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Journal of Virology, January 2005, p. 717-724, Vol. 79, No. 2
0022-538X/05/$08.00+0     doi:10.1128/JVI.79.2.717-724.2005
Copyright © 2005, American Society for Microbiology. All Rights Reserved.

Virus-Like Particles as Carriers for T-Cell Epitopes: Limited Inhibition of T-Cell Priming by Carrier-Specific Antibodies

Christiane Ruedl, Katrin Schwarz, Andrea Jegerlehner, Tazio Storni, Vania Manolova, and Martin F. Bachmann*

Cytos Biotechnology AG, Schlieren-Zürich, Switzerland

Received 15 July 2004/ Accepted 4 August 2004


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ABSTRACT
 
Virus-like particles (VLPs) are able to induce cytotoxic T-cell responses in the absence of infection or replication. This makes VLPs promising candidates for the development of recombinant vaccines. However, VLPs are also potent inducers of B-cell responses, and it is generally assumed that such VLP-specific antibodies interfere with the induction of protective immune responses, a phenomenon summarized as carrier suppression. In this study, we investigated the impact of preexisting VLP-specific antibodies on the induction of specific cytotoxic T-cell and Th-cell responses in mice. The data show that VLP-specific antibodies did not measurably reduce antigen presentation in vitro or in vivo. Nevertheless, T-cell priming was slightly reduced by antigen-specific antibodies; however, the overall reduction was limited and vaccination with VLPs in the presence of VLP-specific antibodies still resulted in protective T-cell responses. Thus, carrier suppression is unlikely to be a limiting factor for VLP-based T-cell vaccines.


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INTRODUCTION
 
Cytotoxic T cells (CTL) are key lymphocytes for the clearance of many viral and bacterial infections and for the eradication of tumors. The goal of most therapeutic vaccination strategies is, therefore, the induction of specific CTLs. However, in the absence of intracellular replication, it is usually difficult to induce efficient CTL responses (2, 7, 37). An important reason for this inefficiency is the difficulty for an exogenous antigen to reach the major histocompatibility complex (MHC) class I pathway. In general, exogenous antigens reach the MHC class II pathway while only endogenous antigens efficiently fuel the MHC class I pathway (7, 13). Accordingly, immunization with inactivated viral particles usually fails to induce CTL responses (3, 18). This makes it thorny to generate powerful vaccines based on recombinant proteins. Virus-like particles (VLPs) are an interesting exception, since they are able to efficiently reach the MHC class I pathway (4, 14, 24, 27, 29) in the absence of infection or intracellular replication. Thus, VLPs are therefore promising therapeutic vaccine candidates that may induce efficient T-cell responses in the absence of viral replication.

It is generally assumed that vaccine-specific antibodies impair the induction of protective immune responses upon vaccination. A basis for this assumption may be that many vaccines are based on attenuated, but replication competent, viral strains (1, 22). Under these conditions, the attenuated virus may be neutralized by the antibodies, resulting in reduced replication and antigen load. As a consequence, T-cell induction is impaired. The situation for nonreplicating vaccines is less clear, and reports of reduced T-cell responses in the presence of specific antibodies are rare (8, 30). In fact, it may be expected that antibodies enhance opsonization of the vaccine, leading to increased antigen presentation. Thus, it seems possible that the presence of specific antibodies may facilitate CTL activation. In support of this, tumor-specific T-cell responses were reported to be enhanced rather than reduced by the presence of specific antibodies (9, 15). Moreover, immune complexes efficiently reach the MHC class I pathway upon binding to Fc{gamma} receptors, which facilitates induction of CTL responses (25).

