Antiviral CD8-mediated responses in chronic HCV carriers with HBV superinfection

Authors

  • Carolina Boni,

    1. Divisione Malattie Infettive ed Epatologia (Division of Infectious Diseases and Hepatology), Azienda Ospedaliera Universitaria di Parma, Parma, Italy
    Search for more papers by this author
  • Barbara Amadei,

    1. Divisione Malattie Infettive ed Epatologia (Division of Infectious Diseases and Hepatology), Azienda Ospedaliera Universitaria di Parma, Parma, Italy
    Search for more papers by this author
  • Simona Urbani,

    1. Divisione Malattie Infettive ed Epatologia (Division of Infectious Diseases and Hepatology), Azienda Ospedaliera Universitaria di Parma, Parma, Italy
    Search for more papers by this author
  • Paola Fisicaro,

    1. Divisione Malattie Infettive ed Epatologia (Division of Infectious Diseases and Hepatology), Azienda Ospedaliera Universitaria di Parma, Parma, Italy
    Search for more papers by this author
  • Alessandro Zerbini,

    1. Divisione Malattie Infettive ed Epatologia (Division of Infectious Diseases and Hepatology), Azienda Ospedaliera Universitaria di Parma, Parma, Italy
    Search for more papers by this author
  • Cristina Mori,

    1. Divisione Malattie Infettive ed Epatologia (Division of Infectious Diseases and Hepatology), Azienda Ospedaliera Universitaria di Parma, Parma, Italy
    Search for more papers by this author
  • Gabriele Missale,

    1. Divisione Malattie Infettive ed Epatologia (Division of Infectious Diseases and Hepatology), Azienda Ospedaliera Universitaria di Parma, Parma, Italy
    Search for more papers by this author
  • Roberto Bertoni,

    1. Divisione Malattie Infettive ed Epatologia (Division of Infectious Diseases and Hepatology), Azienda Ospedaliera Universitaria di Parma, Parma, Italy
    Search for more papers by this author
  • Annalisa Azzurri,

    1. Dipartimento di Medicina Interna (Department of Internal Medicine), Università di Firenze, Firenze, Italy
    Search for more papers by this author
  • Gianfranco Del Prete,

    1. Dipartimento di Medicina Interna (Department of Internal Medicine), Università di Firenze, Firenze, Italy
    Search for more papers by this author
  • Carlo Ferrari

    Corresponding author
    1. Divisione Malattie Infettive ed Epatologia (Division of Infectious Diseases and Hepatology), Azienda Ospedaliera Universitaria di Parma, Parma, Italy
    • Divisione Malattie Infettive ed Epatologia, Azienda Ospedaliera e Universitaria di Parma, Via Gramsci 14, 43100 Parma, Italy
    Search for more papers by this author
    • fax: +39 521-988706.


Abstract

Hepatitis B virus (HBV) superinfection in chronic hepatitis C represents a natural model to investigate whether or not hepatitis C virus (HCV) can influence priming and maturation of antiviral T cells; whether or not HBV superinfection, which is known to determine control of HCV replication, can restore HCV-specific T cell responsiveness; and whether or not cytokines stimulated by HBV infection can contribute to HCV control. To address these issues, the function of CD8 cells specific for HBV and HCV was studied longitudinally in two chronic HCV patients superinfected with HBV. Patients with acute hepatitis B were also examined. Frequency and function of HBV tetramer+ CD8 cells were comparable in patients acutely infected with HBV with or without chronic HCV infection. HBV-specific CD8 cell function was efficiently expressed irrespective of serum HCV-RNA levels. Moreover, fluctuations of HCV viremia at the time of HBV superinfection were not associated with evident changes of CD8 responsiveness to HCV. Finally, no correlation was found between serum levels of interferon α, interleukin (IL)-12, IL-10, or IL-18 and control of HCV replication. In conclusion, HCV did not affect the induction of primary and memory HBV-specific CD8 responses. HCV-specific CD8 responses were undetectable when HCV-RNA was negative, showing that inhibition of HCV replication in the setting of a HBV superinfection was not sufficient to induce a restoration of CD8 reactivity against HCV. (H EPATOLOGY 2004;40:289–299.)

Combined hepatitis C virus (HCV) and hepatitis B virus (HBV) infection is a frequent clinical finding that can be associated with severe forms of chronic liver disease. Each of the two viruses can inhibit the replication of the other,1–4 and acute HBV infection has been reported to induce transient or even persistent HCV clearance in patients with chronic HCV infection.5–9 Although a direct interference of HBV on HCV replication has been postulated in this situation of dual infection, the antiviral immune response to each of these viruses may also influence the rate of HCV replication.5 Conversely, the presence of HCV at the time of HBV infection might exert a negative effect on the priming of the immune response to HBV, as suggested by the observation that HCV or HCV gene products can influence dendritic cell maturation and T cell function.10–16 Through these mechanisms, HCV may interfere with the function of HCV-specific CD8 cells which has been reported to be defective at the early stages of HCV infection.17–21 In addition, the high HCV load is believed to be a primary determinant of the persistent collapse of the HCV-specific T cell response observed in chronic HCV infection.17 If this is the case, inhibition of HCV replication associated with HBV superinfection may allow reconstitution of T cell responsiveness. Therefore, HBV superinfected patients with pre-existing chronic HCV infection provide an opportunity to address a series of important and still unsolved issues regarding virus–host interplay during HBV and HCV infections.

