Abstract
Background
Hepatitis C virus (HCV) frequently causes chronic hepatitis, cirrhosis, and hepatocellular carcinoma after long-term persistent infection. Among various genotypes of HCV, HCV1b is resistant to standard interferon therapy, and thus the development of new treatment modality is needed.
Results
To provide a scientific basis for specific immunotherapy for HCV1b, we investigated HCV1b-derived epitope peptides recognized by human leukocyte antigen (HLA)-A11, -A31, or -A33-restricted cytotoxic T-lymphocytes (CTLs), and report here three novel vaccine candidate peptides selected by both antibody screening and CTL-inducing capacity from among 46 peptides of conserved regions of HCV1b sequences with binding motifs to HLA-A11, -A31, and -A33. Significant levels of IgG reactive to each of the three peptides were detected in the plasma of more than 50% of the HCV1b+ patients. One peptide at positions 30–39 of the core protein induced peptide-specific CTLs from peripheral blood mononuclear cells (PBMCs) of HLA-A11+, -A31+, and -A33+ patients. The other two peptides at positions 35–43 of the core protein and at positions 918–926 of the non-structural protein 2 also induced peptide-specific CTLs from the PBMCs of HLA-A11+ and -A33+ patients.
Conclusion
Therefore, the peptide at positions 30–39 of the core protein could be an appropriate target molecule of specific immunotherapy for all HLA-A11+, -A31+, and -A33+ patients with HCV1b-related diseases.
Keywords: Hepatitis, HCV, Peptide vaccine, CTL, Epitope, HLA-A3
Introduction
Hepatitis C virus (HCV) is a major public health problem, and World Health Organization estimates 180 million people, some of 3% of the world’s population, are infected by the virus [26]. Most of the cases are with chronic hepatitis, and major outcomes of chronic hepatitis are liver cirrhosis and hepatocellular carcinoma. Recent study indicated that spontaneous eradication of the virus occurred in up to 50% of acute infections and this viral clearance was associated with specific immune responses to the virus [17]. Therefore, a determination of immunogenic epitopes has been conducted during the past decade to develop vaccines against HCV, with a focus on cellular immune responses restricted by human leukocyte antigen (HLA)-A2 and -A24 alleles, due to the relatively high worldwide frequency of these alleles. Thus, many HCV-derived peptides capable of inducing cytotoxic T-lymphocyte (CTL) activity have been obtained to date [3–5, 7, 8, 15, 20, 22, 24, 25]. However, these peptides have thus far failed to provide clinical benefits to HCV-infected patients. This failure might be in part due to the weakness of these peptides to elicit protective immunity against HCV. To identify a potential peptide capable of inducing protective immunity, we investigated epitopes recognized by both the cellular and humoral immunities of HCV1b-infected patients with HLA-A11, -A31, and -A33, which are major members of A3 supertype [18, 19].
Materials and methods
Subjects
The Institutional Ethical Review Board of Kurume University approved this study protocol (Protocol # 2289), and informed written consent was obtained from all subjects from whom blood samples were taken in this study. Peripheral blood mononuclear cells (PBMCs) were obtained from 13 HCV1b+ patients possessing the HLA-A3 supertype including HLA-A11, -A31, and -A33 patients. This study did not include HLA-A3+ or -A68.1+ patients, since the frequency of these two HLA phenotypes is extremely low (1.6 and 0.5%) in the Japanese population [1]. None of the participants was infected with HIV and hepatitis B virus. Twenty milliliters of peripheral blood were obtained from each subject, and the PBMCs were prepared by Ficoll-Conray density gradient centrifugation. All of the samples were cryopreserved in liquid nitrogen until use for the experiments. The expression of HLA-A11, -A31, and -A33 molecules on the PBMCs was determined by flow cytometry using the following antibodies: anti-HLA-A11 monoclonal antibody (mAb) (Cat# 0284HA; One Lambda, Canoga, CA, USA), anti-HLA-A31 mAb (Cat# 0273HA; One Lambda), and anti-HLA-A33 mAb (Cat# 0612HA; One Lambda).
Cell lines
C1R-A11, -A31, and -A33 are sublines of C1R cells that have been stably transfected with the HLA-A1101, -A3101, and -A3303 genes, respectively. The expressions of HLA-A11, -A31, and -A33 molecules on these cell lines has been reported previously [21]. All of the cell lines used here were maintained in RPMI 1640 (Invitrogen) with 10% FCS.
