scholarly journals Characterization of a Monoclonal Antibody and Its Single-Chain Antibody Fragment Recognizing the Nucleoside Triphosphatase/Helicase Domain of the Hepatitis C Virus Nonstructural 3 Protein

2000 ◽  
Vol 7 (1) ◽  
pp. 58-63 ◽  
Author(s):  
Zhu-Xu Zhang ◽  
Una Lazdina ◽  
Margaret Chen ◽  
Darrell L. Peterson ◽  
Matti Sällberg
Keyword(s):  
Monoclonal Antibody ◽  
Hepatitis C Virus ◽  
Hepatitis C ◽  
Antibody Fragment ◽  
Expression Vectors ◽  
Helicase Domain ◽  
Single Chain ◽  
Single Chain Antibody ◽  
Variable Regions

ABSTRACT We have produced a murine monoclonal antibody (MAb), ZX10, recognizing the NTPase/helicase domain of the hepatitis C virus (HCV) nonstructural 3 protein (NS3), from which we designed a single-chain variable fragment (ScFv). The ZX10 MAb recognized a discontinuous epitope of the NTPase/helicase domain, of which the linear sequence GEIPFYGKAIPL at residues 1371 to 1382 constitutes one part. cDNAs from variable regions coding for the heavy and light chains were cloned, sequenced, and assembled into the NS3-ScFv, which was inserted into procaryotic and eucaryotic expression vectors.Escherichia coli-expressed NS3-ScFv inhibited the binding of the ZX10 MAb to NS3, confirming a retained specificity. However, the ability to bind the peptide 1371–1382 had been lost. In vitro-translated NS3-ScFv and HCV NS3/NS4A were coprecipitated by antibodies to HCV NS4A, confirming the in vitro activity of the NS3 ScFv. Thus, we have designed a functional NS3 NTPase/helicase domain-specific ScFv which should be evaluated further with respect to disturbing enzymatic functions of the NS3 protein.

scholarly journals Prospective Characterization of Full-Length Hepatitis C Virus NS5A Quasispecies during Induction and Combination Antiviral Therapy

2000 ◽  
Vol 74 (19) ◽  
pp. 9028-9038 ◽  
Author(s):  
J.-B. Nousbaum ◽  
S. J. Polyak ◽  
S. C. Ray ◽  
D. G. Sullivan ◽  
A. M. Larson ◽  
...  
Keyword(s):  
Hepatitis C Virus ◽  
Hepatitis C ◽  
Amino Acid ◽  
Antiviral Therapy ◽  
Variable Region ◽  
Response To Therapy ◽  
Full Length ◽  
Variable Regions ◽  
C Terminus

ABSTRACT The hepatitis C virus (HCV) nonstructural 5A (NS5A) protein has been controversially implicated in the inherent resistance of HCV to interferon (IFN) antiviral therapy in clinical studies. In this study, the relationship between NS5A mutations and selection pressures before and during antiviral therapy and virologic response to therapy were investigated. Full-length NS5A clones were sequenced from 20 HCV genotype 1-infected patients in a prospective, randomized clinical trial of IFN induction (daily) therapy and IFN plus ribavirin combination therapy. Pretreatment NS5A nucleotide and amino acid phylogenies did not correlate with clinical IFN responses and domains involved in NS5A functions in vitro were all well conserved before and during treatment. A consensus IFN sensitivity-determining region (ISDR237–276) sequence associated with IFN resistance was not found, although the presence of Ala245 within the ISDR was associated with nonresponse to treatment in genotype 1a-infected patients (P < 0.01). There were more mutations in the 26 amino acids downstream of the ISDR required for PKR binding in pretreatment isolates from responders versus nonresponders in both HCV-1a- and HCV-1b-infected patients (P < 0.05). In HCV-1a patients, more amino acid changes were observed in isolates from IFN-sensitive patients (P < 0.001), and the mutations appeared to be concentrated in two variable regions in the C terminus of NS5A, that corresponded to the previously described V3 region and a new variable region, 310 to 330. Selection of pretreatment minor V3 quasispecies was observed within the first 2 to 6 weeks of therapy in responders but not nonresponders, whereas the ISDR and PKR binding domains did not change in either patient response group. These data suggest that host-mediated selective pressures act primarily on the C terminus of NS5A and that NS5A can perturb or evade the IFN-induced antiviral response using sequences outside of the putative ISDR. Mechanistic studies are needed to address the role of the C terminus of NS5A in HCV replication and antiviral resistance.

scholarly journals A novel occludin-targeting monoclonal antibody prevents hepatitis C virus infection in vitro

Oncotarget ◽  
2018 ◽  
Vol 9 (24) ◽  
pp. 16588-16598 ◽  
Author(s):  
Ken Okai ◽  
Naoki Ichikawa-Tomikawa ◽  
Akira C. Saito ◽  
Tetsuya Watabe ◽  
Kotaro Sugimoto ◽  
...  
Keyword(s):  
Monoclonal Antibody ◽  
Hepatitis C Virus ◽  
Hepatitis C ◽  
Virus Infection ◽  
Hepatitis C Virus Infection

A Novel Anti CD23 Fully Human Monoclonal Antibody Potentially Useful for B-CLL Therapy.