To further define the reciprocal influence between HCV and cellular components of the immune system, we asked whether or not HCV can influence T cell response to a different but closely related pathogen, such as HBV, and whether the CD8 response to HCV can change in relation with fluctuations of viremia and superinfection by HBV. Moreover, some parameters of innate immunity, such as serum levels of interferon (IFN)-α, interleukin (IL)-12, IL-10, and IL-18, were analyzed to assess whether or not a correlation can be found between spontaneous control of HCV replication and activation of the innate immune system caused by simultaneous acute HBV infection. Addressing these issues may provide important information relative to disease pathogenesis and development of therapeutic approaches for HBV and HCV infections.

Abbreviations

HBV, hepatitis B virus; HCV, hepatitis C virus; IL, interleukin; IFN, interferon; HLA, human leukocyte antigen; PBMC, peripheral blood mononuclear cell; ELISpot assay, enzyme-linked immunosorbent spot assay; SFC, spot-forming cell; ICS, intracellular cytokine staining.

Patients and Methods

Patients.

Two human leukocyte antigen (HLA)-A0201 positive patients with chronic hepatitis C (the first infected by HCV of genotype 1a and the second infected by genotype 2c) and acute HBV superinfection and 5 HLA-A0201–positive patients with acute hepatitis B enrolled at the Department of Infectious Diseases and Hepatology of the University Hospital of Parma were studied.

The diagnosis of chronic HCV infection was based on the following criteria: documented seroconversion to anti-HCV antibodies, detection of HCV-RNA, and levels of serum alanine aminotransferase at least 1.5 times the upper limit of normal (50 U/L) for at least 1 year prior to HBV superinfection, in the absence of other possible causes of liver disease (e.g., viruses, toxins, alcohol, autoimmunity, and metabolic factors). The diagnosis of acute HBV infection was made based on the detection of elevated alanine aminotransferase levels (at least 10 times the upper limit of normal), hepatitis B surface antigen, and immunoglobulin M anti–hepatitis B core antigen antibodies in the serum. All patients were negative for anti–human immunodeficiency virus 1 and 2 antibodies and for other markers of viral or autoimmune hepatitis. All patients gave written informed consent before entering the study, and the study protocol conformed to the ethical guidelines of the 1975 Declaration of Helsinki.

Virological Assessment.

Hepatitis B surface antigen, anti–hepatitis B surface antigen, total and immunoglobulin M anti-–hepatitis B core antigen, hepatitis B e antigen, anti–hepatitis B e antigen, anti–hepatitis D virus, anti-HCV, anti–human immunodeficiency virus 1 and 2 were determined by commercial enzyme immunoassay kits (Abbott Laboratories, North Chicago, IL; Ortho Diagnostic Systems, Raritan, NJ; Sanofi Diagnostics Pasteur, Marnes-la-Coquette, France). Serum HBV-DNA was quantified by PCR (Cobas Amplicor Test, Roche Diagnostic, Basel, Switzerland). Serum HCV-RNA was analyzed by branched-DNA assay (Bayer System 340bDNA Analyzer, Bayer Corporation, Tarrytown NY, USA).

Synthetic Peptides, Peptide-HLA Class I Tetramers, and Antibodies.

Synthetic peptides and the corresponding phycoerythrin-labeled tetrameric peptide-HLA Class I complexes representing the HLA-A2 restricted epitopes HBV core 18-27 (FLPSDFFPSV), polymerase 575-583 (FLLSLGIHL), polymerase 816-824 (SLYADSPSV), envelope 183-191 (FLLTRILTI), envelope 335-343 (WLSLLVPFV), envelope 348-357 (GLSPTVWLSV) and the HCV peptides NS3 1073-1081 (CINGVCWTV), NS3 1406-1415 (KLVALGINAV), NS4B 1992-2000 (VLSDFKTWL), and NS5 2594-2602 (ALYDVVTKL) of genotype 1a were purchased from Chiron Mimotopes (Victoria, Australia) and from Proimmune LTD (Oxford, UK). The corresponding HCV peptides of genotype 2c as well as a panel of 601 15-mer peptides overlapping by 10 residues and covering the overall HCV-1 sequence were also purchased from Chiron Mimotopes. Anti-CD8 (conjugated with Quantum Red or fluorescein isothiocyanate) and anti–IFN-γ fluorescein isothiocyanate were purchased from Sigma Aldrich (St. Louis, MO). Anti-perforin (fluorescein isothiocyanate) was purchased from BD Pharmingen (San Jose, CA).

