Isolation and functional characterization of hepatitis B virus-specific T-cell receptors as new tools for experimental and clinical use

T-cell therapy of chronic hepatitis B is a novel approach to restore antiviral T-cell immunity and cure the infection. We aimed at identifying T-cell receptors (TCR) with high functional avidity that have the potential to be used for adoptive T-cell therapy. To this end, we cloned HLA-A*02-restricted, hepatitis B virus (HBV)-specific T cells from patients with acute or resolved HBV infection. We isolated 11 envelope- or core-specific TCRs and evaluated them in comprehensive functional analyses. T cells were genetically modified by retroviral transduction to express HBV-specific TCRs. CD8+ as well as CD4+ T cells became effector T cells recognizing even picomolar concentrations of cognate peptide. TCR-transduced T cells were polyfunctional, secreting the cytokines interferon gamma, tumor necrosis factor alpha and interleukin-2, and effectively killed hepatoma cells replicating HBV. Notably, our collection of HBV-specific TCRs recognized peptides derived from HBV genotypes A, B, C and D presented on different HLA-A*02 subtypes common in areas with high HBV prevalence. When co-cultured with HBV-infected cells, TCR-transduced T cells rapidly reduced viral markers within two days. Our unique set of HBV-specific TCRs with different affinities represents an interesting tool for elucidating mechanisms of TCR-MHC interaction and dissecting specific anti-HBV mechanisms exerted by T cells. TCRs with high functional avidity might be suited to redirect T cells for adoptive T-cell therapy of chronic hepatitis B and HBV-induced hepatocellular carcinoma.


Introduction
Chronic hepatitis B affects 257 million people worldwide and a curative treatment does not exist [1].A cure of HBV infection is characterized by a full control of virus replication and disappearance of circulating hepatitis B surface (HBs) antigen [2].Adoptive T-cell therapy, in which the viral transcription template covalently closed circular DNA (cccDNA) is eliminated or controlled by the immune system, might allow HBV cure or at least functional cure.
In chronic infection T cells fail to control HBV in infected hepatocytes so that the virus can persist [3].Adoptive T-cell therapy intends to mimic the T-cell response that is mounted during naturally resolving, acute hepatitis B [4] by infusion of HBV-specific T cells that can secrete antiviral cytokines and/or kill infected hepatocytes [5].To this end, the patient´s T cells can be rendered HBV-specific by introduction and expression of receptors that target the infected cells [6].These receptors can either be chimeric antigen receptors (CAR) or cloned, natural TCRs.CARs are artificial molecules with an antibody domain binding HBs protein on infected cells and intracellular TCR stimulation domains that activate the T cell.CARs function independently from major histocompatibility complex (MHC)-presentation and can theoretically be used in every patient [7,8].On the other hand, certain pairs of natural α and β TCR chains bind an MHC-molecule loaded with an HBV peptide.They can be isolated, cloned and used to genetically engineer T cells [9,10].A natural TCR is restricted to a distinct peptide and a distinct MHC molecule, but has the advantage of activating the T cell in its physiological way and is presumably more sensitive.
Adoptive transfer of T cells equipped with TCRs recognizing tumor antigens showed promising results in the treatment of malignant diseases [11][12][13].The strength of a T-cell response and hence its antitumor or antiviral activity are defined by the avidity of the TCRs [14,15].TCRs can not only be expressed in CD8 + but also in CD4 + T cells.This allows CD4 + T cells to recognize MHC-I-restricted epitopes, which likely increases the success of adoptive T-cell therapy [16,17].In this regard, it is important that the TCR is of high affinity and thus does not depend on CD8 co-receptor binding [18].
In the present study, we aimed at generating a set of HBV-specific TCRs with high avidity to identify promising candidates that are suitable for adoptive T-cell therapy.We identified 11 TCRs specific for three different HBV peptides presented on HLA-A Ã 02.A thorough characterization of these 11 HBV-specific TCRs allowed us to define their specificity and unravel their functional avidity in assays that take into account TCR affinity, TCR avidity and clustering, co-receptor binding and physiological peptide presentation [19].Some of the TCRs displayed a high functional avidity in both CD8 + and CD4 + T cells and allowed T cells transduced with these TCRs to achieve a rapid antiviral activity against HBV infection.

