Cellular immune responses in patients with hepatitis B surface antigen seroclearance induced by antiviral therapy
Cellular immune responses in patients with hepatitis B surface antigen seroclearance induced by antiviral therapy
Minfeng Liang 0
Shiwu Ma 0
Xiaoxiong Hu 0
Bin Zhou 0
Junchang Zhang 2
Jinjun Chen 0
Zhanhui Wang 0
Jian Sun 0
Xiaolin Zhu 0
William Abbott 0 1
Jinlin Hou 0
0 Hepatology Unit and Key Lab for Organ Failure Research, Nanfang Hospital, Southern Medical University , Guangzhou 510515 , P.R. China
1 New Zealand Liver Transplant Unit, Auckland Hospital , Private Bag 92-024, Auckland , New Zealand
2 Department of Liver Diseases, Zhuhai Branch Guangdong Provincial Hospital of Traditional Chinese Medicine , Zhuhai 519015 , P.R. China
Background: The mechanisms by which chronic hepatitis B is completely resolved through antiviral therapy are unknown, and the contribution of acquired T cell immunity to hepatitis B surface antigen (HBsAg) seroclearance has not been investigated. Therefore, we measured the T-cell responses to core and envelope antigens in patients with HBsAg seroclearance. Methods: Fourteen subjects with HBsAg seroclearance following antiviral treatment for chronic hepatitis B, 7 HBeAg-positive immunotolerant HBV carriers and 9 HBeAg-negative inactive HBsAg carriers were recruited. HBVspecific T-cell responses to recombinant HBV core (rHBcAg) and envelope (rHBsAg) proteins and pools of core and envelope peptides were measured using an ELISPOT assay detecting interferon-gamma and intracellular cytokine staining (ICS) assays detecting interferon-gamma or interleukin 2. Results: Interferon-gamma ELISPOT assays showed a low frequency of weak responses to the rHBsAg and S peptide pool in the HBsAg seroclearance group, and the response frequency to the rHBcAg and the C peptide pool was higher than to the rHBsAg (P < 0.001) and S peptide pool (P = 0.001) respectively. A higher response frequency to C than S peptide pools was confirmed in the interferon-gamma ICS assays for both CD4+ (P = 0.033) and CD8+ (P = 0.040) T cells in the HBsAg seroclearance group. The responses to C and S antigens in the inactive carriers were similar. Conclusions: There was a low frequency of CD4+ and CD8+ T cell immune responses to envelope antigens in Chinese subjects with HBsAg seroclearance following antiviral therapy. It is unlikely that these immune responses are responsible for HBsAg seroclearance in these subjects.
Major advances have been made in the treatment of
hepatitis B during the past 10 years. However, much
remains to be accomplished because current antiviral
therapy does not permanently eradicate infection. A
permanent suppression of HBV replication following
treatment will require generation of a robust acquired
immune response against peptides contained within the
HBV core and envelope genes . There is some
evidence that drug-induced suppression of the HBV results
in increased acquired immunity against the virus [2-4];
possibly because the decrease in the levels of core and
particularly envelope antigens allows T cells to recover
from exhaustion . The possibility that this may be an
important mechanism leading to complete recovery from
chronic hepatitis B has not yet been explored in vivo.
A small number of patients taking modern antiviral
therapies achieve a complete loss of both core and
envelope proteins from serum. This process is known as
HBsAg seroclearance. It would be of interest to know if
there was an increase in acquired immunity to envelope
antigens in these subjects, as this would allow two
hypotheses to be tested. The first hypothesis is that the
acquired immune response to the envelope protein
contributes to HBsAg seroclearance. The second
hypothesis is that the decrease in serum HBsAg levels
would reverse the exhaustion of CD8+ T cells that
respond to envelope peptides . In this study, we have
investigated the T-cell responses to peptides encoded by
the HBV C and S open reading frames in patients with
HBsAg seroclearance following antiviral therapy.
