0014-2980/02/1111-3305$17.50+.50/0© 2002 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim

Regulation of IgG antibody responses by epitopedensity and CD21-mediated costimulation

Andrea Jegerlehner1, Tazio Storni1, Gerd Lipowsky1, Markus Schmid1, Paul Pumpens2

and Martin F. Bachmann1

1 Cytos Biotechnology AG, Schlieren, Switzerland2 Research and study center, University of Latvia, Riga, Latvia

Epitope density and organization have been shown to be important factors for B cell activa-tion in many animal model systems. However, it has been difficult to separate the role of anti-gen organization from the role of local antigen concentrations because highly organized anti-gens are usually particulate whereas non-organized antigens are more soluble. Hence,highly organized and non-organized antigens may interact with different cell types and in dif-ferent locations within lymphoid organs. In order to assess the role of antigen organization inregulating B cell responses, we immunized mice with highly repetitive virus-like particles,which exhibit different epitope densities covalently attached to them. Therefore, the sameparticulate structure was used to present identical epitopes that differed in their degree oforganization. Induction of epitope-specific IgM titers, reflecting early B cell activation, wereunaffected by the degree of epitope density. Furthermore, the absence of Th cells or CD21/CD35 did not reduce the IgM response. In contrast, the degree of organization was a criticalfactor influencing the magnitude of the epitope-specific IgG response. Moreover, the thresh-old for IgG responses was shifted in the absence of CD21/CD35, resulting in the requirementfor higher epitope densities to allow efficient IgG responses. Thus, IgG but not IgMresponses are regulated by epitope density and B cell costimulatory thresholds.

Key words: Antibody response / Virus-like particle / Epitope density / CD19/CD21/CD81 complex/ T cell help

Received 8/8/02Accepted 18/9/02

[I 23419]

Abbreviations: BCR: B cell receptors HBc: Hepatitis Bcore HBcAg: Hepatitis B core antigen OD: Optical densityRT: Room temperature Sulfo-MBS: Maleimidobenzoic acidsulfosuccinimidyl ester VLP: Virus-like particles

1 Introduction

Induction of B cell responses is a carefully controlledmultistep process. Cross-linking of B cell receptors byantigen is the initial step in B cell activation. If the antigenis multivalent, B cells may be sufficiently activated forproliferation and production of IgM antibodies. In fact, 20epitopes presented within a space of approximately5–10 nm suffice to trigger T-cell-independent IgMresponses [1]. Many viruses exhibit such repetitive sur-face structures and are thus able to trigger T-cell-independent IgM responses [2, 3]. However, unless veryhigh antigen doses are used for immunization [4], theresponse is limited at this stage and the subsequentswitch from IgM to IgG does not occur, unless specificTh cells are present [5–7]. As a result, long-lived IgG

responses are generally Th-cell-dependent. Althoughonly multivalent antigens and polyclonal B cell activa-tors, but not proteins in adjuvants, are able to trigger T-cell-independent IgM responses, the influence of epi-tope density on the magnitude of T-cell-dependent IgGresponses is less clear. There is ample evidence to sug-gest that soluble and partially aggregated antigensinduce poor IgG responses in the absence of adjuvantswhereas highly organized antigens, such as bacterial[8–10] or viral [11–14] surface proteins, can inducestrong IgG responses under the same conditions.Although these data probably reflect an important rolefor antigen-organization, it remains possible that alteredcellular targeting of antigens within lymphoid organs, orother factors, are responsible for the striking differencesin IgG titers. The observation that particulate antigens,unlike soluble proteins, efficiently trigger splenic mar-ginal zone B cells may support this hypothesis [15].

