Adjuvants that enhance priming of cytotoxic T cells to a Kb-restricted epitope processed from exogenous but not endogenous hepatitis B surface antigen
Reinhold Schirmbeck,
Karl Melber1 and
Jörg Reimann
Institute for Medical Microbiology and Immunology, University of Ulm, Helmholtzstrasse 8/1, 89081, Ulm, Germany
1 Rhein-Biotech GmbH, Eichsfelderstr. 11, 40595 Düsseldorf, Germany
Correspondence to:
J. Reimann
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Abstract
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Intramuscular (i.m.) or s.c. injection of plasmid DNA encoding hepatitis B small surface antigen (HBsAg) primes potent MHC I-restricted cytotoxic T lymphocyte (CTL) responses in H-2d (BALB/c) and H-2b (C57BL/6) mice. In contrast, i.m. or s.c. injection of exogenous HBsAg particles without adjuvants primes CTL responses in `high responder' H-2d but not `low responder' H-2b mice. We have shown that processing of exogenous but not endogenous HBsAg generates the Kb-binding S208215 peptide ILSPFLPL. This system allowed us to optimize conditions for stimulating murine CTL responses to exogenous antigen by identifying adjuvants that facilitate priming of Kb-restricted CTL by injecting recombinant HBsAg particles into `low responder' H-2b mice. Synthetic oligodeoxynucleotides with immunostimulating sequences or the recombinant cytokine IL-12 efficiently enhanced priming of CTL to exogenous HBsAg. Hence, the adjuvanticity of DNA sequences that induce Th1 cytokines facilitate priming of MHC I-restricted T cell responses to exogenous antigen and are therefore of potential value in formulating vaccines designed to enhance CTL priming to exogenous antigen.
Keywords: cytotoxic T lymphocyte, hepatitis B surface antigen, MHC class I, mouse
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Introduction
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A major aim in the design of anti-viral and anti-bacterial recombinant subunit vaccines is the induction of MHC I-restricted cytotoxic T lymphocyte (CTL) responses by exogenous antigens. In particular, there is a need to design such strategies for the prophylactic vaccination of `low responder' individuals or the therapeutic vaccination of persistently infected individuals. There is therefore an interest in adjuvants that efficiently support CTL priming to exogenous antigen. A major problem is the design of preclinical experimental systems that allow the selective read out of successful CTL priming by exogenous antigen, especially under `low responder' conditions.
CTL responses to hepatitis B surface antigen (HBsAg) are primed in H-2d mice vaccinated by a single low dose of either native HBsAg lipoprotein particles or HBsAg-encoding plasmid DNA (17). We have described the alternative processing of exogenous HBsAg for Ld-restricted epitope presentation (811). CTL from H-2d mice specifically recognize the immunodominant Ld-restricted epitope S2839 of HBsAg generated during processing of exogenous as well as endogenous HBsAg (8). We have reported that MHC I-restricted, HBsAg-specific CTL are primed in H-2b mice by DNA vaccination but not by immunization with exogenous HBsAg particles (5). H-2b mice were therefore considered `low-responders' with respect to CTL priming to exogenous HBsAg while H-2d mice were considered `high responders'. An adjuvant effect of synthetic oligodeoxynucleotides (ODN) on the immune response of `high responder' BALB/c mice to HBsAg delivered by the systemic or mucosal route has recently been published (12,13).