A total of 30 patients were enrolled in the study. They
were recruited from the Hepatology Unit of Nanfang
Hospital in Southern Medical University, and the
Department of Liver Diseases in Zhuhai Branch
Guangdong Provincial Hospital of Traditional Chinese
Medicine. There were 14 patients with sustained HBsAg
seroclearance (HBsAg < 0.05 IU/mL) following
treatment of their HBeAg-positive chronic hepatitis B with
either pegylated interferon a (n = 3), nucleot(s)ide
analogue treatment (n = 8) or sequential treatement with
pegylated interferon a and nucleot(s)ide analogue
(n = 3). They were off-treatment when the blood was
taken, and all subjects had an ALT of < = 60 IU/L. At
baseline, they were all positive for HBeAg, had an HBV
DNA level > 5.0 log10 copies/mL and a serum ALT > 80
IU/L. The controls were 9 inactive HBsAg carriers
(HBsAg positive, anti-HBe positive, anti-HBc positive,
ALT < 40 IU/L, HBV DNA < 3.0 log10 copies/mL;
representing the inactive hepatitis B phase) and 7
immunotolerant HBV carriers (HBsAg positive, HBeAg positive,
anti-HBc positive, ALT < 40 IU/L, HBV DNA > 4.0 log10
copies/mL; representing the immune tolerant phase).
The presence of HCV/HIV infection, hepatocellular
carcinoma, autoimmune liver disease, other malignant
tumors and severe metabolic diseases was excluded in all
patients. The clinical characteristics of these patients are
summarized in Table 1 and Table 2. In this investigation,
we enrolled the patients with normal ALT level in sera
(only one patient in HBsAg seroclearance group with
ALT at 60 IU/L), in order to ensure that all subjects were
under homogeneity of hepatic inflammation.
The quantitation of HBsAg in patients with HBsAg
seroclearance was determined by the Abbott Architect®
i2000 System, and the ARCHITECT HBsAg assay was
obtained from Abbott Ireland Diagnostics Division,
which was a chemiluminescent microparticle
immunoassay for quantitative determination of HBsAg in human
serum and plasma. The ARCHITECT HBsAg assay has
Table 1 The HBV seromarkers, genotypes and mutations in patients with HBsAg seroclearance
Patient Genotype Drugs HBsAga HBsAba HBeAga HBeAba HBcAba
Note. SC, patient with HBsAg seroclearance; UD, HBV DNA undetectable by nested PCR; ETV, entecavir; LAM, lamivudine; Peg-IFN, pegylated IFN; NEG, negative;
POS, positive; RT, reverse transcriptase; S, S open reading frame; HBsAb, antibody to HBsAg; HBeAb, antibody to HBeAg; HBcAb, antibody to HBcAg.
a HBV serum markers were all determined by chemiluminescent microparticle immunoassay through Abbott ARCHITECT® i2000 system.
b Two of these were seropositive for HBeAg at the time we collected the blood sample. However, both subjects became HBeAg-negative during follow-up.
Table 2 The demographic and clinical features of all subjects in this investigation
Note. a Kruskal-Wallis H test; b Fisher’s exact test; c HBV DNA undetectable by real-time PCR.
a sensitivity of ≤ 0.05 IU/mL. The most prevalent
HBsAg mutants, such as the G145A mutant, are readily
detected in the ARCHITECT HBsAg assay with a
sensitivity equivalent to detection of wild type HBsAg.
The study was conducted according to the guidelines
of the Declaration of Helsinki, and was approved by the
Ethical Committee of Nanfang hospital in Southern
Medical University. All subjects gave informed consent.
HBV Genotyping in Patients with HBsAg Seroclearance
HBV genotypes in patients with HBsAg seroclearance
were assigned as described previously . Briefly, HBV
DNA was extracted from serum samples with the
QIAGEN MinElute Viral Spin kit (QIAGEN, Germany)
following the manufacturer’s recommendations.
Amplimers covering AA46-217 of the P open reading frame
or AA38-208 of the S open reading frame were
amplified. Sequence alignments were performed using
ClustalX v1.8. Phylogenetic tree analysis was performed
using the Tamura-Nei model of evolutionary distance,
and the topology was evaluated by bootstrap analysis
(1,000 replicates) using the neighbor joining method.
All amplifications were performed by nested-PCR.