Efficient cross-linking of the surface Ig on B cells is animportant step in B cell activation. In addition, costimula-tory molecules, in particular the CD19–CD21–CD81complex, may facilitate the process. It has been shown

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Fig. 1. VLP decorated with different densities of a modelantigen. (A) Schematic diagram of the coupling strategy andcarrier structure [55]. (B) A model peptide (D2) was coupledto VLP (HBc) in such way that peptide density on the VLPwas low, medium, or high and VLP were subsequently ana-lyzed by SDS-PAGE gel electrophoresis (15%) under reduc-ing conditions. The relative intensity of the upper bands tothe lower bands indicates how many subunits are coupledto a peptide (upper band) and how many are not coupled toa peptide (lower band). (C) The second coupling reaction toVLP (HBc): peptide density on the VLP was very low, low,medium, or high. M.W., molecular weight.

that antibody-mediated cross-linking of CD19 enhancesactivation of B cells in vitro [16], and mice deficient inCD19, CD21 or CD81 exhibit severely reduced T-cell-dependent IgG responses [17–22]. Surprisingly, T-cell-independent IgM responses, induced by haptenatedpolymers, were enhanced rather then reduced inCD19–/– or CD81–/– mice [17, 19, 21]. Thus, these costi-mulatory molecules may exhibit a more important func-tion during the later phase of the immune response. Thisnotion is further supported by the finding that CD21enhances survival of germinal center B cells derived fromBCR-transgenic mice [23]. Interestingly, both in vitro andin vivo studies indicate that expression of CD21 and/orCD35, a splice variant of CD21, is important not only onB cells, but also on follicular dendritic cells (FDC) [24,25]. These receptors on FDC most likely facilitateantigen-trapping [26, 27], which is important for the ger-minal center reaction and perhaps the maintenance ofIgG titers [28].

To date, CD21 is the only molecule within theCD19–CD21–CD81 complex with a known receptorfunction: it binds C3d, a degradation product of the com-plement component C3 [29]. C3d preferentially deco-rates pathogens, which allows CD21 to bridge the innateand specific immune response, by facilitating the induc-tion of specific antibodies. Hence, CD21 may play a keyrole in the regulation of pathogen-specific antibodyresponses [29, 30]. Nevertheless, many viruses, includ-ing vesicular stomatitis virus [31] and influenza virus [32],are able to generate efficient IgG responses in theabsence of CD21, indicating that other factors may beable to compensate for the absence of CD21 during viralinfections. Furthermore, B cells from transgenic miceexpressing a BCR specific for hen egg lysozymeresponded normally to a high affinity ligand but failed torespond to a low affinity ligand, suggesting that a highaffinity of the BCR for its antigen may be able to com-pensate for the lack of CD21 [23].

In order to be able study the role of epitope density andCD21-mediated costimulation in regulating B cellresponses to one and the same particulate antigen, wegenerated antigenic preparations exhibiting differentdegrees of epitope repetitiveness, by coupling peptidesat various densities to virus-like particles (VLP) derivedfrom the hepatitis B core antigen (HBcAg) [33] or thebacteriophage Q g [34]. Since the size of the particlesvaries minimally with addition of the small peptides, itcan be expected that the trafficking to, and within, lym-phoid organs is consistent for the various VLP-preparations. Differences in B cell responses may there-fore be directly attributed to variations in epitope repeti-tiveness. Using this experimental system, we haveshown that epitope density is a key parameter for effi-

cient IgG, but not IgM, responses. Moreover, CD21 facili-tated immune responses by lowering the epitope densityrequired for optimal IgG responses. Hence, IgGresponses are tightly regulated by the level of antigenorganization and CD21 fine-tunes the cross-linkingthreshold required for optimal activation.

2 Results

2.1 Generation of VLP exhibiting various epitopedensities

In order to be able to specifically couple antigenic epi-topes to VLP derived from HBcAg, a chemically reactiveamino acid (lysine) was introduced into the immuno-dominant region of HBcAg [33]. Using a bivalent cross-linker, the peptide D2 (CGGTSNGSNPSTSYGFAN) con-taining a free cysteine could be covalently attached tothe lysine residue on the VLP (Fig. 1A, [33]). By titratingthe peptide concentration used for coupling, it was pos-sible to modulate the number of peptides coupled perVLP. Fig. 1B and C show examples of SDS-PAGE analy-sis of such preparations. The lower bands represent VLPsubunits without peptide attached to them whereas theupper bands consist of subunits covalently attached to

