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Characterization of humoral and cell-mediated immune responses directed against hepatitis C virus F protein in subjects co-infected with hepatitis C virus and HIV-1

Troesch, Myriama,d; Jalbert, Emiliea,c,d; Canobio, Sophiea,d; Boulassel, M Rachidg; Routy, Jean-Pierreg; Bernard, Nicole Fg; Bruneau, Julieh; Lapointe, Normandb,e; Boucher, Marcb,f; Soudeyns, Hugoa,d,e

Basic Science

Background: Hepatitis C virus (HCV) F protein is encoded in an alternate reading frame overlapping the core protein region. Its precise sequence, biological function and mode of expression are currently unclear. This study was conducted to examine the prevalence and characteristics of host humoral and cell-mediated immune responses directed against F protein in patients co-infected with HCV and HIV-1.

Methods: Mutations were introduced to allow the expression of HCV-1a F protein in the absence of core. This recombinant and a truncated form lacking the first 11 amino acid residues shared with core were expressed in Escherichia coli, and their amino acid sequences were verified by mass spectrometry. Vaccinia-F protein recombinants were used to test F protein-specific cytotoxic T lymphocyte (CTL) activity. The binding of F protein-derived peptides to HLA-A*0201 was studied to identify putative CTL epitopes.

Results: Sera from 23 of 39 patients infected with various HCV genotypes recognized the truncated form, including 13 of 25 subjects co-infected with HIV-1, indicative of antigenic crossreactivity and consistent with the conservation of F protein coding sequences between HCV genotypes. Crossreactive F protein-specific CTL precursors were detected in nine of 11 HCV-infected subjects, including seven of nine patients co-infected with HCV and HIV-1. Finally, three novel putative HLA-A*0201-restricted CTL epitopes were identified.

Conclusion: These results indicate that patients co-infected with HCV and HIV-1 can mount immunoglobulin and CTL responses directed against HCV F protein that are fully comparable in scope and magnitude with those observed in individuals infected with HCV alone.

From aUnité d’Immunopathologie Virale

bCentre Maternel et Infantile sur le SIDA, and Hôpital Sainte-Justine, Montreal, Quebec, Canada

cProgram in Infectious Diseases, Clinical Research Division, Fred Hutchinson Cancer Research Center, Seattle, WA, USA

dDepartments of Microbiology and Immunology


fObstetrics and Gynecology, Université de Montréal, Montreal, Quebec, Canada

gMcGill University Health Center, Montreal, Quebec, Canada

hCHUM, Hôpital Saint-Luc, Montreal, Quebec, Canada.

Correspondence to Hugo Soudeyns, Unité d’Immunopathologie Virale, Centre de Recherche, l’Hôpital Sainte-Justine, 3175 Côte Sainte-Catherine, room 6735, Montréal, Québec H3T 1C5, Canada. Tel: +1 514 345 4931 (ext. 3907); fax: +1 514 345 4794; e-mail:

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Cell-mediated immune responses composed of CD4 T helper and CD8 cytotoxic T lymphocytes (CTL) constitute the main mechanisms by which hepatitis C virus (HCV) replication can be controlled in the host, although innate immunity, including natural killer cells, interferons, and chemokines are also involved in protective responses [1–7]. As a result of overlapping transmission routes and target populations, a large proportion of chronic HCV carriers are also infected with HIV-1, which markedly worsens the prognosis of HCV disease in adults [8,9]. The pathology of HCV–HIV co-infection is complex but results at least partly from the inhibition of HCV-specific humoral and cell-mediated immunity by HIV-1 [10–12]. Cryptic epitopes generated from the translation of viral or cellular gene products using alternate reading frames are known to elicit such responses, and were recently shown to represent an important source of tumour-specific antigens in certain forms of human cancer [13,14]. Various RNA and DNA viruses of bacteria, plants and animals use overlapping open reading frames to compensate for size limitations imposed on viral genomes or to regulate viral protein expression. Likewise, HCV encodes an alternative 144–162 amino acid, 17 000 Mr polypeptide termed F protein or alternative reading frame protein from the 5′ moiety of the core gene [15–17]. The function and biological significance of F protein are unclear [15–19]. It is expressed in transfected cells [20] and can be recognized by sera and T cells isolated from HCV-infected patients, suggesting that it is produced in vivo [16,17,21,22]. Here we explore the prevalence and characteristics of host immune responses directed against F protein in patients co-infected with HCV and HIV-1.