HBV DNA extraction, preparation of PCR cocktails, and
addition of templates were performed in separate areas,
and negative controls in duplicate were included in each
Preparation of Peripheral Blood Mononuclear Cells
Peripheral blood mononuclear cells (PBMCs) were
freshly isolated from heparinized blood by
Ficoll-Hypaque density gradient centrifugation with Lymphoprep
(Axis-Shield, Oslo, Norway) as previously described .
Subsequently, the cells were resuspended in complete
RPMI-1640 medium (Gibco®, Invitrogen, Beijing, P.R.
China), which contained 10% heat-inactivated fetal
bovine serum (Gibco®, Invitrogen, Australia), 2 mM
Lglutamine, 100 U/mL penicillin and 100 μg/mL
streptomycin. After isolation, PBMCs were cryopreserved in a
medium containing 90% fetal bovine serum and 10%
dimethyl sulfoxide (Sigma-Aldrich, St. Louis, MO), and
then stored in liquid nitrogen.
Synthetic Peptides and Antibodies
A panel of 56 18-mer peptides overlapping by 10
residues and covering the full S and C open reading frames
of the HBV was obtained from Sigma-Aldrich. These
peptides were dissolved in dimethyl sulfoxide (analytic
reagent obtained from Sigma-Aldrich) and were pooled
in 2 mixtures, named the S peptide pool (30 peptides
covering the whole HBV S-ORF) and the C peptide pool
(26 peptides representing HBV C-ORF). The purity of
these peptides was more than 90%. Recombinant HBsAg
and HBcAg were purchased from American Research
Products Inc (Belmont, MA, USA).
Peridinin-chlorophyll-protein (PerCP) labeled
antiCD8 antibody (SK1, Mouse IgG1), allophycocyanin
(APC) labeled anti-CD3 antibody (UCTH1, Mouse
IgG1), fluorescein isothiocyanate (FITC) labeled anti-IL2
antibody (5344.111, Mouse IgG1), phycoerthrin (PE)
labeled anti-IFN-g (25723.11, Mouse IgG2b) and their
isotypes (FITC labeled Mouse IgG1 and PE labeled
Mouse IgG2b) were obtained from BD Bioscience (San
Jose, CA). Purified anti-CD28 monoclonal antibody was
obtained from BD Pharmingen (San Diego, CA) for use
in the intracellular cytokine staining assays.
Ex vivo ELISPOT Assay for Interferon-gamma
The antigens for the human IFN-g ELISPOT assays 
were the 2 pools of 18-mer peptides, the rHBsAg and
the rHBcAg as described above. Multiscreen-IP 96-well
plates (Millipore, Billerica, MA) were coated overnight
at 4°C with 10 μg/mL anti-human IFN-g monoclonal
antibody (1-DIK; Mabtech, Sweden). Plates were then
washed seven times with DPBS, and blocked with RPMI
1640 supplemented with 10% fetal bovine serum for 2
hours at room temperature. Freshly isolated or
frozenthawed PBMCs (2.5 × 105/well) were seeded in duplicate
for either each individual peptide mixture (2 μg/mL per
peptide) or each recombinant HBV antigen (10 μg/mL).
Plates were incubated for 32 hours at 37°C with 5%
CO2. After washing, 50 μL of 1 μg/mL biotinylated
monoclonal antibody (7-B6-1; Mabtech, Sweden) was
added to each well. After 2 hours of incubation at room
temperature, plates were washed seven times; 50 μl per
well of 1 μg/mL Streptavidin-Alkaline phosphatase
(Mabtech, Sweden) was added, and the plates were
incubated for a further 1 hour at room temperature. Plates
were then washed seven times, and 100 μL per well of
BCIP/NBT (diluted by distilled water; Zymed® BCIP/
NBT SUBSTRATE KIT, Invitrogen, Camarillo, CA) was
added. After 10 minutes, the colorimetric reaction was
stopped by distilled water and, washed three times with
distilled water. Plates were air dried, and the
Spot® S4 Macro Analyzer (Cellular Technology Ltd,
USA) was used for spot counting. Results were
expressed as numbers of spot-forming cells (SFC) per
106 PBMCs. The number of specific IFN-g-secreting
cells was calculated by subtracting the value of the
unstimulated control from the value of the stimulated
sample. The positive control consisted of PBMCs
stimulated with phytohemagglutinin (PHA, 10 μg/mL; from
Sigma-Aldrich). The criteria for a positive response for
the ex vivo ELISPOT assays was more than 5 SFC per
well and more than twice the number of SFC than the
unstimulated control wells [9,10]. Therefore, in this
study, more than 20 SFC per 106 PBMCs would be a
Intracellular Cytokine Staining for Interferon-g and
Interleukin 2 in vitro
Briefly, freshly isolated or frozen-thawed PBMCs were
seeded into 96-well round bottom culture plates (1
million PBMCs per well). The 2 pools of 18-mer
overlapping peptides covering the S and C open reading frames
of the HBV genome were used as specific stimuli.