Processing, i.e. the partial proteolytic degradation of antigen, is an essential event in MHC-restricted antigen presentation. The major routes of MHC I- and MHC II-restricted antigen processing and presentation have been charted. The degradation of endogenous antigen in the cytosol generates peptides that bind to nascent MHC I molecules in the lumen of the endoplasmic reticulum (ER). The cytosolic ubiquitin/proteasomal pathway has been identified as the main site of proteolysis in the endogenous processing pathway that generates peptides presented by MHC I molecules (1416). The endolysosomal degradation of exogenous antigens generates peptides mainly for loading to MHC II molecules. Recently, attention has shifted to examples that break the established rules. We are interested in the MHC I-restricted presentation of peptides derived from endocytosed, exogenous antigens (reviewed in 17,18). We have identified the Kb-binding S208215 peptide ILSPFLPL of HBsAg (subtype ayw) as a main target in the CTL response of H-2b mice to exogenous HBsAg and demonstrated that this epitope is generated during alternative processing of exogenous HBsAg but not during conventional processing of endogenous HBsAg (19). Here, we confirm the generation of this peptide in an alternative, brefeldin A (BFA)-resistant, exogenous processing pathway in transporter associated with antigen processing (TAP)-deficient cells pulsed with exogenous HBsAg particles. Very little is known about adjuvants that enhance priming of MHC I-restricted CTL responses to exogenous antigens in vivo. This system thus offers the unique chance to investigate in vivo priming of CTL to epitopes of exogenous antigen generated exclusively by its alternative processing in an endolysosomal pathway.
We designed experiments to identify adjuvants that enhance the immunogenicity of exogenous HBsAg for MHC I-restricted CTL precursors from `low responder' H-2b mice. We found that bacterial plasmid DNA itself facilitates CTL priming to exogenous HBsAg in H-2b mice. In line with this finding, ODN containing immunostimulating sequences (ISS) most efficiently support CTL priming to exogenous HBsAg in H-2b mice. Non-methylated ODN containing ISS have been shown to be potent adjuvants for cellular and humoral immune responses in mice (reviewed in 2023). Cytokines, in particular IL-12, have been shown to enhance CTL priming in different experimental systems (2434). Here, we show that agents that induce this Th1 cytokines or IL-12 itself facilitate priming of CTL responses to exogenous antigen in vivo.
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Methods
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Mice
C57BL/6J (B6) mice (H-2b) and BALB/cJ mice (H-2d) were bred and kept under standard pathogen-free conditions in the animal colony of Ulm University (Ulm, Germany). Breeding pairs of these mice were obtained from Bomholtgard (Ry, Denmark). Female mice were used at 1016 weeks of age.
Cell lines
The H-2d mastocytoma cell line P815 (TIB-64) and the H-2b thymoma line EL4 (TIB-39) were obtained from the ATCC (Rockville, MD). The H-2b (C57BL/6-derived) T lymphoma cell line RBL5 was obtained from Dr H.-U. Weltzien (Freiburg, Germany) and its TAP-deficient subline RMA-S was obtained from Dr K. Kärre (Stockholm, Sweden). The P1 (H-2d) cell line and its transfected subclone P1.Kb was generously provided by Drs H. Schild and H. G. Rammensee (Tübingen, Germany). The human cell line T2 and its transfected sublines T2.Ld and T2.Kb were a generous gift of Dr P. Cresswell (New Haven, CT).
Recombinant HBsAg
Yeast-derived HBsAg particles (subtype ayw4; EMBL sequence: HBVGen1, accession no. Z35719) containing only the small surface protein of hepatitis B virus were obtained from Rhein-Biotech (Düsseldorf, Germany). HBsAg was produced in the Hansenula polymorpha host strain RB10 as described (35). HBsAg particles were purified from crude yeast extracts by adsorption to silica gel, column chromatography and isopycnic ultracentrifugation.
ODN
All ODN were synthesized with a nuclease-resistant phosphorothionate backbone (PTO-modified) by MWG-Biotech (Ebersberg, Germany) and used in single-stranded form. The sequence used in the present work was 5'-TCATTGGAAAACGTTCTTCGGGGCG-3' (36). The active sequence motif (underlined) contains a central CpG dinucleotide flanked by two purines 5' and two pyrimidines 3'. Control ODN containing CG to GG substitutions 5'-TCATTGGAAAAGGTTCTTGGGGGGG-3' (ISS*) or methylated C 5'-TCmATTGGAAAACmGTTCTTCmGGGGCmG-3' (ISSM) were used. HBsAg particles were mixed with 50 µg ODN and injected into mice without adding further adjuvants.
