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Antibody response to hypervariable region of HCV differs between genotype 1b and 2a
HEPATOLOGY
897
Vol.
22, NO.
4, Pt.
2, 1995
AASLD
EXPRESSION OF ENZYMATICALLY ACTIVE HCV NS3 PROTEASE (GENOTYPE lb) AND THE DEVELOPMENT OF AN IN-VITRO PROTRASE ASSAY SYSTEM Feller JA, Carroll AR1. Sanear DV1. Lau JYN. Clarke BE’. Section of Hepatobiiary Diseases, Department of Medicine, University of Florid& Gainesville, FL, and Wellcome Research Laboratories’, Kent, UK.
898
EXPRESSION AND CHAFUCTERIZATION OF THE NSSB (BNADEPENDENT RNA POLYMERASE) GENE OF HEPATITIS C VIRUS. RH Al. Y Wang. . Y Xie. CH Haeedom. Division of Digestive Diseases, Emory University School of Medicine and the VA Medical Center, Atlanta. The development of vaccines using recombinant hepatitis C virus (HCV) proteins appears to be difficult and suggests that the development of new antiviral agents should be explored. Nucleoside analogue inhibitors of viral DNA polymerases have been effective in treating some chronic viral infections. In addition, many novel nucleoside analogue inhibitors remain to be tested in different systems (Antimicrob Agents Chemother 1993;37:1720-2). The NSSB gene of HCV encodes a protein that contains sequence motifs that are highly conserved in other viral RNA-dependent RNA polymerases (RDRP). We have subcloned the region encoding the NSSB gene into a variety of prokaryotic expression vectors and successfully expressed a protein of approximately 68 kDa in E. coli that encodes the putative HCV RDRP. E. coli maximally express this protein 2 hours after induction with IPTG as determined by Coomassie Blue staining of SDS-PAGE analyzed cell lysates. In addition, methods to solubilize recombinant NSSB protein under nondenaturing conditions have been determined and used to partially purify the enzyme. Recombinant protein purified by SDS-PAGE has been used to raise polyclonal rabbit antisera that identifies the NSSB protein by Western blotting methods. In addition, sera from patients with chronic HCV infection react with the recombinant NSSB protein using Western blotting methods. Current efforts arc directed at determining if additional co-factors are required for RNA-dependent RNA polymerase activity of purified recombinant NSSB or if it can catalyze this critical step in viral replication without other protein co-factors. Conclusion: recombinant NS5B has been expressed in E. coli, partially purified and reacts with antiserum from patients chronically infected with HCV. This protein will permit detailed studies of the enzyme that catalyzes the formation of replicative intermediate forms of the HCV genome and positive strands of the genome that are used for HCV gene expression or packaged into mature virions. In addition, specific nucleoside analogues that inhibit this enzyme in vitro may prove to be useful in treating patients with chronic hepatitis C virus infections. (Supported by the American Gastroenterological Association Foundation)
ANTIBODY
RESPONSE
TO
HYPERVARIABLE
REGION
OF
HCV DIFFERS BETWEEN GENOTYPE lb AND 2a K Yoshioka. T. Aivama. A. Okumura. M. Takavanaai. K. lwata T: Ishikawa. S Kakumu. Third Department of Internal Medicine, Nagoya University School of Medicine, Nagoya, Japan
Backmound The HCV genome is believed to contain one open reading frame encoding both structural (core, El, EZ) and non-structural proteins (NSZ-NS5). NS3 has been shown to function as a serine protease which is important for the processing of the non-structural proteins. These properties make the NS3 protease a good target for the development of specific anti-viral therapy. A high-level efficiency expression system for the production of native NS3 is critical for this endeavor. Hvllothesis The vaccinia virus mammalian cell expression system can be used for high-level production of native and enzymatically active NS3 for the processing of the HCV NS proteins.