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J Virol, July 1998, p. 6083-6091, Vol. 72, No. 7
Institute of Virology1
and
Institute of Immunology,2 University
of Essen, D-45122 Essen, Germany
Received 29 December 1997/Accepted 1 April 1998
The infection of woodchucks with woodchuck hepatitis virus (WHV)
provides an experimental model to study early immune responses during
hepadnavirus infection that cannot be tested in patients. The T-cell
response of experimentally WHV-infected woodchucks to WHsAg, rWHcAg,
and WHcAg peptides was monitored by observing 5-bromo-2'-deoxyuridine
and [2-3H]adenine incorporation. The first T-cell
responses were directed against WHsAg 3 weeks after infection; these
were followed by responses to rWHcAg including the immunodominant
T-cell epitope of WHcAg (amino acids 97 to 110). Maximal proliferative
responses were detected when the animals seroconvered to anti-WHs and
anti-WHc (week 6). A decrease in the T-cell response to viral antigens coincided with clearance of viral DNA. Polyclonal rWHcAg-specific T-cell lines were established 6, 12, 18, and 24 weeks postinfection, and their responses to WHcAg peptides were assessed. Five to seven peptides including the immunodominant epitope were recognized throughout the observation period (6 months). At 12 months after infection, T-cell responses to antigens and peptides were not detected.
Reactivation of T-cell responses to viral antigens and peptides
occurred within 7 days after challenge of animals with WHV. These
results demonstrate that a fast and vigorous T-cell response to WHsAg,
rWHcAg, and amino acids 97 to 110 of the WHcAg occurs within 3 weeks
after WHV infection. The peak of this response was associated with
viral clearance and may be crucial for recovery from infection. One
year after infection, no proliferation of T cells in response to
antigens was observed; however, the WHV-specific T-cell response was
reactivated after challenge of woodchucks with WHV and may be
responsible for protection against WHV reinfection.
Infection with hepatitis B virus
(HBV) often results in an acute bout of hepatitis followed by clinical
recovery, but progress to chronic infection and disease such as liver
cirrhosis and hepatocellular carcinoma is sometimes observed. Several
studies indicate that cellular immune responses to HBV proteins have a
major influence on the clinical course of HBV infection: vigorous and
multispecific T helper (Th)-cell and cytotoxic T-lymphocyte responses
to HBV surface protein (HBsAg), core protein (HBcAg), polymerase
protein, and X protein (HBxAg) have been associated with recovery from acute HBV infection and viral clearance (4, 11, 12, 17, 18,
21, 26, 29; for reviews, see references 6
and 10). In contrast, low or undetectable T-cell
responses to these proteins were associated with viral persistence and
chronic hepatitis (3, 27; reviewed in references
6 and 13). However, the exact immunologic mechanisms which contribute to viral elimination or persistence during the natural course of hepadnavirus infection are
unknown. Because there are no clinical symptoms immediately after HBV
transmission, T-cell-mediated immune responses during the incubation
period and the early phase of hepatitis in patients are difficult to
analyze. It is also unknown to what extent a long-lasting T-cell
response may support protection from viral reinfection after resolution
of an acute, self-limited HBV infection (30, 34).
Woodchucks (Marmota monax) infected with woodchuck hepatitis
virus (WHV) represent the animal model closest to humans for studying
the cell-mediated immune response during hepadnavirus infections. WHV
and HBV exhibit a high degree of homology in their nucleotide
sequences, genomic organizations, and replication and expression
mechanisms (5, 8, 14, 19, 35, 39). Furthermore, the humoral
immune responses during the course of acute or chronic infections of
HBV-infected humans and WHV-infected woodchucks are similar
(31-33, 41).
There are similarities in the cellular immune responses of woodchucks
to WHV and the responses of humans infected with HBV. In WHV-infected
adult woodchucks, strong T-cell responses to surface protein (WHsAg)
and recombinant core protein (rWHcAg) are found (7, 23, 25).
