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Roth KDR, Wenzel EV, Ruschig M, Steinke S, Langreder N, Heine PA, Schneider KT, Ballmann R, Fühner V, Kuhn P, Schirrmann T, Frenzel A, Dübel S, Schubert M, Moreira GMSG, Bertoglio F, Russo G, Hust M. Developing Recombinant Antibodies by Phage Display Against Infectious Diseases and Toxins for Diagnostics and Therapy. Front Cell Infect Microbiol 2021; 11:697876. [PMID: 34307196 PMCID: PMC8294040 DOI: 10.3389/fcimb.2021.697876] [Citation(s) in RCA: 47] [Impact Index Per Article: 11.8] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/06/2021] [Accepted: 06/21/2021] [Indexed: 12/30/2022] Open
Abstract
Antibodies are essential molecules for diagnosis and treatment of diseases caused by pathogens and their toxins. Antibodies were integrated in our medical repertoire against infectious diseases more than hundred years ago by using animal sera to treat tetanus and diphtheria. In these days, most developed therapeutic antibodies target cancer or autoimmune diseases. The COVID-19 pandemic was a reminder about the importance of antibodies for therapy against infectious diseases. While monoclonal antibodies could be generated by hybridoma technology since the 70ies of the former century, nowadays antibody phage display, among other display technologies, is robustly established to discover new human monoclonal antibodies. Phage display is an in vitro technology which confers the potential for generating antibodies from universal libraries against any conceivable molecule of sufficient size and omits the limitations of the immune systems. If convalescent patients or immunized/infected animals are available, it is possible to construct immune phage display libraries to select in vivo affinity-matured antibodies. A further advantage is the availability of the DNA sequence encoding the phage displayed antibody fragment, which is packaged in the phage particles. Therefore, the selected antibody fragments can be rapidly further engineered in any needed antibody format according to the requirements of the final application. In this review, we present an overview of phage display derived recombinant antibodies against bacterial, viral and eukaryotic pathogens, as well as microbial toxins, intended for diagnostic and therapeutic applications.
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Affiliation(s)
- Kristian Daniel Ralph Roth
- Institut für Biochemie, Biotechnologie und Bioinformatik, Abteilung Biotechnologie, Technische Universität Braunschweig, Braunschweig, Germany
| | - Esther Veronika Wenzel
- Institut für Biochemie, Biotechnologie und Bioinformatik, Abteilung Biotechnologie, Technische Universität Braunschweig, Braunschweig, Germany.,Abcalis GmbH, Braunschweig, Germany
| | - Maximilian Ruschig
- Institut für Biochemie, Biotechnologie und Bioinformatik, Abteilung Biotechnologie, Technische Universität Braunschweig, Braunschweig, Germany
| | - Stephan Steinke
- Institut für Biochemie, Biotechnologie und Bioinformatik, Abteilung Biotechnologie, Technische Universität Braunschweig, Braunschweig, Germany
| | - Nora Langreder
- Institut für Biochemie, Biotechnologie und Bioinformatik, Abteilung Biotechnologie, Technische Universität Braunschweig, Braunschweig, Germany
| | - Philip Alexander Heine
- Institut für Biochemie, Biotechnologie und Bioinformatik, Abteilung Biotechnologie, Technische Universität Braunschweig, Braunschweig, Germany
| | - Kai-Thomas Schneider
- Institut für Biochemie, Biotechnologie und Bioinformatik, Abteilung Biotechnologie, Technische Universität Braunschweig, Braunschweig, Germany
| | - Rico Ballmann
- Institut für Biochemie, Biotechnologie und Bioinformatik, Abteilung Biotechnologie, Technische Universität Braunschweig, Braunschweig, Germany
| | - Viola Fühner
- Institut für Biochemie, Biotechnologie und Bioinformatik, Abteilung Biotechnologie, Technische Universität Braunschweig, Braunschweig, Germany
| | | | | | | | - Stefan Dübel
- Institut für Biochemie, Biotechnologie und Bioinformatik, Abteilung Biotechnologie, Technische Universität Braunschweig, Braunschweig, Germany.,Abcalis GmbH, Braunschweig, Germany.,YUMAB GmbH, Braunschweig, Germany
| | - Maren Schubert
- Institut für Biochemie, Biotechnologie und Bioinformatik, Abteilung Biotechnologie, Technische Universität Braunschweig, Braunschweig, Germany
| | | | - Federico Bertoglio
- Institut für Biochemie, Biotechnologie und Bioinformatik, Abteilung Biotechnologie, Technische Universität Braunschweig, Braunschweig, Germany
| | - Giulio Russo
- Institut für Biochemie, Biotechnologie und Bioinformatik, Abteilung Biotechnologie, Technische Universität Braunschweig, Braunschweig, Germany.,Abcalis GmbH, Braunschweig, Germany