Peptides
The 8mer Kb-binding S208215 peptide ILSPFLPL was synthesized in an Applied Biosystems peptide synthesizer model 431A and purified by reverse-phase HPLC. Peptides were dissolved in a DMSO solution at a concentration of 10 mg/ml and diluted with culture medium before use. 51Cr-labeled cells (2x105) were suspended in 250 µl serum-free UltraCulture medium (cat. no. 12-725F; BioWhittaker, Walkersville, MD). Cells were incubated with the indicated amounts of recombinant HBsAg particles or HBsAg-specific peptides for 12 h (811). Cells were washed and used as targets in cytotoxic assays.
HBsAg-encoding plasmid DNA used for nucleic acid vaccination
The HBsAg-encoding XhoIBglII fragment of hepatitis B virus (subtype ayw; EMBL sequence: XXHEPA, accession no. V01460) was obtained from plasmid pTKTHBV2 (a generous gift of Dr M. Meyer, Munich, Germany) and cloned into the XhoIBamHI-cut pCI vector (cat. no. E1731, Promega, Heidelberg, Germany). This generated plasmid pCI/S in which HBsAg is expressed under control of the human cytomegalovirus immediate early promoter. The amino acid sequence of Hansenula-expressed HBsAg ayw4 and the pCI/S-encoded HBsAg are identical (Swiss-Prot, accession no. P03138).
Vaccination of mice
Mice were immunized once with the indicated dose of recombinant HBsAg in 100 µl PBS by different routes: intramuscularly (i.m.; into both tibialis anterior muscles), s.c. (into the base of the tail) or i.p. In some experimental groups, HBsAg was co-administered with 50 µg ISS-containing ODN, 50 µg ISS*-containing ODN, 50 µg ISSM-containing ODN or 100 ng recombinant murine IL-12 (cat. no. 19361V; PharMingen, Hamburg, Germany). We injected 50 µl of 1 µg/µl plasmid DNA in PBS into each non-pretreated tibialis anterior muscle or 100 µl (1 µg/µl) into the base of the tail. All mice received only one injection.
In vitro stimulation of primed, HBsAg-specific CTL
Spleens were removed from immunized mice 116 weeks post-vaccination. Single-cell suspensions were prepared in
-MEM tissue culture medium supplemented with 10 mM HEPES buffer, 5x105 M 2-mercaptoethanol, antibiotics and 10% v/v FCS (Gibco/BRL, Eggenstein, Germany). A selected batch of concanavalin A-stimulated rat spleen cell supernatant (2% v/v) was added to the culture medium. Responder cells (3x107) were co-cultured with 1x106 irradiated, syngeneic target cells pulsed for 12 h with HBsAg particles as described (811). Co-culture was performed in 10 ml medium in upright 25 cm2 tissue culture flasks in a humidified atmosphere containing 7% CO2 at 37°C. After 5 days of culture, cells were harvested, washed and either re-stimulated under the same stimulation conditions or used as effector cells in the cytotoxic assay.
Cytotoxic assay
Serial dilutions of effector cells were cultured with 2x103 51Cr-labeled targets in 200 µl round-bottom wells. Specific cytolytic activity of cells was tested in a 51Cr-release assay against HBsAg- or peptide-pulsed targets or non-treated control targets. After a 4 h incubation at 37°C, 100 µl of supernatant was collected for
-radiation counting. The percentage specific release was calculated as [(experimental release spontaneous release)/(total release spontaneous release)]x100. Total counts were measured by resuspending target cells. Spontaneously released counts were always <15% of the total counts. Data shown are the mean of triplicate cultures. The SEM of triplicate data was always <20% of the mean.
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Results
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Alternative processing of exogenous HBsAg generates the Kb-restricted epitope S208215 recognized by CTL from H-2b mice
Using a library of overlapping synthetic peptides, we have mapped an epitope of HBsAg recognized by CTL from C57BL/6 (H-2b) mice immunized with pCI/S plasmid DNA as peptide S208215 with the sequence ILSPFLPL. In pCI/S-primed spleen cells re-stimulated in vitro with HBsAg-pulsed syngeneic tumor cells, specific cytolytic reactivity was detected in a standard 4 h cytotoxic assay using either HBsAg particle-pulsed targets (Fig. 1A
) or S208215 peptide-pulsed targets (Fig. 1B
). The S208215-specific CTL did not recognize transfected RBL5/S or RBL5/LS targets processing endogenous HBsAg (Fig. 1C
).