The T-cell responses in these woodchucks to WHcAg are predominantly to
specific peptides of the antigen, whereas in chronically WHV-infected
woodchucks an undetectable or weak T-cell response to these proteins
and peptides is observed (23-25). The importance of T-cell
responses for the elimination of WHV has been demonstrated by
protective immunization with a single WHcAg peptide (amino acids 97 to
110) which contains a major T-cell epitope (23). It was
demonstrated in vitro that proliferating cells from these animals were
T cells, as shown by staining with a monoclonal antibody against CD3 The experimental WHV infection of woodchucks is an important model with
which to examine the role of cellular immune responses during the early
phase of acute hepadnavirus infection. During this crucial phase, the
course of infection resulting in viral elimination or persistence may
be determined. This study investigated the kinetics of T-cell responses
to WHsAg, rWHcAg, and WHcAg peptides during the early acute phase of
self-limited WHV infection. rWHcAg-specific polyclonal T-cell lines
were established at different times after infection, and their
responses to WHcAg-related peptides were measured. Recognition of
rWHcAg and several WHcAg-related peptides was shown to occur over a
period of 6 months after inoculation, as was recognition of the
immunodominant WHcAg epitope (amino acids 97 to 110) described
previously (23). The T-cell responses after convalescence (1 year after infection) and upon challenge with WHV were also examined.
No T-cell proliferation in response to WHV antigens could be detected 1 year after infection. Reactivation of the T-cell responses to viral
antigens and to the previously recognized WHcAg epitopes was
demonstrated after challenge with WHV.
Animals.
Four WHV-negative woodchucks, trapped in the state
of New York, were purchased from North Eastern Wildlife (Ithaca, N.Y.) and maintained in our facility. Before experimentation, all the animals
were clinically examined and tested for parasitic infections including
intestinal worms. Serologic testing was also performed, and all adult
animals, of either sex, were found to be negative for WHV DNA, WHsAg,
and antibodies against WHsAg (anti-WHs) and WHcAg (anti-WHc).
Serologic testing, virus detection, and liver function
assay.
Serologic testing for markers of WHV replication during the
course of acute WHV infection and convalescence and after viral challenge was performed weekly. WHsAg, anti-WHs, and anti-WHc were
determined by an enzyme-linked immunosorbent assay (ELISA) as described
previously (36, 37). WHV DNA was detected by PCR with two
WHV core gene-specific oligonucleotide primers (nucleotides 2015 to
2038 and 2570 to 2595) (23) or by nested PCR with four oligonucleotide primers (nucleotides 2015 to 2038, 2630 to 2656, 2129 to 2148, and 2597 to 2618) after extraction of DNA from serum or from
PBMC. Additionally, WHV DNA was detected by a dot blot technique as
described previously (36). Sorbitol dehydrogenase (SDH)
activity, a marker of acute liver damage in humans (2, 43)
and in woodchucks (16), was assessed by a commercial enzyme assay (Sigma, Deisenhofen, Germany).
Monitoring of T-cell proliferation by BrdU and
[2-3H]adenine incorporation.
Blood was drawn weekly
from anesthetized woodchucks via the vena saphena and collected in EDTA
monovettes (Sarstedt, Nuembrecht, Germany). Peripheral blood
mononuclear cells (PBMC) were separated by Ficoll-Paque (Pharmacia,
Freiburg, Germany) density gradient centrifugation and resuspended in
0.9% NaCl. Cell counting was performed with a Thoma hemocytometer or
an electronic cell counter (Sysmex, Hamburg, Germany). Triplicate
samples of 5 × 104 PBMC were cultured in 96-well
microtiter plates (Falcon; Becton Dickinson, Paramus, N.J.) at 37°C
in a humidified atmosphere containing 5% CO2. AIM-V medium
(Gibco BRL, Eggenstein Leopoldshafen, Germany) (200 µl) supplemented
with 4 mM L-glutamine (Sigma), 12.5 mM gentamicin sulfate
(Sigma), and 10% fetal calf serum (Gibco) was added to each well (this
is referred to below as complete medium). Woodchuck PBMC were
stimulated with 2 µg of WHsAg per ml, 1 µg of rWHcAg per ml, or 1 µg of peptide of WHcAg per ml. These concentrations give optimal
proliferation results (23).
