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*
Epimmune, Inc., San Diego, CA 92121; and
Department of Molecular and Experimental Medicine, The Scripps Research Institute, La Jolla, CA 92121
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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350 million people
worldwide (1, 2). Adult onset infection usually results in
self-limited acute hepatitis followed by viral clearance, although up
to 5% of infected adults become chronically infected (2).
In contrast, neonatal infection is seldom cleared and a majority of
infected children develop persistent infection (2).
Patients chronically infected with HBV are predisposed to developing
cirrhosis of the liver and hepatocellular carcinoma
(2).
CD8+ T lymphocytes are an important component of
host defense mechanisms responsible for HBV clearance in patients with
acute hepatitis (3, 4). In particular, multispecific
responses appear to be associated with spontaneous resolution of acute
infection. In this respect, elicitation of responses directed against
multiple epitopes may be of therapeutic interest (2). It
has been proposed that elimination of virus is due to CTL-mediated
lysis of infected hepatocytes and/or antiviral effects of CTL-derived
cytokines, such as IFN-
and TNF-
(5, 6). Recent data
demonstrate that, in acutely infected chimpanzees, HBV DNA is
eliminated from the liver before the peak of T cell infiltration and
the associated liver disease (7). These effects coincide
with the appearance of IFN- in the liver, suggesting that
IFN--producing non-T cells, presumably NK and NKT cells, as well as
T cells, play a key role in the control of HBV infection. Thus,
noncytopathic mechanisms that eliminate replicative HBV DNA
intermediates from the cytoplasm and covalently closed circular DNA
from the nucleus (7) appear to be crucial for clearance of
HBV from the hepatocyte. Therapeutic induction of this kind of
noncytopathic, antiviral activity in the liver of chronically infected
patients would, therefore, lead to viral clearance, thereby preventing
cirrhosis and hepatocellular carcinoma.
Unfortunately, chronic HBV infection, like many other chronic viral diseases and cancers (2), is associated with T cell hyporesponsiveness or tolerance. The exact molecular mechanisms associated with this phenomenon are not entirely clear but negative selection, peripheral anergy, and imbalances in lymphokine production all appear to contribute to maintaining the hyporesponsive state of the host chronically exposed to viral or cancer Ags (2).
Transgenic mice of the 1.3.32 lineage contain the complete HBV genome, express all HBV gene products, and replicate HBV in their hepatocytes similar to chronically infected patients, but they do not develop chronic hepatitis because they are immunologically tolerant to the viral Ags (5, 8). The availability of these mice provides a model system to evaluate immunotherapeutic strategies to break tolerance and terminate persistent HBV infection.
Thus far, several approaches to overcome CTL tolerance in
HBV-transgenic mice have been investigated. In previous studies, five
different lineages of HBV-transgenic mice, including lineage 1.3.32,
were immunized with either plasmid DNA encoding whole HBV Ags or
activated dendritic cells. DNA immunization induced Ab responses in
some of the lineages, but no CTL responses (8). Dendritic
cell immunization generated CTLs but they had no effect on viral
replication and they did not cause hepatitis (8). These
results are in contrast with a previous study which had indicated that
tolerance could be broken by DNA immunization in a single lineage of
transgenic mice that contain a subgenomic fragment of HBV and express
the major envelope (Env) protein within hepatocytes (9).
In these animals, DNA immunization was associated with a reduction of
HBV RNA expression in hepatocytes (9) and this effect was
shown to be dependent on IFN-. The immunological, virological, and
clinical relevance of those results is uncertain; however, the
transgene in the particular lineage of mice used in those experiments
is susceptible to genomic imprinting and silencing of transgene
expression by methylation (10). Therefore, these events
may have been triggered by immunization but do not occur during
infection.