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Fig. 2. IgG but not IgM response was highly dependent onpeptide density on VLP. (A–H) Groups of three six-week-oldfemale C57BL/6 mice were immunized i.v. with 20 ? g of HBc(carrier) coated with various densities of peptide. (A, B, E, F)Low, medium or high densities of peptide were used (forcoupling-reaction see Fig 1B) and serum samples taken onday 4 and day 46. (C, D, G, H) Very low, low, medium, or highdensities of peptide were used from the second couplingreaction (for coupling-reaction see Fig 1C) and serum sam-ples taken on day 4 and day 17. (A) IgM titers against pep-tide on day 4. (B) IgM titers against HBc on day 4. (C) IgMtiters against peptide on day 4, using the second couplingreaction. (D) IgM titers against HBc on day 4, using the sec-ond coupling reaction. (E) IgG titers against peptide onday 46. (F) IgG titers against HBc on day 46. (G) IgG titersagainst peptide on day 17, using the second coupling reac-tion. (H) IgG titers against HBc on day 17, using the secondcoupling reaction. (A–H) Mean values ± SEM of three miceare indicated. Two representative experiments are shown.

the peptide. It is important to note that internal cysteineswere removed genetically from the VLP in order to pre-vent internal cross-linking, which would preclude suchan analysis. Similar VLP preparations were analyzed onagarose gels, where native VLP could be visualized [33].The results indicated that the various VLP preparationswere relatively homogenous and appeared as a singleband (not shown). Thus, this method allowed for the pro-duction of VLP displaying different densities of antigenicpeptides on their surface.

2.2 Only highly repetitive peptide epitopes areable to trigger IgG responses

Mice were immunized i.v. with the different VLP prepara-tions in the absence of adjuvant, and specific IgM andIgG antibody responses were determined. IgMresponses against the peptide were generally low andwere not affected by the epitope density (Fig. 2A, C). Incontrast, specific IgG responses were highly dependenton epitope density. VLP preparations with the lowest epi-tope densities essentially failed to trigger significant IgGresponses (Fig. 2E, G). As was expected, the responseagainst the VLP itself was not affected by the peptidedensity on the surface, demonstrating that VLP exhibit-ing low peptide densities were non-immunogenic per se(Fig. 2 B, D, F, H). Moreover, normal carrier-specific IgGresponses further indicated that Th cell responsesinduced with the different VLP preparations were com-parable. Induction of comparable T cell help was con-firmed by the observation that normal numbers of VLP-specific Th cells were induced (as assessed by IFN- +and IL-4 ELISPOT assays) by the various VLP prepara-tions. In addition, peptide-specific T help could not bedetected (not shown).

To exclude the possibility that the results were a particu-lar property of the HBc VLP or of the specific peptideused, the experiments were repeated with a second VLP,derived from the bacteriophage Q g (Fig. 3) and a secondpeptide (not shown). Comparable results were obtained,suggesting that the importance of epitope density wasnot restricted to the particular VLP or peptide used.

VLP preparations with low peptide density also containless peptide antigen. In order to test whether increasingdoses of VLP used in immunization could compensatefor poor epitope density, titration experiments were per-formed in vivo. As shown in Fig. 4A, even a 30-foldincrease in the dose of VLP could not compensate theinefficient IgG response. As expected, the responseagainst the carrier was independent of the peptide den-sity. Hence, epitope density critically determines the effi-ciency of IgG responses.

2.3 CD21/CD35 fine-tunes thresholds of IgGresponses in vivo

The CD19–CD21–CD81 complex is known to be impor-tant for optimal B cell responses against soluble anti-

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Fig. 3. Influence of peptide density on antibody response isindependent of carrier. (A) The model peptide (D2) was cou-pled to VLP (Q g ) in such way that peptide density on the VLPwas low or high and VLP were subsequently analyzed bySDS-PAGE gel electrophoresis (15%) under reducing condi-tions. The relative intensity of the upper bands to the lowerbands indicates how many subunits are coupled to two pep-tides (highest bands), how many are coupled to one peptide(middle bands), and how many are not coupled to a peptide(lowest bands). (B–E) Groups of three six-week-old femaleC57BL/6 mice were immunized intravenously with 20 ? g ofQ g coated with low or high peptide density. Serum sampleswere taken on day 4 and day 46. (B) IgM titers against pep-tide on day 4. (C) IgM titers against Q g on day 4. (D) IgGtiters against peptide on day 46. (E) IgG titers against Q g onday 46. (B–E) Mean values ± SEM of three mice are indi-cated.

