a three-dimensional icosahedral image reconstruction. By combining an inner shell-biased reconstruction with another one based on a single particle approach emphasizing the outer OspC densities, we arrived at a model that allowed us to locate the approximate positions of the core dimer spikes of the inner shell (supplemental Fig. S1). Notably, except for weak densities above the 2-fold axes, no evidence for an accumulation of OspC densities directly above the core dimer spikes was seen as would be expected if the corresponding two OspC chains had dimerized. Instead, ~300 rather than 180 OspC-related densities (60 dimer-like densities each around the 5-fold and 3-fold axes, plus two OspC monomer-like densities per each of the 30 2-fold axes) were observed, with approximately half the density as that in the inner shell. Hence these densities likely reflect averaged probability volumes for OspC; in an individual real particle only about half of the densities would be occupied (supplemental Fig. S2).

A plausible model compatible with all the data, including the solubility of the CLPs that likely would be compromised by a significant portion of monomeric OspC moieties, is that OspC dimerization occurs between OspC chains from different, neighboring core dimers. The N atom of Pro-79 of the core protein is roughly at the center of the OspC insertion site. The distance to the Pro-79 N atom within the same core dimer is 10.9 Å, and that to the closer of the two Pro-79 N atoms in a neighboring core dimer is 43.7 Å. Assuming that in an extended conformation each aa residue contributes close to 3 Å in length, spanning that distance requires ~15 residues total. The N-terminal connector sequence between core and OspC consists of 5 linker aa plus 19 OspC residues not ordered in the x-ray structure; at the C-terminal junction, there are 16 non-ordered OspC residues plus 2 extra glycines. Hence two OspC chains could well meet halfway between neighboring core dimers. The presence of two closest neighbors for each of the 90 core protein dimers per T 3 particle allows for an enormous number of possible inter-core-dimer OspC dimers, such that each single CLP could have a unique OspC-dimer pattern. Regardless of how exactly the OspC chains are arranged, the important conclusion for the use of HBcAg as carrier for foreign protein antigens is that even a dimerization-competent protein with non-matching topology of its termini can successfully be presented on HBcAg CLPs.

Enhanced OspC Type-specific yet Cross-reactive Immunogenicity of CoreOspC CLPs—Previous studies demonstrated that CLPs displaying truncated or full-length protein antigens, including a Hantavirus N protein segment (7) and B. burgdorferi OspA (9), are highly immunogenic and readily induce Ab to the foreign polypeptide chains. In line with these results, both coreOspCa and coreOspCb CLPs induced strong OspC-specific antibody responses in mice and in rabbits (not shown). The Ab titers obtained with the two CLP preparations significantly exceeded those obtained with recGST-OspCb (Fig. 5 and Refs. 40, 41), further substantiating the adjuvant potential of the HBV capsid. Furthermore, the Ab levels against the HBcAg carrier were at least one log lower compared with the OspC-specific responses, a desired feature for the use of CLPs as vaccine candidates (54, 55) and probably a consequence of breaking up the immunodominant authentic c/e1 epitope by the insertions.

The anti-coreOspC responses were specific for OspCa and OspCb in that the highest antibody titers were always evoked against the homologous OspC. Hence, whereas nearly all previous work focused on epitopes in the conserved stalk region (56, 57), this demonstrates the presence of immunogenic epitopes in the more variable head region of OspC. These data are fully in line with a very recent study that defined linear type-specific epitopes in a surface-exposed loop preceding helix {alpha}5 ("loop 5") (36), exactly where OspCa and OspCb differ most in sequence (Fig. 2B). According to the typing system used there, the corresponding region in OspCa belongs to type B and that in OspCb to type Q. However, the anti-coreOspC IS also displayed substantial cross-reactivity, indicating that the CLP preparations were able to induce a multispecific response directed against different, namely conserved and variant, parts of the two displayed antigens, illustrating the distinct advantage of CLPs displaying a whole foreign protein rather than a small peptide segment.

Most remarkably, both coreOspC-CLP preparations induced protective Ab that, upon passive transfer, prevented experimental infection and disease in B. burgdorferi-challenged C.B-17 mice. Given that a denatured OspC, though not of identical sequence and in a different mouse strain, induced OspC-specific yet non-protective antibodies (58, 59), the coreOspC CLPs are highly likely to express authentic native OspC epitopes, a prerequisite for the induction of protective immunity, on their surface.

The exact nature of the protective OspC epitopes is not clear. The stalk has been suggested to contain a dominant neutralizing epitope (60). It is presently unclear how this region, containing the lipid anchor attaching OspC to the spirochete membrane, induces and is targeted by antibodies; similarly, this particular region is buried in the CLPs between the outer OspC and inner HBcAg shell. One interpretation is that, despite these steric constraints, antibodies against the conserved stalk part are formed that are neutralizing, similar to the mAb B5 directed against a conformational epitope in this region (57). However, it is equally likely that other, more accessible regions on the coreOspC CLPs contribute to the neutralizing activity.

At any rate, HBV CLPs displaying two OspC variants, which with respect to their loop 5 epitopes (36) belong to distant OspC types, both induced protective immunity. This may give rise to cautious optimism that a coreOspC-based vaccine encompassing a confined number of OspC variants could protect from infection by more than one strain of B. burgdorferi.


   FOOTNOTES
 
* This work was supported by the Deutsche Forschungsgemeinschaft through the Priority Programme 1089, Novel Vaccination Strategies. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Back

Formula The on-line version of this article (available at http://www.jbc.org) contains supplemental text and supplemental Figs. S1 and S2. Back

1 To whom correspondence should be addressed: University Hospital Freiburg, Internal Medicine II/Molecular Biology, Hugstetter Strasse 55, D-79106 Freiburg, Germany. Tel. and Fax: 49-761-270-3507; E-mail: nassal2{at}ukl.uni-freiburg.de.

2 The abbreviations used are: HBV, hepatitis B virus; aa, amino acid; Ab, antibody; mAb, monoclonal Ab; CLP, capsid-like particle; EM, electron microscopy; GFP, green fluorescent protein; HBcAg, hepatitis B core antigen; IS, immune serum; Osp, outer surface protein; PBS, phosphate-buffered saline; recGST-OspCb, recombinant glutathione S-transferase-OspCb fusion protein; ELISA, enzyme-linked immunosorbent assay. Back

3 R. Wallich, T. Stehle, C. Brenner, A. Siebers, L. Gern, and M. Simon, unpublished data. Back


   ACKNOWLEDGMENTS
 
We thank Jolanta Vorreiter, Ida Wingert, and Simone Prinz for excellent technical assistance.



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 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
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