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Materials and methods

Study subjects and clinical parameters

For studies of humoral immune responses, 39 female patients were selected among the participants of the Centre Maternel et Infantile sur le SIDA mother–child cohort (Hôpital Sainte-Justine, Montreal, Canada). The mean age was 29.6 ± 5.75 years (range 20.1–41.3). HCV infection was confirmed by enzyme-linked immunosorbent assay (ELISA) and recombinant immunoblot assays, in accordance with clinical practice and diagnostic algorithms used in the province of Quebec. The mean HCV-RNA level (COBAS Amplicor HCV Monitor assay version 2.0; Roche Diagnostics, Montreal, Canada) was 5.37 log IU/ml plasma (range 2.78–7.09). HCV genotyping was performed by sequence analysis of the 5′ non-coding region [23]. Co-infection with HIV-1 was confirmed by ELISA and non-quantitative polymerase chain reaction (PCR) in 25 of the 39 subjects. In co-infected patients, the mean CD4 cell count, measured using flow cytometry, was 509 ± 243 cells/mm3 (range 33–1287), and the mean HIV-1 viral load (Versant HIV RNA version 3.0 assay; Bayer, Pittsburgh, PA, USA) was 2.81 log RNA copies/ml plasma (range 1.70–4.73). Nineteen of the 25 subjects were treated with antiretroviral therapy, including single agent (n = 3), double combination therapy (n = 6), and triple combination therapy (n = 11). The reported HIV risk categories included injection drug use (n = 22), probable heterosexual transmission (n = 7), as well as surgical procedures or transfusions performed in an HIV-endemic region (n = 3). For studies of cell-mediated immunity, patients were selected from among participants in the St Luc intravenous drug user cohort (CHUM, Hôpital St-Luc, Montreal, Canada) (n = 7; HND, HTM, and PSL codes) or from the Centre Maternel et Infantile sur le SIDA cohort (n = 4; TVC codes). The clinical characteristics of subjects in this second study group are summarized in Table 1. Alanine aminotransferase (ALT) and aspartate aminotransferase (AST) levels were assayed on a Synchron LX20 system (Beckman Coulter, Palo Alto, CA, USA). None of the subjects in either group had been treated with anti-HCV therapy at the time of the study. Full informed consent was obtained from all study participants. Counseling and appropriate medical treatment were provided. This research protocol was approved by the Ethics Review Board of the Montreal Chest Institute, CHUM Hôpital Saint-Luc, and Hôpital Sainte-Justine, Montreal, Canada, where patients were enrolled and the study was conducted.

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Hepatitis C virus F protein gene cloning and mutagenesis

