Download The Characteristics of the Cell-Mediated Immune Response Identify

GASTROENTEROLOGY 2008;134:1470 –1481
The Characteristics of the Cell-Mediated Immune Response Identify
Different Profiles of Occult Hepatitis B Virus Infection
ALESSANDRO ZERBINI,* MASSIMO PILLI,* CAROLINA BONI,* PAOLA FISICARO,* AMALIA PENNA,*
PAOLA DI VINCENZO,* TIZIANA GIUBERTI,* ALESSANDRA ORLANDINI,* GIUSEPPINA RAFFA,‡ TERESA POLLICINO,‡
GIOVANNI RAIMONDO,‡ CARLO FERRARI,* and GABRIELE MISSALE*
*Laboratory of Viral Immunopathology, Azienda Ospedaliero-Universitaria di Parma, Parma, Italy; and the ‡Laboratory of Molecular Biology and Hepatology,
Department of Internal Medicine, University of Messina, Italy
BASIC–LIVER,
PANCREAS, AND
BILIARY TRACT
Background& Aims: Hepatitis B virus (HBV) DNA
detection in serum and/or in the liver of hepatitis B
surface antigen (HBsAg)-negative patients with or
without serologic markers of previous viral exposure
is defined as occult HBV infection. Because the role of
the adaptive response in keeping HBV replication
under control in occult infection still is undefined,
this study was performed to characterize the features
of the HBV-specific T-cell response in this condition.
Methods: HBV-specific T-cell frequency and function
were tested ex vivo and after in vitro expansion in 32
HBsAg-negative patients undergoing diagnostic liver
biopsy for chronic hepatitis C: 18 with occult HBV
infection (11 anti-HBc–negative and 7 anti-HBc–positive patients) defined by the detection of intrahepatic
HBV DNA by polymerase chain reaction; 14 without
detectable intrahepatic HBV DNA (5 anti-HBc–positive and 9 anti-HBc–negative patients). Six patients
with chronic hepatitis B and 7 HBsAg-inactive carriers were studied for comparison. Results: The presence or absence of serologic HBV markers defined 2
profiles of HBV-specific T-cell responses in occult
infection. Anti-HBc–positive patients showed a T-cell
response typical of protective memory, suggesting
that this condition represents a resolved infection
with immune-mediated virus control. In contrast,
HBV-specific T cells in anti-HBc–negative patients
did not readily expand and produce interferon-␥ in
vitro, suggesting the possibility of a low-dose infection insufficient to allow maturation of protective
memory. Conclusions: Our results suggest different
mechanisms of control of viral replication in seropositive and seronegative occult infections. Additional
studies aimed at understanding possible different
clinical implications are needed.
O
ne of the fundamental steps of the hepatitis B virus
(HBV) life cycle is the conversion of the 3.2-Kb
relaxed circular DNA in a covalently closed-circular DNA
in the nucleus of the infected hepatocyte, where it then is
conjugated with nuclear proteins forming a minichromosome. Covalently closed-circular DNA is the template for
transcription leading to the production of new infectious
virions in the infected cell. The highly stable covalently
closed-circular DNA, resistant to cell enzymes digestion,
is probably the basis for life persistence of HBV infection,
even after complete clinical recovery from acute hepatitis
B.1 Thus, an overt HBV infection can persist in association with a chronic active or inactive disease with the
presence of hepatitis B surface antigen (HBsAg) in the
serum, but HBV also can persist decades after acute
hepatitis along with a readily detectable memory T-cell
response,2,3 despite a profound down-regulation of HBV
gene expression4 – 6 under the effect of the protective
antiviral immune response. This type of condition with
persistence of minute amounts of virus in the liver
and/or serum and with possible detection of HBV DNA
also in peripheral blood mononuclear cells (PBMCs)
likely can be identified with the so-called occult HBV
infection and it generally is characterized by the presence
of serum anti-HBV antibodies. However, occult infection
with negative HBsAg can be present also in completely
seronegative patients.7
The lack of HBsAg detection in occult infections may
depend on mutations in the “a” determinant, but this
condition only accounts for a minority of cases in the
Mediterranean area and recent studies tend to exclude
that HBV genetic mutations are responsible for the
strong suppression of viral replication typical of occult
HBV infection.8 Several other mechanisms could be involved, such as viral interference by other viruses, including hepatitis C virus (HCV), down-regulation of HBV
gene expression by an undefined cellular mechanism, and
virus control by the adaptive T-cell response.
In the clinical setting, occult HBV infection represents
not only a condition at risk of HBV reactivation, but it is
also a cofactor of liver disease progression and hepatoAbbreviations used in this paper: CH-B, chronic hepatitis B; CH-C,
chronic hepatitis C; ELISPOT, enzyme-linked immunosorbent spot;
FITC, fluorescein isothiocyanate; IFN, interferon; IL, interleukin;
PBMCs, peripheral blood mononuclear cells; PCR, polymerase chain
reaction.
© 2008 by the AGA Institute
0016-5085/08/$34.00
doi:10.1053/j.gastro.2008.02.017
carcinogenesis in patients with chronic HCV infection.9 –12 Moreover, outcome of interferon (IFN) treatment for chronic HCV infection could be influenced by
the concomitant presence of occult HBV infection, even
if definitive conclusions have not been achieved.9,10,13–17
To elucidate the possible pathogenetic mechanisms
responsible for occult HBV infection, we have analyzed
the features of the HBV-specific cell-mediated immune
response in completely seronegative and in anti-HBc–
positive patients with occult HBV infection. Results show
different profiles of T-cell responses according to the
serologic status of occult infection.