In the present study, we used the complete HBV genome transgenic mouse model to evaluate the relative immunogenicity of a pool of HBV-specific lipopeptides with optimized helper T lymphocyte (HTL) function for their capacity to break T cell tolerance and to down-regulate HBV gene expression and replication.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Peptides were synthesized according to standard F-moc solid-phase synthesis methods (11). In HTL-CTL peptide constructs, the HTL epitope was placed at the amino terminus of the CTL epitope. In the case of the lipopeptide synthesis, each amino acid coupling was followed by a capping cycle with 10% acetic anhydride: dimethylformamide (v/v) to facilitate subsequent purification. Lipopeptides were prepared by coupling preformed symmetrical anhydride of palmitic acid to the amino terminus of the resin-bound KSS-elongated peptide. The peptides and the protecting groups on the amino acids were cleaved using trifluoroacetic acid, ethanedithiol, water (9.5:2.5:2.5, v/v/v), or trifluoroacetic acid, thioanisole, phenol, ethanedithiol, water (10 ml:0.5 ml:0.75 g:0.25 ml:0.5 ml), depending upon the specific protecting group present on the various amino acids.
Unlipidated peptides were purified using conventional HPLC methods.
Lipopeptides were purified by repetitive washes with 50% acetic
acid:water (v/v), followed by two washes with H2O
and lyophilization. Lipopeptides were characterized by reverse
phase-HPLC and mass spectrometry. Reverse phase-HPLC was performed
using Waters analytical HPLC (polymer reverse phase 4.6 x 150 mm,
5 µM, 300 Å, column; Polymer Laboratories, Shropshire, U.K.) at a
column temperature of 80°C and detection at 214 nm. The mobile phase
buffers were A ( water with 0.1% trifluoroacetic acid) and B
(acetonitrile with 0.1% trifluoroacetic acid), and the elution was
effected with a gradient of 20–100% B over 30 min at a flow rate of 1
ml min-1. Purity and identity of all compounds
were checked by analytical HPLC and mass spectrometry. Purity was
50% for lipidated peptides and 90% for unlipidated
peptides.
Cell lines, Abs, and MHC purification
The mouse lymphoma EL-4 was used as the source of Kb and Db class I molecules. EL-4 cells were maintained as described previously (12, 13). The mAbs used for purification were Y3 (anti-Kb) and 28-14-8S (anti-Db, Ld, and Dq; American Type Culture Collection, Manassas, VA). The mouse mastocytoma cell line P815 was used as the source of Dd, Kd, and Ld class I molecules. P815 cells were maintained as described previously (12, 13). The mAbs used for purification were 34-5-8S (anti-H-2Dd), SF1.1.1.1 (anti-H-2Kd), and 28-14-8S (anti-Dd, Ld, and Dq). Cell lysates for MHC purification were prepared as previously described (12, 13). Briefly, cells were lysed at a concentration of 108 cells/ml in 50 mM Tris-HCl (pH 8.5) containing 1% Nonidet P-40 (Fluka Biochemika, Buchs, Switzerland), 150 mM NaCl, 5 mM EDTA, and 2 mM PMSF. The lysates were then passed through 0.45-µm filters and cleared of nuclei and debris by centrifugation at 10,000 x g for 20 min. MHC molecules were then purified by repeated sequential passage over either 34-5-8S, SF1.1.1.1, or 28-14-8S columns. Protein purity and effectiveness of depletion steps were monitored by SDS-PAGE.
In vitro MHC peptide-binding assays
Quantitative assays for the binding of peptides to
detergent-solubilized H-2 MHC class I molecules, on the basis of the
inhibition of binding of a radiolabeled standard probe peptide, were
performed as previously described for HLA class I molecules
(12, 13, 14, 15, 16). Briefly, 1–10 nM radiolabeled probe peptide,
iodinated by the chloramine-T method (12), was coincubated
for 2 days at room temperature with various amounts of MHC in the
presence of 1 µM human 
2-microglobulin
(Scripps Laboratories, San Diego, CA) and a mixture of protease
inhibitors. At the end of the incubation period, the percentage of
MHC-bound radioactivity was determined by size exclusion gel filtration
chromatography on a TSK 2000 column (TosoHaas, Montgomeryville,
PA).
