1 Institute of Clinical Medicine, National Yang-Ming University, Division of Gastroenterology, Department of Medicine, Taipei Veterans General Hospital, 201 Shih-Pai Road, Sec. 2, Taipei 112, Taiwan, Republic of China
2 Division of Cancer Research, Institute of Biomedical Science, Academia Sinica, Taipei, Taiwan, Republic of China
3 Department of Research and Education, Taipei Veterans General Hospital, Taipei, Taiwan, Republic of China
4 Institute of Microbiology and Immunology, National Yang-Ming University, Taipei, Taiwan, Republic of China
Correspondence
Jaw-Ching Wu
jcwu{at}vghtpe.gov.tw
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ABSTRACT |
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INTRODUCTION |
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CD8+ cytotoxic T lymphocytes (CTLs) play a key role in immunity against intracellular pathogens. In HBV and hepatitis C virus (HCV) infection, multi-specific CD8+ T-cell responses to HBV and HCV are related strongly to virus clearance (Chisari & Ferrari, 1995; Maini et al., 2000
; Thimme et al., 2001
); however, the pathogenic mechanism of chronic HDV infection is undetermined. HDV transgenic mice that express hepatitis delta antigen (HDAg) in the liver do not develop liver damage, providing evidence that HDV is not a cytopathic virus (Guilhot et al., 1994
). The facts that a high HDV load was detected in HIV-infected CHD patients whose circulating T-cell numbers were suppressed and that activity of HDV-induced liver disease is related to CD4+ T-cell response to HDV suggest that immune mechanisms play a significant role in chronic HDV infection (Roingeard et al., 1992
; Nisini et al., 1997
). Strategies to induce an HDV-specific CTL response are promising ways to control chronic HDV infection.
HDV has two forms of viral proteins: large and small HDAgs (Casey & Gerin, 1995). These two antigens are identical in sequence, except that the large HDAg (L-HDAg) contains an additional 19 aa at the C-terminus compared to the small HDAg (S-HDAg). The immunogenic domains of HDAg, recognized by anti-HDV antibodies derived from chronic HDV-infected patients, include aa 27, 6374, 8691, 94100, 159172, 174195 and 197207 (Wang et al., 1990
). Epitopes of HDAg that are recognized by CD4+ T-cells of HDV-infected patients are aa 2641, 5065, 6681 and 106121 (Nisini et al., 1997
). The CD8+ T-cell epitopes on HDAg have never been defined; however, the S-HDAg C-terminus, aa 77195, has been suggested to contain possible epitopes (Karayiannis et al., 1993
). Identification of the CTL epitopes on HDV is crucial work in vaccine design and study of HDV pathogenesis.
A prophylactic or therapeutic HDV vaccine has potential use for HBV carriers who are at risk of HDV superinfection and for CHD patients. Our previous study demonstrated that HDV DNA vaccines can produce a T helper (Th) 1 immune response and that cellular immunity can be generated by DNA vaccines that encode L- or S-HDAg (Huang et al., 2000, 2003
). CTL response against HDAg can also be induced by DNA vaccines in mice (Mauch et al., 2001
). HLA-A*0201 transgenic mice provide a useful model for characterizing CTL epitopes of HLA-restricted viral antigen and evaluating CTL responses. In this study, we used an HDV DNA vaccine in HLA-A*0201 transgenic mice and identified novel CTL epitopes that are restricted by the HLA-A*0201 molecule on HDAg. HDV-specific CTLs were also detected in two chronically infected HDV patients without active disease.
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METHODS |
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Screening for HLA-A*0201-restricted peptides.
Twelve potentially HLA-A*0201-restricted peptides from sequence TWD2667 (GenBank accession no. AF104263) were selected by using the SYFPEITHI database (Table 1) (http://www.syfpeithi.de/; Rammensee et al., 1999
). One HBV core peptide, aa 1827 (FLPSDFFPSI, a common sequence for HBV genotypes B and C), which is known to be capable of binding to HLA-A*0201, was used as a positive control. All peptides were synthesized commercially by Sigma-Genosys.
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Target cells.
A human B lymphoblastoid cell line (B-LCL) that carries the HLA-A2 molecule (wu-LCL) and T2 cells were used as target cells. Another B-LCL (fmc-LCL) that carries the HLA-A11 molecule served as a control. HLA phenotypes were classified by using Micro SSP HLA DNA typing trays (One Lambda).
Animal model and immunization.