Materials and Methods
Patients
A total of 78 patients with chronic HCV infection
undergoing liver biopsy before IFN treatment and without evidence of chronic HBV infection (HBsAg negative)
were enrolled at the Unit of Infectious Diseases and
Hepatology of the Azienda Ospedaliero-Universitaria of
Parma (Italy).
Among these 78 patients, 18 subjects were positive for
intrahepatic HBV DNA (Table 1, patients 1–18). HBVspecific HLA class I and class II restricted T-cell responses
were studied in these 18 subjects and also in 5 anti-HBc–
positive patients negative for intrahepatic HBV DNA
(Table 1, patients 19 –23) and in 9 chronic hepatitis C
(CH-C) patients negative for HBV serum markers and for
intrahepatic HBV DNA (Table 1, patients 37– 45). The
latter were selected randomly (first available patients)
from the overall group of the 60 CH-C patients negative
for intrahepatic HBV DNA. Liver histology of CH-C patients with and without occult HBV infection also is
shown in Table 1.
Six anti-HCV–negative, highly viremic (HBV-DNA
level, ⬎105 copies/mL), anti-HBe–positive patients and 7
anti-HCV–negative, HBsAg-inactive carriers (HBV-DNA
level, ⬍104 copies/mL) were studied for comparison. All
patients were anti– human immunodeficiency virus negative.
This study was approved by the Ethical Committee of
the Azienda Ospedaliero-Universitaria of Parma, and all
subjects gave written informed consent.
HBV-DNA Analyses
DNA was extracted from the frozen liver specimens
and the PBMCs by standard procedures, as described in the
supplementary material (see supplementary materials and
methods online at www.gastrojournal.org).
Occult HBV infection was identified as previously
described with slight modification.11 All liver and
PBMC DNA extracts were analyzed for the presence of
HBV genomes by performing 4 different in-house single-step or nested polymerase chain reaction (PCR)
amplification assays to detect preS-S, precore-core, Pol,
T–CELL RESPONSE IN OCCULT HBV INFECTION
1471
and X regions. A sample was scored as HBV-DNA
positive when amplification products were detected
using at least 2 different sets of primers in 2 or more
independent experiments. Moreover, direct sequencing
of all amplified HBV products confirmed the specificity
of the reactions. All negative cases were tested twice. In
each PCR experiment, the following were included as
negative controls: (1) serum and tissue DNA extracts
from subjects known to be negative for HBV infection,
(2) DNA-free reaction buffer, and (3) water. The limit
of sensitivity of our single-step and nested PCR methods was in the range of 103 and 10 genome equivalents/mL, respectively, which approximately correspond to 2–200 IU/mL.
Serum HBV DNA was quantified by the COBAS TaqMan Hepatitis B Virus Test (Roche Diagnostics, Mannheim, Germany).
Detection of Amplification Products by
Southern Blotting
The PCR products were separated on a 1% agarose
gel and transferred onto a nylon Hybond N⫹ membrane
(Amersham, Buckinghamshire, England). The membrane
was hybridized at 65°C overnight with a 32P random
prime-labeled (Amersham) full-length HBV genome
probe and then was washed and exposed to X-Omat film
(Kodak, Rochester, NY) at ⫺80°C.
Analysis of the Entire PreC-C Genomic
Region of HBV DNA by PCR and Direct
Sequencing
Liver DNA extracts from a selected number of
subjects were amplified by a nested PCR technique using
oligonucleotide primers specific for HBV-DNA sequences
flanking the entire preC-C genomic region and the Expand High Fidelity PCR System (Roche Diagnostics) according to the manufacturer’s instructions. Technical
details are reported in the supplementary material (see supplementary materials and methods online at www.gastrojournal
.org).
Synthetic Peptides, Peptide-HLA Class I