Peptides
Forty-six synthetic peptides derived from the consensus sequence of HCV1b with binding motifs for HLA-A3 supertype molecules (including motifs of valine, isoleucine, leusine, methionine, serine, or threonine, at position 2, and of arginine or lysine at the C-terminus), previously described in the literature [14, 18, 19], were identified with BIMAS software (Center for Information Technology, NIH, Bethesda, MD, USA) [13]. These peptides were purchased from Biologica (Nagoya, Japan). HLA-A3 supertype binding-motif possessing peptides derived from influenza virus (Flu) (NVKNLYEKVK) [11], Epstein-Barr virus (EBV) (AVFDRKSDAK) [11], and HIV (RLRDLLLIVTR) [2] were also used as controls. Precise information regarding these peptides is provided in Table 1. The purities of all the peptides used here was >90%, as determined by HPLC. All peptides were dissolved in dimethylsulfoxide at a concentration of 10 mg/ml, and stored at −70°C.
Table 1.
Characteristics and plasma IgG reactivity of HCV1b-derived putative A3-supertype binding peptides
| Region | Sequence | Binding scorea | Ab positive rate in HCV1b+ patients (positive/total) | |||
|---|---|---|---|---|---|---|
| A11 | A31 | A33 | ||||
| HCV | ||||||
| Core | 30–39 | IVGGVYLLPR | 0.8 | 8 | 15 | 7/12 |
| 35–43 | YLLPRRGPR | 0.12 | 4 | 9 | 6/12 | |
| 43–51 | RLGVRATRK | 1.2 | 0.6 | – | 5/12 | |
| 96–104 | WLLSPRGSR | 0.12 | 4 | 9 | 1/12 | |
| E1 | 288–296 | SQLFTFSPR | 0.18 | 8 | 3 | 2/12 |
| 289–297 | QLFTFSPRR | 0.16 | 6 | 9 | 1/12 | |
| 331–339 | ALVVSQLLR | 0.24 | 8 | 9 | 3/12 | |
| E2 | 401–410 | SLFSSGAQQK | 0.8 | 0.6 | – | 2/12 |
| 415–424 | NTNGSWHINR | 0.4 | 2 | 3 | 1/12 | |
| 535–543 | DVLLLNNTR | 0.18 | 1.2 | 45 | 1/12 | |
| 554–562 | WMNSTGFTK | 1.2 | 0.6 | – | 2/12 | |
| 630–639 | RMYVGGVEHR | 0.48 | 36 | 4.5 | 1/12 | |
| 721–730 | LLFLLLADAR | 0.16 | 6 | 9 | 1/12 | |
| NS2 | 858–867 | QVWIPPLNVR | 0.8 | 12 | 15 | 3/12 |
| 917–926 | GLIRACMLVR | 0.72 | 16 | 9 | 3/12 | |
| 918–926 | LIRACMLVR | 0.16 | 8 | 15 | 9/12 | |
| NS3 | 1042–1050 | CIITSLTGR | 0.12 | 8 | 15 | 2/12 |
| 1147–1156 | DSRGSLLSPR | 45 | – | 45 | 3/12 | |
| 1178–1187 | GIFRAAVCTR | 0.48 | 12 | 15 | 2/12 | |
| 1261–1270 | TLGFGAYMSK | 0.8 | 0.4 | – | 2/12 | |
| 1369–1378 | NTGEIPFYGK | 2 | 1 | – | 2/12 | |
| 1529–1538 | ELTPAETSVR | 0.024 | 0.6 | 27 | 1/12 | |
| NS4a | 1694–1703 | VIPDREVLYR | 0.16 | 8 | 15 | 4/12 |
| 1721–1729 | GMQLAEQFK | 1.2 | 0.2 | – | 1/12 | |
| 1723–1731 | QLAEQFKQK | 0.2 | 0.2 | – | 2/12 | |
| 1895–1903 | GVVCAAILR | 3.6 | 8 | 15 | 2/12 | |
| 1950–1959 | SLTITQLLKR | 0.16 | 8 | 9 | 2/12 | |
| 1951–1959 | LTITQLLKR | 0.6 | 8 | 3 | 1/12 | |
| 1969–1978 | STPCSGSWLR | 0.4 | 5 | 3 | 0/12 | |
| NS5a | 1994–2002 | WLQSKLLPR | 0.16 | 8 | 9 | 0/12 |
| 2124–2132 | ELDGVRLHR | 0.048 | 1.2 | 27 | 1/13 | |
