Therapeutic Vaccination of Chronic Hepatitis C Nonresponder Patients With the Peptide Vaccine IC41
2008; Elsevier BV; Volume: 134; Issue: 5 Linguagem: Inglês
10.1053/j.gastro.2008.02.058
ISSN1528-0012
AutoresChristoph Klade, Heiner Wedemeyer, Thomas Berg, Holger Hinrichsen, Grażyna Cholewińska, Stefan Zeuzem, Hubert E. Blum, Michael Buschle, Sandra Jelovcan, Vera Buerger, Erich Tauber, Juergen Frisch, Michael P. Manns,
Tópico(s)Monoclonal and Polyclonal Antibodies Research
ResumoBackground & Aims: IC41 is a synthetic peptide vaccine containing 7 relevant hepatitis C virus (HCV) T-cell epitopes and the T helper cell (Th)1/Tc1 adjuvant poly-L-arginine. IC41 has been shown to be safe and to induce HCV-specific interferon (IFN)-γ-secreting CD4+ and CD8+ T cells in healthy volunteers. We aimed to investigate whether IC41 is able to induce HCV-specific T-cell responses also in chronic hepatitis C patients. Methods: Sixty HLA-A2-positive chronic HCV patients not responding to or relapsing from standard therapy were randomized in a double-blind phase II study into 5 groups to receive 6 vaccinations of IC41 (3 different dose groups), HCV peptides alone, or poly-L-arginine alone. Results: IC41 was well tolerated, and no drug-related serious adverse events or induction of hepatitis were observed. T-cell proliferation was recorded in up to 67% of patients in the 3 IC41 vaccine groups but only in 17% of patients treated with peptides alone. IFN-γ enzyme-linked immunospot assay responses were observed exclusively in the IC41 groups with response rates up to 42%. There were 3 RNA responders with transient >1-log declines of HCV serum RNA associated with the strongest IFN-γ enzyme-linked immunospot assay values within all 60 patients. Conclusions: This study showed that the HCV peptide vaccine IC41 can induce HCV-specific Th1/Tc1 responses in a subset of difficult to treat HCV nonresponder patients despite persisting viremia. However, changes in HCV RNA occurred only in single patients. Because strongest T-cell responses were associated with HCV RNA decline, further studies with optimized vaccine regimens and combination therapies have been initiated. Background & Aims: IC41 is a synthetic peptide vaccine containing 7 relevant hepatitis C virus (HCV) T-cell epitopes and the T helper cell (Th)1/Tc1 adjuvant poly-L-arginine. IC41 has been shown to be safe and to induce HCV-specific interferon (IFN)-γ-secreting CD4+ and CD8+ T cells in healthy volunteers. We aimed to investigate whether IC41 is able to induce HCV-specific T-cell responses also in chronic hepatitis C patients. Methods: Sixty HLA-A2-positive chronic HCV patients not responding to or relapsing from standard therapy were randomized in a double-blind phase II study into 5 groups to receive 6 vaccinations of IC41 (3 different dose groups), HCV peptides alone, or poly-L-arginine alone. Results: IC41 was well tolerated, and no drug-related serious adverse events or induction of hepatitis were observed. T-cell proliferation was recorded in up to 67% of patients in the 3 IC41 vaccine groups but only in 17% of patients treated with peptides alone. IFN-γ enzyme-linked immunospot assay responses were observed exclusively in the IC41 groups with response rates up to 42%. There were 3 RNA responders with transient >1-log declines of HCV serum RNA associated with the strongest IFN-γ enzyme-linked immunospot assay values within all 60 patients. Conclusions: This study showed that the HCV peptide vaccine IC41 can induce HCV-specific Th1/Tc1 responses in a subset of difficult to treat HCV nonresponder patients despite persisting viremia. However, changes in HCV RNA occurred only in single patients. Because strongest T-cell responses were associated with HCV RNA decline, further studies with optimized vaccine regimens and combination therapies have been initiated. See editorial on page 1601. See editorial on page 1601. Pegylated interferon (IFN)/ribavirin combination treatment elicits