Tetramers, and Antibodies
A panel of 315 peptides (15-mer) overlapping by
10 residues and covering the overall sequence of HBV
genotype D were purchased from Chiron Mimotopes
(Victoria, Australia). Fifteen-mer peptides were pooled in
16 mixtures (supplementary Table 1; see supplementary
material online at www.gastrojournal.org). Recombinant
S protein (Glaxo Smith Kline, Uxbridge, UK) and
pre-S1 and pre-S2 polypeptides (Merck Sharp &
Dohme, Whitehouse Station, NJ) also were used for the
ex vivo enzyme-linked immunosorbent spot (ELISPOT) assay. Phycoerythrin-labeled tetrameric peptideHLA class I complexes, representing the HLA-A2 restricted epitopes HBV core 18 –27 (FLPSDFFPSV),
BASIC–LIVER,
PANCREAS, AND
BILIARY TRACT
May 2008
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ZERBINI ET AL
GASTROENTEROLOGY Vol. 134, No. 5
Table 1. Patient Characteristics
Patient
Age
Sex
HBsAg
BASIC–LIVER,
PANCREAS, AND
BILIARY TRACT
HBsAg-negative/anti-HBc–negative
1
38
M
Neg
2
61
M
Neg
3
65
F
Neg
4
63
F
Neg
5
56
F
Neg
6
28
M
Neg
7
38
M
Neg
8
52
M
Neg
9
41
M
Neg
10
61
M
Neg
11
54
F
Neg
HBsAg-negative/anti-HBc–positive
12
65
F
Neg
13
57
F
Neg
14
52
M
Neg
15
63
F
Neg
16
38
M
Neg
17
47
F
Neg
18
39
M
Neg
19
39
M
Neg
20
66
F
Neg
21
64
F
Neg
22
49
M
Neg
23
40
M
Neg
HBsAg-inactive carriers
24
58
F
Pos
25
54
F
Pos
26
48
M
Pos
27
73
F
Pos
28
49
M
Pos
29
64
F
Pos
30
40
M
Pos
CH-B
31
63
M
Pos
32
31
F
Pos
33
56
M
Pos
34
45
M
Pos
35
55
M
Pos
36
57
F
Pos
CH-C
37
56
F
Neg
38
49
F
Neg
39
44
M
Neg
40
61
F
Neg
41
44
F
Neg
42
57
F
Neg
43
45
F
Neg
44
44
M
Neg
45
42
M
Neg
PBMCs HBV
DNA
Liver HBV
DNA
HCV
antibody
HLA-A2
Liver histology
S/Ga
⬍6
⬍6
⬍6
⬍6
⬍6
⬍6
UI/mL
UI/mL
UI/mL
UI/mL
ND
UI/mL
UI/mL
UI/mL
UI/mL
UI/mL
UI/mL
Neg
Neg
Neg
Neg
ND
Neg
ND
Neg
Neg
Neg
Neg
Pos
Pos
Pos
Pos
Pos
Pos
Pos
Pos
Pos
Pos
Pos
Pos
Pos
Pos
Pos
Pos
Pos
Pos
Pos
Pos
Pos
Pos
Neg
Pos
Pos
Neg
Pos
Pos
Neg
Pos
Neg
Pos
Pos
I/I
III/I
III/I
III/II
I/I
II/II
II/II
II/II
0/I
II/II
III/I
Pos
Pos
Pos
Pos
Pos
Pos
Pos
Pos
Pos
Pos
Pos
Pos
⬍6
⬍6
⬍6
⬍6
⬍6
⬍6
⬍6
⬍6
⬍6
⬍6
⬍6
⬍6
UI/mL
UI/mL
UI/mL
UI/mL
UI/mL
UI/mL
UI/mL
UI/mL
UI/mL
UI/mL
UI/mL
UI/mL
ND
Neg
Neg
Neg
Neg
Neg
ND
Neg
Neg
Neg
ND
Neg
Pos
Pos
Pos
Pos
Pos
Pos
Pos
Neg
Neg
Neg
Neg
Neg
Pos
Pos
Pos
Pos
Pos
Pos
Pos
Pos
Pos
Pos
Pos
Pos
Neg
Neg
Neg
Pos
Pos.
Neg
Pos
Pos
Neg
Neg
Neg
Pos
II/II
II/II
I/I
II/II
I/I
I/I
III/II
I/I
II/II
II/II
I/I
III/II
Neg
Neg
Neg
Neg
Neg
Neg
Pos
Pos
Pos
Pos
Pos
Pos
Pos
Pos
705 UI/mL
8740 UI/mL
1910 UI/mL
300 UI/mL
60 UI/mL
7740 UI/mL
1130 UI/mL
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
Neg
Neg
Neg
Neg
Neg
Neg
Neg
Neg
Neg
Neg
Neg
Pos
Neg
Pos
NA
NA
NA
NA
NA
NA
NA
Neg
Neg
Neg
Neg
Neg
Neg
Pos
Pos
Pos
Pos
Pos
Pos
597,000 UI/mL
2,700,000 UI/mL
77,100,000 UI/mL
1,180,000 UI/mL
968,000 UI/mL
1,410,000 UI/mL
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
Neg
Neg
Neg
Neg
Neg
Neg
Neg
Neg
Neg
Neg
Neg
Neg
NA
NA
NA
NA
NA
NA
Neg
Neg
Neg
Neg
Neg
Neg
Neg
Neg
Neg
Neg
Neg
Neg
Neg
Neg
Neg
Neg
Neg
Neg
ND
ND
ND
ND
ND
ND
ND
ND
ND
Neg
Neg
Neg
Neg
Neg
Neg
Neg
Neg
Neg
Neg
Neg
Neg
Neg
Neg
Neg
Neg
Neg
Neg
Pos
Pos
Pos
Pos
Pos
Pos
Pos
Pos
Pos
Neg
Pos
Neg
Neg
Neg
Pos
Neg
Neg
Neg
0/I
I/I
ND/I
II/II
III/II
I/I
I/I
III/II
I/I
HBsAb
HBcAb
Neg
Neg
Neg
Neg
Neg
Neg
Neg
Neg
Neg
Neg
Neg
Neg
Neg
Neg
Neg
Neg
Neg
Neg
Neg
Neg
Neg
Neg
Neg
Pos
Pos
Pos
Pos
Neg
Pos
Pos
Pos
Pos
Neg
Neg
Serum HBV DNA
⬍6
⬍6
⬍6
⬍6
Neg, negative; Pos, positive; ND, not determined.
aS/G staging and grading according to Sheuer histologic score.
envelope 183–191 (FLLTRILTI), envelope 335–343
(WLSLLVPFV), envelope 348 –357 (GLSPTVWLSV), polymerase 575–583 (FLLSLGIHL), polymerase 816 – 824
(SLYADSPSV) of genotype D, and the corresponding
synthetic peptides were purchased from Proimmune (Oxford, UK) and from Beckman-Coulter, Inc (Fullerton, CA).