| 2226–2234 | LIEANLLWR | 0.16 | 8 | 15 | 2/12 | |
| 2267–2276 | EVSVAAEILR | 0.24 | 1.2 | 45 | 1/12 | |
| NS5b | 2475–2484 | RLQVLDDHYR | 0.24 | 6 | 2.7 | 1/12 |
| 2500–2509 | KLLSVEEACK | 1.8 | 1.2 | – | 1/12 | |
| 2509–2517 | KLTPPHSAK | 1.2 | 0.6 | – | 1/12 | |
| 2578–2587 | LIVFPDLGVR | 0.12 | 8 | 15 | 1/12 | |
| 2579–2587 | IVFPDLGVR | 0.8 | 12 | 15 | 1/12 | |
| 2605–2614 | VMGSSYGFQY | – | 0.06 | – | 1/12 | |
| 2622–2631 | FLVNAWKSKK | 0.6 | 0.4 | – | 2/12 | |
| 2644–2653 | DSTVTENDIR | 45 | – | 45 | 1/12 | |
| 2756–2764 | RVFTEAMTR | 4.8 | 36 | 4.5 | 1/12 | |
| 2900–2909 | EINRVASCLR | 0.024 | 0.6 | 45 | 1/12 | |
| 2912–2920 | GVPPLRVWR | 1.2 | 2 | 15 | 2/12 | |
| 2978–2987 | DIYHSLSRAR | – | 0.18 | 45 | 1/12 | |
| 3001–3010 | GVGIYLLPNR | 1.2 | 4 | 15 | 2/12 | |
| EBV | ||||||
| EBNA3B | 399–408 | AVFDRKSDAK | 4 | 0.6 | 0.5 | |
| Flu | ||||||
| HA | 458–467 | NVKNLYEKVK | 1 | 0.1 | 0.5 | |
| HIV | ||||||
| Env gp41 | 770–780 | RLRDLLLIVTR | n.a. | n.a. | n.a. | |
aThe peptide binding score was calculated based on the predicted half-time of dissociation from HLA class I molecules as obtained from a web site (Bioinformatics and Molecular Analysis Section, Computational Bioscience and Engineering Laboratory, Division of Computer Research and Technology, NIH). The binding score of the HIV peptide was not calculated since the 11-mer peptide was not applicable for the software
Induction of peptide-specific CTLs from PBMCs
Assays for the detection of peptide-specific CTLs were performed according to a previously reported method with minor modifications [20]. In brief, PBMCs (1 × 105 cells/well) were incubated with 10 μl/ml of each peptide in quadruplicate in a 96-well U-bottom-microculture plate (Nunc, Roskilde, Denmark) in 200 μl of culture medium. EBV and Flu peptides were also used as positive controls for the induction of specific T cells. The culture medium consisted of 45% RPMI 1640, 45% AIM-V medium (GIBCO-BRL, Gaithersburg, MD, USA), 10% FCS, 100 U/ml of interleukin-2 (IL-2), and 0.1 mM MEM non-essential amino acid solution (GIBCO-BRL). Half of the culture medium was removed and replaced with new medium containing a corresponding peptide (10 μg/ml) every 3 days. On the 15th day of culture, half of the cultured cells were stimulated with the corresponding peptide-pulsed C1R-A11, or -A31, or -A33 cells, and the other half of the cells were cultured with the negative control HIV peptide-pulsed C1R-A11, or -A31, or -A33 cells. After an 18-h incubation, the supernatant was collected, and the level of interferon (IFN)-γ was determined by enzyme-linked immunosorbent assay. The HCV1b peptide-stimulated PBMCs were further cultured with irradiated HLA-A matched buffycoat PBMCs as feeder cells for approximately 2–3 weeks to obtain a sufficient number of cells for the cytotoxicity assay.
