Hepatitis C Virus Continuously Escapes From Neutralizing Antibody and T-Cell Responses During Chronic Infection In Vivo
2006; Elsevier BV; Volume: 132; Issue: 2 Linguagem: Inglês
10.1053/j.gastro.2006.12.008
ISSN1528-0012
AutoresThomas von Hahn, Joo Chun Yoon, Harvey J. Alter, Charles M. Rice, Barbara Rehermann, Peter Balfe, Jane A. McKeating,
Tópico(s)HIV Research and Treatment
ResumoBackground & Aims: Broadly reactive neutralizing antibodies (nAbs) and multispecific T-cell responses are generated during chronic hepatitis C virus (HCV) infection and yet fail to clear the virus. This study investigated the development of autologous nAb and HCV-glycoprotein–specific T-cell responses and their effects on viral sequence evolution during chronic infection in order to understand the reasons for their lack of effectiveness.Methods: Numerous E1E2 sequences were amplified and sequenced from serum samples collected over a 26-year period from patient H, a uniquely well-characterized, chronically infected individual. HCV pseudoparticles (HCVpp) expressing the patient-derived glycoproteins were generated and tested for their sensitivity to neutralization by autologous and heterologous serum antibodies.Results: A strain-specific nAb response developed early in infection (8 weeks postinfection), whereas cross-reactive antibodies able to neutralize HCVpp-bearing heterologous glycoproteins developed late in infection (>33 wk postinfection). The humoral response continuously failed to neutralize viruses bearing autologous glycoprotein sequences that were present in the serum at a given time. The amplified glycoprotein sequences displayed high variability, particularly in regions corresponding to defined linear B-cell epitopes. Mutations in defined neutralizing epitopes were associated with a loss of recognition by monoclonal antibodies against these epitopes and with decreased neutralization of corresponding HCVpp. Viral escape from CD4 and CD8 T-cell responses also was shown for several novel epitopes throughout the glycoprotein region.Conclusions: During chronic infection HCV is subjected to selection pressures from both humoral and cellular immunity, resulting in the continuous generation of escape variants. Background & Aims: Broadly reactive neutralizing antibodies (nAbs) and multispecific T-cell responses are generated during chronic hepatitis C virus (HCV) infection and yet fail to clear the virus. This study investigated the development of autologous nAb and HCV-glycoprotein–specific T-cell responses and their effects on viral sequence evolution during chronic infection in order to understand the reasons for their lack of effectiveness. Methods: Numerous E1E2 sequences were amplified and sequenced from serum samples collected over a 26-year period from patient H, a uniquely well-characterized, chronically infected individual. HCV pseudoparticles (HCVpp) expressing the patient-derived glycoproteins were generated and tested for their sensitivity to neutralization by autologous and heterologous serum antibodies. Results: A strain-specific nAb response developed early in infection (8 weeks postinfection), whereas cross-reactive antibodies able to neutralize HCVpp-bearing heterologous glycoproteins developed late in infection (>33 wk postinfection). The humoral response continuously failed to neutralize viruses bearing autologous glycoprotein sequences that were present in the serum at a given time. The amplified glycoprotein sequences displayed high variability, particularly in regions corresponding to defined linear B-cell epitopes. Mutations in defined neutralizing epitopes were associated with a loss of recognition by monoclonal antibodies against these epitopes and with decreased neutralization of corresponding HCVpp. Viral escape from