1. Top of page
  2. Abstract
  3. Patients and Methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. References
  8. Supporting Information

The hepatitis B X (HBx) protein is a crucial component in HBV infection in vivo and has been implicated in HCC. In this study, we aimed to detect and characterize peripheral HBx-specific T cells in chronically infected patients at the inactive carrier state of the disease. HBx-specific IFN-γ–secreting T cells were found in 36 of 52 patients (69%), and 78% (28/36) of responding patients had T cells targeting epitopes in the carboxy-terminal part of HBx. IL-10 secretion after the stimulation of T cells with HBx-derived peptides was weak or undetectable. IFN-γ–secreting T cells recognized a previously unknown immunodominant CD4+ T cell epitope, HBx 126–140 (EIRLKVFVLGGCRHK), in 86% (24 of 28) of patients. This peptide bound several HLA-DR molecules (HLA-DRB1*0101, HLA-DRB1*0401, HLA-DRB1*1301, and HLA-DRB5*0101). Its coding sequence overlaps a domain of the HBV genome encompassing the basic core promoter (BCP) region. Taking into account the selection of viral core promoter mutants during HBV infection, we found that HBV variants with BCP mutations were present in patient sera. We further demonstrated that these viral mutant sequences activated T cells specific for the immunodominant epitope only weakly, if at all. This is the first study linking BCP mutations and HBx-specific T cell responses. Conclusion: Wild-type and variant peptides may represent potential tools for monitoring the HBV-specific T cell responses involved in sequence evolution during disease progression. Finally, the degenerate HLA-DR binding of this promiscuous, immunodominant peptide would make it a valuable component of vaccines for protecting large and ethnically diverse patient populations. (HEPATOLOGY 2007;45:1199–1209.)

An effective vaccine against HBV infection has been available for more than 2 decades, but 400 million people—more than 5% of the world's population—are chronically infected with HBV, and more than 1 million people die each year of HBV-related liver cirrhosis and HCC.1, 2

The hepatitis B X (HBx) protein is a key element in HBV infection in vivo and has been implicated in HCC development. HBx is well conserved among mammalian hepadnaviruses and is produced very early after infection and throughout chronic infection. The potentially oncogenic functions of HBx include the transcriptional activation of genes encoding proteins regulating cell growth, apoptosis modulation, and the inhibition of nucleotide excision repair after DNA damage. HBx exerts its effects by interacting with cellular proteins and activating cell signaling pathways.3, 4

The pathogenesis of HBV infection involves the selection and expression of several common viral mutants. HBV genes have overlapping open reading frames. A mutation in the HBV genome may therefore have effects on several proteins. The HBx gene overlaps regions of crucial importance for viral replication, such as the direct repeat sequences DR1 and DR2, the preC/C gene promoter, and the enhancer II region. The common double mutation in the HBV basic core promoter (BCP) region A1762T/G1764A corresponds to a double mutation in codons 130 and 131 of the HBV X gene. The change in HBx amino-acid sequence (K130M and V131I) resulting from these T-A point mutations is associated with severe liver damage and HCC.5–8 These substitutions may be associated with an additional mutation at position 127 in the HBx protein, which has been detected in patients with HCC or fulminant hepatitis.9, 10 Natural mutations in the HBx gene are thought to lead to progression to chronic disease because of the abolition of antiproliferative and apoptotic effects, causing uncontrolled growth and multistep hepatocarcinogenesis.11 The selection and expression of natural HBx mutants may have major implications for T cell recognition of this protein.

Because HBV is mainly not directly cytopathic, the immune response to viral antigens is thought to be responsible for both liver disease and viral clearance after HBV infection. Patients with acute viral infection who successfully clear the virus display a multispecific polyclonal cytotoxic T lymphocyte response specific for a number of epitopes within the core, polymerase, and envelope proteins.12–15 HBV-specific T helper (Th) cells are also activated, and multispecific Th1-like responses are detected in patients successfully clearing HBV after acute infection.16 This HBV-specific T cell response is weak or undetectable in patients who develop chronic infection.17 Little is known about the cytotoxic T lymphocyte directed against HBx protein in HBV-infected individuals18, 19 or about HBx-specific CD4+ T cells and their cytokine profile during the course of viral infection.20

We characterized peripheral HBx-specific T cells in 52 patients with chronic HBV infection at the inactive carrier state of the disease, by measuring interferon gamma (IFN-γ) and interleukin-10 (IL-10) secretion after the activation of peripheral blood mononuclear cells (PBMC) with 15-mer peptides spanning the HBx sequence. We identified an immunodominant, promiscuous T cell epitope, HBx 126–140, located in the carboxy-terminal part of the protein and recognized by IFN-γ–secreting CD4+ T cells in most patients. HBV core promoter mutations, which frequently occur during chronic infection, modify the sequence of this HBx-derived immunodominant CD4+ T cell epitope. These mutant viral sequences were recognized by T cells specific for the HBx wild-type epitope only weakly, if at all.