0022-538X/98/$04.00+0
Copyright © 1998, American Society for Microbiology. All rights reserved.
T-Cell Response to Woodchuck Hepatitis Virus (WHV)
Antigens during Acute Self-Limited WHV Infection and Convalescence
and after Viral Challenge
ABSTRACT
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References
INTRODUCTION
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References
(23). Although further characterization of T cells in the
woodchuck is not yet possible, the proliferation assays were analogous
to those used in humans and the use of linear peptides suggested that
the T cells were Th cells.
MATERIALS AND METHODS
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References
-D-thiogalactopyranoside (IPTG) and isolated to a purity of about 95% (23). The concentrations
of WHsAg and rWHcAg were determined by a protein assay (Pierce,
Rockford, Ill.), and the particulate nature of the purified proteins
was analyzed by electron microscopy.
Synthetic peptides of WHcAg were purchased from Genosys (Cambridge,
United Kingdom). Two panels of peptides were used (see Table 2)
(23). Panel A contained 16 peptides (overlapping each other
by 6 to 10 amino acids) covering the entire WHcAg, and panel B
contained 6 peptides (overlapping each other by 3 to 11 amino acids)
spanning amino acids 97 to 140 of the WHcAg.
Spontaneous proliferation of PBMC in complete medium served as a
background control. T-cell responses to WHV antigens were assessed
after stimulation for 5 days including a 16- to 20-h pulse with 10 µM
bromodeoxyuridine (BrdU) (BrdU cell proliferation ELISA kit
[colorimetric] [Boehringer, Mannheim, Germany]) or with 1 µCi of
[2-3H]adenine (Amersham, Braunschweig, Germany) as
described previously (20, 23-25).
Results for triplicate cultures (5 × 104 PBMC) are
presented as the mean stimulation index (SI); for the BrdU assay, the
mean total absorption for stimulated PBMC was divided by the mean total absorption for controls (background), and for the
[2-3H]adenine assay the mean total counts per minute
(cpm) for stimulated PBMC was divided by the mean total cpm for
controls (background). An SI of 
2.1 was considered significant for
T-cell proliferation in the BrdU assay (23), and an SI of
3.1 was considered significant in the [2-3H]adenine
assay. In the BrdU assay, the standard deviations were less than 10%
of the mean (range, 5 to 20%), and in the [2-3H]adenine
assay, they were less than 30% of the mean (range, 15 to 50%).
For the establishment of rWHcAg-specific polyclonal T-cell lines,
woodchuck PBMC were obtained at 6-week intervals after experimental WHV
infection (weeks 6, 12, 18, and 24), 1 year after experimental WHV
infection, and 1 week after viral challenge. A total of 6 × 106 PBMC (1.5 × 106/ml) were cultured in
tissue culture flasks (Becton Dickinson) in the presence of 10 µg of
rWHcAg per ml in complete medium. After 1 week, the PBMC were expanded
by adding 20 IU of recombinant human interleukin-2 (IL-2; EuroCetus,
Ratingen, Germany) per ml for an additional week. The PBMC were then
separated on nylon wool columns (Polyscience, Inc., Warrington, Pa.)
into adherent and nonadherent lymphocytes. The nonadherent T cells
(23) were restimulated weekly over a period of 5 to 6 weeks
with 10 µg of rWHcAg per ml plus autologous irradiated (3,000 rads)
PBMC (5 × 105/ml) as antigen-presenting cells (APC)
in complete medium supplemented with 20 IU of recombinant IL-2 per ml.
