1 Division of Clinical Virology, F68, Karolinska Institutet at Huddinge University Hospital, S 141 86 Stockholm, Sweden
2 Biomedical Research and Study Center, University of Latvia, Riga, Latvia
3 Department of Biochemistry, Commonwealth University, Richmond, VA, USA
4 Vaccine Research Institute of San Diego, San Diego, CA, USA
Correspondence
Matti Sällberg
Matti.Sallberg{at}impi.ki.se
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ABSTRACT |
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Introduction |
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Methods |
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Recombinant proteins, peptides and plasmid DNA vectors.
Recombinant particulate HBcAg (ayw subtype) encompassing residues 1183 were produced in Escherichia coli and purified as described previously (Schodel et al., 1993).
Recombinant HBcAg (subtype ayw) encompassing residues 1183 and with an Ile at position 80 and a Gly at position 74 was designated HBcAg-Ile (Bichko et al., 1985). Full-length mutants of HBcAg-Ile lacking the regions 7985 (HBc
7985) and 7685 (HBc
7685), respectively, were expressed and purified from E. coli. In addition, a mutant of HBcAg-Ile in which residues 6789 had been replaced by the same region from HBcAg subtype adw was designated HBcAg-Ala (Table 1
). E. coli-expressed HBcAg of subtype ayw with low endotoxin content was purchased from Advanced ImmunoChemical. Particle formation of HBcAg-Ile, HBc
7985 and HBc
7685 was confirmed by electron microscopy using a JEM-100C electron microscope at an accelerating voltage of 80 kV. The non-structural 3 protein (NS3) of hepatitis C virus (HCV) was used as a recombinant control antigen (Jin & Peterson, 1995
).
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Construction of a eukaryotic vector expressing HBcAg was performed as follows. DNA was extracted from the tail of an HBeAg transgenic mouse (Milich et al., 1990) and the 552 bp long HBcAg gene fragment was amplified by PCR using primers C5 (5'-ATGGACATCGACCCTTATAAAGAA-3') and CE3 (5'-CTAACATTGAGATTCCCGAGATTG-3'). ApaI and HindIII sites were introduced using primers ClonC1 (5'-AACTTAAGCTTATGGACATCGACC-3') and ClonCE1 (5'-AAACGGGCCCTAACATTGAGATTC-3'). The resulting amplicon was ligated into the HindIII/ApaI-digested pVAX vector (Invitrogen). The HBcAg DNA plasmid was sequenced to ensure the correct reading frame of the insert. In vitro translation of the HBcAg DNA plasmid was performed using the T7-Coupled Reticulocyte Lysate system (Promega) and expression of the 21 kDa HBcAg protein was confirmed (data not shown). The plasmid DNA used for in vivo injections was purified using DNA Purification Columns (Qiagen), according to the manufacturer's instructions.
Recombinant soluble dimeric mouse H-2KbIg fusion protein used for flow cytometry analysis was purchased from Becton Dickinson. Ionomycin and phorbol myristate 13-acetate were purchased from Sigma.
Antibodies.
Traditional anti-HBc mouse monoclonal antibodies (mAbs) 32/312, recognizing amino acid residues 7883, and HBe/cAg-specific mAb 57/8, recognizing an epitope at residues 128133, have been described in detail previously (Pushko et al., 1994; Sallberg et al., 1991
, 1993
).
The following anti-mouse mAbs were used for flow cytometry analysis: anti-CD19 (clone 1D3), anti-B220 (clone RA3-6B2) and anti-CD8 (clone 53-6.7) (all purchased from Becton Dickinson).
Enzyme immunoassays (EIAs).
The panel of HBcAg recombinant proteins was tested for recognition by previously characterized mAbs 35/312 (specific for residues 7685 of HBcAg) and 57/8 (specific for residues 128133 of HBc/eAg) (Pushko et al., 1994; Sallberg et al., 1993
) by solid phase EIAs. In brief, recombinant proteins were coated overnight on microplates at 1 µg ml-1 in sodium bicarbonate buffer (pH 9·6) at 4 °C. Plates were incubated with primary antibodies 57/8 or 35/312 diluted in PBS containing 1 % BSA, 2 % goat sera and 0·05 % Tween 20 for 1 h. Thereafter plates were incubated with anti-mouse immunoglobulin peroxidase conjugate (P260) (Dako). All incubation steps were carried out at room temperature. Plates were developed using O-phenylenediamine substrate for 30 min and the reactions were stopped using 2 M H2SO4. Optical density measurements were determined at 490 nm.