Isolation of Peripheral Blood Mononuclear Cells and In Vitro Expansion of HBV and HCV-specific CD8 Cells.

Peripheral blood mononuclear cells (PBMCs) were isolated from fresh heparinized blood by Ficoll-Hypaque density gradient centrifugation and resuspended in RPMI 1640 supplemented with 25 mmol/L hydroxyethylpiperazine-N-2 ethanesulfonic acid, 2 mmol/L L-glutamine, 50 μg/mL gentamycin, and 8% human serum (complete medium). For cytotoxic T lymphocyte expansion, PBMCs were resuspended in 96-well plates at a concentration of 2 × 106/mL in complete medium, supplemented with IL-7 (5 ng/mL) and IL-12 (100 pg/mL), and stimulated with HBV and HCV peptides at 1 μM final concentration. Recombinant IL-2 was added on day 4 of culture (50 UI/mL), and the immunological assays were performed on day 10.

Cell Surface and Intracellular Staining, Enzyme-Linked Immunosorbent Spot Assay.

Staining with tetramers and other surface markers and perforin and IFN-γ staining were performed as described previously.21 Enzyme-linked immunosorbent spot (ELISpot) assays were performed using a panel of 601 15-mer peptides based on a genotype 1a sequence (HCV-1) covering all structural (core, E1, E2) and nonstructural (NS2, NS3, NS4, NS5) HCV regions, pooled in 119 mixtures. HCV-specific T cell responses were analyzed using ELISpot assay after overnight incubation with individual peptide mixtures of either PBMCs (ex vivo analysis) or short-term polyclonal T cell lines previously expanded in vitro by 10 days stimulation with the same peptide mixtures used for the ELISpot assay (in vitro analysis). Briefly, 96-well plates (Multiscreen-IP-Millipore S.A.S., Malshelm, France) were coated overnight at 4°C as recommended by the manufacturer with 5 μg/mL capture mouse anti-human IFN-γ monoclonal antibody (1 DIK, Mabtech, Sweden). Plates were then washed 7 times with phosphate-buffered saline/0.05% Tween 20, blocked with RPMI/10% fetal calf serum for 2 hours at 37°C. 2 × 105 PBMCs or 8 × 104 to 5 × 104 T cells from short-term polyclonal T cell lines were seeded per well. Plates were incubated for 18 hours at 37°C in the presence or absence of peptides. After washing with phosphate-buffered saline/0.05% Tween 20, 50 μL of 1 μg/mL biotinylated secondary mouse anti–human IFN-γ monoclonal antibody (7B6-1, Mabtech, Sweden) was added. After 3 hours of incubation at room temperature, plates were washed 4 times and 100 μL goat alkalin phosphatase anti-biotin Ab (Vector Laboratories Inc. Burlingame, CA) was added to wells, and the plates were incubated for a further 2 hours at room temperature. Plates were then washed 4 times, and 75 μL of alkalin phosphatase conjugate substrate (5-bromo-4-chloro-3-indolyl phosphate, Biorad Laboratories, Hercules, CA) was added. After 4 to 7 minutes, the colorimetric reaction was stopped by washing with distilled water. Plates were air-dried and spots were counted using an automated ELISpot reader (AID ELISpot Reader System, Autoimmune Diagnostika Gmbh, Strassberg, Germany). IFN-γ–producing cells were expressed as spot-forming cells (SFC) per well. The number of specific IFN-γ–secreting cells was calculated by subtracting the unstimulated control value from the stimulated sample. While unstimulated controls for ex vivo analysis were the same for all stimulated PBMC samples, distinct controls were set up for each individual polyclonal T cell line derived from 10 days of PBMC stimulation with individual peptide mixtures. Positive controls consisted of PBMCs stimulated with phytohemagglutin. Wells were considered positive if they were at least 2 times above background. To avoid the possibility that positive wells were missed because of a high background level, all in vitro experiments with an SFC ratio between peptide-stimulated and unstimulated wells above 1.5 were retested with intracellular cytokine staining (ICS).

Chromium Release Assay.

Cytotoxic activity was assessed by incubating peptide-stimulated PBMCs with peptide-pulsed 51Cr-labeled, HLA-A2–matched or –mismatched Epstein Barr virus–transformed B cells as targets for 4 hours in round-bottom 96-well plates. Percent specific lysis was calculated as described previously.21 Significant cytotoxic T lymphocyte responses to synthetic peptides were defined by testing a group of 10 healthy HCV and HBV seronegative subjects who never showed levels of specific lysis above 3%. To adopt stringent criteria and avoid the risk of false positive results, only levels of cytotoxic T lymphocyte lysis equal to or greater than 13% were considered significant.