a sustained response, defined as lack of detectable viremia 6 months after end of treatment, in up to 80% of patients infected with genotypes 2 and 3 but only in 43% to 50% of patients infected with genotype 1, which is the most prevalent in Europe, the United States, and Canada.1Manns M.P. Wedemeyer H. Cornberg M. Treating viral hepatitis C: efficacy, side effects, and complications.Gut. 2006; 55: 1350-1359Crossref PubMed Scopus (565) Google Scholar In addition to limited efficacy, standard therapy is associated with substantial adverse effects, severely influencing the quality of life of the patients under treatment and considerable costs resulting from long-term pegylated IFN/ribavirin combination treatment.1Manns M.P. Wedemeyer H. Cornberg M. Treating viral hepatitis C: efficacy, side effects, and complications.Gut. 2006; 55: 1350-1359Crossref PubMed Scopus (565) Google Scholar Furthermore, a large number of HCV-infected patients are not suitable candidates for antiviral IFN-based therapy for reasons including nonadherence to evaluation procedures; medical or psychiatric contraindications; ongoing substance or alcohol abuse; or patient preference after considering possible adverse effects, duration of treatment, family planning issues, and route of administration.2Falck-Ytter Y. Kale H. Mullen K.D. et al.Surprisingly small effect of antiviral treatment in patients with hepatitis C.Ann Intern Med. 2002; 136: 288-292Crossref PubMed Scopus (287) Google Scholar Because of these limitations of standard therapy, new treatment options for chronic HCV infection are urgently required. This study was designed to evaluate the safety and immunogenicity of IC41, a novel vaccine designed to induce HCV-specific T-cell immunity.3Firbas C. Jilma B. Tauber E. et al.Immunogenicity and safety of a novel therapeutic hepatitis C virus (HCV) peptide vaccine: a randomized, placebo controlled trial for dose optimization in 128 healthy subjects.Vaccine. 2006; 24: 4343-4353Crossref PubMed Scopus (128) Google Scholar Primary HCV infection can cause broad and multispecific both CD4+ and CD8+ T-cell responses during acute infection. It has been reported that stronger, broader, and more sustained T helper cell (Th)1/Tc1 (IFN-γ) responses are associated with resolving infection.4Rehermann B. Nascimbeni M. Immunology of hepatitis B virus and hepatitis C virus infection.Nat Rev Immunol. 2005; 5: 215-229Crossref PubMed Scopus (1409) Google Scholar Indeed T-cell responses can readily be detected in humans in the absence of viremia many years after clearing infection.5Lechner F. Wong D.K.H. 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Nascimbeni M. et al.Impaired effector function of hepatitis C virus-specific CD8+ T cells in chronic hepatitis C virus infection.J Immunol. 2002; 169: 3447-3458Crossref PubMed Scopus (583) Google Scholar The high mutation rate of an RNA virus and the existence of quasispecies in the same individual facilitate mutational epitope escape mechanisms that can undermine productive T-cell responses.14Bowen D.G. Walker C.M. Mutational escape from CD8+ T cell immunity: HCV evolution, from chimpanzees to man.J Exp Med. 2005; 201: 1709-1714Crossref PubMed Scopus (186) Google Scholar Additional potential immune deviations in chronic HCV include dysfunction of dendritic cells15Kanto T. Hayashi N. Takehara T. et al.Impaired allostimulatory capacity of peripheral blood dendritic cells recovered from hepatitis C virus-infected individuals.J Immunol. 1999; 162: 5584-5591Crossref PubMed Google Scholar, 16Bain C. Fatmi A. Zoulim F. et al.Impaired allostimulatory function of dendritic cells in chronic hepatitis C infection.Gastroenterology. 2001; 120: 512-524Abstract Full Text Full Text PDF PubMed Scopus (388) Google Scholar, 17Ciesek S. Liermann H. Cornberg M. et al.Impaired TRAIL-dependent cytotoxic activity of CD1c-positive dendritic cells in chronic hepatitis C virus infection.J Viral Hepat. 