| | - Michael Hust
- Institut für Biochemie, Biotechnologie und Bioinformatik, Abteilung Biotechnologie, Technische Universität Braunschweig, Braunschweig, Germany.,YUMAB GmbH, Braunschweig, Germany
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Lai JY, Lim TS. Infectious disease antibodies for biomedical applications: A mini review of immune antibody phage library repertoire. Int J Biol Macromol 2020; 163:640-648. [PMID: 32650013 PMCID: PMC7340592 DOI: 10.1016/j.ijbiomac.2020.06.268] [Citation(s) in RCA: 13] [Impact Index Per Article: 2.6] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/18/2020] [Revised: 06/21/2020] [Accepted: 06/28/2020] [Indexed: 12/18/2022]
Abstract
Antibody phage display is regarded as a critical tool for the development of monoclonal antibodies for infectious diseases. The different classes of antibody libraries are classified based on the source of repertoire used to generate the libraries. Immune antibody libraries are generated from disease infected host or immunization against an infectious agent. Antibodies derived from immune libraries are distinct from those derived from naïve libraries as the host's in vivo immune mechanisms shape the antibody repertoire to yield high affinity antibodies. As the immune system is constantly evolving in accordance to the health state of an individual, immune libraries can offer more than just infection-specific antibodies but also antibodies derived from the memory B-cells much like naïve libraries. The combinatorial nature of the gene cloning process would give rise to a combination of natural and un-natural antibody gene pairings in the immune library. These factors have a profound impact on the coverage of immune antibody libraries to target both disease-specific and non-disease specific antigens. This review looks at the diverse nature of antibody responses for immune library generation and discusses the extended potential of a disease-specified immune library in the context of phage display.
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Affiliation(s)
- Jing Yi Lai
- Institute for Research in Molecular Medicine, Universiti Sains Malaysia, 11800 Penang, Malaysia
| | - Theam Soon Lim
- Institute for Research in Molecular Medicine, Universiti Sains Malaysia, 11800 Penang, Malaysia; Analytical Biochemistry Research Centre, Universiti Sains Malaysia, 11800 Penang, Malaysia.
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Patel J, Sharma P. Design of a novel rapid immunoassay for simultaneous detection of hepatitis C virus core antigen and antibodies. Arch Virol 2020; 165:627-641. [PMID: 31965313 DOI: 10.1007/s00705-019-04518-0] [Citation(s) in RCA: 6] [Impact Index Per Article: 1.2] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/14/2019] [Accepted: 12/05/2019] [Indexed: 01/04/2023]
Abstract
HCV is a potential cause of viral hepatitis, which leads to blood-borne chronic hepatitis, liver cirrhosis and hepatocellular carcinoma. Anti-HCV antibody detection assays detect HCV infection after approximately 70 days. HCV core antigen can be detected much earlier than anti-HCV antibodies. However, it disappears soon after the appearance of anti-HCV antibodies. Thus, there is a need for a rapid assay for simultaneous detection of HCV core antigen and anti-HCV antibodies for early diagnosis of HCV infection. A rapid diagnostic assay (HCV Ag-Ab Combo) for simultaneous detection of HCV core antigen and anti-HCV antibodies for early diagnosis of HCV infection was developed. HCV Ag-Ab Combo was evaluated in order to determine its potential for detection of HCV infection earlier than anti-HCV antibody detection assays. We compared the sensitivity of the newly developed assay with anti-HCV antibody detection assays (ELISA [HCV Ab ELISA] and rapid test [HCV Ab Rapid]) and HCV core antigen/anti-HCV antibody detection ELISA (HCV Ag-Ab ELISA). This study included 11 samples that were found positive in HCV RNA detection and HCV Ag-Ab ELISA but negative in HCV antibody detection assays (HCV Ab ELISA and rapid), 10 samples that were found positive in HCV Ag-Ab ELISA and HCV Ab ELISA but negative in HCV Ab Rapid, 69 samples that were found positive in HCV Ag-Ab ELISA, HCV Ab ELISA, and HCV Ab Rapid, and 509 samples that were found negative in HCV Ag-Ab ELISA, HCV Ab ELISA, and HCV Ab Rapid. Three seroconversion panels, PHV 913, PHV 911 (M) and PHV904-00-1.0, and a HCV RNA genotype qualification panel (2400-0182) acquired from Seracare Life Sciences (USA) were also tested. HCV Ag-Ab Combo showed a combined sensitivity and specificity of 100% when tested with 90 samples that were