CD4 and CD8 T-cell responses also was shown for several novel epitopes throughout the glycoprotein region. Conclusions: During chronic infection HCV is subjected to selection pressures from both humoral and cellular immunity, resulting in the continuous generation of escape variants. See editorial on page 801. See editorial on page 801. Hepatitis C virus (HCV) is an important human pathogen infecting about 170 million people worldwide. In the United States, it is the single most common cause of chronic liver disease requiring liver transplantation.1Alter M. Hepatitis C virus infection in the United States.J Hepatol. 1999; 31: 88-91Abstract Full Text PDF PubMed Google Scholar, 2Alter M. Margolis H. Krawczynski K. Judson F. Mares A. Alexander W. Hu P. Miller J. Gerber M. Sampliner R. et al.The Sentinel Counties Chronic non-A, non-B Hepatitis Study TeamThe natural history of community-acquired hepatitis C in the United States.N Engl J Med. 1992; 327: 1899-1905Crossref PubMed Scopus (1646) Google Scholar Cellular and humoral responses are generated during acute infection, however, they are insufficient to achieve viral clearance in the majority of individuals, with approximately 60%–80% of new infections becoming persistent. In vivo, HCV replicates to high levels using an error-prone viral RNA polymerase, which leads to a spectrum of related but distinct sequences within infected individuals, often referred to as a quasispecies.3Martell M. Esteban J. Quer J. Genesca J. Weiner A. Esteban R. Guardia J. Gomez J. Hepatitis C virus (HCV) circulates as a population of different but closely related genomes: quasispecies nature of HCV genome distribution.J Virol. 1992; 66: 3225-3229Crossref PubMed Google Scholar The immune system is thought to exert unequal selective pressure on variants within the circulation, favoring the rapid emergence of escape mutants.4Brown R. Juttla V. Tarr A. Finnis R. Irving W. Hemsley S. Flower D. Borrow P. Ball J. Evolutionary dynamics of hepatitis C virus envelope genes during chronic infection.J Gen Virol. 2005; 86: 1931-1942Crossref PubMed Scopus (30) Google Scholar Escape from CD8 T-cell responses by mutation is well documented and an important predictor of progression to chronic HCV infection.5Weiner A. Erickson A. Kansopon J. Crawford K. Muchmore E. Hughes A. Houghton M. Walker C. Persistent hepatitis C virus infection in a chimpanzee is associated with emergence of a cytotoxic T lymphocyte escape variant.Proc Natl Acad Sci U S A. 1995; 92: 2755-2759Crossref PubMed Scopus (312) Google Scholar, 6Chang K. Rehermann B. McHutchison J. Pasquinelli C. Southwood S. Sette A. Chisari F. Immunological significance of cytotoxic T lymphocyte epitope variants in patients chronically infected by the hepatitis C virus.J Clin Invest. 1997; 100: 2376-2385Crossref PubMed Scopus (299) Google Scholar, 7Erickson A. Kimura Y. Igarashi S. Eichelberger J. Houghton M. Sidney J. McKinney D. Sette A. Hughes A. Walker C. The outcome of hepatitis C virus infection is predicted by escape mutations in epitopes targeted by cytotoxic T lymphocytes.Immunity. 2001; 15: 883-895Abstract Full Text Full Text PDF PubMed Scopus (359) Google Scholar, 8Seifert U. Liermann H. Racanelli V. Halenius A. Wiese M. Wedemeyer H. Ruppert T. Rispeter K. Henklein P. Sijts A. Hengel H. Kloetzel P.M. Rehermann B. Hepatitis C virus mutation affects proteasomal epitope processing.J Clin Invest. 2004; 114: 250-259Crossref PubMed Scopus (129) Google Scholar In keeping with this, a vigorous and broad CD8 T-cell response during the acute phase of infection is associated with viral clearance.9Cooper S. Erickson A. Adams E. Kansopon J. Weiner A. Chien D. Houghton M. Parham P. Walker C. Analysis of a successful immune response against hepatitis C virus.Immunity. 