2008; 15: 200-211Crossref PubMed Scopus (17) Google Scholar and suppressor T cells.18Sugimoto K. Ikeda F. Stadanlick J. et al.Suppression of HCV-specific T cells without differential hierarchy demonstrated ex vivo in persistent HCV infection.Hepatology. 2003; 38: 1437-1448PubMed Google Scholar, 19Cabrera R. Tu Z. Xu Y. et al.An immunomodulatory role for CD4(+)CD25(+) regulatory T lymphocytes in hepatitis C virus infection.Hepatology. 2004; 40: 1062-1071Crossref PubMed Scopus (482) Google Scholar, 20Rushbrook S.M. Ward S.M. Unitt E. et al.Regulatory T cells suppress in vitro proliferation of virus-specific CD8+ T cells during persistent hepatitis C virus infection.J Virol. 2005; 79: 7852-7859Crossref PubMed Scopus (250) Google Scholar, 21Boettler T. Spangenberg H.C. Neumann-Haefelin C. et al.T cells with a CD4+CD25+ regulatory phenotype suppress in vitro proliferation of virus-specific CD8+ T cells during chronic hepatitis C virus infection.J Virol. 2005; 79: 7860-7867Crossref PubMed Scopus (371) Google Scholar Importantly, IFN-α-based therapy may not necessarily restore cellular immune responses in acute and chronic hepatitis C infection.22Barnes E. Harcourt G. Brown D. et al.The dynamics of T-lymphocyte responses during combination therapy for chronic hepatitis C virus infection.Hepatology. 2002; 36: 743-754Crossref PubMed Scopus (142) Google Scholar, 23Lauer G.M. Lucas M. Timm J. et al.Full-breadth analysis of CD8+ T-cell responses in acute hepatitis C virus infection and early therapy.J Virol. 2005; 79: 12979-12988Crossref PubMed Scopus (100) Google Scholar, 24Wiegand J. Cornberg M. Aslan N. et al.Fate and function of peripheral HCV-specific T-cells during peginterferon α-2b therapy of acute hepatitis C.Antivir Ther. 2007; 12: 303-316Crossref PubMed Google Scholar For these reasons, eliciting anti-HCV immune response based on the induction of epitope-specific CD8+ cytotoxic T lymphocytes (CTLs) and CD4+ responses may be highly beneficial for an HCV-infected patient. It may be advantageous to concentrate on well-conserved regions within the HCV polyprotein. Choosing CTL epitopes restricted to HLA-A*0201 is mandated because most knowledge has accumulated for these epitopes, and the allele is frequently expressed in approximately 45% of white populations.25Gjertson D.W. Terasaki P.L. HLA 1998. American Society for Histocompatibility and Immunogenetics, Lenexa, Kansas1998Google Scholar IC41 was designed to contain 5 synthetic peptides from core, NS3, and NS4. They harbor at least 4 HLA-A*0201 restricted CTL epitopes and 3 highly promiscuous CD4+ helper T-cell epitopes, all of which have been described in detail26Ward S. Lauer G. Isba R. et al.Cellular immune responses against hepatitis C virus: the evidence base 2002.Clin Exp Immunol. 2002; 128: 195-203Crossref PubMed Scopus (141) Google Scholar and were independently confirmed to be targeted in patients responding to standard treatment or spontaneously recovering from HCV (own unpublished data, Klade, 2004). With one exception, peptide sequences are highly conserved in genotype 1. T helper epitopes for the peptides included in the IC41 vaccine have been shown to be active in the context of at least HLA-DRB1*0101, DRB1*0401, DRB1*0404, DRB1*0408, DRB1*0701, DRB1*0802, DRB1*0901, DRB1*1101, DRB1*1302, DRB1*1501, and DRB5*0101. Collectively, these account for at least 70% to 80% population coverage. IC41 contains poly-L-arginine as synthetic adjuvant, which has been shown to augment Th1/Tc1 (IFN-γ) responses in animal studies.27Buschle M. Schmidt W. Zauner W. et al.Transloading of tumor antigen-derived peptides into antigen-presenting cells.Proc Natl Acad Sci U S A. 1997; 94: 3256-3261Crossref PubMed Scopus (51) Google Scholar, 28Schmidt W. Buschle M. Zauner W. et al.Cell-free tumor antigen peptide-based cancer vaccines.Proc Natl Acad Sci U S A. 1997; 94: 3262-3267Crossref PubMed Scopus (52) Google Scholar, 29Mattner F. Fleitmann J.K. Lingnau K. et al.Vaccination with poly-L-arginine as immunostimulant for peptide vaccines: induction of potent and long-lasting T-cell responses against cancer antigens.Cancer Res. 2002; 62: 1477-1480PubMed Google Scholar, 30Luhrs P. Schmidt W. Kutil R. et al.Induction of specific immune responses by polycation-based vaccines.J Immunol. 