positive for HCV by HCV Ag-Ab ELISA and 509 HCV-negative samples. Its positive predictive value (PPV) and negative predictive value (NPV) were found to be 100%. It detected HCV infection approximately 7 to 12 days earlier than the HCV Ab detection assays, and its performance was not affected when testing different genotypes of HCV. HCV Ag-Ab Combo did not detect HCV infection as early as HCV RNA or HCV Ag-Ab ELISA. HCV Ag-Ab Combo provided a significant improvement for the early detection of HCV infection during the preseroconversion phase when compared with anti-HCV antibody detection assays. It could be a useful screening assay, and an alternative to HCV RNA detection or HCV Ag-Ab ELISA when nucleic acid technologies cannot be implemented.
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Affiliation(s)
- Jayendrakumar Patel
- Department of Biotechnology, Veer Narmad South Gujarat University, Surat, Gujarat, 395007, India. .,Research and Development Department, ARKRAY Healthcare Pvt. Ltd. (Formerly Span Diagnostics Ltd.), Surat, Gujarat, India.
| | - Preeti Sharma
- Department of Biotechnology, Veer Narmad South Gujarat University, Surat, Gujarat, 395007, India
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Rafik M, Bakr S, Soliman D, Mohammed N, Ragab D, ElHady WA, Samir N. Characterization of differential antibody production against hepatitis C virus in different HCV infection status. Virol J 2016; 13:116. [PMID: 27357382 PMCID: PMC4928299 DOI: 10.1186/s12985-016-0572-9] [Citation(s) in RCA: 13] [Impact Index Per Article: 1.4] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/06/2016] [Accepted: 06/22/2016] [Indexed: 01/05/2023] Open
Abstract
BACKGROUND The Centers for Disease Control and Prevention (CDC) issued an update on hepatitis C virus (HCV) testing approach, in which it omitted the use of recombinant immunoblot assay (RIBA) in the diagnostic algorithm and recommended that future studies are needed to evaluate the performance of HCV testing without RIBA. As Egypt has the highest prevalence of HCV worldwide, we aimed to evaluate the value of RIBA in HCV testing in a high prevalence population. Our objective was to clarify whether enzyme linked immunosorbent assay (ELISA) anti-HCV signal-to-cutoff (S/CO) ratios were able to discriminate true positive from false positive anti-HCV antibody status and to evaluate the role of RIBA in solving this problem which may lead to a redefined strategy for diagnosis of HCV infection. Our second objective was to elucidate the effects of different HCV peptides of both structural and non-structural proteins on the humoral immune response to HCV infection. METHODS The current study drew results from 167 individuals divided into three groups: Group I: included 77 HCV antibody positive (ELISA) high risk health care workers (HCW), Group II: included 56 presumably uninfected individuals who showed normal liver enzymes, negative HCV RNA and were asymptomatic. Their ELISA HCV antibody S/C ratio ranged from 0.9 to <5. Group III: included 34 patients enrolled from outpatient clinics of Ain Shams Hospital with persistent viral replication, elevated liver enzymes, and chronic HCV related liver disease. All study participants were assessed for the presence of anti-HCV antibodies by 3(rd) generation ELISA which was confirmed by RIBA. RESULTS Interpreting the results of both ELISA and RIBA together, false positive results were highly significantly increased in HCW when compared with the other two groups. Indeterminate and false negative results were only found in the presumably uninfected group. For differentiated antibody responses by RIBA, chronic HCV cases had the highest frequency of positive antibody response to core peptides while the presumably uninfected group had the lowest. Antibody response to E2 was found less frequently in chronic cases than Core 1, Core 2 and NS3. The specific antibody response to the different HCV peptides showed the same distribution of frequencies in both chronic HCV cases and the presumably uninfected individuals with the chronic cases having the highest frequencies. This distribution was different from the HCW. The most evident difference was the reaction towards NS3 which was the highest antibody producing peptide in chronic HCV and presumably uninfected individuals whereas in HCW Core1 was the highest. CONCLUSION The HCV antibody immunoblot assay (RIBA) is still necessary for the detection of false positive cases which can occur quite frequently in countries of high prevalence as Egypt. Indeterminate RIBA results indicate a waning antibody response in elderly individuals who recovered from previous or distant HCV infection.