Fig. 4. Antigen repetitiveness is more important for theinduction of a strong IgG response than antigen dose.Groups of three six-week-old female C57BL/6 mice wereimmunized with 60, 30, 20, 10, 6, 1 or 0.1 ? g of VLP coatedwith low peptide density or 30, 20, 10, 6, 2, 1, 0.1 ? g of VLPcoated with high peptide density (for coupling-reaction seeFig 1B). Serum samples were taken on day 14 and day 30.(A) IgG titers against peptide on day 14. (B) IgG titers againstHBc on day 14. Similar titers were measured on day 30 (datanot shown). ELISA titers are indicated as dilution at whichhalf-maximal optical density was reached. Mean values ±SEM of three mice are indicated. nd, not determined.

gens in adjuvant, particularly against low affinity ligands[18–23]. However, the role of this complex in drivingresponses against antigens with different epitope densi-ties had not yet been addressed. Cr2–/– mice, which aredeficient in both CD21 and CD35 [19], were immunizedwith various VLP preparations differing in epitope den-sity. Specific IgM and IgG responses were measured onday 4 and day 17, respectively (Fig. 5). IgM responseswere essentially normal in Cr2–/– mice and almost inde-pendent of peptide density (Fig. 5A). In contrast,epitope-specific IgG titers were extremely dependentupon the presence of CD21/CD35 and were significantly

reduced even at high epitope density (Fig. 5C). Thus,absence of CD21/CD35 dramatically increased thethreshold of B cell activation required to induce IgGresponses. In contrast, IgG titers against the carrier wereclose to normal (Fig. 5B, D), indicating that by achievingmaximal epitope densities one can largely overcome theabsence of CD21/CD35. This was confirmed by immu-nizing mice with a new preparation of VLP exhibiting avery high peptide density. Under these conditions,Cr2–/– mice mounted strong, albeit slightly reduced,peptide-specific IgG responses (Fig. 6B, D).

2.4 Limited role of Th cells in regulating IgMresponses

We next determined whether the absence of Th cellscould affect peptide-specific IgM responses. MHC classII–/– mice [35], which lack Th cells, and control micewere immunized with various VLP preparations, and IgMand IgG responses were assessed. Surprisingly, theabsence of Th cells had little or no influence on the IgMresponse, even at low epitope density (Fig. 7A). In con-trast, and as expected, specific IgG responses were dra-matically reduced (Fig. 7C, D).

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Fig. 5. CD21/CD35 is not required for IgM responses against peptide irrespective of peptide density on VLP, but IgG responsesagainst peptide are highly dependent on CD21/CD35. (A–D) Groups of three six-week-old Cr2–/– or C57BL/6 mice were immu-nized with 20 ? g of HBc decorated with very low, low, medium, or high peptide density (for coupling reaction see Fig 1C). Serumsamples were taken on day 4 and day 17. (A) IgM titers against peptide on day 4. (B) IgM titers against HBc on day 4. (C) IgGtiters against peptide on day 17. (D) IgG titers against HBc on day 17. (A–D) ELISA titers are indicated as dilution at which half-maximal OD was reached in the assay. Mean values ± SEM of three mice are indicated. Comparable results were obtained atlater time-points. One representative experiment of two is shown.

P

Fig. 6. Limited IgG response against peptide in Cr2 –/– micecan be overcome if peptide density on VLP is very high. (A)The model peptide (D2) was coupled to VLP (HBc) in suchway that peptide density on the VLP was very high and VLPwere subsequently analyzed by SDS-PAGE gel electropho-resis (15%) under reducing conditions. The relative intensityof the upper bands to the lower band indicates how manysubunits are coupled to two peptides (highest band), howmany are coupled to one peptide (middle band) and howmany are not coupled to a peptide (lowest band). (B–E)Groups of three six-week-old female C57BL/6 mice orCr2–/– mice were immunized with 20 ? g of HBc decoratedwith a very high density of peptide. Serum samples weretaken on day 4 and day 41. (B) IgM titers against peptide onday 4. (C) IgM titers against HBc on day 4. (D) IgG titersagainst peptide on day 41. (E) IgG titers against HBc onday 41. Mean values ± SEM of three mice are indicated.Comparable results were obtained at earlier time points (notshown).