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Fig. 1. Generation of CTL specific for exogenous HBsAg. C57BL/6 mice were vaccinated with HBsAg-encoding pCI/S plasmid DNA. Their spleens were removed 4 weeks post-vaccination, re-stimulated in vitro with inactivated, HBsAg-pulsed EL4 cells and tested against non-pulsed RBL5 control targets or RBL5 targets pulsed for 2 h with indicated amounts of HBsAg particles (A) or RBL5 targets pulsed with the S208215 peptide (B). Specific lysis values at an E/T ratio of 20 are plotted. (C) CTL generated as described above specifically lysed HBsAg particle- and S208215 peptide-pulsed RBL5 cells but not endogenous HBsAg-expressing RBL5/S and RBL5/LS transfectants.
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The antigenic 8mer peptide ILSPFLPL contains the Kb-binding motif xxxxFxxL (F at position 5, L at position 8) (37,38). We confirmed the Kb restriction specificity of this peptide (Fig. 2
). EL4 cells (H-2b) but not P1 cells (H-2d) pulsed with HBsAg particles or the S208215 peptide were lysed by CTL. Transfected P1.Kb cells (i.e. P1 cells that express a functional Kb molecule) pulsed with HBsAg particles or the S208215 peptide were specifically and efficiently lysed by these CTL. Human T2 cells transfected with the murine Kb gene (T2.Kb) but not T2 cells transfected with the murine Ld gene (T2.Ld) presented the S208215 epitope to CTL after a pulse with the peptide S208215. The S208215 peptide thus stimulates Kb-restricted CTL responses to HBsAg.

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Fig. 2. Kb-restricted recognition of the S208215 epitope of HBsAg by CTL. H-2b EL4 cells, H-2d P1 cells, Kb-transfected P1.Kb cells and TAP-deficient human T2 cells transfected with Kb (T2.Kb) or Ld (T2.Ld) were pulsed for 1 h with 10 µg HBsAg particles or 108 M of the 8mer S208215 peptide ILSPFLPL. Cells were washed and used as targets in the CTL assay. Effector cells were derived from pCI/S immunized C57BL/6 mice that were in vitro re-stimulated with HBsAg-pulsed RBL5 cells. Specific lysis values at the indicated E/T ratios are shown.
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The Kb-restricted presentation of the S208215 epitope processed from exogenous HBsAg particles was TAP independent. TAP-competent EL4 and RMA cells or TAP-deficient T2.Kb and RMA-S cells were efficiently sensitized for S208215-specific CTL lysis by a pulse with exogenous HBsAg particles (Figs 2 and 3
). RMA-S cells metabolically blocked by low temperature (4°C) failed to take up HBsAg particles and did not present the Kb-restricted epitope to CTL while sensitization of targets with the antigenic S208215 peptide operated efficiently at 4°C (Fig. 3
). The failure of exogenous HBsAg particles to sensitize metabolically inactive target cells indicated that antigenic peptides did not contaminate the HBsAg preparations as most exogenous peptides do not require intracellular processing before sensitizing metabolically inactive targets. Processing of HBsAg particles was blocked by the protease inhibitor leupeptin (Fig. 3
). Regurgitation of antigenic, MHC I-binding peptides by cells processing exogenous HBsAg particles was not detectable (data not shown). Hence, a striking similarity in the Kb-restricted presentation of the S208215 epitope and the Ld-restricted presentation of the S2839 epitope processed from exogenous HBsAg particles was apparent (811).

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Fig. 3. Alternative processing of exogenous HBsAg for the Kb-restricted presentation of the S208215 epitope. TAP-competent RMA and TAP-deficient RMA-S cells were pulsed for 1 h with 10 µg HBsAg particles or 109 M of the 8mer S208215 peptide ILSPFLPL at 37°C or at 4°C. Furthermore, cells pulsed at 37°C were treated with leupeptin for 1 h before the pulse, during the pulse and during the subsequent cytotoxic assay as described previously (8). Pulsed cells were washed and used as targets in the cytotoxic assay using effector cells specific for a Kb/S208215-specific CTL from pCI/S-immunized mice. Specific lysis values at the indicated E/T ratios are shown.