1999; 10: 439-449Abstract Full Text Full Text PDF PubMed Scopus (728) Google Scholar, 10Lechner F. Wong D. Dunbar P. Chapman R. Chung R. Dohrenwend P. Robbins G. Phillips R. Klenerman P. Walker B. Analysis of successful immune responses in persons infected with hepatitis C virus.J Exp Med. 2000; 191: 1499-1512Crossref PubMed Scopus (1132) Google Scholar, 11Thimme R. Oldach D. Chang K. Steiger C. Ray S. Chisari F. Determinants of viral clearance and persistence during acute hepatitis C virus infection.J Exp Med. 2001; 194: 1395-1406Crossref PubMed Scopus (1038) Google Scholar Studies of acute HCV infection in chimpanzees previously exposed to the virus provide compelling evidence that protective CD8 T-cell–mediated immunity exists.12Shoukry N.H. Grakoui A. Houghton M. Chien D.Y. Ghrayeb J. Reimann K.A. Walker C.M. Memory CD8+ T cells are required for protection from persistent hepatitis C virus infection.J Exp Med. 2003; 197: 1645-1655Crossref PubMed Scopus (546) Google Scholar CD4 T cells also are required for control of HCV on re-exposure, but their role is less well defined.13Grakoui A. Shoukry N.H. Woollard D.J. Han J.H. Hanson H.L. Ghrayeb J. Murthy K.K. Rice C.M. Walker C.M. HCV persistence and immune evasion in the absence of memory T cell help.Science. 2003; 302: 659-662Crossref PubMed Scopus (708) Google Scholar Even less is known about the impact of the humoral immune response on HCV pathobiology. Without the ability to culture HCV, there was, until recently, no simple in vitro method to evaluate viral escape from the antibody-mediated immune response. The development of HCV glycoprotein-bearing retroviral pseudoparticles (HCVpp) has made it possible to assess antibody-dependent neutralization of HCV entry.14Hsu M. Zhang J. Flint M. Logvinoff C. Cheng-Mayer C. Rice C. McKeating J. Hepatitis C virus glycoproteins mediate pH-dependent cell entry of pseudotyped retroviral particles.Proc Natl Acad Sci U S A. 2003; 100: 7271-7276Crossref PubMed Scopus (698) Google Scholar, 15Bartosch B. Bukh J. Meunier J. Granier C. Engle R. Blackwelder W. Emerson S. Cosset F. Purcell R. In vitro assay for neutralizing antibody to hepatitis C virus: evidence for broadly conserved neutralization epitopes.Proc Natl Acad Sci U S A. 2003; 100: 14199-14204Crossref PubMed Scopus (289) Google Scholar, 16Drummer H.E. Maerz A. Poumbourios P. Cell surface expression of functional hepatitis C virus E1 and E2 glycoproteins.FEBS Lett. 2003; 546: 385-390Abstract Full Text Full Text PDF PubMed Scopus (171) Google Scholar, 17Logvinoff C. Major M. Oldach D. Heyward S. Talal A. Balfe P. Feinstone S. Alter H. Rice C. McKeating J. Neutralizing antibody response during acute and chronic hepatitis C virus infection.Proc Natl Acad Sci U S A. 2004; 101: 10149-10154Crossref PubMed Scopus (357) Google Scholar, 18McKeating J. Zhang L. Logvinoff C. Flint M. Zhang J. Yu J. Butera D. Ho D. Dustin L. Rice C. Balfe P. Diverse hepatitis C virus glycoproteins mediate viral infection in a CD81-dependent manner.J Virol. 2004; 78: 8496-8505Crossref PubMed Scopus (141) Google Scholar Neutralizing antibody (nAb) responses often provide the first-line adaptive defense against infection by limiting virus spread. Serum antibodies from chronically HCV-infected individuals show broadly reactive neutralizing properties and yet fail to clear viral infection.15Bartosch B. Bukh J. Meunier J. Granier C. Engle R. Blackwelder W. Emerson S. Cosset F. Purcell R. In vitro assay for neutralizing antibody to hepatitis C virus: evidence for broadly conserved neutralization epitopes.Proc Natl Acad Sci U S A. 2003; 100: 14199-14204Crossref PubMed Scopus (289) Google Scholar, 17Logvinoff C. Major M. Oldach D. Heyward S. Talal A. Balfe P. Feinstone S. Alter H. Rice C. McKeating J. Neutralizing antibody response