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Affiliation(s)
- Mona Rafik
- Clinical and Chemical Pathology Department, Faculty of Medicine, Ain Shams University, Children village, POB 9505, Nasr city, Cairo, Egypt
| | - Salwa Bakr
- Clinical and Chemical Pathology Department, Faculty of Medicine, Ain Shams University, Children village, POB 9505, Nasr city, Cairo, Egypt
| | - Dina Soliman
- Clinical and Chemical Pathology Department, Faculty of Medicine, Ain Shams University, Children village, POB 9505, Nasr city, Cairo, Egypt
| | - Nesrine Mohammed
- Clinical and Chemical Pathology Department, Faculty of Medicine, Ain Shams University, Children village, POB 9505, Nasr city, Cairo, Egypt
| | - Dina Ragab
- Clinical and Chemical Pathology Department, Faculty of Medicine, Ain Shams University, Children village, POB 9505, Nasr city, Cairo, Egypt.
| | - Walid Abd ElHady
- Clinical and Chemical Pathology Department, Faculty of Medicine, Ain Shams University, Children village, POB 9505, Nasr city, Cairo, Egypt
| | - Nancy Samir
- Clinical and Chemical Pathology Department, Faculty of Medicine, Ain Shams University, Children village, POB 9505, Nasr city, Cairo, Egypt
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Kuhn P, Fühner V, Unkauf T, Moreira GMSG, Frenzel A, Miethe S, Hust M. Recombinant antibodies for diagnostics and therapy against pathogens and toxins generated by phage display. Proteomics Clin Appl 2016; 10:922-948. [PMID: 27198131 PMCID: PMC7168043 DOI: 10.1002/prca.201600002] [Citation(s) in RCA: 56] [Impact Index Per Article: 6.2] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/11/2016] [Revised: 03/30/2016] [Accepted: 05/17/2016] [Indexed: 12/11/2022]
Abstract
Antibodies are valuable molecules for the diagnostic and treatment of diseases caused by pathogens and toxins. Traditionally, these antibodies are generated by hybridoma technology. An alternative to hybridoma technology is the use of antibody phage display to generate recombinant antibodies. This in vitro technology circumvents the limitations of the immune system and allows—in theory—the generation of antibodies against all conceivable molecules. Phage display technology enables obtaining human antibodies from naïve antibody gene libraries when either patients are not available or immunization is not ethically feasible. On the other hand, if patients or immunized/infected animals are available, it is common to construct immune phage display libraries to select in vivo affinity‐matured antibodies. Because the phage packaged DNA sequence encoding the antibodies is directly available, the antibodies can be smoothly engineered according to the requirements of the final application. In this review, an overview of phage display derived recombinant antibodies against bacterial, viral, and eukaryotic pathogens as well as toxins for diagnostics and therapy is given.