Moreover, for phenotypic and functional characterization of HBV-specific T cells, anti-CD8 peridinin chlorophyll protein-labeled (PerCP) or allophycocyanin-labeled
(APC), anti-CD3 PerCP, anti-CD4 phycoerytrin-labeled
(PE), anti-CD107a phycoerytrin Cy5-labeled, anti-perforin fluorescein isothiocyanate (FITC), anti–program
death 1 (PD-1) FITC, anti-CD127, goat anti-mouse FITC
or APC (BD Biosciences–Pharmingen, San Jose, CA), anti–IFN-␥–FITC (Sigma–Aldrich, St Louis, MO) were used.
In Vitro Expansion of HBV-Specific T Cells
For CD8 expansion, PBMCs of HLA-A2–positive
patients were cultured with interleukin (IL)-7 (5 ng/mL)
and IL-12 (100 pg/mL) at a concentration of 2 ⫻ 106/mL,
seeded at 200 ␮L/well in 96-well plates, and stimulated
with HLA-A2–restricted HBV peptides at 1 ␮mol/L final
concentration. Recombinant IL-2 (50 IU/mL) was added
on day 3 of culture, and the immunologic assays were
performed on day 10. For PBMC stimulation with overlapping 15-mer peptides covering the overall HBV sequence, only IL-2 was added at day 3.
Cell Surface and Intracellular Staining
Staining with tetramers and other surface markers. PBMCs, either freshly isolated or after in vitro ex-
pansion for 10 days, were stained with PE-labeled tetramers. Tetramer-positive responses are reported as a
percentage of tetramer-positive T cells on total CD8 population. Frequencies of tetramer-positive CD8 cells 0.01%
or greater were considered positive. This threshold was
determined as the background signal plus 3 SD after
staining PBMCs from 5 HLA-A2–positive healthy uninfected controls.
Perforin staining. Tetramer-CD8 –stained cells
were fixed and permeabilized with Fix and Perm (Caltag
Laboratories, Burlington, CA) buffer in the presence of
antiperforin FITC. The cells were washed and immediately analyzed by flow cytometry.
IFN-␥ staining. Cells were incubated in medium
alone (unstimulated control) or with viral peptides (1
␮mol/L) for 1 hour; brefeldin A (10 ␮g/mL) was added
for an additional 15 hours of incubation. At the end of
the incubation, the cells were stained with tetramers,
anti-CD8 PerCP, and CD4-PE for 30 minutes at 4°C, and
then fixed and permeabilized in the presence of anti–
IFN-␥ FITC. Finally, cells were washed and analyzed by
flow cytometry.
Degranulation experiments. For CD107a staining, the specific antibody was added to effector CD8
cells at the beginning of the incubation time with or
without the relevant peptide. Staining with tetramers,
anti-CD3, and anti-CD8 was performed 2 hours later.
The degranulation capacity of the antigen-specific cytotoxic T lymphocytes was expressed as the frequency
of CD107a-positive cells.
ELISPOT Assay
ELISPOT assays were performed using the panel
of 315 peptides (15-mer) pooled in 16 mixtures. HBVspecific T-cell responses were analyzed after overnight
incubation with individual peptide mixtures of either
PBMCs (ex vivo analysis) or short-term polyclonal T-cell
lines previously expanded in vitro by 10 days’ stimulation
T–CELL RESPONSE IN OCCULT HBV INFECTION
1473
with the same peptide mixtures used for the ELISPOT
assay (in vitro analysis). Technical details are reported in
the supplementary material (see supplementary materials
and methods online at www.gastrojournal.org).
Statistical Analysis
Statistical analysis of mean spot-forming units
and frequencies of positive T-cell responses generated by
ELISPOT assay were compared by t test for unpaired data
and by chi-square analysis, respectively. The mean of
IFN-␥ positive in vitro expanded CD4 and CD8 cells also
were compared by t test for unpaired data. A P value of
less than .05 was considered significant.
Results
Identification of Occult HBV Infection
Intrahepatic HBV DNA was analyzed in 78
HBsAg-negative patients with CH-C who underwent liver
biopsy for IFN/ribavirin treatment. Eighteen of them
(23%) were positive for HBV DNA by nested PCR on at
least 2 different HBV genomic regions and confirmed by
sequencing of the amplified DNA fragments. Eleven of
the 18 patients with occult HBV infection were negative
for serum anti-HBV antibodies (anti-HBs and anti-HBc),
whereas the remaining 7 patients were seropositive for
anti-HBc and 5 of them also for anti-HBs (Table 1). Age
and sex distribution within the groups of patients with
and without occult HBV infection were similar. Prevalence of occult HBV infection in our cohort of CH-C was
in line with previously published results on patients from
similar geographic areas.9,11 Among the 60 CH-C patients
negative for intrahepatic HBV DNA, 5 anti-HBc–positive
and 9 seronegative subjects were studied for comparison
(Table 1).
The possible presence of HBV DNA in PBMC samples from 27 of the 32 patients tested for intrahepatic
HBV DNA also was examined (Table 1). Nested PCR
amplification and subsequent Southern blot analysis
did not reveal occult HBV in any of the samples examined. Even if a sensitive methodologic approach was
followed, the possibility that the lack of HBV-DNA
detection was influenced by heparin traces should be
considered because PBMCs were derived from heparinized blood. This possibility, however, does not
cast doubts on the accuracy of patient selection because occult infection was defined by analysis of liver
tissue.