during acute and chronic hepatitis C virus infection.Proc Natl Acad Sci U S A. 2004; 101: 10149-10154Crossref PubMed Scopus (357) Google Scholar, 19Meunier J.C. Engle R.E. Faulk K. Zhao M. Bartosch B. Alter H. Emerson S.U. Cosset F.L. Purcell R.H. Bukh J. Evidence for cross-genotype neutralization of hepatitis C virus pseudo-particles and enhancement of infectivity by apolipoprotein C1.Proc Natl Acad Sci U S A. 2005; 102: 4560-4565Crossref PubMed Scopus (229) Google Scholar The reasons for their lack of effect are understood poorly, however, one possible explanation is that Ab response(s) are less able to neutralize autologous glycoprotein species circulating within an individual at the time of sampling. In setting out to address these questions we were fortunate to have access to sequential serum samples from patient H, an individual who was infected with HCV in 1977 and has been meticulously followed-up since then. Moreover, patient H was the source of the prototype HCV strains H and H77, and thus a wide range of tailor-made reagents are available for virologic and immunologic analyses.20Yanagi M. Purcell R. Emerson S. Bukh J. Transcripts from a single full-length cDNA clone of hepatitis C virus are infectious when directly transfected into the liver of a chimpanzee.Proc Natl Acad Sci U S A. 1997; 94: 8738-8743Crossref PubMed Scopus (467) Google Scholar, 21Kolykhalov A. Agapov E. Blight K. Mihalik K. Feinstone S. Rice C. Transmission of hepatitis C by intrahepatic inoculation with transcribed RNA.Science. 1997; 277: 570-574Crossref PubMed Scopus (637) Google Scholar, 22Ogata N. Alter H. Miller R. Purcell R. Nucleotide sequence and mutation rate of the H strain of hepatitis C virus.Proc Natl Acad Sci U S A. 1991; 88: 3392-3396Crossref PubMed Scopus (534) Google Scholar For these reasons this patient is a unique subject for studies into the immunologic history of chronic HCV infection. To assess the impact of the HCV quasispecies on the nAb response we generated HCVpp-bearing glycoprotein variants cloned from sequential samples from chronically infected patient H. By using a series of samples obtained between 3 weeks postinfection and throughout the 26 years thereafter, we sought to investigate the process of antigenic escape in the viral glycoproteins from humoral and cellular immune surveillance. We found compelling evidence for repeated mutational change resulting in loss of recognition of the HCV glycoprotein by the cognate antibody response and escape from antibody-mediated neutralization. Similarly, mapping of T-cell responses to the E1E2 region identified 4 novel T-cell epitopes in which mutations occurred, leading to escape from CD4 and CD8 T-cell recognition. Both 293T and Hep3B cells were propagated in Dulbecco’s modified Eagle medium with 10% fetal bovine serum. Antibodies against HCV E2 have been described previously.14Hsu M. Zhang J. Flint M. Logvinoff C. Cheng-Mayer C. Rice C. McKeating J. Hepatitis C virus glycoproteins mediate pH-dependent cell entry of pseudotyped retroviral particles.Proc Natl Acad Sci U S A. 2003; 100: 7271-7276Crossref PubMed Scopus (698) Google Scholar, 23Flint M. Maidens C. Loomis-Price L.D. Shotton C. Dubuisson J. Monk P. Higginbottom A. Levy S. McKeating J.A. Characterization of hepatitis C virus E2 glycoprotein interaction with a putative cellular receptor, CD81.J Virol. 