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Abstract

  1. Top of page
  2. Abstract
  3. Materials and Methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. References
  8. Supporting Information

The development of human cultured hepatitis C virus (HCV) replication-permissive hepatocarcinoma cell lines has provided important new virological tools to study the mechanisms of HCV infection; however, this experimental model remains distantly related to physiological and pathological conditions. Here, we report the development of a new ex vivo model using human adult liver slices culture, demonstrating, for the first time, the ability of primary isolates to undergo de novo viral replication with the production of high-titer infectious virus as well as Japanese fulminant hepatitis type 1, H77/C3, and Con1/C3. This experimental model was employed to demonstrate HCV neutralization or HCV inhibition, in a dose-dependent manner, either by cluster of differentiation 81 or envelope protein 2–specific antibodies or convalescent serum from a recovered HCV patient or by antiviral drugs. Conclusion: This new ex vivo model represents a powerful tool for studying the viral life cycle and dynamics of virus spread in native tissue and also allows one to evaluate the efficacy of new antiviral drugs. (HEPATOLOGY 2012;56:861–872)

Studies on the mechanisms of hepatitis C virus (HCV) infection have been hampered by limited in vivo and ex vivo models1-4 as the consequence of poor replication of primary viral isolates and restriction of susceptible hosts (i.e., humans and chimpanzees). Recent advances have yielded cell-culture replication-competent chimeric viruses that have the capacity to achieve the complete infectious cycle. Of note, the infectious clone of genotype 2a HCV, Japanese fulminant hepatitis type 1 (JFH-1),5, 6 allowed us to develop chimeric viruses with exceptionally efficient replication,7 because so-called cell-culture–grown hepatitis C virus (HCVcc) can be propagated, with infectivity titers in the range of 105 focus-forming units (ffu)/mL in the highly permissive Huh-7 subline, Huh-7.5.1.8, 9 However, this system has several limitations that include the inability to study the effects of pharmacologic inhibitors targeting the nonstructural proteins of the most prevalent, problematic viral strains (e.g., genotypes 1a and 1b). Moreover, the study of virus/host cell interactions is limited, because the permissive cell lines are transformed and poorly differentiated. Attempts to infect primary hepatocytes using primary HCV isolates resulted in limited poor viral replication and low production of de novo infectious virus particles.10, 11 Other models using primary human HCVcc-infected hepatocytes12 or HCV-transfected hepatocytes cocultured with stellate cell lines13 have been developed, which allowed virus replication of the entire viral life cycle with relatively high viral titers.

Herein, we report on the development and analysis of an ex vivo model of HCV infection of adult human liver slices. The system is based on precision-cut tumor slices or human liver slices that could be infected and maintained ex vivo for the evaluation of viral replication.14 The infection of adult human liver slices culture allowed us to achieve the robust replication of the HCVcc genotype 2a, 1a, and 1b genome and to obtain infectivity titers above 105 ffu/mL. In addition, we report on productive infection using human primary isolates of HCV genotype 1b. Altogether, this new model offers a powerful ex vivo system for the preclinical analysis of infectivity and evaluation of antiviral therapy efficacy.

Taken together, we report on a new ex vivo experimental model for studying HCV replication in native tissue and antiviral drug efficacy.

Materials and Methods

  1. Top of page
  2. Abstract
  3. Materials and Methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. References
  8. Supporting Information

For details of Materials and Methods, please see Supporting Materials.

Human Liver Tissue Specimens.

Adult human primary liver tissue samples were obtained from 25 HCV seronegative patients who underwent liver resection surgery, mainly for liver metastasis, in the absence of underlying liver disease. The patients were seronegative for hepatitis B virus, and human immunodeficiency virus (Department of Digestive Surgery, La Pitié-Salpétrière Hospital and Cochin Hospital, Assistance Publique–Hôpitaux de Paris [AP-HP], Paris, France). Experimental procedures were carried out in accord with French laws and regulations. Because liver tissue is strongly sensitive to ischemia and rapidly deteriorates, human liver slices preparation required an appropriate coordination and logistics between surgeons, nurses and the laboratory. Samples were transported to the laboratory as quickly as possible. Immediately after surgical resection, all liver pieces were harvested and kept on ice in sterile University of Wisconsin solution (ViaSpan; Barr Laboratories, Inc., Pomona, NY) until slicing. Time from harvest to slicing was kept at an absolute minimum (<2 hours). The criteria of human liver sample selection are in the Supporting Materials.

Primary Virus Isolates.

Sera from patients were obtained at the Department of Hepatology, Cochin Hospital, AP-HP (Paris, France). For human liver slices infection, fresh (i.e., nonpreviously frozen) sera from HCV-infected patients (genotype 1b) with a high viral load (>106 IU/mL) (Table 1) who had not received previous antiviral treatment were used. Serum from an HCV-infected patient (genotype 1b) cured spontaneously was used for neutralization experiments. Control sera were obtained from 3 healthy blood donors. All patients provided informed consent for blood sampling in accord with the rules of the local ethics committee.