To analyze the role of specific antibodies in regulating VLP-induced T-cell responses, we used VLPs based on the hepatitis B virus core antigen (HBcAg) fused to lymphocytic choriomeningitis virus (LCMV)-derived MHC class I-restricted peptide p33 (23) or MHC class II-restricted peptide p13 (20). p33-VLPs have been previously shown to be efficiently cross-presented by dendritic cells and macrophages partly by a transporter associated with antigen processing (TAP)-independent mechanism (27). In this study, we assessed the influence of specific antibodies on the presentation of peptides p33 (MHC class I) and p13 (MHC class II) in vitro and in vivo and on the induction of specific T-cell responses. We observed that antigen presentation was not affected in vitro or in vivo by the presence of specific antibodies. Moreover, protective immunity could be established in carrier vaccinated animals. Thus, carrier suppression by VLP-specific antibodies was of minor importance for VLP-based vaccination.


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MATERIALS AND METHODS
 
Viruses and virus-like particles. LCMV isolate WE was originally obtained from R. M. Zinkernagel (Institute of Experimental Immunology, University Hospital, Zürich, Switzerland) and propagated on L929 cells (6). Virus titers were determined by a focus-forming assay on MC57 fibroblasts.

The generation, production, and purification of p33-VLPs have been described earlier (33). p13-VLPs, to which the LCMV-derived p13 epitope (sequence GLNGPDIYKGVYQFKSVEFD) was genetically fused via a 6-amino-acid linker (RSSGMY) to the C terminus of the HBcAg. Production and purification of p13-VLPs was performed as described previously (32).

Mice and carrier immunization. Female C57BL/6 mice aged between 8 and 12 weeks were purchased from Harlan Netherlands B.V. (Horst, The Netherlands). Transgenic mice expressing a T-cell receptor (TCR) specific for peptide p33 (23) or p13 (21) in association with H-2Db and H-2-Ab have been described previously. They were bred and kept at Cytos Biotechnology AG.

C57BL/6 mice were vaccinated subcutaneously (s.c.) with 50 µg of wild-type VLP/mouse (carrier-vaccinated mice) or kept untreated (controls). Twenty-one days later, both groups of mice were immunized with peptide-specific VLPs as described in more detail below.

Measurement of anti-VLP antibodies by ELISA. Anti-VLP antibody titers were measured in the serum of mice vaccinated 21 days earlier with wild-type VLP. Nonimmunized mice were used as controls. Ninety-six-well plates (NuncImmuno Maxisorp; Nunc) were coated overnight with 10 µg of wild-type VLP, p33-VLP, or p13-VLP/ml, respectively. After blocking for 2 h with 2% bovine serum albumin-phosphate-buffered saline (PBS), serum obtained from vaccinated or control mice (diluted 1:500 to 1:12,500) was added and plates were incubated for 2 h at room temperature. After washing the plates three times with PBS-0.05% Tween, peroxidase-labeled goat anti-mouse immunoglobulin G (IgG) was added for 1 h, followed by the addition of orthophenylendiamine-HCl as a substrate before reading the optical density at 450 nm (OD450). Titers are expressed as serum dilutions at the half-maximal OD. To test whether native VLPs were recognized, a sandwich enzyme-linked immunosorbent assay (ELISA) was performed. In brief, plates were coated with goat anti-rabbit antibodies, washed, and incubated with a rabbit anti-HBcAg antiserum. Plates were washed and incubated with HBcAg (10 µg/ml), washed again, and incubated with the murine anti-HBcAg or preimmune serum at a dilution previously shown to yield optimal results (1:320). After a further washing step, bound murine antibodies were detected with horseradish peroxidase-conjugated goat anti-mouse IgG antibodies.

Isolation and staining of DCs and macrophages. Dendritic cells (DCs) and macrophages were isolated as previously described (26). In brief, lymph nodes (LNs) and spleens were collected and digested twice for 30 min at 37°C in Iscove's modified Dulbecco's medium supplemented with 5% fetal calf serum and 100 µg of collagenase D (Boehringer Mannheim, Mannheim, Germany)/ml. Released cells were recovered and resuspended in an Optiprep gradient (Nycomed, Asker, Norway) and centrifuged at 600 x g for 15 min. Low-density cells were collected and stained with phycoerythrin-labeled anti-CD11c and allophycocyanin-labeled anti-CD11b antibodies. DCs and macrophages were sorted by using a FACSStarplus (Becton Dickinson) on the basis of CD11chigh CD11bneg/pos (DCs) and CD11cint CD11bhigh (macrophages) expression (98% purity).