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Affiliation(s)
- Philipp Kuhn
- Technische Universität Braunschweig, Institut für Biochemie, Biotechnologie und Bioinformatik, Abteilung Biotechnologie, Braunschweig, Germany
| | - Viola Fühner
- Technische Universität Braunschweig, Institut für Biochemie, Biotechnologie und Bioinformatik, Abteilung Biotechnologie, Braunschweig, Germany
| | - Tobias Unkauf
- Technische Universität Braunschweig, Institut für Biochemie, Biotechnologie und Bioinformatik, Abteilung Biotechnologie, Braunschweig, Germany
| | | | - André Frenzel
- Technische Universität Braunschweig, Institut für Biochemie, Biotechnologie und Bioinformatik, Abteilung Biotechnologie, Braunschweig, Germany.,YUMAB GmbH, Braunschweig, Germany
| | - Sebastian Miethe
- Technische Universität Braunschweig, Institut für Biochemie, Biotechnologie und Bioinformatik, Abteilung Biotechnologie, Braunschweig, Germany
| | - Michael Hust
- Technische Universität Braunschweig, Institut für Biochemie, Biotechnologie und Bioinformatik, Abteilung Biotechnologie, Braunschweig, Germany.
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Quiroga JA, Avellón A, Bartolomé J, Andréu M, Flores E, González MI, González R, Pérez S, Richart LA, Castillo I, Alcover J, Palacios R, Carreño V, Echevarría JM. Detection of hepatitis C virus (HCV) core–specific antibody suggests occult HCV infection among blood donors. Transfusion 2016; 56:1883-90. [DOI: 10.1111/trf.13645] [Citation(s) in RCA: 9] [Impact Index Per Article: 1.0] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/21/2016] [Revised: 03/28/2016] [Accepted: 03/30/2016] [Indexed: 12/14/2022]
Affiliation(s)
- Juan A. Quiroga
- Fundación para el Estudio de las Hepatitis ViralesMadrid Spain
| | - Ana Avellón
- Department of VirologyCentro Nacional de Microbiología, Instituto de Salud Carlos IIIMadrid Spain
| | | | - María Andréu
- Centro de Transfusión de la Cruz RojaMadrid Spain
| | - Elena Flores
- Centro de Transfusión de la Comunidad de MadridMadrid Spain
| | - María I. González
- Centro de Hemoterapia y Hemodonación de Castilla y LeónValladolid Spain
| | | | - Sonia Pérez
- Centro de Hemoterapia y Hemodonación de Castilla y LeónValladolid Spain
| | | | | | | | | | - Vicente Carreño
- Fundación para el Estudio de las Hepatitis ViralesMadrid Spain
| | - José M. Echevarría
- Department of VirologyCentro Nacional de Microbiología, Instituto de Salud Carlos IIIMadrid Spain
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Comparative Immunogenicity in Rabbits of the Polypeptides Encoded by the 5' Terminus of Hepatitis C Virus RNA. J Immunol Res 2015; 2015:762426. [PMID: 26609538 PMCID: PMC4644844 DOI: 10.1155/2015/762426] [Citation(s) in RCA: 2] [Impact Index Per Article: 0.2] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/22/2015] [Accepted: 09/29/2015] [Indexed: 12/26/2022] Open
Abstract
Recent studies on the primate protection from HCV infection stressed the importance of immune response against structural viral proteins. Strong immune response against nucleocapsid (core) protein was difficult to achieve, requesting further experimentation in large animals. Here, we analyzed the immunogenicity of core aa 1–173, 1–152, and 147–191 and of its main alternative reading frame product F-protein in rabbits. Core aa 147–191 was synthesized; other polypeptides were obtained by expression in E. coli. Rabbits were immunized by polypeptide primes followed by multiple boosts and screened for specific anti-protein and anti-peptide antibodies. Antibody titers to core aa 147–191 reached 105; core aa 1–152, 5 × 105; core aa 1–173 and F-protein, 106. Strong immunogenicity of the last two proteins indicated that they may compete for the induction of immune response. The C-terminally truncated core was also weakly immunogenic on the T-cell level. To enhance core-specific cellular response, we immunized rabbits with the core aa 1–152 gene forbidding F-protein formation. Repeated DNA immunization induced a weak antibody and sustained proliferative response of broad specificity confirming a gain of cellular immunogenicity. Epitopes recognized in rabbits overlapped those in HCV infection. Our data promotes the use of rabbits for the immunogenicity tests of prototype HCV vaccines.