For further characterization of T-cell lines, T cells were washed four
times with 0.9% NaCl to remove IL-2. Subsequently, triplicate samples
of 5 × 104 T cells/well were added to a microtiter
plate and incubated for 5 days with 105 autologous
irradiated (3,000 rads) PBMC in complete medium in the presence of 1 µg of rWHcAg or WHcAg peptides per ml, and T-cell proliferation was
assessed by [2-3H]adenine incorporation.
Design of experimental WHV infection and viral challenge in woodchucks. Infection of woodchucks was performed by intravenous inoculation with 105 50% woodchuck infectious doses (ID50) of a WHV serum pool of known titer (37). Blood samples for proliferation assays and for detection of viral DNA, WHsAg, anti-WHs, and anti-WHc were drawn weekly over a total of 12 weeks. One year after experimental WHV infection, woodchucks were challenged by inoculation with 200 µl of autologous serum, containing 109 woodchuck ID50. This serum was obtained during the highly viremic phase of acute, self-limited WHV infection (i.e., 4 to 5 weeks after experimental infection).
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RESULTS |
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Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References
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Kinetics of WHV serological markers, WHV DNA, and T-cell responses to viral antigens during acute, self-limited WHV infection. Markers of WHV infection and T-cell proliferation in response to WHV antigens were analyzed in four experimentally WHV-infected adult woodchucks (NW7029, NW7030, NW7031, and NW7032) to characterize the appearance and duration of humoral and cellular immune responses to WHsAg, rWHcAg, and WHcAg-related peptides during the incubation period and the early phase of acute WHV infection. Humoral and cellular immune responses were determined weekly for a total of 12 weeks.
Markers of WHV infection. Woodchucks NW7029, NW7030, and NW7032 showed a similar pattern of viral markers, i.e., appearance and duration of viremia (WHV DNA, WHsAg) and onset of convalescence (anti-WHs and anti-WHc). WHV DNA and WHsAg were detected in the serum from week 2 or 3 to week 7 (Fig. 1). Anti-WHc and anti-WHs were detected in the serum beginning at week 4 or weeks 5 to 6, respectively, and continued throughout the study (more than 52 weeks). A positive signal for anti-WHc at week 1 is a result of infection with an inoculum containing a high titer of anti-WHc. The SDH level in serum increased from normal values (86 to 182 IU) beginning at week 4 and reached its peak value at week 7 (1,185 to 1,325 IU). The SDH activity subsequently decreased and returned to normal values by week 9.
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Cellular immune responses to WHV antigens. (i) T-cell response to WHsAg. By using both a BrdU assay and a [2-3H] adenine incorporation assay, the first T-cell response detected was directed against WHsAg in woodchucks NW7029, NW7030, and NW7032 (Fig. 1). This T-cell response was detected 3 weeks after experimental infection and correlated with the detection of WHV DNA and WHsAg in the serum as well as with an increase in SDH levels. A maximum T-cell response to WHsAg was observed at week 4. The mean SI was 2.3 to 2.6 in the BrdU assay and 4.2 to 4.9 in the [2-3H]adenine assay. By weeks 5 to 6, the T-cell response to WHsAg decreased, and it was below the cutoff value by week 8. The decrease in the T-cell response to WHsAg coincided with the detection of anti- WHs in serum (weeks 5 to 6). Clearance of WHV DNA (week 8) and normalization of SDH levels (week 9) followed shortly. Some differences in the course of the T-cell response to WHsAg were evident between woodchuck NW7031 and the other three animals (Fig. 1). Depending on the assay, a T-cell response to WHsAg was detected 1 to 3 weeks later in the course of infection beginning on weeks 4 to 6. The T-cell response reached its maximum level 7 weeks after infection (mean SI, 2.5 in the BrdU assay and 4.9 in the [2-3H]adenine assay). Similar to the other three animals, the T-cell response decreased afterwards (weeks 8 and 9), coincident with the detection of anti-WHs (week 8). Clearance of WHV DNA and normalization of SDH levels were observed at week 11.