In the case of bulk DCs, the total CD11c+ population was isolated by magnetic bead separation (anti-CD11c, MACS; Miltenyi, Bergisch Gladbach, Germany) by following the manufacturer's instructions.

CD8+ and CD4+ T cells, used as responder cells, were isolated by magnetic bead isolation (anti-CD8 and CD4, respectively, MACS; Miltenyi) according to manufacturer's instructions.

Influence of anti-VLP antibodies on the presentation capacity of DC and macrophages. For analysis of the anti-VLP antibody effect on in vitro T-cell activation, purified DCs were obtained from spleens and pulsed for 2 h with various concentrations of p33-VLP or p13-VLP (2 to 0.12 µg/ml) in the presence or absence of anti-wild-type VLP antiserum or normal mouse serum (1:100). After three washes, presenter cells were cocultured together with antigen-specific transgenic CD8+ or CD4+ T cells, respectively. Two days later, T-cell proliferation was measured by [3H]thymidine uptake in a 16-h pulse (1 µCi/well).

For the in vivo effect of anti-VLP antibodies, mice were immunized s.c. with 50 µg of wild-type VLP/mouse (carrier-vaccinated mice) or kept untreated (control mice). Twenty-one days later, peptide-specific VLPs (p33- and p13-VLP) were injected together intradermally (i.d.) (50 µg of each/ear) in carrier-vaccinated or untreated mice. After 1 day, DCs and macrophages were isolated from draining LNs as described above. Their capacity to prime antigen-specific transgenic CD8+ or CD4+ T cells by their in vivo-loaded antigen cargo was measured without any additional antigen supply in vitro. Two days later, T-cell proliferation was measured by [3H]thymidine uptake in a 16-h pulse (1 µCi/well).

In vivo activation of adoptively transferred CFSE-labeled transgenic T cells. TCR-transgenic CD8+ or CD4+ T cells were labeled with the green fluorescent dye 5-(and 6-)carboxyfluorescein diacetate, succinimidyl ester (CFSE) (Molecular Probes, Eugene, Oreg.) as described previously (19). Labeled cells (5 x 106 to 10 x 106) were injected into the tail vein of sex-matched C57BL/6 recipients. After 16 h, recipients were vaccinated i.d. with p33- or p13-VLP at the indicated doses. To analyze cell proliferation, single-cell suspensions were prepared from draining LNs 4 days later. Cells were stained with anti-V{alpha}2 and anti-Vß8 antibodies in combination with anti-CD8 or anti-CD4 antibodies to visualize the injected transgenic T cells. For the analysis of intracellular gamma interferon (IFN-{gamma}) expression of the transferred p33- or p13-specific T cells, LN cells were resuspended in Iscove's modified Dulbecco's medium with 10% fetal calf serum and restimulated in vitro with 5 µM p33 or p13 for 6 h. Cultures were supplemented with 10 µg of brefeldin A (Sigma)/ml for the last 4 h of incubation. Restimulated cells were stained with cytochrome-labeled anti-CD8 or anti-CD4 antibodies (BD PharMingen, San Diego, Calif.). Cells were fixed in 2% formaldehyde for 15 min and permeabilized in 0.5% saponin-PBS for a further 30 min at room temperature. During permeabilization, cells were stained with anti-IFN-{gamma} antibodies (BD PharMingen). Cells were acquired in a FACSCalibur device and analyzed with CellQuest software (BD Biosciences, Mountain View, Calif.).

In vivo activation of p33-specific CD8+ T cells. Wild-type VLP-vaccinated (50 µg s.c. between days 20 and 40) and control C57BL/6 mice were immunized s.c. with increasing doses of p33-VLP-CpG (1 to 100 µg/mouse) in the presence of 10 nmol of phosphothioester-stabilized oligonucleotides (1668, 5'-TCC ATG ACG TTC CTG AAT AAT-3'). Seven days later, blood was collected and analyzed for the presence of p33-specific CD8+ T cells by staining with phycoerythrin-labeled p33-H-2Db tetrameric complexes (20 min at 37°C) and subsequently with anti-CD8 cytochrome-conjugated antibodies. Live cells (5 x 104) were acquired in a FACSCalibur device and analyzed with CellQuest software. The same staining procedure was applied to blood cells isolated 5 days after infection with LCMV.