HBV-Specific T-Cell Responses Can Be
Detected Ex Vivo in HBsAg-Negative Patients
With Occult HBV Infection
First, we asked whether a HBV-specific T-cell response is detectable in patients with occult HBV infection. To get a global representation of the overall T-cell
response against HBV, HBV-specific T-cell responses were
analyzed with a panel of 315 peptides (15-mer) covering
BASIC–LIVER,
PANCREAS, AND
BILIARY TRACT
May 2008
1474
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GASTROENTEROLOGY Vol. 134, No. 5
BASIC–LIVER,
PANCREAS, AND
BILIARY TRACT
Figure 1. Ex vivo IFN-␥ ELISPOT responses to individual HBV antigens in patients with and without occult HBV infection. (A) ELISPOT results obtained in
the different patient populations are illustrated according to responses obtained by stimulation with HBV peptides spanning individual HBV regions (x, core,
envelope, polymerase) and with recombinant envelope antigens corresponding to preS1, preS2, and S regions. (B) Comparisons of mean spots of the
positive tests and frequency of positive responses (calculated on all tests) obtained in the different patients’ groups are presented: □, HBV-DNA positive
and anti-HBc negative; , HBV-DNA positive and anti-HBc positive; □, HBV-DNA negative and anti-HBc positive; , CH-C.
the whole HBV sequence of genotype D and also with
recombinant HBV proteins expressing the preS1, preS2,
and S envelope regions. Synthetic peptides were pooled in
18 mixtures, 2 of them covering the X region, 2 the core
region, 4 the envelope region, and 8 the polymerase
region. Peptide pools and recombinant proteins were
used to stimulate PBMCs in an ELISPOT assay. By this
approach, HBV-specific T cells were detected in patients
with occult HBV infection at a frequency ranging between 30 and 120 specific T cells/106 PBMCs (supplementary Figure 1A and B; see supplementary material
online at www.gastrojournal.org). The frequency of HBVspecific T cells did not differ between anti-HBc–positive
and anti-HBc–negative subjects (Figure 1A and B). By the
same methodologic approach we also tested 5 anti-HBc–
positive CH-C patients negative for intrahepatic HBV
DNA (supplementary Figure 1C and Figure 1A; see supplementary material online at www.gastrojournal.org)
and 9 CH-C patients negative for HBV serum markers
and for intrahepatic HBV DNA, as controls (Figure 1A
and B). The mean values of spot-forming cells and the
mean frequencies of positive responses detected in seronegative and seropositive patients with and without occult HBV infection were not significantly different, although no T-cell reactivity could be detected in the
control group (Figure 1A and B).
Interestingly, anti-HBc–negative patients with occult
infection did not show T-cell responses to HBV core
(peptide mixtures 3 and 4; supplementary Figure 1A
and Figure 1A; see supplementary material online at
www.gastrojournal.org), whereas 4 of 6 anti-HBc–positive subjects with occult HBV infection (supplementary Figure 1B; see supplementary material online at
www.gastrojournal.org; and Figure 1A) and 2 of 5
anti-HBc–positive subjects with undetectable intrahepatic HBV-DNA (supplementary Figure 1C; see supplementary material online at www.gastrojournal.org; and
Figure 1A) displayed T-cell reactivity to the peptide
pools representing the HBV core region.
HBV-specific T-cell responses also were confirmed by
intracellular IFN-␥ staining in 2 anti-HBc–negative patients with occult infection from whom a sufficient number of frozen PBMCs was available (patients 7 and 8)
(supplementary Figure 2; see supplementary material online at www.gastrojournal.org), showing a CD4-mediated
T-cell response directed to envelope (patient 7) and to
polymerase (patient 8) sequences.
Ex Vivo Frequencies of HBV-Specific T Cells
Are Comparable in Patients With Occult
Infection and Inactive HBsAg Carriers
The ELISPOT results obtained in patients with
occult HBV infection also were compared with T-cell
responses generated with the same peptides and HBV
envelope antigens in 7 inactive HBsAg carriers (HBVDNA level, ⬍104 copies/mL) and in 6 anti-HBe–positive
patients with chronic hepatitis B (CH-B) (viral load, ⬎105
copies/mL). Inactive carriers showed frequencies of circulating HBV-specific T cells similar to the patients with
occult HBV infection (Figure 1A), whereas T-cell responses were almost undetectable in anti-HBe–positive
CH-B patients (Figure 1A).
HBV-Specific T-Cell Responses After In Vitro
Expansion Are Different in Anti–HBcNegative and Anti–HBc-Positive Patients
With Occult HBV Infection
By the use of the same peptide pools, short-term
T-cell lines were generated and tested in a culture ELISPOT.
To confirm the positive tests, T-cell lines showing at least
1.5 times the number of spots obtained with medium were
analyzed by intracellular IFN-␥ staining; this experimental
approach also allowed identification of the T-cell subset
responsible for the antigen-specific T-cell response. AntiHBc–positive subjects with or without detectable HBV DNA
in the liver and inactive HBsAg carriers showed the most
intense T-cell responses (Figure 2A), whereas lower levels of
T-cell expansion, which was sustained mainly by CD4 cells,
was detected in anti-HBc–negative patients with occult HBV
infection and in anti-HBe–positive CH-B patients (Figure
2A). In agreement with ELISPOT results generated ex vivo,
HBV core was the most immunogenic antigen for all patient groups with the exception of anti-HBc–negative pa-
T–CELL RESPONSE IN OCCULT HBV INFECTION
1475
tients with occult HBV infection. Envelope-specific T-cell
responses were almost totally undetectable in HBsAg-positive subjects. No HBV-specific T-cell expansion was observed in the control group, confirming the antigen specificity of the T-cell responses detected in the other patient
groups (Figure 2A).