Table 1. Specific Infectivity of HCV Produced by Huh-7.5.1 Cells and Human Liver Slices. Specific Infectivity Appears Higher in the Later than in the Former
  HCVcc (Huh-7.5.1)HCVpc* (Liver Slices)HCVrepc (Liver Slices)HCVrecc (Huh-7.5.1)
Liver SamplesLiver Slices Viability (%)VirusStockHCVRNA (log10 IU/mL)Specific lnfectivity§HCV RNA (log10 IU/mL)Specific lnfectivity§Fold Increase in Specific InfectivitySpecific lnfectivity§Fold Increase In Specific InfectivitySpecific lnfectivity§Fold Reduction in Specific Infectivity
  • *

    Progeny virus recovered from liver slices inoculated with HCVcc or primary HCV isolates at the end of the 10-day culture period.

  • Progeny virus recovered from human liver slices inoculated with HCVpc at the end of the 10-day culture period.

  • Progeny virus recovered from Huh-7.5.1 cells inoculated with HCVpc at the end of the 10-day culture period.

  • §

    HCV RNA levels and infectivity titers in filtered culture supernatants were measured with the Abbott viral load test (Abbot laboratories, Abbott Park, IL) and focus-formation assay, respectively, and specific infectivity values were calculated as follows: 103 × infectivity titer (ffu)/HCV RNA level (IU).

  • Relative to HCVcc.

  • Relative to HCVpc.

  • Abbreviation: G1b; HCV genotype 1b.

188JFH-1J17.79166.37774981051
286JFH-1J17.79166.9479250800502729
385JFH-1J17.79165.956724269844
485JFH-1J17.79165.52512325333
588JFH-1J27.82158.4665043655432131
686JFH-1J27.821577004773049
790H77H18106.2580080840842236
888H77H18106.17047073073
988H77H18107.381581835831845
  Conl/C3C17.912785071875731944
1090Conl/C3C17.9126.18336984070
1190Conl/C3C28.1868301038651071943
1288Patient sera G1bP16.39.86107001061
1385Patient sera G1bP26105125601437
1490Patient sera G1bP368.56596801255

Slices Preparation and Culture.

Slices were prepared from the 0.5-cm3 cube (∼16 mg) of human liver. Cubes were embedded into 5% low-gelling-temperature agarose (type VII-A; Sigma-Aldrich Chemie GmbH, Seelze, Germany) prepared in phosphate-buffered saline (PBS), cooled until solid, and sliced into 350-μm-thick slices in an ice-cold PBS bath with a vibratome (Leica, Milton Keynes, UK). Human liver slices were transferred to 0.4-μm organotypic culture inserts (Millicell; Millipore, Bedford, MA) in 12-well plates (one slice/well) containing 2 mL of complete Dulbecco's modified Eagles's medium culture media and maintained at 37°C under a constant flow of humidified 95% O2/5% CO2 for up to 24 hours before viral infection, as described in the Supporting Materials. Cell number for tissue slices was estimated at ∼2.7 106 cells/slice based on a 14-cell-thick slice (cell diameter, ∼25 μm).15

Liver Slices Infection.

Liver slices were infected either with the infectious clone, JFH-1 (genotype 2a),6 or H77/C3 (genotype 1a) or Con1/C3 (genotype 1b) (JFH1-derived chimeric viruses whose structural proteins are encoded either by the genotype 1a/HCV sequence, H77, or the genotype 1b HCV sequence, Con1)7 or fresh (i.e., nonpreviously frozen) HCV-positive patient sera (genotype 1b) (Table 1), as described in the Supporting Materials.

HCV RNA Transfection and Virus Production.

To produce HCVcc, viral RNAs were transcribed in vitro and electroporated into the Huh-7.5.1 cell line, which was kindly provided by Francis V. Chisari (The Scripps Research Institute, La Jolla, CA), as previously described.8 The infectious titer of cell-culture supernatants was evaluated by classical titration assay, as described in the Supporting Materials.

Quantification of HCV Strand RNA and Liver-Specific Genes by Quantitative Real-Time Reverse-Transcription Polymerase Chain Reaction.

A strand-specific quantitative reverse-transcription polymerase chain reaction (qRT-PCR) technique, to quantify the intracellular levels of positive- and negative-strand HCV RNA, was performed as previously described.16 The relative expression of each liver-specific transcript (i.e., albumin, hepatocyte nuclear factor [HNF]-1β, [HNF]-4αtranscription factors, cytochrome P450 [CYP] enzymes CYP2E1 and CYP3A4) was quantified by qRT-PCR, as described in the Supporting Materials.

Western Blotting.

Western blotting was performed as described in the Supporting Materials. Mouse monoclonal antibodies (mAbs) to HCV core protein (C7-50; Affinity BioReagents, Golden, CO) and to HCV nonstructural protein 3 (NS3) (clone1847; ViroStat, Portland, ME) were used as primary antibodies.

Neutralization of HCV Infection.

Neutralization effect of mAbs against human cluster of differentiation (CD)81 or HCV envelope protein 2 (E2) and neutralizing human serum on human liver slices infection with HCVcc Con1 was tested as described in the Supporting Materials.

Drug Inhibition of HCV Replication and Cytotoxicity Assays.

Pegylated interferon (Peg-IFN) and telaprevir (VX-950) inhibition of HCVcc Con1 liver slices infection and cytotoxicity assays were performed as described in the Supporting Materials.