Phorbol 12-myristate 13 acetate (PMA, 50 ng/ml; Merck,
Darmstadt, Germany) and ionomycin (1 μmol/L; Merck,
Darmstadt, Germany) were used as positive controls.
Anti-CD28 antibody was added to each well for
amplification. After 2 hours of incubation at 37°C in 5% CO2,
the transport inhibitor BD GolgiPlug™ (containing
Brefeldin A; BD Pharmingen, San Diego, CA) was added for
an additional 4 hours of incubation. At the end of the
culture, the cells were treated with 25 mM EDTA at
37°C for 15 minutes, and then harvested. Subsequently,
the cells were stained with APC labeled anti-CD3
antibody and PerCP labeled anti-CD8 antibody for 30
minutes at room temperature in darkness, and then fixed by
Medium A (Caltag™, Fix&Perm® reagents, Invitrogen)
for 15 minutes at room temperature. Cells were then
permeabilized by Medium B (Caltag™, Fix&Perm®
reagents, Invitrogen) in the presence of FITC labeled
anti-IL2 antibody and PE labeled anti-IFN-g or the
respective isotype control antibody at room temperature
for 25 minutes. Finally, the cells were washed by DPBS,
containing 1% bovine serum albumin and 0.1% sodium
azide, and resuspended with 1% paraformaldehyde. Two
hundred thousand (2 × 105) cells were acquired by BD
FACSCanto™ II (BD Bioscience, San Jose, CA) and
analysed with BD FACSDiva software (BD Bioscience,
San Jose, CA). A positive response was considered to be
0.05% cytokine+, CD4+/CD8+T cells above the
background response (response to medium and
co-stimulatory molecular stimulation) in this
Statistical analysis was performed using the
MannWhitney U test or Kruskal-Wallis H test for comparison
of continuous variables between study groups. The rates
of positive response were compared by Fisher’s exact test.
The SPSS statistical package version 15.0 (SPSS Inc.,
Chicago, IL, USA) was used for analysis while statistical
significance was assessed at the 0.05 level (P < 0.05).
The Demographic and Serological Characteristics of
Patients with HBsAg Seroclearance
The age, gender, HBV DNA levels and ALT levels in all
subjects are shown in Table 2. The patients with HBsAg
seroclearance showed a variety of patterns of serological
HBV markers, but they were all positive for anti-HBc
antibody. Eight patients were anti-HBs positive, and 2 of
these were seropositive for HBeAg at the time we
collected the blood sample. However, both subjects became
HBeAg-negative during follow-up (Table 1).
Virological Analysis in Patients with HBsAg Seroclearance
A 583 bp fragment from the HBV S open reading frame
was amplified and sequenced from 6 of the 14 subjects
with HBsAg seroclearance. The HBV genotypes and the
repertoire of amino acid mutations in the reverse
transcriptase region of the P and overlapping S polypeptides
were identified by aligning the sequences with 242
genotype C sequences from GenBank. Five of the 6 sequences
were genotype C and one was genotype B. We found that
two lamivudine-treated patients infected with a genotype
C HBV developed L80I+M204I+L180M triploid mutants
in the reverse transcriptase (RT) region. The W196L
mutation in the S region in these two patients corresponded to
M204I in the RT region (Table 1). The other variants
identified in the RT region and S regions (Table 1), have not
been previously reported in occult HBV infection, and no
evidence have proved that they would be attributed to the
loss of HBsAg in vitro or in vivo.