To examine systemic antiviral immunity in these vaccinated mice, immunized mice were infected intravenously with 200 PFU of LCMV WE. The animals were sacrificed 5 days later, and spleens were collected. LCMV titers were determined by an LCMV focus-forming assay as described previously (6).


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RESULTS
 
Limited influence of VLP-specific antibodies on VLP processing in vitro. To generate VLP-specific antibodies, mice were immunized with wild-type (WT) HBcAg. Antibody responses against WT VLPs and VLPs fused to the LCMV-derived MHC class I and MHC class II epitopes peptides p33 (p33-VLPs) and p13 (p13-VLPs) were assessed 3 weeks later by ELISA. All three VLPs were efficiently recognized by the antisera (Fig. 1A). The dominant subclass induced by vaccination was IgG2a, with a contribution by IgG1, IgG2b, and almost no IgG3 (Fig. 1B). In addition, the immune sera recognized native VLPs as determined in a sandwich ELISA (Fig. 1C). As expected, the response was rather long-lived and declined with a half-life of about 2 to 3 months (not shown).



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  		FIG. 1. Immunization with WT VLP induces strong anti-VLP antibody responses. (A) C57BL/6 mice were vaccinated s.c. with 50 µg of WT VLP/mouse (carrier-vaccinated mice) or kept untreated (controls). Twenty-one days later, blood was collected and the anti-VLP antibody titers (WT VLP, p33-VLP, and p13-VLP) were measured from the serum. The ELISA was performed as described in Materials and Methods. Each group consisted of 24 independent mice. Bars are representative of the means ± standard deviations. (B) Mice (6 per group) were immunized s.c. with 50 µg of WT VLPs or left untreated. VLP-specific antibody subclasses were determined 5 weeks later. Dots represent endpoint ELISA titers (OD, >0.2 above background) for individual mice. Preimmune sera had titers of <1:100 (C) Immune sera from panel B were tested for the ability to recognize antibody-bound native WT VLPs in a sandwich ELISA (see figure for setup). Bars represent mean OD values ± standard errors of the means.

Antibodies from 24 mice were pooled to perform the subsequent in vitro experiments with a standardized immune serum. The influence of specific antibodies, which may affect VLP processing, was assessed next in vitro. Dendritic cells were isolated from the spleen and incubated with titrated amounts of p13-VLPs and p33-VLPs to assess processing of MHC class II- and MHC class I-associated peptides, respectively (Fig. 2). To determine the influence of specific antibodies on processing, the incubation was performed in the presence of preimmune control or of specific antiserum (diluted 1:100). After 2 h of pulsing, cultures were washed and T cells from transgenic mice expressing a receptor specific for peptide p13 or p33 were added. Proliferation of T cells was measured 48 h later by [3H]thymidine incorporation. The influence of antibodies was minimal, indicating that VLPs opsonized by antibodies are processed in a fashion similar to that of untreated VLPs (Fig. 2).



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  		FIG. 2. Effect of anti-VLP antibodies on the presentation capacity of DC in vitro. Spleen CD11c+ cells were pulsed for 2 h at 37°C with different concentrations of p33-VLP (A) or p13-VLP (B) (2 to 0.12 µg/ml) in the presence of anti-VLP antiserum (white circles) or control preimmune serum (black circles) (both diluted 1:100). After three washes, 5 x 104 presenter cells were cocultured with CD8+ or CD4+ antigen-specific T cells (105) for 2 days. Proliferation was measured by a thymidine incorporation assay. Means of triplicate experiments are shown. Background values obtained with T cells stimulated with unpulsed DCs are <2,000 cpm in this assay. On the y axes, thymidine incorporation is measured in 103 cpm.

