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Characterizing Antibody Responses and Blocking Viral Entry in Hepatitis C Viral Infection

Permanent Link: http://ufdc.ufl.edu/UFE0022807/00001

Material Information

Title: Characterizing Antibody Responses and Blocking Viral Entry in Hepatitis C Viral Infection
Physical Description: 1 online resource (100 p.)
Language: english
Creator: Bess, Jennifer
Publisher: University of Florida
Place of Publication: Gainesville, Fla.
Publication Date: 2008

Subjects

Subjects / Keywords: antibodies, hcv, hepatitis
Immunology and Microbiology (IDP) -- Dissertations, Academic -- UF
Genre: Medical Sciences thesis, Ph.D.
bibliography   ( marcgt )
theses   ( marcgt )
government publication (state, provincial, terriorial, dependent)   ( marcgt )
born-digital   ( sobekcm )
Electronic Thesis or Dissertation

Notes

Abstract: Hepatitis C virus (HCV) infection is a global infectious disease without an effective vaccine. The aim of this study was to establish a method of measuring and quantifying HCV-specific neutralizing antibodies and autoantibodies, as well as determine the role they play in clinical outcome of disease. Further investigation into alternative methods of blocking viral infection led us to utilize the HCV cell culture based system to test novel small molecule candidates as inhibitors. To develop a quantitative and reproducible assay, numerous dilutions of HCV-positive human serum samples were incubated with the JFH1 virus. Huh7.5 cells were then inoculated with the virus/sera solution and cells remained in culture for 7 days. An indirect immunofluorescence assay (IIF) was performed using a monoclonal anti-HCV NS5A antibody. The percentage of HCV-positive cells was recorded for each dilution of serum and the Ec50 was calculated to be the dilution at which the viral infection was decreased by 50%. These patients were also screened for HCV autoantibodies by IIF using Hep-G2 cells. The in vitro results were then correlated with patient characteristics, including liver tissue damage, viral load, and overall clinical outcome. Overall, 150 different chronic HCV patients were examined and the results showed that neutralizing antibodies were present in 65.3% of the patients. This is indicated by a reduction in ?-NS5A cellular staining in contrast to positive controls and measured by the half maximal effective concentration (Ec50). Genotype 1 patients exhibited an average Ec50 of 1:5, whereas genotype 2 patients had an average Ec50 of 1:25, therefore requiring less serum to neutralize virus. High levels of ?-nuclear, ?-cytoplasmic or ?-mitochondrial antibodies were observed in 92.3% of chronic HCV patients in this study. CD81, the cellular surface protein, was recognized as the first entry factor for HCV and has the capacity to bind specifically and with high affinity to the E2 glycoprotein. The crystal structure of the CD81 receptor was used to identify small molecule modulators by a high throughput molecular docking method. Molecular docking (DOCK5.1.0) identified ten best molecules from the NCI database directed to the HCV binding target site. Huh7.5 cells were first treated with various concentrations of the compounds, followed by HCV infection. Total cellular RNA was extracted from the cells after 5 days of incubation and IIF was also performed to determine if the drugs were able to block HCV infection. Our data show that two compounds consistently inhibit HCV infection in nanomolar range. The inhibitory effect is dose-dependent and no cellular toxicity was noted in these compounds. Our preliminary study validates this novel approach to develop anti-HCV therapeutic agents. If neutralization could be enhanced in vivo by using drugs or exogenous antibodies that increase the effectiveness of native neutralizing antibodies, these findings would provide the basis for the development of vaccines and other antibody dependent immunotherapy for treatment of HCV infection.
General Note: In the series University of Florida Digital Collections.
General Note: Includes vita.
Bibliography: Includes bibliographical references.
Source of Description: Description based on online resource; title from PDF title page.
Source of Description: This bibliographic record is available under the Creative Commons CC0 public domain dedication. The University of Florida Libraries, as creator of this bibliographic record, has waived all rights to it worldwide under copyright law, including all related and neighboring rights, to the extent allowed by law.
Statement of Responsibility: by Jennifer Bess.
Thesis: Thesis (Ph.D.)--University of Florida, 2008.
Local: Adviser: Liu, Chen.
Electronic Access: RESTRICTED TO UF STUDENTS, STAFF, FACULTY, AND ON-CAMPUS USE UNTIL 2009-12-31

Record Information

Source Institution: UFRGP
Rights Management: Applicable rights reserved.
Classification: lcc - LD1780 2008
System ID: UFE0022807:00001

Permanent Link: http://ufdc.ufl.edu/UFE0022807/00001

Material Information

Title: Characterizing Antibody Responses and Blocking Viral Entry in Hepatitis C Viral Infection
Physical Description: 1 online resource (100 p.)
Language: english
Creator: Bess, Jennifer
Publisher: University of Florida
Place of Publication: Gainesville, Fla.
Publication Date: 2008

Subjects

Subjects / Keywords: antibodies, hcv, hepatitis
Immunology and Microbiology (IDP) -- Dissertations, Academic -- UF
Genre: Medical Sciences thesis, Ph.D.
bibliography   ( marcgt )
theses   ( marcgt )
government publication (state, provincial, terriorial, dependent)   ( marcgt )
born-digital   ( sobekcm )
Electronic Thesis or Dissertation

Notes

Abstract: Hepatitis C virus (HCV) infection is a global infectious disease without an effective vaccine. The aim of this study was to establish a method of measuring and quantifying HCV-specific neutralizing antibodies and autoantibodies, as well as determine the role they play in clinical outcome of disease. Further investigation into alternative methods of blocking viral infection led us to utilize the HCV cell culture based system to test novel small molecule candidates as inhibitors. To develop a quantitative and reproducible assay, numerous dilutions of HCV-positive human serum samples were incubated with the JFH1 virus. Huh7.5 cells were then inoculated with the virus/sera solution and cells remained in culture for 7 days. An indirect immunofluorescence assay (IIF) was performed using a monoclonal anti-HCV NS5A antibody. The percentage of HCV-positive cells was recorded for each dilution of serum and the Ec50 was calculated to be the dilution at which the viral infection was decreased by 50%. These patients were also screened for HCV autoantibodies by IIF using Hep-G2 cells. The in vitro results were then correlated with patient characteristics, including liver tissue damage, viral load, and overall clinical outcome. Overall, 150 different chronic HCV patients were examined and the results showed that neutralizing antibodies were present in 65.3% of the patients. This is indicated by a reduction in ?-NS5A cellular staining in contrast to positive controls and measured by the half maximal effective concentration (Ec50). Genotype 1 patients exhibited an average Ec50 of 1:5, whereas genotype 2 patients had an average Ec50 of 1:25, therefore requiring less serum to neutralize virus. High levels of ?-nuclear, ?-cytoplasmic or ?-mitochondrial antibodies were observed in 92.3% of chronic HCV patients in this study. CD81, the cellular surface protein, was recognized as the first entry factor for HCV and has the capacity to bind specifically and with high affinity to the E2 glycoprotein. The crystal structure of the CD81 receptor was used to identify small molecule modulators by a high throughput molecular docking method. Molecular docking (DOCK5.1.0) identified ten best molecules from the NCI database directed to the HCV binding target site. Huh7.5 cells were first treated with various concentrations of the compounds, followed by HCV infection. Total cellular RNA was extracted from the cells after 5 days of incubation and IIF was also performed to determine if the drugs were able to block HCV infection. Our data show that two compounds consistently inhibit HCV infection in nanomolar range. The inhibitory effect is dose-dependent and no cellular toxicity was noted in these compounds. Our preliminary study validates this novel approach to develop anti-HCV therapeutic agents. If neutralization could be enhanced in vivo by using drugs or exogenous antibodies that increase the effectiveness of native neutralizing antibodies, these findings would provide the basis for the development of vaccines and other antibody dependent immunotherapy for treatment of HCV infection.
General Note: In the series University of Florida Digital Collections.
General Note: Includes vita.
Bibliography: Includes bibliographical references.
Source of Description: Description based on online resource; title from PDF title page.
Source of Description: This bibliographic record is available under the Creative Commons CC0 public domain dedication. The University of Florida Libraries, as creator of this bibliographic record, has waived all rights to it worldwide under copyright law, including all related and neighboring rights, to the extent allowed by law.
Statement of Responsibility: by Jennifer Bess.
Thesis: Thesis (Ph.D.)--University of Florida, 2008.
Local: Adviser: Liu, Chen.
Electronic Access: RESTRICTED TO UF STUDENTS, STAFF, FACULTY, AND ON-CAMPUS USE UNTIL 2009-12-31

Record Information

Source Institution: UFRGP
Rights Management: Applicable rights reserved.
Classification: lcc - LD1780 2008
System ID: UFE0022807:00001


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1 CHARACTERIZING ANTIBODY RESPONSES AND BLOCKING VIRAL ENTRY IN HEPATITIS C VIRAL INFECTION By JENNIFER R. BESS A DISSERTATION PRESENTED TO THE GRADUATE SCHOOL OF THE UNIVERSITY OF FLOR IDA IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY UNIVERSITY OF FLORIDA 2008

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2 2008 Jennifer R. Bess

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3 ACKNOWLEDGMENTS I would like to dedicate this work to all the people who have helped m e throughout my entire graduate career. First, I am especia lly thankful for my ment or, Dr. Chen Liu. His guidance and continual s upport throughout the completion of my projects have been invaluable to my success. I would also like to thank my committee members for their advice and support, Dr. Sally Litherland, Dr. Bryon Petersen, and Dr. David Nelson. My collaborators, Dr. Ed Chan and Dr. David Ostrov were instrumental in the succe ss of my research projects. I feel fortunate to have had the ability to work with such talented researchers. I must also express gratitude to my cowo rkers, who have trained me and provided guidance in my years as a gra duate student. Dr. Haizhen Zhu, Dr. John Elyar, Dr. Hui-Jia Dong and Dr. Rafal Witek were instrumental teachers in my early years as a graduate student. Erika Eksioglu has also been a much apprecia ted friend and colleague in the lab. I would like to thank my parents, Jim and Li sa, who have always believed that I could accomplish anything I set out to do. I thank my siblings, Matt and Jackie for moral support and for setting the bar high for themselves while follo wing their own path. I also owe thanks to my extended family members that have provided me a backbone of support. Lastly, I would like to extend many thanks to my husband, Adam Bess, for standing by me and believing in me through all these years. I am greatly appreciative of his ability to motivate me, be the driving force behind my success, and fo r taking me out for dinner when I had a hard day.

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4 TABLE OF CONTENTS page ACKNOWLEDGMENTS ............................................................................................................... 3 LIST OF TABLES ...........................................................................................................................6 LIST OF FIGURES .........................................................................................................................7 ABSTRACT ...................................................................................................................... ...............8 CHAP TER 1 INTRODUCTION .................................................................................................................. 10 Hepatitis C Virus ....................................................................................................................10 Envelope Structure ..................................................................................................................12 Binding and Entry Receptors .................................................................................................. 13 Model Systems ........................................................................................................................15 HCV Envelope Glycoproteins ......................................................................................... 16 HCV Replicon System .................................................................................................... 16 HCV Like Particles (HCV-LPs) ......................................................................................16 HCV Psuedoparticle System (HCVpp) ........................................................................... 17 HCV Cell Culture System (HCVcc) ................................................................................ 18 Chimpanzee Model ..........................................................................................................18 Neutralizing Antibodies ..........................................................................................................19 Non-Organ Specific Autoantibodies .......................................................................................27 2 NEUTRALIZING ANTIBODIES IN HCV INFECTION .....................................................30 Experimental Rationale ........................................................................................................ ..30 Materials and Methods ...........................................................................................................30 HCV Constructs and Viral Particle Generation ............................................................... 30 Cell Culture .....................................................................................................................31 Patient Sera ......................................................................................................................31 Optimal Incubation Period .............................................................................................. 32 Serum Controls ................................................................................................................ 32 Indirect Immunofluor escence Microscopy ...................................................................... 32 Scoring Process ...............................................................................................................33 Results .....................................................................................................................................33

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5 3 NON-ORGAN-SPECIFIC ANTIBODIES IN HCV INFECTION ........................................ 69 Experimental Rationale ........................................................................................................ ..69 Materials and Methods ...........................................................................................................69 Patient Sera ......................................................................................................................69 Indirect Immunofluor escence Microscopy ...................................................................... 69 Scoring Process ...............................................................................................................70 Results .....................................................................................................................................70 4 NOVEL SMALL MOLECULES BLOCKING HCV ENTRY .............................................. 74 Experimental Rationale ........................................................................................................ ..74 Materials and Methods ...........................................................................................................74 Molecular Docking .................................................................................................................74 Novel Small Molecules ...................................................................................................75 HCV Constructs and Viral Particle Generation ............................................................... 75 Cell Culture .....................................................................................................................76 RNA Isolation ..................................................................................................................76 Reverse Transcription and Poly m erase Chain Reaction (PCR) ...................................... 77 Indirect Immunofluor escence Microscopy ...................................................................... 78 Scoring Process ...............................................................................................................78 Results .....................................................................................................................................78 5 DISCUSSION AND CONCLUSION .................................................................................... 85 Development of a Novel Assay to Analyze Neutralizing Antibodies in HCV Infection ....... 85 Non-Organ Specific Autoantibodies are Highl y P revalent in Chronic HCV Infected Patients ...................................................................................................................... ..........87 Novel Anti-Viral Small Molecules Can Block HCV Cellular Entry Through the CD81 Receptor ..............................................................................................................................88 LIST OF REFERENCES ...............................................................................................................89 BIOGRAPHICAL SKETCH .......................................................................................................100

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6 LIST OF TABLES Table page 2-1 Patient characteristics and immunofluores cence m icroscopy of non-organ specific autoantibodies.. .............................................................................................................. ....37 3-1 Correlation between non-organ spec ific antibodies a nd HCV genotype ........................... 72 3-2 Correlation between non-organ specific antibodies and neutralizing antibodies in HCV positive patients.. ......................................................................................................72 3-3 Average viral load and liver enzyme le vels of HCV positive patien ts with non-organ specific antibodies. .............................................................................................................73 3-4 Statistical correlation between non-orga n specific antibod ies and HCV positive patient characteristics. ........................................................................................................73

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7 LIST OF FIGURES Figure page 2-1 IIF of HCV-infected Huh7.5 cells as reflected in Patient 59. ......................................... 35 2-2 The Ec50 of HCV positive patients in each genotype.. ..................................................... 36 4-1 Molecular docking strategy fo r binding inhibition of CD81. ............................................80 4-2 DOCK Scores for the top 10 scoring compounds. .............81 4-3 Complementary Determining Region-81 interacts with novel sm all molecules to inhibit HCV infection in hu man hepatoma cell lines. ........................................................ 82 4-4 Histogram summary of the percentage of positive cells in the immuno fluorescence assay for 10 drugs at concentratio ns of 1 nm, 5 nm and 20 nm. ........................................ 83 4-5 Small novel molecules can inhibit HCV RNA replication in Huh7.5 cells in a dosedependent m anner. ............................................................................................................. 84

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8 Abstract of Dissertation Pres ented to the Graduate School of the University of Florida in Partial Fulfillment of the Requirements for the Degree of Doctor of Philosophy CHARACTERIZING ANTIBODY RESPONSES AND BLOCKING VIRAL ENTRY IN HEPATITIS C VIRAL INFECTION By Jennifer R. Bess December 2008 Chair: Chen Liu Major: Medical Sciences--Immunology and Microbiology Hepatitis C virus (HCV) infection is a globa l infectious disease without an effective vaccine. The aim of this study was to estab lish a method of measur ing and quantifying HCVspecific neutralizing antibodies and autoantibodies, as well as determine the role they play in clinical outcome of disease. Fu rther investigation into altern ative methods of blocking viral infection led us to utilize the HCV cell culture based system to test novel small molecule candidates as inhibitors. To develop a quantitative and reproducible assay, numerous dilutions of HCV-positive human serum samples were incubated with the JF H1 virus. Huh7.5 cells were then inoculated with the virus/sera solution and cells remain ed in culture for 7 days. An indirect immunofluorescence assay (IIF) was performed using a monoclonal an ti-HCV NS5A antibody. The percentage of HCV-positive cells was recorded for each dilution of serum and the Ec50 was calculated to be the di lution at which the viral infection was decreased by 50%. These patients were also screened for HCV autoantibodies by IIF using Hep-G2 cells. The in vitro results were then correlated with patient characteristics, incl uding liver tissue damage, viral load, and overall clinical outcome.