Cytokines.

Levels of IFN-α, IL-12 (p40 + p70), IL-10, and IL-18 were measured in the serum of the 2 HCV/HBV infected patients, 4 patients with acute HBV infection, and 4 patients with chronic HCV infection using commercial enzyme-linked immunosorbent assay kits according to the manufacturer's instructions (Technogenetics–Bouty SPA, Milan, Italy).

Statistical Analysis.

Perforin expression ex vivo and after peptide stimulation detected in patients with chronic hepatitis C and acute HBV superinfection and in patients with acute HBV monoinfection were compared using Student's t test for unpaired data.

Results

Frequency and Function of HBV-Specific CD8 Cells.

To assess whether or not CD8 cells primed by HBV can be influenced by the simultaneous presence of HCV, the CD8 responses of 2 patients with chronic HCV infection who were acutely infected with HBV were compared with those of 5 patients with acute HBV infection alone. Serum HCV-RNA, that was undetectable at the time of the clinical onset of acute hepatitis B when HBV-DNA was positive, became strongly positive concurrently with the decline (patient 2) or the disappearance (patient 1) of serum HBV-DNA (Table 1). Serum HBV-DNA levels were comparable in both groups of patients with combined HBV/HCV infection (see Table 1) and with HBV infection alone (HBV-DNA ranging from 13,800 to 44,800 copies/mL) at the time of clinical presentation when the first immunological analyses were performed. This suggests that HBV-specific responses were studied in both groups at a similar time following infection. Six HLA-A2/peptide tetramers containing 6 distinct HBV epitopes known to be frequently recognized by patients with acute hepatitis B were used to quantify HBV-specific CD8 cells and to analyze their phenotype and function. Each patient was tested at the time of clinical presentation with acute hepatitis and then subsequently for a follow-up period ranging from 18–48 weeks in dually infected patients to 13–40 weeks in monoinfected patients. At least 2 of the 6 CD8 epitopes tested were recognized by each patient. Frequencies ex vivo of CD8 cells specific for individual epitopes ranged from 0.04%–2.2% and from 0.06%–1.1% of the total CD8 cells in patients with associated HBV/HCV infections or with HBV infection alone, respectively. By staining CD8 cells simultaneously with tetramers and antibodies to CCR7, CD45RA, and CD27 molecules, the great majority of HBV-specific cells in both HBV superinfected and HBV monoinfected patients were CCR7-negative at the time of acute HBV infection; most CCR7 cells did not express CD45RA (Table 2). The CCR7/CD45RA subset was also predominantly CD27-positive and homogeneously HLA-DR–positive, showing that circulating HBV tetramer-positive cells mostly belong to an effector-memory subset at this stage of infection.

Table 1. Clinical and Virological Features of Patients With Chronic HCV Infection and HBV Superinfection
Weeks from the OnsetALTHBV-DNA (Copies/mL)HCV-RNA (Copies/mL)HBsAgIgManti-HBcHCV-Ab
  1. Abbreviations: ALT, alanine aminotransferase; HBsAg, hepatitis B surface antigen; IgManti-HBc, immunoglobulin M anti-hepatitis B core antigen antibodies; HCV-Ab, antibodies to hepatitis C virus; ND, not done.

Patient 1      
−1272NDND  +
01,574NDND+++
1708NDNDNDND 
241436,000<3,200+++
37468,480<3,200++ 
9128<2001,247,257+ 
1361<200243,708+
18208<200948,769 
Patient 2      
−1268NDND  +
1451134,000<3,200+++
215413,800<3,200++ 
6261,44024,648,270+ND+
1063<2009,215,501++ 
1165<20012,141,213+ 
15ND<20011,379,514ND 
1944<2009,320,441ND 
24ND<20014,721,935NDND 
3835NDNDNDND 
4835<20011,997,155 
Table 2. Phenotypic Analysis of HBV-Specific CD8 Cells at the Time of Acute Hepatitis in Patients With Chronic HCV Infection and HBV Superinfection and in Two Acute HBV Monoinfected Patients
 CD45RA+ CCR7+CD45RA CCR7+CD45RA CCR7CD45RA+ CCR7
Combined HBV/HCV infection
 Patient 1124.382.21.5
 Patient 201.397.41.3
 Mean6 ± 8.52.78 ± 2.189.8 ± 10.71.34 ± 0.08
HBV monoinfection
 Patient 14.85.982.46.9
 Patient 20.11.589.49
 Mean2.45 ± 3.33.7 ± 3.185.9 ± 4.97.95 ± 1.48

The capacity of CD8 cells to proliferate in vitro to the relevant peptides was similar in both groups of patients, and the frequencies of tetramer-positive CD8 cells after peptide stimulation were up to 100 times higher than the initial values detected in ex vivo samples (Fig. 1). It is important to note that the capacity of expansion upon peptide stimulation was not affected by the presence of high HCV viremia, because CD8 expansion was not less efficient when HCV viremia was high after the resolution of HBV infection than when HCV viremia was low at the time of the acute stage of hepatitis B (see Fig. 1).