Patients and Methods

  1. Top of page
  2. Abstract
  3. Patients and Methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. References
  8. Supporting Information

Patient Population.

Fifty-two subjects with chronic HBV infection, in the inactive carrier state of the disease21 with less than 100,000 HBV copies/ml, were enrolled (Supplementary Table 1). All were hepatitis B surface antigen–positive, hepatitis B e antigen (HBeAg)–negative, anti–HBe antibodies-positive, and had normal transaminase levels and no or low underlying liver disease. This group of patients is heterogeneous because it contains (i) patients with persistently low HBV DNA levels, even falling to undetectable levels (<200 cp/ml) either spontaneously or after effective antiviral treatment and (ii) patients with fluctuating levels of HBV DNA being nevertheless <100,000 cp/ml. This sub-group may include patients carrying HBV viruses with preC or BCP mutations.22 Patients had received no antiviral treatment for at least 6 months before inclusion. All patients were 18 to 60 years old, had no immunosuppression or infections associated with human immunodeficiency virus, HCV, or hepatitis D virus, or liver diseases other than HBV infection, and consumed less than 40 g alcohol/day. HLA-DR genotyping was carried out with the Olerup SSP Genovision kit (Saltsjöbaden, Sweden). Two blood samples were collected from each patient at a mean of an 8 ± 4-month interval. This study was approved by the ethics committee of the hospital, and all participants gave informed, written consent for participation, in line with French ethical guidelines.

Synthetic Peptides.

Synthetic peptides were purchased from NeoMPS (Strasbourg, France). The consensus sequence of the HBx protein, obtained by comparing published HBx-encoding sequences in Genbank—MAARLCCQLDPARDVLCLRPVGAESRGRPLSGPLGTLSSPSPSAVPTDHGAHLSLRGLPVCAFSSAGPCALRFTSARRMETTVNAHQILPKVLHKRTLGLSAMSTTDLEAYFKDCLFKDWEELGEEIRLKVFVLGGCRHKLVCAPAPCNFFTSA— was covered by 29 15-mer peptides with 10-residue overlaps. Individual or pooled peptides were used to stimulate PBMCs in vitro and for the Elispot assay. Three peptide pools were used: pool A (peptides x1 to x10), pool B (peptides x11 to x20), and pool C (peptides x21 to x29). Two additional peptides corresponding to variant sequences of the wild-type x26 peptide (HBx 126–140, EIRLKVFVLGGCRHK) were used: V2 (EIRLMIFVLGGCRHK) and V3 (ETRLMIFVLGGCRHK). Peptides were prepared at 1 mg/ml in water or 20% DMSO if required and stored at −20°C until use.

HLA-DR Peptide-Binding Assays.

HLA-DR molecules were purified from homologous EBV cell lines by affinity chromatography, as previously described.23, 24 Binding to various HLA-DR molecules was assessed by competitive enzyme-linked immunosorbent assay, as previously described.23, 24 We used the individual peptides of pool C and a 20-mer HBc-derived peptide (core 50–69; PHHTALRQAILCWGELMTLA) as competitors.25 Maximal binding was determined by incubating the biotinylated peptide with the MHC class II molecule in the absence of competitor. Binding specificity for each HLA-DR molecule was ensured by the choice of the biotinylated peptides as described.23, 24 Concentration of the peptide that prevented 50% of binding of the biotinylated peptide was evaluated (concentration that inhibits 50%; IC50). The reference peptide is the unlabeled form of the biotinylated peptide and corresponds to high binder. Their IC50 are the following: 2 nM for DRB1*0101; 403 nM for DRB1*0301; 38 nM for DRB1*0401; 6 nM for DRB1*0701; 6 nM for DRB1*1101; 170 nM for DRB1*1301; 26 nM for DRB1*1501; 15 nM for DRB3*0101; 10 nM for DRB4*0101, and 6 nM for DRB5*0101. Data are expressed as relative activity: ratio of the IC50 of the peptide by the IC50 of the reference peptide.

In Vitro Expansion of the PBMC.