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Priming MHC I-restricted CTL responses to HBsAg in H-2b and H-2d mice by injections of either recombinant HBsAg particles or plasmid DNA encoding HBsAg
Either recombinant HBsAg particles (without adjuvants) or HBsAg encoding pCI/S plasmid DNA were injected i.m. into BALB/c (H-2d) or C57BL/6 (H-2b) mice. Immunization by a single i.m. injection of 120 µg HBsAg particles or 100 µg `naked' HBsAg-encoding plasmid DNA induced long-lasting serum antibody titers specific for HBsAg in both mouse strains (data not shown) confirming previously published data (1,5).
A single i.m. injection of 10 µg HBsAg particles without adjuvants into BALB/c mice efficiently primed a CTL response (Fig. 4B
). This response is primed by injecting 0.110 µg HBsAg, is mediated by S2839-specific, Ld-restricted CD8+ CTL and is detectable from 1 week post-vaccination to at least 4 months post-vaccination (1,5). In addition, the i.m. injection of 100 µg pCI/S plasmid DNA efficiently primed HBsAg-specific CTL in `high responder' H-2d mice (Fig. 4B
). A different picture emerged when we tried to induce HBsAg-specific CTL reactivity in H-2b mice by either particle injection or DNA-based vaccination. Only DNA vaccination (but not HBsAg particle vaccination) primed CTL in this mouse strain (Fig. 4A
), confirming our previously published data (1,5). No specific CTL reactivity was detectable in lymph node or spleen cells from H-2b mice primed with even high doses of yeast-derived HBsAg (without adjuvants). Thus, although HBsAg-specific CTL responses are potentially inducible in H-2b mice (by DNA-based vaccination), this is difficult to achieve using recombinant HBsAg particles without adjuvants.

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Fig. 4. HBsAg-specific CTL responses of C57BL/6 (H-2b) (A) and BALB/c (H-2d) mice (B) vaccinated with either recombinant HBsAg particles (HBsAg) or HBsAg-encoding plasmid DNA (pCI/S). Mice were either not immunized (non-immunized), or injected i.m. with 10 µg recombinant HBsAg (without adjuvants), or injected i.m. with 100 µg HBsAg-encoding pCI/S plasmid DNA. Splenic CTL reactivity was measured in primed mice 3 weeks post-vaccination after re-stimulating cells in vitro for 5 days with syngeneic HBsAg-pulsed RBL5 cells (A) or P815 cells (B); their specific cytolytic reactivity was measured using RBL5 and P815 targets pulsed in vitro with recombinant HBsAg. Specific lysis values at the indicated E/T ratios are plotted.
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This system has three features that make it attractive for the study of CTL priming by exogenous antigen: (i) HBsAg is a virus-like lipoprotein particle that can prime MHC I-restricted CTL when delivered as an exogenous antigen in its native form without adjuvants; (ii) a high and low responder status with respect to CTL induction to exogenous HBsAg is defined; and (iii) the epitope specificity of the CTL response is an indicator that the MHC I-binding peptide is exclusively generated in an alternative vesicular pathway (and not in the conventional endogenous pathway). Because of these features, the HBsAg system offers a unique chance to define in vivo adjuvants that enhance priming of cytotoxic T cell populations to epitopes generated during processing of exogenous antigen. We succeeded in identifying adjuvants that enhanced CTL priming in low responder mice to exogenous HBsAg to a level comparable to that in high responder mice.
Bacterial DNA facilitates priming of CTL to exogenous HBsAg in H-2b mice
An i.m. injection of 100 µg, but not 1 or 10 µg `naked' pCI/S plasmid DNA into H-2b mice reproducibly primed HBsAg-specific CTL (Fig. 5A
). While the i.m. injection of 20 µg HBsAg particles into H-2b mice did not prime CTL, the same dose of HBsAg mixed with 100 µg (but not 10 µg) pCI plasmid DNA (vector DNA without insert) induced a detectable CTL response (Fig. 5B
). Control experiments showed that injection of 100 µg non-coding pCI DNA alone did not induce HBsAg-specific CTL (Fig. 5A and B
). The data suggested an adjuvant effect of plasmid DNA on CTL priming by exogenous HBsAg.