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Fig. 7. T help is not required for IgM responses against peptide even at low peptide density on VLP. Groups of three six-week-oldMHCII–/– mice or C57BL/6 mice were immunized intravenously with 20 ? g of HBc coated with very low, low, medium, or highdensities of peptide (for coupling reaction see Fig 1C). Serum samples were taken on day 4 and day 60. (A) IgM titers againstpeptide on day 4. (B) IgM titers against HBc on day 4. (C) IgG titers against peptide day 60. (D) IgG titers against HBc on day 60.(A–D) ELISA titers are indicated as dilutions at which half-maximal OD was reached in the assay. Mean values ± SEM of threemice are indicated. (A, B) One representative experiment of three is shown.

3 Discussion

The present study demonstrates that antigen organiza-tion regulates IgG but not IgM responses. Moreover,CD21/CD35 is shown to fine-tune the minimal cross-linking threshold required for production of IgG anti-bodies in vivo.

The role of epitope repetitiveness and organization hadbeen previously studied using essentially two types ofapproaches: (1) organic, non-peptidic polymers weredecorated with haptens, at various densities, which ledto the finding that approximately 20–25 haptens spacedby 5–10 nm were sufficient for T-cell-independent B cellactivation [1, 36–38]; (2) viral or bacterial proteins werepresented to the immune system in a highly organizedfashion on viral surfaces or bacterial flagella, or in adepolymerized, soluble form [9, 11–14]. These studiesrevealed a fundamental role for antigen-organization inthe regulation of B cell responses, because highly repeti-tive proteins on viral surfaces were more immunogenicthan their soluble counterparts. Moreover, highly orga-nized antigens were able to overcome B cell unrespon-siveness and to activate self-specific B cells [11, 14].However, these experiments could not take into accountthe role of Th cells in B cell activation. On the one hand,

organic, haptenated polymers do not contain Th cell epi-topes and therefore fail to induce a Th cell response; onthe other hand, particulate antigens, such as viruses andbacteria, are not presented to Th cells in the same wayas soluble proteins. Indeed, highly organized antigens onviruses or bacteria are usually administered withoutadjuvant; however, soluble proteins fail to induce anti-body responses under these conditions. Although thisdifference may illustrate the importance of epitope den-sity in driving B cell responses, it may also reflect ineffi-cient processing of soluble antigens for antigen-presentation in association with MHC class II moleculespresent on DC and macrophages. It is therefore difficultto distinguish between direct effects on B cells and indi-rect effects on Th cells. Moreover, targeting within lym-phoid organs may be different, since particulate anti-gens, such as viruses or bacteria, appear to be filteredfrom the circulation by marginal-zone macrophages [15,39]. Furthermore, the B cell response against such anti-gens has been reported to originate in the marginal zone[15, 39] and primarily induce marginal zone B cells andB1 cells [15]. In contrast, B cell responses against pro-teins administered in adjuvant are initiated at the inter-face of the T and B cell zones [40–42]. As a further com-plicating factor, repetitive surfaces may preferentiallyactivate the complement cascade [43], potentially