The concentration of peptide yielding 50% inhibition of the binding of
the radiolabeled probe (IC50) in competitive
inhibition assays was calculated. Peptides were usually tested at one
or two high doses, and the IC50 values of
peptides yielding positive inhibition were determined in subsequent
experiments, in which two to six additional dilutions were tested, as
necessary. MHC concentrations yielding 15% binding of the
radiolabeled probe peptide were used for all competition assays. Each
competitor peptide was tested in two to four independent experiments.
The radiolabeled probes used, and their average
IC50 values in the respective assays, were as
follows: HIV-IIIB Env gpG4 > Y analogue (sequence RGPYRAFVTI),
3.7 nM for Dd; 1079.03 (sequence KFNPMKTYI), 1.1
nM for Kd; and B35CON2 (sequence FPFKYAAAF), 30
nM for Ld; adenovirus E1A
P7 Y analogue (sequence SGPSNTYPEI) 4.4 nM for
Db; and VSV NP52–59 (sequence RGYVFQGL) 3.1 nM
for Kb.
Mice
Eight- to 12-wk-old mice were used in all experiments.
(C57BL/6 x B10D2)F1
(B6D2F1) and (C57BL/6 x
BALB/c)F1 (CB6F1) were
purchased from The Jackson Laboratory (Bar Harbor, ME). HBV-transgenic
mice from lineage 1.3.32 (official designation Tg(HBV 1.3
genome)
32) (inbred C57BL/6) were bred at The
Scripps Research Institute. Transgenic mice from lineage 1.3.32 have
been previously described (17). The hepatocytes from these
animals express all of the HBV gene products and replicate HBV at high
levels in the liver without any evidence of cytopathology. Lineage
1.3.32 mice C57BL/6 (H-2b) were backcrossed one
generation against BALB/c (H-2d) mice to produce
H-2bxd F1
(CB6F1) hybrids for this study.
Peptide formulations and immunizations
Peptides and lipopeptides were resuspended at 20 mg ml-1 and 10 mg ml-1, respectively, in DMSO/0.1% trifluoroacetic acid and stored as stock solutions at -20°C. Two different formulations were used for immunizations. For experiments involving IFA, each peptide (or peptide mixture) stock solution was diluted after vortexing for 30 s with appropriate amounts of DMSO and PBS at room temperature to obtain the desired peptide concentrations in a final buffer composition of 90% PBS, 10% DMSO/0.1% trifluoroacetic acid. These samples were emulsified with an equal volume of IFA (Difco, Detroit, MI) for 20 min in a 5100 Spexmixer/mill (Spex Industries, Metuchen, NJ). In experiments involving lipopeptides, stock solutions were heated for 10 min at 45°C (to ensure solubilization of the lipopeptides in DMSO) and then vortexed for 30 s before dilution with PBS at room temperature. Eight- to 12-wk-old mice were immunized s.c. in the base of the tail with the formulations described above.
Assay of CTL activity
Eleven to 14 days after immunization, splenocytes were harvested from individual mice and 3 x 107 cells were stimulated with specific HBV peptides at 10.0 µg ml-1 in the presence of 107 irradiated (4500 rad) syngeneic LPS blasts. To obtain LPS blast cells, syngeneic splenocytes were resuspended at a concentration of 1–1.5 x 106 cells ml-1 in complete media in the presence of 25 µg ml-1 of LPS (Sigma, St. Louis, MO) and 7 µg ml-1 of dextran sulfate (Pharmacia Biotech, Uppsala, Sweden) and kept in culture for 72 h at 37°C. After 5–6 days, splenocytes from each flask were collected and assayed for cytolytic activity using a standard 4-h 51Cr release assay.
