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S1 and S2. Phylogenetic analyses of viral sequences detected before and 7 d after transplantation revealed a marked and abrupt decrease of diversity bottleneck effect at day 7 in four out of six patients P01, P02, P03, and P06 , with selected variants clustering in a single branch Fig. Sequence analysis of serum samples at month 6 and 12 after transplantation available from three patients demonstrated that variants selected during the early phase of transplantation P01VL, P04VE, and P06VI had the same or similar envelope glycoprotein sequence as predominant variants present at later time points after transplantation unpublished data. To investigate the evolutionary dynamics of the envelope quasispecies evolution, distribution of viral quasispecies was analyzed as described previously for other cohorts Farci et al. Infection efficiency was 10—fold lower in human hepatocytes, as indicated by the level of reporter gene expression in cells infected with the same HCVpp preparations. Viral isolates used for functional analysis are indicated with capital letters. Circle graphs represent the percentage of each clone detected. Variants containing stop codons, insertions, or deletions altering the HCV open reading frame are depicted with a number sign and were not further analyzed in HCVpp assays. These findings provide significant insights into the molecular mechanisms of viral evasion during HCV reinfection and suggest that viral entry is a viable target for prevention of HCV reinfection of the liver graft. HCVpp—antibody complexes were then added to Huh7 cells, and infection was analyzed as described in Fig. Indeed, the rapid induction of cross-neutralizing antibodies in the very early phase of infection has been suggested to contribute to control of HCV infection Lavillette et al. S5 , the ratio of highly infectious subvariants compared with the less infectious ones was high in HVR1 variants with a high infectivity phenotype VL and low in HVR1 variants with a low infectivity phenotype the ratio of high to low infectivity subvariants was for VA, for VB, and for VK subvariants. Because viral entry is a major target of neutralizing antibodies and HCV reinfection of the liver graft occurs efficiently despite the presence of high titer of anti-HCV antibodies, we investigated whether neutralizing antibodies present in pretransplantation serum were able to neutralize infection of patient-derived HCVpps. Sequences isolated at day 7 and month 1 after LT were clustered in the same branch of phylogenetic trees Fig. HCV has a very high replication rate, and the highly error-prone viral polymerase allows for rapid production of minor viral variants that may outpace humoral and cellular immune responses Bowen and Walker, ; Ray et al. Sequence analysis demonstrated the presence of different viral variants in the serum before and after transplantation Figs. Compared with the marked decrease in diversity of variants in the majority of patients immediately after transplantation Fig. Viral isolates are indicated by an individual color and capital letters. Sequences of variants isolated before LT and not detected on day 7 after transplantation are depicted as open black circles, sequences of variants isolated before LT and reinfecting the graft are depicted as open blue circles, and sequences isolated 7 d after LT are depicted as blue squares. The mechanisms by which viral variants are selected and HCV evades host immunity to establish persistence in transplanted patients are not understood. To address whether the efficiency of infection was different in strains reinfecting the liver graft selected variants and strains not detected during the first days of LT nonselected variants , we performed a comprehensive analysis of viral entry of all patient-derived HCVpps containing functional full-length HCV envelope glycoproteins. These variants are under constant immune pressure in the infected host, and selection processes lead to domination of the viral quasispecies by the most fit virus that can also evade immune recognition Uebelhoer et al. Phylogenetic analyses of viral quasispecies evolution before and 7 d after LT. To determine the mechanism of selection and evasion of viral variants during reinfection, we produced HCVpps harboring full-length E1E2 glycoproteins of all detected and grouped variants before transplantation nonselected variants; Figs. These data indicate that the new host environment leads to an abrupt and marked change in the composition and diversity of the viral quasispecies in the majority of patients, which remains relatively stable after transplantation. A Transfection efficiency was analyzed for each variant and control Ctrl; empty vector without E1E2 by quantifying luciferase expression expressed as normalized percentage of transfection efficiency based on the predominant selected variant. S1 and not depicted. HCVpps bearing patient-derived HCV envelope glycoproteins were added to Huh7 cells, and infection was analyzed by luciferase reporter gene expression. The percentage of patient-derived viral isolates resulting in infectious HCVpps was higher than in a recent longitudinal study of a single HCV-infected patient von Hahn et al. Several variants e. Distribution of full-length E1E2 variants mean number of clones per patient, 24; range, 20—31 depicted in Fig. These analyses corroborate that phylogenetic grouping of full-length E1E2 genomes by HVR1 analysis is valid and allows a functional analysis of full-length clones whose function is representative of the entire E1E2 quasispecies population.