HBV-specific T-cell clones can be isolated from peripheral blood mononuclear cells of individuals with resolved HBV infection
Three HLA-A Ã 02 + donors with already resolved or acute, resolving HBV infection were selected to clone high-affinity TCRs (Table 1).Donor 1 had anti-HBs titers of >1000 IU/ml, was HBV-DNA-negative and had resolved HBV infection around 25 years before.Donor 2 had a prolonged course of an acute hepatitis B (2.9x10 7 IU/ml HBV-DNA), which was cleared after one year receiving entecavir treatment.Patient 3 had an acute infection with 1.9x10 6 IU/ ml HBV-DNA, which spontaneously decreased to 48.8 IU/ml ten weeks later, pointing at a resolving course of infection (Table 1).
HBV-specific T cells were expanded with HBV peptides C18 (Core 18-27 ), S20 (S 20-28 ) and S172 (S 172-180 ) of proven immunogenicity [20].After two weeks, cells were stained with reversible HLA-A Ã 02 multimers (Streptamers) [21], isolated using flow cytometry-based cell sorting and clonally expanded thereafter (Fig 1A,S1 Table).In total, about 400 T-cell clones were obtained, 100 of which showed HBV-specific cytotoxicity and were subjected to PCR-based TCR sequence analyses.Sequence comparison of individual TCR chains revealed that several T-cell clones from one patient shared an identical TCR indicating clonal expansion from the same precursor cell (Fig 1B).Interestingly, for the 11 different TCRs defined, we could detect a common usage of TCR chains among different donors, i.e.Vα17 and Vβ12 recognizing peptide S20, or Vα12/13 and Vβ27 for C18 (S2 Table ).Functional avidity of 11 different T-cell clones was determined by titrating the corresponding peptide in a chromium release killing assay (S1 Fig) .Activation by nano-or picomolar concentration of peptide suggested that we had obtained potent HBV-specific T-cell clones, with C18-specific clones being the most sensitive ones.
We next sought to compare the functionality of the TCRs independently from the general fitness of the T-cell clone.First, complete TCR α and β chains were cloned into separate retroviral vectors and expression after retroviral transduction of peripheral blood mononuclear cells (PBMC) was confirmed by MHC multimer staining (S2 Fig) .Secretion of interferon (IFN)-γ in a co-culture assay revealed that cells loaded with HBV-derived peptides specifically activated transduced T cells.We noticed that the specific response of T cells primarily correlated with the expression level of the respective TCR with e.g.G6 and D1 being expressed at low and FLP14 and FLP122 at high levels, respectively (S2 Fig) .Therefore, in a next step TCR expression was improved by optimizing codon-usage, inserting an additional disulfide bond, exchanging constant regions for their murine counterparts and ensuring equimolar translation of both TCR chains from one construct using a P2A element [22]

T cells genetically engineered to express HBV-specific TCRs become polyfunctional
To study the functional profile and sensitivity that our TCRs conferred to transduced T cells, we loaded T2 cells with decreasing amounts of peptide and co-cultured them with TCR + T cells.All TCRs conferred polyfunctionality in terms of cytokine secretion to transduced CD8 +

Transduction with HBV-specific TCRs renders CD4 + and CD8 + T cells cytotoxic
To further dissect TCR functionality, we analyzed the killing capacity of T cells transduced with our HBV-specific TCRs (Fig 3).Hereby, C18-specific TCRs conferred the highest sensitivity.With a half maximum lysis at a peptide concentration of below 100 pM, C18-specific cells were about ten-fold more sensitive than S20-or S172-specific CD8 + T cells (Fig 3A).The killing profile of CD8 + T cells was similar when they belonged to a group with the same peptide specifictiy (Fig 3A Overall, nine of the 11 TCRs had a high functional avidity and their expression resulted in a polyfunctional response of the transduced T cells upon antigen recognition, comprising cytokine production and cytotoxicity in CD8 + as well as CD4 + T cells.