The following cell lines were used as target cells in the cytotoxicity assays: EL4 (a murine lymphoma), P815 (a mouse mastocytoma), and P815-S (P815 cells that stably express all the HBV Env protein (17). All of the cell lines were grown in RPMI 1640 (Life Technologies, Grand Island, NY) containing 10% FCS (Gemini Bioproduct, Calabasas, CA), 4 nM L-glutamine (Irvine Scientific, Santa Ana, CA), 10 µg ml-1 of gentamicin (Irvine Scientific), and 5 x 10-5 nM 2-ME (Sigma). Target cells (3 x 106) were labeled with 300 µCi of 51Cr sodium chromate (NEN Research Products, Boston, MA) for 60 min at 37°C, washed three times, and resuspended in RPMI 1640/10% FCS at a concentration of at 105 cells ml-1 in the absence or presence of 2 µg ml-1 of the appropriate peptides. To assay for CTL activity, 100 µl of target cells was incubated with 100 µl of different numbers of effectors cells in U-bottom 96-well plates. Supernatants (100 µl) were removed after 4 h at 37°C and the percent lysis was determined by the following formula: percent release = 100 x [(experimental release - spontaneous release) ÷ (maximum release - spontaneous release)].
To allow averaging of different experiments, specific CTL activity was also expressed in LU (LU30), in which 1 LU (LU30) corresponds to the number of effector cells required to induce 30% lysis of 104 51Cr-labeled target cells during the 4-h assay (18). Thus, in the conditions used in the assay 1 LU30 represents 30% lysis at the 100:1 E:T ratio. Ten LU30 represents 30% lysis at the 10:1 ratio, 100 LU30 represents 30% lysis at the 1:1 ratio, and so on.
CTL activity was also measured by IFN- production. A total of 1
x 105 CTLs were cultured with
105 target cells (HBV-Env-transfected or
-untransfected P815 cells), with or without relevant peptide, in
96-well Immulon-2 plates (Dynex Technologies, Chantilly, VA) that were
precoated with a capture anti-mouse IFN- Ab (clone R4-6A2;
PharMingen, San Diego, CA). After a 20-h incubation at 37°C, wells
were washed with PBS/0.5%Tween 20 and incubated sequentially with a
biotinylated secondary anti-mouse IFN- Ab (XMG1.2; PharMingen),
followed by streptavidin-HRP (Zymed, San Francisco, CA), and finally
substrate (3,3',5,5'-tetramethylbenzidine +
H2O2; PharMingen).
Absorbance of each well was measured spectrophotometrically at 450 nm
using an automated ELISA reader and the picograms per milliliter of
IFN- captured in each well was determined by extrapolating from an
IFN- standard curve.
Injection of CTL lines
Short-term CTL lines generated, as described above, were expanded in vitro in 6-well plates, each well containing 4 x 10 6 CTLs and 1.4 x 107 LPS/dextran sulfate-activated splenocytes previously pulsed with 100 µg/ml peptide for 1 h at 37°C and irradiated with 3000 rad. Eighteen hours later, ConA-activated splenocyte supernatant (10% final concentration, v/v) was added to cultures that were fed and expanded as necessary. Five days after the last stimulation, cells were then washed, counted, suspended in PBS containing 2% FCS, and injected i.v. into recipient 1.3.32 HBV-transgenic mice. One day after injection, mice were sacrificed and their liver tissue biopsies were harvested for histological and immunohistochemical analyses or snap frozen in liquid nitrogen and stored at -80°C for subsequent molecular analyses (see below).
Tissue DNA and RNA analyses
Frozen liver tissue was mechanically pulverized under liquid nitrogen and total genomic DNA and RNA were isolated for Southern and Northern blot analyses exactly as previously described (17). Nylon membranes were analyzed for HBV DNA, HBV RNA, GAPDH and 2',5'-oligoadenylate synthetase as described elsewhere (19). Quantitation of cytokine, T lymphocyte, and macrophage marker mRNAs was performed by RNase protection assay exactly as previously described (6, 19).