Figure 1.

Frequency of HBV-specific CD8 cells in acute HBV infection alone or superimposed to a pre-existing chronic HCV infection. Representative dot plot analysis of HBV core 18-27, envelope 335-343, envelope 348-357, and polymerase 575-583 tetramer+ cells stained with anti-CD8 monoclonal antibody in 2 patients with acute hepatitis B and chronic HCV infection and in 2 representative patients with acute HBV infection alone. The analysis was performed ex vivo and after 10 days of peptide stimulation in vitro. Two representative time points corresponding to the acute phase of infection and to a later time during the follow-up are illustrated. The percentage of tetramer+ cells among the total population of CD8+ lymphocytes is indicated at the top of each dot plot. HBV, hepatitis B virus; HCV, hepatitis C virus; Pt, patient; ENV, envelope; POL, polymerase.

To characterize further the functional features of HBV-specific CD8 cells, we analyzed the perforin content ex vivo and following peptide stimulation in vitro. In both experimental conditions, intracellular staining of tetramer-positive cells with anti-perforin antibodies revealed similar proportions of perforin-positive cells in patients with combined HBV/HCV infection and in HBV-monoinfected patients (Figs. 2 and 3). A substantial increase in perforin expression was induced in both groups of patients by peptide stimulation. Moreover, no difference in perforin expression was observed in conditions of high or low HCV-RNA load in patients with combined HBV and HCV infections.

Figure 2.

Analysis of the perforin content in HBV-specific CD8 cells ex vivo and after peptide stimulation. Representative dot plot analysis of HBV core 18-27, envelope 335-343, envelope 348-357, and polymerase 575-583 tetramer+ cells in 2 patients with chronic HCV infection and acute HBV superinfection and in 2 patients with acute HBV infection alone stained with anti-perforin monoclonal antibodies ex vivo and after 10 days of peptide stimulation in vitro, at two different time points corresponding to the acute phase of infection and to a later time of the follow-up. The percentage of perforin+ cells among tetramer+ CD8 lymphocytes is indicated at the top of each dot plot. *Frequency of tetramer+ cells < 0.01% of the total CD8+ cells. HBV, hepatitis B virus; HCV, hepatitis C virus; Pt, patient; ENV, envelope; ND, not done; FITC, fluorescein isothiocyanate; POL, polymerase.

Figure 3.

Level of perforin expression ex vivo and after peptide stimulation by the overall population of patients with combined HBV/HCV infection or with HBV infection alone. Each symbol represents the percentage of tetramer+ cells expressing perforins assessed ex vivo or after 10 days of peptide stimulation in vitro in each subject of both categories of patients at two different time points corresponding to the acute phase of infection when HCV-RNA was below the sensitivity threshold of the method and at a later time point of the follow-up when HCV-RNA was more than 3,200 copies/mL. No significant difference was observed between the two groups of patients either ex vivo or after peptide stimulation (Student's t test). HBV, hepatitis B virus; HCV, hepatitis C virus; Pt, patient.

The CD8 function was next analyzed in terms of expression of IFN-γ and lytic activity induced by in vitro peptide stimulation. Both patients with chronic HCV infection and acute HBV superinfection showed a strong expression of IFN-γ in response to multiple peptides that was maintained with similar intensity throughout the follow-up, irrespective of serum HCV-RNA levels (Figs. 4 and 5). No difference in IFN-γ expression was observed between patients with chronic HCV infection who were HBV-superinfected and patients with acute HBV infection alone (data not shown).

Figure 4.

IFN-γ production upon peptide stimulation of HBV-specific short-term T cell lines. IFN-γ production by HBV-specific CD8 cells of the 2 patients with chronic HCV and acute HBV superinfection was analyzed after 10 days of peptide stimulation at two different time points corresponding to the acute phase of infection when HCV-RNA was undetectable and at a later time point of the follow-up when HCV-RNA was more than 3,200 copies/mL. Each bar represents the percentage of IFN-γ production by HBV core 18-27, envelope 183-191, envelope 335-343, envelope 348-357, polymerase 575-583, and polymerase 816-824 tetramer+ cells. Two representative dot plot analyses of tetramer+ cells stained with anti–IFN- γ monoclonal antibody are shown at the bottom of each panel. *Frequency of tetramer+ cells < 0.01 of CD8+ cells. HCV, hepatitis C virus; PT, patient; IFN-γ, interferon γ; pol, polymerase; env, envelope.

Figure 5.

