Volume 50, Issue 4, Pages 693-704 (April 2009) Acute infection with a single hepatitis C virus strain in dialysis patients: Analysis of adaptive immune response and viral variability  Lukas Weseslindtner, Christoph Neumann-Haefelin, Sergei Viazov, Anita Haberstroh, Josef Kletzmayr, Judith H. Aberle, Joerg Timm, Stefan R. Ross, Renate Klauser-Braun, Thomas F. Baumert, Michael Roggendorf, Robert Thimme, Heidemarie Holzmann  Journal of Hepatology  Volume 50, Issue 4, Pages 693-704 (April 2009) DOI: 10.1016/j.jhep.2008.11.023 Copyright © 2009 European Association for the Study of the Liver Terms and Conditions

Fig. 1 Time table indicating time of exposure, HCV RNA and anti-HCV testing and CD4+ T-cell analyses. Four patients were dialyzed at the same time and in the same room for a period of 6 weeks (period of exposure). Positive HCV RNA PCR results were obtained for all patients at week 6 post-exposure (p.e.). Acute HCV infection could be verified by seroconversion in patients #1, #2 and #4 (week 7, 17 and 6 p.e., respectively). In patient #3, HCV antibodies were never detected. Journal of Hepatology 2009 50, 693-704DOI: (10.1016/j.jhep.2008.11.023) Copyright © 2009 European Association for the Study of the Liver Terms and Conditions

Fig. 2 (A) Phylogenetic consensus tree of partial HCV core sequences from the patients, local control (LC) isolates, and epidemiologically unlinked strains from Genbank (accession numbers given). (B) Alignment of HCV subtype 1b hypervariable region 1 (HVR1) sequences isolates from the patients. The high relatedness of the partial HCV core sequences (A) and the high similarity of HVR1 (B) compared to reference sequences and viral strains from five local controls (LC1-5) indicate that HCV infection in dialysis patients #1–4 had occurred from the same source. Journal of Hepatology 2009 50, 693-704DOI: (10.1016/j.jhep.2008.11.023) Copyright © 2009 European Association for the Study of the Liver Terms and Conditions

Fig. 3 Kinetics of HCV RNA, alanine aminotransferase (ALT) levels and HCV antibodies in dialysis patients (#1–4) infected with the same HCV strain. The patients exhibited differences in HCV RNA- and ALT levels as well as the time point of seroconversion. While patient #1 achieved transient control of infection with undetectable HCV RNA levels and normalization of ALT at week 17 p.e., patients #2 and #3 displayed persistently high viral loads. In patient #4, HCV RNA and ALT levels fluctuated until therapy was initiated. Journal of Hepatology 2009 50, 693-704DOI: (10.1016/j.jhep.2008.11.023) Copyright © 2009 European Association for the Study of the Liver Terms and Conditions

Fig. 4 Non-structural protein (NS) 3 and 4-specific T-helper cells type 1 (TH1) cytokine production (tumor necrosis factor-α [TNF-α]: black bars, interferon-γ [IFN-γ]: grey bars) of CD4+ T-cells in relation to HCV RNA levels. Although all 4 dialysis patients were infected with a single HCV strain, the vigour of TH1 response varied among the patients. In correlation to the decrease of viremia, CD4+ T-cells from patient #1 initially displayed robust production of TNF-α and IFN-γ, which were detected by FACS analysis. When HCV RNA recurred, TH1 responses weakened, with frequencies of IFN-γ-producing CD4+ T-cells falling below the cut-off value (determined by mean+3×SD in 10 non-HCV-infected dialysis patients). In contrast, TH1 responses were completely absent in patients #2 and #3. Patient #4 showed low frequencies of TNF-α- producing CD4+ T-cells but no IFN-γ-producing cells. Journal of Hepatology 2009 50, 693-704DOI: (10.1016/j.jhep.2008.11.023) Copyright © 2009 European Association for the Study of the Liver Terms and Conditions

Fig. 5 CD8+ T-cell responses in patients #1 and #4 at week 42 post-exposure. CD8+ T-cell responses to peptides covering the whole HCV polyprotein were investigated for patients #1 and #4 at week 42 p.e. While CD8+ T-cells from patient #1 produced interferon-γ (IFN-γ) upon stimulation with peptides non-structural protein (NS)3 aa1268–1285 and NS5 aa2227–2244, patient #4 did not show any CD8+ T-cell responses to these HCV peptides. Journal of Hepatology 2009 50, 693-704DOI: (10.1016/j.jhep.2008.11.023) Copyright © 2009 European Association for the Study of the Liver Terms and Conditions

Fig. 6 Sequences of non-structural (NS) proteins NS3, NS4 and NS5a (aa2185–2270) from patients #1 and #3. Sequences of NS3, NS4 and a part of NS5a from patient #1 (week 38 p.e.), who displayed significant NS3/NS4-specific CD4+ T-cell responses and whose CD8+ T-cells targeted peptides NS3 aa1268–1285 and NS5 aa2227–2244, were compared to sequences obtained from patient #3 (week 27 p.e.), who was the probable source of the infection and showed no detectable T-cell responses. Two amino acids, NS3 aa1046 and aa1222, differed between the patients. The sequences of the immunodominant CD8+ T-cell epitopes from patient #1 were identical to those from patient #3. Journal of Hepatology 2009 50, 693-704DOI: (10.1016/j.jhep.2008.11.023) Copyright © 2009 European Association for the Study of the Liver Terms and Conditions

Fig. 7 Antibody-mediated neutralization of HCV pseudotype particles (HCVpp). Antibody-mediated neutralization was analyzed using retroviral HCVpp derived of the HCV-J strain as described previously [14,33]. In patients #1, #2, #3 and #4, levels of neutralization were only detectable at the lowest serum dilution (1/20) and did not exceed the cut-off value. Journal of Hepatology 2009 50, 693-704DOI: (10.1016/j.jhep.2008.11.023) Copyright © 2009 European Association for the Study of the Liver Terms and Conditions