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Sample records for antibodies display broad

  1. VLPs displaying a single L2 epitope induce broadly cross-neutralizing antibodies against human papillomavirus.

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    Ebenezer Tumban

    Full Text Available BACKGROUND: Virus-like Particles (VLPs display can be used to increase the immunogenicity of heterologous antigens. Here, we report the use of a bacteriophage MS2-based VLP display platform to develop a monovalent vaccine targeting a broadly neutralizing epitope in the minor capsid protein human papillomavirus (HPV that provides broad protection from diverse HPV types in a mouse pseudovirus infection model. METHODOLOGY/PRINCIPAL FINDINGS: Peptides spanning a previously described cross-neutralizing epitope from HPV type 16 were genetically inserted at the N-terminus of MS2 bacteriophage coat protein. Three of the four recombinant L2-coat proteins assembled into VLPs. L2-VLPs elicited high-titer anti-L2 antibodies in mice, similar to recombinant VLPs that we had previously made in which the L2 peptide was displayed on a surface-exposed loop on VLPs of a related bacteriophage, PP7. Somewhat surprisingly, L2-MS2 VLPs elicited antibodies that were much more broadly cross-reactive with L2 peptides from diverse HPV isolates than L2-PP7 VLPs. Similarly, mice immunized with L2-MS2 VLPs were protected from genital and cutaneous infection by highly diverse HPV pseudovirus types. CONCLUSION/SIGNIFICANCE: We show that peptides can be displayed in a highly immunogenic fashion at the N-terminus of MS2 coat protein VLPs. A VLP-based vaccine targeting HPV L2 elicits broadly cross-reactive and cross-protective antibodies to heterologous HPV types. L2-VLPs could serve as the basis of a broadly protective second generation HPV vaccine.

  2. Broadly Neutralizing Antibodies Display Potential for Prevention of HIV-1 Infection of Mucosal Tissue Superior to That of Nonneutralizing Antibodies.

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    Cheeseman, Hannah M; Olejniczak, Natalia J; Rogers, Paul M; Evans, Abbey B; King, Deborah F L; Ziprin, Paul; Liao, Hua-Xin; Haynes, Barton F; Shattock, Robin J

    2017-01-01

    Definition of the key parameters mediating effective antibody blocking of HIV-1 acquisition within mucosal tissue may prove critical to effective vaccine development and the prophylactic use of monoclonal antibodies. Although direct antibody-mediated neutralization is highly effective against cell-free virus, antibodies targeting different sites of envelope vulnerability may display differential activity against mucosal infection. Nonneutralizing antibodies (nnAbs) may also impact mucosal transmission events through Fc-gamma receptor (FcγR)-mediated inhibition. In this study, a panel of broadly neutralizing antibodies (bnAbs) and nnAbs, including those associated with protection in the RV144 vaccine trial, were screened for the ability to block HIV-1 acquisition and replication across a range of cellular and mucosal tissue models. Neutralization potency, as determined by the TZM-bl infection assay, did not fully predict activity in mucosal tissue. CD4-binding site (CD4bs)-specific bnAbs, in particular VRC01, were consistent in blocking HIV-1 infection across all cellular and tissue models. Membrane-proximal external region (MPER) (2F5) and outer domain glycan (2G12) bnAbs were also efficient in preventing infection of mucosal tissues, while the protective efficacy of bnAbs targeting V1-V2 glycans (PG9 and PG16) was more variable. In contrast, nnAbs alone and in combinations, while active in a range of cellular assays, were poorly protective against HIV-1 infection of mucosal tissues. These data suggest that tissue resident effector cell numbers and low FcγR expression may limit the potential of nnAbs to prevent establishment of the initial foci of infection. The solid protection provided by specific bnAbs clearly demonstrates their superior potential over that of nonneutralizing antibodies for preventing HIV-1 infection at the mucosal portals of infection.

  3. Broadly Neutralizing Antibodies Display Potential for Prevention of HIV-1 Infection of Mucosal Tissue Superior to That of Nonneutralizing Antibodies

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    Cheeseman, Hannah M.; Olejniczak, Natalia J.; Rogers, Paul M.; Evans, Abbey B.; King, Deborah F. L.; Ziprin, Paul; Liao, Hua-Xin; Haynes, Barton F.

    2016-01-01

    ABSTRACT Definition of the key parameters mediating effective antibody blocking of HIV-1 acquisition within mucosal tissue may prove critical to effective vaccine development and the prophylactic use of monoclonal antibodies. Although direct antibody-mediated neutralization is highly effective against cell-free virus, antibodies targeting different sites of envelope vulnerability may display differential activity against mucosal infection. Nonneutralizing antibodies (nnAbs) may also impact mucosal transmission events through Fc-gamma receptor (FcγR)-mediated inhibition. In this study, a panel of broadly neutralizing antibodies (bnAbs) and nnAbs, including those associated with protection in the RV144 vaccine trial, were screened for the ability to block HIV-1 acquisition and replication across a range of cellular and mucosal tissue models. Neutralization potency, as determined by the TZM-bl infection assay, did not fully predict activity in mucosal tissue. CD4-binding site (CD4bs)-specific bnAbs, in particular VRC01, were consistent in blocking HIV-1 infection across all cellular and tissue models. Membrane-proximal external region (MPER) (2F5) and outer domain glycan (2G12) bnAbs were also efficient in preventing infection of mucosal tissues, while the protective efficacy of bnAbs targeting V1-V2 glycans (PG9 and PG16) was more variable. In contrast, nnAbs alone and in combinations, while active in a range of cellular assays, were poorly protective against HIV-1 infection of mucosal tissues. These data suggest that tissue resident effector cell numbers and low FcγR expression may limit the potential of nnAbs to prevent establishment of the initial foci of infection. The solid protection provided by specific bnAbs clearly demonstrates their superior potential over that of nonneutralizing antibodies for preventing HIV-1 infection at the mucosal portals of infection. IMPORTANCE Key parameters mediating effective antibody blocking of HIV-1 acquisition within mucosal

  4. Comprehensive mapping of functional epitopes on dengue virus glycoprotein E DIII for binding to broadly neutralizing antibodies 4E11 and 4E5A by phage display.

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    Frei, Julia C; Kielian, Margaret; Lai, Jonathan R

    2015-11-01

    Here we investigated the binding of Dengue virus envelope glycoprotein domain III (DIII) by two broadly neutralizing antibodies (bNAbs), 4E11 and 4E5A. There are four serotypes of Dengue virus (DENV-1 to -4), whose DIII sequences vary by up to 49%. We used combinatorial alanine scanning mutagenesis, a phage display approach, to map functional epitopes (those residues that contribute most significantly to the energetics of antibody-antigen interaction) on these four serotypes. Our results showed that 4E11, which binds strongly to DENV-1, -2, and -3, and moderately to DENV-4, recognized a common conserved core functional epitope involving DIII residues K310, L/I387, L389, and W391. There were also unique recognition features for each serotype, suggesting that 4E11 has flexible recognition requirements. Similar scanning studies for the related bNAb 4E5A, which binds more tightly to DENV-4, identified broader functional epitopes on DENV-1. These results provide useful information for immunogen and therapeutic antibody design.

  5. Engineering antibodies by yeast display.

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    Boder, Eric T; Raeeszadeh-Sarmazdeh, Maryam; Price, J Vincent

    2012-10-15

    Since its first application to antibody engineering 15 years ago, yeast display technology has been developed into a highly potent tool for both affinity maturing lead molecules and isolating novel antibodies and antibody-like species. Robust approaches to the creation of diversity, construction of yeast libraries, and library screening or selection have been elaborated, improving the quality of engineered molecules and certainty of success in an antibody engineering campaign and positioning yeast display as one of the premier antibody engineering technologies currently in use. Here, we summarize the history of antibody engineering by yeast surface display, approaches used in its application, and a number of examples highlighting the utility of this method for antibody engineering.

  6. Broadly Neutralizing Antibodies for HIV Eradication.

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    Stephenson, Kathryn E; Barouch, Dan H

    2016-02-01

    Passive transfer of antibodies has long been considered a potential treatment modality for infectious diseases, including HIV. Early efforts to use antibodies to suppress HIV replication, however, were largely unsuccessful, as the antibodies that were studied neutralized only a relatively narrow spectrum of viral strains and were not very potent. Recent advances have led to the discovery of a large portfolio of human monoclonal antibodies that are broadly neutralizing across many HIV-1 subtypes and are also substantially more potent. These antibodies target multiple different epitopes on the HIV envelope, thus allowing for the development of antibody combinations. In this review, we discuss the application of broadly neutralizing antibodies (bNAbs) for HIV treatment and HIV eradication strategies. We highlight bNAbs that target key epitopes, such as the CD4 binding site and the V2/V3-glycan-dependent sites, and we discuss several bNAbs that are currently in the clinical development pipeline.

  7. Methods for Selecting Phage Display Antibody Libraries.

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    Jara-Acevedo, Ricardo; Diez, Paula; Gonzalez-Gonzalez, Maria; Degano, Rosa Maria; Ibarrola, Nieves; Gongora, Rafael; Orfao, Alberto; Fuentes, Manuel

    2016-01-01

    The selection process aims sequential enrichment of phage antibody display library in clones that recognize the target of interest or antigen as the library undergoes successive rounds of selection. In this review, selection methods most commonly used for phage display antibody libraries have been comprehensively described.

  8. Cross-reactive broadly neutralizing antibodies: timing is everything

    OpenAIRE

    Euler, Zelda; Schuitemaker, Hanneke

    2012-01-01

    The recent surge of research into new broadly neutralizing antibodies in HIV-1 infection has recharged the field of HIV-1 vaccinology. In this review we discuss the currently known broadly neutralizing antibodies and focus on factors that may shape these antibodies in natural infection. We further discuss the role of these antibodies in the clinical course of the infection and consider immunological obstacles in inducing broadly neutralizing antibodies with a vaccine.

  9. Cross-reactive broadly neutralizing antibodies: timing is everything.

    Science.gov (United States)

    Euler, Zelda; Schuitemaker, Hanneke

    2012-01-01

    The recent surge of research into new broadly neutralizing antibodies in HIV-1 infection has recharged the field of HIV-1 vaccinology. In this review we discuss the currently known broadly neutralizing antibodies and focus on factors that may shape these antibodies in natural infection. We further discuss the role of these antibodies in the clinical course of the infection and consider immunological obstacles in inducing broadly neutralizing antibodies with a vaccine.

  10. Baculovirus display of functional antibody Fab fragments.

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    Takada, Shinya; Ogawa, Takafumi; Matsui, Kazusa; Suzuki, Tasuku; Katsuda, Tomohisa; Yamaji, Hideki

    2015-08-01

    The generation of a recombinant baculovirus that displays antibody Fab fragments on the surface was investigated. A recombinant baculovirus was engineered so that the heavy chain (Hc; Fd fragment) of a mouse Fab fragment was expressed as a fusion to the N-terminus of baculovirus gp64, while the light chain of the Fab fragment was simultaneously expressed as a secretory protein. Following infection of Sf9 insect cells with the recombinant baculovirus, the culture supernatant was analyzed by enzyme-linked immunosorbent assay using antigen-coated microplates and either an anti-mouse IgG or an anti-gp64 antibody. A relatively strong signal was obtained in each case, showing antigen-binding activity in the culture supernatant. In western blot analysis of the culture supernatant using the anti-gp64 antibody, specific protein bands were detected at an electrophoretic mobility that coincided with the molecular weight of the Hc-gp64 fusion protein as well as that of gp64. Flow cytometry using a fluorescein isothiocyanate-conjugated antibody specific to mouse IgG successfully detected the Fab fragments on the surface of the Sf9 cells. These results suggest that immunologically functional antibody Fab fragments can be displayed on the surface of baculovirus particles, and that a fluorescence-activated cell sorter with a fluorescence-labeled antigen can isolate baculoviruses displaying specific Fab fragments. This successful baculovirus display of antibody Fab fragments may offer a novel approach for the efficient selection of specific antibodies.

  11. Simultaneous expression of displayed and secreted antibodies for antibody screen.

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    Yuanping Zhou

    Full Text Available The display of full-length antibody on the cell surface was achieved by fusing a transmembrane domain of the platelet-derived growth factor receptor (PDGFR to the C-terminus of the heavy chain constant region. We also incorporated a furin cleavage site between the constant region and PDGFR transmembrane domain to obtain secreted antibodies. As a result, antibodies can be expressed simultaneously on the cell surface in a membrane-anchored version for screening and selecting through fluorescence-activated cell sorting (FACS analysis, as well as in conditioned medium in a secreted version for function analysis.

  12. Antiviral Therapy by HIV-1 Broadly Neutralizing and Inhibitory Antibodies

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    Zhiqing Zhang

    2016-11-01

    Full Text Available Human immunodeficiency virus type 1 (HIV-1 infection causes acquired immune deficiency syndrome (AIDS, a global epidemic for more than three decades. HIV-1 replication is primarily controlled through antiretroviral therapy (ART but this treatment does not cure HIV-1 infection. Furthermore, there is increasing viral resistance to ART, and side effects associated with long-term therapy. Consequently, there is a need of alternative candidates for HIV-1 prevention and therapy. Recent advances have discovered multiple broadly neutralizing antibodies against HIV-1. In this review, we describe the key epitopes on the HIV-1 Env protein and the reciprocal broadly neutralizing antibodies, and discuss the ongoing clinical trials of broadly neutralizing and inhibitory antibody therapy as well as antibody combinations, bispecific antibodies, and methods that improve therapeutic efficacy by combining broadly neutralizing antibodies (bNAbs with latency reversing agents. Compared with ART, HIV-1 therapeutics that incorporate these broadly neutralizing and inhibitory antibodies offer the advantage of decreasing virus load and clearing infected cells, which is a promising prospect in HIV-1 prevention and treatment.

  13. Antiviral Therapy by HIV-1 Broadly Neutralizing and Inhibitory Antibodies.

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    Zhang, Zhiqing; Li, Shaowei; Gu, Ying; Xia, Ningshao

    2016-11-18

    Human immunodeficiency virus type 1 (HIV-1) infection causes acquired immune deficiency syndrome (AIDS), a global epidemic for more than three decades. HIV-1 replication is primarily controlled through antiretroviral therapy (ART) but this treatment does not cure HIV-1 infection. Furthermore, there is increasing viral resistance to ART, and side effects associated with long-term therapy. Consequently, there is a need of alternative candidates for HIV-1 prevention and therapy. Recent advances have discovered multiple broadly neutralizing antibodies against HIV-1. In this review, we describe the key epitopes on the HIV-1 Env protein and the reciprocal broadly neutralizing antibodies, and discuss the ongoing clinical trials of broadly neutralizing and inhibitory antibody therapy as well as antibody combinations, bispecific antibodies, and methods that improve therapeutic efficacy by combining broadly neutralizing antibodies (bNAbs) with latency reversing agents. Compared with ART, HIV-1 therapeutics that incorporate these broadly neutralizing and inhibitory antibodies offer the advantage of decreasing virus load and clearing infected cells, which is a promising prospect in HIV-1 prevention and treatment.

  14. Human broadly neutralizing antibodies to the envelope glycoprotein complex of hepatitis C virus

    DEFF Research Database (Denmark)

    Giang, Erick; Dorner, Marcus; Prentoe, Jannick C

    2012-01-01

    Abs recognizing five distinct antigenic regions on the virus envelope glycoprotein complex E1E2 from an HCV-immune phage-display antibody library by using an exhaustive-panning strategy. Many of these mAbs were broadly neutralizing. In particular, the mAb AR4A, recognizing a discontinuous epitope outside the CD81......bs on the E1E2 complex, has an exceptionally broad neutralizing activity toward diverse HCV genotypes and protects against heterologous HCV challenge in a small animal model. The mAb panel will be useful for the design and development of vaccine candidates to elicit broadly neutralizing antibodies...

  15. Broadly protective influenza vaccines: Redirecting the antibody response through adjuvation

    NARCIS (Netherlands)

    Cox, F.