Viral genomic RNA was isolated from 500 μl serum obtained from a subject (TVC33) infected with HCV-1a, and was reverse transcribed and amplified using the QIAamp procedure (Qiagen, Mississauga, Ontario, Canada) with primers HCV 1a 1-16 (5′–GCC AGC CCC CTG ATG G–3′) and HCV 988-970 (5′–GCC TCG TAC ACA ATA CTC G–3′) (Alpha DNA, Montreal, Canada). Reverse transcription (RT)–PCR conditions were 50°C/30 min, 95°C/15 min denaturation, 40 cycles of 94°C/30 s, 55°C/1 min, and 72°C/1 min, followed by a 72°C/10 min extension cycle, in a T3 thermal cycler (Biometra, Goettingen, Germany). The amplicon was cloned into the SrfI site of pCRScript Amp SK+ (Stratagene Cloning Systems, La Jolla, CA, USA; pCore1a33). Core sequences were then modified by mutagenesis to force-shift translation into the (+2) reading frame, and lock translation by introducing three silent mutations within the frameshift-associated slippery-like sequence (Fmut8). This was done by first inserting the AatII-NotI fragment from pCore1a33 into the pSC11ss vaccinia transfer vector [24] cleaved with StuI and NotI (pSC11ssF16). pCore1a33 was reamplified using primers Fmut S Sal (5′–GAC CGT CGA CCA TGA GCA CGA ATC CTA AAC CTC AGA GGA AGA CCC CAA ACG TAA–3′) and Fmut AS Kpn (5′–AAG GGT ACC CGG GCT GAG CCC AGG TCC TGC CCT CGG G–3′). PCR conditions were 94°C/3 min, 25 cycles of 94°C/30 s, 60–72°C/1 min, and 72°C/1 min, followed by 72°C/15 min extension in a TGradient thermal cycler (Biometra). The amplicon was then cut with SalI and KpnI, and was shuffled into pSC11ssF16 to form pSC11ssFmut8. A truncated form of the F protein lacking the 11 first N-terminal amino acids shared with core (Fmut8Δ11) was similarly generated using primers HCV1a Sal I (5′–TCA AGT CGA CCC AAA CGT AAC ACC AAC CG–3′) and pCRS1 (5′–GGA AAC AGC TAT GAC CAT GAT TAC GCC AAG C–3′). PCR conditions were 25 cycles of 94°C/30 s, 53°C/1 min, and 72°C/1 min, followed by 72°C/15 min extension. Finally, Fmut8 and Fmut8Δ11 were also subcloned into SalI–NotI-digested pET-30c and pET-30b, respectively (Novagen, Madison, WI, USA), for inducible, N-terminal His-tagged expression in Escherichia coli. The structure of all constructs was verified by automated DNA sequencing.

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Production of recombinant F protein

pET-30cFmut8 and pET-30bFmut8Δ11 were expressed in E. coli BL21 cells, and F protein was purified under denaturing conditions on Ni-NTA His-Bind resin (Novagen), followed by preparative polyacrylamide gel electrophoresis using a Mini Prep cell (Bio-Rad Laboratories, Hercules, CA, USA). The Fmut8Δ11 gene product was used to raise rabbit antiserum with no crossreactivity against core protein. For mass spectrometry (MS), Coomassie-stained protein gel bands were rehydrated and trypsinized. Extracted peptides were then eluted from a nanoscale C18 reverse-phase high-pressure liquid chromatography capillary column, and were subjected to electrospray ionization followed by MS using an LCQ DECA ion-trap mass spectrometer (ThermoFinnigan, San Jose, CA, USA). Protein identity was assessed by sequence comparison with protein or translated nucleotide databases with the use of the SEQUEST program [25].

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F protein enzyme-linked immunosorbent assay

Antigen (2 μg/ml Ni-NTA-purified Fmut8Δ11) was incubated overnight at 4°C in flat-bottomed 96-well polystyrene plates in 100 μl 0.1 M sodium bicarbonate pH 8.6. Plates were blocked for 2 h/4°C with 200 μl per well of 5 mg/ml bovine serum albumin in 100 mM sodium bicarbonate pH 8.6, and were washed six times with 10 mM tris base, 150 mM sodium chloride, 0.05% Tween-20 pH 7.4 (1× TBST). Dilutions of the patient's sera (1/50 to 1/6400 in 100 μl 1× TBST) were then added and incubated for 2 h at room temperature with gentle shaking. Wells were washed six times with 1× TBST and 100 μl/well of alkaline phosphatase-coupled anti-human IgG antibody (1/3000; Sigma, Saint Louis, MO, USA) or anti-rabbit antibody (1/1000; Biosys, Compiegne, France) was added in 1× TBST + 5 mg/ml bovine serum albumin. After 1 h incubation at room temperature with gentle agitation, wells were washed six times, and antibody binding was revealed with 50 mM sodium bicarbonate, 1 mM magnesium chloride, and 1 mg/ml p-nitrophenyl-phosphate. After 90 min, optical density was measured at 410 nm in a MR7000 spectrophotometer (Dynatec, Chantilly, VA, USA). Rabbit antisera raised against Fmut8Δ11 or purified core protein (kindly provided by D. Leclerc, Centre de Recherche du CHUL, Quebec, Canada) were used as positive and negative controls.