Results

  1. Top of page
  2. Abstract
  3. Materials and Methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. References
  8. Supporting Information

Maintenance of Hepatocyte Differentiation in Human Liver Slices Culture.

Liver slices cultures were established according to the protocol detailed above. Cell viability and tissue morphology was assessed daily until day 10. Liver slices presented a normal architecture (Fig. 1A) and retained their differentiation status over the entire period of investigation (Fig. 1B). Human liver slices were shown to express albumin (marker for liver synthesis function), HNF transcription factors HNF-1β and HNF-4α (key regulators for the maintenance of hepatocyte differentiation), and CYP enzymes (CYP2E1,17, 18 involved in alcohol metabolism, and CYP3A4, which is the predominant isoform expressed in adult human liver and is involved in the metabolism of over 50% of all clinically used drugs).12 As previously reported, messenger RNA (mRNA) expression of albumin increased during week 2,12, 19, 20 as well as other liver markers (i.e., HNF-4α and HNF-1β21 and CYP isoenzymes (i.e., CYP2E122 and CYP3A4).23, 24 Cell viability and expression of hepatocyte-specific genes were also evaluated postinfection, showing similar results to uninfected tissue and thus indicating that there was no evident cytopathic effect (data not shown).12 Comparison of hepatocyte-specific gene expression in uninfected liver slices and Huh-7.5.1 cells showed increased expression levels in liver slices at day 10 of culture, compared to that in Huh-7.5.1 cells, either in an exponential growth phase or at confluence (Supporting Fig. 1). Expression of CYP3A4 was undetected in Huh-7.5.1 cells, whatever the growth stage. The increasing level of albumin and urea secretion over the experiments indicated that liver slices had retained normal physiological and biochemical parameters (Fig. 1C,D).

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Figure 1. Maintenance of phenotypic characteristics of human liver slices during culture. (A) Light microscopy of human liver tissue 7-μm-thick section stained with hematoxylin and eosin showing normal liver lobular architecture at day 5. (1) magnification ×3.5, (2) magnification ×20, and (3) magnification ×40. (B) Maintenance of hepatocyte-specific gene-expression patterns in human noninfected liver slices during culture. qRT-PCR analysis was performed on mRNA from noninfected liver slices prepared from 3 different donors. All liver-specific gene-expression values were normalized to 18S RNA as an internal standard and expressed relative to the zero time point. qRT-PCR experiments were performed with five independent human livers using slices in triplicate from each liver. Values are expressed as means ± standard error of the means (SEMs). Results were compared using the two-paired Student t test (albumin: ****P < 0.0001; CYP2E1: **P < 0.001; CYP3A4: ***P < 0.0003; HNF-1β: *P < 0.01; HNF-4α: **P < 0.008). (C and D) Albumin and urea production by human cultured liver slices (n = 4). Assays were performed as described in Supporting Materials. Studies were done in triplicate and repeated twice for each liver sample. Values are expressed as means ± SEMs. (C) Albumin production expressed as per milligrams of tissue and per hour (*P < 0.02). (D) Urea production expressed per milligrams of tissue and per hour (**P < 0.001).

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Robust Replication of HCVcc and Primary Human Viral Isolates in Primary Human Liver Slices and Production of Infectious Viral Particles.

HCVcc infection of human liver slices was performed using JFH-1, H77/C3, and Con1/C3 at multiplicity of infection (MOI) = 1. Immunofluorescence (IF) using specific mouse mAbs to HCV proteins (e.g., core or NS3) revealed infection in the HCV-infected liver slices (Fig. 2A). The intracellular co-localization of virus core protein either with NS3 (Fig. 2A, I) or BODIPY-labeled lipid droplets (Fig. 2A, II, III, IV) or calnexin, an endoplasmic reticulum (ER) marker (Fig. 2A, V), provided compelling evidence for the specificity of detection of HCV infection, as described previously.25