As a control for the observed specific T-cell response to WHsAg that was purified from the sera of chronically WHV-infected woodchucks, the studied animals were tested in parallel for their response to pooled serum from WHV-negative woodchucks. Neither the four woodchucks (NW7029, NW7030, NW7031, and NW7032 [data not shown]) nor several WHV-negative woodchucks (Table 1) showed a T-cell response to these serum in the [2-3H]adenine assay. Stimulation of PBMC from WHV-negative woodchucks with WHsAg led to no proliferative response (Table 1).
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(ii) T-cell response to rWHcAg. The T-cell responses to rWHcAg in woodchucks NW7029, NW7030, and NW7032 were detected 1 week after the T-cell response to WHsAg (Fig. 1). The T-cell response to rWHcAg reached maximal levels at week 6 and decreased thereafter (weeks 7 to 9). The maximum mean SI was 2.9 to 3.3 in the BrdU assay and 6.8 to 7.5 in the [2-3H]adenine assay. The maximum T-cell response appeared 2 weeks later but was stronger than the T-cell response to WHsAg. It appeared that the increase, maximum, and decrease in the T-cell response to WHcAg coincided with the detection of anti-WHc in the serum beginning at week 4. The maximum T-cell response to rWHcAg in woodchuck NW7031 occurred later during the course of acute infection (week 9) than did the response to WHsAg (Fig. 1). However, the maximum T-cell response was similar to that in the other three woodchucks (SI = 3.2 in the BrdU assay and 7.3 in the [2-3H]adenine assay) and a decrease in the T-cell response to rWHcAg was observed at weeks 10 to 12. As with the other animals, anti-WHc was detected in the serum beginning at a point coincident with the maximum rWHcAg-specific T-cell response.
Stimulation of PBMC from WHV-negative woodchucks with rWHcAg led to no significant proliferative response (Table 1).(iii) T-cell response to WHcAg-derived peptides. In addition to the T-cell responses to WHsAg and rWHcAg, the T-cell response to the peptide from amino acids 97 to 110 (peptide 97-110) representing an immunodominant epitope of the WHcAg (23), was monitored. As a control for the peptide-specific proliferative response, based on previous experiments (23), the WHcAg peptide 129-140 was used. Stimulation of PBMC from WHV-negative woodchucks with peptide 97-110 or 129-140 resulted in no T-cell response (Table 1).
T-cell responses to peptide 97-110 were detected in woodchucks NW7029, NW7030, and NW7032 3 to 4 weeks after infection (Fig. 1). A maximum T-cell response was seen at week 6 and decreased thereafter (weeks 7 to 10). The maximum mean SI was 4.2 to 4.9 in the BrdU assay and 8.2 to 9.1 in the [2-3H]adenine assay. A T-cell response to peptide 97-110 in woodchuck NW7031 became detectable at weeks 4 to 6 (Fig. 1). The maximum T-cell response was seen at weeks 9 to 10 (mean SI = 4.2 in the BrdU assay and 8.6 in the [2-3H]adenine assay). As expected, stimulation with peptide 129-140 did not result in T-cell proliferation in the tested animals at any time during acute WHV infection (Fig. 1). Based on these results, we decided to assess T-cell responses in the following experiments, e.g., of polyclonal T-cell lines or after WHV reinoculation, only by [2-3H]adenine incorporation.Recognition of WHcAg epitopes during acute WHV infection and convalescence. Epitopes recognized by Th cells are major histocompatibility complex (MHC) class II restricted (42), and it has previously been shown that T cells of outbred woodchucks recognize different epitopes of WHcAg (23). However, one epitope located between amino acids 97 and 110 appeared promiscuous, since it has so far been recognized by all the woodchucks during acute self-limited WHV infections. PBMC from experimentally WHV-infected animals were stimulated with rWHcAg-related peptides 6 weeks after infection. It was found that PBMC from woodchucks NW7029, NW7030, and NW7032 recognized six or seven WHcAg peptides of panel A (peptides 1-20, 28-47, 28-57, 70-89, 90-109, 100-119, 112-131 and 120-139) whereas woodchuck NW7031 showed PBMC proliferation only to three peptides of panel A (Table 2). Four peptides of panel B (peptides 97-110, 100-113, 111-124, and 120-131) induced proliferation of PBMC from the four woodchucks tested, whereas several peptides from panel A (peptides 15-34, 50-69, 61-80, 82-101, 131-150, and 146-165) were not stimulatory for PBMC from any of these animals.
