Volume 141, Issue 3, Pages 890-899.e4 (September 2011) A Poxvirus Vaccine Is Safe, Induces T-Cell Responses, and Decreases Viral Load in Patients With Chronic Hepatitis C  François Habersetzer, Géraldine Honnet, Christine Bain, Marianne Maynard–Muet, Vincent Leroy, Jean–Pierre Zarski, Cyrille Feray, Thomas F. Baumert, Jean–Pierre Bronowicki, Michel Doffoël, Christian Trépo, Delphine Agathon, Myew–Ling Toh, Martine Baudin, Jean–Yves Bonnefoy, Jean–Marc Limacher, Geneviève Inchauspé  Gastroenterology  Volume 141, Issue 3, Pages 890-899.e4 (September 2011) DOI: 10.1053/j.gastro.2011.06.009 Copyright © 2011 AGA Institute Terms and Conditions

Figure 1 Study design. Patients (3 cohorts) received 3 subcutaneous injections of TG4040 on days 1, 8, and 15 at doses of 106 PFU, 107 PFU, and 108 PFU. Because the dosage of 108 PFU was well tolerated, 6 additional patients were treated in the 108 dose group. All patients were followed up at least 6 months after the last injection. Patients treated at the dosage of 108 PFU received a TG4040 boost injection 6 months after the first injection and were further followed up an additional 6 months period. Gastroenterology 2011 141, 890-899.e4DOI: (10.1053/j.gastro.2011.06.009) Copyright © 2011 AGA Institute Terms and Conditions

Figure 2 MVA-specific immune responses. Circulating MVA-specific total (A) and neutralizing (B) antibody titers were evaluated by enzyme-linked immunosorbent assay or by the capacity of patient's serum to inhibit MVA-GFP infection of BHK-21 cells, respectively. MVA-specific IFN-γ producing cells (C) were evaluated by IFN-γ ELISpot assay. Each symbol represents the mean score of triplicate values for each patient at each time point. Cut-off value corresponding to 95% of specificity is shown as a dotted line. TG4040 injections are indicated by arrows. Gastroenterology 2011 141, 890-899.e4DOI: (10.1053/j.gastro.2011.06.009) Copyright © 2011 AGA Institute Terms and Conditions

Figure 3 Analysis of viral load evolution with respect to HCV-specific immune responses. For immunologic studies, blood samples were taken at different time points before (M-1 and D1) and after vaccination: 5 time points were typically assessed, ie, on days 8, 22, and 37 and at 2 and 6 months, and in cohort 3 at days 8 and 22 after the boost. Nonetheless, a number of time points could not be evaluated because of technical reasons (eg, volume of blood samples taken too low, number of viable cells recovered after freezing too low). This was the case for the following: Pt 1: M-1, D22, M6; Pt 2: M-1; Pt 3: D37, M2; Pt 4: D22, M2; Pt 5: M2; Pt 8: M-1; Pt 18: D8. Evolution of the viral load and the IFN-γ ELISpot in comparison with D1 baseline measure over time is demonstrated in cohorts 1 (A) and 2 (B) and in patients 15 and 18 in cohort 3 (C). Viral load is expressed as IU/mL. IFN-γ ELISpot assay was performed on PBMC after restimulation with peptide pools specific of vaccine immunogens (NS3/1, NS3/2, and NS4B) and from TG4040-unrelated peptide pools NS5/1 and NS5/2 (represented by different box symbols are shown at the bottom of Figures). Results are expressed as delta spots (measured specific spots in response to HCV antigens minus background spots in control dimethyl sulfoxide × 106 PBMC). The cut-off values corresponding to the geometric mean value leading to 95% specificity was 28. ND, not done; Pt, patient. Arrows indicate vaccine injections. Gastroenterology 2011 141, 890-899.e4DOI: (10.1053/j.gastro.2011.06.009) Copyright © 2011 AGA Institute Terms and Conditions

Figure 4 Kinetics of serum HCV RNA after TG4040 administration. HCV RNA levels were measured at screening time points (shown here for D1, see also Supplementary Table 1) as well as days 8, 15, 22, and 37 and then once a month until 6 months after the last injection. In patients who received a boost injection, additional measurements were performed on days 8, 22, and 3 and 6 months (M9 and M12) after the booster administration. The Figure represents the fold change of serum HCV RNA in the different patients after administration of TG4040 in comparison with baseline level. Panels A, B, and C represent patients from cohorts 1, 2, and 3, respectively. Arrows indicate vaccine injections. Gastroenterology 2011 141, 890-899.e4DOI: (10.1053/j.gastro.2011.06.009) Copyright © 2011 AGA Institute Terms and Conditions

Figure 5 Cytokine and chemokine profiles in response to HCV peptide pools in TG4040 treated patients. The cytokine/chemokine response to HCV peptide pools NS3, NS4, and NS5A in patient's PBMC supernatant was determined by Luminex. Comparative analysis of cytokine profiles at (A) baseline between vaccine “responders” and “nonresponders” defined on the basis of > or < 0.5-log viral load decrease is shown and (B) following vaccination in cohort 1. Gastroenterology 2011 141, 890-899.e4DOI: (10.1053/j.gastro.2011.06.009) Copyright © 2011 AGA Institute Terms and Conditions

Supplementary Figure 1 (A–C) Fluctuation of absolute RNA levels following TG4040 administration. Changes in absolute viral load titers are shown for patients of cohorts 1, 2, and 3 in panels A, B, and C, respectively. Arrows indicate vaccine injections. Gastroenterology 2011 141, 890-899.e4DOI: (10.1053/j.gastro.2011.06.009) Copyright © 2011 AGA Institute Terms and Conditions

Supplementary Figure 2 (A and B) Cytokine and chemokine profiles in response to hepatitis C virus peptide pools in TG4040-treated patients. Tested as described in Figure 5. Comparative analysis of cytokine profiles between vaccine “responders” and “nonresponders” defined on the basis of > or < 0.5-log viral load decrease tested as described in Figure 5 following vaccination for cohorts 2 and 3 (panels A and B, respectively). Gastroenterology 2011 141, 890-899.e4DOI: (10.1053/j.gastro.2011.06.009) Copyright © 2011 AGA Institute Terms and Conditions

Supplementary Figure 2 (A and B) Cytokine and chemokine profiles in response to hepatitis C virus peptide pools in TG4040-treated patients. Tested as described in Figure 5. Comparative analysis of cytokine profiles between vaccine “responders” and “nonresponders” defined on the basis of > or < 0.5-log viral load decrease tested as described in Figure 5 following vaccination for cohorts 2 and 3 (panels A and B, respectively). Gastroenterology 2011 141, 890-899.e4DOI: (10.1053/j.gastro.2011.06.009) Copyright © 2011 AGA Institute Terms and Conditions