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enhancing immune responses against such antigens.Thus, it was important to study B cell responses againsthighly/poorly organized antigens presented on identical,highly repetitive particles. Under these conditions,induction of T cell help, which is directed against the car-rier, is identical and trafficking within lymphoid organs isexpected to be comparable. Using this system, we madetwo clear-cut observations: (1) at the epitope densitiesstudied, Th-cell-independent IgM responses were littleaffected; (2) Th-cell-dependent IgG responses were dra-matically affected. Thus, the early signs of B cell activa-tion, such as IgM production and also B cell proliferation(it is difficult to imagine the production of measurableIgM antibody titers in the absence of specific B cellamplification) were not significantly affected by epitopedensity. In contrast, the isotype switching from IgM toIgG was highly dependent on epitope density and Thcells. Isotype switching is under very tight control duringB cell responses [44–47], most likely because its conse-quences lead to long-term activation of the immune sys-tem and the generation of long-lived antibodyresponses. Current models stipulate that this tight regu-lation is brought about by Th cell tolerance. The datapresented here are consistent with this model but alsodemonstrate an additional mechanism: B cells have anintrinsic control device that tightly regulates the induc-tion of long-lived IgG responses. B cells failed to mountan IgG response at low epitope density, despite compa-rable early B cell activation (IgM production) and thepresence of functional T cell help (indicated by normalcarrier-specific IgG). As previously pointed out [6], theevolutionary explanation for this strict dependence onhigh epitope density for induction of IgG responses maybe the observation that most prokaryotic pathogensexhibit highly repetitive surfaces, whereas eukaryoticcells do not. Thus, a highly organized antigen is morelikely to be a pathogen and a protective and long-livedIgG response will therefore be more important. Hence, itis evolutionarily justified.

The CD19–CD21–CD81 complex is thought to amplifysignals from the BCR, enhancing B cell activation [48]. Inconcordance with this, we found that CD21/CD35-deficient mice mounted significantly reduced IgGresponses at low epitope densities. However, at veryhigh epitope densities, CD21/CD35-defiency could beovercome, at least in part, and robust IgG responseswere induced. This indicated that the CD21-mediatedenhancement of BCR signaling was not absolutely nec-essary under these conditions. This observation wasconsistent with relatively normal IgG responsesobserved against vesicular stomatitis virus [31] and influ-enza virus [32] in these mice. Nevertheless, specific IgMresponses were essentially unaffected in the absence ofCD21/CD35, even at low epitope densities, suggesting

that early events of B cell activation were independent ofCD21 and that BCR-mediated signaling may not be limit-ing during this early phase of the immune response.There is an interesting parallel to CD28-mediated costi-mulation of T cells. Similar to the CD19–CD21–CD81 complex [29], CD28 enhances TCR-mediated signaling [49]. Nevertheless, early in vivoresponses against high affinity peptide ligands may benormally induced in CD28-deficient mice but are subse-quently terminated prematurely, resulting in T cell unre-sponsiveness [50]. This may correspond to normalinduction of IgM responses along with severely reducedIgG response in the absence of CD21. In addition, viralinfections may overcome both CD28 and CD21 defi-ciency [31, 32, 50], indicating that optimal immune stim-ulation overcomes costimulation dependence.

The mechanism for the strict dependence of IgGresponses on high epitope density and CD21/CD35,despite seemingly normal IgM responses, is presentlyunknown. It is possible that B cells, activated by strongcross-linking of their receptors, are more susceptible toCD40L-mediated T cell help. This may be due toenhanced processing leading to increased presentationof carrier epitopes on MHC class II molecules. In fact, ithas been shown that activated B cells are more efficientat loading MHC class II molecules with peptide althoughthe observed effects were subtle [51]. Alternatively, it ispossible that strongly activated B cells may be morereceptive to CD40-mediated stimulation. In vitro experi-ments using anti-IgM and anti-CD40 antibodies supportthis latter possibility [52, 53]. Finally it is also possiblethat different subsets of B cells are able to mount eitheran IgM or IgG response following immunization with VLP,and this is supported by the observation that particulateantigens preferentially activate marginal zone B cells andB1 cells [15] whereas production of IgG antibodies maybe restricted to follicular B2 cells.

Taken together, the data presented in this study indicatea critical role for antigen-organization in the regulation ofIgG responses and demonstrate, in vivo, that B cellresponsiveness is governed by thresholds of epitopedensity and costimulation.