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T-cell response to WHV antigens 1 year after infection.
During
the acute phase of WHV infection (week 6), PBMC of all woodchucks
recognized WHsAg, rWHcAg, and certain WHcAg epitopes. To determine
whether these woodchucks possess a long-lasting cellular immune
response, PBMC from woodchucks NW7030 and NW7031 were stimulated with
viral proteins and WHcAg peptides 1 year after the experimental WHV
infection. At this time, both animals were positive for anti-WHs and
anti-WHc and negative for WHsAg. WHV DNA was detectable neither in the
sera nor in the PBMC by nested PCR (data not shown). Furthermore, PBMC
of both animals were not specifically stimulated by any of the previous
recognized epitopes or rWHcAg (SI 
3.1) (Fig.
3).
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T-cell response to WHV antigens after viral challenge. At 1 year after experimental WHV infection, woodchucks NW7030 and NW7031 were challenged with a higher dose of WHV than for the primary experimental infection to analyze whether reinfection and/or reactivation of a cellular immune response to WHV antigens and WHcAg epitopes may occur. Before challenge (week zero), the animals showed no specific T-cell responses to WHsAg, rWHcAg, or peptide 97-110 (Fig. 4). At 1 week after WHV challenge, T-cell responses to WHsAg, rWHcAg, and peptide 97-110 were detected. For woodchuck NW7030 the SI was 3.4 for WHsAg, 5.3 for rWHcAg, and 6.9 for peptide 97-110, and for woodchuck NW7031 the SI was 3.2, 3.4, and 4.7, respectively. Control peptide 129-140 did not stimulate T cells from either animal. T-cell responses were detected for 2 weeks and decreased thereafter, approaching cutoff values by week 4. At the time of viral challenge, both woodchucks were positive for anti-WHs and anti-WHc and negative for WHsAg. WHV DNA and WHsAg were not detectable in the sera by nested PCR or ELISA, respectively, at the time of challenge and thereafter.
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DISCUSSION |
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Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References
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Appropriate Th and cytotoxic T-lymphocyte responses during the early phase of an acute HBV infection are regarded as crucial for determining either recovery from infection or progression to chronicity (6, 13). However, studies on the earliest immunological events occurring after HBV infection in humans are limited because monitoring first becomes possible only at the onset of symptoms, not immediately after exposure. Thus, studies in animal models are required to examine the asymptomatic incubation period of hepadnavirus infection. The experimental WHV infection of woodchucks is an excellent model with which to study cellular and humoral immune responses during the earliest phase after inoculation and to determine their role in disease progression.
In this study, T-cell responses to viral proteins and peptides after experimental WHV infection of adult woodchucks were examined. T-cell responses were assessed by BrdU and [2-3H] adenine incorporation (20, 23) due to low [3H]thymidine uptake by woodchuck PBMC (22). The molecular mechanism by which BrdU that is used as a thymidine analog in proliferation assays is incorporated into cellular DNA has not yet been characterized and needs further investigation (25). For the kinetics of T-cell responses after infection, we used both assays in parallel to compare their sensitivity (25). The response of woodchuck T-cells to WHsAg, rWHcAg, and WHcAg peptides could be monitored by both assays (Fig. 1). However, the [2-3H]adenine assay had higher sensitivity in that the T-cell responses were detected up to 3 weeks earlier and up to 2 weeks later than by the BrdU assay (Fig. 1).