Biochemical and histological analyses
The extent of hepatocellular injury was monitored by measuring serum alanine aminotransferase (sALT) activity at multiple time points after treatment with saline or the CTL lines were measured in a Paramax chemical analyzer (Baxter Diagnostics., McGaw Park, IL), exactly as previously described (6). For histological analysis, liver tissue samples were fixed in 10% zinc-buffered Formalin (Anatech, Battle Creek, MI), embedded in paraffin, sectioned (3 µm), and stained with hematoxylin and eosin exactly as described elsewhere (17). To detect 5-bromo-2'-deoxyuridine-positive cells in tissue, Formalin-fixed paraffin-embedded tissue sections were stained with anti-5-bromo-2'-deoxyuridine Abs, as previously described (20).
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Previous studies have characterized an HBV-Env-derived,
H-2Ld-restricted dominant CTL epitope (17, 21, 22). To identify additional epitopes and thus being able to
generate responses of maximum breadth, the HBV-Env protein sequence was
scanned for the presence of H-2d and
H-2b motif peptides (Table I
). The corresponding peptides were
tested for binding to purified MHC molecules in vitro. Consistent with
previous studies (23, 24), a peptide was classified as a
good binder when associated with an IC50 of 500
nM or less.
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Next, the immunogenicity of H-2b and
H-2d class I binding peptides was evaluated in
B6D2F1 mice. Analogous to previous studies
(26, 27), peptides were emulsified in IFA in the presence
of an excess (140 µg) of the I-Ab/d-restricted
helper epitope OVA.323–336. Herein, to allow averaging and combination
in a single consistent format in several different experiments,
specific CTL activity is expressed in LU (LU30),
in which 1 LU (LU30) corresponds to the number of
effector cells required to induce 30% lysis of 104
51Cr-labeled target cells during the 4-h assay
(18). Thus, in the conditions used in the assay 1
LU30 represents 30% lysis at the 100:1 E:T
ratio. Ten LU30 represent 30% lysis at the 10:1
ratio, 100 LU30 represent 30% lysis at the 1:1
ratio, and so on. Also, as defined previously (23, 24, 26), CTL responses of 2
LU30/106 cells were
considered positive.
The results of testing the three newly identified
H-2d-binding peptides and the known dominant
Env.28 epitope for immunogenicity are shown in Table II. As expected, vigorous responses were
observed in the case of Env.28 (17/18 positive mice, 11.2 LU average
response). Similar responses were observed in the case of Env.364,
(12/14 mice yielded recall CTL responses, with an average of 13.6 LU).
Lower but still appreciable responses were noted in the case of Env.362
(recall CTL responses in 7/12 wild-type mice, with an average of 5.7
LU). Finally, in the case of Env.281, recall CTL responses were
observed in only 2 of 10 mice tested (6.2 LU average magnitude).
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Recognition of naturally processed HBV-ENV Ag by CTLs specific for the Env.362 and Env.364 epitopes
To determine whether the Env.362 and the Env.364 epitopes were generated in the course of natural processing of the HBV-Env Ag, short-term CTL lines were derived by peptide immunization of nontransgenic (B6D2F1) mice and tested for their capacity to recognize P815-S cells that express all HBV-Env proteins (17).
As shown in Fig. 1, CTLs specific for
control epitopes were completely devoid of cytotoxicity for the
HBV-Env-transfected P815 target cells in multiple independent
experiments (Fig. 1, a and b). As expected, high
levels of specific killing were detected in the case of Env.28 (Fig. 1c), with 51Cr release approaching
100% at an E:T of 10:1. Recognition of P815-S target cells was also
detected in the case of both Env.364- and Env.362-specific CTL lines
(Fig. 1, d and e) with 51Cr
release approaching 50% at an E:T of 10:1 for both cultures.