HBV-specific TCRs recognize peptides from different HBV genotypes presented on various HLA-A*02 subtypes
With regard to a potential clinical application of HBV-specific TCRs for T-cell therapy it is of interest to have TCRs available for a broad spectrum of patients.The HLA background of all our original donors was HLA-A Ã 02:01 (Table 1) and they were stimulated with peptides derived from HBV genotype (gt) D. Therefore, we first addressed the suitability of our TCRs to recognize peptides presented on other HLA-A Ã 02 subtypes using lymphoblastoid cell lines (LCL).In addition, we analyzed whether our TCRs would recognize the corresponding peptides derived from HBV gt B and C, which are dominant in Asia, on the most frequent HLA-A Ã 02 subtypes Ã 02:01, Ã 02:06 and Ã 02:07.All C18-and all S172-specific TCRs, as well as the S20-specific TCRs G6 and 4G recognized their cognate peptides derived from HBV gt A, B, C or D not only when presented on HLA-A Ã 02:01, but also when presented on Ã 02:07 (Fig 5A -5C).On HLA-A Ã 02:06, all C18-TCRs recognized the gt A-and D-variant of the C18 peptide, and all but 6K the B-and C-variant (Fig 5A).In contrast, S20 and S172 peptides were only weakly recognized by any of our TCRs on HLA-A Ã 02:06 except 4G, which recognized at least the gt A-and D-variant of S20.Thus, HBV gt A-and D-derived peptides were recognized on all HLA-A Ã 02 subtypes except Ã 02:08, and HBV gt B and C peptides on HLA-A Ã 02:01, Ã 02:06, and Ã 02:07 by at least one of our TCRs.

TCR-transduced T cells recognize endogenously processed peptide
External loading of peptides on target cells allows quantifying the magnitude of a T-cell response to a defined peptide concentration.However, in vivo HBV proteins will be processed within the infected cell and will be loaded on MHC-I molecules in the endoplasmatic reticulum.To investigate whether endogenously processed HBV peptides are recognized by our TCRs, we co-cultured TCR-transduced T cells with an HLA-A Ã 02 + human hepatoma cell line that replicates HBV.CD8 + T cells expressing any C18-or S20-specific TCR killed HBV-replicating HepG2 cells and secreted high amounts of IFN-γ, even at relatively low effector to target (E:T) cell ratios (Fig 6A).They did not react unspecifically to the parental cell line that does not contain HBV (S5 Fig) .Expression of the C18-specific TCR 5E, 6K or 7D (all derived from donor 1, who had resolved his HBV infection), but not of TCR FLP14 or FLP122 (derived from donor 2, who had a protracted course of HBV infection), conferred effector function also to CD4 + T cells enabling them to specifically kill HBV-replicating HepG2 cells (Fig 6B, S5 Fig) .In contrast, S20-specific TCRs (except TCR 4G) were not able to activate CD4 + T cells (Fig 6B).S172-specific T cells were not activated at all upon co-culture with HBV-positive HepG2 hepatoma cells, but were activated by epithelial cells that had been transfected with an S-plasmid (S6 Fig) .This indicates a certain defect in antigen processing or presentation of this peptide in HepG2 hepatoma cells, but still allows to conclude that processed antigen may be recognized [23].Thus, all HBV-specific TCRs were able to detect HBV peptides presented after intracellular processing.

Treatment of HBV-infected cells with gene-modified T cells leads to a rapid reduction of viral markers
Our next step was to assess the antiviral capacity of TCR-transduced T cells (CD8 + and CD4 + combined).To this end, we generated a stable HLA-A Ã 02 expressing HepaRG cell line (Fig 7A ), cells with an antibody against the murine constant domain of the transduced TCR.1x10 5 T2 cells loaded with decreasing amounts of peptide were co-cultured with 1x10 5 CD8 + or CD4 + T cells expressing (A) C18-specific, (B) S20-specific, or (C) S172-specific TCRs.Cytokine + cells were detected by intracellular cytokine staining and are given as % of TCR-transduced T cells.Data are presented as mean values from triplicate (CD8 + ) or single (CD4 + ) co-cultures.