    2016-01-01

    Influenza virus infections are responsible for significant morbidity worldwide and current vaccines have limited coverage, therefore it remains a high priority to develop broadly protective vaccines. With the discovery of broadly neutralizing antibodies (bnAbs) against influenza these vaccines becam

  16. Engineering broadly neutralizing antibodies for HIV prevention and therapy.

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    Hua, Casey K; Ackerman, Margaret E

    2016-08-01

    A combination of advances spanning from isolation to delivery of potent HIV-specific antibodies has begun to revolutionize understandings of antibody-mediated antiviral activity. As a result, the set of broadly neutralizing and highly protective antibodies has grown in number, diversity, potency, and breadth of viral recognition and neutralization. These antibodies are now being further enhanced by rational engineering of their anti-HIV activities and coupled to cutting edge gene delivery and strategies to optimize their pharmacokinetics and biodistribution. As a result, the prospects for clinical use of HIV-specific antibodies to treat, clear, and prevent HIV infection are gaining momentum. Here we discuss the diverse methods whereby antibodies are being optimized for neutralization potency and breadth, biodistribution, pharmacokinetics, and effector function with the aim of revolutionizing HIV treatment and prevention options.

  17. Phage display-derived human antibodies in clinical development and therapy.

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    Frenzel, André; Schirrmann, Thomas; Hust, Michael

    2016-10-01

    Over the last 3 decades, monoclonal antibodies have become the most important class of therapeutic biologicals on the market. Development of therapeutic antibodies was accelerated by recombinant DNA technologies, which allowed the humanization of murine monoclonal antibodies to make them more similar to those of the human body and suitable for a broad range of chronic diseases like cancer and autoimmune diseases. In the early 1990s in vitro antibody selection technologies were developed that enabled the discovery of "fully" human antibodies with potentially superior clinical efficacy and lowest immunogenicity. Antibody phage display is the first and most widely used of the in vitro selection technologies. It has proven to be a robust, versatile platform technology for the discovery of human antibodies and a powerful engineering tool to improve antibody properties. As of the beginning of 2016, 6 human antibodies discovered or further developed by phage display were approved for therapy. In 2002, adalimumab (Humira®) became the first phage display-derived antibody granted a marketing approval. Humira® was also the first approved human antibody, and it is currently the best-selling antibody drug on the market. Numerous phage display-derived antibodies are currently under advanced clinical investigation, and, despite the availability of other technologies such as human antibody-producing transgenic mice, phage display has not lost its importance for the discovery and engineering of therapeutic antibodies. Here, we provide a comprehensive overview about phage display-derived antibodies that are approved for therapy or in clinical development. A selection of these antibodies is described in more detail to demonstrate different aspects of the phage display technology and its development over the last 25 years.

  18. Identification and specificity of broadly neutralizing antibodies against HIV

    OpenAIRE

    McCoy, Laura E.; Burton, Dennis R.

    2017-01-01

    Summary Beginning in 2009, studies of the humoral responses of HIV‐positive individuals have led to the identification of scores, if not hundreds, of antibodies that are both broadly reactive and potently neutralizing. This development has provided renewed impetus toward an HIV vaccine and led directly to the development of novel immunogens. Advances in identification of donors with the most potent and broad anti‐HIV serum neutralizing responses were crucial in this effort. Equally, developme...

  19. Broad epitope coverage of a human in vitro antibody library

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    Sivasubramanian, Arvind; Lynaugh, Heather; Yu, Yao; Miles, Adam; Eckman, Josh; Schutz, Kevin; Piffath, Crystal; Boland, Nadthakarn; Durand, Stéphanie; Boland, Todd; Vásquez, Maximiliano; Xu, Yingda; Abdiche, Yasmina

    2017-01-01

    ABSTRACT Successful discovery of therapeutic antibodies hinges on the identification of appropriate affinity binders targeting a diversity of molecular epitopes presented by the antigen. Antibody campaigns that yield such broad “epitope coverage” increase the likelihood of identifying candidates with the desired biological functions. Accordingly, epitope binning assays are employed in the early discovery stages to partition antibodies into epitope families or “bins” and prioritize leads for further characterization and optimization. The collaborative program described here, which used hen egg white lysozyme (HEL) as a model antigen, combined 3 key capabilities: 1) access to a diverse panel of antibodies selected from a human in vitro antibody library; 2) application of state-of-the-art high-throughput epitope binning; and 3) analysis and interpretation of the epitope binning data with reference to an exhaustive set of published antibody:HEL co-crystal structures. Binning experiments on a large merged panel of antibodies containing clones from the library and the literature revealed that the inferred epitopes for the library clones overlapped with, and extended beyond, the known structural epitopes. Our analysis revealed that nearly the entire solvent-exposed surface of HEL is antigenic, as has been proposed for protein antigens in general. The data further demonstrated that synthetic antibody repertoires provide as wide epitope coverage as those obtained from animal immunizations. The work highlights molecular insights contributed by increasingly higher-throughput binning methods and their broad utility to guide the discovery of therapeutic antibodies representing a diverse set of functional epitopes. PMID:27748644

  20. Escherichia coli surface display of single-chain antibody VRC01 against HIV-1 infection

    Energy Technology Data Exchange (ETDEWEB)

    Wang, Lin-Xu [Nebraska Center for Virology, Lincoln, NE (United States); School of Biological Sciences, University of Nebraska—Lincoln, Lincoln, NE 68583 (United States); Mellon, Michael [Nebraska Center for Virology, Lincoln, NE (United States); School of Veterinary Medicine and Biomedical Sciences, Lincoln, NE (United States); Bowder, Dane [Nebraska Center for Virology, Lincoln, NE (United States); School of Biological Sciences, University of Nebraska—Lincoln, Lincoln, NE 68583 (United States); Quinn, Meghan [Nebraska Center for Virology, Lincoln, NE (United States); School of Veterinary Medicine and Biomedical Sciences, Lincoln, NE (United States); Shea, Danielle; Wood, Charles [Nebraska Center for Virology, Lincoln, NE (United States); School of Biological Sciences, University of Nebraska—Lincoln, Lincoln, NE 68583 (United States); Xiang, Shi-Hua, E-mail: sxiang2@unl.edu [Nebraska Center for Virology, Lincoln, NE (United States); School of Veterinary Medicine and Biomedical Sciences, Lincoln, NE (United States)

    2015-01-15

    Human immunodeficiency virus type 1 (HIV-1) transmission and infection occur mainly via the mucosal surfaces. The commensal bacteria residing in these surfaces can potentially be employed as a vehicle for delivering inhibitors to prevent HIV-1 infection. In this study, we have employed a bacteria-based strategy to display a broadly neutralizing antibody VRC01, which could potentially be used to prevent HIV-1 infection. The VRC01 antibody mimics CD4-binding to gp120 and has broadly neutralization activities against HIV-1. We have designed a construct that can express the fusion peptide of the scFv-VRC01 antibody together with the autotransporter β-barrel domain of IgAP gene from Neisseria gonorrhoeae, which enabled surface display of the antibody molecule. Our results indicate that the scFv-VRC01 antibody molecule was displayed on the surface of the bacteria as demonstrated by flow cytometry and immunofluorescence microscopy. The engineered bacteria can capture HIV-1 particles via surface-binding and inhibit HIV-1 infection in cell culture. - Highlights: • Designed single-chain VRC01 antibody was demonstrated to bind HIV-1 envelope gp120. • Single-chain VRC01 antibody was successfully displayed on the surface of E. coli. • Engineered bacteria can absorb HIV-1 particles and prevent HIV-1 infection in cell culture.

  1. An efficient method for isolating antibody fragments against small peptides by antibody phage display

    DEFF Research Database (Denmark)

    Duan, Zhi; Siegumfeldt, Henrik

    2010-01-01

    We generated monoclonal scFv (single chain variable fragment) antibodies from an antibody phage display library towards three small synthetic peptides derived from the sequence of s1-casein. Key difficulties for selection of scFv-phages against small peptides were addressed. Small peptides do....... The scFvs were sequenced and characterized, and specificity was characterized by ELISA. The methods developed in this study are universally applicable for antibody phage display to efficiently produce antibody fragments against small peptides....

  2. Recognition determinants of broadly neutralizing human antibodies against dengue viruses.

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    Rouvinski, Alexander; Guardado-Calvo, Pablo; Barba-Spaeth, Giovanna; Duquerroy, Stéphane; Vaney, Marie-Christine; Kikuti, Carlos M; Navarro Sanchez, M Erika; Dejnirattisai, Wanwisa; Wongwiwat, Wiyada; Haouz, Ahmed; Girard-Blanc, Christine; Petres, Stéphane; Shepard, William E; Desprès, Philippe; Arenzana-Seisdedos, Fernando; Dussart, Philippe; Mongkolsapaya, Juthathip; Screaton, Gavin R; Rey, Félix A

    2015-04-02

    Dengue disease is caused by four different flavivirus serotypes, which infect 390 million people yearly with 25% symptomatic cases and for which no licensed vaccine is available. Recent phase III vaccine trials showed partial protection, and in particular no protection for dengue virus serotype 2 (refs 3, 4). Structural studies so far have characterized only epitopes recognized by serotype-specific human antibodies. We recently isolated human antibodies potently neutralizing all four dengue virus serotypes. Here we describe the X-ray structures of four of these broadly neutralizing antibodies in complex with the envelope glycoprotein E from dengue virus serotype 2, revealing that the recognition determinants are at a serotype-invariant site at the E-dimer interface, including the exposed main chain of the E fusion loop and the two conserved glycan chains. This 'E-dimer-dependent epitope' is also the binding site for the viral glycoprotein prM during virus maturation in the secretory pathway of the infected cell, explaining its conservation across serotypes and highlighting an Achilles' heel of the virus with respect to antibody neutralization. These findings will be instrumental for devising novel immunogens to protect simultaneously against all four serotypes of dengue virus.

  3. Structural basis of hepatitis C virus neutralization by broadly neutralizing antibody HCV1

    Energy Technology Data Exchange (ETDEWEB)

    Kong, Leopold; Giang, Erick; Robbins, Justin B.; Stanfield, Robyn L.; Burton, Dennis R.; Wilson, Ian A.; Law, Mansun (Scripps)

    2012-10-29

    Hepatitis C virus (HCV) infects more than 2% of the global population and is a leading cause of liver cirrhosis, hepatocellular carcinoma, and end-stage liver diseases. Circulating HCV is genetically diverse, and therefore a broadly effective vaccine must target conserved T- and B-cell epitopes of the virus. Human mAb HCV1 has broad neutralizing activity against HCV isolates from at least four major genotypes and protects in the chimpanzee model from primary HCV challenge. The antibody targets a conserved antigenic site (residues 412-423) on the virus E2 envelope glycoprotein. Two crystal structures of HCV1 Fab in complex with an epitope peptide at 1.8-{angstrom} resolution reveal that the epitope is a {beta}-hairpin displaying a hydrophilic face and a hydrophobic face on opposing sides of the hairpin. The antibody predominantly interacts with E2 residues Leu{sup 413} and Trp{sup 420} on the hydrophobic face of the epitope, thus providing an explanation for how HCV isolates bearing mutations at Asn{sup 415} on the same binding face escape neutralization by this antibody. The results provide structural information for a neutralizing epitope on the HCV E2 glycoprotein and should help guide rational design of HCV immunogens to elicit similar broadly neutralizing antibodies through vaccination.

  4. Broadly neutralizing antibodies: An approach to control HIV-1 infection.

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    Yaseen, Mahmoud Mohammad; Yaseen, Mohammad Mahmoud; Alqudah, Mohammad Ali

    2017-01-02

    Although available antiretroviral therapy (ART) has changed human immunodeficiency virus (HIV)-1 infection to a non-fatal chronic disease, the economic burden of lifelong therapy, severe adverse ART effects, daily ART adherence, and emergence of ART-resistant HIV-1 mutants require prospecting for alternative therapeutic modalities. Indeed, a growing body of evidence suggests that broadly neutralizing anti-HIV-1 antibodies (BNAbs) may offer one such feasible alternative. To evaluate their therapeutic potential in established HIV-1 infection, we sought to address recent advances in pre-clinical and clinical investigations in this area of HIV-1 research. In addition, we addressed the obstacles that may impede the success of such immunotherapeutic approach, suggested strategic solutions, and briefly compared this approach with the currently used ART to open new insights for potential future passive immunotherapy for HIV-1 infection.

  5. HIV-1 envelope glycoprotein immunogens to induce broadly neutralizing antibodies.

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    Sliepen, Kwinten; Sanders, Rogier W

    2016-01-01

    The long pursuit for a vaccine against human immunodeficiency virus 1 (HIV-1) has recently been boosted by a number of exciting developments. An HIV-1 subunit vaccine ideally should elicit potent broadly neutralizing antibodies (bNAbs), but raising bNAbs by vaccination has proved extremely difficult because of the characteristics of the HIV-1 envelope glycoprotein complex (Env). However, the isolation of bNAbs from HIV-1-infected patients demonstrates that the human humoral immune system is capable of making such antibodies. Therefore, a focus of HIV-1 vaccinology is the elicitation of bNAbs by engineered immunogens and by using vaccination strategies aimed at mimicking the bNAb maturation pathways in HIV-infected patients. Important clues can also be taken from the successful subunit vaccines against hepatitis B virus and human papillomavirus. Here, we review the different types of HIV-1 immunogens and vaccination strategies that are being explored in the search for an HIV-1 vaccine that induces bNAbs.

  6. Construction of human antibody gene libraries and selection of antibodies by phage display.

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    Frenzel, André; Kügler, Jonas; Wilke, Sonja; Schirrmann, Thomas; Hust, Michael

    2014-01-01

    Antibody phage display is the most commonly used in vitro selection technology and has yielded thousands of useful antibodies for research, diagnostics, and therapy.The prerequisite for successful generation and development of human recombinant antibodies using phage display is the construction of a high-quality antibody gene library. Here, we describe the methods for the construction of human immune and naive scFv gene libraries.The success also depends on the panning strategy for the selection of binders from these libraries. In this article, we describe a panning strategy that is high-throughput compatible and allows parallel selection in microtiter plates.

  7. Discovery and Characterization of Phage Display-Derived Human Monoclonal Antibodies against RSV F Glycoprotein.

    Directory of Open Access Journals (Sweden)

    Zhifeng Chen

    Full Text Available Respiratory syncytial virus (RSV is a leading cause of lower respiratory tract infection in infants, the elderly and in immunosuppressed populations. The vast majority of neutralizing antibodies isolated from human subjects target the RSV fusion (F glycoprotein, making it an attractive target for the development of vaccines and therapeutic antibodies. Currently, Synagis® (palivizumab is the only FDA approved antibody drug for the prevention of RSV infection, and there is a great need for more effective vaccines and therapeutics. Phage display is a powerful tool in antibody discovery with the advantage that it does not require samples from immunized subjects. In this study, Morphosys HuCAL GOLD® phage libraries were used for panning against RSV prefusion and postfusion F proteins. Panels of human monoclonal antibodies (mAbs against RSV F protein were discovered following phage library panning and characterized. Antibodies binding specifically to prefusion or postfusion F proteins and those binding both conformations were identified. 3B1 is a prototypic postfusion F specific antibody while 2E1 is a prototypic prefusion F specific antibody. 2E1 is a potent broadly neutralizing antibody against both RSV A and B strains. Epitope mapping experiments identified a conformational epitope spanning across three discontinuous sections of the RSV F protein, as well as critical residues for antibody interaction.

  8. Ricin Detection Using Phage Displayed Single Domain Antibodies

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    Ellen R. Goldman

    2009-01-01

    Full Text Available Phage-displayed single domain antibodies (sdAb were compared to monomeric solubly expressed sdAb and llama polyclonal antibodies for the detection of ricin. SdAb are comprised of the variable domain derived from camelid heavy chain only antibodies (HcAb. Although HcAb lack variable light chains, they as well as their derivative sdAb are able to bind antigens with high affinity. The small size of sdAb (~16 kDa, while advantageous in many respects, limits the number of labels that can be incorporated. The ability to incorporate multiple labels is a beneficial attribute for reporter elements. Opportunely, sdAb are often selected using phage display methodology. Using sdAb displayed on bacteriophage M13 as the reporter element gives the potential for incorporating a very high number of labels. We have demonstrated the use of both sdAb and phage- displayed sdAb for the detection of ricin using both enzyme linked immunosorbent assays (ELISAs and Luminex fluid array assays. The phage-displayed sdAb led to five to ten fold better detection of ricin in both the ELISA and Luminex assays, resulting in limits of detection of 1 ng/mL and 64 pg/mL respectively. The phage-displayed sdAb were also dramatically more effective for the visualization of binding to target in nitrocellulose dot blot assays, a method frequently used for epitope mapping.