Limited influence of VLP-specific antibodies on VLP processing in vivo. The influence of VLP-specific antibodies on antigen processing in vivo was subsequently assessed. Mice were immunized as described for Fig. 1. Three weeks later, p33-VLPs and p13-VLPs were injected together (50 µg of each/ear) i.d., and macrophages and DCs were isolated from draining lymphoid organs 24 h later by single-cell sorting (Fig. 3A). The isolated antigen-presenting cells (APCs) were subsequently cocultured with T cells from transgenic mice expressing a TCR specific for peptide p33 or p13. The proliferation of T cells was assessed 48 h later (Fig. 3B). Both DCs and macrophages were able to present peptide p13 and peptide p33, confirming earlier results (27). The presence of VLP-specific antibodies did not significantly alter the capacity of either macrophages or DCs to stimulate specific CD8+ or CD4+ T cells. Thus, antigen processing in vivo was not significantly affected by VLP-specific antibodies.



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  		FIG. 3. Effect of anti-VLP antibodies on the presentation capacity of DC and macrophages in vivo. Carrier-immunized or control mice were injected i.d. with a mixture of 50 µg of p33-VLP and p13-VLP/ear. One day later, draining LNs were processed for isolation of DCs and macrophages. CD11chigh CD11bneg/pos cells (DCs) and CD11cint CD11bhigh (macrophages) were sorted and used as purified presenters in an in vitro T-cell proliferation assay. DCs (2 x 104) or macrophages (105) obtained by single-cell sorting from carrier-vaccinated mice (white bars) or from untreated mice (black bars) were cocultured with 105 antigen-specific CD4+ or CD8+ T cells, respectively. Proliferation was measured by a thymidine incorporation assay 2 days later. Background values obtained with T cells stimulated with unpulsed DCs or macrophages are <2,000 cpm in this assay. On the y axis in panel B, thymidine incorporation is measured in 103 cpm.

Limited influence of VLP-specific antibodies in vivo on T-cell expansion and effector cell induction. To test the influence of antibodies on T-cell proliferation and effector cell induction in vivo, mice were vaccinated as described for Fig. 1 prior to transfer of CFSE-labeled T cells from transgenic mice expressing a TCR specific for peptide p33 or p13. One day later, mice were immunized with p33-VLPs or p13-VLPs, respectively, and proliferation was assessed after 3 days (Fig. 4, left panel). Within 3 days, both CD8+ p33-specific T cells and CD4+ p13-specific T cells had proliferated extensively. In addition, the presence of antibodies had limited influence on the extent of cell cycling. We quantified the numbers (data not shown) and the fraction of TCR-transgenic T cells that had proliferated three to six times. On average, 30% (range, 26 to 35%) of the CD8+ T cells in control mice and 28% (range, 26 to 30%) in prevaccinated mice had undergone proliferation. For the CD4+ T cells, 29% (range, 26 to 31%) had proliferated in control mice and slightly more (36% [range, 34 to 38%]) had proliferated in prevaccinated mice. Since numbers of spleen cells were similar for all mice, the data demonstrate that preexisting antibodies also do not affect absolute numbers of proliferating T cells. The presence of effector cells was assessed by restimulating T cells for 6 h with specific peptide before performing an intracellular cytokine staining (Fig. 4, right panel). No significant influence of VLP-specific antibodies on effector cell generation could be identified (Fig. 4, right panel), and similar frequencies and numbers of CD8+ T cells produced IFN-{gamma} in all mice. Note that the large CFSE-negative population in the right panel is in part due to endogenous V{alpha}2Vß8+ T cells that do not produce IFN-{gamma}. Surprisingly, the peptide-specific CD4+ T cells efficiently proliferated but essentially failed to produce IFN-{gamma} upon stimulation. We are currently investigating whether this reflects a real difference between CD4+ and CD8+ T cells or whether this is due to the particular transgenic mouse lines used for the experiment.