1999; 73: 6235-6244Crossref PubMed Google Scholar To generate soluble CD81 an expression plasmid encoding the CD81 large extracellular loop fused to Glutathione S-transferase (GST) was transformed into Rosetta-gami Escherichia coli (Novagen, La Jolla, CA). Fusion proteins were prepared by lysis with an Avestin (Avestin, Ottawa, Canada) air emulsifier (3 passages at 15,000 psi) and subsequent centrifugation (25,000 × g for 30 min at 4°C). Cleared lysates were purified over a GSTrap FF affinity column according to the manufacturer’s instructions (Amersham Biosciences, Piscataway, NJ). For the initial screening of T-cell responses against the HCV E1 and E2 proteins, 15-mer peptides (total, 111) (Mimotopes, Clayton, Australia), overlapping by 10 amino acids and covering the complete HCV H77 (genotype 1a) E1E2 sequence, were resuspended at 20 mg/mL in dimethyl sulfoxide and further diluted with phosphate-buffered saline (PBS) solution to obtain a final concentration of 1 μg/mL. For direct comparison of responses against the mutant epitopes, the corresponding mutant peptides were synthesized at Rockefeller University (New York, NY) to more than 95% purity. The plasmid encoding H77 E1E2 has been described previously.14Hsu M. Zhang J. Flint M. Logvinoff C. Cheng-Mayer C. Rice C. McKeating J. Hepatitis C virus glycoproteins mediate pH-dependent cell entry of pseudotyped retroviral particles.Proc Natl Acad Sci U S A. 2003; 100: 7271-7276Crossref PubMed Scopus (698) Google Scholar, 21Kolykhalov A. Agapov E. Blight K. Mihalik K. Feinstone S. Rice C. Transmission of hepatitis C by intrahepatic inoculation with transcribed RNA.Science. 1997; 277: 570-574Crossref PubMed Scopus (637) Google Scholar, 24Flint M. Logvinoff C. Rice C. McKeating J. Characterization of infectious retroviral pseudotype particles bearing hepatitis C virus glycoproteins.J Virol. 2004; 78: 6875-6882Crossref PubMed Scopus (81) Google Scholar Total RNA was prepared from HCV-infected plasma using commercial kits (Qiagen, Valencia, CA).25Hammond A. Lewis J. May J. Albert J. Balfe P. McKeating J. Antigenic variation within the CD4 binding site of human immunodeficiency virus type 1 gp120: effects on chemokine receptor utilization.J Virol. 2001; 75: 5593-5603Crossref PubMed Scopus (8) Google Scholar Briefly, complementary DNA (cDNA) was synthesized in a reaction volume of 20 μL, containing 2–5 μL of template RNA, 2.5 U of Multiscribe MMuLV reverse transcriptase with 400 μmol/L each of the 4 deoxynucleoside triphosphates and 200 nmol/L of the antisense primer p7-2710 (AGC AGG AGG AGN GGC CAY ATC CCR TAG A, Y = C/T mixture, R = A/G, N = A/G/C/T) in the manufacturer’s recommended buffer (N808-0234; ABI, Foster City, CA) for 2 hours at 42°C. This cDNA was used as the template for polymerase chain reaction (PCR) amplification of the E1E2 region as previously described.24Flint M. Logvinoff C. Rice C. McKeating J. Characterization of infectious retroviral pseudotype particles bearing hepatitis C virus glycoproteins.J Virol. 2004; 78: 6875-6882Crossref PubMed Scopus (81) Google Scholar Briefly, a 50-μL PCR was set up containing 2.5 μL of cDNA, 2.5 U of the proofreading Expand polymerase mixture (1 681 834; Roche, Mannheim, Germany) in 1× Expand buffer 3 (2.25 mmol/L Mg2+), with 400 μmol/L each of the 4 deoxynucleoside triphosphates and 200 nmol/L each of the primers core+813 (GAG GAC GGY RTR AAY TAY GCA ACA GG; sense) and p7-2710. The PCR consisted of 30 cycles at 92°C for 45 seconds, 45°C for 45 seconds, and 68°C for 300 seconds, and was performed in an Eppendorf (Westbury, NY) thermal cycler. A total of 2 μL of the completed reaction was used as template for a second amplification, containing the same reaction components as described previously with 200 nmol/L of the primers core+843 (CACC ATG GGT TGC TCT TTC TCT ATC TT; sense) and E2-2580H (CTA CTA CGC CTC CGC TTG GGA TAT GAG TAA CAT CAT CCA, antisense). This second round of PCR comprised 25 cycles at 92°C for 35 seconds, 55°C for 35 seconds, and 68°C for 150 seconds. In those cases in which the input RNA was more than 2000 viral copies, full-length E1E2 was amplified readily. PCR products were cloned into pcDNA3.1D-TOPO (K4900-01; Invitrogen, Carlsbad, CA) and