  9. Antibody repertoire profiling using bacterial display identifies reactivity signatures of celiac disease.

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    Spatola, Bradley N; Murray, Joseph A; Kagnoff, Martin; Kaukinen, Katri; Daugherty, Patrick S

    2013-01-15

    A general strategy to identify serum antibody specificities associated with a given disease state and peptide reagents for their detection was developed using bacterial display peptide libraries and multiparameter flow cytometry (MPFC). Using sera from patients with celiac disease (CD) (n = 45) or healthy subjects (n = 40), bacterial display libraries were screened for peptides that react specifically with antibodies from CD patients and not with those from healthy patients. The libraries were screened for peptides that simultaneously cross-react with CD patient antibodies present in two separate patient groups labeled with spectrally distinct fluorophores but do not react with unlabeled non-CD antibodies, thus affording a quantitative separation. A panel of six unique peptide sequences yielded 85% sensitivity and 91% specificity (AUC = 0.91) on a set of 60 samples not used for discovery, using leave-one-out cross-validation. Individual peptides were dissimilar with known CD-specific antigens tissue transglutaminase (tTG) and deamidated gliadin, and the classifier accuracy was independent of anti-tTG antibody titer. These results demonstrate that bacterial display/MPFC provides a highly effective tool for the unbiased discovery of disease-associated antibody specificities and peptide reagents for their detection that may have broad utility for diagnostic development.

  10. A recombinant, fully human monoclonal antibody with antitumor activity constructed from phage-displayed antibody fragments

    NARCIS (Netherlands)

    Huls, GA; Heijnen, IAFM; Cuomo, ME; Koningsberger, JC; Boel, E; de Vries, ARV; Loyson, SAJ; Helfrich, W; Henegouwen, GPV; van Meijer, M; de Kruif, J; Logtenberg, T

    1999-01-01

    A single-chain Fv antibody fragment specific for the tumor-associated Ep-CAM molecule was isolated from a semisynthetic phage display library and converted into an intact, fully human IgG1 monoclonal antibody (huMab), The purified huMab had an affinity of 5 nM and effectively mediated tumor cell kil

  11. Antibody engineering using phage display with a coiled-coil heterodimeric Fv antibody fragment.

    Directory of Open Access Journals (Sweden)

    Xinwei Wang

    Full Text Available A Fab-like antibody binding unit, ccFv, in which a pair of heterodimeric coiled-coil domains was fused to V(H and V(L for Fv stabilization, was constructed for an anti-VEGF antibody. The anti-VEGF ccFv showed the same binding affinity as scFv but significantly improved stability and phage display level. Furthermore, phage display libraries in the ccFv format were constructed for humanization and affinity maturation of the anti-VEGF antibody. A panel of V(H frameworks and V(H-CDR3 variants, with a significant improvement in affinity and expressibility in both E. coli and yeast systems, was isolated from the ccFv phage libraries. These results demonstrate the potential application of the ccFv antibody format in antibody engineering.

  12. Peptides of the constant region of antibodies display fungicidal activity.

    Directory of Open Access Journals (Sweden)

    Luciano Polonelli

    Full Text Available Synthetic peptides with sequences identical to fragments of the constant region of different classes (IgG, IgM, IgA of antibodies (Fc-peptides exerted a fungicidal activity in vitro against pathogenic yeasts, such as Candida albicans, Candida glabrata, Cryptococcus neoformans, and Malassezia furfur, including caspofungin and triazole resistant strains. Alanine-substituted derivatives of fungicidal Fc-peptides, tested to evaluate the critical role of each residue, displayed unaltered, increased or decreased candidacidal activity in vitro. An Fc-peptide, included in all human IgGs, displayed a therapeutic effect against experimental mucosal and systemic candidiasis in mouse models. It is intriguing to hypothesize that some Fc-peptides may influence the antifungal immune response and constitute the basis for devising new antifungal agents.

  13. A broad set of different llama antibodies specific for a 16 kDa heat shock protein of Mycobacterium tuberculosis.

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    Anke K Trilling

    Full Text Available BACKGROUND: Recombinant antibodies are powerful tools in engineering of novel diagnostics. Due to the small size and stable nature of llama antibody domains selected antibodies can serve as a detection reagent in multiplexed and sensitive assays for M. tuberculosis. METHODOLOGY/PRINCIPAL FINDINGS: Antibodies for Mycobacterium tuberculosis (M. tb recognition were raised in Alpaca, and, by phage display, recombinant variable domains of heavy-chain antibodies (VHH binding to M. tuberculosis antigens were isolated. Two phage display selection strategies were followed: one direct selection using semi-purified protein antigen, and a depletion strategy with lysates, aiming to avoid cross-reaction to other mycobacteria. Both panning methods selected a set of binders with widely differing complementarity determining regions. Selected recombinant VHHs were produced in E. coli and shown to bind immobilized lysate in direct Enzymelinked Immunosorbent Assay (ELISA tests and soluble antigen by surface plasmon resonance (SPR analysis. All tested VHHs were specific for tuberculosis-causing mycobacteria (M. tuberculosis, M. bovis and exclusively recognized an immunodominant 16 kDa heat shock protein (hsp. The highest affinity VHH had a dissociation constant (KD of 4 × 10(-10 M. CONCLUSIONS/SIGNIFICANCE: A broad set of different llama antibodies specific for 16 kDa heat shock protein of M. tuberculosis is available. This protein is highly stable and abundant in M. tuberculosis. The VHH that detect this protein are applied in a robust SPR sensor for identification of tuberculosis-causing mycobacteria.

  14. Phage Display for the Generation of Antibodies for Proteome Research, Diagnostics and Therapy

    Directory of Open Access Journals (Sweden)

    Michael Hust

    2011-01-01

    Full Text Available Twenty years after its development, antibody phage display using filamentous bacteriophage represents the most successful in vitro antibody selection technology. Initially, its development was encouraged by the unique possibility of directly generating recombinant human antibodies for therapy. Today, antibody phage display has been developed as a robust technology offering great potential for automation. Generation of monospecific binders provides a valuable tool for proteome research, leading to highly enhanced throughput and reduced costs. This review presents the phage display technology, application areas of antibodies in research, diagnostics and therapy and the use of antibody phage display for these applications.

  15. Phage display for the generation of antibodies for proteome research, diagnostics and therapy.

    Science.gov (United States)

    Schirrmann, Thomas; Meyer, Torsten; Schütte, Mark; Frenzel, André; Hust, Michael

    2011-01-10

    Twenty years after its development, antibody phage display using filamentous bacteriophage represents the most successful in vitro antibody selection technology. Initially, its development was encouraged by the unique possibility of directly generating recombinant human antibodies for therapy. Today, antibody phage display has been developed as a robust technology offering great potential for automation. Generation of monospecific binders provides a valuable tool for proteome research, leading to highly enhanced throughput and reduced costs. This review presents the phage display technology, application areas of antibodies in research, diagnostics and therapy and the use of antibody phage display for these applications.

  16. Tetanus Neurotoxin Neutralizing Antibodies Screened from a Human Immune scFv Antibody Phage Display Library.

    Science.gov (United States)

    Wang, Han; Yu, Rui; Fang, Ting; Yu, Ting; Chi, Xiangyang; Zhang, Xiaopeng; Liu, Shuling; Fu, Ling; Yu, Changming; Chen, Wei

    2016-09-11

    Tetanus neurotoxin (TeNT) produced by Clostridium tetani is one of the most poisonous protein substances. Neutralizing antibodies against TeNT can effectively prevent and cure toxicosis. Using purified Hc fragments of TeNT (TeNT-Hc) as an antigen, three specific neutralizing antibody clones recognizing different epitopes were selected from a human immune scFv antibody phage display library. The three antibodies (2-7G, 2-2D, and S-4-7H) can effectively inhibit the binding between TeNT-Hc and differentiated PC-12 cells in vitro. Moreover, 2-7G inhibited TeNT-Hc binding to the receptor via carbohydrate-binding sites of the W pocket while 2-2D and S-4-7H inhibited binding of the R pocket. Although no single mAb completely protected mice from the toxin, they could both prolong survival when challenged with 20 LD50s (50% of the lethal dose) of TeNT. When used together, the mAbs completely neutralized 1000 LD50s/mg Ab, indicating their high neutralizing potency in vivo. Antibodies recognizing different carbohydrate-binding pockets could have higher synergistic toxin neutralization activities than those that recognize the same pockets. These results could lead to further production of neutralizing antibody drugs against TeNT and indicate that using TeNT-Hc as an antigen for screening human antibodies for TeNT intoxication therapy from human immune antibody library was convenient and effective.

  17. Tetanus Neurotoxin Neutralizing Antibodies Screened from a Human Immune scFv Antibody Phage Display Library

    Science.gov (United States)

    Wang, Han; Yu, Rui; Fang, Ting; Yu, Ting; Chi, Xiangyang; Zhang, Xiaopeng; Liu, Shuling; Fu, Ling; Yu, Changming; Chen, Wei

    2016-01-01

    Tetanus neurotoxin (TeNT) produced by Clostridium tetani is one of the most poisonous protein substances. Neutralizing antibodies against TeNT can effectively prevent and cure toxicosis. Using purified Hc fragments of TeNT (TeNT-Hc) as an antigen, three specific neutralizing antibody clones recognizing different epitopes were selected from a human immune scFv antibody phage display library. The three antibodies (2-7G, 2-2D, and S-4-7H) can effectively inhibit the binding between TeNT-Hc and differentiated PC-12 cells in vitro. Moreover, 2-7G inhibited TeNT-Hc binding to the receptor via carbohydrate-binding sites of the W pocket while 2-2D and S-4-7H inhibited binding of the R pocket. Although no single mAb completely protected mice from the toxin, they could both prolong survival when challenged with 20 LD50s (50% of the lethal dose) of TeNT. When used together, the mAbs completely neutralized 1000 LD50s/mg Ab, indicating their high neutralizing potency in vivo. Antibodies recognizing different carbohydrate-binding pockets could have higher synergistic toxin neutralization activities than those that recognize the same pockets. These results could lead to further production of neutralizing antibody drugs against TeNT and indicate that using TeNT-Hc as an antigen for screening human antibodies for TeNT intoxication therapy from human immune antibody library was convenient and effective. PMID:27626445

  18. Tetanus Neurotoxin Neutralizing Antibodies Screened from a Human Immune scFv Antibody Phage Display Library

    Directory of Open Access Journals (Sweden)

    Han Wang

    2016-09-01

    Full Text Available Tetanus neurotoxin (TeNT produced by Clostridium tetani is one of the most poisonous protein substances. Neutralizing antibodies against TeNT can effectively prevent and cure toxicosis. Using purified Hc fragments of TeNT (TeNT-Hc as an antigen, three specific neutralizing antibody clones recognizing different epitopes were selected from a human immune scFv antibody phage display library. The three antibodies (2-7G, 2-2D, and S-4-7H can effectively inhibit the binding between TeNT-Hc and differentiated PC-12 cells in vitro. Moreover, 2-7G inhibited TeNT-Hc binding to the receptor via carbohydrate-binding sites of the W pocket while 2-2D and S-4-7H inhibited binding of the R pocket. Although no single mAb completely protected mice from the toxin, they could both prolong survival when challenged with 20 LD50s (50% of the lethal dose of TeNT. When used together, the mAbs completely neutralized 1000 LD50s/mg Ab, indicating their high neutralizing potency in vivo. Antibodies recognizing different carbohydrate-binding pockets could have higher synergistic toxin neutralization activities than those that recognize the same pockets. These results could lead to further production of neutralizing antibody drugs against TeNT and indicate that using TeNT-Hc as an antigen for screening human antibodies for TeNT intoxication therapy from human immune antibody library was convenient and effective.

  19. Signature biochemical properties of broadly cross-reactive HIV-1 neutralizing antibodies in human plasma.

    Science.gov (United States)

    Sajadi, Mohammad M; Lewis, George K; Seaman, Michael S; Guan, Yongjun; Redfield, Robert R; DeVico, Anthony L

    2012-05-01

    The common properties of broadly cross-reactive HIV-1 neutralization antibodies found in certain HIV-1-infected individuals holds significant value for understanding natural and vaccine-mediated anti-HIV immunity. Recent efforts have addressed this question by deriving neutralizing monoclonal anti-envelope antibodies from memory B cell pools of selected subjects. However, it has been more difficult to identify whether broadly neutralizing antibodies circulating in plasma possess shared characteristics among individuals. To address this question, we used affinity chromatography and isoelectric focusing to fractionate plasma immunoglobulin from 10 HIV-1-infected subjects (5 subjects with broad HIV-1 neutralizing activity and 5 controls). We find that plasma neutralizing activity typically partitions into at least two subsets of antibodies. Antibodies with restricted neutralization breadth have relatively neutral isoelectric points and preferentially bind to envelope monomers and trimers versus core antigens from which variable loops and other domains have been deleted. In comparison, broadly neutralizing antibodies account for a minor fraction of the total anti-envelope response. They are consistently distinguished by more basic isoelectric points and specificity for epitopes shared by monomeric gp120, gp120 core, or CD4-induced structures. Such biochemical properties might be exploited to reliably predict or produce broad anti-HIV immunity.

  20. Co-evolution of a broadly neutralizing HIV-1 antibody and founder virus.

    Science.gov (United States)

    Liao, Hua-Xin; Lynch, Rebecca; Zhou, Tongqing; Gao, Feng; Alam, S Munir; Boyd, Scott D; Fire, Andrew Z; Roskin, Krishna M; Schramm, Chaim A; Zhang, Zhenhai; Zhu, Jiang; Shapiro, Lawrence; Mullikin, James C; Gnanakaran, S; Hraber, Peter; Wiehe, Kevin; Kelsoe, Garnett; Yang, Guang; Xia, Shi-Mao; Montefiori, David C; Parks, Robert; Lloyd, Krissey E; Scearce, Richard M; Soderberg, Kelly A; Cohen, Myron; Kamanga, Gift; Louder, Mark K; Tran, Lillian M; Chen, Yue; Cai, Fangping; Chen, Sheri; Moquin, Stephanie; Du, Xiulian; Joyce, M Gordon; Srivatsan, Sanjay; Zhang, Baoshan; Zheng, Anqi; Shaw, George M; Hahn, Beatrice H; Kepler, Thomas B; Korber, Bette T M; Kwong, Peter D; Mascola, John R; Haynes, Barton F

    2013-04-25

    Current human immunodeficiency virus-1 (HIV-1) vaccines elicit strain-specific neutralizing antibodies. However, cross-reactive neutralizing antibodies arise in approximately 20% of HIV-1-infected individuals, and details of their generation could provide a blueprint for effective vaccination. Here we report the isolation, evolution and structure of a broadly neutralizing antibody from an African donor followed from the time of infection. The mature antibody, CH103, neutralized approximately 55% of HIV-1 isolates, and its co-crystal structure with the HIV-1 envelope protein gp120 revealed a new loop-based mechanism of CD4-binding-site recognition. Virus and antibody gene sequencing revealed concomitant virus evolution and antibody maturation. Notably, the unmutated common ancestor of the CH103 lineage avidly bound the transmitted/founder HIV-1 envelope glycoprotein, and evolution of antibody neutralization breadth was preceded by extensive viral diversification in and near the CH103 epitope. These data determine the viral and antibody evolution leading to induction of a lineage of HIV-1 broadly neutralizing antibodies, and provide insights into strategies to elicit similar antibodies by vaccination.

  1. Two Escape Mechanisms of Influenza A Virus to a Broadly Neutralizing Stalk-Binding Antibody.

    Science.gov (United States)

    Chai, Ning; Swem, Lee R; Reichelt, Mike; Chen-Harris, Haiyin; Luis, Elizabeth; Park, Summer; Fouts, Ashley; Lupardus, Patrick; Wu, Thomas D; Li, Olga; McBride, Jacqueline; Lawrence, Michael; Xu, Min; Tan, Man-Wah

    2016-06-01

    Broadly neutralizing antibodies targeting the stalk region of influenza A virus (IAV) hemagglutinin (HA) are effective in blocking virus infection both in vitro and in vivo. The highly conserved epitopes recognized by these antibodies are critical for the membrane fusion function of HA and therefore less likely to be permissive for virus mutational escape. Here we report three resistant viruses of the A/Perth/16/2009 strain that were selected in the presence of a broadly neutralizing stalk-binding antibody. The three resistant viruses harbor three different mutations in the HA stalk: (1) Gln387Lys; (2) Asp391Tyr; (3) Asp391Gly. The Gln387Lys mutation completely abolishes binding of the antibody to the HA stalk epitope. The other two mutations, Asp391Tyr and Asp391Gly, do not affect antibody binding at neutral pH and only slightly reduce binding at low pH. Interestingly, they enhance the fusion ability of the HA, representing a novel mechanism that allows productive membrane fusion even in the presence of antibody and hence virus escape from antibody neutralization. Therefore, these mutations illustrate two different resistance mechanisms used by IAV to escape broadly neutralizing stalk-binding antibodies. Compared to the wild type virus, the resistant viruses release fewer progeny viral particles during replication and are more sensitive to Tamiflu, suggesting reduced viral fitness.