Discussion
In this report, we isolated 11 TCRs that are specific for three different HBV peptides presented on HLA-A Ã 02 (Core 18-27 , S 20-28 , S 172-180 ).Using genetic engineering, the HBV-specific TCRs could be expressed on the surface of CD8 + and CD4 + T cells and induced typical T-cell effector functions upon specific recognition of exogenously added peptides and endogenously processed antigens.
HBV-specific T cells can be detected by MHC multimer staining at frequencies of about 0.1-1.1% in acute HBV infection, but not in chronic hepatitis B patients [24].We therefore isolated TCRs from donors with either acute or resolved HBV infection.The TCRs with the highest functional avidity originated from memory T cells that had probably undergone a selection for high avidity TCRs due to repeated exposure to antigen [25] in the donor who had resolved the infection.TCRs isolated from donors with acute, resolving hepatitis B by contrast had a lower functional avidity.
Among the identified TCR chains we could detect a common usage of Vβ12 for recognition of the S20 peptide presented on HLA-A Ã 02:01.The fact that the Vβ12 chain of two TCRs from different donors had almost identical CDR3 regions could indicate the existence of a public TCR as described for other viral infections [26].Although a skewed TCR Vβ gene usage was found in patients who had recovered from acute hepatitis B [27], Vβ12 usage was not reported.This could be attributed to the cohort containing only few subjects with HLA-A Ã 02:01 [27], which might also influence the choice of TCR variables because their CDR1 and CDR2 regions interact with the MHC molecule [28].Hence, whether Vβ12 usage is associated with recovery from hepatitis B in HLA-A Ã 02:01 subjects remains to be confirmed in larger cohorts.
One challenge of TCR gene therapy is the potential mispairing of endogenous and introduced TCR chains, which could result in autoimmunity.We therefore improved TCR expression and specific pairing by murinization of constant domains, codon-optimization, inserting additional disulfide bonds, and ensuring equimolar translation of both TCR chains from one construct.All of these measures have been shown to greatly reduce the risk of mispairing of endogenous and introduced TCR chains [29][30][31].As a consequence, our transduced T cells did not react to HBV-negative cells, which only present peptides from self-antigens.
In general, cross-reactivity of the transduced, correctly paired TCRs is highly unlikely as they were isolated from patients controlling HBV and underwent thymic selection.Recognition of self-antigens by the TCRs would hence have caused autoimmunity in the donors, which remained healthy after resolution of HBV.In our experiments, however, we detected a cross-reactivity of C18-specific T cells against high peptide concentrations of the S20 peptide but not against other control peptides tested.We assume that this is an exclusive phenomenon that might even increase sensitivity of "C18-specific" T cells towards HBV-infected cells.
The strength of a T-cell response correlates with TCR avidity [14].The functional avidity of our TCRs was in the picomolar range, which is similar to or even higher than what has been reported for high-avidity TCRs against other viral antigens [21,32].Sensitivity of TCRs directed against the core epitope C18 was higher than that against S-derived peptides.While S-specific responses are also associated with resolution of infection [33], C18-specific CD8 + T cells represent the dominant effector cell population in patients with acute, resolving hepatitis B [34,35].This may be due to a high binding affinity of C18 to HLA-A Ã 02:01 (IC 50 2.5 nM) [36] leading to a better presentation and hence to a more effective T-cell response [37].
Polyfunctionality of T cells is associated with long-term control of viral infections [38].Our data demonstrate that T cells redirected to express HBV-specific receptors are polyfunctional in terms of secretion of multiple cytokines and cytotoxicity.Treatment of HBV-infected HepaRG cells with TCR-transduced T cells lead to a rapid reduction of the HBV persistence form, the viral cccDNA, which can be explained both by killing of infected cells or by its cytokine-induced non-cytolytic removal after APOBEC3-induced deamination [39,40].However, after two days, the co-culture with T cells had to be ended because HepaRG cells did not tolerate the change of medium.Hence, a more robust target cell line that can be infected with HBV is necessary to analyze the antiviral activity of TCR-transduced T cells [41].In this setting, TCR-transduced T cells could be used as an experimental tool not only to define their antiviral mechanisms, but also to study virus-host cell interactions that may for example result in alteration in immune pathways and antigen presentation.
Efficacy of adoptive T-cell therapy is likely to be enhanced, if antigen-specific CD4 + T cells are co-infused to provide help to cytotoxic CD8 + T cells [17].A prerequisite for MHC-Irestricted TCRs to function in CD4 + T cells is that the TCR is of high affinity and does not need CD8 co-receptor binding [18].We found several TCRs specific for HBV antigens that were functional without CD8 co-receptor expression arguing for a strong interaction of MHC and TCR.However, MHC multimer staining did not predict functionality of TCR-transduced CD4 + T cells.A discrepancy between MHC multimer binding and effector function [42] but not TCR-ligand K off -rate [43] has been reported for CD8 + T cells and might be explained by the density of the MHC:peptide complex [44] or additional molecules involved in formation of the immunological synapse.
Binding of the TCR to its cognate peptide depends on effective presentation by the MHC-I molecule.We found that TCRs, which were isolated from HLA-A Ã 02:01 donors, were able to recognize peptides presented on other HLA-A Ã 02 subtypes.This is of interest for a clinical application of T-cell therapy, since a broader range of HLA-A Ã 02 subtypes recognized will increase its applicability.Importantly, TCRs should recognize the HLA alleles most common in populations where hepatitis B is endemic.For instance, up to one third of the Chinese population carry the alleles HLA-A Ã 02:03, Ã 02:06, or Ã 02:07 (www.allelefrequencies.net).Our C18-specific TCRs could recognize Ã 02:06 and Ã 02:07, but not Ã 02:03 bound C18 peptide.Although a high binding affinity of C18 to Ã 02:03 was predicted in a molecular binding assay [36], analysis of the crystal structure revealed a steric hindrance between the Ã 02:03 binding groove and the C18 peptide resulting in reduced thermostability [45].Remarkably enough, while a T-cell response to S20 was only found for Ã 02:01 in a Chinese cohort [33], our highavidity TCR 4G recognized S20 also when presented on HLA-A subtype Ã 02:03 or Ã 02:07.Importantly, most of our TCRs recognized peptides from HBV gt A and D strains, most frequent in America and Europe, but also peptides from HBV gt B and C, most frequently found in Asia [33], indicating that the isolated TCRs could also be applied to a significant number of Asian patients.
As an alternative approach, HBV-specific CARs could be used, which would be applicable irrespective of the patient's MHC haplotype [8].While their broader applicability and a nonexistent risk for mispairing with endogenous TCR chains are clear advantages, their antibodybased antigen-recognition domain is usually not as sensitive as a natural T-cell receptor and needs higher antigen loads for T-cell activation.
In conclusion, we identified TCRs with a high functional avidity for HBV S-and corederived peptides with a comprehensive analysis of a unique set of TCRs.They can be employed as an experimental tool to study immune pathways in viral diseases ranging from antigen recognition to antiviral mechanisms exerted by TCR-transduced T cells.Since the TCRs are functional in CD8 + as well as CD4 + T cells and recognize peptides presented on a broad range of HLA-A Ã 02 molecules, they are interesting for clinical application of adoptive T-cell therapy.