& To express NS3-5 at a high level in mammalian cells and to determine the enzymatic activity of the NS3 protease. Methods and Results NS3NS5 derived from a British genotype lb isolate was cloned into pTM1 (under the T7 promoter) by a combination of short fragment PCR and triple ligation. The sequence of the PCR amplified fragment was confirmed by dideoxy chain termination sequencing to ensure fidelity. Recombinant vaccinia tins (vNS3-5) was produced by transfection of Western Reserve vaccinia virus infected CV-1 cells. The recombinant vaccinia were confirmed, plaque-purified through three rounds and expanded in CV-1 cells. For the expression of the HCV proteins, Bl7H cells (BSC1 cells expressing T7 RNA polymerase) were infected with vNS3-5 at a multiplicity of infection of 10. The expression of HCV proteins was checked by radiolabeliig of the protein, Western blotting (for NS3) and radioimmunoprecipitation (RIPA). Interestingly, high level expression of mature HCV non-structural proteins was detected by radiolabeliig but minimal vaccinia virus proteins were seen. Detection of mature non-structural proteins indicated that the expressed NS3 protease \yas enzymatically active. In further experiments, infected cells were lysed with NP40 and the cell lysate test d against an in vitro translated NSSA/NS5B substrate radiolabeled with 39S (around 350 amino acids long). Efficient cleavage of the NSSA/NSSB to the predicted products was obtained. Conclusion A high level mammalian cell expression system for the production of enzymatically active native NS3 was achieved and an in vitro system for testing this protease developed. Imtdication This system will allow for the screening of protease inhibitors, with the ultimate goal of developing HCV specific therapy.
8%
331A
ABSTRACTS
It has been suggested that antibody to the hypervariable region (HVR) of HCV has neutralizing activity, and that the mutations in HVR provide the virus a way to escape from host immunity. HCV genotype has been reported to affect the response to IFN therapy and the clinical course of infection. We investigated the antibody response to HVR and its relationship to the HCV genotypes in nineteen patients with chronic hepatitis C. HVRs amplified by polymerase chain reaction from serum HCV were cloned into an expression vector pGEX, and were expressed as glutathion S-transferase (GST) fusion proteins. 8-20 clones per serum were obtained, and the reactivity with an individual serum was assessed by Western blot. In six patients with genotype 2a, 94.019.7% of HVR fusion proteins were reacted with the corresponding serum and the reactions were constantly potent. On the other hand, in 13 patients with genotype 1 b, sera were reacted with 58.6+ 38.5% of HVR fusion proteins, and the reactions were usually very week. Thus the incidence of HVR fusion proteins reactive with the corresponding serum was significantly higher in genotype 2a than in genotype 1 b (~~0.05). These results suggest that antibody response to HVR differs considerably between genotype 1 b and 2a. This difference may be related to the distinct clinical course between patients infected with these two HCV genotypes.
900
EFFICIENT HEPATITIS MURINE PROTEIN:
PRODUCTION OF A PORTION OF THE C CORE PROTEIN AND ANALYSIS OF THE IMMUNE RESPONSE TO RECOMBINANT TOWARD A VACCINE FOR HEPATITIS C. 1
Hitomil. WM McDonnell**3,JR Department of Internal Medicine,
Baker.
Jr.zA and
FK
Askaril
Divisions of Gastroenterologyl and Allergy and Immunology~~ University of Michigan Medical Center, Ann Arbor, Michigan, and the Veterans Administration Medical Cent&, Ann Arbor Michigan. The present study was undertaken to optimize the production of HCV core protein to facilitate the study of the immune response to recombinant protein. As the most highly conserved and least variable structural protein in the HCV genome, the core protein is one reasonable target for vaccine production. Presented here are data on the manufacture of recombinant core protein containing partial carboxy terminus deletions in an effort to increase the efficiency of core expression. The maltose binding protein (MBP) and glutathione S-transferase (GST) protein prokaryotic expression systems were used to study two different constructs, expressing the first 140 amino acids and 163 amino acids of the core region. Deletion of the 23 amino acids from aa140-163 led to a marked increase in the efficiency of protein production from
897
Vol.