the sense and antisense strands were sequenced (Big Dye 3.1 Terminator Kit; ABI). All sequences were deposited with Genbank and have the accession numbers DQ897773–DQ897818. Several sequences for the E1E2 region of HCV within this patient have been deposited in Genbank previously. For comparison with the new sequences described here we included the following sequences from 1977 in our analyses: H77C (AF01175120Yanagi M. Purcell R. Emerson S. Bukh J. Transcripts from a single full-length cDNA clone of hepatitis C virus are infectious when directly transfected into the liver of a chimpanzee.Proc Natl Acad Sci U S A. 1997; 94: 8738-8743Crossref PubMed Scopus (467) Google Scholar), HPCST77 (M6238122Ogata N. Alter H. Miller R. Purcell R. Nucleotide sequence and mutation rate of the H strain of hepatitis C virus.Proc Natl Acad Sci U S A. 1991; 88: 3392-3396Crossref PubMed Scopus (534) Google Scholar), H77IMC (AF00960621Kolykhalov A. Agapov E. Blight K. Mihalik K. Feinstone S. Rice C. Transmission of hepatitis C by intrahepatic inoculation with transcribed RNA.Science. 1997; 277: 570-574Crossref PubMed Scopus (637) Google Scholar), H21 (AF01175320Yanagi M. Purcell R. Emerson S. Bukh J. Transcripts from a single full-length cDNA clone of hepatitis C virus are infectious when directly transfected into the liver of a chimpanzee.Proc Natl Acad Sci U S A. 1997; 94: 8738-8743Crossref PubMed Scopus (467) Google Scholar), H (M6746326Inchauspe G. Zebedee S. Lee D. Sugitani M. Nasoff M. Prince A. Genomic structure of the human prototype strain H of hepatitis C virus: comparison with American and Japanese isolates.Proc Natl Acad Sci U S A. 1991; 88: 10292-10296Crossref PubMed Scopus (251) Google Scholar), and H11 (AF01175220Yanagi M. Purcell R. Emerson S. Bukh J. Transcripts from a single full-length cDNA clone of hepatitis C virus are infectious when directly transfected into the liver of a chimpanzee.Proc Natl Acad Sci U S A. 1997; 94: 8738-8743Crossref PubMed Scopus (467) Google Scholar). An additional clone, H90, obtained in 1990, was available and was included in the analysis (M6238222Ogata N. Alter H. Miller R. Purcell R. Nucleotide sequence and mutation rate of the H strain of hepatitis C virus.Proc Natl Acad Sci U S A. 1991; 88: 3392-3396Crossref PubMed Scopus (534) Google Scholar). In addition to the sequences recorded in Genbank, several sequences for the E2 hypervariable region (HVR) were reported by Ogata et al,22Ogata N. Alter H. Miller R. Purcell R. Nucleotide sequence and mutation rate of the H strain of hepatitis C virus.Proc Natl Acad Sci U S A. 1991; 88: 3392-3396Crossref PubMed Scopus (534) Google Scholar and several matched the HVR sequences of clones obtained in this study (data not shown). The nucleotide sequences were aligned and translated using the SeAL2.0 program (A. Rambaut, Oxford University, available at: http://evolve.zoo.ox.ac.uk). Synonymous and nonsynonymous distances were estimated using the Nei and Gojobori27Nei M. Gojobori T. Simple methods for estimating the numbers of synonymous and nonsynonymous nucleotide substitutions.Mol Biol Evol. 1986; 3: 418-426PubMed Google Scholar method implemented in the PAML3.14 program.28Yang Z. Maximum likelihood estimation on large phylogenies and analysis of adaptive evolution in human influenza virus A.J Mol Evol. 2000; 51: 423-432Crossref PubMed Scopus (183) Google Scholar Phylogenetic analyses were performed using the PAUP 4.0 software package29Rogers J. Swofford D. A fast method for approximating maximum likelihoods of phylogenetic trees from nucleotide sequences.Syst Biol. 1998; 47: 77-89Crossref PubMed Scopus (155) Google Scholar using a modified HKY85 evolutionary model for the data, selected by hierarchic likelihood testing (program Modeltest 3.630Posada D. Crandall K. MODELTEST: testing the model of DNA substitution.Bioinformatics. 