C57BL/6-TgN (HLA-A2.1)1Enge transgenic mice (Jackson Laboratory, ME, USA) were used in this study. These homozygous mice had previously been used to predict HCV CTL epitopes (Shirai et al., 1995). Control C57BL/6 mice were obtained from the National Laboratory for Animal Breeding and Research Center, Taipei, Taiwan. Mice were housed at the SPF room, Laboratory Animal Facility, Taipei Veterans General Hospital. Animals received humane care and the protocol was approved by the Animal Committee of Taipei Veterans General Hospital. Groups of four mice were immunized with DNA at 810 weeks of age. Mice were anaesthetized and given intramuscular injections into the bilateral quadriceps with a total dose of 100 µg plasmid DNA dissolved in 100 µl sterilized normal saline. All mice received an injection of cardiotoxin (Sigma) 1 week before DNA immunization. Each mouse was given booster doses at 3 and 6 weeks after the first immunization. Mice were immunized as follows: group 1 (HLA-A2.1 transgenic mice) with p2667L; group 2 (HLA-A2.1 transgenic mice) with pcDNA3.1(); and group 3 (C57BL/6 mice) with p2667L. Mice were sacrificed 2 weeks after the last immunization. Splenocytes from immunized mice were stained directly ex vivo with HLA-A*0201peptide tetramers and rat anti-mouse CD8 antibody (Pharmingen). Samples were run on a FACScan flow cytometer and the data were analysed by using CellQuest software (Becton Dickinson). Animal experiments were repeated twice to verify their validation.
Induction of peptide-specific CTL lines.
Spleen cells from immunized mice were suspended in RPMI medium that contained 10 % fetal bovine serum. For stimulation in vitro, red blood cell-depleted spleen cells (5x106 ml1) were mixed with each of the HDV peptides (0·1 µg ml1) and incubated at 37 °C for 2 days. Splenocytes were transferred to anti-mouse CD3 antibody-coated dishes in the presence of recombinant mouse interleukin 2 (IL2, 10 U ml1) and anti-mouse CD28 antibody, then incubated for another 10 days. Splenocytes were restimulated with each of the peptides and mouse IL2 for 10 days. CTL lines were confirmed by staining with HLA-A*0201peptide tetramers.
Cytotoxic assays.
CTL lines were evaluated for their cytotoxic response in the presence of specific peptides (1 µg ml1) and target cells (5x104 ml1, 100 µl). A 4 h 51Cr-release assay was performed in 96-well V-bottom plates by using 51Cr-labelled target cells. The percentage of specific lysis was calculated by the following formula: (experimental releasespontaneous release)/(maximum releasespontaneous release). Experimental release represented mean counts min1 released by target cells in the presence of effector cells. Total release represented the radioactivity released after lysis of target cells with 1 % Triton X-100. Spontaneous release represented the radioactivity counted in medium derived from target cells alone.
Chronic HDV-infected patients and peripheral blood mononuclear cell (PBMC) preparation.
Nine chronically infected HDV patients were screened for their HLA phenotypes. Among them, four patients who expressed HLA-A2 phenotype were found and these were recruited in this study. The study was approved by the Institutional Review Board of Taipei Veterans General Hospital. All four patients tested positive for serum HBsAg and anti-HDV antibodies by using radioimmunoassay kits (Abbott Laboratories) and tested negative for anti-HCV antibodies. Two patients had abnormal serum alanine transaminase (ALT) levels and were chronically positive for HDV RNA. The other two patients, who had been positive for HDV RNA in the past, but whose serum ALT levels had become normal and whose HDV RNA was undetectable since 1996, were defined as being in disease remission. PBMCs were purified from venous blood by using FicollHistopaque gradient centrifugation techniques. PBMCs (2x106 ml1) were stimulated with human anti-CD3 antibody in the presence of recombinant human IL2 (50 U ml1) and incubated in RPMI 1640 medium (Gibco) for 7 days. The expanded PBMCs were used for HLA-A*0201peptide tetramer and mouse anti-human CD8 antibody (Serotec) staining. Stimulated PBMCs from patient 1 were further restimulated with cognate peptide (HBV core 1827, HDV 2634 or HDV 4351) at 0·1 µg ml1 for 7 days to derive short-term lines. The PBMCs from three non-HLA-A2 chronically HDV-infected patients (two HLA-A11 and one HLA-A24) and two healthy individuals who were positive for hepatitis B surface antibodies (anti-HBs Ab) after successful HBV vaccinations served as controls. Control PBMCs were stimulated with human anti-CD3 antibody and IL2 and stained under the same conditions as described above.
HBV DNA and HDV RNA detection.