22, NO.
4, Pt.
2, 1995
AASLD
EXPRESSION OF ENZYMATICALLY ACTIVE HCV NS3 PROTEASE (GENOTYPE lb) AND THE DEVELOPMENT OF AN IN-VITRO PROTRASE ASSAY SYSTEM Feller JA, Carroll AR1. Sanear DV1. Lau JYN. Clarke BE’. Section of Hepatobiiary Diseases, Department of Medicine, University of Florid& Gainesville, FL, and Wellcome Research Laboratories’, Kent, UK.
898
EXPRESSION AND CHAFUCTERIZATION OF THE NSSB (BNADEPENDENT RNA POLYMERASE) GENE OF HEPATITIS C VIRUS. RH Al. Y Wang. . Y Xie. CH Haeedom. Division of Digestive Diseases, Emory University School of Medicine and the VA Medical Center, Atlanta. The development of vaccines using recombinant hepatitis C virus (HCV) proteins appears to be difficult and suggests that the development of new antiviral agents should be explored. Nucleoside analogue inhibitors of viral DNA polymerases have been effective in treating some chronic viral infections. In addition, many novel nucleoside analogue inhibitors remain to be tested in different systems (Antimicrob Agents Chemother 1993;37:1720-2). The NSSB gene of HCV encodes a protein that contains sequence motifs that are highly conserved in other viral RNA-dependent RNA polymerases (RDRP). We have subcloned the region encoding the NSSB gene into a variety of prokaryotic expression vectors and successfully expressed a protein of approximately 68 kDa in E. coli that encodes the putative HCV RDRP. E. coli maximally express this protein 2 hours after induction with IPTG as determined by Coomassie Blue staining of SDS-PAGE analyzed cell lysates. In addition, methods to solubilize recombinant NSSB protein under nondenaturing conditions have been determined and used to partially purify the enzyme. Recombinant protein purified by SDS-PAGE has been used to raise polyclonal rabbit antisera that identifies the NSSB protein by Western blotting methods. In addition, sera from patients with chronic HCV infection react with the recombinant NSSB protein using Western blotting methods. Current efforts arc directed at determining if additional co-factors are required for RNA-dependent RNA polymerase activity of purified recombinant NSSB or if it can catalyze this critical step in viral replication without other protein co-factors. Conclusion: recombinant NS5B has been expressed in E. coli, partially purified and reacts with antiserum from patients chronically infected with HCV. This protein will permit detailed studies of the enzyme that catalyzes the formation of replicative intermediate forms of the HCV genome and positive strands of the genome that are used for HCV gene expression or packaged into mature virions. In addition, specific nucleoside analogues that inhibit this enzyme in vitro may prove to be useful in treating patients with chronic hepatitis C virus infections. (Supported by the American Gastroenterological Association Foundation)
ANTIBODY
RESPONSE
TO
HYPERVARIABLE
REGION
OF
HCV DIFFERS BETWEEN GENOTYPE lb AND 2a K Yoshioka. T. Aivama. A. Okumura. M. Takavanaai. K. lwata T: Ishikawa. S Kakumu. Third Department of Internal Medicine, Nagoya University School of Medicine, Nagoya, Japan
Backmound The HCV genome is believed to contain one open reading frame encoding both structural (core, El, EZ) and non-structural proteins (NSZ-NS5). NS3 has been shown to function as a serine protease which is important for the processing of the non-structural proteins. These properties make the NS3 protease a good target for the development of specific anti-viral therapy. A high-level efficiency expression system for the production of native NS3 is critical for this endeavor. Hvllothesis The vaccinia virus mammalian cell expression system can be used for high-level production of native and enzymatically active NS3 for the processing of the HCV NS proteins.