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Fig. 5. Plasmid DNA facilitates priming of CTL from `low responder' C57Bl/6 mice to exogenous HBsAg. (A) C57Bl/6 mice were i.m. injected with 1, 10 or 100 µg pCI/S plasmid DNA (encoding HBsAg), or with 100 µg pCI plasmid DNA (vector without insert, negative control). (B) C57Bl/6 mice were i.m. injected with 20 µg HBsAg particles either without adjuvants, or with 10 µg pCI plasmid DNA, or with 100 µg pCI plasmid DNA. Controls included non-immunized mice or mice injected with 100 µg pCI plasmid DNA (not encoding antigen). Spleens were removed from mice 3 weeks post-vaccination, re-stimulated in vitro with inactivated, HBsAg-pulsed EL4 cells and tested against HBsAg-pulsed RBL5 targets (and non-pulsed RBL5 control targets) in a 4 h 51Cr-release assay. Specific lysis values at the indicated E/T ratios are plotted.
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Bacterial plasmid DNA is known to contain ISS. In the next series of experiments we used a synthetic 25mer ODN 5'-TCATTGGAAAACGTTCTTCGGGGCG-3' containing an non-methylated, immunostimulating CpG motif (36). HBsAg particles were injected once either without ODN (HBsAg), or with 50 µg immunostimulating ODN (HBsAg + ISS), or with 50 µg methylated ODN 5'-TCmATTGGAAAACmGTTCmTTCmGGGGCmG-3' (HBsAg + ISSM), or with 50 µg mutated ODN 5'-TCATTGGAAAAGGTTCTTGGGGGGG-3' (HBsAg + ISS*). These formulations as well as control plasmid pCI/S DNA were injected i.m., s.c. or i.p. The s.c., i.m. or i.p. injections of exogenous HBsAg did not prime HBsAg-specific CTL in H-2b mice (Fig. 6A
C) although all three routes of vaccination induced readily detectable CTL responses in H-2d mice (data not shown). The i.m. and s.c., but not the i.p. injection of HBsAg-encoding pCI/S DNA and the mixture of exogenous HBsAg with ISS-containing ODN into H-2b mice primed efficiently a CTL response to HBsAg (Fig. 6A
C). Addition of methylated ISSM or mutant ISS*-containing ODN did not support induction of a CTL response against exogenous HBsAg particles (Fig. 6A and B
). ISS-containing ODN can therefore induce specific CTL priming to recombinant HBsAg in `low responder' mice.

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Fig. 6. ODN containing ISS facilitate priming of CTL to exogenous HBsAg in `low responder' C57Bl/6 mice. C57Bl/6 mice were vaccinated i.m. (A), s.c. (B) or i.p. (C). Mice were injected with either 100 µg pCI/S DNA, or 20 µg HBsAg (HBsAg), or 20 µg HBsAg mixed with 50 µg ISS-containing ODN (HBsAg + ISS), or 20 µg HBsAg mixed with 50 µg non-stimulating ISS* (HBsAg + ISS*) or methylated ISSM (HBsAg + ISSM). Spleens removed from immunized mice 3 weeks post-vaccination were re-stimulated in vitro with inactivated, HBsAg-pulsed cells and tested against HBsAg-pulsed RBL5 targets (and non-pulsed RBL5 control targets) in a 4 h 51Cr-release assay. Specific lysis values at the indicated E/T ratios are plotted.
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IL-12 mimicks the adjuvant effect of ISS-containing DNA
We tested if co-administration of recombinant cytokines with HBsAg particles enhances its immunogenicity for CTL. C57Bl/6 mice were tested for splenic MHC I-restricted, HBsAg-specific CTL reactivity 4 weeks after i.m. injections of different HBsAg-containing formulations. C57BL/6 mice vaccinated once with exogenous HBsAg/IL-12 developed specific cytolytic reactivity against HBsAg-pulsed and S208215 peptide-pulsed RBL5 targets in a standard 4 h cytotoxic assay (Fig. 7C
). Hence, immunization of mice with exogenous HBsAg adjuvanted with either IL-12 or ISS-containing ODN generated CTL with the S208215 epitope and Kb restriction specificity (Fig. 7
). No (or only low) CTL reactivity was detectable after vaccinations with HBsAg particles co-administered with recombinant IFN-
, IL-2 or IL-4 (data not shown).