(A) Sequential determinations of HBV-DNA (•), HCV-RNA (▪), and alanine aminotransferase (white columns) in 2 patients with chronic HCV infection and acute HBV superinfection. (B–D) Sequential analysis from the time of infection of perforin expression (B) and IFN-γ production (C) by tetramer+ cells and cytolitic activity (D) after 10 days of peptide stimulation. Each bar represents the mean percentage of cells positive for perforin (B) and IFN-γ (C) among the overall population of tetramer+ lymphocytes and the mean 51Cr release (D) induced by stimulation with the different peptides at the indicated time points. HBV, hepatitis B virus; HCV, hepatitis C virus; NT, not tested; IFN-γ, interferon γ.

In addition, lytic activity was efficiently expressed by CD8 cells of HBV-superinfected patients and was maintained at similar levels in conditions of either low or high HCV replication, as indicated by serum HCV-RNA levels (see Fig. 5).

Longitudinal Analysis of HCV-Specific, CD8-Mediated Responses With Selected HLA-A2 Epitopes.

To assess whether or not the fluctuations of HCV viremia associated with HBV superinfection have an effect on HCV-specific CD8 cells, the CD8 response was studied with 4 HLA-A2/HCV tetramers containing 4 HLA-A2–restricted HCV epitopes (NS3 1073-1081, NS3 1406-1415, NS4B 1992-2000, and NS5 2594-2602) known to be frequently recognized in acute and chronic HCV infection. Tetramer staining as well as perforin and IFN-γ expression ex vivo and in vitro tested at 6 (patient 1) and 5 (patient 2) sequential time points from clinical presentation remained constantly negative in both patients, with the exception of a transient increase in frequency of NS3 1406–specific CD8 cells in patient 1, associated with the detection of significant proliferation, perforin, and IFN-γ expression (data not shown). However, this recovery of CD8 responsiveness was not observed when serum HCV-RNA was negative at the time of HBV superinfection, but only at later time points, suggesting that this transient elevation of CD8 reactivity likely reflected the periodic fluctuations of T cell responses typical of chronic HCV patients. Responses were also negative in patient 2 (infected by genotype 2c) when PBMCs were tested using ELISpot assay with synthetic peptides of genotype 2c (data not shown).

Analysis of the Global CD8 Reactivity to HCV by Overlapping Peptides Covering the Entire HCV Sequence.

Possible fluctuations of the HCV-specific CD8 reactivity could be missed by using only 4 HLA-A2 restricted epitopes that may give only a partial representation of the overall CD8 response against HCV. To overcome this problem, we analyzed CD8 responses with a panel of 601 15-mer peptides covering the whole HCV sequence. Through this approach, a preselection of the epitopes used for T cell analysis is avoided and responses to structural and nonstructural HCV proteins can be identified with no limitations imposed by HLA restriction. Peptides were pooled in 119 mixtures and responses were tested with ELISpot assay; positive results were further assessed with intracellular cytokine staining. Both patients were tested at an early time when HCV-RNA was either low or undetectable and at later time points when HCV-RNA was high. Responses were totally negative ex vivo after overnight PBMC stimulation (data not shown). Therefore, both patients were also tested after 10 days of PBMC stimulation to assess whether or not the decline of viremia was associated with recovery of T cell responsiveness to HCV upon a longer time of stimulation in vitro. Few positive responses were detected with ELISpot assay at each time point analyzed (Fig. 6), and some of them were subsequently confirmed by ICS analysis (Fig. 6, Table 3). The lack of difference in CD8 responsiveness between time points with high or negative serum HCV-RNA was further confirmed with additional ELISpot assays with the overall panel of peptides, which were repeated in both patients during acute HBV infection and after resolution of hepatitis B (data not shown). Similar to that illustrated in Fig. 6, patient 1 showed positive responses to 3 and 4 different mixtures at the 2 time points with negative and positive (948,769 copies/mL) HCV-RNA, respectively, while in patient 2 positive responses were detected against one mixture (when HCV-RNA was negative) and eleven mixtures (HCV-RNA 12,141,213 copies/mL) in each of the two sequential experiments (data not shown).

Figure 6.

ELISpot assay and ICS analysis after 10 days of PBMC stimulation in vitro. T cell lines were produced via in vitro PBMC stimulation with 119 pools of HCV overlapping peptides at two different time points corresponding to the acute phase of infection when HCV-RNA was undetectable and at a later time point of the follow-up when HCV-RNA was more than 3,200 copies/mL. After 10 days of culture, T cells were tested with ELISpot assay. The results are expressed as SFCs per well. Variable levels of spontaneous IFN-γ production were observed with a mean background SFCs per well generally between 20 and 100 (patient 1: experiment 1, 98 SFCs; experiment 2, 54 SFCs; patient 2: experiment 1, 168 SFCs; experiment 2, 24 SFCs). The background in ex vivo experiments (data not shown) was generally lower and ranged from 12 (patient 1, experiment at the time of negative HCV-RNA) to 33 (patient 1, experiment at high HCV-RNA levels) SFCs per well. At the top of each bar representing positive ELISpot assay results confirmed by ICS, the T cell subset (CD4 or CD8) responsible for the observed IFN-γ production is indicated. The internal squares represent IFN-γ expression by HCV-specific CD8 cells detected by intracellular cytokine staining. The figure illustrates the dot plot analysis of the positive ELISpot assay results that were confirmed upon ICS and shown to be CD8-mediated. Pt, patient; HCV, hepatitis C virus; ELISpot, enzyme-linked immunosorbent spot assay; ICS, intracellular cytokine staining; SFC, spot-forming cells; IFN-γ, interferon γ; FITC, fluorescein isothiocyanate. [Color figure can be viewed in the online issue, which is available at www.interscience.wiley.com.]