1998; 14: 817-818Crossref PubMed Scopus (18327) Google Scholar), the transition/transversion ratio (7.04), proportion of invariable sites (.46), and gamma distribution shape parameter (α) for variable sites (.86) were estimated by maximum likelihood methods; distance estimates were averaged within and between groups using Excel 2001 (Microsoft, Redmond, WA). Pseudoparticles were generated by transfection of 293T cells with pNL4-3.Luc.R−E− plasmid containing the env-defective proviral genome and an expression plasmid encoding the HCV glycoproteins (gps), as previously described.14Hsu M. Zhang J. Flint M. Logvinoff C. Cheng-Mayer C. Rice C. McKeating J. Hepatitis C virus glycoproteins mediate pH-dependent cell entry of pseudotyped retroviral particles.Proc Natl Acad Sci U S A. 2003; 100: 7271-7276Crossref PubMed Scopus (698) Google Scholar The virus containing extracellular media was collected 48 and 72 hours after transfection. Heat-inactivated sera, monoclonal antibodies (MAbs), or soluble CD81 were incubated with virus at their appropriate dilution in 3% fetal bovine serum/Dulbecco’s modified Eagle medium plus 4 μg/mL polybrene at 37°C for 1 hour. The virus-Ab mixture was transferred to Hep3B cells seeded in 96-well plates (8 × 103 cells/well) and infections were centrifuged at 400g for 1 hour, incubated at 37°C for 6 hours, and unbound virus was removed and incubated for a total of 72 hours. Cells were lysed with cell lysis buffer (Promega, Madison, WI) and tested for luciferase activity as previously described.14Hsu M. Zhang J. Flint M. Logvinoff C. Cheng-Mayer C. Rice C. McKeating J. Hepatitis C virus glycoproteins mediate pH-dependent cell entry of pseudotyped retroviral particles.Proc Natl Acad Sci U S A. 2003; 100: 7271-7276Crossref PubMed Scopus (698) Google Scholar The percentage neutralization was determined by comparing pseudoparticle infectivity (luciferase relative light units) in the presence of a test serum or MAb with infection in the presence of a control HCV-negative serum or an irrelevant isotype-matched immunoglobulin (Ig)G at the same dilution. GNA lectin (Sigma, St. Louis, MO) was used to coat Immulon II enzyme-linked immunosorbent assays (EIA) plates (Nunc, Rochester, NY) at 1 μg/mL for 4 hours at 37°C. After washing with PBS, the plates were blocked with 5% bovine serum albumin/PBS and lysates of cells expressing HCV E1E2 or pelleted virus allowed to bind overnight at 4°C. A preparation of truncated E2661 was used as an internal calibrator in all EIAs and allowed comparison of data between different assays. Bound antigen was visualized with MAbs specific for E2 or pooled HCV-positive human sera, an antispecies IgG–horseradish-peroxidase conjugate (Jackson, West Grove, PA) and tetramethylbenzidene (BioFX Laboratories, Owings Mills, MD). Absorbance values were measured at 450 nm (fusion plate reader; Perkin Elmer, Boston, MA). Peripheral blood mononuclear cells (PBMCs) were isolated by density gradient centrifugation as previously described.31Takaki A. Wiese M. Maertens G. Depla E. Seifert U. Liebetrau A. Miller J. Manns M. Rehermann B. Cellular immune responses persist and humoral responses decrease two decades after recovery from a single-source outbreak of hepatitis C.Nat Med. 2000; 6: 578-582Crossref PubMed Scopus (679) Google Scholar For selected experiments, CD8 T cells were isolated from PBMCs using CD8 microbeads and the autoMACS separator (Miltenyi, Bergisch-Gladbach, Germany). Subsequently, CD4 T cells were isolated from the CD8-negative population using CD4 microbeads. The purity of the CD8 T-cell population was 95% and the purity of the CD4 T-cell population was 92%, as analyzed by flow cytometry. The negatively selected CD4−CD8− cells were irradiated (3000 rad) and 105 cells per well were used as feeder cells in enzyme-linked immunospot assays. Interferon-γ enzyme-linked immunospot assays were performed as described32Rahman F. Heller T. Sobao Y. Mizukoshi E. Nascimbeni M. Alter H. Herrine S. Hoofnagle J. Liang T. Rehermann B. Effects of antiviral therapy on the cellular immune response in acute hepatitis C.Hepatology. 