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9 Overall, 150 different chronic HCV patients we re examined and the results showed that neutralizing antibodies were present in 65.3% of the patients. This is indicated by a reduction in -NS5A cellular staining in contrast to positiv e controls and measured by the half maximal effective concentration (Ec50). Genotype 1 patie nts exhibited an average Ec50 of 1:5, whereas genotype 2 patients had an aver age Ec50 of 1:25, therefore requi ring less serum to neutralize virus. High levels of -nuclear, -cytoplasmic or -mitochondrial antibodies were observed in 92.3% of chronic HCV pa tients in this study. CD81, the cellular surface protein, was recognized as the first entry factor for HCV and has the capacity to bind specifically and with high affinity to th e E2 glycoprotein. The crystal structure of the CD81 receptor was used to identify small molecule modulators by a high throughput molecular docking method. Molecular docking (DOCK5.1.0) identified ten best molecules from the NCI database directed to the HCV binding target site. Huh7.5 cells were first treated with various concentrations of the compounds, followed by HCV infection. Total cellular RNA was extracted from the cells after 5 days of incubation and IIF was also performed to determine if the drugs were able to block HCV infection. Ou r data show that two compounds consistently inhibit HCV infecti on in nanomolar range. The inhib itory effect is dose-dependent and no cellular toxicity was not ed in these compounds. Our prel iminary study validates this novel approach to develop anti-HCV therapeutic agents. If neutralization could be enhanced in vivo by using drugs or exogenous antibodies that increase the effectiveness of native neutralizing antibodies, these findings would provide the basis for the development of vaccines and other antibody dependent immunotherapy for treatment of HCV infection.

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10 CHAPTER 1 INTRODUCTION Hepatitis C Virus With an estimated 120-170 million people infe cted world wide, hepatitis C virus (HCV) is one of the most pervasive causes of chronic liver disease, and has a serious impact on public health.1,2 HCV affects approximately 3% of the worl ds population and at this time there is no effective vaccine.2,3 Initially isolated in 19894, HCV is an enveloped positive sense RNA virus that is classified as a Hepacivirus of the Flaviviridae family.1,2,5 The genome encodes a single polyprotein 3000 amino acids in length that is processed by host and viral proteases to release ten structural and non-structur al proteins. These proteins make up th e viral particle and facilitate the viral replication machinery.1,2 HCV viral RNA is associated with a nucleocapsid surrounded by a lipid-containing envelope and two glycoproteins, E1 and E2.2,3 HCVs RNA-dependent RNA polymerase lacks proofreading capabilities, thus promoting a high rate of mutation in the viral genome. Viral production in a chronically infected patient can be as high as 1012 virions per day.2,6 There are six genotypes and over 100 subtypes of HCV which vary in their clinical presentation, geographic dist ribution, and ability to respond to anti-viral therapy.2,3 HCV is a blood-borne pathogen and was the most prominent source of post-transfusion hepatitis until the i nduction of blood screening for HCV an tibodies became available in 1990.3 Since the induction of screening processes, HCV infection is now primarily spread through IVdrug use.2 The liver is the principal target organ of HCV and it preferen tially replicates in hepatocytes1. Humans and chimpanzees remain as the only host species that can sustain HCV infection. In the majority ( 50-80%) of infected i ndividuals, HCV establishes a persistent infection even though it is recogn ized by the hosts immune system.2,3 Chronic carriers typically

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11 display minimal to mild hepatitis, which leads to the development of end stage liver disease.3 The development of liver disease varies in different individuals and may exceed 30-40 years. During the majority of this time, many indivi duals are not aware of their infection. HCV infection is currently the leading cause of liv er transplantation in the United States, and represents a major health care concern.2 Variable degrees of hepatic inflammation accompany chronic HCV infections, which can progress to liver cirrhosis, fibrosis, and hepatocellular carcinoma (HCC).1,5 In hepatitis C research, there is a large body of evidence that suggests the leve l and form of activation by the immune response is the determining f actor for control of viral infection.5 The virus is recognized by both the innate and adaptive immune mechan isms, but only when a robust and multispecific response is mounted, can th is virus be controlled.1,2 Most occurrences of chronic HCV develop despite the induction of antibod ies to both structural and non -structural viral proteins. Treatments for infected individuals are inadequate and a vaccine to prevent occurrence of HCV has yet to be established.1 Many studies during the last decade of HCV research have provided increased knowledge of the immunological and viral factors that de termine persistence, clearance, and disease progression in HCV infection. In the liver, viral mechanisms are mediated by dynamic effector functions including support from both cytotoxic and non-cytotoxic T cells.7,8 Nevertheless, HCV persistence occurs in approximately 80% of infected patients and the mechanisms contributing to the failure of virus-specific T cell responses are not yet clear. HCV utilizes numerous strategies to evade host response and contribut e to viral persistence, therefor e more research is required to create and implement successful immunothera py for chronically infected HCV patients.8 Through analysis of how HCV interacts and evad es the adaptive immune response, an improved

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12 understanding of how liver fibrosis, cirrhosis and HCC are induced can be achieved. The development of universally effective therapies an d vaccines for HCV are a main goal in research HCV today.9 Envelope Structure The HCV ge nome contains three structural proteins and se ven non-structural proteins. The genome includes both structural and non-structural proteins, th e core protein formulates the viral nucleocapsid, while E1 and E2 make up the envelope glycoproteins.10 The envelope glycoproteins are cleaved from the pol yprotein by a host cell signal peptidase.11,12 E1 and E2 are type-I transmembrane proteins and their glyc osylation sites are highly conserved, which indicates that they are critical for both envelope glycoprotein structure and function.10 The lipid membranes of HCV envelope glycoproteins are m odified by N-linked glycans. E1 and E2 have up to 6 and 11 glycosylation sites respectively.11 Certain glycans are kno wn to function in HCV glycoprotein folding or in virus entry.12,13 Glycans can alter the abil ity of a virus to elicit an immune response and impede its ability to be recognized by host receptors.14,15 Even though HCV envelope glycoproteins can be found at the plasma membrane when they are expressed in high quantities,12,16-18 the E1 and E2 proteins form a heterodimer and are accumulated in the endoplasmic reticulum (ER). 19 The accumulation of structural proteins on the ER membrane implies that this is the location where th e viral capsid and envelope are assembled.10,11 In spite of the complexity in visualizing the envelope proteins in clinical HCV isolates, data suggest that E1 and E2 are present in infectious particles. 20 Antibodies specific for E2 can inhibit the binding of HCV from hu man cell lines to infected serum.20,21 Chimpanzees who were vaccinated with recombinant E1 and E2, were protected from subsequent HCV infection or resolved the infection.4 In addition, antibodies against E1 and E2 block the interaction of HCV and host cells, and thus may provide a target for therapeutic treatment.10

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13 A section of the 27 amino acid long N-terminal region of the E2 glycoprotein forms a hypervariable region (HVR-1). Variability of this region may affect antibody selection for immune-escape variants. Anti-HCV antibodies specific for one variant are only partially able to neutralize other HCV variants.11 When chimpanzees were infect ed with a strain of HCV not including the HVR-1 region, the virus was infec tious, but the strength of its virulence was reduced.22 The structure and arrang ement of HVR-1 residues are highly conserved amongst the different genotypes, and its presence at the surface of the viral particle su ggests that it might be involved in viral entry.11,23 Both E1 and E2 have essential roles in vari ous parts of the HCV life cycle. Early in infection, E1 and E2 are vital for virus entry24,25, and subsequently partic ipate in the packaging and assembly of HCV particles.26 The E1-E2 heterodimer is locat ed at the surface of infectious particles, therefore it represents a candidate ligand for host receptors.11,12 The envelope proteins are targets of neutralizing antibodies because th ey are exposed at the surface of the virion. By utilizing the HCV pseudoparticle system (H CVpp), many studies to find and characterize neutralizing antibody response to HCV have been initiated. As determined with this system and validated by the HCV cell culture system (HCVcc), it is apparent that the majority of chronically infected HCV patients have neutralizing antibodie s that are broad and cr oss-reactive. Because they are essential for virus entr y, HCV envelope glycoprot eins remain as an attractive target for the development of antiviral molecules that block HCV entry.12,27 Binding and Entry Receptors Three cell surface molecules, CD81, human scav enger receptor class B type 1 (SR-BI) and claudin-1, are reported to pl ay a role in HCV infection.15 CD81 was recognized as the first entry factor for HCV by its capacity to bind specif ically and with high affinity to a soluble form of the E2 glycoprotein.15,28 CD81 is a member of the tetraspanin family, whose attributes

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14 include involvement in a variety of cellular f unctions such as motility, adhesion, metastasis, differentiation, and morphology.29,30 This receptor is found in n early all human tissues, except platelets and red blood cells.10 CD81 is composed of four transmembrane domains, a small extracellular loop, and a large ex tracellular loop, which is suffi cient to mediate binding to recombinant E2 and is mainly responsible for HCVpp entry.10,27 Monoclonal antibodies to CD81, as well as a soluble form of the large extracellular loop of CD 81, are able to reduce HCVpp and HCVcc infectivity in vitro .16,17,26,30 In addition, liver cells with CD81 knockeddown are no longer permissi ve to HCVpp and HCVcc.30,31 Cells that do not express CD81 become permissive to HCVpps and HC Vccs after surface expression of CD81.30,32 Research has demonstrated that susceptibility of hepatoma cel ls to HCV infection is intertwined with the expression level of CD81. However, the precise function of this tetraspanin in HCV entry is incompletely understood.27,30 Furthermore, it was revealed that CD81 expression is essential but not entirely sufficient for viral entry into targ et cells, signifying that additional factors are necessary for HCV entry.16,17,33 SR-BI is another cell surface protein whic h has been identified as a putative HCV receptor.30,34 SR-BI is a 509-amino-acid protein cont aining two transmembrane passages, two short cytoplasmic domains, and one large extracellular loop.30,35,36 It is primarily expressed in the liver and other tissues.10 It is a multi-ligand receptor, whic h can bind a variety of lipoproteins including high density lipoproteins (HDL), low de nsity lipoproteins (LDL) and very low density lipoprotiens (VLDL).30,34 SR-BI was recognized as potential HCV receptor and it is believed to interact with HVR-1. Antibodi es to HVR-1 compete with SR-BI for E2 binding, and recombinant E2 without HVR-1 does not bind to SR-BI.10,34,37

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15 The participation of SR-BI in HCV entry has been validated by both the HCVpp and HCVcc systems.16,38 Prior incubation of Huh-7 cells with anti-SR-BI antibodies considerably reduces HCV entry.38 SR-BI knock-down hepatoma cells are le ss tolerant to pa rticles from both HCVpps and HCVccs.39 Although direct interaction between SR-BI and the viral particle has been suggested40, but HCV also may act together with SR-BI through its associated lipoproteins.41 SR-BI is found in many types of ma mmalian tissues, although it is found in a greater extent in the liver.25,27 In a screen for genes that provide cells susceptibility to HCVpp infection, Evans et al 42 identified claudin-1 as a new protein involved in HCV entry.30 Claudin-1 is largely expressed in the liver43 and belongs to a family of proteins invol ved in the developmen t of tight junctions. This receptor contains four transmembran e passages, two extracellular loops, and three intracellular domains.30,42,43 Expression of claudin-1 in variou s cell lines makes them permissive for HCVpps and HCVccs. Overexpression of cl audin-1 in permissive cells does not augment infectivity, but claudin-1 knock-down cells are less tolerant to in fection by HCVpps and HCVccs.43 The first extracellular loop of cl audin-1 is involved in HCV entry.27 Antibodies directed against this epitope of claudin-1 inhi bits HCV infectivity. It has been proposed that claudin-1 has delayed participation in the entry process, at a point after the virus binds and interacts with CD81. No direct contact betw een the HCV particle a nd claudin-1 has been reported, but this interaction may require a conformational change in the envelope glycoproteins prompted by contact between E2 and another entry factor, such as CD81 or SR-BI. Model Systems Before developm ent of the HCV-culture system in 200526, specialized in vitro neutralizing antibody assays were unavailable and virus-antibody interactions were difficult to study.