PBMC were suspended at 3 × 106 cells per milliliter in complete medium (RPMI 1640 medium, Life Technologies, Gaithersburg, MD) supplemented with 2 mM L-glutamine, 1 mM sodium pyruvate, nonessential amino acids, 100 U/ml penicillin, 100 μg/ml streptomycin, and 10% human AB serum (Institut Jacques Boy, Reims, France) plus 20 ng/ml IL-7 (Roche, Meylan, France) and 100 pg/ml IL-12 (R&D Systems Inc., Minneapolis, MN), in 24-well plates. Cells were stimulated by incubation with peptide pools A, B, and C (1 μg/ml of each peptide) or with individual peptides (10 μg/ml). Half the medium was replaced every 3 to 4 days with complete medium supplemented with recombinant IL-2 (50 IU/ml) (Roche, Meylan, France). After 10 to 14 days of culture, HBx-specific IFN-γ– and IL-10–producing cells were quantified by Elispot assays and intracellular cytokine staining.

Elispot Assay.

Sterile nitrocellulose HA 96-well plates (Millipore, Bedford, MA) were coated with 15 μg/ml anti–IFN-γ monoclonal antibody (clone 1-DIK; Mabtech, Stockholm, Sweden) in 0.1 M bicarbonate buffer (pH 9.6) or with 10 μg/ml anti-IL-10 monoclonal antibody (clone B-N10, Diaclone, Besançon, France) in PBS (pH 7.0). The wells were blocked and washed,26 then filled, in triplicate, with in vitro stimulated cells (1 to 2 × 105/well) in complete medium and the appropriate peptides (1 μg/ml), with medium alone used as a negative control and phorbol myristate acetate (25 ng/ml)/ionomycin (2 mg/ml), or staphylococcal enterotoxin B (500 ng/ml) (Sigma, St. Louis, MO), as positive control. After 20 hours of incubation at 37°C, plates were washed and incubated with 1 μg/ml biotinylated anti–IFN-γ monoclonal antibody (clone 7B6-1; Mabtech) or with 20 μg/ml biotinylated anti–IL-10 monoclonal antibody (clone B-T10; Diaclone) for 2 hours at room temperature. Plates were then washed, and antibody binding was detected as described.26 A Zeiss Elispot automatic counter was used to score the number of spots.

The specificity and cutoff of Elispot assays were determined with PBMCs from healthy individuals (n = 9) and with PBMC from hemochromatosis patients (n = 2). These PBMCs were subjected to in vitro expansion with HBx-derived peptides and tested in Elispot assays in experimental conditions identical to those used for PBMC from chronic HBV carriers. The cutoff of Elispot assays were 62 IFN-γ and 40 IL-10–spot-forming cells (SFC) per million PBMCs, calculated as mean + 2 SD SFC per million PBMCs from HBV-negative subjects. The response was considered positive if the median number of SFC in triplicate wells was at least twice than in control wells without peptide and was superior to the cutoff values.

Inhibition of Elispot Assays.

Class II HLA-restriction was determined, after in vitro expansion, by incubating PBMC for 90 minutes at 37°C with 10 μg/ml anti–class II HLA antibodies: anti–HLA-DR (L243) from ATCC, anti–HLA-DQ (SPVL3), and anti–HLA-DP (B7/21) provided by Dr. Y. van de Wal (Department of Immuno-hematology and Blood Bank, Leiden, The Netherlands). Anti–class I HLA-A2 antibody (BB7-2) was used as a negative control. Preincubated PBMCs were then tested in Elispot assays, as described above.

Intracellular Staining.

Populations of PBMCs expanded in vitro were incubated overnight either with 500 ng/ml staphylococcal enterotoxin B (Sigma) as a positive control, with medium alone as a negative control, or with individual HBx-derived peptides (1 μg/ml) and brefeldin A (2 μg/ml) (Sigma). After washing, cells were stained with appropriate combinations of monoclonal antibodies—anti–CD3-APC (clone HIT3a; BD Pharmingen), anti–CD4-PE (clone RPA-T4; BD Pharmingen), anti–CD4-APC (clone RPA-T4; Serotec), and/or anti–CD8-PerCP (clone SK1; BD Biosciences)— for 30 minutes at 4°C and washed again. Cells were fixed, permeabilized, and stained with anti-human IFN-γ–FITC antibody (clone 4S.B3; BD Biosciences) and with anti-human IL-10–PE antibody (clone JES3-9D7; Serotec) for 30 minutes at 4°C and washed again. At least 50,000 lymphocyte-gated events were acquired on a FACSCalibur flow cytometer (BD Biosciences) and analyzed with Cellquest (BD Biosciences). Background staining was assessed with an isotype-matched control monoclonal antibody and subtracted from all values.


  1. Top of page
  2. Abstract
  3. Patients and Methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. References
  8. Supporting Information

Presence of HBx-Specific IFN-γ–Secreting T Cells in Chronic HBV Carriers.