  2. Enhanced clearance of HIV-1-infected cells by broadly neutralizing antibodies against HIV-1 in vivo.

    Science.gov (United States)

    Lu, Ching-Lan; Murakowski, Dariusz K; Bournazos, Stylianos; Schoofs, Till; Sarkar, Debolina; Halper-Stromberg, Ariel; Horwitz, Joshua A; Nogueira, Lilian; Golijanin, Jovana; Gazumyan, Anna; Ravetch, Jeffrey V; Caskey, Marina; Chakraborty, Arup K; Nussenzweig, Michel C

    2016-05-20

    Antiretroviral drugs and antibodies limit HIV-1 infection by interfering with the viral life cycle. In addition, antibodies also have the potential to guide host immune effector cells to kill HIV-1-infected cells. Examination of the kinetics of HIV-1 suppression in infected individuals by passively administered 3BNC117, a broadly neutralizing antibody, suggested that the effects of the antibody are not limited to free viral clearance and blocking new infection but also include acceleration of infected cell clearance. Consistent with these observations, we find that broadly neutralizing antibodies can target CD4(+) T cells infected with patient viruses and can decrease their in vivo half-lives by a mechanism that requires Fcγ receptor engagement in a humanized mouse model. The results indicate that passive immunotherapy can accelerate elimination of HIV-1-infected cells.

  3. Yeast display of antibody fragments: a discovery and characterization platform

    Energy Technology Data Exchange (ETDEWEB)

    Feldhaus, Michael; Siegel, Robert W.

    2004-07-01

    This review will focus on some of the novel attributes of the yeast surface display platform for the discovery and characterization of novel affinity reagents, optimization of those reagents, and novel uses of the platform. This is not intended to serve as an exhaustive review on the broader topic of general scFv technologies (see Winter et al., 1994; Smith and Petrenko, 1997; Bradbury et al., 2003) Furthermore, the scFv format of antibodies are easily manipulated through molecular cloning into a number of other formats such IgG, Fab, diabodies and such, for use in down steam applications and the reader is encouraged to read ?IgG?, ?Fab?, or your favorite format whenever scFv is seen in this review. This review is presented in 5 parts; (1) description of yeast display and its components, (2) library types and construction methods, (3) screening approaches for non-immune libraries and benefits, (4) screening approaches for directed evolution, kinetic on and off rates and (5) epitope complementation binning of clones.

  4. Specifically modified Env immunogens activate B-cell precursors of broadly neutralizing HIV-1 antibodies in transgenic mice.

    Science.gov (United States)

    McGuire, Andrew T; Gray, Matthew D; Dosenovic, Pia; Gitlin, Alexander D; Freund, Natalia T; Petersen, John; Correnti, Colin; Johnsen, William; Kegel, Robert; Stuart, Andrew B; Glenn, Jolene; Seaman, Michael S; Schief, William R; Strong, Roland K; Nussenzweig, Michel C; Stamatatos, Leonidas

    2016-02-24

    VRC01-class broadly neutralizing HIV-1 antibodies protect animals from experimental infection and could contribute to an effective vaccine response. Their predicted germline forms (gl) bind Env inefficiently, which may explain why they are not elicited by HIV-1 Env-immunization. Here we show that an optimized Env immunogen can engage multiple glVRC01-class antibodies. Furthermore, this immunogen activates naive B cells expressing the human germline heavy chain of 3BNC60, paired with endogenous mouse light chains in vivo. To address whether it activates B cells expressing the fully humanized gl3BNC60 B-cell receptor (BCR), we immunized mice carrying both the heavy and light chains of gl3BNC60. B cells expressing this BCR display an autoreactive phenotype and fail to respond efficiently to soluble forms of the optimized immunogen, unless it is highly multimerized. Thus, specifically designed Env immunogens can activate naive B cells expressing human BCRs corresponding to precursors of broadly neutralizing HIV-1 antibodies even when the B cells display an autoreactive phenotype.

  5. Trimerization of the HIV Transmembrane Domain in Lipid Bilayers Modulates Broadly Neutralizing Antibody Binding.

    Science.gov (United States)

    Reichart, Timothy M; Baksh, Michael M; Rhee, Jin-Kyu; Fiedler, Jason D; Sligar, Stephen G; Finn, M G; Zwick, Michael B; Dawson, Philip E

    2016-02-18

    The membrane-proximal external region (MPER) of HIV gp41 is an established target of antibodies that neutralize a broad range of HIV isolates. To evaluate the role of the transmembrane (TM) domain, synthetic MPER-derived peptides were incorporated into lipid nanoparticles using natural and designed TM domains, and antibody affinity was measured using immobilized and solution-based techniques. Peptides incorporating the native HIV TM domain exhibit significantly stronger interactions with neutralizing antibodies than peptides with a monomeric TM domain. Furthermore, a peptide with a trimeric, three-helix bundle TM domain recapitulates the binding profile of the native sequence. These studies suggest that neutralizing antibodies can bind the MPER when the TM domain is a three-helix bundle and this presentation could influence the binding of neutralizing antibodies to the virus. Lipid-bilayer presentation of viral antigens in Nanodiscs is a new platform for evaluating neutralizing antibodies.

  6. Yeast surface display for antibody isolation: library construction, library screening, and affinity maturation.

    Science.gov (United States)

    Van Deventer, James A; Wittrup, Karl Dane

    2014-01-01

    Antibodies play key roles as reagents, diagnostics, and therapeutics in numerous biological and biomedical research settings. Although many antibodies are commercially available, oftentimes, specific applications require the development of antibodies with customized properties. Yeast surface display is a robust, versatile, and quantitative method for generating these antibodies and is accessible to single-investigator laboratories. This protocol details the key aspects of yeast surface display library construction and screening.

  7. HIV therapy by a combination of broadly neutralizing antibodies in humanized mice

    Science.gov (United States)

    Klein, Florian; Gruell, Henning; Scheid, Johannes F.; Bournazos, Stylianos; Mouquet, Hugo; Spatz, Linda A.; Diskin, Ron; Abadir, Alexander; Zang, Trinity; Dorner, Marcus; Billerbeck, Eva; Labitt, Rachael N.; Gaebler, Christian; Marcovecchio, Paola; Incesu, Reha-Baris; Eisenreich, Thomas R.; Bieniasz, Paul D.; Seaman, Michael S.; Bjorkman, Pamela J.; Ravetch, Jeffrey V.; Ploss, Alexander; Nussenzweig, Michel C.

    2013-01-01

    Summary Human antibodies to HIV-1 can neutralize a broad range of viral isolates in vitro and protect non-human primates against infection1,2. Previous work showed that antibodies exert selective pressure on the virus but escape variants emerge within a short period of time3,4. However, these experiments were performed before the recent discovery of more potent anti-HIV-1 antibodies and their improvement by structure-based design5-9. Here we re-examine passive antibody transfer as a therapeutic modality in HIV-1-infected humanized mice (hu-mice). Although HIV-1 can escape from antibody monotherapy, combinations of broadly neutralizing antibodies (bNAbs) can effectively control HIV-1 infection and suppress viral load to levels below detection. Moreover, in contrast to antiretroviral therapy (ART)10-12, the longer half-life of antibodies led to viremic control for an average of 60 days after cessation of therapy. Thus, combinations of potent monoclonal antibodies can effectively control HIV-1 replication in hu-mice, and should be re-examined as a therapeutic modality in HIV-1-infected individuals. PMID:23103874

  8. Roles of glycans in interactions between gp120 and HIV broadly neutralizing antibodies.

    Science.gov (United States)

    Qi, Yifei; Jo, Sunhwan; Im, Wonpil

    2016-03-01

    Many novel broadly neutralizing antibodies against human immunodeficiency virus (HIV) have been identified during the past decade, providing promising templates for the development of an effective HIV-1 vaccine. Structural studies reveal that the epitopes of some of these antibodies involve one or more crucial glycans, without which the binding is completely abolished. In this study, we have investigated the critical roles of glycans in interactions between HIV-1 gp120 and two broadly neutralizing antibodies PG9 (targeting V1/V2) and PGT128 (targeting V3) that are able to neutralize more than 70% of HIV-1 isolates. We have performed molecular dynamics simulations of a number of systems including antibody-gp120 complex with and without glycans, antibody, gp120 with and without glycans, and glycan-only systems. The simulation results show that the complex structures are stabilized by the glycans, and the multivalent interactions between the antibody and gp120 promote cooperativities to further enhance the binding. In the free gp120, the glycans increase the flexibility of the V1/V2 and V3 loops, which likely increases the entropy cost of the antibody recognition. However, the antibodies are able to bind the flexible interface by recognizing the preexisting glycan conformation, and penetrating the glycan shield with flexible complementarity determining region loops that sample the bound conformations occasionally.

  9. Efficient method to optimize antibodies using avian leukosis virus display and eukaryotic cells.

    Science.gov (United States)

    Yu, Changming; Pike, Gennett M; Rinkoski, Tommy A; Correia, Cristina; Kaufmann, Scott H; Federspiel, Mark J

    2015-08-11

    Antibody-based therapeutics have now had success in the clinic. The affinity and specificity of the antibody for the target ligand determines the specificity of therapeutic delivery and off-target side effects. The discovery and optimization of high-affinity antibodies to important therapeutic targets could be significantly improved by the availability of a robust, eukaryotic display technology comparable to phage display that would overcome the protein translation limitations of microorganisms. The use of eukaryotic cells would improve the diversity of the displayed antibodies that can be screened and optimized as well as more seamlessly transition into a large-scale mammalian expression system for clinical production. In this study, we demonstrate that the replication and polypeptide display characteristics of a eukaryotic retrovirus, avian leukosis virus (ALV), offers a robust, eukaryotic version of bacteriophage display. The binding affinity of a model single-chain Fv antibody was optimized by using ALV display, improving affinity >2,000-fold, from micromolar to picomolar levels. We believe ALV display provides an extension to antibody display on microorganisms and offers virus and cell display platforms in a eukaryotic expression system. ALV display should enable an improvement in the diversity of properly processed and functional antibody variants that can be screened and affinity-optimized to improve promising antibody candidates.

  10. Molecular evolution of broadly neutralizing Llama antibodies to the CD4-binding site of HIV-1.

    Directory of Open Access Journals (Sweden)

    Laura E McCoy

    2014-12-01

    Full Text Available To date, no immunization of humans or animals has elicited broadly neutralizing sera able to prevent HIV-1 transmission; however, elicitation of broad and potent heavy chain only antibodies (HCAb has previously been reported in llamas. In this study, the anti-HIV immune responses in immunized llamas were studied via deep sequencing analysis using broadly neutralizing monoclonal HCAbs as a guides. Distinct neutralizing antibody lineages were identified in each animal, including two defined by novel antibodies (as variable regions called VHH identified by robotic screening of over 6000 clones. The combined application of five VHH against viruses from clades A, B, C and CRF_AG resulted in neutralization as potent as any of the VHH individually and a predicted 100% coverage with a median IC50 of 0.17 µg/ml for the panel of 60 viruses tested. Molecular analysis of the VHH repertoires of two sets of immunized animals showed that each neutralizing lineage was only observed following immunization, demonstrating that they were elicited de novo. Our results show that immunization can induce potent and broadly neutralizing antibodies in llamas with features similar to human antibodies and provide a framework to analyze the effectiveness of immunization protocols.

  11. Molecular Evolution of Broadly Neutralizing Llama Antibodies to the CD4-Binding Site of HIV-1

    Science.gov (United States)

    McCoy, Laura E.; Rutten, Lucy; Frampton, Dan; Anderson, Ian; Granger, Luke; Bashford-Rogers, Rachael; Dekkers, Gillian; Strokappe, Nika M.; Seaman, Michael S.; Koh, Willie; Grippo, Vanina; Kliche, Alexander; Verrips, Theo; Kellam, Paul; Fassati, Ariberto; Weiss, Robin A.

    2014-01-01

    To date, no immunization of humans or animals has elicited broadly neutralizing sera able to prevent HIV-1 transmission; however, elicitation of broad and potent heavy chain only antibodies (HCAb) has previously been reported in llamas. In this study, the anti-HIV immune responses in immunized llamas were studied via deep sequencing analysis using broadly neutralizing monoclonal HCAbs as a guides. Distinct neutralizing antibody lineages were identified in each animal, including two defined by novel antibodies (as variable regions called VHH) identified by robotic screening of over 6000 clones. The combined application of five VHH against viruses from clades A, B, C and CRF_AG resulted in neutralization as potent as any of the VHH individually and a predicted 100% coverage with a median IC50 of 0.17 µg/ml for the panel of 60 viruses tested. Molecular analysis of the VHH repertoires of two sets of immunized animals showed that each neutralizing lineage was only observed following immunization, demonstrating that they were elicited de novo. Our results show that immunization can induce potent and broadly neutralizing antibodies in llamas with features similar to human antibodies and provide a framework to analyze the effectiveness of immunization protocols. PMID:25522326

  12. Molecular evolution of broadly neutralizing Llama antibodies to the CD4-binding site of HIV-1.

    Science.gov (United States)

    McCoy, Laura E; Rutten, Lucy; Frampton, Dan; Anderson, Ian; Granger, Luke; Bashford-Rogers, Rachael; Dekkers, Gillian; Strokappe, Nika M; Seaman, Michael S; Koh, Willie; Grippo, Vanina; Kliche, Alexander; Verrips, Theo; Kellam, Paul; Fassati, Ariberto; Weiss, Robin A

    2014-12-01

    To date, no immunization of humans or animals has elicited broadly neutralizing sera able to prevent HIV-1 transmission; however, elicitation of broad and potent heavy chain only antibodies (HCAb) has previously been reported in llamas. In this study, the anti-HIV immune responses in immunized llamas were studied via deep sequencing analysis using broadly neutralizing monoclonal HCAbs as a guides. Distinct neutralizing antibody lineages were identified in each animal, including two defined by novel antibodies (as variable regions called VHH) identified by robotic screening of over 6000 clones. The combined application of five VHH against viruses from clades A, B, C and CRF_AG resulted in neutralization as potent as any of the VHH individually and a predicted 100% coverage with a median IC50 of 0.17 µg/ml for the panel of 60 viruses tested. Molecular analysis of the VHH repertoires of two sets of immunized animals showed that each neutralizing lineage was only observed following immunization, demonstrating that they were elicited de novo. Our results show that immunization can induce potent and broadly neutralizing antibodies in llamas with features similar to human antibodies and provide a framework to analyze the effectiveness of immunization protocols.

  13. Elicitation of broadly neutralizing influenza antibodies in animals with previous influenza exposure.

    Science.gov (United States)

    Wei, Chih-Jen; Yassine, Hadi M; McTamney, Patrick M; Gall, Jason G D; Whittle, James R R; Boyington, Jeffrey C; Nabel, Gary J

    2012-08-15

    The immune system responds to influenza infection by producing neutralizing antibodies to the viral surface protein, hemagglutinin (HA), which regularly changes its antigenic structure. Antibodies that target the highly conserved stem region of HA neutralize diverse influenza viruses and can be elicited through vaccination in animals and humans. Efforts to develop universal influenza vaccines have focused on strategies to elicit such antibodies; however, the concern has been raised that previous influenza immunity may abrogate the induction of such broadly protective antibodies. We show here that prime-boost immunization can induce broadly neutralizing antibody responses in influenza-immune mice and ferrets that were previously infected or vaccinated. HA stem-directed antibodies were elicited in mice primed with a DNA vaccine and boosted with inactivated vaccine from H1N1 A/New Caledonia/20/1999 (1999 NC) HA regardless of preexposure. Similarly, gene-based vaccination with replication-defective adenovirus 28 (rAd28) and 5 (rAd5) vectors encoding 1999 NC HA elicited stem-directed neutralizing antibodies and conferred protection against unmatched 1934 and 2007 H1N1 virus challenge in influenza-immune ferrets. Indeed, previous exposure to certain strains could enhance immunogenicity: The strongest HA stem-directed immune response was observed in ferrets previously infected with a divergent 1934 H1N1 virus. These findings suggest that broadly neutralizing antibodies against the conserved stem region of HA can be elicited through vaccination despite previous influenza exposure, which supports the feasibility of developing stem-directed universal influenza vaccines for humans.