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Immunogenicity of HBV-Env CTL epitopes in 1.3.32 HBV-transgenic mice
Next, we compared the immunogenicity of the two newly identified
H-2d-restricted epitopes (Env.362 and Env.364)
and the known dominant Env.28 epitope in H-2bxd
CB6F1-nontransgenic mice and
CB6F1 hybrids of 1.3.32 lineage HBV-transgenic
mice. Specifically, we used a pool of HBV-Env-specific lipopeptides
consisting of three constructs, each composed of a two palmitic acid
lipid tail, the optimized PADRE HTL epitope, and an HBV-specific CTL
epitope (PAM2-PADRE-Env.28,
PAM2-PADRE-Env.362, and
PAM2-PADRE-Env.364) to prime responses for the
three HBV-Env epitopes. Eleven to 14 days after injection of a total of
30 µg of the pool of lipopeptides, splenocytes were cultured for 6
days with the appropriate CTL epitopes and then tested for activity in
the chromium release assay. As expected, all of the nontransgenic
H-2bxd F1 mice tested
responded with CTL responses in the 38–88 LU range (Table III). Interestingly, recall CTL responses
were observed for a majority of the transgenic animals tested, with
magnitudes in the 10–86 LU range for the three different HBV-specific
CTL epitopes (Table III), comparable to those induced in nontransgenic
mice. These results demonstrate that T cell tolerance can be overcome
by lipopeptide immunization at the level of induction of recall CTL
activity. However, unlike the CB6F1-derived CTLs,
the CTLs derived from the HBV-transgenic mice did not recognize
endogenously synthesized Ag since they failed to kill
HBV-Env-transfected target cells (Table IV). In the HBV-transgenic mouse system,
in addition to monitoring for the induction of recall memory CTL
responses after immunization, the capacity of the induced CTLs to lyse
HBV-transgenic hepatocytes in the immunized mice in vivo can be
monitored biochemically by measuring sALT activity, a marker of
hepatocellular lysis. Despite detectable recall memory CTLs, indicative
of in vivo CTL induction, sALT activity remained at background levels
in all of the mice (50–60 U/L; data not shown). Furthermore, no
significant inflammatory foci were detected by immunohistochemistry,
and no down-regulation of viral DNA was detected in the liver of these
animals (data not shown). This was in keeping with their failure to
kill endogenously synthesized Ags in vitro (see above).
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In the next series of experiments, we compared the avidity of the
CTLs generated in nontransgenic CB6F1 mice and in
1.3.32 lineage CB6F1 mice using a dose titration
of the various epitopes. One of two independent experiments yielding
similar results is shown in Fig. 2. In
the case of the Env.28 epitope, the concentration of peptide necessary
to achieve 30% lysis by CTLs from 1.3.32
CB6F1-transgenic mice and nontransgenic mice was
5.9 and 0.13 pg/ml, respectively. In the case of the Env.364 epitope,
the concentrations necessary for 30% lysis were 276 and 0.256 ng/ml
for 1.3.32 and CB6F1-derived CTL lines,
respectively. Thus, in both cases, CTL lines derived from transgenic
mice displayed 100- to 1000-fold lower avidity for their targets. In
contrast, little or no difference in avidity was detected in the case
of the Env.362 epitope.
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Adoptive CTL transfer experiments were performed next to further characterize the quality of the recall CTL activity elicited by lipopeptide immunization in the transgenic and nontransgenic mice. Groups of three CB6F1 mice were immunized with a pool of the three lipidated PADRE-CTL constructs encompassing the Env.28, 362, and 364 CTL epitopes. After 1 mo, all groups were boosted with the same immunogens. Eleven to 14 days later, splenocytes were harvested and CTL-specific lines were expanded in vitro with the appropriate CTL epitopes.
Five days after the second in vitro stimulation, CTL cultures from each
group were tested for activity in a 51Cr release
assay. Data from one representative experiment is shown in Table V. Cultures derived from normal
CB6F1 mice yielded strong CTL activity in the
60–220 LU range, and cultures derived from 1.3.32-transgenic mice
yielded slightly less CTL activity (in the 40–160 LU range) against
P815 target cells pulsed with the various HBV-Env-derived epitopes.