We analyzed the HBx-specific IFN-γ–secreting T cell response by Elispot assays and the use of three peptide pools (A, B, C) covering the entire HBx sequence to stimulate PBMCs from 52 chronic inactive carriers of HBV. HBx-specific IFN-γ–secreting T cells were found in 36 of the 52 patients studied (69%): 28 (78%) had T cells recognizing epitopes in the carboxy-terminal part of HBx (pool C), 17 (42%) had T cells specific for the central region of the protein (pool B), and nine (25%) had T cells specific for epitopes in the amino-terminal part of the protein (pool A) (Fig. 1A). The diversity of HBx-specific IFN-γ–secreting T cell responses to pools A, B, and C is shown in Fig. 1B. No more than 10% of patients displayed specific responses to all 3 peptide pools.

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Figure 1. Presence and diversity of HBx-specific IFN-γ–secreting T cells in 52 patients with chronic HBV infection. PBMCs were stimulated in vitro with HBx-derived 15-mer peptides covering the whole HBx sequence, divided into pools A, B, and C. IFN-γ–secreting T cells were determined by Elispot, using the same peptide pools. The proportion of patients testing positive is indicated on the top of each column. (A) Percentage of patients with undetectable HBx-specific T cells (white column) and with HBx-specific T cells activated with each peptide pool (gray columns); (B) diversity of recognition of regions within the HBx protein by HBx-specific T cells (gray striped columns).

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Mapping of the IFN-γ–Secreting T Cell Response to Individual Pool C Peptides.

Because most HBx-specific IFN-γ–secreting T cells recognized the carboxy-terminal part of the protein, we mapped the single epitopes targeted by T cells in this region. PBMC from the 28 patients with pool C–specific T cells were stimulated with the entire peptide pool C and with individual pool C peptides, and then tested in Elispot assays (Fig. 2). T cell activation after PBMC culture with the entire peptide pool resulted from specific stimulation with a single peptide, as 24 of the 28 patients (86%) had T cells specific for the x26 epitope (compare left and middle panels of Fig. 2). Moreover, x26-specific T cells were the only T cells reactive against the domain of the protein covered by peptide pool C in at least 13 of the 28 patients (46%), and an absence of T cells recognizing the x26 epitope was noted in only 4 of these patients (patients P62, P34, P53, and P38). We analyzed PBMCs from 11 uninfected individuals to check the specificity of these responses. None had T cells responding to pool C or x26 peptides in Elispot assays after in vitro expansion (data not shown).

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Figure 2. IFN-γ–secreting T cells upon stimulation with peptide pool C and mapping of the T cell response to single peptides. Number of IFN-γ–secreting T cells determined by Elispot and expressed as the number of specific spot-forming cells (SFC)/106 PBMCs after in vitro stimulation with peptide pool C (left panel), individual peptide x26 (middle panel), and single peptides or groups of peptides from pool C with the exception of x26 peptide (right panel). In the right panel, the number of SFC is indicated on each bar. The scale of the right panel differs from that of the left and central panels. ND: not done.

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T cells specific for individual pool C peptides other than x26 were detected in eight patients (Fig. 2, right panel). In the absence of x26-specific T cell reactivity, T cells recognized the x22 peptide (patients P62, P53 and P38). In 5 of the 24 patients with x26-specific T cells, weak reactivity to x21-x25 region and x27 was also observed (patients P31, P46, P59, P28, and P23; right panel, Fig. 2). With the exception of patient P23, whose T cells were more strongly activated with x25 than with x26, specific T cell reactivity was 5 to 10 times higher for x26 than for other pool C single peptides (compare middle and right panels).

In conclusion, IFN-γ–secreting T cells recognizing the carboxy-terminal domain of HBx targeted a single immunodominant epitope. Because this epitope was recognized by T cells from a large number of patients expressing different HLA molecules, x26 peptide may be considered a promiscuous epitope.

HBx-Derived Peptides Activated IFN-γ– and IL-10–Secreting T Cells in Chronic HBV Carriers.

Even if HBx-specific IFN-γ–secreting T cells were found in PBMCs from most of the studied patients, we asked whether IL-10 secretion can be detected in PBMCs from IFN-γ–Elispot-negative patients. We quantified and compared IFN-γ and IL-10 secretion after the in vitro expansion of PBMCs from 31 chronic HBV carriers with and without pool C–specific IFN-γ–secreting T cell responses. In 13 of the 31 patients (42%), neither IFN-γ– nor IL-10–producing specific T cells were detected. No IL-10 secretion was observed in 11 patients (35%) with IFN-γ–secreting T cell responses of various magnitudes (Fig. 3A). Finally, only 7 of the 31 patients (23%) with generally strong IFN-γ–secreting T cell responses had detectable numbers of specific IL-10–secreting T cells (Fig. 3B). Except in patient P26, the frequency of IFN-γ–secreting T cells was always higher than that of IL-10–secreting T cells (Fig. 3B). The IFN-γ–secreting T cell response was approximately 10 times higher than the IL-10–producing T cell response in Elispot (range, 4.8-fold to 27.4-fold; median, 11.5-fold).