Immunizations.
To study humoral responses, groups of C57BL/6 mice were immunized intraperitoneally with 20 µg HBcAg, HBcAg-Ile, HBc7985 and HBc
7685 in PBS. Mice were boosted 4 weeks later with the same dose of antigen. Sera were collected at weeks 2 and 6, pooled and antibody titres were determined by EIA.
To prime CTLs, groups of C57BL/6 and µMT.B6 mice were immunized with a single injection of the 100 µg HBcAg93100 peptide in complete Freund's adjuvant (CFA) (Sigma), or 20 µg of recombinant protein in incomplete Freund's adjuvant (IFA), subcutaneously at the base of the tail. Mice were sacrificed 913 days later.
For DNA immunizations, mice were immunized by needle injections of 100 µg of plasmid DNA encoding HBcAg. Plasmid DNA reconstituted in PBS was given intramuscularly to the tibialis anterior (TA) muscle (Lazdina et al., 2001b). At 5 days prior to DNA immunization, mice were injected intramuscularly with 50 µl per TA muscle of 0·01 mM cardiotoxin (Latoxan) in 0·9 % sterile saline (Lazdina et al., 2001b
). Mice were boosted at intervals of 4 weeks.
B cell enrichment.
To prepare enriched naive B cells, spleens were removed from non-immune C57BL/6 mice. Single cell suspensions were prepared and depleted of red blood cells using Red Blood Cell Lysing Buffer (Sigma). T cells were depleted using a 1 : 1 : 1 ratio of supernatants from hybridomas 31M (anti-CD8), RL172.4 (anti-CD4) and AT83 (anti-Thy1.2) (kindly provided by Eva Severinson and Lena Ström, CMB, Karolinska Institutet, Sweden) plus low-toxicity rabbit complement (Saxon Europe). The cells were incubated for 1 h at 37 °C. Resulting T cell-depleted cell populations consisted of 85 % CD19-positive cells, as determined by flow cytometry.
Direct binding of HBcAg to B cells.
A total of 1x106 T cell-depleted spleen cells from non-immune C57BL/6 mice were incubated on ice for 20 min with Fc-receptor blocking reagent (Fc-block) (Becton Dickinson) diluted in PBS containing 1 % foetal bovine serum (hereafter referred to as FACS buffer). Cells were then incubated with 1 µg of recombinant HBcAg, HBcAg-Ile, HBc7985, HBc
7685, HBcAg-Ala or the control antigen HBeAg for 30 min on ice. Cells were washed and incubated with HBc/eAg-specific mAb 57/8 conjugated with sulfo-NHS-LC-biotin (Pierce). Biotinylation was done according to the protocol provided by the manufacturer. After two washes, cells were incubated on ice for 30 min with allophycocyanin-conjugated streptavidin (Becton Dickinson) and phycoerythrin (PE)-conjugated CD19 antibody (Becton Dickinson). Samples were applied to a FACSCalibur flow cytometer (Becton Dickinson) and analysed using CELLQUEST software (Becton Dickinson).
Detection of HBcAg-specific CTLs.
Spleen cells from mice immunized with recombinant protein or peptide were harvested 13 days after immunization. DNA-immunized spleens were obtained 2 weeks after the second boost (6 weeks after the first injection). Single cell suspensions were prepared as described and 25x106 splenocytes were restimulated with 25x106 syngeneic irradiated (20 Gy) splenocytes pulsed with 0·05 µM of peptide, as described previously (Sandberg et al., 2000). Restimulation cultures were set in 12 ml complete RPMI medium. After 5 days, effector cells were harvested and washed twice. RMA-S target cells (Karre et al., 1986
) were pulsed with 50 µM peptide for 90 min at 5 % CO2 and 37 °C. Serial dilutions of effector cells were incubated with 5x103 chromium-labelled peptide-pulsed RMA-S target cells in a final volume of 200 µl per well in 96-well plates. After a 4 h incubation at 5 % CO2 and 37 °C, 100 µl of supernatant was collected and the radioactivity was determined using a
-counter. The percentage of specific release was calculated according to the formula: (experimental release-spontaneous release/total release-spontaneous release) xtimes;100. Results are shown as the mean of triplicate values.