HBV Tetramer Analysis Reveals the Presence
of Functional Memory HBV-Specific CD8
Cells in Seropositive Patients With Occult
Infection
To analyze phenotypic and functional features of
HBV-specific CD8 cells ex vivo and after short-term in
vitro expansion in the different patient groups, 6 HLA-A2
tetramers containing HBV epitopes known to be recognized frequently in HBV infection were used to identify
circulating HBV-specific CD8 cells. Seven patients with
occult HBV infection (4 anti-HBc–negative and 3 antiHBc–positive), 2 anti-HBc–positive subjects negative for
intrahepatic HBV DNA, and 2 HBsAg-inactive carriers
were tested with HBV–HLA-A2 tetramers. Ex vivo analysis
showed very low frequencies of circulating HBV-specific
CD8 cells in the 7 anti-HBc–negative patients with occult
infection that did not efficiently expand in vitro on peptide stimulation (Figure 2B). The low frequencies of tetramer-positive cells did not allow a reliable phenotypic
analysis of HBV-specific CD8 cells in this patient group.
Anti-HBc–positive subjects and HBsAg-inactive carriers
showed the highest frequencies of HBV-specific CD8
cells, ranging from 0.01% up to 0.185% of the total CD8
population (Figures 2B and 3). Phenotypic analysis, performed on 4 anti-HBc–positive subjects (2 positive and 2
negative for intrahepatic HBV DNA) showed a prevalent
memory phenotype characterized by the co-expression of
CCR7 and CD127 on the majority (from 83% up to 100%)
of HBV-specific CD8 cells, whereas PD-1 was expressed
on a minor fraction of them (from 21% to 29%) (Table 2).
Intracellular expression of perforin and granzyme also
was very low (Figure 3 and Table 2), indicating a prevalent central memory phenotype.18 In vitro stimulation
with specific peptides expanded HBV-specific CD8 cells
very efficiently, generating T-cell lines containing up to
70% of tetramer-positive cells. Moreover, expanded CD8
cells were functional in terms of IFN-␥ secretion and
cytotoxicity analyzed as surface CD107a up-regulation
(Figure 3).
In contrast to HBsAg-negative/anti-HBc–positive patients, ex vivo phenotypic analysis performed in HBsAginactive carriers showed a lower frequency of doublepositive CCR7/CD127 cells and a higher intracellular
perforin and granzyme content (Figure 3). HBV-specific
CD8 cells also expanded efficiently in these patients.
Analysis of the Entire PreC-C Genomic
Region of HBV DNA
Because of the lack of core-specific T-cell responses in anti-HBc–negative patients with occult infec-
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Figure 2. Intensity of the HBV-specific CD4- and CD8-mediated T-cell reactivity. After 10 days of in vitro stimulation with the same peptide pools
used for ex vivo analysis, expanded T cells were tested in a culture IFN-␥ ELISPOT assay. Tests showing at least 1.5-fold the number of spot-forming
colonies obtained with medium were confirmed by IFN-␥ intracellular staining (rate of confirmation of the positive ELISPOT results ranging from 75%
to 82% in the different patients’ groups). (A) Mean IFN-␥ production induced by HBV peptides is illustrated for patients with occult HBV infection
negative or positive for anti-HBc, for patients positive for anti-HBc and negative for intrahepatic HBV-DNA, for HBsAg-inactive carriers, for
anti-HBe–positive CH-B patients, and for CH-C patients negative for HBV serum markers and intrahepatic HBV DNA. Each bar in the upper part of
A represents the mean percentage ⫾ standard error of IFN-␥–positive CD4⫹ and CD8⫹ cells reactive to all peptide mixtures representing each HBV
antigen. Comparisons of mean frequencies of IFN-␥–positive CD4 and CD8 cells and frequencies of positive responses obtained in the different
patient groups to all peptide pools is presented at the bottom. , CD8⫹; □, CD4⫹. (B) The sum of the frequencies of tetramer-positive CD8 cells
measured ex vivo and after in vitro expansion in HLA-A2–positive patients is presented. Seven patients with occult HBV infection (4 anti-HBc–
negative and 3 anti-HBc–positive patients), 2 anti-HBc–positive patients with undetectable intrahepatic HBV DNA, and 2 HBsAg-inactive carriers
were studied. □, Ex vivo; , T-cell line.
tion, in a selected number of subjects with a sufficient
amount of liver-extracted DNA, preC-C genomic region
sequencing was performed. Sequences derived from 3
anti-HBc–negative patients (patients 1, 3, and 7) were
compared with sequences derived from 4 anti-HBc–positive patients (patients 12, 13, 15, and 16) all with occult
HBV infection (Figure 4). HBV sequences derived from 6
patients were of genotype D, whereas the HBV sequence
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Table 2. Phenotypic and Functional Analysis of Tetramer-Positive CD8 Cells
Ex vivo
Patient
Epitope
CCR7⫹/CD127⫹
PD1
Intrahepatic HBV-DNA–positive/anti-HBc–positive
16
ENV. 335–43
83%
21%
ENV 183–191
ND
ND
Core 18–27
ND
ND
18
Core 18–27
ND
ND
ENV 183–191
100%
ND
ENV. 335–43
100%
24%
POL 816–24
ND
ND
Intrahepatic HBV-DNA–negative/anti-HBc–positive
19
ENV. 335–43
91%
26%
23
Core 18–27
93%
29%
ENV. 335–43
ND
ND
HBsAg-positive inactive carriers
28
Core 18–27
69%
76%
30
Core 18–27
20%
ND
ENV. 335–43
ND
ND
POL 816–24
ND
ND
Short-term T-cell line
Perforin⫹ granzyme B⫹
 IFN-␥ ⫹
 CD107a
 IFN-␥ ⫹
 CD107a
Perforin⫹
ND
ND
ND
ND
ND
0%
ND
ND
ND
ND
ND
ND
26%
ND
65%
88%
ND
ND
ND
ND
ND
89%
95%
96%
71%
95%
62%
53%
57%
33%
ND
58.20%
88.10%
21.30%
52.30%
71%
64%
ND
9%
21%
15%
53%
5%
0%
ND
ND
28%
ND
ND
ND
ND
73%
52%
80%
ND
ND
ND
ND
89%
95%
9%
26%
ND
ND
23%
5%
ND
ND
ND
ND
ND
ND
96%
98%
63%
27%
ND
ND
ND
ND
76.8%
98%
72%
78%
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NOTE. Percentages indicate the frequencies of tetramer-positive CD8 cells expressing the indicated phenotypic and functional parameters.