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Figure 3. IFN-γ– and IL-10–secreting T cells determined by Elispot assays after in vitro stimulation of PBMCs with the entire peptide pool C or x26 alone. Number of IFN-γ– or IL-10 spot-forming cells (SFC)/106 PBMCs (black and gray columns, respectively) of 11 patients with only IFN-γ–secreting T cells (A panel) and seven patients with both IFN-γ– and IL-10–secreting T cells (B panel). Number of SFC is indicated on the top of each column. The cutoff points for Elispot assays are 62 IFN-γ– and 40 IL-10–SFC/106 PBMCs.

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IL-10 secretion was observed in only 2 and 1 of the 24 studied patients after the activation of PBMC with peptide pools A and B, respectively. IFN-γ secretion by activated T cells was detected simultaneously in these three patients with IL-10–producing T cells. Peptide pool A activated 505 IFN-γ– and 170 IL-10–secreting T cells per million PBMCs from patient P7 and 104 IFN-γ– and 313 IL-10–SFC per million PBMC from patient P27. Finally, 652 IFN-γ– and 79 IL-10–SFC pool B-specific T cells per million PBMC were detected for patient P41.

Overall, IL-10 secretion after T cell activation with HBx-derived peptides was weak or undetectable. Studies focusing on the carboxy-terminal region showed that peptide pool C or x26 activated IFN-γ production more efficiently than IL-10 production.

Phenotype of x26-Specific T Cells.

We investigated whether the x26 epitope activated CD4+ or CD8+ T cells, by intracellular IFN-γ staining of PBMC stimulated in vitro with x26. The phenotype of CD3+ IFN-γ–producing x26-specific T cells from a representative patient (P30) is shown in Fig. 4A. The promiscuous, immunodominant x26 peptide specifically stimulated CD4+ but not CD8+ T cells. This result was confirmed in 10 patients with x26-positive response.

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Figure 4. Phenotype of x26-specific T cells after in vitro expansion from PBMC with x26. (A) Percentages of IFN-γ–secreting CD3+ CD4+ (left panel) or CD3+ CD8+ (right panel) specific T cells are shown. (B) Within the CD4+ T cell population, the percentages of x26-specific CD4+ T cells secreting IFN-γ (left panel), IL-10 (central panel), and IL-10 and/or IFN-γ (right panel) are shown.

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The x26-specific T cell response of patient P26 was further characterized by intracellular staining of both IFN-γ and IL-10, to identify more precisely the T cells producing either or both cytokines. We found that 7.66% of CD4+ T cells produced IFN-γ and 0.73% produced IL-10 (Fig. 4B, left and central panels) However, most CD4+ T cells activated by x26 (6.95% of total CD4+ T cells) produced IFN-γ but not IL-10 (Fig. 4B, right panel). Less than 10% of the x26-specific CD4+ T cell population (0.73% of total CD4+ T cells) produced IL-10 together with IFN-γ. No activated T cells producing IL-10 only were observed.

This is consistent with the small number of IL-10–producing T cells identified by Elispot and highlights the more precise quantification of IFN-γ and IL-10 by intracellular staining than by Elispot assays when these cytokines are produced simultaneously (compare Figs. 3B and 4B).

HLA Class II Restriction of x26 Peptide.

We used 3 experimental approaches to characterize the HLA class II restriction of x26. After in vitro expansion, PBMC were incubated with anti–HLA-DR, anti–HLA-DQ, or anti–HLA-DP antibodies and tested in Elispot assays. Prior incubation with anti–HLA-DR antibodies inhibited IFN-γ secretion upon stimulation with x26 by at least 80%. No such effect was observed after the prior incubation of PBMCs with anti–HLA-DP or anti–HLA-DQ antibodies, or with irrelevant control anti–HLA-A2 antibodies (Fig. 5). The x26 epitope is therefore presented by HLA-DR molecules.

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Figure 5. Anti-MHC class II antibody-mediated inhibition of IFN-γ secretion by x26-specific T cells. PBMCs expanded in vitro with peptide x26 were first incubated with anti-class II HLA antibodies—anti–HLA-DR, anti–HLA-DQ, or anti–HLA-DP—or with an irrelevant antibody (anti–HLA-A2). PBMC were then tested in Elispot assays as described in Patients and Methods. Results obtained with 3 representative patients are shown.

