Memory T-cell-mediated immune responses specific to an alternative core protein in hepatitis C virus infection

Abstract

In vitro studies have described the synthesis of an alternative reading frame form of the hepatitis C virus (HCV) core protein that was named F protein or ARFP (alternative reading frame protein) and includes a domain coded by the +1 open reading frame of the RNA core coding region. The expression of this protein in HCV-infected patients remains controversial. We have analyzed peripheral blood from 47 chronically or previously HCV-infected patients for the presence of T lymphocytes and antibodies specific to the ARFP. Anti-ARFP antibodies were detected in 41.6% of the patients infected with various HCV genotypes. Using a specific ARFP 99-amino-acid polypeptide as well as four ARFP predicted class I-restricted 9-mer peptides, we show that 20% of the patients display specific lymphocytes capable of producing gamma interferon, interleukin-10, or both cytokines. Patients harboring three different viral genotypes (1a, 1b, and 3) carried T lymphocytes reactive to genotype 1b-derived peptides. In longitudinal analysis of patients receiving therapy, both core and ARFP-specific T-cell- and B-cell-mediated responses were documented. The magnitude and kinetics of the HCV antigen-specific responses differed and were not linked with viremia or therapy outcome. These observations provide strong and new arguments in favor of the synthesis, during natural HCV infection, of an ARFP derived from the core sequence. Moreover, the present data provide the first demonstration of the presence of T-cell-mediated immune responses directed to this novel HCV antigen.

FIG. 1.

Detection of IFN-γ- and IL-10-producing cells specific to the ARFP. ELISPOT assays were performed with PBMC from different cohorts of patients chronically infected with HCV and control subjects including nontreated carriers (NT), nonresponding patients (NR), responding patients (R), sustained virological responding patients (SVR), and HCV-seronegative subjects (HCV⁻). The number of IFN-γ (A)- and IL-10 (B)-producing cells per 10⁶ PBMC following in vitro stimulation by the G97A peptide (empty circles) and of IFN-γ-producing cells in response to TT (black dots) (A) were evaluated by ELISPOT on the different cohorts of patients. (C) The presence of anti-ARFP Abs in the serum or plasma of 36 of these 47 patients was assessed and is reported as OD values on the graph (saturating OD values are marked by asterisks on the graph). The dotted line on each graph represents the cutoff value of each assay, determined as described in Materials and Methods.

FIG. 2.

IFN-γ- and IL-10-producing cells are detected in response to class I predicted 9-mer epitopes derived from ARFP. The presence of IFN-γ-producing (A) and IL-10-producing (B) cells in the peripheral blood of 5 chronic HCV patients (patient 1, NT, genotype 1, HLA.A1, HLA.A24, HLA.B14, HLA.B44; patient 2, NT, genotype 1b, HLA.A1, HLA.A3, HLA.B7, HLA.B8; patient 3, NR, genotype 1, HLA.A2, HLA.B18, HLA.B35; patient 4, R, genotype 1b, HLA.A2, HLA.B18, HLA.B35; patient 5, SVR, genotype 1a, HLA.A24, HLA.A69, HLA.B51) was determined following in vitro stimulation with the following class I predicted HLA-A2- or HLA-B7-restricted peptide epitopes: peptide R7V (black bars), W7L (hatched bars), A7L (dotted bars), and G7L (white bars). The reactivity of PBMC from HCV-negative individuals against these same epitopes was evaluated and represented as the mean ± standard deviation of the results from nine and seven experiments with IFN-γ-producing cells and IL-10-producing cells, respectively. The dotted line on each graph represents the cutoff value of each assay determined as described in Materials and Methods.

FIG. 3.

Conservation of ARFP sequences between HCV-JA and genotype 1a, 1b/4, and 3 consensus sequences. (A) The amino acid sequence of the G97A peptide derived from the HCV-JA isolate (genotype 1b) was aligned with the consensus sequences derived from the dominant circulating viral variants carried by the different patients enrolled in the study. The positions of the 4 class I restricted epitopes used in the study (W7L, G7L, A7L, and R7V) are indicated by shaded boxes. Differing amino acids are indicated in boldface type. (B) The 20 sequences of the 4 class I restricted epitopes derived from each patient are aligned with the genotype 1b sequence from which these 9-mer epitopes were derived. Common amino acids between these patients' derived sequences and the corresponding synthetic genotype 1b peptide are represented by dashes while differing amino acids are specified.