{/INSERTKEYS}{/PARAGRAPH} HCC, hepatocellular carcinoma; tac, tacrolimus; rap, rapamycin; cor, corticosteroids. In this study, we aimed to investigate whether viral entry and escape from neutralizing antibodies are determinants for viral evasion and persistence during the very early phase of graft infection. Bootstrap values are expressed as percentages per 1, replicates. Viral entry is important for selection of viral variants after LT. These variants were not further analyzed for HCVpp production and are indicated by a number sign in the respective pie charts of Fig. In contrast, HCVpps derived from HCV variants that were not detected after transplantation were efficiently neutralized by autologous host-neutralizing antibodies mean neutralizing titer, ,; range, to , Furthermore, nonselected isolates showing high infectivity e. Patient-derived HCVpps were preincubated with autologous pretransplantation serum, and their subsequent infection levels were quantified. The number of each clone is indicated. Alternatively, technical factors such as the use of more permissive Huh7 cells may have resulted in the isolation of more variants with more easily detectable HCVpp infection in this study. HCV isolates in plasma and liver samples were cloned and sequenced as described in Materials and methods 30 clones per sample in one animal. In a longitudinal analysis of six HCV-infected patients undergoing LT, we demonstrate that HCV variants reinfecting the liver graft were characterized by efficient entry and poor neutralization by antibodies present in pretransplant serum compared with variants not detected after transplantation. This result corroborates the presumed selective advantage of the variant reinfecting the graft and demonstrates the relevance of our analysis and validity of the HCVpp model system for HCV infection in vivo. Hepatitis C virus HCV —related cirrhosis and hepatocellular carcinoma are leading indications for liver transplantation LT. NI, noninfectious. These data demonstrate that escape from antibody-mediated neutralization represents an important mechanism for viral evasion from host immunity during LT. Both viral and host factors are potential determinants for evasion from host responses and adaptation of the virus after transplantation. Grouping of full-length E1E2 quasispecies Figs. Circle graphs represent the percentage of each clone detected, and numbers of clones are shown. Using HCVpps bearing viral envelope glycoproteins derived from patients undergoing LT, we show that efficient viral entry and escape from antibody-mediated neutralization are key determinants for selection of viral variants reinfecting the liver graft. Patient number, age of recipient R and donor D , recipient gender, indication of transplantation, model of end-stage liver disease MELD score before transplantation, HCV genotype, viral load before [BT] and day 7 [D7] and month 1 [M1] after transplantation , and immunosuppressive IS treatment are shown. This approach allowed us to address the question whether all the subvariants harboring the same HVR1 have the same level of infectivity and if not, what is the ratio of highly infectious subvariants compared with the less infectious ones. Infectious retroviral HCV pseudoparticles HCVpps have been shown to represent a robust and valid system for the study of HCV entry and antibody-mediated neutralization in clinical cohorts Lavillette et al. Monoclonal antibodies directed against HCV envelope glycoproteins or a cellular entry factor efficiently cross-neutralized infection of human hepatocytes by patient-derived viral isolates that were resistant to autologous host-neutralizing responses. To study the impact of viral entry and neutralization on viral evasion during LT, we investigated viral quasispecies evolution in six patients infected with HCV genotype 1b undergoing LT Table I. End point dilution titers are indicated for each variant. Sequence analysis of viral isolates showed that 13 out of 63 isolates contained stop codons or insertions altering the open reading frame. Similar to results obtained with hepatoma cells, HCVpps of the selected variants showed more efficient entry than HCVpps produced from the nonselected variants Fig. Moreover, viral entry is a major target of neutralizing antibodies, a first-line host defense inhibiting viral spread. These observations suggest that HCV has developed efficient strategies to evade host immunity and adapt rapidly to the new host environment. Variants