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  		FIG. 4. In vivo T-cell proliferation and IFN-{gamma} production is not influenced by anti-VLP antibodies. CFSE-labeled CD8+ and CD4+ transgenic T cells were transferred into carrier-vaccinated mice (a, c, e, g) or untreated recipient mice (b, d, f, h) one day before i.d. vaccination with p33-VLP or p13-VLP, respectively. Four days after injection, cells obtained from the draining LN were stained with anti-CD8 (a, b) or anti-CD4 (c, d) antibody in combination with anti-V{alpha}2 and anti-Vß8.1 for analysis of T-cell proliferation. Cells were gated on V{alpha}2+ Vß8+ and shown as CFSE-labeled cells (x axis) versus CD8 or CD4 (y axis). For measurement of IFN-{gamma} production, cells were restimulated for 6 h with the corresponding peptide p33 or p13, fixed, and permeabilized, as described in Material and Methods. Cells were stained with anti-CD8 (e, f) or anti-CD4 (g, h) antibody in combination with anti-IFN-{gamma}. Cells were gated on CD4 or CD8 and illustrated as CFSE-labeled cells (x axis) versus IFN-{gamma}-producing cells (y axis). Numbers (mean in brackets) in the quadrant indicate the percentage of CD8/CD4+ V{alpha}2+ T cells secreting IFN-{gamma}. Note that the large CFSE-negative population in the right panel is in part due to endogenous V{alpha}2 Vß8+ T cells that do not produce IFN-{gamma}. A representative of at least two experiments is shown.

VLP-specific antibodies partially impair induction of protective T-cell responses. The induction of protective CTL responses was assessed in a nontransgenic system. Our laboratory has previously shown that vaccination with p33-VLPs results in minimal responses in normal mice unless APCs are activated by the addition of CpGs or anti-CD40 antibodies (33). To test whether CpGs would similarly affect the magnitude of the VLP-specific IgG response, mice were immunized with VLPs alone or mixed with CpGs and IgG responses were assessed subsequently 10 days later (Fig. 5A). Unexpectedly, IgG titers were not enhanced by the CpGs but rather slightly reduced, most likely due to alterations in the splenic architecture induced by these CpGs (35). To test whether preexisting antibodies may inhibit the induction of protective immunity by CpGs plus VLPs, prevaccinated and control mice were immunized with p33-VLPs in the presence of CpGs. As expected, immunization of WT VLP-prevaccinated mice with p33-VLPs boosted the VLP-specific antibody response by about a factor of 10 (Fig. 5B), further indicating that WT VLPs and p33-VLP induce cross-reactive antibodies. In a next experiment, mice prevaccinated with WT VLPs and control mice were immunized with 100, 10, or 1 µg of p33-VLPs in the presence of CpGs or left untreated and challenged 7 days later intravenously with LCMV (200 PFU WE) and viral titers were determined in the spleen 5 days later. Mice immunized with 10 or 100 µg of p33-VLPs loaded with CpGs were fully protected (Fig. 5B). Importantly, mice prevaccinated with the WT VLP were also protected. In contrast, mice immunized with 1 µg of p33-VLPs were only partially protected and prevaccination with the carrier resulted in further reduced protection. This difference was, however, barely statistically significant (P = 0.04, one-sided t test; P = 0.07, two-sided t test) (Fig. 5C). Thus, the presence of specific antibodies had minimal influence on the induction of protective T-cell responses.



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  		FIG. 5. Systemic antiviral immunity is not influenced by anti-VLP antibodies. (A) CpGs slightly reduce the IgG response induced by the carrier. Mice (3 per group) were immunized with WT VLPs (100 µg/mouse) in the presence or absence of CpGs. IgG responses were measured 10 days later. (B) Carrier-vaccinated and control C57BL/6 mice (6 mice per group) were boosted s.c. with p33-VLP (100 µg/mouse) mixed with CpGs, and the antibody response was measured 7 days later. (C) Carrier-vaccinated and control C57BL/6 mice were immunized s.c. with increasing concentrations of p33-VLP (1 to 100 µg/mouse) mixed with CpGs (10 nmol). Seven days later, mice were infected intravenously with 200 PFU of LCMV WE. The animals were sacrificed 5 days later (indicated by cross), spleens were collected, and LCMV titers were determined by an LCMV focus-forming assay. Each group included three mice.