4 Materials and methods

4.1 Construction of plasmid ab1

Plasmid ab1 was constructed as also described previously[33]. Hepatitis B clone pEco63 containing the complete viralgenome of hepatitis B virus was purchased from ATCC. Thegene encoding HBcAg was introduced into the EcoRI/HindIIIrestriction sites of expression vector pkk223.3 (Amersham

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Pharmacia Biotechnology AB, Uppsala, Sweden) under thecontrol of a strong tac promoter. The C terminus (residues150–183) of HBcAg, which contains the RNA/DNA-bindingsite of the viral capsid, was removed to prevent such binding.Subsequently, a sequence of five amino acids that contains aLys residue was introduced into the c/e1 epitope of HBcAg.The c/e1 epitope (residues 72–88) of HBcAg is located on thesurface of the hepatitis B core (HBc). A part of this region,Pro79 and Ala80, was replaced by the peptide Gly–Gly–Lys–-Gly–Gly. The introduced Lys residue contains a reactive 4amino group in its side-chain that facilitates intermolecularchemical cross-linking of HBcAg with any antigen containinga free Cys group via a hetero-bifunctional cross-linker likemaleimidobenzoic acid sulfosuccinimidyl ester (sulfo-MBS).The modified HBcAg was named HBcAg(1–149)-Lys. In addi-tion, we mutated both Cys48 and Cys107 to Ser using stan-dard PCR methods. The final plasmid was named ab1.

4.2 Expression and purification of HBcAg(1–149)-Lys-2Cys-Mut

Expression and purification steps were performed as alsodescribed previously [33]. A culture of Escherichia coliK802d, containing the plasmid ab1, was grown in LBmedium (containing 100 ? g/ml ampicillin) at 37°C and at125 rpm until optical density (OD)600 0.6–0.8 was reached.Expression of the protein was induced by 1 mM isopropyl- g -D-thiogalactopyranoside. The cells were solubilized andsonicated, the suspension was centrifuged and the super-natant was precipitated using ammonium sulfate. After incu-bation for 30 min on ice and centrifugation for 15 min at47,800×g at 4°C the supernatant was discarded and the pel-let resuspended in PBS, pH 7.2.

The solution containing the HBc was loaded onto a Sephac-ryl® S-400 HR column (Amersham) and the peak fractionswere further purified using a CHT® ceramic hydroxyapatitecolumn (Bio-Rad, CA, USA). The flow-through (which con-tains purified HBc) was collected. The protein concentrationwas determined by Bradford assay. From one liter of culture2–5 mg of purified HBc could be produced.

4.3 Expression and purification of Q I

Capsids of the RNA-phage Q g were expressed using theexpression vector, pQ g 10, and purified as described previ-ously [54]. In brief, E. coli lysates containing the expressedcoat protein were cleared by centrifugation. After centrifuga-tion, proteins from the supernatant were fractionated byammonium sulfate precipitation (at final concentrations of20% and 40% saturation, respectively). The precipitatedcapsids were resuspended in a minimal volume of gel filtra-tion buffer containing 20 mM Tris-HCl pH 7.8, 5 mM EDTAand 150 mM NaCl, and were purified over a sepharose CL-4B column (Amersham). Eluted capsids were precipitatedwith PEG-6000 at 13.3% saturation and repurified on asepharose CL-4B column. Capsids present in the peak frac-

tions were precipitated with ammonium sulfate at 60% satu-ration. Sedimented VLP were resuspended in gel filtrationbuffer and loaded onto a sepharose CL-6B column (Amers-ham). Fractions containing the Q g were pooled, concen-trated by ammonium sulfate precipitation and dialyzedagainst 20 mM HEPES, 150 mM NaCl, pH 7.4.

4.4 Procedure for coupling to HBc

HBc (1.2 mg/ml) was mixed with a 50-fold molar excess ofsulfo-MBS (Pierce, Rockford, USA) and incubated for 30 minat room temperature. The reaction was performed in PBS(pH 7.2). Free, unreacted cross-linker was removed by dialy-sis with SnakeSkinTM tubing (Pierce) against coupling-buffer(20 mM HEPES, 150 mM NaCl, pH 7.2) overnight at 4°C.Derived HBc was mixed with different molar excesses of thepeptide D2 (CGGTSNGSNPSTSYGFAN; 100 mM in DMSO),ranging from 1 to 20 in comparison with HBc subunits, toobtain HBc with different densities of peptide, and incu-bated for 4 h at RT. Uncoupled peptide was removed bydialysis with a Spectra/Por® 6 50 MWCO dialysis membrane(Spectrum® Laboratories Inc., CA, USA). Coupling effi-ciency was determined by SDS-PAGE analysis.