2002; 169: 5217-5226Crossref PubMed Scopus (31) Google Scholar, 31Lingnau K. Egyed A. Schellack C. et al.Poly-L-arginine synergizes with oligodeoxynucleotides containing CpG-motifs (CpG-ODN) for enhanced and prolonged immune responses and prevents the CpG-ODN-induced systemic release of pro-inflammatory cytokines.Vaccine. 2002; 20: 3498-3508Crossref PubMed Scopus (54) Google Scholar Data from a phase I study with IC41 indicated that administration of the vaccine is safe and well tolerated and that IC41 can induce HCV-specific Th1/Tc1 responses in healthy volunteers.3Firbas C. Jilma B. Tauber E. et al.Immunogenicity and safety of a novel therapeutic hepatitis C virus (HCV) peptide vaccine: a randomized, placebo controlled trial for dose optimization in 128 healthy subjects.Vaccine. 2006; 24: 4343-4353Crossref PubMed Scopus (128) Google Scholar Here, we report a double-blind, randomized, multicenter, phase II study of immunization with IC41 HCV peptide vaccine together with poly-L-arginine, HCV peptide vaccine alone, or poly-L-arginine alone, in 60 patients with chronic HCV who did not respond to or relapsed from primary standard HCV therapy. This study was a double-blind, randomized, parallel group, multicenter comparison of immunization with IC41, HCV peptide vaccine alone, and poly-L-arginine alone, in HLA-A2-positive patients with chronic HCV who did not respond to or relapsed from primary standard HCV therapy. Inclusion criteria included a documented course of chronic hepatitis C (HCV RNA and antibody positive) for at least 6 months, nonresponse/relapse from primary standard HCV therapy of 6 to 12 months, and liver biopsy within 30 months before inclusion, demonstrating hepatic inflammation and/or fibrosis. Major exclusion criteria included cirrhosis or fibrosis with Ishak score ≥4 and any liver disease other than hepatitis C (Table 1). The IC41 HCV vaccine (Intercell AG, Vienna, Austria) contains 5 synthetic peptides (Ipep83, 84, 87, 89, 1426) derived from HCV genotype 1 core23–44 and 132–140, NS3 1073–1081 and 1248–1261, and NS4 1764–1786. These peptides contain 4 HLA-A*0201 CTL epitopes (core35–44 and 132–140, NS3 1073–1081, NS4 1764–1772) and 3 helper epitopes (core23–44, NS3 1248–1261, NS4 1767–1786). Sequences are conserved in the most prevalent HCV genotypes 1a (100%, 100%, 83%, 100%, 100% for the respective 5 peptides), 1b (98%, 90%, 15%, 94%, 88%), and 2 (91%, 96%, 13%, 91%, 87%). Three different formulations of IC41, peptide vaccine alone, and poly-L-arginine alone as controls were tested in different groups (Table 2). Six vaccinations were administered as a 0.5-mL subcutaneous injection in the upper arm every 4 weeks, and 3 follow-up visits 4, 12, and 24 weeks after the last vaccination were done. Peripheral blood mononuclear cells (PBMC) for T-cell analysis were obtained at baseline; at the time of the fourth, fifth, and sixth vaccination; and at the 3 follow-up visits. Objectives of the study were (1) to determine the immunologic profile (HCV peptide-specific CD4+ and CD8+ T-cell responses) induced by immunization, (2) to document virologic (HCV RNA) and biochemical (alanine aminotransferase) responses, and (3) to assess the safety of immunization with IC41 (Supplemental Table).Table 1Patient Baseline CharacteristicsVaccine groups n = 36Control groups n = 24Total n = 60Age (y), mean46.1 (range, 23−63)47.3 (range, 23−65)46.5 (range, 23−65)Sex, n (%) Male2055.61666.73660.0 Female1644.4833.32440.0Race, n (%) White3597.224100.05998.3 Oriental12.800.011.7Weight (kg), mean74.6 (range, 50−99)75.7 (range, 57−105)75.0 (range, 50−105) Open table in a new tab Table 2Dosing GroupsPeptide dose/injectionPoly-L-arginine dose/injectionVaccine