  14. Envelope deglycosylation enhances antigenicity of HIV-1 gp41 epitopes for both broad neutralizing antibodies and their unmutated ancestor antibodies.

    Directory of Open Access Journals (Sweden)

    Ben-Jiang Ma

    2011-09-01

    Full Text Available The HIV-1 gp41 envelope (Env membrane proximal external region (MPER is an important vaccine target that in rare subjects can elicit neutralizing antibodies. One mechanism proposed for rarity of MPER neutralizing antibody generation is lack of reverted unmutated ancestor (putative naive B cell receptor antibody reactivity with HIV-1 envelope. We have studied the effect of partial deglycosylation under non-denaturing (native conditions on gp140 Env antigenicity for MPER neutralizing antibodies and their reverted unmutated ancestor antibodies. We found that native deglycosylation of clade B JRFL gp140 as well as group M consensus gp140 Env CON-S selectively increased the reactivity of Env with the broad neutralizing human mAbs, 2F5 and 4E10. Whereas fully glycosylated gp140 Env either did not bind (JRFL, or weakly bound (CON-S, 2F5 and 4E10 reverted unmutated ancestors, natively deglycosylated JRFL and CON-S gp140 Envs did bind well to these putative mimics of naive B cell receptors. These data predict that partially deglycoslated Env would bind better than fully glycosylated Env to gp41-specific naïve B cells with improved immunogenicity. In this regard, immunization of rhesus macaques demonstrated enhanced immunogenicity of the 2F5 MPER epitope on deglyosylated JRFL gp140 compared to glycosylated JRFL gp140. Thus, the lack of 2F5 and 4E10 reverted unmutated ancestor binding to gp140 Env may not always be due to lack of unmutated ancestor antibody reactivity with gp41 peptide epitopes, but rather, may be due to glycan interference of binding of unmutated ancestor antibodies of broad neutralizing mAb to Env gp41.

  15. Broadly Neutralizing Alphavirus Antibodies Bind an Epitope on E2 and Inhibit Entry and Egress

    NARCIS (Netherlands)

    Fox, Julie M.; Long, Feng; Edeling, Melissa A.; Lin, Hueylie; van Duijl-Richter, Mareike K. S.; Fong, Rachel H.; Kahle, Kristen M.; Smit, Jolanda M.; Jin, Jing; Simmons, Graham; Doranz, Benjamin J.; Crowe, James E.; Fremont, Daved H.; Rossmann, Michael G.; Diamond, Michael S.

    2015-01-01

    We screened a panel of mouse and human monoclonal antibodies (MAbs) against chikungunya virus and identified several with inhibitory activity against multiple alphaviruses. Passive transfer of broadly neutralizing MAbs protected mice against infection by chikungunya, Mayaro, and O'nyong'nyong alphav

  16. Amended Final Report - Antibodies to Radionuclides. Engineering by Surface Display for Immunosensors

    Energy Technology Data Exchange (ETDEWEB)

    Blake, Diane A. [Tulane Univ., New Orleans, LA (United States)

    2013-06-14

    The relatively new techniques of antibody display, which permit molecular engineering of antibody structure and function, have the potential to revolutionize the way scientists generate binding proteins for specific applications. However, the skills required to efficiently use antibody display techniques have proven difficult for other laboratories to acquire without hands-on training and exchange of laboratory personnel. This research project is designed bring important expertise in antibody display to the State of Louisiana while pursuing a project with direct relevance to the DOE’s EM program.

  17. A Potent and Broad Neutralizing Antibody Recognizes and Penetrates the HIV Glycan Shield

    Energy Technology Data Exchange (ETDEWEB)

    Pejchal, Robert; Doores, Katie J.; Walker, Laura M.; Khayat, Reza; Huang, Po-Ssu; Wang, Sheng-Kai; Stanfield, Robyn L.; Julien, Jean-Philippe; Ramos, Alejandra; Crispin, Max; Depetris, Rafael; Katpally, Umesh; Marozsan, Andre; Cupo, Albert; Maloveste, Sebastien; Liu, Yan; McBride, Ryan; Ito, Yukishige; Sanders, Rogier W.; Ogohara, Cassandra; Paulson, James C.; Feizi, Ten; Scanlan, Christopher N.; Wong, Chi-Huey; Moore, John P.; Olson, William C.; Ward, Andrew B.; Poignard, Pascal; Schief, William R.; Burton, Dennis R.; Wilson, Ian A. (UWASH); (Progenics); (ICL); (Weill-Med); (NIH); (JSTA); (Scripps); (Oxford)

    2015-10-15

    The HIV envelope (Env) protein gp120 is protected from antibody recognition by a dense glycan shield. However, several of the recently identified PGT broadly neutralizing antibodies appear to interact directly with the HIV glycan coat. Crystal structures of antigen-binding fragments (Fabs) PGT 127 and 128 with Man{sub 9} at 1.65 and 1.29 angstrom resolution, respectively, and glycan binding data delineate a specific high mannose-binding site. Fab PGT 128 complexed with a fully glycosylated gp120 outer domain at 3.25 angstroms reveals that the antibody penetrates the glycan shield and recognizes two conserved glycans as well as a short {beta}-strand segment of the gp120 V3 loop, accounting for its high binding affinity and broad specificify. Furthermore, our data suggest that the high neutralization potency of PGT 127 and 128 immunoglobulin Gs may be mediated by cross-linking Env trimers on the viral surface.

  18. Broadly Neutralizing Antibodies Against HIV: New Insights to Inform Vaccine Design.

    Science.gov (United States)

    Sadanand, Saheli; Suscovich, Todd J; Alter, Galit

    2016-01-01

    HIV-1 poses immense immunological challenges to the humoral immune response because of its ability to shield itself and replicate and evolve rapidly. Although most currently licensed vaccines provide protection via the induction of antibodies (Abs) that can directly block infection ( 1 ), 30 years of HIV-1 vaccine research has failed to successfully elicit such Abs against globally relevant HIV strains. However, mounting evidence suggests that these broadly neutralizing antibodies (bNAbs) do emerge naturally in a significant fraction of infected subjects, albeit after years of infection, indicating that these responses can be selected naturally by the immune response but take long periods of time to evolve. We review the basic structural characteristics of broadly neutralizing antibodies and how they recognize the virus, and we discuss new vaccination strategies that aim to mimic natural evolution to guide B cells to produce protective Abs against HIV-1.

  19. Structural Basis for Broad and Potent Neutralization of HIV-1 by Antibody VRC01

    Energy Technology Data Exchange (ETDEWEB)

    Zhou, Tongqing; Georgiev, Ivelin; Wu, Xueling; Yang, Zhi-Yong; Dai, Kaifan; Finzi, Andrés; Kwon, Young Do; Scheid, Johannes F.; Shi, Wei; Xu, Ling; Yang, Yongping; Zhu, Jiang; Nussenzweig, Michel C.; Sodroski, Joseph; Shapiro, Lawrence; Nabel, Gary J.; Mascola, John R.; Kwong, Peter D. (NIH); (Rockefeller); (DFCI)

    2010-08-26

    During HIV-1 infection, antibodies are generated against the region of the viral gp120 envelope glycoprotein that binds CD4, the primary receptor for HIV-1. Among these antibodies, VRC01 achieves broad neutralization of diverse viral strains. We determined the crystal structure of VRC01 in complex with a human immunodeficiency virus HIV-1 gp120 core. VRC01 partially mimics CD4 interaction with gp120. A shift from the CD4-defined orientation, however, focuses VRC01 onto the vulnerable site of initial CD4 attachment, allowing it to overcome the glycan and conformational masking that diminishes the neutralization potency of most CD4-binding-site antibodies. To achieve this recognition, VRC01 contacts gp120 mainly through immunoglobulin V-gene regions substantially altered from their genomic precursors. Partial receptor mimicry and extensive affinity maturation thus facilitate neutralization of HIV-1 by natural human antibodies.

  20. Host-Pathogen Coevolution and the Emergence of Broadly Neutralizing Antibodies in Chronic Infections.

    Science.gov (United States)

    Nourmohammad, Armita; Otwinowski, Jakub; Plotkin, Joshua B

    2016-07-01

    The vertebrate adaptive immune system provides a flexible and diverse set of molecules to neutralize pathogens. Yet, viruses such as HIV can cause chronic infections by evolving as quickly as the adaptive immune system, forming an evolutionary arms race. Here we introduce a mathematical framework to study the coevolutionary dynamics between antibodies and antigens within a host. We focus on changes in the binding interactions between the antibody and antigen populations, which result from the underlying stochastic evolution of genotype frequencies driven by mutation, selection, and drift. We identify the critical viral and immune parameters that determine the distribution of antibody-antigen binding affinities. We also identify definitive signatures of coevolution that measure the reciprocal response between antibodies and viruses, and we introduce experimentally measurable quantities that quantify the extent of adaptation during continual coevolution of the two opposing populations. Using this analytical framework, we infer rates of viral and immune adaptation based on time-shifted neutralization assays in two HIV-infected patients. Finally, we analyze competition between clonal lineages of antibodies and characterize the fate of a given lineage in terms of the state of the antibody and viral populations. In particular, we derive the conditions that favor the emergence of broadly neutralizing antibodies, which may have relevance to vaccine design against HIV.

  1. Host-Pathogen Coevolution and the Emergence of Broadly Neutralizing Antibodies in Chronic Infections.

    Directory of Open Access Journals (Sweden)

    Armita Nourmohammad

    2016-07-01

    Full Text Available The vertebrate adaptive immune system provides a flexible and diverse set of molecules to neutralize pathogens. Yet, viruses such as HIV can cause chronic infections by evolving as quickly as the adaptive immune system, forming an evolutionary arms race. Here we introduce a mathematical framework to study the coevolutionary dynamics between antibodies and antigens within a host. We focus on changes in the binding interactions between the antibody and antigen populations, which result from the underlying stochastic evolution of genotype frequencies driven by mutation, selection, and drift. We identify the critical viral and immune parameters that determine the distribution of antibody-antigen binding affinities. We also identify definitive signatures of coevolution that measure the reciprocal response between antibodies and viruses, and we introduce experimentally measurable quantities that quantify the extent of adaptation during continual coevolution of the two opposing populations. Using this analytical framework, we infer rates of viral and immune adaptation based on time-shifted neutralization assays in two HIV-infected patients. Finally, we analyze competition between clonal lineages of antibodies and characterize the fate of a given lineage in terms of the state of the antibody and viral populations. In particular, we derive the conditions that favor the emergence of broadly neutralizing antibodies, which may have relevance to vaccine design against HIV.

  2. Influenza A HA's conserved epitopes and broadly neutralizing antibodies: a prediction method.

    Science.gov (United States)

    Ren, Jing; Ellis, John; Li, Jinyan

    2014-10-01

    A conserved epitope is an epitope retained by multiple strains of influenza as the key target of a broadly neutralizing antibody. Identification of conserved epitopes is of strong interest to help design broad-spectrum vaccines against influenza. Conservation score measures the evolutionary conservation of an amino acid position in a protein based on the phylogenetic relationships observed amongst homologous sequences. Here, Average Amino Acid Conservation Score (AAACS) is proposed as a method to identify HA's conserved epitopes. Our analysis shows that there is a clear distinction between conserved epitopes and nonconserved epitopes in terms of AAACS. This method also provides an excellent classification performance on an independent dataset. In contrast, alignment-based comparison methods do not work well for this problem, because conserved epitopes to the same broadly neutralizing antibody are usually not identical or similar. Location-based methods are not successful either, because conserved epitopes are located at both the less-conserved globular head (HA1) and the more-conserved stem (HA2). As a case study, two conserved epitopes on HA are predicted for the influenza A virus H7N9: One should match the broadly neutralizing antibodies CR9114 or FI6v3, while the other is new and requires validation by wet-lab experiments.

  3. A pilot study on an attenuated Chinese EIAV vaccine inducing broadly neutralizing antibodies.

    Science.gov (United States)

    Meng, Qinglai; Lin, Yuezhi; Ma, Jian; Ma, Yan; Zhao, Liping; Li, Shenwei; Liang, Hua; Zhou, Jianhua; Shen, Rongxian; Zhang, Xiaoyan; Shao, Yiming

    2011-08-01

    The attenuated Chinese equine infectious anemia virus (EIAV) vaccine has successfully protected millions of equine animals from EIA disease in China. In this pilot study, to determine whether this attenuated vaccine can induce broadly neutralizing antibodies, we immunized four horses with the attenuated Chinese vaccine strain EIAVFDDV and then observed the evolution of neutralizing antibodies against different EIAV strains. During the vaccination phase, all vaccinees rapidly developed high levels of neutralizing antibodies against the homologous vaccine strain (pLGFD3V), and 3 out of 4 horses showed a gradual increase in serum neutralizing activity against two relatively heterologous virulent variants of the challenge strain (pLGFD3Mu12V and DLV34). After challenge, the three horses that had developed high levels of neutralizing antibodies against pLGFD3Mu12V and DLV34 did not show signs of infection, which was demonstrated by immune suppression, while the one horse producing serum that could only neutralize pLGFD3V developed a febrile episode during the 8-month observation period. To assess whether the broadly neutralizing activity is associated with immune protection, sera drawn on the day of challenge from these four vaccinees and an additional four EIAVFDDV-vaccinated horses were analyzed for neutralizing antibodies against pLGFD3V, pLGFD3Mu12V and DLV34. Although there was no significant correlation between protection from infection and serum neutralizing activity against any of these three viral strains, protection from infection was observed to correlate better with serum neutralizing activity against the two heterologous virulent strains than against the homologous vaccine strain. These data indicate that EIAVFDDV induced broadly neutralizing antibodies, which might confer enhanced protection of vaccinees from infection by the challenge virus.

  4. N-terminal residues of an HIV-1 gp41 membrane-proximal external region antigen influence broadly neutralizing 2F5-like antibodies

    Institute of Scientific and Technical Information of China (English)

    Dezhi Li; Jie Liu; Li Zhang; Tianshu Xu; Junheng Chen; Liping Wang; Qi Zhao

    2015-01-01

    The Human immunodeficiency virus type 1(HIV-1) gp41 membrane proximal external region(MPER) is targeted by broadly neutralizing antibodies(e.g. 2F5, 4E10, Z13 e and m66.6), which makes this region a promising target for vaccine design. One strategy to elicit neutralizing antibodies against the MPER epitope is to design peptide immunogens mimicking neutralization structures. To probe 2F5-like neutralizing antibodies, two yeast-displayed antibody libraries from peripheral blood mononuclear cells from a HIV-1 patient were screened against the 2F5 epitope peptide SP62. Two 2F5-like antibodies were identified that specifically recognized SP62. However,these antibodies only weakly neutralized HIV-1 primary isolates. The epitopes recognized by these two 2F5-like antibodies include not only the 2F5 epitope(amino acids(aa) 662–667 in the MPER)but also several other residues(aa 652–655) locating at the N-terminus in SP62. Experimental results suggest that residues of SP62 adjacent to the 2F5 epitope influence the response of broadly neutralizing 2F5-like antibodies in vaccination. Our findings may aid the design of vaccine immunogens and development of therapeutics against HIV-1 infection.

  5. N-terminal residues of an HIV-1 gp41 membrane-proximal external region antigen influence broadly neutralizing 2F5-like antibodies.

    Science.gov (United States)

    Li, Dezhi; Liu, Jie; Zhang, Li; Xu, Tianshu; Chen, Junheng; Wang, Liping; Zhao, Qi

    2015-12-01

    The Human immunodeficiency virus type 1 (HIV-1) gp41 membrane proximal external region (MPER) is targeted by broadly neutralizing antibodies (e.g. 2F5, 4E10, Z13e and m66.6), which makes this region a promising target for vaccine design. One strategy to elicit neutralizing antibodies against the MPER epitope is to design peptide immunogens mimicking neutralization structures. To probe 2F5-like neutralizing antibodies, two yeast-displayed antibody libraries from peripheral blood mononuclear cells from a HIV-1 patient were screened against the 2F5 epitope peptide SP62. Two 2F5-like antibodies were identified that specifically recognized SP62. However, these antibodies only weakly neutralized HIV-1 primary isolates. The epitopes recognized by these two 2F5-like antibodies include not only the 2F5 epitope (amino acids (aa) 662-667 in the MPER) but also several other residues (aa 652-655) locating at the N-terminus in SP62. Experimental results suggest that residues of SP62 adjacent to the 2F5 epitope influence the response of broadly neutralizing 2F5-like antibodies in vaccination. Our findings may aid the design of vaccine immunogens and development of therapeutics against HIV-1 infection.