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16 However, the past decade of HCV research ha s provided many model systems for the study of HCV entry and infection. HCV Envelope Glycoproteins The putative envelope glycoproteins, E1 and E 2, are essential in the initial steps of viral infection.44 The study of these proteins has been co mplicated due to the propensity of HCV envelope glycoproteins to misfold and accumulate in the endoplasmic reticulum.24,44 To overcome these problems, solubilized forms of glycoproteins with deleted transmembrane domains have been created. Studies using these recombinant HCV envelope glycoproteins have been successfully used to examine virus-host interactions. These studies have led to the discovery of HCV receptor candidates incl uding human tetraspani n CD81 and the human scavenger receptor SR-BI.1,24 This system was also used to screen for antibodies that inhibited the attachment of virus to host cells.24 Both monoclonal and polyc lonal antibodies directed against the E2 glycoprotein ar e able to neutralize HCV.45 This system provided information on the initial steps of virus assembly and entry, but later events in the maturation of these glycoprotiens was not facilitated by this model. HCV Replicon System An altern ative cell culture system was develope d that included a full-length consensus HCV genome cloned from liver tissue of a chronically in fected patient, used to construct subgenomic selectable replicons. This system allowed scientis ts to define the struct ure of HCV replicons in cell culture, but it was not indicat ive of a true viral infection.46,47 HCV Like Particles (HCV-LPs) To investig ate the structure of HCV in the ab sence of virions, resear chers developed selfassembling virus-like particles th at do not include genomes; and th erefore, lack the ability to replicate. These HCV-like par ticles (HCV-LP) are generated by introducing viral vectors into

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17 insect or mammalian cells.24 For example, HCV viru s-like particles have been generated in these insect cells infected by a r ecombinant baculovirus containing the cDNA of HCV structural proteins.16,25,48 HCV-LPs display antigeni c and morphological properties comparable to those of authentic virions isolated from patients with chronic HCV-infections. Therefore, they may interact with anti-HCV antibodies directed against epitope s of HCV envelope proteins.49-52 HCV structural proteins, E1 and E2, exhibit similar characteristic s of putative vi rions isolated from HCV-infected patients, but no viral particles are released into the supernatant. Contrary to the envelope glycoproteins E1 and E2, the E1-E2 HCV-LP heterodimers are in a native, virionlike structure. These particles have their recepto r-dependent ability to attach and enter human hepatoma cells as well as dendrit ic cells and primary hepatocytes.1 However, while this is a valuable model system to analyze HCV-host cell interactions and attachment, the lack of a genome and ability to produce infections virions reduces the application in infectious assays.1,24 HCV Psuedoparticle System (HCVpp) Retroviral HCV pseudotype part icles (HCVpp) were created by transfecting cells with expression vectors encoding unmodified HCV envel ope glycoproteins E1 and E2, lentiviral or retroviral core particles, and a pack aging component with a marker gene.1,24 HCVpp are infectious in liver cancer cell lines, such as Huh-7 cells, and human primary hepatocytes.1 Transfected cells produce assemble d virus particles into the supe rnatant, which can be produced and used to infect naive target cells.24 Assessing the presence of a luciferase reporter gene or fluorescent protein can monitor in fection of target cells, which provides a dependable method of determining infectivity.24 HCVpp assembly and production is reasonably proficient, with an average of 105 infectious particles per milliliter of supernatant. This system allows investigation of functions mediated by the HCV glycoproteins. This method is able to specifically detect antibodies with in vivo neutralizing potential as well as illustrate the molecular mechanisms of

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18 antibody-mediated neutralization.24 Extensive studies of HCVpp have shown that these pseudoparticles are similar to the serological pr operties and early steps of the HCV life cycle16, 25 and represented the most informative model before the development of the HCV cell culture system.26 HCV Cell Culture System (HCVcc) Recently, a full-leng th genotype 2a HCV genome was established which supports the efficient production of virus particles that are infectious in cell culture.26,32 The system uses a unique clone derived from a viral isol ate of a Japanese patient with fulminant hepatitis C and is designated JFH-1. Infection of highly permissive Huh7derived hepatoma cells is based on the transfection of JFH-1 mRNA into th ese cells, leading to the producti on of viral proteins. This system represents an important breakthrough in HCV research and currently allows the study of the full viral life cycle in vitro .26 Initial research using th e HCVcc system has confirmed previous research on molecular mechanisms and cell entry. However, while HCVcc undoubtedly represents a reliab le system, its use has limitations in terms of safety.53 Efficient viral growth is only observed in Huh7 cells and the genotype is restricted to 2a.26,32,54-55 Chimpanzee Model The lack of a sm all animal model has hindered the progress in HCV research. The chimpanzee remains the only naturally existing in vivo model for the study of HCV. 1 Since blood and tissue samples are accessible from the beginning of inf ection and continue throughout the acute phase of infection, chimpanzees are valuable for gainin g insights into the early events of the immune response to HCV. 56 HCV-specific neutralizing antibodies are found in chimpanzees and the duration of infection is milder and shorter than in humans. 1 Experimentally infected chimpanzees have provided information about various components of the immune response, including protective immun ity against re-infection. 1,2 The biomedical research on chimpanzees

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19 and other primates is reduced by limited availa bility, ethical concerns, and expensive housing costs. 57 Neutralizing Antibodies Recent stud ies suggest that HCV does not dire ctly cause liver cell injury but triggers inflammatory responses. It is this response that either rapidly clears the virus or gradually destroy hepatocytes.58 Thus, the host immune responses to virus-infected hepatocytes plays an essential role in viral pathogenesis.58-60 Virus-neutralizing antibodies often provide the first line of adaptive humoral defense against inf ection by limiting the spread of virus.61 Little is known about the antibody response to HCV infection and despite extensive research in HCV pathogenesis over past decades, the interaction of the virus and host immune responses is still not fully understood.63 When HCV viral proteins are recognized by th e hosts immune system as foreign they can induce the production of virus neutraliz ing antibodies.1 These specific types of antibodies are able to bind exposed epitope s of structural viral proteins and neutralize them by either averting or controlling infection.1,64 Neutralization of the initial binding of virus to hepatocyte receptors represents a prime ta rget for preventing infection.6 The antibody-mediated response plays a central role in vaccination against most infectious diseases61, but the nature of these antibodies and their effective mechanisms of inhi bition in HCV infection ha s yet to be defined. Recent developments have increased understa nding of the HCV life cycle and viral entry mechanisms. Viral attachment to host cells is facilitated through binding of the HCV glycoproteins to cellular receptors present on th e surface of hepatocytes. This process is highly complex and involves a variety of entry factor s including, but not limited to, CD81, SR-BI and claudin-1.25,42,65 The HCV virion is internalized in a pH dependent manner and the genome is

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20 delivered into hepatocytes before replication occurs.1 Neutralizing antibodies may be able to interfere with viral binding to co-receptors and entry into hepatocytes by activating complement, inducing conformational chan ges in the viral proteins, or by sequestering viral proteins in the cytoplasm.66 Direct binding of neutralizing antibodies to the virion may hinder attachment to the host cell, or the antibodies may impede attachment of virus to entry factors. Interfering with the initial steps of viral replication or with viral uncoating may fac ilitate neutralization and antibodies may prevent fusion at the cell surface or in endosomal compartments. Neutralizing antibodies play distinctive roles in viral entry, attachment, and interact with virus in chronic HCV infection.1 Antibodies targeting specific HCV epitopes have been detected in patients approximately two weeks after acute infection oc curs and are instrumental in the diagnosis of HCV infection.67 The HCV envelope glycoproteins are highly glycosylated and ha ve 6 or 11 N-linked glycans on E1 and E2, respectively. Th ese glycosylation sites are hi ghly conserved across various genotypes and some of the glycans have b een shown to be essential in HCV entry.13 The CD81 tetraspanin is the most highly characterized en try factor for HCV and it has been shown to interact with HCV glycoprotein E2.28,68 Numerous viral epitopes th at are possibly targeted by neutralizing antibodies have been identified to be located in the HVR-1 of the E2 protein. Early studies demonstrated that this region of the E2 protein of HC V has the highest variability in the whole viral genome; suggesting it represents a critical region for targeting neutralization.69 This variability of the HVR-1 region may contribute to the ability of HCV to generate quasispecies and, in turn, escape neutralizing antibody responses.67 Sequence changes in the E2 region have been described in acute infections and were found to be associated with the outcome of infection. Most noticeably, these sequence changes c oncurred with anti-HCV seroconversion,

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21 demonstrating that humoral responses adapt to immune selection pressure by escape mutations.70 The presence of neutralizing antibodies direct ed against HVR-1 of the E2 glycoprotein have been shown to decrease viral load and contribute to viral eradication in some patients.71 There is substantial evidence indicating that neutralizing antibodies are involved in disease control. Establishment of persistent infection is facilitated by the inadequate adaptive immune response that the host mounts towards HCV. It still remains to be determined whether neutralizing antibodies can c ontrol of HCV replication.67 In contrast, acute responders to HCV infection do not develop anti-H CV antibodies. The function of HCV specific neutralizing antibodies and their involvement in protection is controversial si nce the mere existence of antiHCV antibodies does not prot ect against re-infection.5 In recent years, many studies have supported the notion that HCVspecific neutralizing antibodies are broad and cross-reactive. Both laboratory and epidemiological studies have suggested that HCV-specific neut ralizing antibodies are important in control and prevention of HCV infection although antibody effects have re mained difficult to study and accurately measure. Using the in vitro system based on neutralization of infectious HCVpp, it was found that broadly reactive neutraliz ing and protective antibodies are present in HCV chronic infection.32,39,62,72 Early detection of E1-E2 specific anti bodies associated with natural resolution of HCV.73-75 Others have shown that early neutra lizing antibody responses during the acute phase of infection is associated with fluctuating HCV RNA levels39 and viral clearance.76 These data suggest that neutralizing an tibodies facilitate contro l of viral replicatio n in early infection stages.77 High-titer neutralizing antibody levels have been detected in blood plasma from chimpanzees and humans with chronic infections.39,72 In a chimpanzee study, Bartosch et al did

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22 not find HCV-specific neutralizi ng antibodies in infections th at resolved, but did discover somewhat high titers of these an tibodies in chronic infections.37 Such neutralizing antibodies were directed against epitopes in HVR-1 and to epitopes in other locations on the viral envelope.6,37 Neutralizing antibodies were determined to be broadly reactive and were able to neutralize HCVpp of two different subgenotypes, 1a and 1b.37 Major et al. studied two previously infected chimpanzees and one nave an imal who were infected with a clonal virus stock that had no quasispecies vari ability. They saw a significant difference in the levels of viremia, HCV-specific antibody re sponses, and ALT levels between the animals rechallenged following clearance and the nave animal. Their results showed that a memory immune response is present in chimpanzees who ar e able to clear infections and that virus can be controlled upon re-challenge.78,79 Neutralization studies have identified neut ralization epitopes in the HVR-1 region of E2. 45,72,80,81 Overall, it has been shown that anti bodies targeted to HVR-1 can effectively neutralize HCVpp and HCVcc in the presence of human serum, but HDL may impede this process. 79 HVR-1 has been shown to hinder the rec ognition of neutralizi ng antibodies in both acutely and chronically infected HCV patie nts. The use of HVR-1 deleted HCVpp in neutralization assays with sera from a cohort of patients shed li ght on the existence of a robust neutralization response against both hom ologous and heterologous HCV sequences. 79 Furthermore, this system led to the discovery of broader, stronger, and neutralizing response in sera from patients. This signifies that HDL may, in some way, mask HCVpp from the crossneutralizing antibodies existing within seru m from chronically infected HCV patients. 82 The

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23 interaction of HDL with SR-BI only interferes w ith antibodies that block E2 binding to CD81, displaying its vital role during the cell entry process. 18,19 Deletion or mutation of HVR-1, which is under intense evolutionary pressure, greatly amplified sensitivity of HCVpp to cross-neutra lizing antibodies present in these sera. These findings support the important ro le HVR-1 plays in the modul ating the effectiveness of antibodies. In patients w ho are unable to resolve the infection, antibody-escape methods contribute to the inadequacy of the host humoral response against chronic HCV infection. 79 Many studies have been performed with sera fr om chronically infected "patient H", from whom the model H77 (genotype 1a) HCV strain was cloned. 72 Patient H is a uniquely welldefined subject who is representative of many pa tients with mild, chronic hepatitis C. Patient Hs serum samples are availa ble from numerous decades. 83 Logvinoff et al. showed that this patient demonstrated an increase in strain -specific neutralizing antibody responses at seroconversion that expanded to neutralize othe r viral strains between 33 and 111 weeks postinfection. 72 These investigators also ev aluated the neutralizing antibody response in chimpanzees infected with clonal H77 virus. Many of the infected chimpanzees developed strain-specific neutralizing antibody response late in disease, but that this response was low-titer and did not correlate with viral clearance. 72,83 Cross-reactive neutralizing antibody responses have been detected after more than 2 years of infection and increased in number and breadth to neutralize othe r genotypes of HCV. These data correspond to reports for human immunodeficiency virus and advocate the notion that neutralizing antibody responses are delayed until the emergence of rapidly evolving glycoproteins sequences present within the quasispecies population. 84,85

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24 Netski et al. examined sera from two different chronically HCV-infected patients and found that sera could neutrali ze all H-derived E1-E2 clones. 62 This demonstrates that neutralizing antibody responses elic ited during chronic infection are broadly reactive to most E1E2 sequences, as well as strains that the host immune system has not come across. Therefore, viruses with autologous E1-E2 sequences are responsive to antibodymediated neutralization. The resistance noticed by viruses expressing gl ycoproteins cloned from samples collected from patient H, and also sequences to antibodies pres ent in earlier serum samples, suggest that virus can escape from the humoral immune responses ma ny years after chronic in fection begins. It is believed that HCV-specific neutralizing anti bodies poorly neutrali ze because the virus continually produces new antigenic proteins derivied from its mutable genome. This and other research on patient H has provided a convincing explanation for the inab ility of the immune response to resolve HCV infection. 62,85 Early experiments involving chimpanzees showed that serum from a chronically infected human patient could neutralize HCV infection in the chimpanzee. 69,70 This suggested the presence of neutralizing antibodies in HCV infection and research ers have continued to utilize this model successfully. Rosa et al. reported that the ex istence of antibodies was able to hamper binding of soluble E2 glycoprotei ns to cells associated with viral clearance in immunized animals. 86 Previously, Shimizu et al. used a lymphoid cell line to demonstrate that neutralizing antibodies to HCV could prevent replication of the virus. 87 The protective ability of HCV-specific neutra lizing antibodies in infection is further supported when HCV pre-treatment with antibodies in vitro prevented infectivity in the chimpanzee model.69,70 Antibody responses have been shown in vivo in the chimpanzee model vaccinated with recombinant E1-E2 proteins of HCV.88-90 In a chimpanzee study, plasma of

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25 chronically infected human patient s prevented HCV infection after in vitro neutralization. The recent results from chimpanzee studies highlight the notion that neutralization of infection in chimpanzees can be achieved.86 Another study measured neutralizing antibodies in commerc ial Gammagard lots created before the screening test for HCV was developed. Yu et al demonstrated that broadly reactive high-titer neutralizing antibodies exist in Gammagard lots prepared from unscreened plasma. Chimpanzees vaccinated with recombinant E1 and E2 glycoproteins were moderately protected from a subsequent homologous challenge with low dose HCV. These results suggest that antibodies to E1 and E2 may protect against HC V infection. Antibodies to the E1-E2 proteins, especially to the HVR-1 region, were suspected to be the principle neutralization epitopes. 91 Occasionally, viral isolate specific outbr eaks will occur in hospitals, and although devastating to patients and the health care comm unity, the study of such occurrences can be of great value for researchers. Lavillette et al examined hemodialysis patients who acquired HCV nosocomially. In this study of patients who became coinfected with two di stinct HCV isolates, it was found that viral load correl ates with the induction of ne utralizing antibodi es throughout the acute phase of infection. 39,76 HCV RNA loads were found to be reduced in some patients, which corresponded to the emergence of specific neutralizing antibodies; contrary to other patients, elevated HCV RNA levels were linked with th e absence of neutralizing antibodies despite seroconversion. 82 Another study of antibody-mediated virus ne utralization in a singl e-source outbreak of HCV infection occurred when a co hort of patients received the same viral inoculum. It was revealed that quick stimulation of neutralizing antibodies is associat ed with viral clearance in the

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26 acute phase of infection and a reduction of neutralizing antibodies after recovery from infection. 76 Although high viral variability in HCV infections occurs, researchers have been able to identify monoclonal antibodies from both imm unized animals and chronic patients that neutralize HCVpp of differe nt subtypes and genotypes.79 Neutralizing antibody responses to HCV during chronic infection have also been associated with the extent of liver injury92, and anti-E2 antibodies with the potential to control damage to the liver by antibody-dependent cellular cytotoxi city only occur after many y ears of HCV infection. Ther efore, the contributions of HCV specific neutralizing anti bodies to viral elimination, co mpared with progression of disease, still remain undefined. Ov erall, these results s uggest that potential va ccines could induce a cross-reactive antibody response.90 HCV has the ability to persever e regardless of the generation of neutralizing antibodies. Petska et al outlined four ways in which viral escape from antibody-mediated neutralization may occur. First, the variability of HCV leads to distinct quasispecies that change constantly throughout the infected patient. The chimpan zee model has led researchers to believe that variability represents a mech anism of escape from antibody-me diated neutralization. Second, the interaction between HCV glycoproteins, HDL, and SR-BI has been shown to induce protection from neutralizing antibod ies present in the serum of acute and chronic HCV-infected individual. Third, similar to vi ruses such as HIV, escape from neutralizing antibodies may occur through a variety of different mechanisms, such as; insertions, deletions, conformational masking of receptor-binding sites after envelopeantibody interaction, point mutations, prevention of neutralizing antibody binding, or cha nges in glycosylation patterns of the viral envelope. Fourth,