Cytotoxicity assay
Peptide-stimulated PBMCs were tested for their cytotoxicity against C1R-A11, -A31, and -A33, which had been pulsed with either the control HIV peptide or a corresponding peptide by a standard 6-h 51Cr-release assay. Two thousand 51Cr-labeled cells per well were cultured with effector cells in 96-round-well plates at the indicated effector/target ratio. The specific 51Cr-release was calculated according to the following formula: (test c.p.m. − spontaneous c.p.m.). Spontaneous release was determined by the supernatant of the culture with no effector cells, and the maximum release was determined by the supernatant of the culture with 1% Triton X-100. In some experiments, 10 μg/ml of either anti-HLA-class I (W6/32—mouse IgG2a), anti-HLA-DR (L243—mouse IgG2a), anti-CD4 (NU-TH/I—mouse IgG1), anti-CD8 (NU-TS/C—mouse IgG2a), or anti-CD14 (H14—mouse IgG2a) mAbs were added to the wells at the initiation of the culture.
Detection of peptide-specific IgG
Plasma levels of peptide-specific IgG were measured by means of the Luminex® method as reported previously [22]. In brief, 200 μl of plasma (1:100 dilution) was incubated with 25 μl of peptide-coupled, color-coded beads (Luminex Corporation, Austin, TX, USA) in a 96-well filter plate (MABVN1250; Millipore Corp., Bedford, MA, USA) for 2 h at room temperature on a plate shaker. Two hours later, the plate was washed with T-PBS and incubated with 100 μl of biotinylated goat anti-human IgG for 1 h at room temperature on a plate shaker. Then the plate was washed, followed by the addition of 100 μl of streptavidin-PE (5 μg/ml) to the wells, and the plate was incubated for 30 min at room temperature on a plate shaker. The fluorescence intensity (FI), as determined using the Luminex system, was used to measure antibody (Ab) level for each peptide, and the cut-off FI value was determined as exceeding the mean plus 2SD of the FI from 14 healthy donor Ab levels. Inclusion criteria of the healthy donors are as follows: (1) levels of liver-function-related biochemical lab markers, such as AST/ALT, are normal, (2) no history of viral hepatitis, and (3) HCV-free has been confirmed by second or third generation antibody test. Allergic individuals were excluded from the healthy donors.
Statistics
Statistical analyses were performed using Student’s t-test. Values of P < 0.05 were considered statistically significant.
Results
Measurement of peptide-specific IgG levels
Using the plasma of 12 patients infected with HCV1b, we first measured the levels of IgG reactive to each of 46 peptides (listed in Table 1) derived from the consensus sequence of HCV1b, and which contained binding motifs for HLA-A3 supertype molecules. The plasma samples obtained from 14 HCV-negative healthy donors served as controls. A summary of the results is provided in Table 1. IgGs reactive to the core 30–39, core 35–43, and NS2 918–926 peptides were frequently detected in patients with HCV1b infection (7, 6, and 9 of 12 patients, respectively), and IgGs reactive to other peptides are rarely observed. The plasma levels of IgG reactive to the HCV 30–39, 35–43, and 918–926 peptides for the 12 patients and 14 HCV-negative healthy donors are also shown in Fig. 1.
Fig. 1.
Plasma levels of IgG reactive to the HCV 30–39, 35–43, and 918–926 peptides in the 12 patients with HCV1b infection and 14 HCV-negative healthy donors were measured by Luminex. The FI values of each of 1:100 diluted plasma samples were blotted in this figure. Mean of the FI values of each group is shown as closed symbols. Asterisk denotes statistically significant at P < 0.05
Induction of peptide-specific CTLs from the PBMCs of HCV1b+ patients
We assessed the ability of the three HCV peptides to induce peptide-specific CTLs in the PBMC cultures from HLA-A11+, -A31+, or -A33+ HCV1b+ patients. The PBMCs were stimulated in vitro with HCV 30–39, 35–43, and 918–926 peptides or control peptides, and their IFN-γ production in response to the corresponding peptide-pulsed C1R-A11 (HLA-A11+), C1R-A31 (HLA-A31+), or C1R-A33 (HLA-A33+) cells were examined (Table 2). Characteristics of the patients are also shown in Table 2. In cases when more than 50 pg/ml of IFN-γ was produced in response to the corresponding peptide-pulsed cells, and when the P-value was less than 0.05, as compared to that of control HIV peptide-pulsed cells, the induction of peptide-specific CTLs was considered to be positive. The three HCV peptides (HCV 30–39, 35–43, and 918–926) were found to induce peptide-specific CTLs from the PBMCs of 2, 0, and 2 of 5 HLA-A11+ patients; 1, 3, and 0 of 5 HLA-A31+ patients; and 3, 3, and 2 of 5 HLA-A33+ patients, respectively (Table 2). No apparent correlation between the clinical lab data and peptide-specific CTL responses was observed.