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Figure 2. Kinetics of HCV RNA replication and core and NS3 protein expression in human infected liver slices, compared to primary human HCVcc JFH-1-infected hepatocytes. Human liver slices were infected overnight either with HVCcc JFH-1 (JFH-1 slices) or HCVcc H77 (H77 slices) or HCVcc Con1 (Con1 slices) (MOI = 0.1). The supernatant was then removed, and the human liver slices were washed and cultured. Liver slices and culture supernatants were collected at different times postinfection. (A) (I) IF in situ with mouse mAbs against core and NS3 proteins in human liver slices at day 5 postinfection with HCVcc Con1. Colocalization of HCV core and NS3 proteins was detected. (II-IV) Infected liver slices with HCVcc Con1 at day 5 of culture: detection of HCV core protein by IF in situ with mAb against HCV core. Intracellular lipid droplets were stained with BODIPY dye 493/503 (1 μM). Colocalization of HCV core proteins and lipid droplets was detected. (III and IV) Enlargement of white squares in Fig. 2A (III and IV, respectively). (V) IF in situ with mAb against core proteins and polyclonal rabbit antibodies against calnexin, an ER marker, in human liver slices at day 5 postinfection with HCVcc Con1. Colocalization of HCV core proteins and calnexin was detected. Scale bar: 10 μm. (VI) Noninfected liver slices at day 5 of culture staining with cyanine 3–conjugated AffiniPure F(ab')2 fragment donkey antimouse immunoglobulin G (IgG) (H+L) antibody (IgG isotype) (red), DAPI, and mouse mAbs against human CD81 (green), Alexa Fluor 488–conjugated goat antirabbit IgG antibody (IgG isotype) (green). Scale bars: 50 μm (I, II, and VI); 20 μm (III); and 10 μm (IV and V). (B) Western blotting analysis of human JFH-1-infected liver slices lysates, compared to primary human JFH-1-infected hepatocytes, with mAbs against HCV NS3 or core proteins at different days postinfection. (4-8) Kinetic of primary human hepatocyte infection (HH) with HCVcc JFH1 supernatant (MOI = 0.1) at days 3, 6, 9, 12, and 15. (9-13) Kinetic of human liver slices infection with HCVcc JFH-1 supernatant (MOI = 0.1) at days 2, 4, 6, 8, and 10. (+) Lysates of HCV-replicating Huh-7.5.1 cells; (1) naïve Huh-7.5.1 cell lysate, (2) naïve human liver slices lysate, and (3) naïve primary human hepatocyte lysate. Lysates were run in parallel to serve as positive and negative controls, respectively. β-actin was used as a loading control. Positions of molecular-weight markers are indicated in kDa. (C) Human HCVcc JFH-1-infected liver slices were lysed to evaluate intracellular levels of positive- and negative-strand HCV RNA by specific-strand qRT-PCR25 at 1, 3, 6, 8, and 10 days postinfection. Values are expressed as means ± standard error of the means (SEMs). Positive strand: *P < 0.03; negative strand: *P < 0.015. Detection of negative strand of HCV RNA evidences active replication as well as increase over time of both positive and negative strands of HCV RNA. Replication was significantly inhibited in a dose-dependent manner in the presence of increasing concentrations of anti-CD81 mAb over 10 days: *P < 0.05. (D) Quantification of intracellular levels of positive- and negative-strand HCV RNA in primary human HCVcc JFH-1-infected hepatocytes by specific-strand qRT-PCR25 at 1, 3, 6, 8, and 10 days postinfection. Values are expressed as means ± SEMs. Positive strand: *P < 0.01; negative strand: *P < 0.04. All results were compared using the two-paired Student t test. DAPI, 4′6-diamidino-2-phenylindole.

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After the inoculation of liver slices with JFH-1/HCVcc, intracellular replication of the viral genome was assessed by a strand-specific qRT-PCR, as previously described.16 HCV RNA–negative strand, a proof of HCV genome replication, could be detected as soon as day 1 postinfection, and moreover, the intracellular levels of both negative and positive strands increased during liver slices culture (Fig. 2C). These results provided evidence of efficient, active viral replication in liver slices as well as it was in primary human HCVcc-infected hepatocytes (Fig. 2D).

Intracellular expression of the viral NS3 and core proteins was confirmed by western blotting. Increased levels of proteins were shown after 2 and 4 days postinfection (Fig. 2B). In HCV-infected human liver slices, the size of the mature form of HCV core protein is 23 kDa, compared to that (21 kDa) in HCV Huh-7.5.1 cells. Indeed, the comparative migration of HCV primary hepatocyte cultures and liver slices lysates gave a similar pattern, compared to that of Huh-7.5.1 cell lines (Fig. 2B). In any case, the HCV polyprotein is processed in the HCV-infected liver slices.26

To determine whether progeny virions produced from infected liver slices were able to replicate, virus titers were estimated in filtered culture supernatants using the classical titration assay, based on the inoculation of naïve Huh-7.5.1 cells. The infectivity titration of viruses in supernatants in the presence of a specific antiviral inhibitor did not yield HCV-positive foci in Huh-7.5.1 cells (data not shown), indicating that a de novo–produced, but not a carried over, virus was detected. Typically (Fig. 3A), infectivity and viral load increased during the culture and reached a peak of up to 1.1 × 105 ffu/mL and 7.8 log10 IU/mL, respectively, by day 10 postinfection. Similar viral loads and infectivity were obtained for using H77 (Fig. 3B) and Con1 (Fig. 3C). Kinetics of HCVcc liver slices infection was similar to that of JFH-1 kinetics after HCVcc primary hepatocyte infection (Fig. 3F). The infectivity titers of these different infections increased in a similar way (Fig. 3D). As a negative control, we performed infections of mouse liver slices with HCVcc supernatant (JFH-1 or Con1) in 2 CD81-deficient mice in the same conditions as those for human liver slices. We obtained negative results (from days 0 to 10) in any case.