Blood ◽  
2005 ◽  
Vol 106 (11) ◽  
pp. 5047-5047
Author(s):  
Marc Delcommenne ◽  
Hans-Georg Klingemann ◽  
Stephanie A. Gregory
Keyword(s):  
Monoclonal Antibody ◽  
B Lymphocytes ◽  
Human Monoclonal Antibody ◽  
Myeloma Cell ◽  
Lymphocytic Leukemia ◽  
Single Chain ◽  
Single Chain Antibody ◽  
Normal Peripheral Blood ◽  
Human Igg1

Abstract B-cell chronic lymphocytic leukemia (B-CLL) is one of the most common hematological malignancies and is, in most cases, characterized by an increased expression of CD23 on the cell surface. Since cross-linking CD23 induces B-CLL apoptosis, it is an attractive target for B-CLL antibody-based immunotherapy. In this study we show that an anti-CD23 human IgG1 monoclonal antibody, C6F5, may be useful in treating B-CLL. This antibody is derived from the human single chain antibody (scFv) C6F5 that was originally raised against the RPMI-8226 multiple myeloma cell line using the antibody phage display technique. While the C6F5 scFv did not bind to other myeloma cell lines, it was able to bind weakly to normal peripheral blood B lymphocytes and strongly to EBV transformed B cells and B-CLL cells. The antigen recognized by C6F5 was also upregulated on B lymphocytes that had been stimulated by CD40 ligand. Immunoprecipitations by the scFv C6F5 identified a protein of 45 kDa which co-migrated with CD23. Furthermore, this protein was recognized by an anti-CD23 mouse mAb in Western blot analyses. Immunofluorescence staining with the C6F5 scFv was inhibited if cells were preincubated with an anti-CD23 polyclonal antiserum. Taken together, these results verify that C6F5 recognizes CD23. The VH and VL regions of C6F5 antibody were then cloned into a baculovirus transfer vector encoding the human IgG1 heavy and light chains so that fully human C6F5 IgG1 antibody could be produced in baculovirus infected SF9 cells. Since C6F5 binding specificity was preserved in the IgG1 format, this antibody is ready to be tested in in vitro cytotoxic assays against B-CLL cells.

scholarly journals Specific targeting of hepatitis C virus core protein by an intracellular single-chain antibody of human origin

Hepatology ◽  
2008 ◽  
Vol 48 (3) ◽  
pp. 702-712 ◽  
Author(s):  
Juliane Karthe ◽  
Kathi Tessmann ◽  
Jisu Li ◽  
Raiki Machida ◽  
Maaike Daleman ◽  
...  
Keyword(s):  
Hepatitis C Virus ◽  
Hepatitis C ◽  
Core Protein ◽  
Human Origin ◽  
Single Chain ◽  
Single Chain Antibody ◽  
Specific Targeting ◽  
Virus Core

scholarly journals Loxoscelism: Advances and Challenges in the Design of Antibody Fragments with Therapeutic Potential

Toxins ◽  
2020 ◽  
Vol 12 (4) ◽  
pp. 256
Author(s):  
Sabrina Karim-Silva ◽  
Alessandra Becker-Finco ◽  
Isabella Gizzi Jiacomini ◽  
Fanny Boursin ◽  
Arnaud Leroy ◽  
...  
Keyword(s):  
Monoclonal Antibody ◽  
Therapeutic Potential ◽  
Antibody Fragment ◽  
Binding Activity ◽  
Antibody Fragments ◽  
Antigen Binding ◽  
Single Chain ◽  
Single Chain Antibody ◽  
Humanized Antibody ◽  
Neutralizing Capacity

Envenoming due to Loxosceles spider bites still remains a neglected disease of particular medical concern in the Americas. To date, there is no consensus for the treatment of envenomed patients, yet horse polyclonal antivenoms are usually infused to patients with identified severe medical conditions. It is widely known that venom proteins in the 30–35 kDa range with sphingomyelinase D (SMasesD) activity, reproduce most of the toxic effects observed in loxoscelism. Hence, we believe that monoclonal antibody fragments targeting such toxins might pose an alternative safe and effective treatment. In the present study, starting from the monoclonal antibody LimAb7, previously shown to target SMasesD from the venom of L. intermedia and neutralize its dermonecrotic activity, we designed humanized antibody V-domains, then produced and purified as recombinant single-chain antibody fragments (scFvs). These molecules were characterized in terms of humanness, structural stability, antigen-binding activity, and venom-neutralizing potential. Throughout this process, we identified some blocking points that can impact the Abs antigen-binding activity and neutralizing capacity. In silico analysis of the antigen/antibody amino acid interactions also contributed to a better understanding of the antibody’s neutralization mechanism and led to reformatting the humanized antibody fragment which, ultimately, recovered the functional characteristics for efficient in vitro venom neutralization.

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