Table 2.
Induction of peptide-specific CTLs in PBMC culture from HCVIb-infected patients
| Patients | HLA | Age | Gender | HCV-RNA (KIU/ml) | AST (IU/ml) | ALT (IU/ml) | IFN-γ production (pg/ml)a | ||||
|---|---|---|---|---|---|---|---|---|---|---|---|
| EBV—AVFDRKSDAK | Flu—NVKNLYEKVK | HCV 30–39—IVGGVYLLPR | HCV 35–43—YLLPRRGPR | HCV 918–926—LIRACMLVR | |||||||
| Pt #1 | A11 | 64 | M | n.t | 39 | 27 | 20 | 0 | 65 | 0 | 52 |
| Pt #2 | A11 | 55 | M | 2,110 | 70 | 99 | 140 | 0 | 37 | 0 | 127 |
| Pt #3 | A11 | 69 | F | n.t | 35 | 32 | 40 | 177 | 216 | 0 | 24 |
| Pt #4 | A11 | 63 | M | n.t | 31 | 30 | 368 | 0 | 32 | 0 | 34 |
| Pt #8 | A11 | 60 | M | <0.05 | 27 | 16 | 215 | 0 | 0 | 0 | 0 |
| Pt #5 | A31 | 51 | M | n.t | 30 | 31 | 13 | 0 | 0 | 0 | 0 |
| Pt #11 | A31 | 61 | F | n.t | 26 | 25 | 112 | 0 | 0 | 0 | 0 |
| Pt #12 | A31 | 61 | F | n.t | 73 | 98 | 0 | 0 | 0 | 63 | 0 |
| Pt #13 | A31 | 49 | M | 4,000 | 41 | 51 | 70 | 591 | 182 | 82 | 0 |
| Pt #14 | A31 | 51 | M | n.t | 51 | 52 | 0 | 238 | 0 | 206 | 0 |
| Pt #3 | A33 | 69 | F | n.t | 35 | 32 | 45 | 179 | 124 | 189 | 115 |
| Pt #6 | A33 | 63 | F | 3,690 | 52 | 66 | 157 | 172 | 51 | 18 | 0 |
| Pt #8 | A33 | 60 | M | <0.05 | 27 | 16 | 116 | 0 | 56 | 72 | 255 |
| Pt #9 | A33 | 67 | F | n.t | 138 | 274 | 0 | 74 | 0 | 0 | 0 |
| Pt #15 | A33 | 33 | M | n.t | 31 | 34 | 113 | 0 | 0 | 112 | 0 |
| CTL induction rate | 8/15 | 6/15 | 6/15 | 6/15 | 4/15 | ||||||
aStatistically significant (P < 0.05) values are italicized
Cytotoxicity of the peptide-induced CTLs from PBMCs of HCV1b+ patients
We examined the cytotoxicity of the peptide-induced CTLs from the PBMCs of HLA-A3 supertype-positive patients. Cytotoxicity against the corresponding peptide-loaded C1R-A11, -A31, or -A33 cells was measured by 51-Cr release assay, and HIV-peptide-loaded cells were used to determine the non-specific cytotoxicity. Representative results are shown in Fig. 2. HCV 30–39 peptide-induced CTLs from HLA-A11+, -A31+, or -A33+ patients specifically lysed the HCV 30–39 peptide-loaded C1R-A11, -A31, or -A33 cells, respectively, in an effector cells/target cells (E/T) ratio-dependent manner (Fig. 2a). The cytotoxic activities of HCV 35–43-induced CTLs from HLA-A31+ or -A33+ patients and that of HCV 918–926-induced CTLs from HLA-A11+ or -A33+ patients were also confirmed. The cytotoxicity of these HCV-peptide induced CTLs against the corresponding peptide-loaded C1R-A11, -A31, or -A33 cells was inhibited by the addition of anti-HLA-class I or CD8 mAb, but not by anti-HLA-class II, CD4, or CD14 mAb in all cases tested (Fig. 2). These results indicated that the specific cytotoxicity of the peptide-induced CTLs was largely mediated by CD8+ T cells in an HLA-class I-restricted manner.