  6. Selection of Ceratitis capitata (Diptera: Tephritidae) specific recombinant monoclonal phage display antibodies for prey detection analysis.

    Science.gov (United States)

    Monzó, César; Urbaneja, Alberto; Ximénez-Embún, Miguel; García-Fernández, Julia; García, José Luis; Castañera, Pedro

    2012-01-01

    Several recombinant antibodies against the Mediterranean fruit fly, Ceratitis capitata (Wiedemann) (Diptera: Tephritidae), one of the most important pests in agriculture worldwide, were selected for the first time from a commercial phage display library of human scFv antibodies. The specificity and sensitivity of the selected recombinant antibodies were compared with that of a rabbit polyclonal serum raised in parallel using a wide range of arthropod species as controls. The selected recombinant monoclonal antibodies had a similar or greater specificity when compared with classical monoclonal antibodies. The selected recombinant antibodies were successfully used to detect the target antigen in the gut of predators and the scFv antibodies were sequenced and compared. These results demonstrate the potential for recombinant scFv antibodies to be used as an alternative to the classical monoclonal antibodies or even molecular probes in the post-mortem analysis studies of generalist predators.

  7. Stapled HIV-1 peptides recapitulate antigenic structures and engage broadly neutralizing antibodies.

    Science.gov (United States)

    Bird, Gregory H; Irimia, Adriana; Ofek, Gilad; Kwong, Peter D; Wilson, Ian A; Walensky, Loren D

    2014-12-01

    Hydrocarbon stapling can restore bioactive α-helical structure to natural peptides, yielding research tools and prototype therapeutics to dissect and target protein interactions. Here we explore the capacity of peptide stapling to generate high-fidelity, protease-resistant mimics of antigenic structures for vaccine development. HIV-1 has been refractory to vaccine technologies thus far, although select human antibodies can broadly neutralize HIV-1 by targeting sequences of the gp41 juxtamembrane fusion apparatus. To develop candidate HIV-1 immunogens, we generated and characterized stabilized α-helices of the membrane-proximal external region (SAH-MPER) of gp41. SAH-MPER peptides were remarkably protease resistant and bound to the broadly neutralizing 4E10 and 10E8 antibodies with high affinity, recapitulating the structure of the MPER epitope when differentially engaged by the two anti-HIV Fabs. Thus, stapled peptides may provide a new opportunity to develop chemically stabilized antigens for vaccination.

  8. A broadly flavivirus cross-neutralizing monoclonal antibody that recognizes a novel epitope within the fusion loop of E protein.

    Directory of Open Access Journals (Sweden)

    Yong-Qiang Deng

    Full Text Available Flaviviruses are a group of human pathogenic, enveloped RNA viruses that includes dengue (DENV, yellow fever (YFV, West Nile (WNV, and Japanese encephalitis (JEV viruses. Cross-reactive antibodies against Flavivirus have been described, but most of them are generally weakly neutralizing. In this study, a novel monoclonal antibody, designated mAb 2A10G6, was determined to have broad cross-reactivity with DENV 1-4, YFV, WNV, JEV, and TBEV. Phage-display biopanning and structure modeling mapped 2A10G6 to a new epitope within the highly conserved flavivirus fusion loop peptide, the (98DRXW(101 motif. Moreover, in vitro and in vivo experiments demonstrated that 2A10G6 potently neutralizes DENV 1-4, YFV, and WNV and confers protection from lethal challenge with DENV 1-4 and WNV in murine model. Furthermore, functional studies revealed that 2A10G6 blocks infection at a step after viral attachment. These results define a novel broadly flavivirus cross-reactive mAb with highly neutralizing activity that can be further developed as a therapeutic agent against severe flavivirus infections in humans.

  9. Broad neutralizing human monoclonal antibodies against influenza virus from vaccinated healthy donors

    Energy Technology Data Exchange (ETDEWEB)

    Kubota-Koketsu, Ritsuko; Mizuta, Hiroyuki [Department of Virology, Research Institute for Microbial Diseases, Osaka University, Suita, Osaka 565-0871 (Japan); Oshita, Masatoshi; Ideno, Shoji [Osaka Research Laboratory, Benesis Corporation, Yodogawa-ku, Osaka 532-6505 (Japan); Yunoki, Mikihiro [Osaka Research Laboratory, Benesis Corporation, Yodogawa-ku, Osaka 532-6505 (Japan); Department of Virology, Research Institute for Microbial Diseases, Osaka University, Suita, Osaka 565-0871 (Japan); Kuhara, Motoki [Ina Laboratory, Medical and Biological Laboratories Corporation, Ltd., Ina, Nagano 396-0002 (Japan); Yamamoto, Naomasa [Department of Biochemistry, School of Pharmaceutical Sciences, Ohu University, Koriyama, Fukushima 963-8611 (Japan); Okuno, Yoshinobu [Kanonji Institute, The Research Foundation for Microbial Diseases of Osaka University, Kanonji, Kagawa 768-0061 (Japan); Ikuta, Kazuyoshi, E-mail: ikuta@biken.osaka-u.ac.jp [Department of Virology, Research Institute for Microbial Diseases, Osaka University, Suita, Osaka 565-0871 (Japan)

    2009-09-11

    Human monoclonal antibodies (HuMAbs) prepared from patients with viral infections could provide information on human epitopes important for the development of vaccines as well as potential therapeutic applications. Through the fusion of peripheral blood mononuclear cells from a total of five influenza-vaccinated volunteers, with newly developed murine-human chimera fusion partner cells, named SPYMEG, we obtained 10 hybridoma clones stably producing anti-influenza virus antibodies: one for influenza A H1N1, four for influenza A H3N2 and five for influenza B. Surprisingly, most of the HuMAbs showed broad reactivity within subtype and four (two for H3N2 and two for B) showed broad neutralizing ability. Importantly, epitope mapping revealed that the two broad neutralizing antibodies to H3N2 derived from different donors recognized the same epitope located underneath the receptor-binding site of the hemagglutinin globular region that is highly conserved among H3N2 strains.

  10. Structural Basis for Recognition of Human Enterovirus 71 by a Bivalent Broadly Neutralizing Monoclonal Antibody

    Science.gov (United States)

    Ku, Zhiqiang; Zuo, Teng; Kong, Liangliang; Zhang, Chao; Shi, Jinping; Liu, Qingwei; Chen, Tan; Zhang, Yingyi; Jiang, Wen; Zhang, Linqi; Huang, Zhong; Cong, Yao

    2016-01-01

    Enterovirus 71 (EV71) is the main pathogen responsible for hand, foot and mouth disease with severe neurological complications and even death in young children. We have recently identified a highly potent anti-EV71 neutralizing monoclonal antibody, termed D5. Here we investigated the structural basis for recognition of EV71 by the antibody D5. Four three-dimensional structures of EV71 particles in complex with IgG or Fab of D5 were reconstructed by cryo-electron microscopy (cryo-EM) single particle analysis all at subnanometer resolutions. The most critical EV71 mature virion-Fab structure was resolved to a resolution of 4.8 Å, which is rare in cryo-EM studies of virus-antibody complex so far. The structures reveal a bivalent binding pattern of D5 antibody across the icosahedral 2-fold axis on mature virion, suggesting that D5 binding may rigidify virions to prevent their conformational changes required for subsequent RNA release. Moreover, we also identified that the complementary determining region 3 (CDR3) of D5 heavy chain directly interacts with the extremely conserved VP1 GH-loop of EV71, which was validated by biochemical and virological assays. We further showed that D5 is indeed able to neutralize a variety of EV71 genotypes and strains. Moreover, D5 could potently confer protection in a mouse model of EV71 infection. Since the conserved VP1 GH-loop is involved in EV71 binding with its uncoating receptor, the scavenger receptor class B, member 2 (SCARB2), the broadly neutralizing ability of D5 might attribute to its inhibition of EV71 from binding SCARB2. Altogether, our results elucidate the structural basis for the binding and neutralization of EV71 by the broadly neutralizing antibody D5, thereby enhancing our understanding of antibody-based protection against EV71 infection. PMID:26938634

  11. Structural Basis for Recognition of Human Enterovirus 71 by a Bivalent Broadly Neutralizing Monoclonal Antibody.

    Science.gov (United States)

    Ye, Xiaohua; Fan, Chen; Ku, Zhiqiang; Zuo, Teng; Kong, Liangliang; Zhang, Chao; Shi, Jinping; Liu, Qingwei; Chen, Tan; Zhang, Yingyi; Jiang, Wen; Zhang, Linqi; Huang, Zhong; Cong, Yao

    2016-03-01

    Enterovirus 71 (EV71) is the main pathogen responsible for hand, foot and mouth disease with severe neurological complications and even death in young children. We have recently identified a highly potent anti-EV71 neutralizing monoclonal antibody, termed D5. Here we investigated the structural basis for recognition of EV71 by the antibody D5. Four three-dimensional structures of EV71 particles in complex with IgG or Fab of D5 were reconstructed by cryo-electron microscopy (cryo-EM) single particle analysis all at subnanometer resolutions. The most critical EV71 mature virion-Fab structure was resolved to a resolution of 4.8 Å, which is rare in cryo-EM studies of virus-antibody complex so far. The structures reveal a bivalent binding pattern of D5 antibody across the icosahedral 2-fold axis on mature virion, suggesting that D5 binding may rigidify virions to prevent their conformational changes required for subsequent RNA release. Moreover, we also identified that the complementary determining region 3 (CDR3) of D5 heavy chain directly interacts with the extremely conserved VP1 GH-loop of EV71, which was validated by biochemical and virological assays. We further showed that D5 is indeed able to neutralize a variety of EV71 genotypes and strains. Moreover, D5 could potently confer protection in a mouse model of EV71 infection. Since the conserved VP1 GH-loop is involved in EV71 binding with its uncoating receptor, the scavenger receptor class B, member 2 (SCARB2), the broadly neutralizing ability of D5 might attribute to its inhibition of EV71 from binding SCARB2. Altogether, our results elucidate the structural basis for the binding and neutralization of EV71 by the broadly neutralizing antibody D5, thereby enhancing our understanding of antibody-based protection against EV71 infection.

  12. Structural Basis for Recognition of Human Enterovirus 71 by a Bivalent Broadly Neutralizing Monoclonal Antibody.

    Directory of Open Access Journals (Sweden)

    Xiaohua Ye

    2016-03-01

    Full Text Available Enterovirus 71 (EV71 is the main pathogen responsible for hand, foot and mouth disease with severe neurological complications and even death in young children. We have recently identified a highly potent anti-EV71 neutralizing monoclonal antibody, termed D5. Here we investigated the structural basis for recognition of EV71 by the antibody D5. Four three-dimensional structures of EV71 particles in complex with IgG or Fab of D5 were reconstructed by cryo-electron microscopy (cryo-EM single particle analysis all at subnanometer resolutions. The most critical EV71 mature virion-Fab structure was resolved to a resolution of 4.8 Å, which is rare in cryo-EM studies of virus-antibody complex so far. The structures reveal a bivalent binding pattern of D5 antibody across the icosahedral 2-fold axis on mature virion, suggesting that D5 binding may rigidify virions to prevent their conformational changes required for subsequent RNA release. Moreover, we also identified that the complementary determining region 3 (CDR3 of D5 heavy chain directly interacts with the extremely conserved VP1 GH-loop of EV71, which was validated by biochemical and virological assays. We further showed that D5 is indeed able to neutralize a variety of EV71 genotypes and strains. Moreover, D5 could potently confer protection in a mouse model of EV71 infection. Since the conserved VP1 GH-loop is involved in EV71 binding with its uncoating receptor, the scavenger receptor class B, member 2 (SCARB2, the broadly neutralizing ability of D5 might attribute to its inhibition of EV71 from binding SCARB2. Altogether, our results elucidate the structural basis for the binding and neutralization of EV71 by the broadly neutralizing antibody D5, thereby enhancing our understanding of antibody-based protection against EV71 infection.

  13. Enhanced neutralization of HIV by antibodies displayed on the S-layer of Caulobacter crescentus.

    Science.gov (United States)

    Duval, Mark; Lewis, Christopher J; Nomellini, John F; Horwitz, Marc S; Smit, John; Cavacini, Lisa A

    2011-12-01

    Innovative methods of prevention are needed to stop the more than two million new HIV-1 infections annually, particularly in women. Local application of anti-HIV antibodies has been shown to be effective at preventing infection in nonhuman primates; however, the concentrations needed are cost prohibitive. Display of antibodies on a particulate platform will likely prolong effectiveness of these anti-HIV agents and lower the cost of goods. Here, we demonstrate that the bacterium Caulobacter crescentus and its highly expressed surface-layer (S-layer) protein can provide this antibody display platform. Caulobacters displaying protein G, alone or with CD4 codisplay, successfully captured HIV-1-specific antibodies and demonstrated functional neutralization. Compared to soluble antibodies, a neutralizing anti-HIV antibody displayed on Caulobacter was as effective or more effective at neutralizing diverse HIV-1 isolates. Moreover, when an antibody reactive with an epitope induced by CD4 binding (CD4i) was codisplayed with CD4, there was significant enhancement in HIV-1 neutralization. These results suggest that caulobacters displaying anti-HIV antibodies offer a distinct improvement in the use of antibodies as microbicides. Furthermore, these reagents can specifically evaluate anti-HIV antibodies in concert with other HIV-1 blocking agents to assess the most suitable tools for conversion to scFvs, allowing for direct display within the S-layer protein and further reducing cost of goods. In summary, C. crescentus, which can be easily produced and chemically stabilized at low cost, is well suited for engineering as an effective platform, offering an inexpensive way to produce and deliver HIV-1-specific microbicides.

  14. Sequential Immunization Elicits Broadly Neutralizing Anti-HIV-1 Antibodies in Ig Knockin Mice.

    Science.gov (United States)

    Escolano, Amelia; Steichen, Jon M; Dosenovic, Pia; Kulp, Daniel W; Golijanin, Jovana; Sok, Devin; Freund, Natalia T; Gitlin, Alexander D; Oliveira, Thiago; Araki, Tatsuya; Lowe, Sarina; Chen, Spencer T; Heinemann, Jennifer; Yao, Kai-Hui; Georgeson, Erik; Saye-Francisco, Karen L; Gazumyan, Anna; Adachi, Yumiko; Kubitz, Michael; Burton, Dennis R; Schief, William R; Nussenzweig, Michel C

    2016-09-08

    A vaccine that elicits broadly neutralizing antibodies (bNAbs) against HIV-1 is likely to be protective, but this has not been achieved. To explore immunization regimens that might elicit bNAbs, we produced and immunized mice expressing the predicted germline PGT121, a bNAb specific for the V3-loop and surrounding glycans on the HIV-1 spike. Priming with an epitope-modified immunogen designed to activate germline antibody-expressing B cells, followed by ELISA-guided boosting with a sequence of directional immunogens, native-like trimers with decreasing epitope modification, elicited heterologous tier-2-neutralizing responses. In contrast, repeated immunization with the priming immunogen did not. Antibody cloning confirmed elicitation of high levels of somatic mutation and tier-2-neutralizing antibodies resembling the authentic human bNAb. Our data establish that sequential immunization with specifically designed immunogens can induce high levels of somatic mutation and shepherd antibody maturation to produce bNAbs from their inferred germline precursors.

  15. Broadly Neutralizing Antibodies against HIV-1 As a Novel Aspect of the Immune Response.

    Science.gov (United States)

    Shcherbakov, D N; Bakulina, A Y; Karpenko, L I; Ilyichev, A A

    2015-01-01

    The human immunodeficiency virus-1 (HIV-1) has the ability to evade the adaptive immune response due to high mutation rates. Soon after the discovery of HIV-1, it was originally proposed that neutralizing of antibodies to the virus occurs rarely or cannot be elicited at all. In the 1990s, there appeared reports that sera of select HIV-1-infected individuals contained antibodies capable of neutralizing different virus subtypes. Such antibodies were named broadly neutralizing antibodies (bNAbs). Since 2009, the development of new cell technologies has intensified research efforts directed at identifying new bNAbs with a neutralization potency of over 90% of primary HIV-1 isolates. These antibodies have unique characteristics which include high levels of somatic mutations and unusually long variable loops that penetrate through the glycan shield of HIV-1 Env to contact the protein surface. In this review, we will attempt to summarize the latest data on bNAbs against HIV-1 in terms of their interactions with the sites of vulnerability on HIV-1 glycoproteins.