HBV DNA was quantified by using a Cobas Amplicor HBV monitor (Roche). The detection limit of this assay was 200 copies ml1. Serum HDV RNA was detected by RT-PCR and sequenced as described previously (Wu et al., 1994). HBV and HDV genotypes were classified by PCR-RFLP as reported previously (Mizokami et al., 1999
; Wu et al., 1995b
).
ELISPOT assay for gamma interferon (IFN-).
A human IFN- ELISPOT assay kit (R&D Systems) was used to determine whether the stimulated PBMCs were functional, by following the manufacturer's protocol. To summarize, stimulated PBMCs were washed and restimulated with the cognate peptide (HBV core 1827, HDV 2634 or HDV 4351) at 0·1 µg ml1. All of the PBMCs (2x104 in 100 µl medium) were pipetted into the wells and incubated at 37 °C overnight. Biotinylated polyclonal antibody that was specific for human IFN-
was added, followed by alkaline phosphatase conjugated to streptavidin. 5-Bromo-4-chloro-3-indolyl phosphate/nitro blue tetrazolium chloride was used as substrate. Images of spots were captured by using a dissection microscope, then counted using ImageMaster TotalLab v1.10 software (Amersham Biosciences) (Huang et al., 2003
). The number of specific spot-forming cells (SFCs) was determined as the mean number of spots in the presence of an antigen minus the mean number of spots in the wells with medium only.
Statistical analysis.
The MannWhitney rank sum test was used to compare the results between the groups. A P value of <0·05 was considered to be significant.
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RESULTS |
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Two weeks after the last immunization, pooled spleen cells from groups of mice were stained directly ex vivo with HLA-A*0201peptide tetrameric complexes. As shown in Fig. 2, five of the seven tetramers gave background levels, but two showed a significantly enhanced response in HLA-A2.1 transgenic mice after HDV DNA vaccination. Tetramer-positive CD8+ T cells accounted for 0·77±0·37 and 0·94±0·09 % of total CD8+ T cells for Td 2634 and Td 4351, respectively, in HLA-A2.1 transgenic mice immunized with p2667L. These numbers were significantly higher than those detected in the control groups (P=0·029 by MannWhitney rank sum test). Nearly 0·9 % of CD8+ T cells in HLA-A*0201 transgenic mice were specific for HDV peptides 2634 and 4351 after DNA immunization.
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DISCUSSION |
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The large form (L-HDAg) plasmid construct, instead of the small form, was adopted in this study as L-HDAg encompasses the whole 214 aa length of HDAg. HDAg is a nuclear protein that is usually detected in the nuclei of infected hepatocytes in chronically infected hepatitis delta patients (Chang et al., 1988). Our findings suggest that HDAg can be processed in the cytoplasm and the resultant peptides presented on the surface of infected cells for recognition by CD8+ T cells.
It is recognized that the HLA-A*0201-restricted peptide-binding motif has unique properties, with leucine, isoleucine or methionine at position 2 and valine or a residue with an aliphatic hydrocarbon side chain at the C-terminus (Falk et al., 1991; Hunt et al., 1992
). We selected the potential HLA-A*0201-restricted peptides in HDAg for analysis by using the SYFPEITHI database (Rammensee et al., 1999
). It is worth noting that not every peptide with a high SYFPEITHI score had binding affinity for T2 cells. In the T2 cell-stabilization assay, HDV 2634 had a mean fluorescence ratio of 1·17, but is an HLA-A*0201-restricted peptide. Both the SYFPEITHI database and T2 cell-stabilization assay can serve as screening tools, but a further confirmation study is needed to identify HLA-A*0201-restricted peptides. In addition, we should point out that our approach for initial screening may have overlooked some epitopes, as the SYFPEITHI database and the T2 cell-stabilization assay are not perfect screening methods.
Recently, HHD mice (which are deficient for the 2-microglobulin gene and mouse MHC class I H-2Db molecules) have been reported to facilitate identification and characterization of HLA class I-restricted virus T-cell epitopes (Pascolo et al., 1997
). In this study, even though C57BL/6-TgN(HLA-A2.1)1Enge transgenic mice were not devoid of H-2 class I molecules, we identified two novel HLA-A*0201-restricted epitopes in HDAg. According to the results in non-transgenic C57BL/6 mice and the control plasmid group, the two epitopes that were identified in HLA-A2.1 transgenic mice were confirmed as being HLA-A2.1-specific. Previous studies suggested that HDAg CTL epitopes are located within the S-HDAg C-terminus (Karayiannis et al., 1993
), but the eight peptides chosen within the region were not the candidate HLA-A*0201-restricted CTL epitopes. We cannot exclude the possibility that other CTL epitopes, in addition to the peptides studied, exist in HDAg; further studies in HHD mice are needed for clarification.