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Figure 3. Production of HCV infectious particles (genotypes 2a, 1a, and 1b) in primary adult human liver slices (A-E), compared to primary human HCVcc JFH-1-infected hepatocytes (F). (A) Viral load (gray line) and infectivity (i.e, viral titer) (black line) of HCVpc JFH1 particles: *P < 0.05. (B) Viral load (gray line) and infectivity (i.e., viral titer) (black line) of HCVpc H77 particles: *P < 0.05. (C) Viral load (gray line) and infectivity (i.e., viral titer) (black line) of HCVpc Con1 particles: *P < 0.05. (D) Kinetics of infectivity titers (i.e., infectivity) of culture supernatants from human liver slices infected either by JFH-1 (gray line) or H77 (dark gray line) or Con1 (black line). Each curve represents the average of three independent infections from 3 different donors. Each kinetic was performed in triplicate. Values are expressed as means ± standard error of the means (SEMs). Results were compared using the two-paired Student t test: JFH-1 (gray line), *P < 0.014; H77 (dark gray line), *P < 0.04; Con1 (black line), *P < 0.02. (E) Infection of naïve liver slices with supernatants from HCV-infected human liver slices culture (HCVpc) clearly indicates the infectivity of extracellular viral particles, which are produced by HVCcc genotype 2a (JFH-1) (gray line), 1a (H77) (dark gray line), or 1b (Con1) (black line) infection. Values are expressed as means ± SEMs. Results were compared using the two-paired Student t test: JFH-1 (gray line), *P < 0.01; H77 (dark gray line), *P < 0.02; Con1 (black line), *P < 0.04. (F) Viral load (gray line; *P < 0.05) and infectivity (black line; *P < 0.03) of HCVpc JFH1 particles in primary human hepatocytes. Values are expressed as means ± SEMs. Results were compared using the two-paired Student t test.

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To provide further evidence that the new progeny virus produced by the human liver slices, called primary-culture–derived HCV (HCVpc), was indeed infectious, human naïve liver slices were infected de novo either with HCVpc JFH-1 or HCVpc H77 or HCVpc Con1 at MOI = 0.1. A productive infection of liver slices was obtained with higher infectivity titers at day 10, both for genotype 2a (120,000 ffu/mL), 1a (150,000 ffu/mL), and 1b (140,000 ffu/mL) (Fig. 3E).

Using the same methodology, human liver slices were infected with primary isolates obtained from 3 different patients chronically infected with HCV genotype 1b with a high viral load (>106 IU/mL) (Table 1) in three independent experiments. None of the patients had specific features, such as fulminant hepatitis. Human liver slices exposed to fresh patient sera showed evidence of intracellular replication of the HCV genome (Fig. 4A) as well as intracellular expression of viral core and NS3 proteins at days 5 and 10 (Fig. 4B). The size of core protein was smaller (∼18 kDa) than that obtained in HCVcc-infected Huh-7.5.1 cells. Infectious viral particles, produced in the culture supernatant (called HCVpc sera supernatants), reached a viral load and an infectivity titer up to ∼9.4 log10 IU/mL and 9 × 104 ffu/mL, respectively, at day 10 (Fig. 4C,D). De novo infection of naïve liver slices with HCVpc sera supernatants gave rise to an HCV production of infectious particles (HCVrepc) with higher infectivity titer (1.1 × 105 ffu/mL) by day 10 postinfection (Fig. 4E). The specific infectivity of the virus produced from the human liver slices (i.e., HCVpc and HCVrepc), compared to the HCVcc virus produced from the Huh-7.5.1 cell line, was higher, whatever the source of human liver slices and HCVcc stock that were used (Table 1). To investigate whether this result was related to the nature of the cells used for infection, naïve Huh-7.5.1 cells were infected with HCVpc. The specific infectivity of progeny virus recovered after 10 days of HCV reculture in Huh-7.5.1 cells (HCVrecc) was lower than that of HCVpc used as viral input and came back to that of HCVcc (Table 1). These results underline the critical role of the host cells used to determine the specific infectivity of the produced virus.

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Figure 4. Production of infectious viral particles by human liver slices infected with primary viral isolates from genotype 1b–infected HCV RNA–positive patient sera (n = 3). (A) Kinetic of HCV RNA replication, (B) expression of HCV core and NS3 proteins, and (C) viral load and infectivity of HCVpc (genotype 1b) particles. (D) Production of extracellular infectious viral particles (HCVpc) by human liver slices infected with genotype 1b–infected HCV RNA–positive patient sera, compared to HCVpc Con1-infected liver slices. (E) Infection of naïve liver slices with HCVpc from human liver slices infected with genotype 1b–infected HCV RNA–positive patient sera. (A) Human liver slices were lysed to evaluate intracellular levels of positive- and negative-strand HCV RNA by specific qRT-PCR25 at 1, 5, and 10 days postinfection. Values are expressed as means ± standard error of the means (SEMs). Results were compared using the two-paired Student t test. Positive and negative strand: *P < 0.05. Detection of a negative strand of HCV RNA evidences active replication as well as increase over time of both positive and negative strands of HCV RNA. (B) Western blotting analysis of human patient sera-infected liver slices for HCV core and NS3 proteins at days 1 (D1), 5 (D5), and 10 (D10) postinfection. Lysates of HCVcc Con1 chronically infected Huh7.5.1 cells (+) and naïve human liver slices (NI) served as positive and negative controls, respectively. β-actin was used as a loading control. Positions of molecular-weight markers are indicated in kDa. (C) Viral load (HCV RNA log10 IU/mL/mg tissue) (gray line; *P < 0.05) and infectivity titer of HCVpc from human liver slices infected by sera from HCV-RNA-positive genotype 1b–infected patients (black line; *P < 0.05). Each curve represents the average of three independent infections from three different liver samples with sera from 3 different patients. Each kinetic was performed in triplicate. Values are expressed as means ± SEMs. Results were compared using the two-paired Student t test. (D) Infectivity titer of HCVpc from human liver slices infected either by sera from HCV-RNA-positive genotype 1b–infected patients (gray line; *P < 0.05) or Con1 HCVcc (black line; *P < 0.01). (E) Infection of naïve liver slices with HCVpc sera-infected culture supernatants (day 10) (gray line; *P < 0.02) gives rise to a production of infectious extracellular viral particles in a similar range as HCVpc Con1 infection of naïve liver slices (black line; **P < 0.007). Values are expressed as means ± SEMs. Results were compared using the two-paired Student t test.