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Preparation of recombinant Vaccinia virus

CV1 cells, maintained in Dulbecco's minimal essential medium (DMEM) supplemented with 10% fetal bovine serum (FBS; Invitrogen, Burlington, Canada), were infected with the Western Reserve strain of Vaccinia virus, and were then transfected with pSC11ssFmut8 using calcium chloride precipitation. Progeny virus was extracted by three freeze–thaw cycles, treatment with 0.25 mg/ml trypsin (Worthington, Lakewood, NJ, USA) and sonication, followed by three rounds of plaque selection on HuTK-143B cells incubated in 2% w/v low melting point agarose (Invitrogen) supplemented with 2× DMEM, 0.05 mg/ml neutral red, 80 μM bromodeoxyuridine, and 400 μM 5′-bromo-4-chloro-3-indolyl-β-D-galactopyranoside (X-Gal) [24]. The identity and structure of plaque-purified Vaccinia recombinants were verified by PCR and automated DNA sequencing.

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Cytotoxic T lymphocyte assays

To serve as targets in cellular cytotoxicity assays, autologous B-lymphoblastoid cell lines were derived from each of the study subjects by incubating patient peripheral blood mononuclear cells (PBMC) isolated on a Ficoll gradient (Amersham Biosciences, Mississauga, Canada) with supernatant from the B95-8 Epstein–Barr virus-infected cell line in the presence of 1 μM cyclosporine A (Sandoz, Vienna, Austria) [26]. For limiting dilution analysis, serial dilutions (500 cells per well–seven cells per well) of patient PBMC were replica-plated onto wells containing 200 000 irradiated (3000 rads) allogeneic feeders cells, and were cultivated for 21 days in RPMI media supplemented with 10% FBS, 1 μg/ml phytohemagglutinin (Sigma) and 80 units/ml recombinant IL-2 (Hoffman-La Roche, Nutley, NJ, USA; obtained through the National Institutes of Health AIDS Research and Reference Reagents Programme). Autologous B-lymphoblastoid cell lines, infected at a multiplicity of infection of 10 with specific Vaccinia recombinants, were labeled with 51Cr-sodium chromate (Amersham Biosciences) and the cytotoxic activity of cultured effectors was tested after a standard 5 h incubation [27]. Wild-type Vaccinia Western Reserve and vaccinia SC59 NNRd expressing amino acid residues 364–1618 (E2, p7, NS2, and NS3) of HCV-1a (obtained from M. Houghton, Chiron Corporation, Emeryville, CA, USA) [28] were used as controls in these experiments. Percentage specific lysis was defined as 100× (test release–spontaneous release)/(total release–spontaneous release), with the significance threshold set at 2.67 standard deviations above negative control values. CTL precursor frequencies and 95% confidence intervals were computed using software kindly provided by Dr Spyros Kalams (Vanderbilt University, Nashville, TN, USA).

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Peptide binding assay

Thirty-seven overlapping peptides (15 amino acid residues with 10 residue overlaps) derived from F protein and five additional 15-mer peptides corresponding to alternative N-terminal frameshift products of the core protein sequence were synthesized using Fmoc chemistry (SynPep, Dublin, CA, USA) based on the sequence of HCV-1a F protein (GenBank accession no. M62321) [29], and were resolubilized in 25% w/v dimethyl sulphoxide in 1× Hank's balanced salts solution. Peptides were diluted to 30, 100, and 200 μg/ml final concentration in DMEM supplemented with 3% w/v FBS, and were incubated with 100 000 serum-depleted (3% FBS for 24 h) T2 cells [30] for 16 h at 37°C in 5% carbon dioxide. T2 cells were then stained (30 min at room temperature) for MHC class I molecules using fluorescein isothiocyanate-conjugated W6/32 monoclonal antibody (Sigma) and were analysed on a FACSCalibur flow cytometer (Becton-Dickinson Immunocytometry Systems, La Jolla, CA, USA) with live gating based on forward and side scatter.

