An hepatitis B virus surface antigen specific single chain of variable fragment derived from a natural immune antigen binding fragment phage display library is specifically internalized by HepG2.2.15 cells

Wen, Weihong; Qin, Weijun; Gao, Hui; Zhao, Jing; Jia, Lintao; Liao, Qimei; Meng, Yanyan; Jin, Bo; Yao, Libo; Chen, S.-Y.; Yang, Aiying

doi:10.1111/j.1365-2893.2007.00843.x

Cited by 14 publications

(14 citation statements)

References 42 publications

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“…Maeda et al studied the B cell receptor (BCR) sequence associated with the response to HBV vaccination and the synthesis of anti-HBsAg monoclonal antibodies, and developed HBsAb transgenic tomatoes [4–6]. Several other studies have utilized BCR sequencing and phage display technology to detect HBsAbs with high affinity to HBsAg [7–9]. Yao et al and Yang et al used gene melting spectral pattern (GMSP) assay to detect clonal expansion in CD4 + T and CD8 + T cells in patients with acute HBV infection.…”

Section: Introductionmentioning

confidence: 99%

Characteristics Peripheral Blood IgG and IgM Heavy Chain Complementarity Determining Region 3 Repertoire before and after Immunization with Recombinant HBV Vaccine

Wang

et al. 2017

PLoS ONE

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Immunization with recombinant HBV vaccine induces specific immune responses in human causing B lymphocytes to produce protective HBsAb, and to form memory B lymphocytes, thereby facilitating HBV immunity in the body. However, B lymphocytes heterogeneity and characteristics are not fully elucidated. In this study, we conducted high-throughput sequencing of BCR heavy chain CDR3 repertoires in 3 healthy volunteers before and after the third immunization with recombinant HBV vaccine. We used Roche 454 Genome Sequencer FLX system to perform a comparative analysis of IgM and IgG H chain CDR3 repertoires. First, we found that the diversity of IgG H chain CDR3 repertoires was 1/6 of IgM on average. Moreover, after the third immunization with HBV vaccine, the diversity of IgG H chain CDR3 repertoires was 1/26 of IgM on average. Second, we detected relatively high levels of HBsAbs in all the healthy volunteers after immunization with HBV vaccine. The volunteers shared a small number of CDR3 sequences before and after immunization, and among each other. However, we did not find completely identical BCR H chain CDR3 amino acid sequences in these volunteers. Lastly, after immunization with recombinant HBV vaccine, the volunteers showed high frequency of IgG H chain CDR3 amino acid sequences mostly resulting from rearrangements of IGHV, IGHD and IGHJ, suggesting that the mechanism of high frequency CDR3 generation might be associated with the maturation of IgG affinity (somatic hypermutation) during the recombinant HBV vaccine-induced B lymphocyte responses. This study identified the characteristics and changes of BCR CDR3 repertoires before and after immunization with HBV vaccine, and evaluated the performance of the sequencing technology for this application. Our findings provide a basis for further research in B lymphocyte generated HBsAb heterogeneity and monitoring the maintenance of memory B lymphocytes.

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Section: Introductionmentioning

confidence: 99%

Characteristics Peripheral Blood IgG and IgM Heavy Chain Complementarity Determining Region 3 Repertoire before and after Immunization with Recombinant HBV Vaccine

Wang

et al. 2017

PLoS ONE

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“…A variety of HBV donor-specific phage display antibody libraries have been constructed in order to screen, select and develop HBsAg-specific monoclonal antibody therapeutics. 12,13 Using phage display technology, the complex structure of full-length antibodies have previously necessitated a cumbersome 'divide and conquer' approach whereby antibodies are first dissected into fragments that can be engineered in microorganisms, followed by 'stitching back' the optimized fragments into the full-length form for production in mammalian cells.…”

Section: Introductionmentioning

confidence: 99%

Identification of HBsAg-specific antibodies from a mammalian cell displayed full-length human antibody library of healthy immunized donor

Liang

Chen

et al. 2011

Cell Mol Immunol

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Hepatitis B immunoglobulin (HBIG) is important in the management of hepatitis B virus (HBV) infection. Aiming to develop recombinant monoclonal antibodies as an alternative to HBIG, we report the successful identification of HBV surface antigen (HBsAg)-specific antibodies from a full-length human antibody library displayed on mammalian cell surface. Using total RNA of peripheral blood mononuclear cells of a natively immunized donor as template, the antibody repertoire was amplified. Combining four-way ligation and the Flp recombinase-mediated integration (Flp-In) system, we constructed a mammalian cell-based, fully human, full-length antibody display library in which each cell displayed only one kind of antibody molecule. By screening the cell library using fluorescence-activated cell sorting (FACS), eight cell clones that displayed HBsAg-specific antibodies on cell surfaces were identified. DNA sequence analysis of the antibody genes revealed three unique antibodies. FACS data indicated that fluorescent strength of expression (FSE), fluorescent strength of binding (FSB) and relative binding ability (RBA) were all different among them. These results demonstrated that by using our antibody mammalian display and screening platform, we can successfully identify antigen-specific antibodies from an immunized full-length antibody library. Therefore, this platform is very useful for the development of therapeutic antibodies.