  16. Ontogeny-based immunogens for the induction of V2-directed HIV broadly neutralizing antibodies.

    Science.gov (United States)

    Moore, Penny L; Gorman, Jason; Doria-Rose, Nicole A; Morris, Lynn

    2017-01-01

    The development of a preventative HIV vaccine able to elicit broadly neutralizing antibodies (bNAbs) remains a major challenge. Antibodies that recognize the V2 region at the apex of the HIV envelope trimer are among the most common bNAb specificities during chronic infection and many exhibit remarkable breadth and potency. Understanding the developmental pathway of these antibodies has provided insights into their precursors, and the viral strains that engage them, as well as defined how such antibodies mature to acquire breadth. V2-apex bNAbs are derived from rare precursors with long anionic CDR H3s that are often deleted in the B cell repertoire. However, longitudinal studies suggest that once engaged, these precursors contain many of the structural elements required for neutralization, and can rapidly acquire breadth through moderate levels of somatic hypermutation in response to emerging viral variants. These commonalities in the precursors and mechanism of neutralization have enabled the identification of viral strains that show enhanced reactivity for V2 precursors from multiple donors, and may form the basis of germline targeting approaches. In parallel, new structural insights into the HIV trimer, the target of these quaternary antibodies, has created invaluable new opportunities for ontogeny-based immunogens designed to select for rare V2-bNAb precursors, and drive them toward breadth.

  17. Viraemia suppressed in HIV-1-infected humans by broadly neutralizing antibody 3BNC117.

    Science.gov (United States)

    Caskey, Marina; Klein, Florian; Lorenzi, Julio C C; Seaman, Michael S; West, Anthony P; Buckley, Noreen; Kremer, Gisela; Nogueira, Lilian; Braunschweig, Malte; Scheid, Johannes F; Horwitz, Joshua A; Shimeliovich, Irina; Ben-Avraham, Sivan; Witmer-Pack, Maggi; Platten, Martin; Lehmann, Clara; Burke, Leah A; Hawthorne, Thomas; Gorelick, Robert J; Walker, Bruce D; Keler, Tibor; Gulick, Roy M; Fätkenheuer, Gerd; Schlesinger, Sarah J; Nussenzweig, Michel C

    2015-06-25

    HIV-1 immunotherapy with a combination of first generation monoclonal antibodies was largely ineffective in pre-clinical and clinical settings and was therefore abandoned. However, recently developed single-cell-based antibody cloning methods have uncovered a new generation of far more potent broadly neutralizing antibodies to HIV-1 (refs 4, 5). These antibodies can prevent infection and suppress viraemia in humanized mice and nonhuman primates, but their potential for human HIV-1 immunotherapy has not been evaluated. Here we report the results of a first-in-man dose escalation phase 1 clinical trial of 3BNC117, a potent human CD4 binding site antibody, in uninfected and HIV-1-infected individuals. 3BNC117 infusion was well tolerated and demonstrated favourable pharmacokinetics. A single 30 mg kg(-1) infusion of 3BNC117 reduced the viral load in HIV-1-infected individuals by 0.8-2.5 log10 and viraemia remained significantly reduced for 28 days. Emergence of resistant viral strains was variable, with some individuals remaining sensitive to 3BNC117 for a period of 28 days. We conclude that, as a single agent, 3BNC117 is safe and effective in reducing HIV-1 viraemia, and that immunotherapy should be explored as a new modality for HIV-1 prevention, therapy and cure.

  18. Isolation of llama antibody fragments for prevention of dandruff by phage display in shampoo

    NARCIS (Netherlands)

    Dolk, E.; Vaart, M. van der; Lutje Hulsik, D.; Vriend, G.; Haard, H. de; Spinelli, S.; Cambillau, C.; Frenken, L.; Verrips, T.

    2005-01-01

    As part of research exploring the feasibility of using antibody fragments to inhibit the growth of organisms implicated in dandruff, we isolated antibody fragments that bind to a cell surface protein of Malassezia furfur in the presence of shampoo. We found that phage display of llama single-domain

  19. Cooperation of B cell lineages in induction of HIV-1-broadly neutralizing antibodies.

    Science.gov (United States)

    Gao, Feng; Bonsignori, Mattia; Liao, Hua-Xin; Kumar, Amit; Xia, Shi-Mao; Lu, Xiaozhi; Cai, Fangping; Hwang, Kwan-Ki; Song, Hongshuo; Zhou, Tongqing; Lynch, Rebecca M; Alam, S Munir; Moody, M Anthony; Ferrari, Guido; Berrong, Mark; Kelsoe, Garnett; Shaw, George M; Hahn, Beatrice H; Montefiori, David C; Kamanga, Gift; Cohen, Myron S; Hraber, Peter; Kwong, Peter D; Korber, Bette T; Mascola, John R; Kepler, Thomas B; Haynes, Barton F

    2014-07-31

    Development of strategies for induction of HIV-1 broadly neutralizing antibodies (bnAbs) by vaccines is a priority. Determining the steps of bnAb induction in HIV-1-infected individuals who make bnAbs is a key strategy for immunogen design. Here, we study the B cell response in a bnAb-producing individual and report cooperation between two B cell lineages to drive bnAb development. We isolated a virus-neutralizing antibody lineage that targeted an envelope region (loop D) and selected virus escape mutants that resulted in both enhanced bnAb lineage envelope binding and escape mutant neutralization-traits associated with increased B cell antigen drive. Thus, in this individual, two B cell lineages cooperated to induce the development of bnAbs. Design of vaccine immunogens that simultaneously drive both helper and broadly neutralizing B cell lineages may be important for vaccine-induced recapitulation of events that transpire during the maturation of neutralizing antibodies in HIV-1-infected individuals.

  20. The HIV glycan shield as a target for broadly neutralizing antibodies.

    Science.gov (United States)

    Doores, Katie J

    2015-12-01

    The HIV envelope glycoprotein (Env) is the sole target for HIV broadly neutralizing antibodies (bnAbs). HIV Env is one of the most heavily glycosylated proteins known, with approximately half of its mass consisting of host-derived N-linked glycans. The high density of glycans creates a shield that impedes antibody recognition but, critically, some of the most potent and broadly active bnAbs have evolved to recognize epitopes formed by these glycans. Although the virus hijacks the host protein synthesis and glycosylation machinery to generate glycosylated HIV Env, studies have shown that HIV Env glycosylation diverges from that typically observed on host-derived glycoproteins. In particular, the high density of glycans leads to a nonself motif of underprocessed oligomannose-type glycans that forms the target of some of the most broad and potent HIV bnAbs. This review discusses the changing perception of the HIV glycan shield, and summarizes the protein-directed and cell-directed factors controlling HIV Env glycosylation that impact on HIV bnAb recognition and HIV vaccine design strategies.

  1. Hydroxytyrosol: a new class of microbicide displaying broad anti-HIV-1 activity

    Science.gov (United States)

    Bedoya, Luis M.; Beltrán, Manuela; Obregón-Calderón, Patricia; García-Pérez, Javier; de la Torre, Humberto E.; González, Nuria; Pérez-Olmeda, Mayte; Auñón, David; Capa, Laura; Gómez-Acebo, Eduardo; Alcamí, José

    2016-01-01

    Objective: To investigate the toxicity and activity against HIV of 5-hydroxytyrosol as a potential microbicide. Design: The anti-HIV-1 activity of 5-hydroxytyrosol, a polyphenolic compound, was tested against wild-type HIV-1 and viral clones resistant to nucleoside reverse transcriptase inhibitors (NRTIs), non-nucleoside reverse transcriptase inhibitors (NNRTIs), protease inhibitors and integrase inhibitors. In addition to its activity against founder viruses, different viral subtypes and potential synergy with tenofovir disoproxil fumarate, lamivudine and emtricitabine was also tested. 5-Hydroxytyrosol toxicity was evaluated in vivo in rabbit vaginal mucosa. Methods: We have cloned pol gene from drug-resistant HIV-1 isolated from infected patients and env gene from Fiebeg III/IV patients or A, C, D, E, F and G subtypes in the NL4.3-Ren backbone. 5-Hydroxytyrosol anti-HIV-1 activity was evaluated in infections of MT-2, U87-CCR5 or peripheral blood mononuclear cells preactivated with phytohemagglutinin + interleukin-2 with viruses obtained through 293T transfections. Inhibitory concentration 50% and cytotoxic concentration 50% were calculated. Synergy was analysed according to Chou and Talalay method. In-vivo toxicity was evaluated for 14 days in rabbit vaginal mucosa. Results: 5-Hydroxytyrosol inhibited HIV-1 infections of recombinant or wild-type viruses in all the target cells tested. Moreover, 5-hydroxytyrosol showed similar inhibitory concentration 50% values for infections with NRTIs, NNRTIs, protease inhibitors and INIs resistant viruses; founder viruses and all the subtypes tested. Combination of 5-hydroxytyrosol with tenofovir was found to be synergistic, whereas it was additive with lamivudine and emtricitabine. In-vivo toxicity of 5-hydroxytyrosol was very low even at the highest tested doses. Conclusion: 5-Hydroxytyrosol displayed a broad anti-HIV-1 activity in different cells systems in the absent of in-vivo toxicity, therefore supporting its

  2. Elicitation of broadly neutralizing antibodies against HIV-1 – the germline/maturation hypothesis

    Directory of Open Access Journals (Sweden)

    Prabakaran ePonraj

    2014-08-01

    Full Text Available We have previously observed that all known broadly neutralizing antibodies (bnAbs against HIV-1 are highly divergent from their putative germline predecessors in contrast to bnAbs against henipaviruses and SARS coronavirus, which are much less divergent from their germline counterparts. We have hypothesized that because the germline antibodies are so different compared to the highly somatically mutated HIV-1 bnAbs they may not bind to the Env. This led us to the hypothesis that the immunogenicity of the highly conserved epitopes on the HIV-1 envelope glycoproteins (Envs is reduced or eliminated by their very weak or absent interactions with germline antibodies. Thus immune responses leading to elicitation of bnAbs may not be initiated and/or sustained; even if they are, the maturation pathways are so complex that prolonged periods of time may be required for elicitation of such bnAbs. In support of the hypothesis, our initial experiments showed that germline-like precursors of several bnAbs do not bind to their epitopes. Recently, a number of research groups working in the HIV vaccine field have obtained data supporting and further expanding the germline/maturation hypothesis. Vaccine immunogens that could bind putative germline antibody predecessors of known bnAbs were successfully generated. However, guiding the immune system through the exceptionally complex antibody maturation pathways in order to elicit those bnAbs remains a major challenge. Here, we summarize developments in the HIV-1 vaccine field based on the germline/maturation hypothesis including our recent data demonstrating germline-like VRC01 antibodies in a human cord blood IgM library.

  3. Peptide Paratope Mimics of the Broadly Neutralizing HIV-1 Antibody b12.

    Science.gov (United States)

    Haußner, Christina; Damm, Dominik; Nirschl, Sandra; Rohrhofer, Anette; Schmidt, Barbara; Eichler, Jutta

    2017-01-26

    The broadly neutralizing HIV-1 antibody b12 recognizes the CD4 binding site of the HIV-1 envelope glycoprotein gp120 and efficiently neutralizes HIV-1 infections in vitro and in vivo. Based on the 3D structure of a b12⋅gp120 complex, we have designed an assembled peptide (b12-M) that presents the parts of the three heavy-chain complementarity-determining regions (CDRs) of b12, which contain the contact sites of the antibody for gp120. This b12-mimetic peptide, as well as a truncated peptide presenting only two of the three heavy-chain CDRs of b12, were shown to recognize gp120 in a similar manner to b12, as well as to inhibit HIV-1 infection, demonstrating functional mimicry of b12 by the paratope mimetic peptides.

  4. Virological features associated with the development of broadly neutralizing antibodies to HIV-1.

    Science.gov (United States)

    Moore, Penny L; Williamson, Carolyn; Morris, Lynn

    2015-04-01

    The development of a preventative HIV-1 vaccine remains a global public health priority. This will likely require the elicitation of broadly neutralizing antibodies (bNAbs) able to block infection by diverse viral strains from across the world. Understanding the pathway to neutralization breadth in HIV-1 infected humans will provide insights into how bNAb lineages arise, a process that probably involves a combination of host and viral factors. Here, we focus on the role of viral characteristics and evolution in shaping bNAbs during HIV-1 infection, and describe how these findings may be translated into novel vaccine strategies.

  5. A stably expressed llama single-domain intrabody targeting Rev displays broad-spectrum anti-HIV activity.

    Science.gov (United States)

    Boons, Eline; Li, Guangdi; Vanstreels, Els; Vercruysse, Thomas; Pannecouque, Christophe; Vandamme, Anne-Mieke; Daelemans, Dirk

    2014-12-01

    The HIV Rev protein mediates the transport of partially and unspliced HIV mRNA from the nucleus to the cytoplasm. Rev multimerizes on a secondary stem-loop structure present in the viral intron-containing mRNA species and recruits the cellular karyopherin CRM1 to export viral mRNAs from the nucleus to the cytoplasm. Previously we have identified a single-domain intrabody (Nb(190)), derived from a llama heavy-chain antibody, which efficiently inhibits Rev multimerization and suppresses the production of infectious virus. We recently mapped the epitope of this nanobody and demonstrated that Rev residues K20 and Y23 are crucial for interaction while residues V16, H53 and L60 are important to a lesser extent. Here, we generated cell lines stably expressing Nb(190) and assessed the capacity of these cell lines to suppress the replication of different HIV-1 subtypes. These cells stably expressing the single-domain antibody are protected from virus-induced cytopathogenic effect even in the context of high multiplicity of infection. In addition, the replication of different subtypes of group M and one strain of group O is significantly suppressed in these cell lines. Next, we analysed the natural variations of Rev amino acids in sequence samples from HIV-1 infected patients worldwide and assessed the effect of Nb(190) on the most prevalent polymorphisms occurring at the key epitope positions (K20 and Y23) in Rev. We found that Nb(190) was able to suppress the function of these Rev variants except for the K20N mutant, which was present in only 0.7% of HIV-1 sequence populations (n = 4632). Cells stably expressing the single-domain intrabody Nb(190) are protected against virus-induced cytopathogenic effect and display a selective survival advantage upon infection. In addition, Nb(190) suppresses the replication of a wide range of different HIV-1 subtypes. Large-scale sequence analysis reveals that the Nb(190) epitope positions in Rev are well conserved across major HIV-1

  6. Antibody affinity maturation through combining display of two-chain paired antibody and precision flow cytometric sorting.

    Science.gov (United States)

    Sun, Shuang; Yang, Xiao; Wang, Haifeng; Zhao, Yun; Lin, Yan; Ye, Chen; Fang, Xiangdong; Hang, Haiying

    2016-07-01

    Recombination of antibody light and heavy chain libraries greatly increases the size of a two-chain paired antibody library, thus easing the construction of large antibody libraries. Here, light and heavy chain variable domains paired by a coiled coil were applied to a bacterial inner membrane display system. However, the probability of the correct pairing of light and heavy chains through random recombination after each round of flow cytometric sorting and cloning was very low in the presence of mostly unmatched light and heavy chain genes, resulting in inefficient enrichment; a target antibody clone in the ratio of 1:100,000 negative control spheroplasts was unable to be enriched by six rounds of sorting and cloning by a conventional sorting strategy (sorting the top 1 %). By just sorting the top 0.000025 % of spheroplasts, we succeeded in enriching the target antibody clone mixed with negative control spheroplasts in a ratio of 1:10(8) by just one round of sorting and cloning. Furthermore, using this gating strategy, we efficiently enriched for an antibody clone with an affinity slightly better than the parent antibody clone from mixed spheroplasts which were present in the ratio of 1 better affinity clone to 10 parent clones to 10(6) negative control clones after just two rounds of sorting and cloning, suggesting that this gating strategy is highly sensitive in distinguishing between clones with a small difference in affinity and also enriching for clones with a higher affinity. Taken together, the combination of the display of a two-chain paired antibody library and the use of stringent gating has significantly increased the efficiency of the antibody maturation system.