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F protein-specific humoral immune responses

Five synonymous nucleotide substitutions were introduced in the putative frameshift-associated slippery-like sequence to force the expression of HCV-1a F protein in the absence of core gene product (Fig. 1a). This recombinant protein (Fig. 1b) and a truncated form lacking the first 11 amino acid residues shared with core (data not shown) were expressed in E. coli and purified by nickel chelation chromatography. Because multiple translational recoding events were reported to occur when F protein was produced in different expression systems [16,31,32], the amino acid sequences of Fmut8 and Fmut8Δ11 were confirmed using MS, with peptide coverage of 87.0 and 68.2% by amino acid count for Fmut8 and Fmut8Δ11, respectively (Fig. 1c and data not shown). Purified Fmut8Δ11 was used in ELISA to measure reactivity against F protein in sera derived from 39 patients infected with various HCV subtypes, including HCV-1a (n = 20), HCV-1b (n = 9), HCV-3a (n = 7), HCV-4a (n = 1), HCV-4c (n = 1), and HCV-5a (n = 1). Of these patients, 25 were also co-infected with HIV-1. Levels of ALT and AST were significantly higher in co-infected patients than in patients infected with HCV alone (P = 0.0464 and P = 0.00542, respectively, Student's t test). The plasma HCV load was also larger in co-infected subjects, but this difference was not statistically significant (P = 0.0778, Student's t test).

Overall, serum samples from 23 of 39 HCV-infected patients (59.0%) had positive reactivity to Fmut8Δ11 at dilutions of 1: 400 or greater (Fig. 2a). These include 10 of 20 patients (50.0%) infected with HCV-1a, seven of nine patients (77.8%) infected with HCV-1b, four of seven patients (57.1%) infected with HCV-3a, two of two patients (100%) infected with HCV-4a or 4c, and none of one patient (0.0%) infected with HCV-5a. Humoral responses directed against F protein were only detected in HCV-infected subjects (Fig. 2a and data not shown). Plasma HCV viral load, ALT and AST levels were not significantly different in subjects with detectable versus undetectable anti-F antibody responses (P = 0.137, 0.112, and 0.105, respectively, Student's t test). Furthermore, there was no correlation in linear regression analysis between titres of anti-F antisera and HCV viral load, ALT, or AST (r2 = 0.124, 0.134, and 0.125, respectively).

The proportion of patients co-infected with HCV and HIV-1 who displayed detectable antibody responses to F protein (13 of 25 subjects; 52.0%) was not significantly different from that observed in subjects infected with HCV alone (10 of 14 subjects; 71.4%) (P = 0.317, Fisher's exact test), and was similar to that reported in a recent survey of HCV-infected subjects [22]. When analysis was restricted to patients with detectable anti-F antibody responses, ELISA titres were not significantly different in co-infected patients (n = 13) than those measured in sera from subjects infected with HCV alone (n = 10) (P = 0.170, Mann–Whitney U test) (Fig. 2a). F protein-specific antibody responses were detected in co-infected patients with CD4 cell counts ranging from 33 cells/mm3 to 1287 cells/mm3 (Fig. 2c). However, there was no significant correlation between the anti-F antibody titre and CD4 cell counts (r2 = 0.146) (Fig. 2b), although the correlation coefficient improved (r2 = 0.205) and bordered statistical significance when subjects with CD4 cell counts less than 200 cells/mm3 were excluded from the analysis (data not shown). There was no significant correlation between the anti-F antibody titre and HIV-1 viral load (r2 = 0.00172) (Fig. 2c). Finally, crossreactive HCV subtype recognition of F protein was observed in sera from co-infected patients (Fig. 2a). ELISA results were corroborated by Western blot (data not shown). Overall, these data indicate that co-infected patients can mount immunoglobulin responses against HCV F protein, and that these responses are crossreactive between HCV subtypes.