To study the T-cell response in greater detail, frequencies of specific T cells were determined by tetramer staining prior to and 5 days after the challenge (Table 1). Mice prevaccinated with WT VLP exhibited reduced frequencies of specific T cells independent of the dose of vaccine used for vaccination. Moreover, specific T-cell frequencies after challenge closely correlated with the frequencies before challenge. The difference between prevaccinated and control groups was surprisingly large given the rather insignificant difference seen in the protection assays. To assess whether a similar reduction in T-cell frequencies could be observed in the spleen, the experiment was repeated and splenic T-cell frequencies were determined in the blood (data not shown) and in the spleen (Table 1). Since the number of splenocytes was not different in the prevaccinated and control mice, reduced frequencies of specific T cells translate into equally reduced absolute numbers of T cells (data not shown). Thus, VLP-specific antibodies have a minor, but significant, influence on the efficiency of T-cell responses after vaccination with VLPs.


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  		TABLE 1. Frequencies of peptide p33-specific CD8+ T cells before and after challenge with LCMVa


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DISCUSSION
 
Maternal antibodies are known to interfere with vaccination efficacy in infants, leading to the general concept that vaccine-specific antibodies reduce vaccine-induced immune responses, a phenomenon often referred to as carrier suppression. Thus, antibodies against a vaccine carrier are suggested to inhibit the establishment of a protective immune response. Many of these observations may be attributed to reduced replication of attenuated vaccine strains in the presence of maternal neutralizing antibodies, which may explain the reduced vaccination efficacy (1, 5, 22). Under these conditions, carrier suppression may simply reflect the neutralization of replication-competent viruses. In addition, preimmunization against carrier proteins often results in reduced hapten-specific antibody responses, but T-cell responses have rarely been examined (12). In fact, maternal antibodies have been reported to primarily affect antibody responses while Th-cell responses may not be reduced dramatically (31). Nevertheless, the concept that carrier-specific immune responses may interfere with de novo induction of T-cell responses specific for epitopes conjugated to the carrier are rather prevalent in the field. Furthermore, there has been a report of reduced T-cell responses induced by nonreplicating VLPs in the presence of neutralizing antibodies (8). The particular VLPs used in this study (chimeric human papillomavirus VLPs carrying human papillomavirus type 16 E7 protein) carry the protein structure responsible for virus-receptor interaction. The reduced T-cell priming in the presence of neutralizing antibodies directed against human papillomavirus type 16 E7 seen in this study was attributed to inhibition of the VLP-receptor interaction. Accordingly, neutralizing antibodies strongly inhibited receptor-mediated cellular uptake of VLPs. Other than this report, evidence for reduced T-cell priming in the presence of specific antibodies is rather scarce. On the contrary, recent results suggest that Fc receptors may enhance rather than inhibit cross-presentation and cross-priming. More specifically, antigen targeted to the Fc{gamma}RI are efficiently cross-presented (36), and tumor-specific antibodies may enhance cross-presentation of tumor-associated antigens (9, 15), particularly if Fc{gamma}RIIB interactions are avoided (15). Moreover, the processing capacities of B cells expressing an antigen-specific immunoglobulin versus B cells expressing an immunoglobulin of unrelated specificity are different by orders of magnitude, indicating that antibodies may be able to focus the antigen and dramatically enhance local antigen concentrations (16). Similarly, mannose receptors on DCs greatly increase antigen uptake and processing, further suggesting that antigen recognized by receptors may be more easily processed, at least for MHC class II presentation (11, 28).

On the other hand, it is conceivable that pathways of cross-presentation may be altered by the presence of antibodies. Cross-presentation of VLPs occurs via a TAP-independent, endosomal pathway and a TAP-dependent, endosome to cytosol pathway (27). For both pathways, it is likely that specialized endosomal compartments are required, either for loading of MHC class I molecules within the endosome or for release of antigen into the cytosol. Thus, FcR-triggering may affect endosomal maturation or target antigens to different endosomal/lysosomal compartments, in turn altering cross-presentation.