ND, not determined.
Figure 3. Phenotypic and functional profiles of tetramer-positive CD8 cells. Top panels illustrate percentages of HBV tetramer-positive CD8
lymphocytes with different specificities, analyzed ex vivo and after 10 days in vitro expansion in representative HBsAg-negative patients with and
without occult HBV infection and in a HBsAg-inactive carrier. Lower panels illustrate the phenotypic and functional analysis of 3 representative
patients (patient 18, occult⫹ anti-HBc⫹; patient 23, occult- anti-HBc⫹; patient 28, HBsAg⫹-inactive carrier). CD127, PD1, CCR7, perforin, and
granzyme-B were tested ex vivo, whereas intracellular IFN-␥ and IL-2 expression as well as CD107a were tested after in vitro expansion.
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Figure 4. Sequencing of the entire preC-C genomic region in patients with occult HBV infection. Sequencing was performed in 3 anti-HBc–negative
(patients 1, 3, and 7) and 4 anti-HBc–positive patients (patients 12, 13, 15, and 16). HBV sequences derived from 6 patients were aligned to a
representative genotype D (GenBank accession number: V01460),19 whereas the HBV sequence derived from patient 12 was aligned to a
representative genotype A (GenBank accession number: X02763).27 The number of variations over the 212 aligned amino acid residues is shown in
parentheses for each patient.
derived from patient 17 aligned to genotype A (Figure 4);
none of the sequenced patients showed the G1896A mutation that determines a stop codon at position 28. AntiHBc–negative patient 3 with occult infection presented
the highest number of AA variations (Figure 4). Overall,
there was no dramatic increase in sequence variation in
the pre-CC region that could explain the reduced T-cell
reactivity to the core antigen in anti-HBc–negative patients.
Discussion
Occult HBV infection is defined by the persistence
of HBV in the serum and/or within the liver in the
absence of serum HBsAg. In this study, occult HBV
infection was defined when at least 2 different HBV
regions among preS-S, precore-core, Pol, and X were
amplified by PCR on liver tissue. Although the prevalence
of occult HBV infection in CH-C has been reported to be
around 30%,7 the prevalence among the general population still is unknown. The natural history is partially
understood because occult infection can represent the
final outcome of a past resolved infection with persistence of viral sequences, which have not been cleared
completely from the liver, or the result of a low-dose
infection without liver cell injury and a partial induction
or a total lack of humoral immunity. In the former
scenario of occult infection in the context of a resolved
hepatitis, initial priming of efficient HBV-specific T-cell
responses, followed by generation of protective memory,
is expected to occur. In the latter scenario of occult HBV
resulting from a low-dose infection, whether a cell-mediated antiviral response is primed successfully and what
type of response is mounted is totally undefined.
To shed further light on the natural history and pathogenesis of occult HBV infection, we compared HBV-specific T-cell responses detected in patients with occult
HBV infection with or without anti-HBc antibodies with
those displayed by HBV-immune subjects (anti-HBc–positive without HBV DNA in the liver) and chronic HBV
patients. In line with the possibility that in occult infection HBV replication is controlled by the cell-mediated
immune response, our study shows the existence of an
HBV-specific T-cell response in patients with occult HBV
infection, even when serum HBV markers are completely
negative. Several viruses, in particular DNA viruses and
retroviruses, can be confined to different compartments
most likely controlled by virus-specific T cells.20,21
In occult HBV infection, 2 different profiles of HBVspecific T-cell responses were displayed by patients with
and without anti-HBc antibodies. Even if in seronegative
patients with cryptic infection circulating HBV-specific T
cells were detected at frequencies comparable with inactive HBsAg carriers and immune subjects (HBsAg negative, anti-HBc positive), in vitro expansion and IFN-␥
production by HBV-specific T cells was much weaker
than in anti-HBc–positive patients with or without intrahepatic HBV DNA. Moreover, T-cell expansion almost
completely was limited to the CD4 cell subset. The length
of the peptides, although not optimal for CD8 cells, was
suitable for CD8 recognition because CD8 cells were
expanded readily in HBsAg-positive inactive carriers and
in patients with acute HBV infection tested for comparison (data not shown). Thus, predominance of CD4mediated responses seems to be a feature of the immune
response in these patients, rather than a technical artifact. In addition, the core antigen, which was the most
immunogenic HBV antigen for the other groups of patients, was not recognized by T cells derived from seronegative occult patients. Even if sequencing was performed in only 3 anti-HBc–negative patients with occult
T–CELL RESPONSE IN OCCULT HBV INFECTION
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HBV infection, this lack of recognition does not seem to
be the result of mutations of the preC-C region.