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Nguyen KTH, Adamkiewicz MA, Hebert LE, Zygiel EM, Boyle HR, Martone CM, Meléndez-Ríos CB, Noren KA, Noren CJ, Hall MF. Identification and characterization of mutant clones with enhanced propagation rates from phage-displayed peptide libraries. Anal Biochem 2014; 462:35-43. [PMID: 24952360 DOI: 10.1016/j.ab.2014.06.007] [Citation(s) in RCA: 26] [Impact Index Per Article: 2.4] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/06/2014] [Revised: 06/04/2014] [Accepted: 06/08/2014] [Indexed: 01/25/2023]
Abstract
A target-unrelated peptide (TUP) can arise in phage display selection experiments as a result of a propagation advantage exhibited by the phage clone displaying the peptide. We previously characterized HAIYPRH, from the M13-based Ph.D.-7 phage display library, as a propagation-related TUP resulting from a G→A mutation in the Shine-Dalgarno sequence of gene II. This mutant was shown to propagate in Escherichia coli at a dramatically faster rate than phage bearing the wild-type Shine-Dalgarno sequence. We now report 27 additional fast-propagating clones displaying 24 different peptides and carrying 14 unique mutations. Most of these mutations are found either in or upstream of the gene II Shine-Dalgarno sequence, but still within the mRNA transcript of gene II. All 27 clones propagate at significantly higher rates than normal library phage, most within experimental error of wild-type M13 propagation, suggesting that mutations arise to compensate for the reduced virulence caused by the insertion of a lacZα cassette proximal to the replication origin of the phage used to construct the library. We also describe an efficient and convenient assay to diagnose propagation-related TUPS among peptide sequences selected by phage display.
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Affiliation(s)
- Kieu T H Nguyen
- Department of Chemistry, Stonehill College, 320 Washington Street, Easton, MA 02357, USA
| | - Marta A Adamkiewicz
- Department of Chemistry, Stonehill College, 320 Washington Street, Easton, MA 02357, USA
| | - Lauren E Hebert
- Department of Chemistry, Stonehill College, 320 Washington Street, Easton, MA 02357, USA
| | - Emily M Zygiel
- Department of Chemistry, Stonehill College, 320 Washington Street, Easton, MA 02357, USA
| | - Holly R Boyle
- Department of Chemistry, Stonehill College, 320 Washington Street, Easton, MA 02357, USA
| | - Christina M Martone
- Department of Chemistry, Stonehill College, 320 Washington Street, Easton, MA 02357, USA
| | - Carola B Meléndez-Ríos
- Department of Chemistry, Stonehill College, 320 Washington Street, Easton, MA 02357, USA
| | - Karen A Noren
- New England Biolabs, Inc., 240 County Road, Ipswich, MA 01938, USA
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Yang T, Yang L, Chai W, Li R, Xie J, Niu B. A strategy for high-level expression of a single-chain variable fragment against TNFα by subcloning antibody variable regions from the phage display vector pCANTAB 5E into pBV220. Protein Expr Purif 2011; 76:109-14. [DOI: 10.1016/j.pep.2010.10.006] [Citation(s) in RCA: 11] [Impact Index Per Article: 0.8] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/02/2010] [Revised: 10/08/2010] [Accepted: 10/08/2010] [Indexed: 10/18/2022]
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10
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Komurian-Pradel F, Rajoharison A, Berland JL, Khouri V, Perret M, Van Roosmalen M, Pol S, Negro F, Paranhos-Baccalà G. Antigenic relevance of F protein in chronic hepatitis C virus infection. Hepatology 2004; 40:900-9. [PMID: 15382175 DOI: 10.1002/hep.20406] [Citation(s) in RCA: 53] [Impact Index Per Article: 2.5] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Indexed: 01/29/2023]
Abstract