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Together, these data demonstrated that our ex vivo model is efficient to analyze HCV replication. Infection of liver slices with both culture-derived viral strains of different genotypes and primary viral isolates resulted in the release of high titers of infectious viruses.

Neutralization of HCV Infection Using HCV Patient Sera, Anti-CD81, and Anti-E2 Antibodies.

To further characterize the model, we assessed an antibody neutralization of HCV infection (Fig. 5A-C). The neutralizing activity of chronically infected patient sera has been reported in experiments using pseudotyped HCV virus and HCVcc infection of Huh-7.5 cells.27, 28 Here, we used serum obtained from a genotype 1b–infected patient who spontaneously resolved his HCV infection. This patient serum neutralized HCVcc Con1 infection of primary liver slices in a dose-dependent manner. At day 5, both infectivity titer and viral load were significantly decreased by 50%-95%, depending on the dilution of sera used (Fig. 5A). A 50% neutralization endpoint was obtained using a 1:1,400 dilution of patient serum. Previously, we checked that this human serum neutralized HCVcc Con1 infection of Huh-7.5.1 cells using the same methodology. Similar results were obtained at days 3 and 10 (data not shown). The specific neutralization of Con1 infectivity with positive human serum confirmed that the infectious particles produced by Con1-infected liver slices seemed genetically related to those produced during human infections with wild-type virus. There was no evidence of viral escape, because the neutralization of the infection was maintained in the same dose-dependent manner at day 10.

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Figure 5. Dose-dependent neutralization of HCVcc Con1 liver slices infection with neutralizing antibodies. HCV Con1 liver slices infection was neutralized either by (A) an HCV-positive patient serum that spontaneously resolved a genotype 1b acute HCV infection or (B) an antihuman CD81 mouse mAb (JS-81) or (C) an anti-HCV E2 protein mouse mAb (AP33). Results, obtained at day 5 postinfection, are representative of viral neutralization, over time (days 3 and 10; data not shown). Neutralization percentages were calculated by comparing infectivity titers (infectivity) in culture supernatants upon incubation with specific antibodies relative to respective control antibodies (CTRL serum: sera from healthy blood donors; CTRL IgG: mouse immunoglobulin G1 isotype). Viral load (log10 IU/mL) decreased in a dose-dependent manner. Data are expressed as means ± standard error of the means (SEMs) of at least three independent experiments, each realized in triplicate. Comparisons between infectivity neutralization or viral load were performed using Mann-Whitney's rank-sum test (*P < 0.05). Neutralization experiments of Con1 viruses, which clearly indicate that human neutralizing antibodies (A) or antihuman CD81 antibodies (B) or anti-HCV E2 antibodies (C) prevent infection of human liver slices in a dose-dependent manner. Infectivity of liver slices requires an active entry of virus into hepatocytes, enhancing intrahepatocyte replication of HCV.

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In addition, antibodies to CD81, an essential component of the HCV cellular entry into both Huh-7 cells and primary human hepatocytes12, 29 and to E2 protein (AP33)30 neutralized the viral replication and infectious particle production in culture supernatants in a dose-dependent manner at day 5 (Fig. 5B,C). A 10-μg/mL concentration of anti-CD81 reduced 50% of liver slices infection, whereas it almost completely abolished the infection of Huh-7.5.1 cells (not shown), in agreement with previous reports.7, 10 Similar results were obtained with antibodies against E2 protein (10 μg/mL) (Fig. 5C).

Other neutralization assays gave the same results at days 3 and 10 (data not shown), showing that (1) neutralization took place at the initiation of the viral infection, (2) the viral infection of liver slices was CD81 dependent, and (3) there was no viral escape mutants competent to evade neutralizing antibodies.

Drug Inhibition Assay of HCV Infectious Particle Production.

To validate our model, antiviral therapies (i.e., Peg-IFN-α2a ± ribavirin [RBV] or telaprevir [VX-950], a peptidomimetic of HCV serine protease31) were tested on the de novo viral production of HCVcc Con1-infected liver slices (Fig. 6A,E). The INF/RBV doses tested were adapted to our model according the standard doses in humans. At day 4 postinfection, we treated HCVcc Con1-infected liver slices either with different doses of Peg-INF-α2a ± 100 μM of RBV or telaprevir for 6 days. At day 4 post-treatment, the production of infectious particles (i.e., infectivity) was inhibited in a dose-dependent manner (75% up to 95%, Fig. 6B; 40% up to 85%, Fig. 6E), confirming the occurrence of de novo infectious particles. At day 6 post-treatment, the infectivity inhibition reached either from 95% up to 98% (Fig. 6B) or 61% up to 95% (Fig. 6E). No cytotoxic effects were observed in human liver slices by lactate dehydrogenase (LDH) leakage assay (Fig. 6C,D,F).