The Production of Interferon-gamma in HBV-Specific T
Cells through Ex Vivo ELISPOT Assay
First, we wondered whether an HBV-specific T-cell
response is detectable in patients with HBsAg
seroclearance. In addition, we asked about the antigen specificity
and the magnitude of the detected responses. Both the
peptide pools and both the recombinant HBV proteins
were used to stimulate PBMCs in an ex vivo ELISPOT
assay for IFN-g. HBV-specific T cells could be detected
in patients with HBsAg seroclearance at a frequency
ranging between 22 and 846 antigen-specific T cells per
106 PBMCs (Figure 1A). The immune responses in
patients with HBsAg seroclearance were mainly to the C
peptide pool and the rHBcAg (9 of 14 and 11 of 14
positive tests respectively). There were only three
positive responses to S open reading frame antigens, and
these were all close to the baseline response (Figure
1A). Consistent with this, patients with HBsAg
seroclearance displayed a more robust T-cell response
against rHBcAg than against rHBsAg (Figure 1A, P <
0.001), and against the C peptide pool relative to the S
peptide pool (Figure 1A, P = 0.001). Furthermore,
rHBcAg could prime a more dramatic HBV-specific
T-cell response than the C peptide pool in patients with
HBsAg seroclearance (Figure 1A, Mean ± SD, 152.1 ±
231.9 VS. 47.1 ± 27.6 SFC pos resp/106 PBMCs,
MannWhitney U test, P = 0.014).
The frequency of positive responses in patients with
HBsAg seroclearance was higher than that in
immunotolerant HBV carriers (37.5% VS. 14.3%, c2 = 4.812, P =
0.028, Figure 1B). The frequency of positive responses in
the inactive carriers was also higher than that in the
immunotolerant HBV carriers (50.0% VS. 14.3%, c2 =
8.905, P = 0.003, Figure 1B). The mean values of
spotforming cells in positive responses from subjects with
HBsAg seroclearance were similar to those from the
inactive HBsAg carriers (Mean ± SD, 113.0 ± 174.6 VS.
169.9 ± 282.7 SFC pos resp/106 PBMCs, Mann-Whitney
U test, P = 0.723). They were both more vigorous than
those from immunotolerant HBV carriers (Figure 1C,
Mean ± SD, 113.0 ± 174.6 VS. 27 ± 6 SFC pos resp/106
PBMCs, P = 0.034 and 169.9 ± 282.7 VS. 27 ± 6 SFC
pos resp/106 PBMCs, P = 0.040; by Mann-Whitney U
The Response of Interferon-gamma in HBV-Specific T Cells
Determined by Intracellular Cytokine Staining in vitro
The purpose of the intracellular cytokine staining assays
was to determine the phenotype of the HBV-antigen
specific, interferon-gamma producing T cells found in
the ELISPOT assay. Both CD8+ (Figure 1A) and CD4+
(Figure 1B) peptide-specific T cells were identified in all
3 groups of subjects. In the HBsAg seroclearance group,
the frequency of both CD8+ (P = 0.040) and CD4+ (P =
Figure 1 Ex vivo IFN-g ELISPOT responses to HBV S and C open reading frame peptide pools and recombinant HBV proteins. (A)
Comparison of subjects with HBsAg seroclearance, immunotolerant HBV carriers and inactive HBsAg carriers. Each line represents the mean
number of spot-forming cells induced by each stimulus. Each dotted line represents the cut-off value for a positive test in the ELISPOT assay. (B)
Comparison of the frequency of positive responses obtained in HBsAg seroclearance and control populations. (C) Comparison of the mean
number of spots/well from the subjects with positive tests in each group. Each bar represents the mean ± SD.