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Fig. 7. IL-12 facilitated priming of CTL by exogenous HBsAg particles in `low responder' C57BL/6 mice. C57BL/6 mice (H-2b) mice were immunized i.m. with: 20 µg HBsAg particles without adjuvants (A), HBsAg mixed with 50 µg immunostimulating ODN + ISS (B) or HBsAg mixed with 100 ng IL-12 + IL-12 (C). Control groups included either non-immunized mice (D) or mice injected with IL-12 without HBsAg (data not shown). Spleen cells obtained from immunized or non-immunized mice 4 weeks post-vaccination were co-cultured in vitro with inactivated, HBsAg-pulsed cells. Specific cytolytic reactivity of CTL was tested in short-term 51Cr-release assays using non-pulsed targets (RBL5), HBsAg-pulsed targets (RBL5/HBsAg) or S208215 peptide-pulsed targets (RBL5 /S208215). Specific lysis values at the indicated E/T ratios are shown.
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Discussion
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Processing of exogenous antigens in a vesicular compartment can generate peptides for MHC I-restricted presentation (reviewed in 17). Little is known about the cell biology of this type of alternative processing. We have investigated the Ld-restricted presentation of the S2839 peptide of HBsAg by cells pulsed with exogenous HBsAg particles. In this system, processing of endocytosed HBsAg by cells of different tissue origin for Ld-restricted epitope presentation is leupeptin and pH sensitive, BFA resistant, and TAP independent (810). To test if endolysosomal processing of HBsAg can generate peptides that bind to other MHC class Ia molecules, we investigated the Kb-restricted presentation of a HBsAg epitope released by processing exogenous HBsAg (17). We show in this paper that the presentation of the Kb-binding S208215 peptide by HBsAg particle-pulsed cells displays similar features as those previously described for the Ld-restricted presentation of the S2839 peptide of HBsAg. Processing of particulate, endocytosed HBsAg in a vesicular compartment for the generation of MHC I-binding peptides is therefore not restricted to the generation of Ld-binding peptides, but has general validity.
The S208215 epitope is localized in the last of four transmembrane domains of the multiple membrane-spanning, small HBsAg protein (39,40). Processing of endogenously expressed LMP2 of EpsteinBarr virus has been shown to generate peptides that are presented in a TAP-independent pathway by HLA-A2 molecules (41). Similar to the Kb-binding peptide of HBsAg, the two peptide epitopes of LMP2 are located in the transmembrane domain of the multiple membrane-spanning LMP2. The antigenic peptides from LMP2 seem to be processed by ER proteases and to be loaded to HLA-A2.1 class I molecules in the ER lumen. In contrast to the Kb-restricted presentation of the S208215 epitope by cells that process exogenous HBsAg (which is BFA resistant, data not shown), HLA-A2-restricted presentation of the LMP2 epitopes is blocked by BFA. HBsAg particles are assembled in the lumen of the ER or the intermediate compartment and then secreted, while LMP2 molecules remain associated with membranes. This difference may influence the distinct processing pattern of these two viral proteins for MHC I-binding peptides. Hence, class I-binding peptides can be processed from transmembrane domains of multiple membrane-spanning proteins.