Fig. 2.
PBMCs of HLA-A3 supertype+ HCV1b+ patients were repeatedly stimulated with HCV 30–39 (a), HCV 35–43 (b), and HCV 918–926 (c) peptides with IL2, and their cytotoxicity to the corresponding peptide-loaded C1R-A11, -A31, or -A33 cells was measured by 6-h 51Cr-release assay (upper panels). HIV peptide-loaded target cells were also used to estimate the background cytotoxicity. Effects of various mAbs on the cytotoxicity of the peptide-induced CTLs was also measured (lower panels). Anti-CD14 mAb was used as negative control. Asterisk denotes statistically significant at P < 0.05
Discussion
Our recent studies regarding peptide-based cancer vaccines have indicated that the majority of vaccine peptides, which had been originally identified as CTL epitopes, induced both cellular and humoral immune responses to the vaccinated peptides in the clinical trials [10, 16, 23]. The IgG class of antibodies to some of the vaccine peptides is not only found in sera of the patients after vaccination but also found in the pre-vaccinated sera [10, 16, 23]. The clinical studies also showed that the pre-existence of antibodies to the vaccine peptides well correlated to subsequent induction of both CTL and IgG responses [10, 23]. The pre-existence of antibodies to the vaccine peptides indicated that presence of memory B cells to the peptides. Furthermore, presence of helper T cells that helped peptide-specific IgG responses was suggested. The helper T cells may also help the peptide-specific CTL responses. Therefore, we used antibody screening to reduce the number of HCV-peptides that possessing HLA-A3 supertype-binding motifs for subsequent selection of vaccine candidate peptides. Finally, we demonstrated that three peptides, HCV core 30–39, core 35–43, and NS2 918–926, had the potential to induce CTLs from PBMCs of HLA-A11, -A31, and -A33 positive HCV1b+ patients. These three alleles, along with the HLA-A3 and -A68 alleles belong to the HLA-A3 supertype [18, 19]. This supertype is relatively dominant in the human population, and the phenotypic frequencies of the HLA-A3 supertype, except for the HLA-A68, are ∼30, ∼35, ∼44, ∼52, and ∼35% among Caucasians, North American African-Americans, Japanese, Chinese, and Hispanics, respectively [19].
There are some reports regarding HLA-A3 supertype-restricted CTL-epitope peptides of HCV. Koziel et al. [7] have reported a peptide at positions 2588–2596 of NS5B as an HLA-A3-restricted epitope peptide with no information of the restriction to the other member of the A3 supertype. Systematic identification of HLA-A3 supertype-restricted epitope peptides of HCV was reported by Chang et al. [5]. They identified eight peptides including two core peptides, one NS1/E2 peptide, two NS3 peptides, and two NS4 peptides. At least three more peptides, one from NS3 and two from NS5A and NS5B, have been reported at the present time [6, 9, 12]. However, none of the three peptides (core 30–39, core 35–43, and NS2 918–926) identified in the present study have been reported previously. Discrepancy between the previous reports and our present results is mainly due to a difference of the HCV genotype. Most of the previous studies referred the amino acid sequence of HCV-1 prototype whose genotype is 1a. In contrast, we referred consensus sequence of HCV genotype 1b in the present study, since the genotype 1b is the dominant genotype in European and Asian countries [27]. Therefore, this is the first report of the identification of HLA-A3 supertype-restricted CTL-epitope peptides derived from HCV genotype 1b at literature levels.
Most dominant HLA allele in the world is HLA-A2 and HLA-A24 allele is also dominant in Asian countries. Namely, the HLA-A2 allele is found in 40% of Japanese, 50% of Caucasians, and 12% of Africans [1]. Similarly, the HLA-A24 allele is found in 60% of Japanese, in 20% of Caucasians, and in 12% of Africans [1]. The previously reported vaccine candidate peptides for HLA-A2+ and HLA-A24+ HCV1b+ patients [20, 22] together with the newly identified peptides for HLA-A3 supertype+ patients are able to cover the majority of HCV1b patients in the world.
In conclusion, we identified one new HCV1b-peptide that may be applicable for use in the development of therapeutic vaccines for HCV1b+ patients with HLA-A11, -A31, and -A33 alleles. The present study should facilitate the development of peptide-based vaccines for HCV1b+ patients throughout the world.
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