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Figure 6. Dose-dependent inhibition of HCVpc production by antiviral drugs in HCVcc Con1-infected liver slices. Human liver slices were inoculated with HCVcc Con1 supernatant (MOI = 0.1) cultured for 10 days. At day 4 postinfection, Peg-IFN-α2a ± RBV (100 μM) or telaprevir (VX-950) were added in the culture medium (black arrow) up to day 10. (A) Representative dose-dependent decrease of infectivity titers released from primary human liver slices infected with HCVcc Con1 viral stock (MOI = 0.1) and treated with increasing amounts of Peg-INF ± RBV (100 μM) for 6 days post-treatment. Primary human liver slices infected with HCVcc Con1 viral stock (MOI = 0.1) and treated with 0.5% dimethyl sulfoxide (DMSO) for 6 days served as the carrier control. Infectivity titers are expressed as ffu/mL/mg tissue. Values are expressed as means ± standard error of the means (SEMs) of at least two independent experiments realized in triplicate each, using the two-paired Student t test. Con1 (dark blue line): ***P < 0.0001; Con1 + Peg-INF (26 nM; red line): *P < 0.05; Con1 + Peg-INF (26 nM) + RBV (100 μM; green line): *P < 0.05; Con1 + RBV (100 μM; black line): ***P < 0.0005; Con1 + Peg-INF (2.6 nM) + RBV (100 μM; pink line): ***P < 0.0002; Con1 + Peg-INF (50 nM) + RBV (100 μM; orange line): *P < 0.05; Con1 + Peg-INF (260 nM) + RBV (100 μM; blue line): *P < 0.05. (B and E) Inhibition of infectivity is expressed as the percentage of the DMSO control. Values are expressed as means ± SEMs of at least two independent experiments, each realized in triplicate. Comparisons were performed using Mann-Whitney's rank-sum test ([B]: *P < 0.03; [E]: *P < 0.02]. (C, D, F) No significant cytotoxic effect of drug treatments in HCVcc Con1-infected liver slices at day 6 post-treatment (day 10 postinfection). Percentages of LDH leakage are relative to DMSO control-treated liver slices (CTRL). All experiments were performed in triplicate. Values are expressed as means ± SEMs. Comparisons were performed using Mann-Whitney's rank-sum test ([C and D): *P < 0.04; [F]: **P < 0.001).

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Discussion

  1. Top of page
  2. Abstract
  3. Materials and Methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. References
  8. Supporting Information

Research on HCV liver infection was seriously retarded because of the absence of an adequate ex vivo model. By all means, the existing “flat” cellular models (i.e., Huh7.5.1, etc.) are incapable of reflecting a full spectrum of events, because sophisticated “three-dimensional (3D) interactions” between different cell types in the liver are destroyed. As a consequence, results obtained on cell lines and on primary cells might suffer from simplification, whereas investigation of the infection and important elements associated with liver infection might simply be missed or wrongly interpreted.

To overcome, at least in part, this problem, we have established a new HCV infection ex vivo model using primary culture of adult human liver slices and provided evidence that the model is feasible and might significantly better reflect the in vivo situation.

We have shown that liver slices infection is possible with HCVcc (e.g., JFH-1, H77/JFH-1, or Con1/JFH-1 chimera) and, what is of special importance, with HCV (genotype 1b) from sera of infected individuals. We described the intracellular replication of HCV RNA and the intracellular expression of HCV proteins as well as the efficient release of HCV infectious particles (e.g., HCVpc) from infected human liver slices. Overall, specific HCVpc infectivity was higher than that of HCVcc particles. Finally, we demonstrated the potential neutralization of HCV infection either by the serum of an HCV-infected patient (spontaneously recovered) or by anti-CD81 or anti-E2 antibodies, as well as the inhibition of HCV replication by antiviral drugs, in a dose-dependent manner.

Compared to models established earlier, such as cell lines and primary hepatocytes, liver slices culture possesses several evident advantages. First, they maintained the original 3D structure of the liver that allows cell cross-talk: the extracellular matrix and Kupffer cells essential for the normal function of hepatocytes and lobular structure. Second, the gene-expression profiles in liver tissue slices (Fig. 1B) were similar to that of in vivo gene expression.32 Third, hepatocyte preparations undergo treatment with collagenase, but liver slices do not. In fact, this treatment might have a negative effect on the integrity of the proteins' repertoire on the cell urface. Of note, using established procedures, the tissue slices remained viable for at least 10 days, as was shown by the secretion of albumin and urea (Fig. 1C,D). However, this secretion is lower than that for the model of micropatterned hepatocyte cocultures.33