& To express NS3-5 at a high level in mammalian cells and to determine the enzymatic activity of the NS3 protease. Methods and Results NS3NS5 derived from a British genotype lb isolate was cloned into pTM1 (under the T7 promoter) by a combination of short fragment PCR and triple ligation. The sequence of the PCR amplified fragment was confirmed by dideoxy chain termination sequencing to ensure fidelity. Recombinant vaccinia tins (vNS3-5) was produced by transfection of Western Reserve vaccinia virus infected CV-1 cells. The recombinant vaccinia were confirmed, plaque-purified through three rounds and expanded in CV-1 cells. For the expression of the HCV proteins, Bl7H cells (BSC1 cells expressing T7 RNA polymerase) were infected with vNS3-5 at a multiplicity of infection of 10. The expression of HCV proteins was checked by radiolabeliig of the protein, Western blotting (for NS3) and radioimmunoprecipitation (RIPA). Interestingly, high level expression of mature HCV non-structural proteins was detected by radiolabeliig but minimal vaccinia virus proteins were seen. Detection of mature non-structural proteins indicated that the expressed NS3 protease \yas enzymatically active. In further experiments, infected cells were lysed with NP40 and the cell lysate test d against an in vitro translated NSSA/NS5B substrate radiolabeled with 39S (around 350 amino acids long). Efficient cleavage of the NSSA/NSSB to the predicted products was obtained. Conclusion A high level mammalian cell expression system for the production of enzymatically active native NS3 was achieved and an in vitro system for testing this protease developed. Imtdication This system will allow for the screening of protease inhibitors, with the ultimate goal of developing HCV specific therapy.
8%
331A
ABSTRACTS
It has been suggested that antibody to the hypervariable region (HVR) of HCV has neutralizing activity, and that the mutations in HVR provide the virus a way to escape from host immunity. HCV genotype has been reported to affect the response to IFN therapy and the clinical course of infection. We investigated the antibody response to HVR and its relationship to the HCV genotypes in nineteen patients with chronic hepatitis C. HVRs amplified by polymerase chain reaction from serum HCV were cloned into an expression vector pGEX, and were expressed as glutathion S-transferase (GST) fusion proteins. 8-20 clones per serum were obtained, and the reactivity with an individual serum was assessed by Western blot. In six patients with genotype 2a, 94.019.7% of HVR fusion proteins were reacted with the corresponding serum and the reactions were constantly potent. On the other hand, in 13 patients with genotype 1 b, sera were reacted with 58.6+ 38.5% of HVR fusion proteins, and the reactions were usually very week. Thus the incidence of HVR fusion proteins reactive with the corresponding serum was significantly higher in genotype 2a than in genotype 1 b (~~0.05). These results suggest that antibody response to HVR differs considerably between genotype 1 b and 2a. This difference may be related to the distinct clinical course between patients infected with these two HCV genotypes.
900
EFFICIENT HEPATITIS MURINE PROTEIN:
PRODUCTION OF A PORTION OF THE C CORE PROTEIN AND ANALYSIS OF THE IMMUNE RESPONSE TO RECOMBINANT TOWARD A VACCINE FOR HEPATITIS C. 1
Hitomil. WM McDonnell**3,JR Department of Internal Medicine,
Baker.
Jr.zA and
FK
Askaril
Divisions of Gastroenterologyl and Allergy and Immunology~~ University of Michigan Medical Center, Ann Arbor, Michigan, and the Veterans Administration Medical Cent&, Ann Arbor Michigan. The present study was undertaken to optimize the production of HCV core protein to facilitate the study of the immune response to recombinant protein. As the most highly conserved and least variable structural protein in the HCV genome, the core protein is one reasonable target for vaccine production. Presented here are data on the manufacture of recombinant core protein containing partial carboxy terminus deletions in an effort to increase the efficiency of core expression. The maltose binding protein (MBP) and glutathione S-transferase (GST) protein prokaryotic expression systems were used to study two different constructs, expressing the first 140 amino acids and 163 amino acids of the core region. Deletion of the 23 amino acids from aa140-163 led to a marked increase in the efficiency of protein production from