reinfecting the liver graft are depicted in blue, and nonselected variants not detected after transplantation are depicted in white, gray, or black. To confirm the results obtained with Huh7 hepatoma cell lines, we infected primary human hepatocytes obtained from four different donors with patient-derived HCVpps from selected and nonselected variants. B Comparative analysis of viral entry of HCVpps containing full-length E1E2 functional envelope proteins from pretransplant variants in Huh7 cells depicted in Fig. Due to viral evasion from host immune responses and the absence of preventive antiviral strategies, reinfection of the graft is universal. S1 , and variants selected at day 7 remained predominant 1 mo after transplantation in all patients Fig. A major limitation is the universal HCV reinfection of the graft followed by an accelerated course of virus-induced liver disease Brown, A prophylactic strategy for prevention of reinfection is lacking, and interferon-based antiviral therapies have limited efficacy and tolerability in LT recipients Brown, Thus, recurrent liver disease with poor outcome has become an increasing problem facing hepatologists and transplant surgeons, underlying the urgent need for novel strategies for prevention of reinfection. Complete sequences of isolates characterized in functional experiments are shown in Fig. The difference in viral entry observed between selected and nonselected variants did not result from variations in transfection efficiency Fig. Calculation of neutralization and determination of background and thresholds for neutralization are described in Materials and methods. The strains reinfecting the graft always produced functional E1E2 Fig. Results are expressed in relative light units RLU plotted in a logarithmic scale. Finally, grouping of viral quasispecies on complete envelope glycoprotein sequences with subsequent functional analysis of all individual full-length clones in patient 01 again demonstrated that selected strains were characterized by efficient viral entry and escape from neutralizing responses, whereas nonselected strains exhibited a poor entry and neutralization phenotype unpublished data. These results define the molecular mechanisms of viral evasion during HCV reinfection and suggest that viral entry is a viable target for the prevention of HCV reinfection of the liver graft. A human liver—chimeric mouse was challenged by intraperitoneal injection of pretransplant serum of patient P01 infectious dose administered, 3. Viral entry is the very first step of HCV infection Evans et al. The development of preventive antiviral strategies has been hampered by a limited understanding of the mechanisms leading to HCV reinfection. A total of clones mean, 24 per time point and patient; range, 20—31 was obtained by RT-PCR from serum before and 7 d and 1 mo after transplantation. As shown in Fig. The mechanisms by which the virus evades host immunity to reinfect the liver graft are unknown. Escape from antibody-mediated neutralization is a key determinant for selection of viral variants after LT. The threshold for a detectable infection in this system is indicated by dashed lines. Reinfection occurs within a few hours of graft reperfusion despite the presence of anti-HCV antibodies Brown, Evolution of viral quasispecies rapidly changes after transplantation, and only a small fraction of viral variants present before transplantation is selected after LT Moreno Garcia et al. {PARAGRAPH}{INSERTKEYS}Baumert; Viral entry and escape from antibody-mediated neutralization influence hepatitis C virus reinfection in liver transplantation. Furthermore, we demonstrate that mAbs directed against viral or host entry factors efficiently cross-neutralized infection of human hepatocytes by patient-derived viral isolates that were resistant to autologous host-neutralizing responses. Most importantly, we demonstrate that all of the nonselected E1E2 subvariants exhibiting high infectivity were efficiently neutralized by autologous patient serum e. J Exp Med 30 August ; 9 : — End-stage liver disease caused by chronic hepatitis C virus HCV infection is a leading cause for liver transplantation LT. Variants containing stop codons, insertions, or deletions altering the HCV open reading frame are depicted with a number sign. The viral diversity in the other two patients P04 and P05 , which was low before transplantation, remained stable before and after transplantation Fig. Collectively, these data suggest that the efficiency of viral entry into naive hepatocytes of the liver graft is important for selection of viral variants after LT. These results demonstrate that viral isolates that are selected during transplantation are characterized by more efficient entry compared with isolates not detected after transplantation. Background levels of the assay were determined in each experiment. Dashed lines indicate the threshold for a positive neutralization titer corresponding to Means from at least four independent experiments performed in triplicate are shown.