groups (n) Group 1 (12)2.5 mg1.25 mg Group 2 (12)2.5 mg2 mg Group 3 (12)5 mg2 mgControl groups (n) Poly-L-arginine (12)0 mg2 mg Peptides (12)5 mg0 mg Open table in a new tab The study protocol, amendments, and informed consent form were approved by independent ethics committees and by the authorities according to the country specific laws prior to commencement of the study. The study was conducted in compliance and in accordance with the protocol, the current revision of the Declaration of Helsinki, Good Clinical Practice, and the applicable regulatory requirements and all relevant guidelines of the International Conference for Harmonization. Prior to exposure to any study-related procedures, all patients had to give their written informed consent. Patients were informed by the investigator about the aims, methods, anticipated benefits, and potential hazards of participating in the study (Appendix 1). To assess vaccine immunogenicity, IFN-γ enzyme-linked immunospot assay (ELIspot), T-cell proliferation, and HLA tetramer-binding assays were carried out with cryopreserved PBMC using validated T-cell assays and conforming to good laboratory practice, essentially as described.3Firbas C. Jilma B. Tauber E. et al.Immunogenicity and safety of a novel therapeutic hepatitis C virus (HCV) peptide vaccine: a randomized, placebo controlled trial for dose optimization in 128 healthy subjects.Vaccine. 2006; 24: 4343-4353Crossref PubMed Scopus (128) Google Scholar Response was scored if any peptide tested at any time point during or after vaccination was at least 3-fold above the baseline value or at least significantly positive if baseline was zero. Accordingly, proliferation class II (any of the 3 class II T helper epitopes), ELIspot class I (any of the 4 class I = HLA-A*0201 epitopes), ELIspot vaccine (any of the 5 peptides of IC41), and fluorescence-activated cell sorter (FACS) class I (any of the 3 class I = HLA-A*0201 epitopes tested) responders were defined. Responder definitions were purposely set as low as possible to avoid underestimation of responder rates because of false-negative results because direct ex vivo assays without in vitro prestimulation and using cryopreserved PBMC may give very low yet meaningful antigen-specific T-cell responses. The number of patients per group (n = 12) was empiric and based on results seen in a small first in human trial (own unpublished data). No power calculations were performed, and only descriptive statistics were applied. Complete HLA class I and class II typing was performed at the Department of Blood Group Serology and Transfusion Medicine, Medical University of Vienna. Besides intention-to-treat (ITT) analysis, immunologic results were also analyzed based on each subject's HLA class I (HLA-A*0201) and class II (HLA-DR) background. Because both analyses yielded comparable results, here, consistent ITT analysis is presented. HCV RNA was determined at baseline (3 times within 28 days prior to first vaccination); at each vaccination visit; and 1, 3, and 6 months after the last vaccination. As a standard test, the Cobas Amplicor was applied. Data monitoring and data management were performed by ICON Clinical Research GmbH (Frankfurt, Germany). PBMC were isolated from acid citrate dextrose blood on Lymphoprep (Nycomed Pharma AS, Oslo, Norway) using Leuco-sep tubes (Greiner, Frickenhausen, Germany) and cryopreserved in liquid nitrogen at a concentration of 2 × 107/mL in fetal bovine serum (FBS; from PAA, Linz, Austria)/dimethyl sulfoxide (DMSO) (Sigma, Deisenhofen, Germany, 9:1). After thawing, PBMC were incubated overnight (37°C, 5% CO2) in assay medium (RPMI 1640, 1 mmol/L sodium pyruvate, 2 mmol/L L-glutamine, 0.1 mmol/L nonessential amino acids, 50 μmol/L 2-mercaptoethanol, 1% antibiotic/antimycotic, all from Invitrogen Life Technologies, and 5% human serum type AB from PAA). The next day, cells were plated at 50,000 PBMC/well (96-well plates) and cocultivated in 4 replicates