The first T-cell responses were directed against WHsAg and were detected 3 weeks after WHV inoculation (Fig. 1). The maximum T-cell response to WHsAg occurred when WHsAg was detected in serum and decreased upon seroconversion to anti-WHs. The development of anti-WHs, which is regarded as a virus-neutralizing antibody (40), was associated with the elimination of WHsAg from serum. These findings suggest that T-cell responses to WHsAg occur prior to liver cell damage, as shown by the presence of normal SDH levels in serum (Fig. 1). WHsAg may be secreted at high abundance early after infection and therefore is the first antigen presented to T cells by MHC class II molecules of APC. In addition, the differential abundance of WHsAg and WHcAg could explain why T-cell responses to rWHcAg and the corresponding immunodominant epitope (peptide 97-110) became detectable later than the WHsAg-specific T-cell response (Fig. 1).
The T-cell response in woodchucks to recombinant WHV core protein was similar to the response observed in HBV-infected patients, whose HBcAg-specific T-cell responses were stronger and occurred later during the course of infection than did the HBsAg-specific T-cell responses (12, 17, 21). This was likely for the stronger T-cell proliferation to major epitopes of the HBcAg compared to the entire rHBcAg. In all four woodchucks, stimulation of T cells with peptide 97-110 resulted in a greater proliferation than with rWHcAg. The maximum T-cell responses to rWHcAg and peptide 97-110 coincided with the peaks of SDH activities.
The more pronounced T-cell response to rWHcAg than to WHsAg may depend on the higher immunogenicity of this internal viral protein (23, 28). The vigorous T-cell response to WHcAg epitopes (e.g., peptide 97-110) is probably based on their linear features facilitating internalization and presentation by MHC class II molecules of APC.
The T-cell responses to the peptides described previously (23) and used in this study indicate that a variety of epitopes, located throughout the entire core protein, are recognized. The subset of WHcAg epitopes recognized by each animal was different, however (Table 2) (23). One reason for this may be that the animals used in these studies were outbred and unrelated to each other in terms of MHC. This was confirmed by a one-way mixed lymphocyte reaction (data not shown) and characterization of MHC class I patterns (unpublished results). Peptide 97-110 was recognized by all four woodchucks tested in this study, strengthening previous findings that this epitope is promiscuous and immunodominant (23). So far, epitopes covering amino acids 15 to 34, 50 to 69, 61 to 80, 82 to 101, 131 to 150, and 146 to 165 were not recognized (Table 2).
Proliferation of PBMC and of T cells from rWHcAg-specific polyclonal T-cell lines to rWHcAg- and WHcAg-related peptides demonstrated that the rWHcAg, the immunodominant epitope (peptide 97-110), and other epitopes (peptides 100-119, 112-131, and 120-139) were commonly recognized during the early phase of acute WHV infection by all woodchucks included in this study (week 6) (Fig. 2; Table 2). However, the pattern of peptide-induced T-cell response and its magnitude varied among all the woodchucks tested (Table 2). T-cell responses of woodchucks NW7029, NW7030, and NW7032 were directed against five to eight WHcAg epitopes, whereas woodchuck NW7031 showed responses to only three epitopes (Fig. 2). In the woodchuck model, immunological factors such as MHC class II patterns, MHC restriction, and cytokine expression have not been defined; therefore, mechanisms that may have led to the delay in the humoral and cellular immune response in woodchuck NW7031 but resulted in the resolution of WHV infection remain unknown.