Flow cytometry
Staining was done for 30' on ice in the dark using primary antibodies (eBioscience or BD Biosciences, Heidelberg, Germany) diluted in FACS buffer (0.1% BSA/PBS).Transduction efficiency was assessed one day after the 2nd transduction by staining the TCR with MHC Streptamers (see above) or an anti-mTRBC antibody.For intracellular cytokine staining, Brefeldin A (8 μg/ml, Sigma-Aldrich) was added for 4 hours during antigen stimulation.After staining of dead cells with EMA (Life Technologies) and cell surface molecules, intracellular cytokines were stained using the Cytofix/Cytoperm Kit (BD Biosciences).CD4 + T cells were stained with antibodies against IL-2, IFN-γ and TNF-α; CD8 + T cell with antibodies against IFN-γ and TNF-α, but not IL-2 as it was hardly detected in our previous experiments.Cells were analyzed using a FACSCanto II flow cytometer (BD Biosciences) and data were analyzed with FlowJo 9.2 software.

Retroviral transduction
Plasmids were amplified using Stbl3 bacteria (Life Technologies) and purified with a Midiprep Plasmid DNA Endotoxin-free Kit (Sigma-Aldrich).293T cells were transfected in a 6 well plate with 4 μg of plasmid DNA (2 μg TCR plasmids, 1 μg pcDNA3.1-MLVg/p, 1 μg pALF-10A1-MLVenv) and 10 μl of Lipofectamin 2000 (Life Technologies).After 6 hours, the medium was replaced with full DMEM medium.After 24 and 48 hours, the retrovirus supernatant was collected and filtered through a 0.45 μm filter.PBMC were pre-stimulated for 2 days in TCM supplemented with 300 U/ml IL-2 on non-tissue culture plates, which were coated with 5 μg/ml OKT-3 and 0.05 μg/ml anti-CD28 antibody (eBioscience) for 2 hours at 37˚C, blocked with 2% BSA for 30 min and washed with PBS.Retrovirus supernatant was centrifuged at 2000 x g, 32˚C for 2 hours on non-tissue culture plates coated with 20 μg/ml Retro-Nectin (Takara, St. Germain en Laye, France).The retrovirus supernatant was removed and PBMC were spinoculated on the virus-coated plate at 1000 x g for 10 min.A second transduction was performed after 24 hours.
Co-culture with T2 cells CD8 + and CD4 + T cells were isolated out of total transduced PBMC via positive selection magnetic activated cell sorting (MACS, Miltenyi, Bergisch-Gladbach, Germany).For intracellular cytokine staining 1x10 5 TCR + T cells were incubated with 1x10 5 peptide-loaded T2 cells.After 1 hour, Brefeldin A was added to arrest cytokine secretion.