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Transfer of nontransgenic HBV-specific CTL lines into transgenic mice
was associated with increased levels of sALT activity (Table VI), and the presence of small, scattered
necroinflammatory foci in the liver parenchyma (data not shown), both
at day 1 and day 3 after transfer (data not shown). As shown in Fig. 3, 1
day after injection, HBV DNA
replication was strongly inhibited in the liver of these animals when
compared with saline-injected controls. This was associated with the
intrahepatic induction of IFN-, TNF-, CD8, CD4, CD3, and F480
mRNA (Fig. 3). These results are not surprising since using the same
mouse model we have previously shown that the antiviral potential of
the CTLs is primarily mediated by noncytolytic mechanisms that involve
the intrahepatic production of IFN- (6, 7).
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). This is consistent with their lower
avidity (except Env 362) and poorer recognition of endogenously
synthesized Ag than the nontransgenic CB6F1 CTLs.
Nonetheless, HBV replication was strongly inhibited in the animals that
received transgenic CTLs, almost to the same degree observed in the
mice that received nontransgenic CTLs. This was associated with a
moderate induction of IFN-, indicating that Ag recognition has
occurred in vivo (Fig. 3). Finally, no liver disease was detected in
the control group that received LCMV-specific CTLs. As expected, HBV
replication was not affected and IFN- production was not induced in
the liver of these mice (Fig. 3).
These in vivo observations were paralleled by in vitro experiments
which demonstrated that CTL derived from HBV-transgenic mice did not
kill HBV-Env-transfected target cells, but were capable of secreting
IFN-, albeit in small amounts, when confronted with
HBV-Env-transfected targets. Control CTL lines derived from normal mice
were capable, by contrast, of both killing and IFN- secretion in
response to HBV-Env-transfected p815 target cells (Fig. 4).
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Relative to Env.28, recognition of endogenously synthesized Ag was less efficient in the case of the subdominant epitopes (Env.364 and Env.362), suggesting that these epitopes are produced less efficiently by natural HBV-Env processing. This, along with their lower binding affinity relative to Env.28, might explain their subdominant status (30). Analysis of lineage 1.3.32 HBV-transgenic mice, in terms of CTL responsiveness directed against the new H-2d-restricted epitopes and the previously defined dominant Env.28 epitope, demonstrated T cell hyporesponsiveness in 1.3.32 mice for the Env.28 and Env.364, but less so in the case of the Env.362 epitope. It is possible that this subdominant epitope might be a less effective tolerogen because it is naturally expressed in lower amounts in hepatocytes of 1.3.32 HBV-transgenic mice. These results suggest that targeting subdominant epitopes may be an effective way to overcome CTL tolerance.
Studies in nontransgenic H-2d mice demonstrated
that a mixture of HTL-CTL epitope lipoconstructs was capable of
simultaneously inducing responses for each of these CTL epitopes. In
1.3.32 HBV-transgenic mice, this immunization strategy was also
effective in inducing recall CTL activity. These results are
significant because previous attempts aimed at overcoming CTL tolerance
in this lineage of transgenic mice had not met with success
(8). A crucial element of this strategy appears to be the
use of preprocessed epitopes, as in this system whole HBV-Env DNA
immunization does not break CTL tolerance (8), but
epitope-based minigenes do (A. D. Sette, C. Oseroff, and J.
Alexander, unpublished observations). However, the induction of
HBV-specific CTLs was not associated with either down-regulation of
expression of HBV Ags in the liver or with increased sALT activity,
indicating lack of hepatocyte damage. Thus, the induced CTLs either did
not reach the liver in sufficient numbers to exert detectable effector
functions, or the CTLs were functionally defective in the transgenic
mice, or the target Ags were not efficiently processed. The last
explanation is unlikely since all three epitopes are presented by
Env-transfected target cells (Fig. 1) and because Env.28 CTLs are known
to recognize their target epitopes very efficiently in vivo (6, 20, 21).