In contrast to seronegative occult patients, HBV-specific T cells were able to expand and to produce IFN-␥
efficiently in vitro in anti-HBc–positive subjects, either
positive or negative for intrahepatic HBV DNA, with a
phenotypic and functional profile of fully competent
memory T cells, as confirmed also ex vivo by the high
level of CD127 and CCR7 expression and the low rate of
PD-1–positive cells. Trace amounts of virus have been
reported in previous studies to persist even years after
resolution of acute HBV infection,2,3 showing that clinical recovery does not imply complete HBV clearance but
rather reflects the capacity of the immune system to keep
under tight control minute amounts of virus that are
expected to remain after clinical resolution of disease. If
this is the case, the likelihood of detecting HBV DNA in
liver and serum thus may vary over time in individual
resolved acute infections in relation to the level of efficiency of immune control.
These distinct behaviors of cell-mediated immune responses in seropositive and seronegative occult HBV infections may reflect different modalities of HBV transmission. As shown for the woodchuck HBV, exposure to
low woodchuck HB doses (⬍103 virions) may give rise to
a persistent infection without serum markers. Interestingly, this woodchuck primary occult infection does not
confer protective immunity, suggesting that also in this
animal model a functional memory T-cell response is
generated only after infection with a higher dose of
inoculum.22 Unfortunately, only a single study tried to
characterize this type of HBV transmission in the chimpanzee model of infection. Human sera derived from
subjects negative for serum HBV markers caused acute
hepatitis when inoculated into 2 chimpanzees, one of
which developed HBsAg and anti-HBc and the second
one developed only anti-HBs.23 Immune responses, however, were not characterized and additional studies have
not been reported in this animal model.
Long-lasting memory CD4 and CD8 cell responses
detectable several years after recovery from acute hepatitis B have been reported previously in anti-HBc–positive
patients, with HBV-DNA traces in PBMCs and serum.2,3
In our study, ex vivo and in vitro T-cell responses in
anti-HBc–positive subjects with or without HBV DNA in
the liver were comparable. This supports the possibility
that anti-HBc–positive occult infections represent the
resolution phase of infections with high doses of virus
that the immune system has been able to control efficiently with successful maturation of protective T-cell
memory.
The finding of a poor in vitro T-cell expansion in
seronegative patients with occult infection raises questions about the mechanisms of control of HBV replication in this condition. Even if experimental data in support of this interpretation are lacking, we are tempted to
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speculate that control of the virus principally is the result
of the innate immune response. This would limit liver
cell damage and the release of inflammatory signals normally involved in activation and maturation of dendritic
cells needed for massive expansion of CD4 and CD8 cell
clones. In particular, the lack of CD8 cell expansion in
seronegative patients could be the result of a dysfunctional CD4 response unable to expand and maintain
memory CD8 cells. Moreover, a dysfunctional core-specific CD4 cell response also could provide insufficient
T-cell help to core-specific B cells with a lack of detectable
anti-HBc in the serum. An alternative hypothesis to explain virus control in these patients, which does not
exclude the previous one, is that co-infection with HCV
or other undefined hepatotropic viruses can interfere
with HBV replication, as shown for HCV core protein,24 –26 and that T-cell responses specific for HCV or
the other viruses can inhibit HBV replication through
local release of cytokines, such as IFN-␥ and tumor necrosis factor-␣, able to affect virus replication by a nonspecific antiviral effect.
In conclusion, in occult HBV infection different mechanisms have been proposed to explain why HBV remains
mainly confined to the liver with only transient low levels
of viremia. These include interference with HBV replication of concomitant viral infections, regulation of HBV
gene transcription by cellular mechanisms, impaired replicative competence of HBV as a result of specific virus
mutations, and control of HBV replication by the immune response. Our study supports this latter mechanism in anti-HBc–positive patients with occult infection
who display features of HBV-specific T cells that are
typical of protective memory. These HBV-specific T-cell
responses are similar to those observed in resolved HBV
infections, supporting the interpretation that anti-HBc–
positive patients with or without detectable intrahepatic
HBV DNA actually represent the same biological entity
corresponding to patients with a prior resolved infection
with variable efficiency of virus control. In contrast, the
type of cell-mediated immune response in patients with
occult infection associated with negative serum HBV
markers suggests the possibility of a low-dose infection
insufficient to allow maturation of an antiviral protective
memory response.
Additional studies are needed to understand whether
the different immune responses and natural histories
associated with the 2 types of occult infections suggested
by our studies also have different clinical implications
with respect to the development of hepatocellular carcinoma and responsiveness to antiviral therapy. Moreover,
the new emerging protocols of immunosuppressive treatments for neoplastic, rheumatic, and hematologic disorders may have a different impact on HBV reactivation in
seronegative and seropositive occult infections.
GASTROENTEROLOGY Vol. 134, No. 5
Supplementary Data
Note: To access the supplementary material accompanying this article, visit the online version of
Gastroenterology at www.gastrojournal.org, and at doi:
10.1053/j.gastro.2008.02.017
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Received August 6, 2007. Accepted January 31, 2008.
Address requests for reprints to: Gabriele Missale, MD, Divisione
Malattie Infettive ed Epatologia, Azienda Ospedaliera e Universitaria di
Parma, Via Gramsci 14, 43100 Parma, Italy. e-mail: [email protected];
fax: (39) 0521-703857.
This study was supported by Schering-Plough S.p.A. (Milan, Italy),
grant RBNE013PMJ_006 from the Fondo per gli Investimenti della
Ricerca di Base, Ministry of Education, University and Research, by the
VIRGIL EC grant QLK2-CT-2002-00700, and by the Fondazione Cassa
Risparmio di Parma (Parma, Italy).
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