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We assessed the capacity of x26 and of other pool C peptides to bind to various purified HLA-DR molecules. A hepatitis B core antigen–derived peptide, c59-60, described as HBV promiscuous epitope,25 was tested in parallel (Table 1). High-affinity binding was observed for x25 peptide with at least 4 of the 10 HLA-DR molecules tested (DRB1*0301, DRB1*1101, DRB1*1301, and DRB1*1501). The x26 peptide could be presented by the HLA-DRB1*0101, HLA-DR01*0401, HLA-DRB1*1301, and HLA-DRB5*0101 molecules. DRB1*1301 molecules could bind x22, x23, and x27 peptides as well, whereas x22 also bound to DRB4*0101. In comparison, the hepatitis B core antigen–derived promiscuous epitope exhibited a good affinity for DRB1*0301, DRB1*1301, and DRB1*1501.

Table 1. Binding of HBx-Derived Peptides to Immunopurified Class II HLA Molecules
PeptidesHLA Class II Molecules
DRB1 *0101DRB1 *0301DRB1 *0401DRB1 *0701DRB1 *1101DRB1 *1301DRB1 *1501DRB3 *0101DRB4 *0101DRB5 *0101
  1. NOTE. Data are expressed as relative activity: ratio of the IC50 of the peptide to the IC50 of the reference peptide. The relative activities of pool C peptides and an HBc-derived peptide (c50–69) are shown. Boldface indicates relative binding affinity below 100 and corresponds to good binders.


Finally, we genotyped the HLA-DR molecules of 30 inactive HBV carriers with or without x26-specific T cells. For the HLA-DRB1 gene, the prevalence of alleles DRB1*0301 (in 9 of 30 patients), DRB1*0401 (9 of 30), DRB1*1101 (9 of 30), and DRB1*1301 (7 of 30) was high among the studied patients. In 16 patients, the presence or absence of HLA-DR alleles binding x26 (HLA-DRB1*0101, HLA-DR01*0401, HLA-DRB1*1301, and HLA-DRB5*0101) was found to be related to specific IFN-γ–secreting T cell reactivity (Table 2).

Table 2. Comparative Analysis of Patients' HLA-DR Genotypes, x26-Binding HLA-DR Molecules and x26-Specific IFN-γ-Secreting T Cell Responses
PatientsHLA-DR genotypePresence of HLA-DR with x26-binding capacity†x26-specific IFN-γ-secreting T cells‡
  1. Brackets for DRB1 genotype indicate that heterozygosity could not be confirmed with the current assay. †HLA-DR molecules binding x26, as shown in Table 1 for DRB1*0101, DRB1*0401, DRB1*1301, and DRB5*0101. ‡Determined by Elispot assays and expressed as ranges of IFN-γ-SFC per million PBMC: + 100–500; ++ 500–1000; +++ 1000–2000 and ++++ >2000.

P32*0701, *0901  Nonegative
P8*0301  Nonegative
P64*0801, *0901  Nonegative
P44*0301  Nonegative
P34*0301, *0701 Nonegative
P3*0301, *1301  Yes++
P41*0101, *0401  Yes++
P45*1101, *1301  Yes++++
P11*0301, *1301  Yes+
P30*0401, *1101 Yes+++
P27*0401, *1301 Yes+
P50*0101, *0301  Yes+
P59*0401, *1101 Yes++++
P46*1101, *1301  Yes++++
P12*0301, *1301  Yes+
P31*0101, *1501  Yes+++
P23*0301 (*1367)  No+
P35*0301, *1101  No++
P6*0701, *1101 No+++
P51*1101, *1201  No+++
P26*1101, *1201  No+++
P47*1101, *1201  No+++
P13*0401, *1301 Yesnegative
P1*0401, *0701  Yesnegative
P2*0801, *1501  Yesnegative
P48*0401, *1401 Yesnegative
P4*0401 (*1367)  Yesnegative
P38*1201, *1501 Yesnegative
P53*0101, *0401  Yesnegative
P62*0101, *1501  Yesnegative

Recognition of Viral Mutants by x26-Specific T Cells.

Some HBx mutations in basic core promoter mutant viruses concern the x26 epitope. An analysis of sequences from 40 cloned HBV genomes published in GenBank ( shows that the frequent codon 130 and 131 (K130M and V131I) mutations were present in 12 of the 40 sequences. Codon 127 (I127T) mutation occurred in 4 of the 40 HBx sequences. We therefore evaluated the recognition of viral sequence variants by x26-specific T cells. For PBMC stimulation in vitro, we used x26 peptide, covering the wild-type epitope, separately or mixed with the peptides V2 and V3, corresponding to viral mutant sequences. No activation of x26-specific T cells by the variant peptides V2 and V3 was found in 10 of 13 patients with x26-specific T cell responses (Fig. 6). When the 3 peptides were used for in vitro expansion (Fig. 6 right panel), markedly fewer cells were recalled in only 3 patients in Elispot assay with the variant peptides. None of 12 patients with x26-negative T cell responses in IFN-γ Elispot assays had T cell responses to variant peptides (not shown).