To address these questions, we used VLPs based on HBcAg fused to MHC class I- or class II-restricted LCMV-derived peptide. Since the core of hepatitis B is not involved in virus-receptor interactions and is not a target of neutralizing antibodies, it was possible to study the influence of VLP-specific antibodies without interference of virus-host interactions. Surprisingly, both MHC class I- and MHC class II-associated presentation of VLP-derived peptides were not significantly affected by the presence of specific antibodies. This was the case for in vitro and in vivo experiments. Hence, VLP-specific antibodies neither increased nor decreased antigen presentation by dendritic cells. The failure of specific antibodies to enhance MHC class II presentation may be explained by the fact that presentation of VLPs is already highly effective in the absence of antibodies. Specifically, VLPs were measurably presented at a VLP concentration of 10–10 to 10–11 M, concentrations similar to those reported for processing of antigens recognized via mannose receptors by DCs (28).

Given the limited influence of VLP-specific antibodies on antigen presentation, it was not surprising that T-cell responses were not dramatically affected. Using an adoptive transfer system where CFSE-labeled specific T cells were transferred into C57BL/6 mice before immunization with the VLPs carrying peptide p33 or p13, T-cell cycling could be measured in detail. No significant differences could be observed between carrier-primed animals and controls. Furthermore, induction of effector cells was also unaffected by the presence of specific antibodies. These results also correlated to protective CTL responses that could be established upon vaccination with p33-VLPs mixed with CpGs. Interestingly, however, in the presence of VLP-specific antibodies, antiviral protection appeared slightly less effective at the lowest vaccine dose. Moreover, the clonal burst size was also reduced, since frequencies of specific T cells were significantly lower in the presence of VLP-specific antibodies. These results seem to contradict the rather normal proliferation of p33-specific TCR-transgenic T cells observed in the adoptive transfer experiments. It should be noted, however, that induction of T-cell proliferation and effector cell induction is generally less demanding in TCR-transgenic systems and it therefore may be easier to achieve efficient responses in the presence of TCR-transgenic T cells (34). In fact, we recently found that p33-VLPs alone were efficient at inducing effector CTLs in the adoptive transfer system, whereas C57BL/6 mice essentially failed to respond unless p33-VLPs were given together with CpGs as adjuvants (32). This is probably due to the fact that specific T cells present at high frequencies serve as their own adjuvants.

In conclusion, the presence of VLP-specific antibodies had a marginal influence on the presentation of VLP-derived MHC class I- and MHC class II-associated peptides, and it was possible to induce protective T-cell responses in the presence of high anti-VLP antibody titers. However, in contrast to observations made with tumor cells, the presence of antibodies did not enhance T-cell responses induced by cross-priming. The difference may be due to the fact that FcR-mediated APC activation seems critical for efficient cross-presentation of tumor-derived antigens (9, 15) while VLPs may directly stimulate activation of APCs (17, 27) or rapidly become decorated with IgM antibodies and/or complement which also facilitates APC activation. Therefore, as previously observed (10, 27), the factors that govern cross-presentation may be, overall, similar for tumor-associated antigens and VLPs, but the precise details may be different.


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ACKNOWLEDGMENTS
 
We thank Tilman Dumrese for MHC tetramers, Anna Flace for excellent technical support, and Alma Fulurija for helpful discussions and for carefully reading the manuscript.


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FOOTNOTES
 
* Corresponding author. Mailing address: Cytos Biotechnology AG, Wagistrasse 25, 8952 Schlieren-Zürich, Switzerland. Phone: 41 1 733 40 23. Fax: 41 1 733 47 04. E-mail: martin.bachmann{at}cytos.com. Back


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REFERENCES
 
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Journal of Virology, January 2005, p. 717-724, Vol. 79, No. 2
0022-538X/05/$08.00+0     doi:10.1128/JVI.79.2.717-724.2005
Copyright © 2005, American Society for Microbiology. All Rights Reserved.



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