Table 3. Elispot and ICS Analysis Carried Out at the Time of Undetectable or Elevated Serum HCV-RNA
Peptide MixturesAA residuesDelta-Spots/WellICS
Ex Vivo*In VitroIFN-γ+/CD4+IFN-γ+/CD8+
  • Abbreviations: Neg, negative; ND, not done.

  • *

    ELISpot assay after overnight incubation with individual peptide mixtures.

  • ELISpot assay after 10 days of stimulation with individual peptide mixtures.

Patient 1
Week 2 (HCV-RNA <3,200 copies/mL)
68NS4A/B 1676-1735Neg154NDND
75NS4B 1851-1910Neg139NegNeg
85NS5A 2101-2160Neg1130.63%Neg
Week 9 (HCV-RNA 1,247,257 copies/mL)
36NS2 876-935Neg1160.63%Neg
39NS2 951-1010Neg1220.59%Neg
76NS4B 1876-1935Neg439.32%Neg
Patient 2
Week 1 (HCV-RNA <3,200 copies/mL)
28E2 676-735Neg423Neg1.5%
37NS2 901-960Neg1500.67%Neg
56NS3 1376-1435Neg248NegNeg
60NS3 1476-1535Neg148NegNeg
Week 10 (HCV-RNA 9,215,501 copies/mL)
28E2 676-735Neg149Neg1.6%
29E2 701-760Neg219Neg14%

Lack of Correlation Between Serum Levels of Cytokines of Innate Immunity and Transient Control of HCV Replication in Patients With Chronic HCV Infection and HBV Superinfection.

To determine if in HCV/HBV-infected patients a correlation could exist between transient control of HCV replication and activation of the innate immune system eventually caused by simultaneous acute HBV infection, serum levels of IFN-α, IL-12, IL-10, and IL-18 were measured in the serum at different time points during follow-up (8 serum samples over the course of 65–75 weeks from each HCV/HBV-infected patient, and 5 samples over the course of 35–40 weeks from each patient with acute HBV infection alone). No significant changes in serum levels of IFN-α, IL-10, or IL-18 were found in either HCV/HBV-infected patients or patients with acute HBV infection.

Discussion

HCV-specific, CD8-mediated responses show different types of functional impairments at the early stages of HCV infection.18–21 Longitudinal analyses from the time of infection in subjects accidentally exposed to HCV-infected blood22 and in HCV-infected chimpanzees23 suggest that circulating HCV-specific CD8 cells detected by tetramer staining in self-limited infection can be transiently unable to produce IFN-γ. Further studies indicate that at the time of clinical presentation, HCV-specific CD8 cells can be also defective in perforin expression, as well as in their capacity to proliferate and to express cytolytic activity upon peptide stimulation in vitro.17, 18, 20, 21 Although these functional defects are transient in patients who recover spontaneously from infection, chronic persistence of the virus seems to be associated with a permanent functional CD8 impairment.21 Different factors have been suggested to influence the T cell function, including the rapid rise of the viral load and the rapid spread of HCV in the infected host after infection,17 an altered priming of T cell responses caused by a defective accessory and antigen-presenting function of dendritic cells,10–13, 24 and a direct effect of the virus on T cells.14–16 Some of these mechanisms of virus interference with the T cell function would imply a generalized effect of HCV on T cells, irrespective of their antigen specificity. This is still an open issue, though the lack of propensity to opportunistic infections of HCV-infected patients is in principle against this possibility.

It has been shown that acute HBV superinfection in patients with a pre-existing chronic HCV infection can interfere with HCV replication causing a transient or permanent HCV clearance.5–9 Moreover, it is well known that HBV-specific CD8 responses in acute symptomatic HBV infection are functionally efficient.17, 21, 25–26 Therefore, we exploited the natural model of HBV infection superimposed to a chronic HCV infection to ask whether or not HBV-specific T cell priming can be affected by the presence of HCV, whether or not fluctuations of HCV replication typical of this condition are associated with changes in vigor and breadth of HCV-specific CD8 responses, and whether or not antiviral cytokines produced by cells of the innate immunity following acute HBV infection can contribute through a bystander effect to the control of HCV replication observed during acute HBV superinfection.