FIG. 4.

Evolution of core- and ARFP-specific cytokine profiles, T-cell proliferation, Abs, and viremia in chronic HCV patients receiving therapy. Immune and virologic readouts were analyzed for six chronically infected HCV patients (patients 5 to 10) from the time of therapy onset (T₀) up to month 12 (M12) following therapy initiation. Patient 5 received only a 6-month course of therapy while all others received a 12-month course. (A) Six cytokines were measured (CBA test) in the supernatants of peripheral blood cells after in vitro stimulation with the G97A peptide, core antigen, or TT. Only cytokines produced at significant levels after antigen stimulation (sensitivity threshold, >80 pg/ml) are represented on the graph (left hand axis). T-cell proliferation, measured by flow cytometry after a 6-day incubation in the presence of CFSE, is reported as the percentage of proliferating T cells (right hand axis) and is represented by bold black crosses. (B) For patients 5 (HLA.A25, HLA.A69, and HLA.B51) and 10 (HLA.A2, HLA.A29, HLA.B7, and HLA.B51), cytokines produced in the supernatant of PBMC stimulated with the corresponding predicted 9-mer peptides were measured with the Th1/Th2 CBA kit. (C) Evolution of anti-ARFP Abs measured by ELISA (OD values are shown on the left hand axis) is reported along with the viremia levels (right hand axis) during antiviral therapy.

FIG. 4.

Evolution of core- and ARFP-specific cytokine profiles, T-cell proliferation, Abs, and viremia in chronic HCV patients receiving therapy. Immune and virologic readouts were analyzed for six chronically infected HCV patients (patients 5 to 10) from the time of therapy onset (T₀) up to month 12 (M12) following therapy initiation. Patient 5 received only a 6-month course of therapy while all others received a 12-month course. (A) Six cytokines were measured (CBA test) in the supernatants of peripheral blood cells after in vitro stimulation with the G97A peptide, core antigen, or TT. Only cytokines produced at significant levels after antigen stimulation (sensitivity threshold, >80 pg/ml) are represented on the graph (left hand axis). T-cell proliferation, measured by flow cytometry after a 6-day incubation in the presence of CFSE, is reported as the percentage of proliferating T cells (right hand axis) and is represented by bold black crosses. (B) For patients 5 (HLA.A25, HLA.A69, and HLA.B51) and 10 (HLA.A2, HLA.A29, HLA.B7, and HLA.B51), cytokines produced in the supernatant of PBMC stimulated with the corresponding predicted 9-mer peptides were measured with the Th1/Th2 CBA kit. (C) Evolution of anti-ARFP Abs measured by ELISA (OD values are shown on the left hand axis) is reported along with the viremia levels (right hand axis) during antiviral therapy.

FIG. 4.

Evolution of core- and ARFP-specific cytokine profiles, T-cell proliferation, Abs, and viremia in chronic HCV patients receiving therapy. Immune and virologic readouts were analyzed for six chronically infected HCV patients (patients 5 to 10) from the time of therapy onset (T₀) up to month 12 (M12) following therapy initiation. Patient 5 received only a 6-month course of therapy while all others received a 12-month course. (A) Six cytokines were measured (CBA test) in the supernatants of peripheral blood cells after in vitro stimulation with the G97A peptide, core antigen, or TT. Only cytokines produced at significant levels after antigen stimulation (sensitivity threshold, >80 pg/ml) are represented on the graph (left hand axis). T-cell proliferation, measured by flow cytometry after a 6-day incubation in the presence of CFSE, is reported as the percentage of proliferating T cells (right hand axis) and is represented by bold black crosses. (B) For patients 5 (HLA.A25, HLA.A69, and HLA.B51) and 10 (HLA.A2, HLA.A29, HLA.B7, and HLA.B51), cytokines produced in the supernatant of PBMC stimulated with the corresponding predicted 9-mer peptides were measured with the Th1/Th2 CBA kit. (C) Evolution of anti-ARFP Abs measured by ELISA (OD values are shown on the left hand axis) is reported along with the viremia levels (right hand axis) during antiviral therapy.