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27 viral escape may occur due to an impaired cap acity of antibodies to cross-neutralize viral variants. 76 Although they appear after the initial HCV infection, neutrali zing antibodies may be an effective way to manage persistent infec tions with poorly or non-cytopathic viruses.93 Now that HCV entry and infection can be examined in vitro many neutralizing antibody assays, examining the function of antibodies, have br ought forth new insights to the host humoral response, viral targets, and possibilities of future vaccine studies.64 Broad cross-reactive neutralizing antibodies are of important clinical relevance, po ssibly to prophylactically decrease the risk of HCV infection after accidental e xposure or needlestick. In addition, passive immunotherapy with such antibodies would be useful in reducing graft rejection.94 The development of cross-reactive neutrali zing antibodies may be delayed until the cellular immune response is impaired and the ability to remove infected hepatocytes is subsequently decreased. To control viral rep lication strategies to increase the neutralizing response during the acute phase of the infection are needed. 8,72 If neutralization could be enhanced in vivo by involving epitopes, drugs, or antibodie s that increase the effectiveness of neutralizing antibodies, the potential for advan ces in vaccine development and antibody-based immunotherapies may be increased. 79 Non-Organ Specific Autoantibodies The occurrence of autoantibodies is significan tly higher in patients with chronic hepatitis C than in patients with other liver diseases, suggesting that HCV may promote the pathogenesis of these autoantibodies.95 A variety of extrahepatic conditions such as autoimmune diseases, are prevalent in about 40% of patients with HCV infections96, but the actual role of HCV is defined in only a few of them. Autoimmune diseases ar e expressed in HCV infect ed patients, including

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28 essential mixed cryoglobulin emia, autoimmune hepatitis97,98, Sjgren's syndrome99, and diabetes mellitus.100 The production of serum autoantibodies is found in 7% of patients with chronic liver disease of different causes, although their mere presence does not define the presence of liver specific autoimmune disease.101 HCV generates an autoimmune response in some individuals that may manifest as non-organ-specific autoantibodies. The autoantibodies found in HCV infection may be either primary or seconda ry, but the link between chronic viral hepatitis and immunologically mediated au toantibody production has not trul y been established. Many of the autoantibodies are believed to be products of tissue damage from the infection, releasing autoantigens. Another explanation implies that a direct reaction against native antigens at the tissue level may occur, and this may explain th e subsequent liver pat hology observed in these HCV patients.102 Antibody seroconversion does not preven t active hepatitis or chronic infection in either chimpanzees or humans. Chronic infect ion supports the persistent cellular and humoral immune responses, and the lack of effective immune responses facilitates HCV infection contributes to this. A combination of viral variability and persistence, damage to hepatocytes, and various HLA genotypes are possibly responsible for the potential role of HCV in inducing autoimmunity.98,102 The therapeutic approach to HCV related ch ronic hepatitis has improved significantly with the application of a combination treatment with pegylated interferon and ribavirin, both of which give superior re sults than IFN alone.103 However, options for patients who have HCVrelated chronic hepatit is and non-organ specific autoanti bodies still remains unresolved. Difficulties in studying this condition are hampered by the limited number of clinical studies, many which have conflicting result s and are limited to IFN monotherapy.104-106

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29 The correlation between non-organ specific au toantibodies and HCV may result from immune modulation induced by th e lymphotropism of HCV itself107 or clinical pathology caused by the genetic variability of the host.108 The relationship between HCV and non-organ specific autoantibodies is solely based on epidemiological evidence and the clinic al significance remains undetermined. It has been observed that HCV patients who are both positive for non-organ specific autoantibodies and treated with interferon have an aggravation of HCV disease activity in during this treatment. This suggests that the immunomodulatory act ivity of interferon may activate an autoimmune reaction and the occurrence of non-organ sp ecific autoantibodies is the trademark of a subclinical autoimmune disease or non-pathological autoimmunity.98,109,110

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30 CHAPTER 2 NEUTRALIZING ANTIBODIES IN HCV INFECTION Experimental Rationale Several recent studies have i ndicated a favorable role of ne utralizing antibodies in acute and chronic HCV infection. Virus neutralizing antibodies react with infectious agents to destroy or inhib it infectiveness and virulence, but the specific role of these antibodies in the context of HCV is unclear. In general, the presence of neutralizing antibodies is essential for a successful vaccination. Therefore, understand ing the role of virus neutralizing antibodies in HCV infection is critical for the design of effective strategi es toward limiting HCV infection and subsequent adverse consequences. There is little information on the specific attributes and clinical relevance of the antibody response against HCV infection.62 This is primarily due to the lack of in vitro cell culture assays capable of m easuring and quantifying such eff ector activity. The objective of our study is to examine the role of HCV neut ralizing antibodies using the newly developed infectious HCV cell culture system, as well as evaluate the impact, clinical significance, and outcome of infection in these patients.1,16 Materials and Methods HCV Constructs and Viral Particle Generation The pJFH-1 plasm id was a gift from Dr. Ta kaji Wakita (Tokyo Metr opolitan Institute for Neuroscience, Tokyo, Japan). The linearized DNA was purified and used as a template for in vitro transcription using MEGAscrip t kit (Ambion, Austin, TX). In vitro transcribed genomic JFH-1 RNA was delivered into Huh-7.5 cells by electroporation. The transfected cells were transferred to complete Dulbeccos Modified Eagle Medium and cultured for the indicated period. Cells were passaged every 3 days, and corresponding supernatants were collected and filtered with a 0.45m filter device and frozen at -80oC. The viral titers were expressed as

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31 focus-forming units per milliliter, determined by the average number of NS5A-positive foci detected through a series of dilutions using the Huh-7.5 cell line as host cells. We have demonstrated efficient viral replication and pr oduction in this cell cu lture system and viral replication is relatively stable in this cell culture system, alt hough fluctuation has been noted. Our NS5A monoclonal antibody (generated by Dr. Johnson Lau, Hybridoma Laboratory, University of Florida) is reactive against this genotype 2a virus. Supernatant from the cell culture contains abundant infectious viral particles (i.e.105-106 ffu/mL), as determined by immunostaining after viral inf ection of a fresh cell culture. Cell Culture Huh-7.5 cells were kindly provided by Dr. Ch arles M. Rice (R ockefeller University, New York, NY). The cell line has been continuously passaged for more than 10 months without obvious morphological changes. All cell lines we re propagated in Dulbeccos Modified Eagle Medium supplemented with 10% fetal bovine serum, 200 mol/L l-glutamine, nonessential amino acids, penicillin, and streptomycin (Invitroge n, Carlsbad, CA). Cells were kept in a 37 C humidified incubator with 5% CO2. For each experiment cells were seeded in 24 well tissueculture dishes containing 1 mL of media. Patient Sera Serum samples were collected with informed consent (IRB 102-2006) from patients at the Health Science Center at the University of Florida. Both HCV-positive and negative serum samples (as determined by PCR) were received on a bi-weekly basis and aliquotted into 1.5 mL microcentrifuge tubes. Each sample was a ssigned a non-identifiable number. Samples were stored at -80 C until use. Medical information was retrieved from the Shands Hospital medical record database after all resu lts were completed (IRB 102-2006).

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32 Twenty-four well tissue culture dishes were used to culture Huh7.5 cells grown to 30-50% were counted from pictures to determine HCV specific antibody titers in patient sera. Optimal Incubation Period To determ ine the appropriate incubation period to provide a measureable virus titer, infectivity titrations of the JFH-1 strain were examined during a period of 3 to 10 days after inoculation. Virus titers of the JFH-1 strain in relation to incubation time and pe rcentage of cell growth were measured. Cell growth reached a plateau at 7 days of incubation and 100 L of JFH-1 virus proved to be effective at inf ecting Huh7.5 cells during this time period. Immunostaining from three experiments done independently gave th e same results. Serum Controls The negative-control serum samples collected from a human who had not been infected with HCV (IRB 102-2006). The positive-control serum sample was a patient who had been exposed to HCV by accidental n eedlestick, had detectable HCV RNA, but then spontaneously cleared the virus. Each assay performed contained internal cont rols, negative (no JFH-1 virus) and positive (100 l JFH-1 virus). Indirect Immunofluorescence Microscopy Cells were fixed with 5% acetic acid in 95% ethan ol for 10 minutes at -20C. Cells were washed with phosphate-buffered saline and in cubated with monoclonal antibody to HCV NS5A protein for 1 hour. The secondary antibody wa s FITC-labeled goat anti-mouse immunoglobulin G antibody (Southern Biotech, Birmingham, AL). The nuclei were counterstained with 4 ,6diamidino-2-phenylindole (DAPIVectaShield, Vector Laboratories, Burlingame, CA), and cells were preserved with 50% glycerol. Examina tion was performed with a fluorescent Leica DMIRE2 microscope and analyzed with the Open Lab 3.1.5 software.

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33 Scoring Process For each serum dilution tested, a photograph co ntaining at least 150 cells was taken within 24 hours of the staining procedure. Positive cells a nd total cells were counted from pictures to determine HCV specific antibody titers. The Ec50 was calculated to be the concentration in which the viral replicat ion is reduced by 50%. Results Huh7.5 cells were plated to approximately 30 -50% confluency in a 24-well tissue culture dish. Initially, various d ilutions of virus were used until an optimal amount, relative to the number of cells, was determined (i.e., 100 L). The experimental basis for this system is to combine JFH-1 virus and dilutions of patient se rum for a period of 30 minutes. This allows sufficient time for association between the virus and potential neutralizing antibodies. Huh7.5 cells were then inoculated with the virus and se ra solution. After 7 days of incubation, the cells were fixed and stained by indirect immunofluorescence with a monoclonal antibody against HCV NS5A protein. Table 2-1 represents the main characteristics of the study population. The number of serum samples tested included 150 HCV-positive patients and 6 negative controls. The study population included one acutely infected patient, who is designated as a positive control. The HCV-positive patients were comprised of 4 ge notypes. Genotype 1a/1b totaled 40 patients, genotype 2a/2b totaled 9 patients, genotype 3a/3b totaled 5 patients and genotype 4a/4b totaled 6 patients. There were 90 patients with unknow n genotypes. The HCV status and patient characteristics of each serum samp le were revealed by database review after all experimental results had been completed. Figure 2-1 shows results obtained from immunofluorescence microscopy. Negative controls were performed on each 24 well plate and all were found to be 100% negative for NS5A

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34 protein. Positive controls consisted of 100 uL of JFH-1 virus. For all 150 HCV-positive patients and 5 HCV negative patients, the serum was dilu ted to 6 concentrations (1:3, 1:5, 1:15, 1:25, 1:125 and 1:625). All patients, once serum was received, were given nonidentifiable numbers and the identity of patients was not determined. Digital images were taken on a Leica fluorescent microscope and the percentage of po sitive cells was determined. The antibody titer is expressed as Ec50, the dilution at which viral infection is decreased by 50%. The Ec50 of HCV-positive patient samples (Pat ients #1-150) show that with an increased amount of serum, more viral particles are neutralized. The HCV-negative patients (Neg Patients #1-6) demonstrates no relationship relative to the amount of serum used. Out of 150 HCV-positive patients, 65.3% of them showed a positive correlation between the amount of serum applied on the cells and the decrease of HCV-positive cells. Figure 2-2 shows that genotype 1 patients required more HCV positive serum to effectively reduce the virus, as the average Ec50 was a 1:5 dilution. Comparatively, the majority of genotype 2 patients displayed an Ec50 of 1:25. The amount of serum required for genotype 3 and 4 patients was in between with an average Ec50 of 1:15. Since the JFH-1 virus is genotype 2a, it is expected that genotype 2 patients woul d display a more positive response to neutralizing antibodies. These results not only validate our assay but also support the notion of a crossprotective antibody. Overall, we have successfully established a reliable and robust assay for neutralizing antibodies in chronic HCV infection and built a large database of HCV positive patient characteristics (1,133 total patients).

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35 Figure 2-1. IIF of HCV-infected Huh7.5 cells as reflected in Patient 59. Cellular nuclei are stained with DAPI (blue) and HCV NS5A pr otein is stained with FITC (green). Pos Neg 1:15 1:5 1:625 1:125 1:25 1:3

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36 0 2 4 6 8 10 12 14 16 1:31:51:151:251:1251:625 Ec50Number of Patients 0 0.5 1 1.5 2 2.5 3 3.5 4 4.5 1:31:51:151:251:1251:625 Ec50Number of Patients 0 0.5 1 1.5 2 2.5 3 3.5 1:31:51:151:251:1251:625 Ec50Number of Patients 0 0.5 1 1.5 2 2.5 3 3.5 1:31:51:151:251:1251:625 Ec50Number of Patients Figure 2-2. The Ec50 of HCV positive patients in each genotype. The Ec50 is defined as the concentration of serum which reduces the infectivity by 50%. Genotype 1 Genotype 2 Genotype 3 Genotype 4

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37 Table 2-1. Patient characteris tics and immunofluorescence micr oscopy of non-organ specific autoantibodies. HepG2 cells were immunostained with goat -human IgG antibody; green DAPI was used for nuclear counterstaining, blue Patient 1Age 57 Sex F HCV StatusPositive Liver EnzymesALT 28 / AST 44 Viral Load Log Viral Load Genotype Ec50 1 to 125 A utoantibodies Patient 2Age 51 Sex M HCV StatusPositive Liver EnzymesALT 102 / AST 179 Viral Load Log Viral Load Genotype Ec50 1 to 125 A utoantibodies Patient 3Age SexF HCV StatusPositive Liver EnzymesALT 45 / AST 63 Viral Load Log Viral Load Genotype Ec501 to 125 A utoantibodiesCytoplasmic Patient 4 Age SexF HCV StatusPositive Liver EnzymesALT 28 / AST 32 Viral Load766333 Log Viral Load5.89 Genotype1a Ec501 to 15 A utoantibodiesCytoplasmic

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38 Table 2-1. Continued Patient 5Age SexM HCV StatusPositive Liver EnzymesALT 26 / AST 29 Viral Load Log Viral Load Genotype1b Ec501 to 5 A utoantibodiesCytoplasmic Patient 6Age 62 Sex M HCV StatusPositive Liver EnzymesALT 34 / AST 30 Viral Load Log Viral Load6.23 Genotype1a Ec50 1 to 125 A utoantibodiesCytoplasmic Patient 7Age SexF HCV StatusPositive Liver EnzymesALT 103 / AST 155 Viral Load94354 Log Viral Load4.98 Genotype Ec501 to 125 A utoantibodiesBoth

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39 Table 2-1. Continued Patient 8Age Sex HCV StatusPositive Liver Enzymes Viral Load Log Viral Load Genotype Ec501 to 25 A utoantibodiesBoth Patient 9Age SexM HCV StatusPositive Liver EnzymesALT 29 / AST 16 Viral Load487869 Log Viral Load5.69 Genotype Ec501 to 25 A utoantibodiesNuclea r Patient 10Age 57 Sex M HCV StatusPositive Liver EnzymesALT 14 / AST 14 Viral Load134759 Log Viral Load5.13 Genotype4c\4d Ec50 1 to 25 A utoantibodiesBoth