The hepatitis C virus (HCV) F protein is a recently described, frameshift product of HCV core encoding sequence of genotype 1a. Its function and antigenic properties are unknown. Using enzyme-linked immunosorbent assay, we assessed the prevalence of anti-F antibodies in 154 patients chronically infected with HCV, 65 patients with other liver diseases, and 121 healthy controls. For this purpose, we expressed a highly purified HCV F recombinant protein from HCV genotype 1a in Escherichia coli. Because the F protein shares the 10 first amino acids with the core protein, the anti-HCV F response was also assessed by a F recombinant protein deleted of its 10 first amino acids [Delta(1-10)-F]. Ninety-six (62%) of the 154 HCV serum samples reacted with the complete F recombinant protein, whereas 39 (25%) showed a weaker anti-Delta(1-10)F reactivity and 150 (97%) had anti-core antibodies. No reactivity against F, Delta(1-10)F, or core was detected in any of the controls. To exclude a potential cross-reaction of anti-F antibodies with anti-core antibodies, a specific enzyme-linked immunosorbent assay was performed for anti-core antibodies. The specificity of anti-F antibodies was confirmed using an F synthetic peptide. The prevalence of anti-F antibodies did not correlate with HCV RNA serum level, genotype, or stage of liver disease. Sequence analysis from 8 anti-F-positive and 5 anti-F-negative serum samples did not reveal any particular difference potentially accounting for their respective anti-F responses. In conclusion, the F protein elicits specific antibodies in 62% of individuals chronically infected with HCV; such anti-F response does not seem to be affected by the F sequence heterogeneity.
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11
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Zhang XX, Deng Q, Zhang SY, Liu J, Cai Q, Lu ZM, Wang Y. Broadly cross-reactive mimotope of hypervariable region 1 of hepatitis C virus derived from DNA shuffling and screened by phage display library. J Med Virol 2004; 71:511-7. [PMID: 14556263 DOI: 10.1002/jmv.10521] [Citation(s) in RCA: 8] [Impact Index Per Article: 0.4] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/15/2022]
Abstract
The hypervariable region 1 (HVR1) is the target of neutralizing antibodies but with isolate specificity. The aim of this study was to obtain immunogenic mimotopes of HVR1, which can react broadly with different HVR1 antibodies and could be one of the candidate immunogens in an effective vaccine against HCV. Thirty-one HVR1 cDNA fragments were digested by DNase I into a pool of random fragments and reassembled by repeated cycles of annealing in the presence of DNA polymerase to their original size. The shuffled HVR1 was then inserted into the gene III phagemid vector pCANTAB-5E and displayed on the surface of the phage. Eight individual phages were selected after four rounds of biopanning against anti-HVR1. ELISA was carried out on immobilized purified phages, respectively, to detect their reactivity with a panel of sera. DNA sequences of the inserts were analyzed and compared with the consensus sequences defined by Puntoriero et al. [(1998) EMBO J 17:3521-3533]. The reactivity of the eight selected clones to the 30 sera were from 53.3 to 80%. Among these, phage 13 (ETYVSGGSAARNAYGLTSLFTVGPAQK, aa 384-410) reacted most broadly. None of the selected sequences encoded for peptides corresponded to known HVR1 from original viral isolates. The two high reactive phages had the similar amino acid sequences with the consensus, which might play a particular role in determining the frequency of reactivity. In conclusion, this study has used effectively DNA shuffling combined with phage display technology to identify broadly cross-reactive mimotopes recognized by human polyclonal antibodies. Mimotope 13 and 23 appeared to be most reactive immunologically and could be candidate immunogens. Efforts are now underway to identify their neutralizing antibodies by immunization of animals.