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Figure 6. Recognition of viral mutants by x26-specific T cells. PBMCs from 13 patients with known x26-specific IFN-γ–producing T cells were expanded in vitro separately with x26 peptide (left panel) or with a mixture of x26, V2, and V3 peptides (right panel). IFN-γ secretion after the activation with each of the 3 peptides in Elispot assays is shown.

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We investigated whether mutant peptides activated IL-10– rather than IFN-γ–secreting T cells, by assessing cytokine secretion after the stimulation of PBMCs with V2 and V3. No IL-10–secreting T cells were found in 15 studied patients with (n = 6) or without (n = 9) x26-specific IFN-γ–secreting T cells. In conclusion, viral mutant sequences activated T cells specific for the x26 epitope much less efficiently, and no cross-recognition of variant sequences by x26-specific T cells was found.

Sequencing of HBV Viral DNA from Patients.

We next investigated whether HBV genome from patients may have viral mutations affecting the x26 epitope. Sequencing of the x26 encoding HBV DNA region could be performed only on 3 samples (P48, P59, P46) with HBV viral load exceeding 2 × 104 copies/ml and detectable HBV DNA after nested PCR (Supplementary Fig. 1). For patients P48 and P59, A-T and G-A mutations at nucleotides 1762 and 1764 were found in HBV genome, changing amino acid in the HBx protein at positions K130M and V131I. Clinical data from patient P48 shows that this patient was infected at birth with an HBeAg-negative mutant (Supplementary Table 1). X26 T cell response was found negative in this patient (Table 2). In contrast, in patient P59 carrying a virus with BCP mutation, x26-specific T cells were detected in PBMCs taken at the time of DNA sequencing (Fig. 2). Finally, the amplified virus from patient P46 with x26-specific T cell response (Fig. 2) showed a wild-type BCP sequence.

This indicates that HBV variants with BCP mutations can be found in some of our patients at the inactive carrier state of the disease.


  1. Top of page
  2. Abstract
  3. Patients and Methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. References
  8. Supporting Information

HBV-specific CD4+ T cells play an important role in HBV infection, secreting the Th1 cytokines that down-regulate HBV replication27 and by contributing to the induction and maintenance of efficient CD8+ T cell and B cell responses.28 CD4+ T cell epitopes have been identified in the core protein,25 HBe antigen,29 envelope,30 and polymerase proteins.31 Immune responses to HBx protein are poorly documented, with only one study dealing with CD4+ T cell responses.20 In an attempt to define more accurately the breadth and epitope specificity of T cell responses to HBx protein, we carried out a systematic analysis of T cells from 52 chronic HBV carriers. After in vitro stimulation with pools of peptides covering the HBx sequence, IFN-γ–secreting T cells specific for HBx were detected in 69% of patients; in 67% (24 of 36 patients), CD4+ T cells could be defined. This contrasts with the low prevalence of the T helper cell responses against structural HBV proteins usually detected during chronic HBV infection. In this study, using the core 50–69 peptide25, 32 for stimulation of PBMCs and our cutured Elispot assay, IFN-γ–secreting T cell response was found in 3 of 8 patients, but with a 10-fold lower number of specific T cells (data not shown). Previous studies found that HLA-class II–restricted nucleocapsid antigen-specific T helper cell responses are only detectable transiently during hepatitis exacerbation.14, 33 The CD4+ T cell response to envelope proteins is markedly reduced during chronic HBV infection.1, 34 The high prevalence of IFN-γ–secreting HBx-specific T cells reported here may be due to the protein itself or to the clinical status of the patients; that is, HBV carriers in the inactive stage of the disease with less than 100,000 HBV copies/ml. The presence of HBx-specific CD4+ T cells during chronic infection may reflect T cell activation because of the release of HBx protein by apoptotic hepatocytes during viral replication. The persistence of HBx-specific T cells could be related to the small amounts of HBx produced by infected hepatocytes, preventing the deletion or anergy of specific T cells occurring with other highly expressed viral antigens, such as hepatitis B surface antigen or hepatitis B core antigen. The impact of viral load on antiviral T cell responses has been characterized in mouse models of chronic infection and in humans.35 T cell responses to HBV antigens are detected more easily in patients with less than 107 copies/ml or after successful antiviral treatment.36, 37 Consistent with these data, we found x26-specific T cells after stimulation of PBMC from only 1 of 13 patients with chronic active hepatitis and >105 copies/ml (data not shown).