T-cell responses to rWHcAg and several epitopes were present beginning 6 weeks after experimental WHV infection and continued to be detected throughout convalescence (weeks 12, 18, and 24). Similar to results with rWHcAg, peptide 97-110 and peptides 100-113, 100-119, and 112-131 were recognized by the T cells of all woodchucks throughout this study. Further WHcAg epitopes, e.g., peptides 1-20, 70-89, and 90-109, which were recognized by woodchucks NW7029, NW7030, or NW7032 at week 6 were also stimulatory at week 24. T-cell proliferation in response to additional WHcAg epitopes during the follow-up was not observed. However, several peptides, e.g., peptides 38-57, 120-131, and 120-139 which induced a strong T-cell response at weeks 6 to 18, were no longer stimulatory for T cells after convalescence (week 24). Consistent responses to different epitopes during a 24-week follow-up have not been detected in studies on human patients (9, 17).
Recent studies have shown a long-term persistence of HBV-specific Th-cell and CTL responses in patients after recovery from HBV infection (30, 34). These results may be due to T-cell memory after initial development of a vigorous T-cell response which led to resolution of HBV infection. They may also be due to the continuous stimulation by residual virus, replicating at very low levels in extrahepatic sites, as demonstrated by the detection of HBV DNA in the sera and/or PBMC of some patients. In contrast to these findings, WHV-specific T-cell responses or WHV DNA were not detected 1 year after the acute self-limited WHV infection in the four woodchucks tested (Fig. 3). This could be due to the short recovery period after infection or to the limited number of animals. However, a decrease in the Th-cell responses to HBV nucleocapsid antigens has been observed in acutely HBV-infected patients simultaneously with or shortly after resolution of infection (11, 12). Especially if HBV DNA was not detected in the serum, the Th-cell and CTL responses to HBV proteins were low (30, 34). These results are explained by the downregulation of the frequency of effector cells toward the end of a successful immune response (1, 38).
The woodchuck model presents a unique opportunity to investigate the cellular immune response upon reexposure to hepadnavirus infection. To test for the presence of memory T cells, which may become reactivated upon challenge with the virus, two animals were inoculated with 109 woodchuck ID50 1 year after recovery from the initial infection. T-cell proliferation in response to WHV antigens was demonstrated 1 week after challenge in both animals (Fig. 4). This T-cell response was detected for 2 to 3 weeks and decreased thereafter. Although circulating anti-WHs may have neutralized most virus particles, a small number of hepatocytes could have been infected, resulting in a limited and low-level viral replication that was responsible for the reactivation of T cells. Interestingly, the reactivated T-cell responses were directed against the same WHcAg epitopes observed during convalescence after the primary WHV infection (Fig. 2 and 5). The similar pattern of T-cell responses found in these woodchucks suggests a hierarchy of specificity in epitope recognition that is maintained unchanged after convalescence until reexposure to the virus. In conclusion, these results demonstrate the development of T-cell responses in the woodchuck directed against WHsAg, rWHcAg, and distinct epitopes of the WHcAg during the early phase of infection and show that they are closely associated with viral clearance and are retained after convalescence, contributing to long-lasting immunity.
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ACKNOWLEDGMENTS |
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We gratefully acknowledge the helpful discussion and critical comments of M.-C. Jung (University of Munich, Munich, Germany) and of J. R. Jacob and B. C. Tennant (Cornell University, Ithaca, N.Y.).
This work was supported by `Deutsche Forschungsgemeinschaft' grant Ro 687/6-1.
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FOOTNOTES |
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* Corresponding author. Mailing address: Institut für Virologie, Universitätsklinikum Essen, Hufelandstr. 55, D-45122 Essen, Germany. Phone: 49-201-723-3550. Fax: 49-201-723-5929. E-mail: roggendorf{at}uni-essen.de.
Present address: Department of Clinical Sciences, Cornell
University, Ithaca, NY 14853.
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REFERENCES |
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Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References
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J Virol, July 1998, p. 6083-6091, Vol. 72, No. 7
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Copyright © 1998, American Society for Microbiology. All rights reserved.
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