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40 Table 2-1. Continued Patient 11Age SexM HCV StatusPositive Liver EnzymesALT 41 / AST 36 Viral Load Log Viral Load<2.79 Genotype3a Ec501 to 25 A utoantibodiesBoth Patient 12Age SexM HCV StatusPositive Liver EnzymesALT 95 / AST 54 Viral Load432754 Log Viral Load Genotype Ec501 to 15 A utoantibodiesBoth Patient 13Age SexF HCV StatusPositive Liver EnzymesALT 7 / AST 15 Viral Load Log Viral Load Genotype Ec501 to 125 A utoantibodiesNuclea r

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41 Table 2-1. Continued Patient 14Age 43 Sex F HCV StatusPositive Liver EnzymesALT 83 / AST 71 Viral Load567123 Log Viral Load5.75 Genotype1b Ec50 1 to 25 A utoantibodiesCytoplasmic Patient 15Age SexM HCV StatusPositive Liver EnzymesALT 72 / AST 44 Viral Load Log Viral Load4.81 Genotype1a Ec501 to 5 A utoantibodiesCytoplasmic Patient 16Age SexM HCV StatusPositive Liver EnzymesALT 82 / AST 102 Viral Load Log Viral Load Genotype Ec501 to 125 A utoantibodiesNeithe r

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42 Table 2-1. Continued Patient 17Age SexF HCV StatusPositive Liver EnzymesALT 16 / AST 23 Viral Load642Log Viral Load<2.79 Genotype2a/2c Ec501 to 5 A utoantibodiesNuclea r Patient 18Age SexM HCV StatusPositive Liver EnzymesALT 34 / AST 39 Viral Load285734 Log Viral Load5.46 Genotype Ec501 to 25 A utoantibodiesBoth Patient 19Age SexF HCV StatusPositive Liver EnzymesALT 36 / AST 29 Viral Load Log Viral Load Genotype Ec501 to 125 A utoantibodiesNuclea r Patient 20Age SexM HCV StatusPositive Liver EnzymesALT 36 / AST 27 Viral Load Log Viral Load Genotype Ec501 to 15 A utoantibodies Patient 21Age SexF HCV StatusPositive Liver EnzymesALT 127 / AST 121 Viral Load89954 Log Viral Load4.95 Genotype1a Ec501 to 5 A utoantibodiesNuclea r

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43 Table 2-1. Continued Patient 22Age SexF HCV StatusPositive Liver EnzymesALT 34 / AST 35 Viral Load Log Viral Load Genotype Ec501 to 5 A utoantibodiesNuclea r Patient 23Age 61 Sex F HCV StatusPositive Liver EnzymesALT 13/ AST 27 Viral Load3156 Log Viral Load Genotype1a Ec50 1 to 3 A utoantibodiesNuclea r Patient 24Age 45 Sex F HCV StatusPositive Liver EnzymesALT 21 / AST 22 Viral Load3861110 Log Viral Load6.59 Genotype1 Ec50 1 to 25 A utoantibodiesBoth Patient 25Age 42 Sex M HCV StatusPositive Liver EnzymesALT 48 / AST 44 Viral Load Log Viral Load Genotype Ec50 1 to 15 A utoantibodiesNuclea r Patient 26Age SexF HCV StatusPositive Liver EnzymesALT 13 / AST 11 Viral Load Log Viral Load Genotype Ec501 to 15 A utoantibodiesNuclea r Patient 27Age 57 Sex M HCV StatusPositive Liver EnzymesALT 11 / AST 12 Viral Load615 Log Viral Load<2.79 Genotype Ec50 1 to 25 A utoantibodiesCytoplasmic

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44 Table 2-1. Continued Patient 28Age SexM HCV StatusPositive Liver Enzymes Viral Load162440 Log Viral Load5.21 Genotype Ec501 to 15 A utoantibodiesNeithe r Patient 29Age SexF HCV StatusPositive Liver EnzymesALT 13 / AST 19 Viral Load615 Log Viral Load Genotype Ec501 to 15 A utoantibodiesNeithe r Patient 30Age SexF HCV StatusPositive Liver Enzymes Viral Load505200 Log Viral Load Genotype1a Ec501 to 25 A utoantibodiesNuclea r Patient 31Age 78 Sex F HCV StatusPositive Liver EnzymesALT 227 / AST 257 Viral Load Log Viral Load Genotype Ec50 1 to 5 A utoantibodiesNuclea r Patient 32Age 65 Sex F HCV StatusPositive Liver EnzymesALT 9 / AST 17 Viral Load615 Log Viral Load<2.79 Genotype Ec50 1 to 5 A utoantibodiesNuclea r Patient 33Age 43 Sex M HCV StatusPositive Liver EnzymesALT 214 / AST 242 Viral Load615 Log Viral Load<2.79 Genotype1b Ec50 1 to 15 A utoantibodiesNuclea r

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45 Table 2-1. Continued Patient 34Age 57 Sex M HCV StatusPositive Liver EnzymesALT 14 / AST 14 Viral Load134759 Log Viral Load5.13 Genotype4c/4d Ec50 1 to 15 A utoantibodiesNuclea r Patient 35Age 50 Sex M HCV StatusPositive Liver EnzymesALT 26 / ALT 40 Viral Load615 Log Viral Load<2.79 Genotype Ec50 1 to 125 A utoantibodiesBoth Patient 36Age SexM HCV StatusPositive Liver EnzymesALT 58 / AST 43 Viral Load25193 Log Viral Load4.4 Genotype3a Ec501 to 15 A utoantibodiesNuclea r Patient 37Age 43 Sex F HCV StatusPositive Liver EnzymesALT 20 / AST 37 Viral Load Log Viral Load Genotype Ec50 1 to 5 A utoantibodiesNeithe r Patient 38Age 40 Sex HCV StatusPositive Liver Enzymes Viral Load Log Viral Load Genotype Ec50 1 to 25 A utoantibodiesNeithe r Patient 39Age 55 Sex F HCV StatusPositive Liver Enzymes Viral Load Log Viral Load Genotype2b Ec50 1 to 15 A utoantibodiesNuclea r

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46 Table 2-1. Continued Patient 40Age 62 Sex F HCV StatusPositive Liver EnzymesALT 122 / AST 109 Viral Load4164870 Log Viral Load Genotype1b Ec50 1 to 3 A utoantibodiesNuclea r Patient 41Age 45 Sex F HCV StatusPositive Liver EnzymesALT 64 / AST 39 Viral Load1606970 Log Viral Load6.21 Genotype1a Ec50 1 to 3 A utoantibodiesNuclea r Patient 42Age 49 Sex M HCV StatusPositive Liver Enzymes Viral Load558272 Log Viral Load6.07 Genotype1b Ec50 1 to 5 A utoantibodiesNeithe r Patient 43Age SexF HCV StatusPositive Liver EnzymesALT 46 / AST 38 Viral Load Log Viral Load Genotype Ec501 to 25 A utoantibodiesNuclea r Patient 44Age SexM HCV StatusPositive Liver EnzymesALT 29 / AST 24 Viral Load2032330 Log Viral Load6.31 Genotype1b Ec501 to 25 A utoantibodiesNeithe r Patient 45Age 49 Sex F HCV StatusPositive Liver EnzymesALT 9 / AST 16 Viral Load Log Viral Load Genotype Ec50 1 to 5 A utoantibodiesNeithe r

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47 Table 2-1. Continued Patient 46Age 35 Sex M HCV StatusPositive Liver EnzymesALT 281 / AST 509 Viral Load1740650 Log Viral Load6.23 Genotype1b Ec50 1 to 15 A utoantibodiesCytoplasmic Patient 47Age 45 Sex M HCV StatusPositive Liver EnzymesALT 64 / AST 48 Viral Load2997210 Log Viral Load6.48 Genotype Ec50 1 to 25 A utoantibodiesNeithe r Patient 48Age SexM HCV StatusPositive Liver EnzymesALT 20 / AST 16 Viral Load Log Viral Load Genotype Ec501 to 15 A utoantibodiesNeithe r Patient 49Age 56 Sex M HCV StatusPositive Liver EnzymesALT 20 / AST 33 Viral Load Log Viral Load Genotype Ec50 1 to 125 A utoantibodiesCytoplasmic Patient 50Age 47 Sex M HCV StatusPositive Liver EnzymesALT 46 / AST 37 Viral Load409457 Log Viral Load Genotype Ec50 1 to 25 A utoantibodiesCytoplasmic Patient 51Age 65 Sex M HCV StatusPositive Liver EnzymesALT 36 / AST 41 Viral Load40351 Log Viral Load4.61 Genotype Ec50 1 to 5 A utoantibodiesNuclea r

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48 Table 2-1. Continued Patient 52Age 70 Sex M HCV StatusPositive Liver EnzymesALT 69 / AST 48 Viral Load Log Viral Load Genotype1a Ec50 1 to 5 A utoantibodiesNeithe r Patient 53Age 36 Sex F HCV StatusPositive Liver EnzymesALT 12 / AST 16 Viral Load Log Viral Load Genotype Ec50 1 to 125 A utoantibodiesCytoplasmic Patient 54Age SexF HCV StatusPositive Liver EnzymesALT 60 / AST 101 Viral Load206092 Log Viral Load5.31 Genotype1a Ec501 to 125 A utoantibodiesBoth Patient 55Age SexM HCV StatusPositive Liver EnzymesALT 146 / AST 168 Viral Load1740650 Log Viral Load5.68 Genotype1b Ec501 to 15 A utoantibodiesNuclea r

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49 Table 2-1. Continued Patient 56Age Sex HCV StatusPositive Liver Enzymes Viral Load Log Viral Load Genotype Ec501 to 3 A utoantibodiesNuclea r Patient 57Age SexM HCV StatusPositive Liver EnzymesALT 22 / AST 30 Viral Load Log Viral Load Genotype Ec501 to 25 A utoantibodiesNuclea r Patient 58Age SexM HCV StatusPositive Liver EnzymesALT 42 / AST 95 Viral Load53714 Log Viral Load4.73 Genotype2b Ec501 to 15 A utoantibodiesNeithe r

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50 Table 2-1. Continued Patient 59Age SexM HCV StatusPositive Liver EnzymesALT 28 / AST 28 Viral Load1227870 Log Viral Load6.51 Genotype1a Ec501 to 25 A utoantibodiesNuclea r Patient 60Age SexF HCV StatusPositive Liver EnzymesALT 11 / AST 12 Viral Load Log Viral Load Genotype Ec501 to 25 A utoantibodiesNuclea r Patient 61Age SexM HCV StatusPositive Liver EnzymesALT 58 / AST 103 Viral Load1451360 Log Viral Load6.16 Genotype2b Ec501 to 25 A utoantibodiesNuclea r

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51 Table 2-1. Continued Patient 62Age SexF HCV StatusPositive Liver EnzymesALT 19 / AST 23 Viral Load<615 Log Viral Load<2.79 Genotype3a Ec501 to 15 A utoantibodiesBoth Patient 63Age SexF HCV StatusPositive Liver EnzymesALT 203 / AST 161 Viral Load352041 Log Viral Load5.55 Genotype2b Ec501 to 25 A utoantibodiesCytoplasmic Patient 64Age SexF HCV StatusPositive Liver EnzymesALT 14 / AST 28 Viral Load615 Log Viral Load<2.79 Genotype Ec501 to 3 A utoantibodiesNeithe r Patient 65Age SexM HCV StatusPositive Liver EnzymesALT 31 / AST 17 Viral Load86727 Log Viral Load4.94 Genotype1a/1b Ec501 to 5 A utoantibodiesNuclea r Patient 66Age SexM HCV StatusPositive Liver EnzymesALT 43 / AST 42 Viral Load409457 Log Viral Load5.61 Genotype Ec501 to 25 A utoantibodiesNeithe r

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52 Table 2-1. Continued Patient 67Age SexF HCV StatusPositive Liver EnzymesALT 27 / AST 34 Viral Load Log Viral Load Genotype Ec501 to 15 A utoantibodiesNuclea r Patient 68Age 46 Sex F HCV StatusPositive Liver EnzymesALT 6 / AST 14 Viral Load Log Viral Load Genotype Ec50 1 to 25 A utoantibodiesCytoplasmic Patient 69Age SexF HCV StatusPositive Liver EnzymesALT 69 / AST 107 Viral Load Log Viral Load Genotype Ec501 to 3 A utoantibodiesCytoplasmic Patient 70Age 47 Sex M HCV StatusPositive Liver EnzymesALT 71 / AST 31 Viral Load<6.15 Log Viral Load<2.79 Genotype Ec50 1 to 5 A utoantibodiesCytoplasmic Patient 71Age 56 Sex M HCV StatusPositive Liver EnzymesALT 31 / AST 17 Viral Load86727 Log Viral Load4.94 Genotype1a/1b Ec50 1 to 15 A utoantibodiesNeithe r Patient 72Age SexF HCV StatusPositive Liver EnzymesALT 43 / AST 47 Viral Load7692310 Log Viral Load>6.89 Genotype1b Ec501 to 15 A utoantibodiesNeithe r

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53 Table 2-1. Continued Patient 73Age SexM HCV StatusPositive Liver EnzymesALT 57 / AST 24 Viral Load615 Log Viral Load<2.79 Genotype Ec501 to 25 A utoantibodiesNeithe r Patient 74Age 52 Sex F HCV StatusPositive Liver EnzymesALT 14 / AST 28 Viral Load Log Viral Load Genotype Ec50 1 to 25 A utoantibodiesNuclea r Patient 75Age SexF HCV StatusPositive Liver EnzymesALT 21 / AST 24 Viral Load984810 Log Viral Load5.99 Genotype2b Ec501 to 5 A utoantibodiesNeithe r Patient 76Age 57 Sex M HCV StatusPositive Liver EnzymesALT 28 / AST 90 Viral Load422225 Log Viral Load5.63 Genotype Ec50 1 to 15 A utoantibodiesNeithe r Patient 77Age 49 Sex M HCV StatusPositive Liver EnzymesALT 20 / AST 20 Viral Load Log Viral Load Genotype Ec50 1 to 15 A utoantibodiesNuclea r Patient 78Age 56 Sex M HCV StatusPositive Liver EnzymesALT 160 / AST 85 Viral Load789660 Log Viral Load5.9 Genotype2b Ec50 1 to 125 A utoantibodiesNeithe r

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54 Table 2-1. Continued Patient 79Age SexF HCV StatusPositive Liver EnzymesALT 63 / AST 45 Viral Load539374 Log Viral Load5.73 Genotype Ec501 to 5 A utoantibodiesNeithe r Patient 80Age 39 Sex F HCV StatusPositive Liver EnzymesALT 153/ AST 71 Viral Load615 Log Viral Load<2.79 Genotype Ec50 1 to 5 A utoantibodiesNuclea r Patient 81Age 53 Sex F HCV StatusPositive Liver EnzymesALT 75 / AST 66 Viral Load3760620 Log Viral Load Genotype Ec50 1 to 15 A utoantibodiesNeithe r Patient 82Age 54 Sex F HCV StatusPositive Liver EnzymesALT 58 / AST 67 Viral Load Log Viral Load5.62 Genotype1a Ec50 1 to 5 A utoantibodiesNeithe r Patient 83Age 50 Sex F HCV StatusPositive Liver EnzymesALT 25 / AST 61 Viral Load Log Viral Load Genotype3a Ec50 1 to 15 A utoantibodiesNeithe r Patient 84Age 49 Sex M HCV StatusPositive Liver EnzymesALT 121 / AST 85 Viral Load1864040 Log Viral Load5.99 Genotype4 Ec50 1 to 15 A utoantibodiesNeithe r