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“…20,21 One of the single-chain antibodies, HBs 15, was effectively internalized into HBsAg-positive cells and was reconstructed into s-tP and sC-tP fusion proteins to evaluate the applicability of antibody-mediated siRNA delivery strategy in HBV-infected cells.…”

mentioning

confidence: 99%

Targeted inhibition of HBV gene expression by single-chain antibody mediated small interfering RNA delivery

et al. 2007

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RNA interference is highly effective at inhibiting HBV gene expression and replication. However, before small interfering RNA (siRNA) can be used in the clinic, it is essential to develop a system to target their delivery. Antibody-mediated delivery is a novel approach for targeting siRNA to appropriate cells. In this report, we asked whether this siRNA delivery strategy would be effective against HBV. Of 5 candidates, a specific siRNA that effectively inhibited HBV gene expression and replication was determined. Two fusion proteins, s-tP and sC-tP, were constructed to contain a single chain of the human variable fragment, scFv, against hepatitis B surface antigen (HBsAg), a truncated protamine (tP), and in the case of sC-tP, a constant region of the chain (C). S-tP and sC-tP were developed to provide targeted delivery of the siRNA, siRNA expressing cassettes (SEC), and siRNA-producing plasmids. Fluorescein isothiocyanate-siRNA, fluorescein isothiocyanate-SEC, and plasmid DNA were specifically delivered into HBsAg-positive cells using the sC-tP fusion protein, and effectively inhibited HBV gene expression and replication. HBV gene expression was also inhibited by siRNA or siRNA-producing plasmids in HBV transgenic mice. Conclusion: Our results describe a potential method for the targeted delivery of siRNA or siRNAproducing plasmids against HBV, using anti-HBsAg fusion proteins. (HEPATOLOGY 2007;46: 84-94.) R ibonucleic acid interference is a naturally occurring mechanism for silencing homologous genes. 1,2 Synthetic and plasmid-based siRNA expression systems are both effective at suppressing HBV infection, in vitro and in vivo, [3][4][5][6][7][8][9] providing the basis for a new therapeutic strategy against HBV. However, the absence of a suitable delivery system presents a major obstacle against their clinical use. [10][11][12] Targeted delivery of small interfering RNA (siRNA) to relevant cell populations should increase the therapeutic index and minimize potential side effects and cellular toxicity.Antibody-mediated delivery is an effective method of targeting siRNA to particular cells. This involves fusing the nucleic acid-binding domain to a Fab fragment or scFv that recognizes a membrane receptor, resulting in a fusion protein that possesses cell recognition and nucleic acid-binding abilities. The fusion protein can then bind nucleic acids and deliver them into target cells through receptor-mediated endocytosis. This antibody-mediated uptake has been successfully used to deliver plasmid DNA and antisense oligonucleotides. [13][14][15][16][17] Song et al. 18 demonstrated that siRNA targeted delivered to human immunodeficiency virus-infected cells and HER2 positive cells by this strategy executed gene silencing. However, whether this promising strategy for siRNA delivery is applicable to other diseases remains unknown. 19 In this report, we provide evidence that antibody-mediated siRNA delivery is effectively targeted to HBV-infected cells.In a previous study, we have obtained 5 human antihepatitis B sur...

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An hepatitis B virus surface antigen specific single chain of variable fragment derived from a natural immune antigen binding fragment phage display library is specifically internalized by HepG2.2.15 cells

Cited by 14 publications

References 42 publications

Characteristics Peripheral Blood IgG and IgM Heavy Chain Complementarity Determining Region 3 Repertoire before and after Immunization with Recombinant HBV Vaccine

Characteristics Peripheral Blood IgG and IgM Heavy Chain Complementarity Determining Region 3 Repertoire before and after Immunization with Recombinant HBV Vaccine

Identification of HBsAg-specific antibodies from a mammalian cell displayed full-length human antibody library of healthy immunized donor

Targeted inhibition of HBV gene expression by single-chain antibody mediated small interfering RNA delivery

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