Interestingly, we observed some differences between the sizes of core protein in HCV-infected liver slices and the size of core protein in HCVcc-infected Huh-7.5.1 cells (Figs. 2B and 4B). The difference may reflect a different processing of core protein in liver slices culture, compared to Huh-7.5.1 cells (Fig. 2) and/or viral genome mutations, resulting in slightly different amino-acid sequences. It is worth mentioning that the sodium dodecyl sulfate polyacrylamide gel electrophoresis migration of proteins from primary hepatocytes and liver slices culture lysates from HCVcc infection gave a similar pattern of core protein. The core proteins from patient sera liver slices infection had a smaller size, compared to that in Huh-7.5.1 cells. This difference might be a result of the minor alteration between the core proteins from the “patient viruses” and prototypes, such as the JFH-1, H77, or Con1 strains. Both short internal deletions in the core protein sequence of primary isolates and variations in phosphorylation (e.g., serine 53 and serine 116), resulting in a different migration profile as was observed previously,34 might have taken place. Thus, sequencing of primary isolates is required to answer this question. In any case, the colocalization of core proteins with lipid droplets (Fig. 2A) and the production of infectious viral particles (Figs. 3D,E and 4D,E) confirmed the complete maturation of core proteins.25, 26

We demonstrated the ability of HCVcc (i.e., JFH-1, H77/JFH-1, or Con1/JFH-1 chimera, although these viral particles are adapted to Huh-7.5 cells) as well as primary viral isolates (genotype 1b) from patient sera to complete the viral replication cycle in human liver slices. Thus, it represents another important step in the development of a liver cell culture model of HCV infection. We have shown that the intracellular replication of HCV RNA and expression of viral proteins could be obtained as well as extracellular production of infective particles (i.e., HCVpc). The infectious viral particles (i.e., HCVpc) produced by the human liver slices were indeed infectious with higher infectivity than that of HCVcc. This higher specific infectivity of HCVpc, compared to HCVcc, reminds one of the reported gain in infectivity of particles produced in the circulating blood of animals infected with HCVcc.35 One challenging explanation may be that highly infectious particles form complexes with lipoproteins and are released from differentiated hepatocytes of liver slices, which retain their competence for very-low-density lipoprotein (VLDL) assembly and secretion. After reculture in Huh-7.5.1 cells, this pattern is lost because the physiological pathways of assembly and maturation of triglyceride-rich VLDL in these transformed cells do not process quite correctly.12, 35

We succeeded at infecting human liver slices with primary isolates from sera of HCV-infected patients. Of note, infected liver slices released infectious particles at the same order of magnitude as HCVcc JFH-1, H77, or Con1. However, the infection of liver slices was not observed if previously frozen HCV-positive sera were used for infection (data not shown). In this regard, we speculate that during the freeze-thawing procedure, lipid rafts on the surface of HCV particles might be disturbed and the recognition of the cellular receptors by E1 and E2 proteins might be significantly reduced.36 It is worth pointing out that using in vitro cellular models (e.g., Huh-7.5 cells) of infection with HCV-positive sera was not obtained. This discrepancy might be attributed to dedifferentiation and/or abnormalities in the physiological pathways of assembly and maturation of triglyceride-rich VLDL.12, 37, 38 The fact that primary human hepatocytes infected with HCV-positive sera released infectious viruses with low-infectivity titer10 could also highlight the important role played by satellite cells in liver infection by primary viral isolates.13, 39 A priori primary human hepatocyte cultures might provide better information on virus infection and propagation, compared to established cell lines. However, primary cells might lack a particular cellular repertoire (i.e., environment), modified cellular interactions, and abrogated signaling cross-talk. At least in part, all these elements and interactions are preserved in liver slices. Taken together, that allows one to examine virus infection precisely and reveal numerous, important details.

Finally, our data either with IFN ± RBV, the basic anti-HCV drug, or telaprevir, a specific HCV protease inhibitor, provide a proof of concept that a proposed ex vivo model can be used to evaluate antiviral activity of new drugs targeting the virus life cycle.40

In conclusion, the established ex vivo model of human liver slices infection with HCV allows one to perform detailed analysis of HCV infection and virus morphogenesis. In addition, the model is suitable for the evaluation of new specific drugs and inhibitors.

Acknowledgements

  1. Top of page
  2. Abstract
  3. Materials and Methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. References
  8. Supporting Information

The authors are grateful to Drs. Francis V. Chisari and Arvind H. Patel for Huh-7.5.1 cells and anti-HCV E2 antibody, respectively, to Drs. Takaji Wakita and Ralf Bartenschlager for kindly providing JFH-1 plasmid and chimeric pFK-H77/C3 and pFK-Con1/C3 plasmids, respectively. The authors acknowledge members of the Department of Digestive Surgery (La Pitié Salpétrière and Cochin-Hôtel Dieu) and the Department of Hepatology in Groupe Hospitalier (Cochin-Hôtel Dieu, AP-HP, Paris, France) for their valuable assistance. The authors are grateful to Dr. Emmanuel Donnadieu for sharing with us the technique for liver slicing. The authors are deeply indebted to Drs. Matthew Albert, Vladimir A. Morozov, and Charles M. Rice for their critical reading of the manuscript for this article.

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  4. Results
  5. Discussion
  6. Acknowledgements
  7. References
  8. Supporting Information
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Supporting Information

  1. Top of page
  2. Abstract
  3. Materials and Methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. References
  8. Supporting Information

Additional Supporting Information may be found in the online version of this article.

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HEP_25738_sm_SuppFig1.tif323KSupporting Information Figure 1.
HEP_25738_sm_SuppInfo.doc89KSupporting Information
HEP_25738_sm_SuppTab1.tif273KSupporting Information Table 1.

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