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55 Table 2-1. Continued Patient 85Age 35 Sex M HCV StatusPositive Liver EnzymesALT 146 / AST 168 Viral Load1740650 Log Viral Load5.68 Genotype1b Ec50 1 to 5 A utoantibodiesNeithe r Patient 86Age 51 Sex M HCV StatusPositive Liver Enzymes Viral Load370361 Log Viral Load Genotype1a Ec50 1 to 3 A utoantibodiesNuclea r Patient 87Age 58 Sex M HCV StatusPositive Liver Enzymes Viral Load619 Log Viral Load Genotype Ec50 1 to 5 A utoantibodiesNeithe r Patient 88Age 52 Sex M HCV StatusPositive Liver Enzymes Viral Load124561 Log Viral Load Genotype1a Ec50 1 to 3 A utoantibodiesNeithe r

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56 Table 2-1. Continued Patient 89Age 58 Sex M HCV StatusPositive Liver Enzymes Viral Load Log Viral Load Genotype Ec50 1 to 5 A utoantibodies Patient 90Age 56 Sex F HCV StatusPositive Liver Enzymes Viral Load Log Viral Load Genotype Ec50 1 to 125 A utoantibodies Patient 91Age Sex HCV StatusPositive Liver Enzymes Viral Load Log Viral Load Genotype Ec501 to 25 A utoantibodies

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57 Table 2-1. Continued Patient 92Age Sex HCV StatusPositive Liver Enzymes Viral Load Log Viral Load Genotype Ec501 to 25 A utoantibodies Patient 93Age Sex HCV StatusPositive Liver Enzymes Viral Load Log Viral Load Genotype Ec501 to 15 A utoantibodies Patient 94Age Sex HCV StatusPositive Liver Enzymes Viral Load Log Viral Load Genotype Ec501 to 3 A utoantibodies

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58 Table 2-1. Continued Patient 95Age Sex HCV StatusPositive Liver Enzymes Viral Load Log Viral Load Genotype Ec501 to 15 A utoantibodies Patient 96Age Sex HCV StatusPositive Liver Enzymes Viral Load Log Viral Load Genotype Ec501 to 15 A utoantibodies Patient 97Age Sex HCV StatusPositive Liver Enzymes Viral Load Log Viral Load Genotype Ec501 to 125 A utoantibodies Patient 98Age Sex HCV StatusPositive Liver Enzymes Viral Load Log Viral Load Genotype Ec501 to 25 A utoantibodies

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59 Table 2-1. Continued Patient 99Age Sex HCV StatusPositive Liver Enzymes Viral Load Log Viral Load Genotype Ec501 to 125 A utoantibodies Patient 100Age 68 Sex HCV StatusPositive Liver Enzymes Viral Load Log Viral Load Genotype1b Ec50 1 to 5 A utoantibodies Patient 101Age Sex HCV StatusPositive Liver Enzymes Viral Load Log Viral Load Genotype Ec501 to 5 A utoantibodies Patient 102Age Sex HCV StatusPositive Liver Enzymes Viral Load Log Viral Load Genotype Ec501 to 15 A utoantibodies Patient 103Age Sex HCV StatusPositive Liver Enzymes Viral Load Log Viral Load Genotype Ec501 to 25 A utoantibodies Patient 104Age Sex HCV StatusPositive Liver Enzymes Viral Load Log Viral Load Genotype Ec501 to 3 A utoantibodies

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60 Table 2-1. Continued Patient 105Age SexM HCV StatusPositive Liver EnzymesALT 172 / AST 93 Viral Load1309300 Log Viral Load612 Genotype2b Ec501 to 25 A utoantibodies Patient 106Age SexM HCV StatusPositive Liver Enzymes Viral Load328456 Log Viral Load5.52 Genotype Ec501 to 25 A utoantibodies Patient 107Age SexF HCV StatusPositive Liver Enzymes Viral Load1957240 Log Viral Load6.29 Genotype Ec501 to 15 A utoantibodies Patient 108Age SexM HCV StatusPositive Liver EnzymesALT 23 / AST 31 Viral Load3002150 Log Viral Load<2.79 Genotype3a Ec501 to 5 A utoantibodies Patient 109Age SexM HCV StatusPositive Liver EnzymesALT 107 / AST 339 Viral Load188924 Log Viral Load5.28 Genotype Ec501 to 15 A utoantibodies Patient 110Age SexF HCV StatusPositive Liver EnzymesALT 38 / AST 62 Viral Load615 Log Viral Load<2.79 Genotype Ec501 to 3 A utoantibodies

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61 Table 2-1. Continued Patient 111Age 42 Sex F HCV StatusPositive Liver EnzymesALT 115 / AST 118 Viral Load661405 Log Viral Load5.82 Genotype1a Ec50 1 to 15 A utoantibodiesNuclea r Patient 112Age SexM HCV StatusPositive Liver EnzymesALT 201 / AST 211 Viral Load Log Viral Load Genotype Ec501 to 15 A utoantibodiesCytoplasmic Patient 113Age SexF HCV StatusPositive Liver EnzymesALT 19 / AST 32 Viral Load Log Viral Load Genotype Ec501 to 5 A utoantibodiesNeithe r Patient 114Age SexM HCV StatusPositive Liver EnzymesALT 40 / AST 31 Viral Load2540240 Log Viral Load6.41 Genotype1a Ec501 to 25 A utoantibodiesNuclea r Patient 115Age SexM HCV StatusPositive Liver EnzymesALT 10 / AST 17 Viral Load615 Log Viral Load<2.79 Genotype Ec501 to 25 A utoantibodiesCytoplasmic Patient 116Age SexM HCV StatusPositive Liver Enzymes Viral Load Log Viral Load Genotype Ec501 to 15 A utoantibodiesNiethe r

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62 Table 2-1. Continued Patient 117Age 54 Sex M HCV StatusPositive Liver EnzymesALT 41 / AST 69 Viral Load Log Viral Load Genotype2b Ec50 1 to 25 A utoantibodiesNuclea r Patient 118Age SexF HCV StatusPositive Liver EnzymesALT 76 / AST 46 Viral Load158300 Log Viral Load5.2 Genotype1a Ec501 to 5 A utoantibodiesNuclea r Patient 119Age SexF HCV StatusPositive Liver EnzymesALT 134 / AST 200 Viral Load280078 Log Viral Load5.45 Genotype Ec501 to 3 A utoantibodiesNuclea r Patient 120Age 54 Sex M HCV StatusPositive Liver EnzymesALT 202 / AST 274 Viral Load27839 Log Viral Load4.45 Genotype Ec50 1 to 25 A utoantibodiesNeithe r Patient 121Age 48 Sex M HCV StatusPositive Liver EnzymesALT 65 / AST 116 Viral Load1246220 Log Viral Load6.1 Genotype1b Ec50 1 to 15 A utoantibodies Patient 122Age SexM HCV StatusPositive Liver EnzymesALT 6 / AST 23 Viral Load615 Log Viral Load<2.79 Genotype Ec501 to 25 A utoantibodiesNeithe r

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63 Table 2-1. Continued Patient 123Age 43 Sex F HCV StatusPositive Liver Enzymes Viral Load615 Log Viral Load<2.79 Genotype Ec50 1 to 25 A utoantibodiesNeithe r Patient 124Age SexM HCV StatusPositive Liver Enzymes Viral Load615 Log Viral Load<2.79 Genotype Ec501 to 15 A utoantibodiesNeithe r Patient 125Age SexF HCV StatusPositive Liver EnzymesALT 25 / AST 26 Viral Load1299570 Log Viral Load6.11 Genotype1a Ec501 to 15 A utoantibodiesNuclea r Patient 126Age SexF HCV StatusPositive Liver EnzymesALT 134 / AST 136 Viral Load183930 Log Viral Load5.27 Genotype3a Ec501 to 25 A utoantibodies Patient 127Age SexF HCV StatusPositive Liver EnzymesALT 37 / AST 112 Viral Load6326440 Log Viral Load6.8 Genotype Ec501 to 125 A utoantibodies Patient 128Age SexM HCV StatusPositive Liver EnzymesALT 93 / AST 70 Viral Load4853690 Log Viral Load6.69 Genotype1a Ec501 to 125 A utoantibodies

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64 Table 2-1. Continued Patient 129Age SexM HCV StatusPositive Liver EnzymesALT 14 / AST 14 Viral Load134759 Log Viral Load5.13 Genotype4c/4d Ec501 to 25 A utoantibodies Patient 130Age SexM HCV StatusPositive Liver EnzymesALT 422 / AST 225 Viral Load2790070 Log Viral Load6.45 Genotype Ec501 to 15 A utoantibodies Patient 131Age SexM HCV StatusPositive Liver EnzymesALT 173 / AST 166 Viral Load215977 Log Viral Load5.34 Genotype1a Ec501 to 15 A utoantibodies Patient 132Age 58 Sex M HCV StatusPositive Liver Enzymes Viral Load449492 Log Viral Load5.65 Genotype Ec50 1 to 25 A utoantibodies Patient 133Age SexF HCV StatusPositive Liver EnzymesALT 36 / AST 32 Viral Load766333 Log Viral Load5.89 Genotype1a Ec501 to 5 A utoantibodies Patient 134Age SexM HCV StatusPositive Liver EnzymesALT 113 / AST 103 Viral Load188168 Log Viral Load5.28 Genotype1a Ec501 to 15 A utoantibodies

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65 Table 2-1. Continued Patient 135Age SexM HCV StatusPositive Liver Enzymes Viral Load449512 Log Viral Load5.65 Genotype Ec501 to 3 A utoantibodies Patient 136Age SexM HCV StatusPositive Liver Enzymes Viral Load188924 Log Viral Load5.28 Genotype Ec501 to 125 A utoantibodies Patient 137Age SexM HCV StatusPositive Liver Enzymes Viral Load75821 Log Viral Load4.88 Genotype Ec501 to 15 A utoantibodies Patient 138Age SexM HCV StatusPositive Liver EnzymesALT 38 / AST 31 Viral Load162440 Log Viral Load5.21 Genotype Ec501 to 3 A utoantibodies Patient 139Age 45 Sex F HCV StatusPositive Liver EnzymesAlt 54 / AST 45 Viral Load124678 Log Viral Load5.1 Genotype4c/4d Ec50 1 to 15 A utoantibodies Patient 140Age SexF HCV StatusPositive Liver EnzymesALT 26 / AST 39 Viral Load478666 Log Viral Load5.68 Genotype1a Ec501 to 25 A utoantibodies

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66 Table 2-1. Continued Patient 141Age SexM HCV StatusPositive Liver EnzymesALT 74 / AST 124 Viral Load688388 Log Viral Load5.84 Genotype1a Ec501 to 5 A utoantibodies Patient 142Age SexM HCV StatusPositive Liver EnzymesALT 65 / AST 51 Viral Load4437640 Log Viral Load6.65 Genotype Ec501 to 125 A utoantibodies Patient 143Age SexM HCV StatusPositive Liver Enzymes Viral Load25193 Log Viral Load4.4 Genotype Ec501 to 5 A utoantibodies Patient 144Age SexM HCV StatusPositive Liver Enzymes Viral Load243568 Log Viral Load5.39 Genotype Ec501 to 25 A utoantibodies Patient 145Age SexM HCV StatusPositive Liver Enzymes Viral Load1363830 Log Viral Load6.14 Genotype Ec501 to 5 A utoantibodies Patient 146Age SexM HCV StatusPositive Liver EnzymesAlt 75 / AST 59 Viral Load340179 Log Viral Load5.53 Genotype Ec501 to 15 A utoantibodies

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67 Table 2-1. Continued Patient 147Age SexM HCV StatusPositive Liver EnzymesALT 44 / AST 59 Viral Load1796940 Log Viral Load6.26 Genotype1b Ec501 to 125 A utoantibodies Patient 148Age SexM HCV StatusPositive Liver EnzymesALT 72 / AST 82 Viral Load7692310 Log Viral Load6.89 Genotype1a Ec501 to 15 A utoantibodies Patient 149Age SexM HCV StatusPositive Liver EnzymesALT 28 / AST 20 Viral Load7299710 Log Viral Load6.86 Genotype1b Ec501 to 5 A utoantibodies Patient 150Age SexM HCV StatusPositive Liver Enzymes Viral Load328456 Log Viral Load5.52 Genotype Ec501 to 3 A utoantibodies

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68 Table 2-1. Continued Negative Patient 1Age 55 Sex M HCV StatusNegative A utoantibodiesNuclea r Negative Patient 2Age 35 Sex F HCV StatusNegative A utoantibodiesBoth Negative Patient 3Age SexF HCV StatusNegative A utoantibodiesNuclea r Negative Patient 4Age SexM HCV StatusNegative A utoantibodiesCytoplasmic Negative Patient 5Age SexM HCV StatusNegative A utoantibodiesNeithe r Negative Patient 6Age SexM HCV StatusNegative A utoantibodiesNeithe r Acute Patient 1Age SexF HCV StatusAcute Responder Ec501:125

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69 CHAPTER 3 NON-ORGAN-SPECIFIC ANTIBODIES IN HCV INFECTION Experimental Rationale Immunological disorders have been frequently described in the c ourse of HCV and nonorgan-specif ic autoantibodies are common exampl es of autoreactivity associated with HCV infection. The pathogenesis and c linical significance, of non-orga n-specific autoantibodies, as well as the relationship to neutra lizing antibodies, in patients w ith chronic hepatitis C are still unclear. The aims of this study were to retrospec tively evaluate the impact, clinical significance, and outcome of infection of nonorgan-specific autoantibodies in patients with chronic HCV. Materials and Methods Patient Sera Serum samples were collected with informed consent (IRB 102-2006) from patients at the Health Science Center at the University of Florida. Both HCV-positive and negative serum samples (as determined by PCR) were received on a bi-weekly basis and aliquotted into 1.5 mL microcentrifuge tubes. Each sample was a ssigned a non-identifiable number. Samples were stored at -80 C until use. Medical information was retrieved from the Shands Hospital medical record database after all resu lts were completed (IRB 102-2006). Indirect Immunofluorescence Microscopy Patient sera with reactivity to non-organ speci fic autoantibodies were observed by Indirect Immunofluorescence (IIF) analyses usi ng commercially prepared HEp-2 cells (Imm unoConcepts, Sacramento, CA) with fl uoresceinconjugated goat anti-human IgG antibodies. Nuclei in the cell substrates were stained with 4 ,6-diamidino-2-phenylindole (DAPI), which was included in the glycer ol mounting medium (VectaShield, Vector

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70 Laboratories, Burlingame, CA). Examination was performed with a Leica confocal microscope. The immunofluorescence was performed by Dr. Ed Chan. Scoring Process To prevent variation from one field to another, at least four overlapping fields of view were exam ined in each patient. Nuclear and cyt oplasmic staining patterns were described and recorded by the intensity of th e staining for FITC by a scale of 0 (negative), 1 (weak), 2 (moderate), 3 (strong), and 4 (s trongest). Other notat ions included the presence and staining patterns of mitotic cells cell cycle variation, nuclear envelo pe, nucleolus, and Golgi apparatus. The scoring was performed by Dr. Ed Chan. Results Tables 3-1 and 3-2 show the quantitative results from the IIF on cryostat sections of HepG2 cell slides. Fluorescence patterns were sc ored on a scale from 0 to 4 for a presence of cytoplasmic, -nuclear antibodies, or -mitochondrial antibodies. Eighteen patients displayed high le vels (score of 2 or above) of -cytoplasmic antibodies. This represents 17.3% of the patient population who were screened for autoantibodies. Six patients were of HCV genotype 1, one patient was genotype 2, one patient was negative for HCV, and 10 patients had indeterminate genotypes. Ten (55.5%) of the patients demonstrating -cytoplasmic antibodies had a high level of neut ralizing antibodies (Ec 50 of 1:125 or 1:25). Forty patients displayed high levels (score 2 or above) of -nuclear antibodies, representing 38.4% of the patien t population who were screened for autoantibodies. Fourteen patients were determined to be HCV genotype 1, four of genotype 2, one of genotype 3, and one of genotype 4. Two patients were negative for HCV and the remaining 18 had indeterminate genotypes. Twelve (30%) of the patients displaying -nuclear antibodies had a high level of neutralizing antibodies (Ec 50 of 1:125 or 1:25).