Quantification of HBcAg93100-specific CTLs by flow cytometry.
The frequencies of HBcAg-specific CD8+ T cells was analysed by ex vivo staining of spleen cells from HBcAg93100 peptide, HBcAg protein and HBcAg DNA-immunized mice using the recombinant soluble dimeric mouse H-2KbIg fusion protein (Becton Dickinson). Spleen cells (12x106) were suspended in 50 µl FACS buffer and pre-incubated with 1 µg of Fc-block per 106 cells on ice for 15 min. After a single wash, cells were incubated on ice for 90 min with 3 µg of H-2KbIg fusion protein per 1x106 cells pre-loaded for 48 h with a 640 M excess of HBcAg93100 peptide (MGLKFRQL) (Kuhober et al., 1996). H-2KbIg fusion protein unloaded or loaded with irrelevant peptide served as control for unspecific staining. In some experiments, naive spleen cells were also included as a control for non-specific staining. After incubation with the H-2KbIg fusion protein, cells were washed twice in FACS buffer and resuspended in 50 µl FACS buffer containing a 1 : 5 dilution of PE-conjugated rat anti-mouse IgG1 antibody (Becton Dickinson) and incubated on ice for 30 min. Cells were washed twice and incubated with 1 µg of FITC conjugated-mouse CD8 antibody per 106 cells for 30 min. After incubation, cells were washed twice and resuspended in 0·5 ml FACS buffer for immediate FACS analysis. Approximately 50100 000 events from each sample were counted on a FACS Calibur.
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Results |
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Discussion |
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It has been shown that exogenous antigen is taken up by endocytosis and can reach the MHC I compartment rendering B cells susceptible to lysis by specific class I-restricted CD8+ T cells (Ke & Kapp, 1996). HBsAg-specific B cells are able to present exogenous HBsAg to HBsAg-specific MHC class I-restricted CD8+ T cells (Barnaba et al., 1990). To study the role of B cells in priming CTLs by endogenously and exogenously produced HBcAg, we immunized wild-type and B cell-deficient mice. HBcAg-specific CTLs were detected by in vitro cytotoxicity assays and quantified directly ex vivo by flow cytometry. These experiments showed that B cells were not required for CTL priming by the HBcAg-derived CTL peptide or endogenously produced HBcAg. In contrast, exogenous HBcAg particles were only able to prime HBcAg-specific CTLs in the presence of B cells. Exogenous HBcAg primed in vitro active CTLs at very low precursor frequencies in wild-type mice. Importantly, the mutant HBcAg particle lacking residues 7685 at the spike region and unable to bind naive B cells could not prime HBcAg-specific CTLs when used as an exogenous immunogen in the presence of B cells. This clearly shows that the priming of HBcAg-specific CTLs by exogenous HBcAg is B cell dependent.
There are several mechanisms by which exogenous HBcAg may prime CTLs in a B cell-dependent fashion. First, the role of HBcAg-binding B cells could be as the primary APC for CD8+ T cells through a leakage between the class I and class II antigen-presenting pathways. Alternatively, B cells may produce HBcAg-specific antibodies that form immune complexes with HBcAg. These immune complexes could then be taken up by macrophages or dendritic cells via Fc-R-mediated endocytosis and thereafter processed and presented to CD8+ T cells (Schuurhuis et al., 2002). Since we know that immunization with the HBc
7685 particle, which is unable to prime CTLs, elicits antibodies in wild-type mice, we would favour the first hypothesis. Additional explanations may be that other cells, such as CD4+ T cells, are participating in CTL priming and the priming of these cells may be insufficient in the absence of B cells or when the particle is unable to engage naive B cells effectively. However, further experiments are needed to fully understand this.
In conclusion, endogenously produced HBcAg primes HBcAg-specific CTLs effectively at a high precursor frequency independently of the presence of B cells. However, binding of particulate HBcAg by naive B cells is pivotal in priming HBcAg-specific CTLs using exogenous HBcAg. These findings have clear implications for vaccine design using recombinant HBcAg particles and suggest that exogenous HBcAg released from infected hepatocytes could possibly regulate the CTL response through B cells.
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ACKNOWLEDGEMENTS |
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Received 28 June 2002;
accepted 11 September 2002.