0.033) T cells responding to the C peptide pool was
higher than the frequency of cells responding to the S
peptide pool. This is consistent with the results of the
The percentage of IFN-g producing CD8+ and CD4+
T cells induced by the C peptide pool in both the
HBsAg seroclearance and inactive HBsAg carrier groups
was higher than that in the immunotolerant HBV
carriers (Figures 2A and 2B). There was also a higher
frequency of positive responses to the C peptide pool in
both the HBsAg seroclearance and inactive carrier
groups than in the immunotolerant group (Figure 2C),
but these differences did not reach statistical
significance. However the frequencies of interferon-gamma
producing CD4+ and CD8+ T cells in the
positivelyresponding subjects was higher in both the HBsAg
seroclearance and inactive carrier groups than in the
immunotolerant group (Figure 2D).
The Production of Interleukin 2 in HBV-Specific T Cells
Determined by in vitro Intracellular Cytokine Staining
Interleukin 2 (IL2) is an alternative index of cell
proliferation and activation capacity. Therefore, in order to
assess the proliferative response to HBV antigen in
vitro, we measured the IL2 responses to the C and S
peptide pools in both CD4+ and CD8+ T cells in the
three groups of subjects. The individual responses of
CD8+ T cells to the C and S peptide pools are shown in
Figure 3A and the individual responses of the CD4+ T
cells are shown in Figure 3B. IL2 was produced by
CD4+ and CD8+ T cells in some subjects from all 3
subject groups, in response to both C and S peptide
pools. There were no differences between the frequency
or magnitude of the IL2 responses to the C and S
peptide pools. There was no difference in the frequency or
magnitude of these responses among the three subject
HBsAg seroclearance following nucleos(t)ide analogue
or pegylated interferon a treatment is a rare event in
chronic hepatitis B. Since HBsAg seroclearance may
result in a decreased risk for hepatocellular carcinoma
in these patients , it will be worthwhile identifying
any immune mechanisms that might contribute to
HBsAg seroclearance, as they may provide targets for
Figure 2 Results of the intracellular cytokine staining assay for interferon gamma. (A) Individual responses of CD8+ T cells stimulated by
HBV peptide pools in subjects with HBsAg seroclearance (Group1), immunotolerant HBV carriers (Group2) and inactive HBsAg carriers (Group3).
(B) Individual responses of CD4+ T cells stimulated by HBV peptide pools in subjects with HBsAg seroclearance, immunotolerant HBV carriers
and inactive HBsAg carriers. (C) The mean frequencies of positive CD4+ and CD8+ T cell responses in the three populations. (D) Comparison of
mean frequencies of IFN-g+ secreting CD8+ and CD4+ T cells in subjects with positive responses to either of the peptide mixtures in the three
groups. Each dotted line in 2A and 2B represents the cut-off value for a positive test. Each bar in 2D represents the mean ± SD percentage of
IFN-g secreting CD4+ and CD8+ cells in subjects with positive responses to either the C or S peptide pools.
Figure 3 Results of the intracellular cytokine staining assay for interleukin 2. (A) Individual responses of CD8+ T cells stimulated by HBV
peptide pools in subjects with HBsAg seroclearance (Group1), immunotolerant HBV carriers (Group2) and inactive HBsAg carriers (Group3). (B)
Individual responses of CD4+ T cells stimulated by HBV peptide pools in subjects with HBsAg seroclearance, immunotolerant HBV carriers and
inactive HBsAg carriers. (C) The mean frequencies of positive CD4+ and CD8+ T cell responses in the three populations. Each bar in 3C
represents the mean ± SD percentage of IL-2 secreting CD4+ and CD8+ cells in subjects with positive responses to either the C or S peptide
immunotherapy. The mechanisms that might contribute
include acquired CD4+ and CD8+ T cell responses ,
innate immune responses , and mutations in the
HBsAg arising from genetic drift . The possibility
that HBsAg seroclearance results from an acquired
immune response to antigens within the envelope
proteins is of particular interest.
In this study, we have focused on the frequency and
magnitude of acquired CD4+ and CD8+ T cell
responses to both the HBV core and envelope proteins.
Our data are consistent with previously published data
[16,17], in that they show both a higher frequency
(Figures 1B and 2C) and magnitude (Figures 1C and 2C) of
antigen-specific, CD4+ and CD8+ T cell responses to
core and envelope antigens in subjects with inactive
HBeAg-negative chronic hepatitis B virus infection
relative to subjects who are immunotolerant. This was
found with both ELISPOT and intracellular cytokine
staining assays for interferon gamma. The frequency
and magnitude of antigen-specific T cell responses was
also higher in the HBsAg seroclearance group than in
subjects with immunotolerance. However the T cell
responses in the seroclearance group were almost
exclusively to core antigens. Only a small number of low
level responses to envelope gene antigens were detected.