4.5 Procedure for coupling to Q I

Q g (2 mg/ml) was mixed with a 20-fold molar excess ofsulfo-MBS and incubated for 30 min at RT. The reaction wasperformed in coupling buffer (20 mM HEPES, 150 mM NaCl,pH 7.2). Free, unreacted cross-linker was removed by pas-sage of the coupling reaction product over a PD-10 desalt-ing column (Amersham). Derived Q g was mixed with differ-ent molar excesses of the peptide D2 (100 mM in DMSO),ranging from 1 to 2 in comparison with Q g -subunits, toobtain Q g with different densities of peptide, and incubatedfor 2 h at RT. Uncoupled peptide was removed by dialysiswith Slide-A-Lyzer® Cassettes (Pierce) against coupling-buffer (20 mM HEPES, 150 mM NaCl, pH 7.2) overnight at4°C. Coupling efficiency was determined by SDS-PAGEanalysis.

4.6 Immunization of mice

Six-week-old female C57BL/6, Cr2–/– [19] or MHCII–/– mice[35] were immunized i.v. with 20 ? g (unless indicated other-wise) of HBc or Q g coupled to different densities of the pep-tide D2. Serum samples were taken at the time-points indi-cated in the figure legends and assessed by ELISA.

4.7 ELISA

Peptide coupled to ribonuclease [33], 10 ? g/ml diluted incoating buffer (0.1 M NaHCO3, pH 9.6), was used for thecoating of ELISA plates (Nunc Immuno Maxisorp). ELISAwere performed according to a standard protocol, usinghorseradish-peroxidase-conjugated secondary antibodies(Sigma). Plates were developed with 1,2-phenylenediamine

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dihydrochloride (OPD) substrate buffer (0.5 mg/ml OPD,0.01 % H2O2, 0.066 M Na2HPO4, 0.038 M citric acid, pH 5.0;100 ? l per well) and were read in an ELISA reader at 450 nm.

4.8 ELISPOT

Six-week-old female C57BL/6 mice were immunized s.c.with 10 ? g HBc or Q g coated with low, medium or high den-sities of the peptide D2 (Fig. 1B, 3A). Seven days afterimmunization mice were killed, spleen and lymph nodes iso-lated and cell suspensions were prepared in RPMI contain-ing 10% FCS. Triplicate wells each containing 5×105 cellswere plated (180 ? l/well) into 96-well plates. Cells were stim-ulated with the peptide D2 (final concentration of 2 or 10 ? M)or Q g /HBc (final concentration of 2 or 10 ? g/ml). As a posi-tive control, cells were stimulated with PMA/ionomycin. As anegative control cells were left unstimulated. All cells wereincubated for 48 h at 37°C and then transferred to ELISPOTplates (Multiscreen plates from Millipore) coated with either500 ng/well of anti-mouse-IFN- + mAb (PharMingen, cata-logue number 554431) or anti-mouse-IL-4 mAb (PharMin-gen, catalogue number 18031D) in filtered PBS and blockedfor 2 h with RPMI containing 10% FCS. Plates were furtherincubated for 14–18 h at 37°C after which they were washedsix times with filtered PBS containing 0.05% Tween 20. Sub-sequently, 100 ng/well of biotinylated anti-mouse-IFN- +mAb (PharMingen, catalogue number 554410) or biotiny-lated anti-mouse-IL-4 mAb (PharMingen, catalogue number18042D) in filtered PBS was added and plates were incu-bated for 2 h at RT. Plates were washed six times and100 ? l/well of streptavidin–alkaline-phosphatase conjugate(diluted 1:1000 in filtered PBS) was added. Plates were incu-bated for 2 h at RT, washed and developed with 100 ? l/wellof alkaline phosphatase reagent (alkaline phosphatase con-jugate substrate kit, Bio-Rad). Plates were rinsed thoroughlywith tap water after the spots became clearly visible. Plateswere left to dry and spots were counted.

Acknowledgements: We are grateful to Anna Flace forexcellent technical assistance in the ELISPOT assay. Wethank Constantino Lopez Macias, Alma Fulurija, ChristianeRuedel, and Manfred Kopf for helpful discussions and criti-cal review of this manuscript and Michael Carroll for provid-ing the Cr2–/– mice.

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