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F protein-specific cytotoxic T-lymphocyte activity

T-cell microcultures were derived from PBMC samples obtained from 11 HCV-infected patients, including nine subjects co-infected with HCV and HIV-1. Vaccinia-Fmut8 recombinant virus was generated and used in 51Cr-release limiting dilution analysis to test F protein-specific CTL activity (Table 1; Fig. 3). Overall, CTL precursors were detected in nine of 11 HCV-infected subjects (81.8%), with precursor frequencies ranging from 1/177 to 1/13372 T cells. These include patients infected with HCV-1a (n = 6), HCV-2a (n = 1) and HCV-3a (n = 2), indicative of the crossreactive recognition of F protein by CTL between HCV subtypes. Although others have reported the production of IFN-γ and IL-10 after the in-vitro stimulation of T cells with F protein-derived peptides [21], our results represent the first direct evidence of cell-mediated cytotoxic activity directed against HCV F protein in HCV-infected individuals. CTL precursors were also detected in seven of nine patients (77.8%) co-infected with HIV-1 (mean CD4 T-cell counts: 519 cells/μl; mean HIV-1 viral load: 3.46 log RNA copies/ml; Table 1). CTL precursors that recognized other HCV proteins (residues 364–1618, i.e. E2, p7, NS2, NS3, but not core or F protein) were also observed in six of eight subjects (75%), with precursor frequencies between 1/1608 and 1/12188, not significantly different from those observed with F protein (P = 0.529, Mann–Whitney U test). Patient HTM319 did not exhibit cytotoxic activity against either of the targets tested, whereas patients HTM325 and PSL19 had discordant responses to F protein and p364-1618 (Table 1; Fig. 3). Finally, there was no correlation between F protein-specific CTL precursor frequencies and patient CD4 cell counts, HIV-1 or HCV viral loads, although such comparisons would be unreliable given the limited number of subjects tested.

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Analysis of F protein peptide binding

Forty-two overlapping 15-mer peptides corresponding to the HCV-1a F protein sequence were tested for interaction with HLA-A*0201 molecules using the T2 peptide binding assay [30]. The HCV-1 NS3 1406-1415 peptide (KLVALGINAV), a well-characterized HLA-A*0201 restricted T-cell epitope [33], was used as a positive control. Mean fluorescence intensity after class I MHC staining by the W6/32 monoclonal antibody was used as the readout. Whereas the efficacy of individual F protein-derived peptides to promote HLA-A*0201 expression at the cell surface varied considerably, six 15-mers stood out as high binders in initial screening experiments (F29–43, F33–47, F37–51, F61–75, F65–79, and F101–115) (200 μg/ml; Fig. 4a). Of those, only peptides F29–43 (VRSLVEFTCCRAGAL), F37–51 (CCRAGALDWVCARRE), and F101–115 (PVALGLAGAPQTPGV) clearly mediated a dose-dependent increase in MHC class I expression at the surface of T2 cells (Fig. 4b). Peak fluorescence obtained with peptides F29–43, F37–51, and F101–115, respectively, reached 87.6, 79.2, and 83.2% of the levels obtained with NS3 1406–1415 (Fig. 4b). The presence of HLA-A*0201-restricted epitopes within peptides F29–43 (VRSLVEFTCCRAGAL) had been predicted in advance based on an analysis of HCV-1a sequences (GenBank accession no. M62321) using the BIMAS web tool (, with a predicted MHC-peptide dissociation time of 20.4 s. Consistent with this, exposure of T2 cells to peptide F31–40 (SLVEFTCCRA) clearly resulted in a dose-dependent increase in cell-surface expression of MHC class I (Fig. 4c). Searches of HCV-1a sequences based using BIMAS also uncovered another potential HLA-A*0201-restricted epitope within peptide F101–115 (PVALGLAGAPQTPGV) (predicted dissociation time of 7.45 s). Within our panel, SLVEFTCCRA and ALGLAGAPQT are only represented in single peptides (i.e. F29–43 and F101–115, respectively). SLVEFTCCRA lies within a region of F protein that is the most conserved between HCV subtypes (Table 2), suggesting that it could display antigenic crossreactivity. In addition, both HLA-A*0201 anchor residues (P2 and P9) are fully conserved across all HCV genotypes examined (Table 2 and data not shown). ALGLAGPQT resides within a section of F protein that is highly variable between genotypes, and this variability also extends to anchor residues. This suggests that it could be HCV-1 subtype specific and that it might not be equally recognized in individuals infected with other HCV strains. Use of the SYFPEITHI algorithm ( revealed other high-scoring potential HLA-A*0201-restricted epitopes, including CRAGALDWV, located within peptides F33–47 and F37–51. In this case, the absence of a clear dose–response with F33–47 compared with F37–51 (Fig. 4b) could be caused by differential peptide processing by T2 cells.