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71 Ten of the 104 patients measured for autoantibodies displayed both -cytoplasmic and nuclear antibodies in high levels (score of 2 or above). This represents 9.6% of the patient population. Two patients were determined to be HCV genotype 1, two of genotype 3, one of genotype 4, and five patients had indetermin ate genotypes. Eight (80%) of the patients displaying both -cytoplasmic and -nuclear antibodies had a hi gh level of neutralizing antibodies (Ec50 of 1:125 or 1:25). Thirty-six patients (34.6%) in the study population displayed neither -cytoplasmic or nuclear antibodies. Five of these patients had strong levels (score 2 or above) of -mitochondrial antibodies. Nine of the patie nts (25%) displaying neither -cytoplasmic or -nuclear antibodies had high levels of neutralizing antibodies (Ec 50 of 1:125 or 1:25). High levels (score 2 or above) of -mitochondrial antibodies were observed in 12 (11.5%) patients in the study population. Overall, 68 patients (65.3%) of patients in the study population disp layed high levels of -cytoplasmic, -nuclear antibodies, or both -cytoplasmic and -nuclear antibodies in addition to chronic hepatitis C viral infection. Eighty patients (76.9%) displayed either -cytoplasmic, nuclear antibodies, both -cytoplasmic and -nuclear, or -mitochondrial antibodies along with chronic hepatitis C. It should be noted that 98 patients (92.3%) of the chronic hepatitis C patients had some level (sco re 1 or above) of either -cytoplasmic, -nuclear antibodies, both cytoplasmic and -nuclear, or -mitochondrial antibodies. A stude nts T test was used to determine the statistical significance of patient vi ral load and liver enzymes. It was determined that chronic HCV patients with hi gh levels of non-organ-specific an tibodies have lower levels of viral load compared to patients with no pr esence of autoantibodies. Patients with -cytoplasmic antibodies had higher levels of liver enzymes than patients with no presence of autoantibodies.

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72 Table 3-1. Correlation between non-organ specif ic antibodies and HCV genotype. Patients positive for -cytoplasmic, -nuclear, or -mitochondrial autoantibodies were matched to their HCV genotype; 1, 2, 3, 4, negative, or indeterminate. Autoantibodies 1234Neg.UnknownTotal -cytoplasmic610011018 -nuclear1441121840 -cytoplasmic and -nuclear20210510 -mitochondrial 50000712 No autoantibodies 631111830 Genotype Table 3-2. Correlation between non-organ specific antibodies and neutralizing antibodies in HCV positive patients. Patients positive for -cytoplasmic, -nuclear, or mitochondrial antibodies. Patients positive for these autoantibodies were matched to the neutralization assay results (Ec50 score). Autoantibodies 1:31:51:151:251:125 -cytoplasmic13364 -nuclear6911102 -cytoplasmic and -nuclear00253 -mitochondrial 16131 no autoantibodies 271262 Ec50

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73 Table 3-3. Average viral load and liver enzyme levels of HCV positive patients with non-organ specific antibodies. AverageAverageAverage Autoantibodiesviral loadALTAST -cytoplasmic76712073.188.8 -nuclear88815161.261.1 -cytoplasmic and -nuclear83580045.853.7 -mitochondrial 846895 56.559.1 All autoantibodies 850223 87.095.0 No autoantibodies 1388369 60.665.4 Patients were screened for -cytoplasmic, -nuclear, and -mitochondrial antibod ies. The viral load and liver enzymes (ALT and AST) were colle cted from the patient database at Shands Hospital in Gainesville, FL. Table 3-4. Statistical correlation between nonorgan specific antibodies and HCV positive patient characteristics. AverageStatistical AverageStatisticalAverageStatistical Autoantibodiesviral loadsignificanceALTsignificanceASTsignificance -cytoplasmic767120p = 0.126373.1p = 0.051188.8p = 0.0583 -nuclear 888151p = 0.068361.2p = 0.063861.1p = 0.1617 -cytoplasmic and -nuclear835800p = 0.137845.8p = 0.600453.7p = 0.5059 -mitochondrial 8 46895p = 0.206956.5p = 0.845559.1p = 0.7846 All autoantibodies 850223p = 0.041187.0p = 0.103995.0p = 0.1731 No autoantibodies 1388369 N/A60.6 N/A65.4 N/A Patients were screened for -cytoplasmic, -nuclear, and -mitochondrial antibod ies. The viral load and liver enzymes (ALT and AST) were colle cted from the patient database at Shands Hospital in Gainesville, FL. A student's T-test was performed to determine the statistical significance of the patient viral load and liver enzymes.

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74 CHAPTER 4 NOVEL SMALL MOLECULES BLOCKING HCV ENTRY Experimental Rationale CD81 plays a critical ro le in HCV entr y into the host cells and blocking CD81 by antibodies in cell culture can impair HCV inf ection. Therefore, the CD81 receptor and HCV envelope protein binding process could be a new target for anti-HCV therapy. The objective of our study is to use the crystal structure of the CD81 receptor to identify small molecule modulators by a high throughput molecular docking method. Mo lecular docking (DOCK5.1.0) identified and ranked the best molecules that interfere with CD81 and HCV E2 binding from the NCI/DTP database. We aim to use the newly de veloped infectious HCV cell culture system to analyze binding inhibition th at these drugs may impose on CD 81 and HCV envel ope proteins. Materials and Methods Molecular Docking All docking calculations were perform ed with the October 15, 2002, development version of DOCK, ver. 5.1.0.25 The general features of DOCK include rigi d orienting of ligands to receptor spheres, AMBER (Assisted Model Build ing with Energy Refinement) energy scoring, GB/SA (GeneralizedBorn Model of Solvation fo r Small Molecules) solv ation scoring, contact scoring, internal nonbonded energy scoring, ligan d flexibility, and both rigid and torsional simplex minimization. Unlike previously distri buted versions, this release incorporates automated matching, internal energy (used in fl exible docking), scori ng function hierarchy, and new minimizer termination criter ia. The coordinates for the crys tal structure of CD81, PDB code 1G8Q, were used in the molecular docking calc ulations. To prepare the site for docking, all water molecules were removed. Protonation of receptor residues was performed with a computational tool kit for molecular analysis (Sybyl; Tripos). The structure was explored by

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75 using sets of spheres to describe potential binding pockets. The number of orientations per molecule was 100. Intermolecular AMBER energy scoring (VDW_coulombi c), contact scoring, and bump filtering were implemented in DOCK5.1.0. SETOR26 and GRASP were used to generate molecular graphic images. The crysta lization and molecular docking was performed by Dr. David Ostrov. Novel Small Molecules The crystal structure of the CD81 receptor was used to iden tify small molecule modulators by a high throughput molecular docking method. Molecular docking (DOCK5.1.0) identified and ranked the best molecules from the NCI/DTP database directed to the HCV binding target site. The molecules were ordered from the De velopmental Therapeutics Program NCI/NIH. The top hits were assayed in the in vitro infectious JFH-1 HCV cell culture system. HCV Constructs and Viral Particle Generation The pJFH-1 plasm id was a gift from Dr. Ta kaji Wakita (Tokyo Metr opolitan Institute for Neuroscience, Tokyo, Japan). The linearized DNA was purified and used as a template for in vitro transcription using MEGAscrip t kit (Ambion, Austin, TX). In vitro transcribed genomic JFH-1 RNA was delivered into Huh-7.5 cells by electroporation. The transfected cells were transferred to complete Dulbeccos Modified Eagle Medium and cultured for the indicated period. Cells were passaged every 3 days, and corresponding supernatants were collected and filtered with a 0.45m filter device and frozen at -80oC. The viral titers were expressed as focus-forming units per milliliter, determined by the average number of NS5A-positive foci detected through a series of dilutions using the Huh-7.5 cell line as host cells. We have demonstrated efficient viral replication and pr oduction in this cell cu lture system and viral replication is relatively stable in this cell culture system, alt hough fluctuation has been noted. Our NS5A monoclonal antibody (generated by Dr. Johnson Lau, Hybridoma Laboratory,

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76 University of Florida) is reactive against this genotype 2a virus. Supernatant from the cell culture contains abundant infectious viral particles (i.e.105-106 ffu/mL), as determined by immunostaining after viral inf ection of a fresh cell culture. Cell Culture Huh-7.5 cells were kindly provided by Dr. Ch arles M. Rice (R ockefeller University, New York, NY). The cell line has been continuously passaged for more than 10 months without obvious morphological changes. All cell lines we re propagated in Dulbeccos Modified Eagle Medium supplemented with 10% fetal bovine serum, 200 mol/L l-glutamine, nonessential amino acids, penicillin, and streptomycin (Invitroge n, Carlsbad, CA). Cells were kept in a 37 C humidified incubator with 5% CO2. For each experiment cells were seeded in 24 well tissueculture dishes containing 1 mL of media. RNA Isolation Cells were lysed by adding 1m L of TRIZOL r eagent and the sample was incubated for 5 minutes at room temperature to permit complete di ssociation of protein comp lexes. The material was pipetted into a microcen trifuge tube, chloroform (200 L) was added, and tubes were shaken vigorously for 15 seconds. Samples undergo inc ubation at room temperature for 3 minutes and then centrifugation at 12,000 x g for 15 minutes at 4C. The RNA was transferred to a new microcentrifuge tube. Isopropanol (500 L) was added, samples were incubated at room temperature for 10 minutes and then centrifuge d at 12,000 x g for 10 minutes at 4 C. 75% ethanol is added to dry the pell et and samples are centrifuged at 7,500 x g for 5 minutes at 4C. Once dry, the RNA is dissolved in 30 L DEPC water and concentration is determined by spectrometer (BioRad, SmartSpec 3000).

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77 Reverse Transcription and Poly merase Chain Reaction (P CR) Reverse-transcription polymera se chain reaction (RT-PCR) wa s performed using reagents from Invitrogen. For each reaction th e following reagents are required; 0.25 L oligomers, 0.25 L random hexamers, 1 L 10x buffer, 2 L 25mM MgCl2, 0.5 L 10mM dNTP, 1 L 0.1 M DTT, 0.5 L RNase out, and 0.5 L Superscript II (Invitrogen). The PCR conditions were as follows: 25C for 10 minutes, 42C for 45 minut es and 70C for 15 minutes for 40 cycles. Quantitative real-time RT-PCR was performed using fluorophore-labeled LUX primers from Invitrogen for HCV. Reactions were conducted in a 96 -well spectrofluorometric thermal cycler (ABI PRISM 7700 Sequence detector system, Applied Bios ystems, Foster City, CA). Fluorescence was monitored during ev ery PCR at the annealing step. The primers that were used for amplification were as follows: for HCV, 5_-CGCTCAATGCCTGGAGATTTG-3_, 5_GCACTCGCAAGCACCCTATC-3_; for GADPH, 5_-TGCTGGCGCTGAGTACGTC-3_,5_GTGCAGGAGGCATTGCTGA-3_. PCR conditions were as follows: 50C for 2 min, 95C for 10 min (95C for 15 sec, 60C for 1 min) for 40 cycles. The negative control was total RNA from Huh7 cells, and the positive control was in vitro transcribed HCV full-length RNA. Glyceraldehyde-3-phosphate dehydr ogenase (GADPH) served as an internal control. The reagents per reaction are as follows; 9 ul DEPC water, 1 L forward primer, 1 L reverse primer, and 15 L of real time PCR buffer (Invitrogen). Samples are run on the Opticon Monitor 3 System in the Molecular Biology Core Facility at the University of Florida. To determine the HCV titer, JFH-1 full-length RNA was transcribed in vitro and used for generation of standard curve. Results were analyzed using SDS 2.0 softwa re (Applied Biosystems, Foster City, CA). Results for all experiments represent triplicate de terminations and results are reported as means SD.

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78 Indirect Immunofluorescence Microscopy Cells were fixed with 5% acetic acid in 95% ethan ol for 10 minutes at -20C. Cells were washed with phosphate-buffered saline and in cubated with monoclonal antibody to HCV NS5A protein for 1 hour. The secondary antibody wa s FITC-labeled goat anti-mouse immunoglobulin G antibody (Southern Biotech, Birmingham, AL). The nuclei were counterstained with 4 ,6diamidino-2-phenylindole (VectaShi eld, Vector Laboratories, Bur lingame, CA), and cells were preserved with 50% glycerol. Examination wa s performed with a fluorescent Leica DMIRE2 microscope and analyzed with the Open Lab 3.1.5 software. Scoring Process For each serum dilution tested, a photograph containing at least 150 cells was taken. Positive cells and total cells were counted fr om pictures to determine HCV specific antibody titer. Results The com bination of the molecular docking approach and the in vitro cell culture system has allowed us to identify novel compounds that ha ve a potential therapeutic application in HCV infection. Figures 4-1 and 4-2 show that the CD81 receptor was successfully crystalized and targeted for novel small molecule inhibition. By measuring the percentage of positive cells in the IIF assay, Figure 4-3 and 4-4 demonstrate the results from the in vitro CD81 binding inhibition study. The average positive control for all ten experiments wa s 74.1% positive. The percentage positive for each drug is as follows; drug 1 (39.8% 1 nm, 19.8% 5 nm, 16.4 % 20 nm); drug 2 (10.8% 1 nm, 25% 5 nm, 6.9% 20 nm); drug 3 (37.8% 1 nm, 26.2% 5 nm, 11.7% 20 nm); drug 4 (41.6% 1 nm, 21.2% 5 nm, 5.8% 20 nm); drug 5 (47.9% 1 nm, 30.9% 5 nm, 60.4% 20 nm); drug 6 (65.5% 1 nm, 48.2% 5 nm, 34.9% 20 nm); drug 7 (46.2% 1 nm, 71.1% 5 nm, 59.4% 20 nm); drug 8 (30.3% 1 nm,

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79 31.6% 5 nm, 54.1% 20 nm); drug 9 (34.8% 1 nm, 51.3% 5 nm, 34.8% 20 nm); drug 10 (77% 1 nm, 52.7% 5 nm, 80.4% 20 nm). Drugs 1, 3, 4 and 6 showed a dose dependent response. Drugs 2, 5, 8 and 9 showed a decrease in HCV infected cells. All negative controls and intern al controls (DMSO only) were 0% positive for virus. Drugs were tested for toxicity prior to this assay. By measuring intracellular RNA, a dose depe ndent response was obser ved in drug 1. The results were also statistically significant (p .008 10 nm, p .011 100 nm, p .007 1000 nm). A dose dependent response was also s een in drug 2 (p .009 10 nm, p .004 100 nm, p .032 1000 nm) and drug 3 (p .011 10 nm, p .026 100 nm, p .002 1000 nm). Drugs 1 and 3 showed a dose dependent res ponse in both the IIF assay and by measuring intracellular HCV RNA. Overall, it was determ ined that our approach validated this novel method to develop anti-HCV therapeutic agents.