As predicted from previous reports, HCV viremia was low or undetectable during the acute phase of HBV superinfection in the 2 patients with combined HBV/HCV infection.5–9 Although sera were not available for HCV-RNA quantitation before the onset of acute hepatitis B, previous detection of anti-HCV antibodies and persistently elevated alanine aminotransferase in the absence of other possible causes of liver disease indicate that both patients were chronic HCV carriers and suggest that the low or undetectable levels of HCV-RNA at the time of acute HBV infection were likely due to suppression of HCV replication by HBV. HCV remained quiescent only transiently, because a sharp elevation of serum HCV-RNA levels was observed 6–9 weeks after clinical presentation of HBV infection, concurrently with or immediately after the decline of serum HBV-DNA.

To determine whether or not HCV can influence the priming and subsequent differentiation of HBV-specific CD8 cells, HBV-specific CD8 responses were analyzed sequentially over time in the 2 chronic HCV carriers acutely infected by HBV and were compared with CD8 responses detected longitudinally in 4 patients with acute HBV infection alone. Frequency of HBV tetramer-positive CD8 cells and perforin expression ex vivo, as well as capacity of expansion, IFN-γ expression, and cytolytic activity following HBV peptide stimulation in vitro were expressed at similar levels by CD8 cells of patients acutely infected with HBV with or without combined chronic HCV infection. CD8 T cell function was efficiently expressed throughout the follow-up period, irrespective of serum levels of HCV-RNA. Furthermore, the phenotype of circulating HBV-specific CD8 cells at the time of acute HBV infection was similar in patients with HBV superinfection or HBV monoinfection, because the great majority of them belonged to an effector-memory subset (CCR7/CD45RA) in both clinical conditions. Therefore, HCV did not affect the differentiation of HBV-specific CD8 cells toward a mature effector memory phenotype and did not influence their function. This finding is apparently different from that recently reported for cytomegalovirus-specific CD8 cells in HCV-infected individuals that show a more immature phenotype compared with healthy individuals.27 This apparent discrepancy may reflect a different effect of HCV infection on primary versus memory T cell responses.

If HBV superinfection can suppress HCV replication as reported previously,5–9 how is the HCV-specific CD8 response at the time of low or undetectable HCV-RNA? Can restoration of HCV-specific CD8 responses, which are generally very weak in chronic HCV carriers, contribute to keep HCV replication under control in this clinical setting? To answer these questions, we used 4 previously described HLA-A2 restricted epitopes and a wide panel of 15-mer peptides covering the whole HCV sequence. Although the use of preselected epitopes can give only a partial representation of the antiviral CD8 response mounted in the context of a single HLA restriction element (HLA-A2), T cell analysis using ELISpot assay with overlapping peptides representing the overall virus can give a comprehensive representation of the global T cell response to HCV, irrespective of the HLA haplotype of the infected patients. Further analysis using ICS of the stimulatory peptides identified with ELISpot assay can also distinguish whether responses are sustained by CD4 or CD8 cells. No significant changes of HCV-specific CD8 reactivity to the 4 known HLA-A2 restricted epitopes was observed throughout the follow-up, with the exception of a transient response to a single HCV epitope (NS3 1406-1415) detected ex vivo and following peptide stimulation in vitro. Identical conclusions derive from the more global analysis of the HCV-specific T cell reactivity with reagents covering the overall HCV sequence, because only a few CD4- and CD8-mediated responses were similarly observed in association with suppressed or elevated HCV viremia. Moreover, no correlation was found between changes in serum levels of IFN-α, IL-10, IL-12, or IL-18 in HCV/HBV-infected patients and changes in HCV viremia. These findings extend what was reported recently by Gruener et al.5 and is in line with the typical profile of T cell responses detectable in HCV viremic carriers who can show sporadic and transient fluctuations from totally negative to weakly positive T cell responses to HCV proteins in vitro. Therefore, a clear restoration of CD8 reactivity was not observed when HCV replication was inhibited. Control of viral replication may not be sufficient to allow recovery of CD8 responsiveness in a condition of long-lasting chronic exposure to high antigen load. Alternately, viral suppression may have been too transient to cause an effect on immune responses. This lack of peripheral blood CD8 responses when HCV viremia was inhibited at the time of HBV superinfection suggests that HCV-specific CD8 cells do not play a major role in HCV control associated with HBV superinfection. Virological rather than immunological factors may be primarily responsible for the changes in HCV titers during HBV superinfection, although the possibility that HCV-specific CD8 responses had peaked before patients presented with acute hepatitis B or had compartmentalized into the liver cannot be ruled out.

In conclusion, HCV seems to be unable to affect the induction of primary CD8 responses and the function of memory T cells specific for a closely related virus, such as HBV. Therefore, if HCV is responsible for the functional impairment of HCV-specific CD8 cells in acute HCV infection, the results of our study suggest that this inhibition is selective for HCV-specific responses.

Ancillary