In this paper we define vaccine strategies that can facilitate CTL priming to exogenous antigen in vivo. Our system based on the non-responsiveness of HBsAg particles in H-2b mice to induce CD8+ CTL. We found that bacterial pCI plasmid DNA mixed with HBsAg particles acts as an `adjuvant' that allowed CTL priming (Fig. 5B
). The finding that only high amounts of HBsAg-encoding pCI/S plasmid DNA reproducibly induced CD8+ CTL in H-2b mice (Fig. 5A
) suggested that plasmid DNA itself acts as an adjuvant for HBsAg-specific CTL priming. Confirming this finding we showed that the plasmid DNA adjuvanticity could be efficiently replaced by synthetic ODN with the sequence 5'-TCATTGGAAAACGTTCTTCGGGGCG-3' with an immunostimulating CpG motif (36) but not by similar ODN containing non-stimulating or methylated motifs. The pCI/S plasmid DNA contains 20 immunostimulating CpG motives (16 within the pCI vector and four within the HBsAg-encoding XhoIBglII fragment). Three of the motifs in the pCI vector DNA contain the 5'-AACGTT-3' sequence identical to the one we used in the ODN. It is difficult to compare the relative efficacy of the adjuvant effects of synthetic nuclease-protected (PTO-modified), single-stranded ODN versus plasmid DNA. Our experiments indicate that
100 µg pCI plasmid DNA has to be used to detect an effect on CTL induction with co-administered HBsAg particles. In contrast 1050 µg synthetic ODN reproducibly enhanced priming of CTL to exogenous HBsAg (Fig. 5
and data not shown). The immunostimulatory effect of ODN may be mediated by activating antigen-presenting cells or by a direct effect on responding T cells (4255).
Co-administration of HBsAg with IL-12 allowed priming of MHC I-restricted CTL responses to exogenous HBsAg particles in H-2b `low responder' mice. IL-12 has been shown to facilitate priming of Th1 T cell responses (24,28,32,5660) and this cytokine may be a major mediator of the `adjuvant effects' of DNA. Specific CTL reactivity was detectable in low responder H-2b mice after a single i.m. or s.c. injection of 20 µg HBsAg particles mixed with IL-12; this HBsAg dose is substantially higher than that required to prime CTL in high responder H-2d mice without cytokines. The cytotoxic effector cells expressed the CD3+ TCR
ß+ CD4CD8+ surface phenotype of conventional CTL. The cytokines IL-2 and IL-4 showed no activity in this system (data not shown). Co-administration of IFN-
with exogenous HBsAg had a low effect in two out of three experiments, but was much less efficient than IL-12 (data not shown). The route of application was critical. The i.m. and s.c. injections of HBsAg mixed with ISS-containing ODN or IL-12-primed CTL, but i.p. injections of the same antigen formulations did not. Similarity, only i.m. and s.c. injections of mice with HBsAg-encoding plasmid DNA elicits CTL responses in vivo. Hence, a critical level of in situ cytokine activity at the site of injection for a critical time period may be required for precursor T cell priming; this level may be maintained in s.c. and muscle tissues, but not in the peritoneal cavity and in blood.
The study contributes to attempts to design vaccination strategies that efficiently prime CTL to exogenous antigen in `difficult' situations, such as, for example, `low responder status' or deviation of cellular immune responses in persistent viral infections. The efficacy of ODN as adjuvants for priming or boosting cellular immune responses to viral antigens in monkeys and humans is under investigation. The aim is to find a rational approach to the design of a therapeutic vaccine against hepatitis B virus based on the immunological characterization of the processing and presentation characteristics of hepatitis B virus antigens.
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Acknowledgments
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The excellent technical assistance of A. Titz and T. Krieg is gratefully appreciated. Drs L. Deml and R. Wagner (Regensburg, Germany) kindly provided some of the ODN. This work was supported by grants from the Deutsche Forschungsgemeinschaft (DFG Schi 505/1-3 and Graduiertenkolleg `Biomolekulare Medizin'), the EC (contract PL 970002) and the Federal Ministry for Research (BMBF/DLR 01 GE9611) to R. S. and J. R.
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Abbreviations
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BFA | brefeldin A |
CTL | cytotoxic T lymphocyte |
ER | endoplasmic reticulum |
HBsAg | small hepatitis B surface antigen |
i.m. | intramuscular |
ISS | immunstimulating sequence |
ODN | oligodeoxynucleotide |
TAP | transporter associated with antigen processing |
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Notes
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Transmitting editor: S. H. Kaufmann
Received 2 December 1998,
accepted 22 March 1999.
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