with individual peptides (10 μg/mL) for 6 days. After adding 1 μCi of [3H]thymidine (Hartmann Analytic, Vienna, Austria) to each well, cells were cultured for a further 16–18 hours. Next, cells were harvested on Multiscreen Harvest plates (Milipore), and incorporated radioactivity was measured using a liquid scintillation β-counter (MicroBeta Jet, Wallac, Perkin-Elmer). An HBV-derived HLA class II peptide was used as negative control. Concanavalin A was used as nonspecific positive control. Tetanus toxoid (Statens Serum Institut, Copenhagen, Denmark) was used as antigen-specific positive control. On each assay plate, a PBMC sample with known reactivity against tetanus toxoid and the HBV negative-control peptide was developed as quality control. Stimulation index (SI) was calculated by dividing the median stimulated culture counts by the median of the negative-control (HBV peptide) culture counts. Each of the 3 T helper peptides of IC41 was tested individually, and results were considered positive when SI was ≥4. Significant SI were added up to yield a calculated "sum of SI" (see Figure 1 and see Table 5). Plates (MAIP S4510; Millipore, Bedford, MA) were coated (overnight/4°C) with a human IFN-γ monoclonal antibody (mAb) (10 μg/mL; Bender Med Systems, Vienna, Austria) and blocked with ELIspot medium (see Proliferation Assay section, except 10% instead of 5% human serum type AB). Cryopreserved PBMC were thawed, incubated overnight (37°C, 5% CO2) and plated at 200,000 PBMC/well. After cocultivation in 6 replicates with individual peptides (10 μg/mL) for 16–20 hours, spots were developed using a biotinylated anti-human IFN-γ mAb (Bender Med Systems), streptavidin-alkaline phosphatase (DAKO, Glostrup, Denmark), and BCIP/NBT alkaline phosphatase substrate (Sigma). Peptides from human immunodeficiency virus (HIV) (HLA-A*0201 restricted) and HBV (promiscuous T helper epitope) or medium without peptide served as negative controls. HLA-A*0201 restricted peptides from cytomegalovirus (CMV), Epstein-Barr virus (EBV), and influenza served as specific positive controls and Concanavalin A as nonspecific positive control. On each assay plate, a PBMC sample with known reactivity against the CMV positive control peptide and the HIV negative control peptide was developed as quality control. All 5 peptides of IC41 and the 2 CTL epitopes contained within longer T helper peptides were tested individually, allowing to discern CD4 from CD8 T-cell responses. Results were considered positive when the median spot number for an individual peptide was at least 3 times greater than the median spot number obtained in the negative control wells (HIV peptide: 0–5 spots per million PBMC in most assays) and at least 15 spots per million PBMC. The identical assay system and responder criteria have been used in a healthy volunteer trial.3Firbas C. Jilma B. Tauber E. et al.Immunogenicity and safety of a novel therapeutic hepatitis C virus (HCV) peptide vaccine: a randomized, placebo controlled trial for dose optimization in 128 healthy subjects.Vaccine. 2006; 24: 4343-4353Crossref PubMed Scopus (128) Google Scholar In that trial, 2 control groups were included receiving only adjuvant (n = 12) or only saline (n = 20), and a total of 256 PBMC samples was tested for HCV-specific T-cell responses in these patients. Applying the above-mentioned criteria, between 0% and 10% showed positive responses, possibly indicating false-positive results.3Firbas C. Jilma B. Tauber E. et al.Immunogenicity and safety of a novel therapeutic hepatitis C virus (HCV) peptide vaccine: a randomized, placebo controlled trial for dose optimization in 128 healthy subjects.Vaccine. 