In contrast to the high prevalence of IFN-γ–secreting HBx-specific T cells, IL-10–secreting T cells were detected in very few of the studied patients, always in the presence of IFN-γ production. This is consistent with previous reports of IL-12–induced IFN-γ/IL-10–secreting T cells generated in response to chronic infection.38, 39 Regulatory T cells specific for HBx, producing IL-10, are therefore unlikely to exist in our group of patients.40 This contrasts with HCV persistent infection, which is associated with enhanced IL-10 production in response to nonstructural HCV antigens such as NS3, IL-10–producing T cells in the liver, and weak CD4+ T helper 1 reactivity in the periphery.16, 41

We observed a high prevalence of peripheral CD4+ T cell responses principally targeting the C-terminal part of HBx. This finding is in agreement with a previous report characterizing T cell clones recognizing peptides within this domain of HBx.20 Remarkably, most of our patients had IFN-γ–secreting CD4+ T cells recognizing a previously unidentified single peptide, HBx 126–140 referred here to x26. In addition, x26 peptide was immunogenic in the context of multiple HLA-class II molecules and therefore may be considered a promiscuous epitope. Nevertheless, some patients with x26-specific T cells lack the HLA-DR molecules that bound the peptide with high affinity in vitro. Other class II molecules not tested here are therefore probably able to bind x26. The partial correlation between the HLA-DR-restriction of the peptides and the pattern of DR alleles from different donors is consistent with published findings.31, 42

Chronic HBV infection evolves from an initial HBeAg-positive phase, through HBe seroconversion, to an HBeAg-negative phase in which replication levels are lower. This process is characterized by a progressive switch from viral quasi-species dominated by “wild-type” variants lacking precore or core promoter mutations, to viral quasi-species in which precore or core promoter variants predominate.43, 44 However, precore and core promoter mutations have been shown to exist in a substantial proportion of patients before HBeAg clearance.22 What role do x26-specific IFN-γ–secreting T cells play against both wild-type and mutated x26-derived sequences? The effector properties of x26-specific T cells may constitute an immune system pressure against which these mutant viral variants are selected.

The lack of recognition of variant HBx peptides, corresponding to BCP mutant sequences, by x26-specfic T cells could not be explained by a decreased binding capacity of these sequences to HLA-DR molecules, as shown by comparative binding studies with the x26, V2, and V3 peptides and purified class II molecules (data not shown). Although core promoter mutations may appear early in HBV infection,8 the possibility of initial infection with the mutant virus cannot be excluded. This is the case for patient P48, who was infected at birth with an HBeAg-negative virus, and lacked for x26-specific T cell response. Conversely, a wild-type sequence virus was found in patient P46 concomitantly with detectable x26-specific T cells. However, for other patients such as P59 who were initially infected with a “wild-type” variant lacking core promoter mutations, x26-specific memory T cells may exist and can be detected in our assay despite the presence of a core promoter variant viral quasi-species at the time of blood collection. Our study shows that HBV strains with mutations affecting the immunodominant HBx epitope are likely to induce weaker T cell responses, favoring the accumulation of such mutant strains.

Mutations resulting in HBx protein truncation also have been associated with low levels of HBV replication and decreases in hepatitis activity in anti-HBe antibody-positive HBV carriers.45 Thus, at least some of the studied chronic carriers may produce truncated HBx. The presence of BCP mutants or truncated HBx mutants might account for the lack of T cell reactivity specific for the wild-type x26 peptide in some of our patients. Therefore, x26 and variant peptides could be used for the immunomonitoring of HBV sequence changes during disease progression. This hypothesis should be tested in longitudinal studies in groups of patients differing in clinical status.

In addition, the immunogenicity, the promiscuous HLA-DR binding and the efficient activation of specific IFN-γ–secreting T cells of this newly described HBx epitope suggest that it is a potential candidate for use in therapeutic vaccines for patients with chronic HBV infection.


  1. Top of page
  2. Abstract
  3. Patients and Methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. References
  8. Supporting Information

We thank Mina Ahloulay, Sandrine Fernandes, and Stéphane Blanchin for their contribution to this work; M-L Chaix for HBV DNA quantification; and Florence Buseyne, Yves Rivière, and Maryline Bourgine-Mancini for helpful discussions. We are indebted to all patients who donated blood samples, and to Françoise Audat from the Unité thérapeutique transfusionnelle, Hôpital Necker Enfants Malades and Etablissement Français du Sang for providing us with control blood samples.


  1. Top of page
  2. Abstract
  3. Patients and Methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. References
  8. Supporting Information
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Supporting Information

  1. Top of page
  2. Abstract
  3. Patients and Methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. References
  8. Supporting Information

Supplementary material for this article can be found on the H EPATOLOGY website ( ).

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