HLA-A*0201peptide tetramers have been used to detect the frequency of epitope-specific CD8+ T cells in this study. About 0·9 % of total CD8+ T cells are specific for epitopes HDV 2634 and 4351 following DNA immunization. This finding corroborates our recent report on BALB/c mice that showed that HDV-specific, IFN--secreting CD8+ splenocytes numbered approximately 0·9 % of the total after DNA-based immunization (Huang et al., 2003
). After stimulation in vitro, both CTL lines were able to trigger specific CTL responses to peptides HDV 2634 or HDV 4351.
The two HLA-A*0201-restricted HDV epitopes that are targeted by the HDV DNA vaccine were identified in mice. We wondered whether these epitope-specific CTLs existed in HLA-A2 HDV-infected patients. Interestingly, of the four patients studied, two had HDV-specific CTLs detected by HLA-A*0201 tetramers. For patient 1, the frequencies of peptide-specific CD8+ T cells increased significantly after peptide stimulation. ELISPOT assays also confirmed that those cells were functional and cytotoxic to HBV and HDV. It is convincing that both epitopes are potentially HLA-A*0201-restricted human HDV epitopes. So far, the pathogenesis of chronic HDV infection is unclear. Dual HBV and HDV infection adds complications to the disease. In chronic HBV infection, an effective HBV-specific CD8+ T-cell response can inhibit virus replication (Maini et al., 2000). Evidence shows that the frequencies of HBV core 1827-specific CD8+ T cells are lower in most chronically infected hepatitis B patients with higher viral load than in patients controlling the virus (Maini et al., 2000
). Coincidentally, we detected HDV 2634- and 4351-specific CTLs in two chronically infected HDV patients without HDV viraemia. From the clinical data, patients 1 and 2 both had persistently normal serum ALT levels and undetectable HDV RNA levels by RT-PCR for at least 3 years. HDV sequences isolated from the stored sera of the two patients from 10 years previously were identical to those of Td 2634 and Td 4351. In addition, not only HDV-, but also HBV-specific CTLs were detectable in patient 1. In contrast, no significant amounts of HDV-specific CTLs were detected in patient 3, who had HBV and HDV viraemia and high serum ALT levels. In patient 4, the HBV load was lower than the detection limit of the assay kit. This HBV DNA may be undetectable, due to the suppression effect of HDV (Wu et al., 1995a
). The divergence in nucleotide sequences among different HDV genotypes ranges from 23 to 34 % (Casey et al., 1993
; Wu et al., 1995c
). Sequence variation between genotype I and IIa HDV may have caused the HDV-specific CD8+ T cells in patient 4 to be undetectable by the tetramers used, which were tailor-made for genotype I HDV. However, the data from patient 4 served as a background control for this study. Genotype I is the most prevalent genotype of HDV in the world and a previous report suggested that genotype I HDV had a worse prognosis than genotype II (Wu et al., 1995b
). There is an urgent need to identify CTL epitopes on genotype I HDV for vaccine design and antiviral therapy; consequently, we focused on genotype I HDV in this study. According to current knowledge on HBV and HCV, it is plausible that an effective HDV-specific CTL response might be related to HDV clearance. Coexistence of HBV- and HDV-specific CTLs is needed to control both viruses in patients with chronic hepatitis delta. If so, strategies to induce CTL responses against HDV epitopes 2634 and 4351 have the potential to treat chronic hepatitis delta. The data from this study also show that the presence of HDV-specific CTLs was coincidentally detectable in two chronically HDV-infected patients without HDV activity. Further study using a larger number of patients is needed to support this idea. However, this kind of study is not easy, due to the rapid decline in the patient population and the fact that only a minority of patients belong to genotype I HDV in Taiwan (Wu et al., 1995b
, 1998
; Huo et al., 1997
).
In conclusion, HDV 2634 and HDV 4351 are novel HLA-A*0201-restricted CTL epitopes in genotype I HDV. HDV 2634- and HDV 4351-specific CTLs happen to be detected in chronically infected hepatitis delta patients without active disease. Evoking CTL responses to HDV by using an HDV DNA vaccine may be an alternative approach to controlling HDV viraemia in patients with chronic hepatitis delta.
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ACKNOWLEDGEMENTS |
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Received 9 April 2004;
accepted 16 June 2004.