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80 Figure 4-1. Molecular docking strategy for binding inhibition of CD81. A) The crystal structure of CD81; -Helices, magenta; binding pocket in the orientation posed by molecular docking; yellow ; the site selected for molecular docking is encompassed by a scoring grid shown as a white box. B) Shows the structure and conformation of the top 6 hits in the binding pocket of CD81. C) Shows th e structure and conformation of the top hit in the binding pocket of CD81. A B C

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81 Drug Mol. Wt. DOCKScore (kcal/mol)1 267 -18.1 2 366 -17.8 3 277 -17.5 4 292 -16.2 5 264 -15.9 6 253 -15.7 7 263 -15.6 8 191 -96.3 9 236 -90.0 10195 -88.5 Figure 4-2. DOCK Scores for the top 10 scoring compounds A) Chemical Structure of Drug 1, C11H11ClN4O2 B) Chemical Structure of Drug 2, C18H26N2O6. A B

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82 Figure 4-3. CD81 interacts with novel small molecules to inhibit HCV infection in human hepatoma cell lines. Cells were immunos tained with mouse monoclonal anti-NS5A antibody; green DAPI was used for nuclear counterstaining, blue (A) Drug 1: Positive control, 75% positive; 1 nm dr ug, 39.8% positive; 5 nm drug 19.8% positive, 20 nm drug, 16.4% positive. (B) Drug 2: Posi tive control, 55% positive; 1 nm drug, 10.8% positive; 5 nm drug 25% positive, 20 nm drug, 6.9% positive. Pos 1nm 5nm Pos 1 nm 5 nm 20nm 20nm Pos 20nm Pos 20nm A B

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83 Figure 4-4. Histogram summary of the percentage of positive cells in the immunofluorescence assay for 10 drugs at concentra tions of 1 nm, 5 nm and 20 nm.

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84 0 0.2 0.4 0.6 0.8 1 1.2 Pos 10 nm 100 nm 1000 nm 0 0.2 0.4 0.6 0.8 1 1.2 Pos 10 nm 100 nm 1000 nm Figure 4-5. Small novel molecules can inhibit HC V RNA replication in Huh7.5 cells in a dosedependent manner. ( A ) Drug 1 ( B ) Drug 2; Total RNA wa s isolated from HCVinfected Huh7.5 cells and vira l RNA kinetics were determined by real-time RT-PCR. Viral RNA was analyzed 5 days after inf ection and data are representative of 3 repeats. The data were normalized with internal control glyc eraldehyde-3-phosphate dehydrogenase. Drug 1 Drug 2 A B

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85 CHAPTER 5 DISCUSSION AND CONCLUSION Development of a Novel Assay to Analyze Neutraliz ing Antibodies in HCV Infection The recent availability of the HCV cell culture system26 allowed us to develop an assay for determining HCV specific neutralizing antibodies in patient serum. A lthough this cell culture system currently represents HCV genotype 2a, we were able to determine that HCV specific neutralizing antibodies were cro ss-reactive to other HCV genotype s. Our results show that genotype 2 patients have greater neutralizing anti body activity than the genotype 1 patients. This is supported by the limitations of the cell culture system as disc ussed previously, and thus we correctly hypothesized that genotype 2 patients would displa y a higher neutralizing antibody response. The data also correlates with clinical presentation of chronic HCV infection due to the notion that genotype 1 patients have higher vi remia and are more difficult to treat when compared to patients harboring other genotypes. This novel method also provides significant insights into the antibody respons e to HCV. Antibodies from ch ronically infected HCV patients demonstrate broadly-reactiv e neutralizing responses in vitro, but fail to control viral infection in vivo as the majority of the patients had been in fected for many years. Even though chronically infected HCV patients may have high titers of ci rculating neutralizing antibodies, it remains to be determined why their effects are limited in vivo111,112. We have shown the existence of in vitro neutralizing antibodies in human patients and opened up discussion about the potential viral-induced mechanisms that suppress this response in th e initial phase of infection. The immune response to HCV w ill result in either viral clearance or chronic inflammation, and the result is most likely determined very ea rly in the course of infection. In chronically infected HCV patients, the virus is able to use multiple complex strategies leading to its control over the host innate and adaptive immune responses5. Therefore, neutralizing antibody

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86 responses have a significant role in limiting viremia and averting infection16,17,39. Patients who have chronic HCV infections display high tite rs of cross-reactive neutralizing antibodies; however, this response is faster and mo re intense in acute responding patients16. The ability of neutralizing antibodies in determ ining the overall humoral immune response to antiviral therapy still remains an important unanswered question113. Acutely infected patients who are capable of clearing the infection, ha ve a confirmed incidence of neutralizing antibodies that contribute to viral eradication and faci litate control of HCV load76. Conversely, chronic HCV infection is exemplified by failure of the humoral immunity to neutralize virus in the early phase of infection114. In a chronic infection, a multispecifi c T cell response and broadly reactive HCV neutralizing antibodies have been found to incompletely control HCV infection, resulting in the continuous production of escape variants85. Control of HCV infecti on is associated with the development of these early and broad cy totoxic T cell and helper T cell responses16. The inability to cross-neutralize vi ral variants that quickly emerge during acute infection may facilitate viral evasion from neutralizi ng responses in chronic HCV infection76. Evidence supports the existence of a cons erved, neutralizing epitope in the E2 glycoprotein and also supports the notion that antibodies to this regi on can be produced in the course of a natural infection. Broad spectrum and cross-neutra lizing antibodies would be beneficial for development of prophylactic immunizations. It has been shown that anti-E2 antibodies are associated with protection in HC V vaccine trials in chimpanzees69,70,112. The mechanism behind their inability to rid the host of virus is st ill poorly understood and it is believed that host neutralizing antibody response lags behind the rapidly mutating HCV envelope glycoproteins115. Our results correlate with others16 and suggest that an early neutraliz ing antibody response that is both strong and broadly reactive ma y contribute to control of HCV. Recent years of research

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87 have shown that HCV interacts with the hosts immune defense by various effector mechanisms. The precise role of humoral immun ity, in particular the role of neutralizing antibodies, remains incomplete.116 We believe our data supports a positiv e role for neutraliz ing antibodies and indicate that a prophylactic vaccine against HCV may be attainable. Non-Organ Specific Autoantibodies are High ly Prevalent in Chronic HCV Infected Patients A variety of non-organ specific autoantibodies are pr esent in the sera of HCV infected patients, but their clinical significance and pa thogenic role remain undetermined. We were successfully able to determine the prevalence of several autoantibodies in chronic HCV patients, and the results were correlated with HCV genot ype. We hypothesized th at viral antigens of various genotypes may elicit different autoan tibodies or other immunological reactions in chronic HCV infected patients. Numerous studies in this field have demonstrated that the serological pattern of non-organ specific autoantibodies does not correlate with HCV genotype and our study found similar results. An explanat ion for this may be our inability to determine many of the patient genotypes in our study, or th ere may not actually be any association between the pattern of autoantibodies and HCV genotype. It remains to be determined whether the serological markers of autoimmunity in HCV infection have clinical implications117. We determined that the majority of chronic HCV pa tients display high leve ls of non-organ specific autoantibodies. Other researchers have confir med our results and found a high prevalence of non-organ specific autoantibodies in patients with chronic hepatitis C118. The most common method for non-organ specific autoantibody detectio n is the indirect immunofluorescence assay on HepG-2 cells slides. Fl uorescent patterns are able to be identified due to the variability of target-autoantigens in the nuclei of HepG-2 cells that have been previously identified. Frequently, a speckle d or homogenous immunofluorescence pattern is

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88 seen and we were able to validate these findings111. The presence of non-organ specific autoantibodies in chronic HCV patients is likely co rrelated with the severity and duration of liver disease. Cross-reactivity against self-antigens may be associated with the degree of chronic liver disease, but may lack an independent role in the pathogenesis of liver damage. Various host and environmental factors help determine pathoge nesis of chronic HCV infection, including autoimmune reactions, but the mechanisms of these autoimmune reactions have not been determined. In autoimmune liver diseases, high titers of non-organ specif ic autoantibodies with restricted epitopes are observed al ong with a homogeneous B cell response117-119. In contrast, the non-organ specific autoantibodies in HCV infe ction are low titer, polymorphic, and not associated with the severity or clinical outcome of liver diseas e. This likely indicates a nonspecific and generalized modification of autoim mune reactivity in the host immune system. Additional studies are needed to investigate the interactions between the mechanisms of autoimmune disease and the host immune system in chronic HCV infection117. Novel Anti-Viral Small Molecules Can Bl ock HCV Cellular Entry Through the CD81 Receptor Numerous examples of successful virtual scr eens that identify hit molecules have been published recently120-121. Docking and scoring t echnology has been recently successful at identifying novel molecules and we demonstrated th at this screening method can effectively lead to potential therapeutic molecules to inhibit binding of HCV to target cells. In this study, we utilized in silico high-throughput computational methods paired w ith in vivo biochemical assays to develop an efficient screening method which was capable of identifying novel small molecules that inhibit binding to the putative HCV receptor, CD81. A great expansion of targets for therapeutic advances may result from resear ch advances with the assistance of computeraided drug design12.

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89 LIST OF REFERENCES 1 Zeisel MB Fafi-Kremer S, Fofana I, Barth H, Stoll-Keller F, Doffoel M, Baumert TF. Neutralizing antibodies in hepatitis C virus infection. World J Gastroenterol 2007; 13: 4824-4830 2 Dustin LB, Rice CM. Flying under the radar: the immunobiology of hepatitis C. Annu Rev Immunol 2007; 25:71-99 3 Wedemeyer H Cornberg M, Manns MP. Immunopa thogenesis and therapy of hepatitis C. In: Gershwin ME, Vierling, JM, Manns, MP, editors. Liver Immunology. Philadelphia. Hanley and Belfus 2003;223-248 4 Choo QL Kuo G, Weiner AJ, Overby LR, Brad ley DW, Houghton M. Isolation of a cDNA clone derived from a blood-borne non-A, non-B viral hepatitis genome. Science 1989; 244:359-362 5 Spengler U Natterman J. Immunopathogenesis in hepatitis C virus cirrhosis. Clin Sci 2007; 112:141 6 Kaplan M Gawrieh S, Cotler SJ, Jensen DM. Ne utralizing antibodies in hepatitis C virus infection: a review of immunologi cal and clinical characteristics. Gastroenterology 2003; 125:597 7 Tan SL Pause A, Shi Y, Sonenberg N. Hepatitis C therap eu tics: current status and emerging strategies. Nat Rev Drug Discov 2002; 1 : 867-881 8 Neumann-Haefelin C Blum HE, Chisari FV, Thimme R. T cell response in hepatitis C virus infection. J Clin Virol 2005; 32 :75-85 9 Hunziker IP Zurbriggen R, Glueck R, Engler OB, Reichen J, Dai WJ, Pichler WJ, Cerny A. Perspectives: towards a peptide-based vaccine against hepatitis C virus. Mol Immunol 2001; 38:475-484 10 Diedrich G How does hepatitis C virus enter cells? FEBS J 2006; 273:3871-85 11 Dubuisson J Hepatitis C virus proteins. World J Gastroenterol 2007; 13 :2406-15 12 Dubuisson J Penin F, Moradpour D. Interaction of hepatitis C virus proteins with host cell membranes and lipids. Trends Cell Biol 2002; 12: 517-523 13 Goffard A Callens N, Bartosch B, Wychowski C, Cosset FL, Montpellier C, Dubuisson J. Role of N-linked glycans in the functions of hepatitis C virus envelope glycoproteins. J Virol 2005; 79: 8400-8409

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91 27 Helle F Wychowski C, Vu-Dac N, Gustafson KR, Voisset C, Dubuisson J. Cyanovirin-N inhibits hepatitis C virus entry by bi nding to envelope protein glycans. J Biol Chem 2006; 281: 25177-25183 28 Pileri P Uematsu Y, Campagnoli S, Galli G, Fal ugi F, Petracca R, Weiner AJ, Houghton M, Rosa D, Grandi G, Abrignani S. Binding of hepatitis C virus to CD81. Science 1998; 282:938 29 Levy S Shoham T. The tetraspanin webmodul ates immune-signalling complexes. Nat Rev Immunol 2005; 5: 136 30 Helle F Dubuisson J. Hepatitis C virus entry into host cells. Cell Mol Life Sci 2008; 65:100-12 31 Akazawa D Date T, Morikawa K, Murayama A, Miyamoto M, Kaga M, Barth H, Baumert TF, Dubuisson J, Wakita T. CD81 expr ession is important forthe permissiveness of huh7 cell clones for heterogene oushepatitis C virus infection. J Virol 2007; 5: 5036 5045 32 Lindenbach BD, Evans MJ, Syder AJ, Wolk B,Tell inghuisen TL, Liu CC, Maruyama T, Hynes RO, Burton DR, McKeating JA, Rice CM. Complete replication of hepatitis C virus in cell culture. Science 2005; 309 :623 33 Cormier EG Tsamis F, Kajumo F, Durso RJ, Ga rdner JP, and Dragic T. CD81 is an entry coreceptor for hepatitis C virus. Proc Natl Acad Sci USA 2004; 101:7270 34 Scarselli E Ansuini H, Cerino R, Roccasecca RM, Acali S, Filocamo G, Traboni C, Nicosia A, Cortese R & Vitelli A. The hum an scavenger receptor class B type I is a novel candidate receptor for the hepatitis C virus. EMBO J 2002; 21:5017 35 Calvo D Vega MA. Identification, primary structure, and distribution of CLA-1, a novel member of the CD36/LIMPII gene family. J Biol Chem 1993; 268:18929 36 Acton SL, Scherer PE, Lodish HF, Krieger M. Expression cloning of SR-BI, a CD36related class B scavenger receptor. J Biol Chem 1994; 269: 21003 37 Bartosch B Vitelli A, Granier C, Goujon C, Dubuisson J, Pascale S, Scarselli E, Cortese R, Nicosia A & Cosset FL. Cell entry of hepa titis C virus re quires a set of co-receptors that include the CD81 tetraspanin and the SR-BI scavenger receptor. J Biol Chem 2003: 278:41624 41630 38 Kapadia SB Barth H, Baumert T, McKeating JA, Chisari FV. Initiation of hepatitis C virus infection is dependent on choleste rol and cooperativity between CD81 and scavenger receptor B type I. J Virol 2007; 81: 374

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100 BIOGRAPHICAL SKETCH Jennifer Rose Bess was raised in Florida a nd graduated cum laude from Niceville High School in 2000. She began her undergraduate car eer at Troy University in Troy, Alabama and was a scholarship athlete in cross country a nd track. There, she majored in cellular and molecular biology and gradated magna cum laude w ith her Bachelor of Science degree in 2004. In 2004 she began the doctoral program in biomed ical science research at the University of Florida. She began her research in the labor atory of Dr. Chen Liu and pursued the immunology and microbiology degree concentration. She gra duated in 2008 with her PhD and will pursue a career in academia.