2006; 24: 4343-4353Crossref PubMed Scopus (128) Google Scholar Thus, the ELIspot assay had a specificity of >90%. A 5-color HLA-A*0201 tetramer-binding assay was used to quantitate HCV-specific CD8+ T cells. After thawing, 106 PBMC were stained with anti-CD8-PC7 mAb (Beckman Coulter, FL); anti-CD4/CD13/CD19-PC5 mAb (i-MascTM Gating Kit; Beckman Coulter Immunomics, Marseille, France); anti-CD45RA-ECD mAb (Beckman Coulter Immunotech, Marseille, France); anti-CCR7-FITC mAb (R&D Systems, Minneapolis, MN); and HLA-A*0201-PE tetramers loaded with HCV, CMV, and HIV epitopes, respectively (Beckman Coulter Immunomics). For analysis (Cytomics FC 500 flow cytometer, Beckman Coulter Immunomics) cells were gated on CD4-, CD13-, and CD19-negative cells first, then on a lymphocyte gate, and finally on CD8-positive cells. A total of 50,000 events were acquired in each analysis. Results were expressed as percentages of tetramer-binding cells in the CD8-positive population and considered positive when ≥0.04% (background of negative control tetramer set to 0.00%–0.01%). In addition to the frequency cut-off, a distinct population (clouds) was required for a positive response. HIV and CMV tetramers served as controls. In each assay, PBMC with known reactivity against control tetramers were developed as assay control. Sixty patients were randomized into 3 IC41 and 2 control groups (see Table 2). Forty-one patients were nonresponders to prior standard IFN/ribavirin therapy (26 or 72% in the IC41 groups; 15 or 62% in the control groups), and 19 were relapsers (10 or 28% in the IC41 groups; 9 or 37.5% in the control groups). Fifty-five patients had HCV genotype 1, and each of the 5 treatment groups included 1 patient with a different genotype (either 3 or 4). There were no clinically significant differences between the IC41 and the control groups (Table 1). There were no serious adverse events considered to be study medication related. Forty-four patients experienced a treatment-emergent adverse event, 86% in the IC41 groups and 54% in the control groups (Table 3). Drug-related adverse events were also more frequent in the IC41 groups (58% vs 41% in the control groups), the most frequent being influenza-like illness (17%), injection site erythema (11%), and induration (11%) compared with dizziness (17%) in the control group. Influenza-like illness was absent in the control groups. Injection site intolerance was greater in the IC41 groups than in the control groups, the most frequent symptoms being erythema (up to 50% of patients in the IC41 groups), followed by edema and induration. Erythema was experienced by a similar proportion of patients in control group poly-L-arginine and was experienced least often in the peptides control group. Symptoms of intolerance persisted for up to 24 hours in the groups that received poly-L-arginine compared with 1 to 3 hours in the peptide control group. There was no exacerbation with increasing number of vaccinations. Mean alkaline phosphatase and bilirubin values were within the normal range at all visits.Table 3Numbers of Patients With Treatment Emergent Adverse Events Experienced by >5% of PatientsVaccine groups n = 36Control groups n = 24Total n = 60n%n%n%Total318613544473Headache71928915Nasopharyngitis71914813Dizziness38417712Influenza-like illness61700610Arthralgia4111458Injection site erythema4111458Nausea4111458Fatigue4110047Injection site induration4110047Injection site pain381447Toothache13312.547Abdominal pain380035Night sweats380035Cholelithiasis260023Cystitis002823Diarrhea260023Erythema260023Influenza260023Injection site edema260023Pruritus260023Pyrexia260023Rigors260023Sinusitis260023 Open table in a new tab Vaccination with IC41 induced T-cell proliferation in up to two thirds of the patients, whereas only 17% in the peptide only and none in the poly-L-arginine control group responded. Proliferation increased over time, peaking after the last vaccination and slowly waning thereafter until the last follow-up 6 months after end of treatment. From the fourth vaccination onward, proliferation was greater in the IC41 groups than in the control groups, with the greatest responses in group 2 (median sum of SI = 5.8, ITT population) and group 3
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