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Taken as a whole, these results indicate that: (i) At the level of humoral immune responses, there is antigenic crossreactivity among F proteins derived from distinct HCV subtypes, consistent with the high degree of intersubtype conservation of F protein amino acid sequences. Although most of the subjects studied had reasonably high CD4 cell counts, significant titres of anti-F antibodies were observed in two patients with less than 200 CD4 T cells/mm3. (ii) F protein-specific cell-mediated immunity can be readily detected in the large majority of, but not all, subjects co-infected with HCV and HIV-1. These responses were crossreactive between HCV subtypes. (iii) Several peptide sequences derived from HCV-1a F protein were capable of binding HLA-A*0201 in cell culture and could therefore correspond to novel HLA-A*0201-restricted CTL epitopes. These differ from those previously reported, with SLVEFTCCRA and CRAGALDWV positioned upstream relative to the sequence corresponding to the 99 amino acid synthetic peptide used by Bain et al. [21].

Because F protein sequences are constrained by the presence of RNA secondary structures and overlapping core sequences, their level of intersubtype conservation is on the same level as that observed with core [34]. CTL epitopes encoded within this region could thus be less susceptible to escape mutation than other segments of the HCV genome, notably E1, E2, and NS3 [35]. This property could be particularly important given the fact that viral escape mutations are believed to contribute to HCV persistence in the host [36]. Also, because F protein coding sequences are located directly downstream from the translational initiation site, and because its production is presumably independent of viral and host proteases (it is not part of the HCV polyprotein per se), it is likely to be one of the first HCV gene products to be expressed after infection of the host cell. This is of significance because immunodominance and epitope hierarchy are influenced not only by the levels of expression of a given viral protein but also by the timing of its expression [37]. Finally, unlike core, F protein has so far not been shown to participate in molecular or cellular pathogenesis [19]. For these reasons, F protein represents an attractive candidate as a component in the development of HCV vaccines, and its immunogenic properties should be investigated further, especially given the fact that vaccination could become an important public health instrument to prevent the acquisition of HCV in patients infected with HIV-1.

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M.T. and E.J. contributed equally to this work. The authors wish to thank Silvie Valois, Martine Caty, Pascal Lapierre and Ampha Khammy for expert technical assistance, Ross Tomaino (Taplin Biological Mass Spectrometry Facility, Boston, MA, USA) for protein sequence analysis, and Naglaa Shoukry for critical reading of the manuscript. H.S. is a Junior-II Scientist of le Fonds de la Recherche en Santé du Québec. M.T. is the recipient of a scholarship from la Fondation de l’Hôpital Sainte-Justine.

Sponsorship: This research was supported by the Canadian Network for Vaccines and Immunotherapeutics, by Valorisation-Recherche Québec, by the FRSQ-SIDA-MI Network, and by grant No28-PG-51355 from the Elizabeth Glases Pediatric AIDS Foundation.

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alternate reading frame protein; co-infection; crossreactivity; cytotoxic T lymphocytes; hepatitis C

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