2004; 40: 87-97Crossref PubMed Scopus (120) Google Scholar using duplicate cultures of either the indicated number of CD4 and CD8 T cells and 105 irradiated (3,000 rad) CD4−CD8− cells, or 3 × 105 CD25-depleted PBMCs. Cells were stimulated with either 1 μg/mL of the individual HCV E1E2 peptides, 10 μg/mL of either wild-type or mutant epitope peptides, 1 μg/mL phytohemagglutinin (Murex Biotech Limited, Dartford, England), or dimethyl sulfoxide control, respectively. The number of spot-forming cells was determined with a KS enzyme-linked immunospot Reader (Zeiss, Thornwood, NY). Numbers of antigen-specific spot-forming cells (in the presence of antigen minus spot-forming cells in dimethyl sulfoxide controls) are shown. Patient H, the subject of this study, was infected with HCV through a blood transfusion in 1977 while undergoing cardiac surgery. After an initial high alanine aminotransferase peak (2112 IU/L), indicative of severe acute hepatitis, he went on to develop mild chronic hepatitis with persistently low alanine aminotransferase levels and HCV RNA levels in the 104 and 106 genome copies/mL range over the following 26 years (Figure 1A). Patient H underwent liver biopsy procedures on 4 occasions, each showing minimal inflammation with no fibrosis and no discernable progression over time. As expected, liver function was well preserved. Thus, patient H is representative of many chronically HCV-infected patients with mild, nonprogressive disease.33Afdhal N.H. The natural history of hepatitis C.Semin Liver Dis. 2004; 24: 3-8Crossref PubMed Scopus (250) Google Scholar Serial serum samples from patient H covering 26 years of chronic HCV infection were used for this study. All samples were characterized for their ability to neutralize HCVpp-bearing autologous H77 gps (a sequence cloned at 3 weeks after infection) and heterologous gps of the closely related genotype 1b (Con1, OH8) and the distant 2a (JFH, J6). In keeping with our previous report,17Logvinoff C. Major M. Oldach D. Heyward S. Talal A. Balfe P. Feinstone S. Alter H. Rice C. McKeating J. Neutralizing antibody response during acute and chronic hepatitis C virus infection.Proc Natl Acad Sci U S A. 2004; 101: 10149-10154Crossref PubMed Scopus (357) Google Scholar HCVpp-H77–specific nAbs were observed at seroconversion (8 weeks after infection) (Figure 1A and data not shown). Neutralization of HCVpp-bearing heterologous gps was first detected at 111 weeks after infection, at a time when chronic infection had been established. The appearance of the early strain-specific nAb response was associated with the detection of antibodies specific for H77 E1E2 and the HVR in an EIA assay (Figure 1B). However, high levels of antibodies capable of binding H77-soluble E2 (sE2) or J6 E1E2 were first detected at 111 weeks, coincident with the appearance of cross-reactive nAbs and an increase in the titer of H77-specific nAbs (Figure 1A and B). In contrast to the other steadily increasing anti-gp responses, the Ig response specific for the H77 HVR peaked at 9 weeks after infection and decreased to undetectable levels by 33 weeks (Figure 1B), suggesting a dynamic and rapidly evolving immune response to the viral gps in the early phase of infection. As previously reported by others,34Chen M. Sallberg M. Sonnerborg A. Weiland O. Mattsson L. Jin L. Birkett A. Peterson D. M
Referência(s)