Chromium release assay
T2 cells were incubated with the indicated concentration of peptide and 50 μCi of 51 Cr for 1 hour at 37˚C and washed 2.5 times.2x10 4 target cells were co-cultured with 2x10 4 T-cell clones or TCR-transduced T cells (E:T ratio = 1:1).The spontaneous (SL) and maximum lyses (ML) were assessed with medium or 2% Triton-X instead of effector cells.The cells were incubated for 4 hours at 37˚C, spun down, and the supernatants were transferred to LUMA-plates (Perkin Elmer, Rodgau, Germany).Radioactivity released into the supernatant was measured with Top Count NXT (Perkin Elmer).Specific lysis was calculated: (counts sample-counts SL)/(counts ML-counts SL)x100.

Co-culture with hepatoma cells
HepG2 and HepG2.2.15 hepatoma cells were kept in full DMEM medium (DMEM, 10% FCS, 1% pen/strep, 1% sodium pyruvate, 1% NEAA; Life Technologies).For co-culture experiments 5x10 4 target cells per well were seeded in collagen-coated (Serva, Heidelberg, Germany) 96-well flat bottom plates.When cells had reached confluence, differentiation medium was applied (Williams medium, 5% FCS, 1% Pen/Strep, 1% sodium pyruvate, 1% NEAA and 0.5% DMSO (Sigma-Aldrich).Target cells were used for experiments after 10 to 14 days of differentiation.The number of effector T cells/well was adjusted according to the transduction efficiency of each receptor to identical numbers of TCR-expressing cells.Viability of target cells was assessed using an XTT assay (Roche) and supernatants were subjected to an IFN-γ ELISA (BioLegend).

Co-culture with HepaRG cells
HepaRG cells were differentiated for 4 weeks with William's medium containing 10% FBS Fetaclone II, 1% pen/strep, 1% glutamine, 0.023 IU/ml insulin (Sanofi-Aventis, Frankfurt, Germany), 4.7 μg/ml hydrocortisone (Pfizer, Berlin, Germany), 80 μg/ml gentamicin (Ratiopharm, Ulm, Germany) and 1.8% DMSO.Cells were infected with HBV (concentrated virus from HepG2.2.15 supernatant) at an MOI of 200 in the presence of 5% PEG overnight.10 days post infection, infected HepaRG cells were used as target cells.When effector T cells were added, the medium was replaced to differentiation medium without hydrocortisone.HBeAg in the supernatant was measured with the Enzygnost HBe monoclonal kit (Siemens Healthcare Diagnostics, Eschborn, Germany).Alanine transaminase activity was determined in 32 μl of supernatant using the Reflotron system (Roche Diagnostics).Total DNA was extracted from cells (Macherey & Nagel, Du ¨ren, Germany) and cccDNA and rcDNA were quantified as described previously [39].HBV DNA quantification was done per well and not relative to the cell number, because of unpredictable numbers of proliferating T cells in the total DNA.
(Fig 1C and S3 Fig).By this, expression was enhanced about two-fold (S3 Fig) and up to 80% of T cells expressed the various TCRs as indicated by MHC multimer staining (Fig 1D) or by staining against the murine constant domain (S3 Fig).Thus, we generated a set of 11 functional TCRs with optimized expression recognizing different HBV proteins.

Fig 1 .
Fig 1. Isolation of HBV-specific T cells.(A) Experimental procedure to isolate HBV-specific T cells.T cells from donor 1 with acute, donor 2 with protracted and donor 3 with resolving HBV infection were stimulated for two weeks with peptide-