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Affiliation(s)
- Xin-Xin Zhang
- State Key Laboratory of Molecular Biology, Institute of Biochemistry and Cell Biology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, Shanghai, People's Republic of China
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Ménez R, Bossus M, Muller BH, Sibaï G, Dalbon P, Ducancel F, Jolivet-Reynaud C, Stura EA. Crystal structure of a hydrophobic immunodominant antigenic site on hepatitis C virus core protein complexed to monoclonal antibody 19D9D6. JOURNAL OF IMMUNOLOGY (BALTIMORE, MD. : 1950) 2003; 170:1917-24. [PMID: 12574359 DOI: 10.4049/jimmunol.170.4.1917] [Citation(s) in RCA: 19] [Impact Index Per Article: 0.9] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Subscribe] [Scholar Register] [Indexed: 11/19/2022]
Abstract
The first crystal structure of a complex between a hepatitis C virus (HCV) core protein-derived peptide (residues 13-40) and the Ab fragment of a murine mAb (19D9D6) has been solved, allowing determination of the recognized epitope and elucidation of its conformation. This Ab, raised against the first 120 residues of the core protein, recognizes core particles and strongly competes with anticore human Abs, suggesting that it is highly representative of the human anti-HCV core response. Its epitope lies within the first 45 aa of the protein, the major antigenic segment of core recognized both by murine and human Abs. Surprisingly, the recognized epitope (29-37: QIVGGVYLL) has an unusual preponderance of hydrophobic residues, some of which are buried in a small hydrophobic core in the nuclear magnetic resonance structure of the peptide (2-45) in solution, suggesting that the Ab may induce a structural rearrangement upon recognition. The flexibility may reside entirely within the Ag, since the Fab'-peptide complex structure at 2.34 A shows that the Ab binding site is hardly perturbed by complexation. Given that the recognized residues are unlikely to be solvent exposed, we are left with the interesting possibility that Ab-core interactions may take place in a nonaqueous environment.
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Affiliation(s)
- Renée Ménez
- Unité Mixte Commissariat à l'Energie Atomique, bioMérieux and Département d'Ingénierie et d'Etudes des Protéines, Commissariat à l'Energie Atomique, Centre d'Etudes de Saclay, Gif-sur-Yvette, France
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Yu ZC, Ding J, Nie YZ, Fan DM, Zhang XY. Preparation of single chain variable fragment of MG 7 mAb by phage display technology. World J Gastroenterol 2001; 7:510-4. [PMID: 11819819 PMCID: PMC4688663 DOI: 10.3748/wjg.v7.i4.510] [Citation(s) in RCA: 3] [Impact Index Per Article: 0.1] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Download PDF] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Indexed: 02/06/2023] Open
Abstract
AIM: To develop the single chain variable fragment of MG7 murine anti-human gastric cancer monoclonal antibody using the phage display technology for obtaining a tumor-targeting mediator.
METHODS: mRNA was isolated from MG7 producing murine hybridoma cell line and converted into cDNA. The variable fragments of heavy and light chain were amplified separately and assembled into ScFv with a specially constructed DNA linker by PCR. The ScFvs DNA was ligated into the phagmid vector pCANTAB5E and the ligated sample was transformed into competent E. Coli TG1. The transformed cells were infected with M13K07 helper phage to form MG7 recombinant phage antibody library. The volume and recombinant rate of the library were evaluated by means of bacterial colony count and restriction analysis. After two rounds of panning with gastric cancer cell line KATOIII of highly expressing MG7-binding antigen, the phage clones displaying ScFv of the antibody were selected by ELISA from the enriched phage clones. The antigen binding affinity of the positive clone was detected by competition ELISA. HB2151 E.coli was transfected with the positive phage clone demonstrated by competition ELISA for production of a soluble form of the MG7 ScFv. ELISA assay was used to detect the antigen-binding affinity of the soluble MG7 ScFv. Finally, the relative molecular mass of soluble MG7 ScFv was measured by SDS-PAGE.
RESULTS: The V-H, V-L and ScFv DNAs were about 340 bp, 320 bp and 750 bp, respectively. The volume of the library was up to 2 × 106 and 8 of 11 random clones were recombinants. Two phage clones could strongly compete with the original MG7 antibody for binding to the antigen expressed on KATOIII cells. Within 2 strong positive phage clones, the soluble MG7 ScFv from one clone was found to have the binding activity with KATOIII cells. SDS-PAGE showed that the relative molecular weight of soluble MG7 ScFv was 32.
CONCLUSION: The MG7 ScFv was successfully produced by phage antibody technology, which may be useful for broadening the scope of application of the antibody.
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Affiliation(s)
- Z C Yu
- Department of Gastroenterology, Xijing Hospital, Fourth Military Medical University, Xi'an 710032, Shaanxi Province, China
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