Direct visualization and/or detection of CTL activity in freshly harvested splenocytes by means of tetrameric staining was not attempted in the current set of experiments. Future studies might investigate the relationship between these CTLs and the functionally impaired CTLs observed in the course of chronic infection with various viruses including LCMV, hepatitis C virus, and HBV (2, 31, 32). It is important to point out, however, that in these cases, functionally impaired CTLs were associated with chronic exposure to viral Ags, whereas in our cases, these CTLs are induced de novo after immunization of animals already chronically expressing large amounts of HBV Ags.
Further adoptive transfer studies characterized the CTLs induced
in 1.3.32-transgenic mice in more detail. It was found that these CTLs
were effective in inhibiting viral replication. However, unlike the
control CTLs induced in nontransgenic mice, they did not cause
hepatitis. This antiviral effect is likely mediated by noncytolytic
mechanisms that involve intrahepatic production of IFN- (6, 33). Peptide titration experiments demonstrated that CTLs
derived from 1.3.32 HBV-transgenic mice were characterized by low
avidity, thus suggesting a possible mechanism to account for these
observations if the lower avidity recognition can trigger the release
of antiviral cytokines without significantly activating the cytolytic
function of the CTLs. The CTLs induced in nontransgenic mice were more
effective than those induced in nontransgenic mice, possibly a
reflection of both cytolytic and noncytolytic mechanisms as well as
potentially higher levels of production of noncytolytic mediators.
Nevertheless, it is obvious that selective induction of antiviral
effects in chronically infected individuals, in the absence of tissue
damage, would be of significant therapeutic interest.
These results are also consistent with previous observations by Sherman and coworkers (34) and von Herrath et al. (35) and demonstrated that low-avidity CTLs can escape tolerance inactivation and might be stimulated by immunization with vaccine constructs using preprocessed optimal epitopes. A crucial point, which remains to be addressed, is why, in the current study, induction of this CTL specificity did not affect viral replication in the immunized animals, but only in the adoptive transfer experiments. Our results are compatible with two nonmutually exclusive explanations. First, it is possible that in vitro expansion might in some way reverse a functional inactivation state. Alternatively, and in our view more likely, it is possible that the magnitude, in terms of the number and expansion of CTL specificities induced in the transgenic mice, is suboptimal and in vitro expansion is necessary to achieve sufficient numbers of CTL effectors.
In this light, we are currently investigating different immunization protocols such as prime/boost strategies (36) and the combined use of multiple epitopes from various HBV Ags as a way to optimize the magnitude and breadth of CTL responses. Other studies have also emphasized the importance of optimized helper activity in overcoming CTL unresponsiveness (37). Taken together, we believe that these results are very encouraging in terms of potential immunotherapy of HBV infection in humans in which, as a result of the recent availability of antiviral compounds such as lamivudine, problems related to viral expression and its suppressive effects might be less severe.
In conclusion, we have shown, for the first time, that immunization of HBV 1.3.32-transgenic mice can result in induction of CTL activity capable, in passive transfer experiments, of down-regulating expression of viral Ags in the absence of hepatocyte damage. These observations have obvious implications for development of therapeutic strategies to treat chronic HBV infection.
Finally, we would also like to point out that these results presented in this manuscript may also have implications for autoimmune disease and cancer treatments. In several instances, it has been reported that T cells involved in autoimmune phenomena (38, 39) or directed against tumor-associated Ags (34) are associated with altered avidity or patterns of cytokine release. Thus, understanding the structural basis of the noncytolytic mechanisms described herein may reveal interesting avenues for treatment of several complex diseases.
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Acknowledgments |
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Footnotes |
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2 Address correspondence and reprint requests to Dr. Alessandro Sette, Epimmune, Inc, 5820 Nancy Ridge Drive, Suite 100, San Diego, CA 92121.
3 Abbreviations used in this paper: HBV, hepatitis B virus; Env, envelope; HTL, helper T lymphocyte; sALT, serum alanine aminotransferase; LCMV, lymphocytic choriomeningitis virus.
Received for publication June 27, 2000. Accepted for publication October 24, 2000.
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) or absence
(
) of the specific peptide or PreS1-transfected P815 target cells
(
).