This is an important negative result for two reasons. It
suggests that acquired T cell responses to envelope
antigens do not contribute to HBsAg seroclearance
following anti-viral therapy in Chinese subjects. In addition, it
does not support the possibility that CD8+ T cell
exhaustion can be reversed by a reduction of antigen
levels in vivo. One possible, but unlikely, limitation to
these conclusions is that this HBsAg seroclearance
group was comprised of subjects with a biased
repertoire of class I and class II alleles that do not present
envelope peptides effectively. It is also possible that a
longer period of antigen loss would be needed to allow
these T cells to recover.
The additional finding that there was no increase in
the responses to core antigens beyond that detected in
inactive carriers suggests that neither acquired immune
response drives HBsAg seroclearance in the
posttreatment situation. However a comparison of anti-core
responses in HBeAg-negative, treated subjects who did
and did not undergo HBsAg seroclearance would be
necessary to fully test this hypothesis.
Thus our data did not provided clues to the cause of
HBsAg seroclearance in this group of subjects. The
possibility that HBsAg seroclearance occurs as a result of
mutations accumulating in the envelope protein as a
result of genetic drift has not been excluded by direct
sequencing of PCR products. There may be a large
number of harmful mutations in the envelope gene that
each occurs at low frequency in the viral population.
Phylogenetic analysis of cloned envelope gene sequences
would be necessary to exclude this possibility. The
possibility that the innate immune system is responsible for
HBsAg seroclearance is currently difficult to test, as the
mechanisms by which the HBV might potentially be
controlled by innate immunity are unknown.
The failure to find evidence that acquired immunity
might drive HBsAg seroclearance in these subjects does
not exclude the possibility that immunotherapy directed
at envelope peptides may eventually have a role in
improving responses to antiviral therapy. The finding
that it is possible to reverse the exhaustion of CD8+ T
cells that respond to envelope antigens in vitro 
suggests that potentially useful CD8+ T cells exist in the
blood of these patients. Further research into how they
might be activated in vivo is still needed.
According to our data, we have been unable to detect an
increase in CD4+ or CD8+ T cell immunity to HBV
envelope proteins in Chinese subjects who have
undergone HBsAg seroclearance following antiviral therapy.
This suggests that acquired immunity to HBV envelope
antigens is not an important cause of HBsAg
seroclearance in these patients. In addition, this study does not
support the hypothesis that a sustained decrease in the
level of the HBV envelope protein in vivo will result in
a reversal of exhaustion of the CD8+ T cells that
respond to envelope peptides.
DPBS: Dulbecco’s phosphatase buffer saline; ELISPOT: enzyme-linked
immunosorbent spot; HBcAg: hepatitis B core antigen; HBeAg: hepatitis B e
antigen; HBsAg: hepatitis B surface antigen; HBV: hepatitis B virus; IFN-γ:
interferon-gamma; IL2: interleukin 2; ORF: open reading frame; PCR:
polymerase chain reaction; rHBcAg: recombinant HBcAg; rHBsAg:
recombinant HBsAg; SFC: spot-forming cell.
MFL carried out the Elispot assays, participated in the design of study and
drafted the manuscript. SWM participated in the design of study, and carried
out the collection of Elispot data. XXH participated in the Elispot and ICS
assays and helped to perform the statistical analysis. BZ participated in the
sequence alignment and helped to draft the manuscript. JCZ participated in
the collection of clinical data and samples. JJC participated in the design of
the study and helped to perform the statistical analysis. ZHW participated in
the sequence alignment and helped to draft the manuscript. JS participated
in the design of the study and helped to draft the manuscript. XL Z
participated in the samples’ collection and a part of immunoassays. WA
conceived of the study and helped to draft the manuscript. JLH obtained
the funding, conceived of the study, and participated in its design and
coordination and helped to draft the manuscript. All authors read and
approved the final manuscript.
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