  7. Generation of human antibody fragments against Streptococcus mutans using a phage display chain shuffling approach

    Directory of Open Access Journals (Sweden)

    Barth Stefan

    2005-01-01

    Full Text Available Abstract Background Common oral diseases and dental caries can be prevented effectively by passive immunization. In humans, passive immunotherapy may require the use of humanized or human antibodies to prevent adverse immune responses against murine epitopes. Therefore we generated human single chain and diabody antibody derivatives based on the binding characteristics of the murine monoclonal antibody Guy's 13. The murine form of this antibody has been used successfully to prevent Streptococcus mutans colonization and the development of dental caries in non-human primates, and to prevent bacterial colonization in human clinical trials. Results The antibody derivatives were generated using a chain-shuffling approach based on human antibody variable gene phage-display libraries. Like the parent antibody, these derivatives bound specifically to SAI/II, the surface adhesin of the oral pathogen S. mutans. Conclusions Humanization of murine antibodies can be easily achieved using phage display libraries. The human antibody fragments bind the antigen as well as the causative agent of dental caries. In addition the human diabody derivative is capable of aggregating S. mutans in vitro, making it a useful candidate passive immunotherapeutic agent for oral diseases.

  8. Preferential germline usage and VH/VL pairing observed in human antibodies selected by mRNA display.

    Science.gov (United States)

    Chen, Lei; Kutskova, Yuliya A; Hong, Feng; Memmott, John E; Zhong, Suju; Jenkinson, Megan D; Hsieh, Chung-Ming

    2015-10-01

    Since the invention of phage display, in vitro antibody display technologies have revolutionized the field of antibody discovery. In combination with antibody libraries constructed with sequences of human origin, such technologies enable accelerated therapeutic antibody discovery while bypassing the laborious animal immunization and hybridoma generation processes. Many in vitro display technologies developed since aim to differentiate from phage display by displaying full-length IgG proteins, utilizing eukaryotic translation system and codons, increasing library size or real-time kinetic selection by fluorescent activated cell sorting. We report here the development of an mRNA display technology and an accompanying HCDR3 size spectratyping monitor for human antibody discovery. Importantly, the mRNA display technology maintains a monovalent linkage between the mRNA (genotype) and display binding protein (phenotype), which minimizes avidity effect common in other display systems and allows for a stringent affinity and off-rate selection. The mRNA display technology successfully identified 100 human antibodies in 15 different selections against various targets from naïve human antibody libraries. These antibodies in general have high affinity and diversity. By analyzing the germline usage and combination of antibodies selected by the mRNA display technology, we identified trends and determined the productivity of each germline subgroup in the libraries that could serve as the knowledge base for constructing fully synthetic, next generation antibody libraries.

  9. New connections: Cell to cell HIV-1 transmission, resistance to broadly neutralizing antibodies, and an envelope sorting motif.

    Science.gov (United States)

    Smith, S Abigail; Derdeyn, Cynthia A

    2017-03-01

    HIV-1 infection from cell to cell may provide an efficient mode of viral spread in vivo and could therefore present a significant challenge for preventative or therapeutic strategies based on broadly neutralizing antibodies. Indeed, Li et al show that the potency and magnitude of multiple HIV-1 broadly neutralizing antibody classes are decreased during cell to cell infection in a context dependent manner. A functional motif in gp41 appears to contribute to this differential susceptibility by modulating exposure of neutralization epitopes.

  10. A pan-HPV vaccine based on bacteriophage PP7 VLPs displaying broadly cross-neutralizing epitopes from the HPV minor capsid protein, L2.

    Directory of Open Access Journals (Sweden)

    Ebenezer Tumban

    Full Text Available BACKGROUND: Current human papillomavirus (HPV vaccines that are based on virus-like particles (VLPs of the major capsid protein L1 largely elicit HPV type-specific antibody responses. In contrast, immunization with the HPV minor capsid protein L2 elicits antibodies that are broadly cross-neutralizing, suggesting that a vaccine targeting L2 could provide more comprehensive protection against infection by diverse HPV types. However, L2-based immunogens typically elicit much lower neutralizing antibody titers than L1 VLPs. We previously showed that a conserved broadly neutralizing epitope near the N-terminus of L2 is highly immunogenic when displayed on the surface of VLPs derived from the bacteriophage PP7. Here, we report the development of a panel of PP7 VLP-based vaccines targeting L2 that protect mice from infection with carcinogenic and non-carcinogenic HPV types that infect the genital tract and skin. METHODOLOGY/PRINCIPAL FINDINGS: L2 peptides from eight different HPV types were displayed on the surface of PP7 bacteriophage VLPs. These recombinant L2 VLPs, both individually and in combination, elicited high-titer anti-L2 IgG serum antibodies. Immunized mice were protected from high dose infection with HPV pseudovirus (PsV encapsidating a luciferase reporter. Mice immunized with 16L2 PP7 VLPs or 18L2 PP7 VLPs were nearly completely protected from both PsV16 and PsV18 challenge. Mice immunized with the mixture of eight L2 VLPs were strongly protected from genital challenge with PsVs representing eight diverse HPV types and cutaneous challenge with HPV5 PsV. CONCLUSION/SIGNIFICANCE: VLP-display of a cross-neutralizing HPV L2 epitope is an effective approach for inducing high-titer protective neutralizing antibodies and is capable of offering protection from a spectrum of HPVs associated with cervical cancer as well as genital and cutaneous warts.

  11. Modular and aggregation resistant Vh antibodies from a phage display library

    DEFF Research Database (Denmark)

    Friis, Niels Anton; Mandrup, Ole Aalund; Lykkemark, Simon

    2012-01-01

    Directed evolution of antibodies through phage display is a powerful technique for producing binders of various biological targets. One of the recent innovations in the fi eld is the domain antibody, an antibody consisting only of a single variable domain. These anti bodies can be obtained either...... through immunisation of sharks or camels, or alternatively from recombinant libraries1. The domain antibodies have certain advantages, both pharmacologically and technically. Here we report the construction of a semi-synthetic and highly modular antibody library, based on a human framework (V3-23/D47......). The antibody scaffold has been codon optimised to improve expression, and the CDR’s have been created using trinucleotide synthesis. These methods give a strict control over the randomisations, and the ability to design a library with minimal aggregation propensity. To facilitate further manipulation, unique...

  12. An Electrochemiluminescence Immunosensor Based on Gold-Magnetic Nanoparticles and Phage Displayed Antibodies.

    Science.gov (United States)

    Mu, Xihui; Tong, Zhaoyang; Huang, Qibin; Liu, Bing; Liu, Zhiwei; Hao, Lanqun; Dong, Hua; Zhang, Jinping; Gao, Chuan

    2016-02-27

    Using the multiple advantages of the ultra-highly sensitive electrochemiluminescence (ECL) technique, Staphylococcus protein A (SPA) functionalized gold-magnetic nanoparticles and phage displayed antibodies, and using gold-magnetic nanoparticles coated with SPA and coupled with a polyclonal antibody (pcAb) as magnetic capturing probes, and Ru(bpy)₃(2+)-labeled phage displayed antibody as a specific luminescence probe, this study reports a new way to detect ricin with a highly sensitive and specific ECL immunosensor and amplify specific detection signals. The linear detection range of the sensor was 0.0001~200 µg/L, and the limit of detection (LOD) was 0.0001 µg/L, which is 2500-fold lower than that of the conventional ELISA technique. The gold-magnetic nanoparticles, SPA and Ru(bpy)₃(2+)-labeled phage displayed antibody displayed different amplifying effects in the ECL immunosensor and can decrease LOD 3-fold, 3-fold and 20-fold, respectively, compared with the ECL immunosensors without one of the three effects. The integrated amplifying effect can decrease the LOD 180-fold. The immunosensor integrates the unique advantages of SPA-coated gold-magnetic nanoparticles that improve the activity of the functionalized capturing probe, and the amplifying effect of the Ru(bpy)₃(2+)-labeled phage displayed antibodies, so it increases specificity, interference-resistance and decreases LOD. It is proven to be well suited for the analysis of trace amounts of ricin in various environmental samples with high recovery ratios and reproducibility.

  13. An Electrochemiluminescence Immunosensor Based on Gold-Magnetic Nanoparticles and Phage Displayed Antibodies

    Directory of Open Access Journals (Sweden)

    Xihui Mu

    2016-02-01

    Full Text Available Using the multiple advantages of the ultra-highly sensitive electrochemiluminescence (ECL technique, Staphylococcus protein A (SPA functionalized gold-magnetic nanoparticles and phage displayed antibodies, and using gold-magnetic nanoparticles coated with SPA and coupled with a polyclonal antibody (pcAb as magnetic capturing probes, and Ru(bpy32+-labeled phage displayed antibody as a specific luminescence probe, this study reports a new way to detect ricin with a highly sensitive and specific ECL immunosensor and amplify specific detection signals. The linear detection range of the sensor was 0.0001~200 µg/L, and the limit of detection (LOD was 0.0001 µg/L, which is 2500-fold lower than that of the conventional ELISA technique. The gold-magnetic nanoparticles, SPA and Ru(bpy32+-labeled phage displayed antibody displayed different amplifying effects in the ECL immunosensor and can decrease LOD 3-fold, 3-fold and 20-fold, respectively, compared with the ECL immunosensors without one of the three effects. The integrated amplifying effect can decrease the LOD 180-fold. The immunosensor integrates the unique advantages of SPA-coated gold-magnetic nanoparticles that improve the activity of the functionalized capturing probe, and the amplifying effect of the Ru(bpy32+-labeled phage displayed antibodies, so it increases specificity, interference-resistance and decreases LOD. It is proven to be well suited for the analysis of trace amounts of ricin in various environmental samples with high recovery ratios and reproducibility.

  14. Broadly Neutralizing Alphavirus Antibodies Bind an Epitope on E2 and Inhibit Entry and Egress.

    Science.gov (United States)

    Fox, Julie M; Long, Feng; Edeling, Melissa A; Lin, Hueylie; van Duijl-Richter, Mareike K S; Fong, Rachel H; Kahle, Kristen M; Smit, Jolanda M; Jin, Jing; Simmons, Graham; Doranz, Benjamin J; Crowe, James E; Fremont, Daved H; Rossmann, Michael G; Diamond, Michael S

    2015-11-19

    We screened a panel of mouse and human monoclonal antibodies (MAbs) against chikungunya virus and identified several with inhibitory activity against multiple alphaviruses. Passive transfer of broadly neutralizing MAbs protected mice against infection by chikungunya, Mayaro, and O'nyong'nyong alphaviruses. Using alanine-scanning mutagenesis, loss-of-function recombinant proteins and viruses, and multiple functional assays, we determined that broadly neutralizing MAbs block multiple steps in the viral lifecycle, including entry and egress, and bind to a conserved epitope on the B domain of the E2 glycoprotein. A 16 Å resolution cryo-electron microscopy structure of a Fab fragment bound to CHIKV E2 B domain provided an explanation for its neutralizing activity. Binding to the B domain was associated with repositioning of the A domain of E2 that enabled cross-linking of neighboring spikes. Our results suggest that B domain antigenic determinants could be targeted for vaccine or antibody therapeutic development against multiple alphaviruses of global concern.

  15. SINGLE CHAIN VARIABLE FRAGMENTS OF ANTIBODIES AGAINST DIPHTHERIA TOXIN B-SUBUNIT ISOLATED FROM PHAGE DISPLAY HUMAN ANTIBODY LIBRARY

    Directory of Open Access Journals (Sweden)

    Oliinyk O. S.

    2014-02-01

    Full Text Available Diphtheria toxin is an exoantigen of Corynebacterium diphtheriae that inhibits protein synthesis and kills sensitive cells. The aim of this study was to obtain human recombinant single-chain variable fragment (scFv antibodies against receptor-binding B subunit of diphtheria toxin. 12 specific clones were selected after three rounds of a phage display naїve (unimmunized human antibody library against recombinant B-subunit. scFv DNA inserts from these 12 clones were digested with MvaI, and 6 unique restriction patterns were found. Single-chain antibodies were expressed in Escherichia coli XL1-blue. The recombinant proteins were characterized by immunoblotting of bacterial extracts and detection with an anti-E-tag antibody. The toxin B-subunit-binding function of the single-chain antibody was shown by ELISA. The affinity constants for different clones were found to be from 106 to 108 М–1. Due to the fact, that these antibody fragments recognized epitopes in the receptor-binding Bsubunit of diphtheria toxin, further studies are interesting to evaluate their toxin neutralization properties and potential for therapeutic applications. Obtained scFv-antibodies can also be used for detection and investigation of biological properties of diphtheria toxin.

  16. CD4 binding site broadly neutralizing antibody selection of HIV-1 escape mutants.

    Science.gov (United States)

    Dreja, Hanna; Pade, Corinna; Chen, Lei; McKnight, Áine

    2015-07-01

    All human immunodeficiency virus type-1 (HIV-1) viruses use CD4 to enter cells. Consequently, the viral envelope CD4-binding site (CD4bs) is relatively conserved, making it a logical neutralizing antibody target. It is important to understand how CD4-binding site variation allows for escape from neutralizing antibodies. Alanine scanning mutagenesis identifies residues in antigenic sites, whereas escape mutant selection identifies viable mutants. We selected HIV-1 to escape CD4bs neutralizing mAbs b12, A12 and HJ16. Viruses that escape from A12 and b12 remained susceptible to HJ16, VRC01 and J3, whilst six different viruses that escape HJ16 remained sensitive to A12, b12 and J3. In contrast, their sensitivity to VRC01 was variable. Triple HJ16/A12/b12-resistant virus proved that HIV-1 could escape multiple broadly neutralizing monoclonal antibodies, but still retain sensitivity to VRC01 and the llama-derived J3 nanobody. This antigenic variability may reflect that occurring in circulating viruses, so studies like this can predict immunologically relevant antigenic forms of the CD4bs for inclusion in HIV-1 vaccines.

  17. Broadly Neutralizing Anti-HIV Antibodies Prevent HIV Infection of Mucosal Tissue Ex Vivo.

    Science.gov (United States)

    Scott, Yanille M; Park, Seo Young; Dezzutti, Charlene S

    2016-02-01

    Broadly neutralizing monoclonal antibodies (nAbs) specific for HIV are being investigated for use in HIV prevention. Due to their ability to inhibit HIV attachment to and entry into target cells, nAbs may be suitable for use as topical HIV microbicides. As such, they would present an alternative intervention for individuals who may not benefit from using antiretroviral-based products for HIV prevention. We theorize that nAbs can inhibit viral transmission through mucosal tissue, thus reducing the incidence of HIV infection. The efficacy of the PG9, PG16, VRC01, and 4E10 antibodies was evaluated in an ex vivo human model of mucosal HIV transmission. nAbs reduced HIV transmission, causing 1.5- to 2-log10 reductions in HIV replication in ectocervical tissues and ≈3-log10 reductions in HIV replication in colonic tissues over 21 days. These antibodies demonstrated greater potency in colonic tissues, with a 50-fold higher dose being required to reduce transmission in ectocervical tissues. Importantly, nAbs retained their potency and reduced viral transmission in the presence of whole semen. No changes in tissue viability or immune activation were observed in colonic or ectocervical tissue after nAb exposure. Our data suggest that topically applied nAbs are safe and effective against HIV infection of mucosal tissue and support further development of nAbs as a topical microbicide that could be used for anal as well as vaginal protection.

  18. Broadly neutralizing human antibody that recognizes the receptor-binding pocket of influenza virus hemagglutinin

    Energy Technology Data Exchange (ETDEWEB)

    Whittle, James R.R.; Zhang, Ruijun; Khurana, Surender; King, Lisa R.; Manischewitz, Jody; Golding, Hana; Dormitzer, Philip R.; Haynes, Barton F.; Walter, Emmanuel B.; Moody, M. Anthony; Kepler, Thomas B.; Liao, Hua-Xin; Harrison, Stephen C. (Harvard-Med); (Novartis); (US-FDA); (Duke)

    2011-09-20

    Seasonal antigenic drift of circulating influenza virus leads to a requirement for frequent changes in vaccine composition, because exposure or vaccination elicits human antibodies with limited cross-neutralization of drifted strains. We describe a human monoclonal antibody, CH65, obtained by isolating rearranged heavy- and light-chain genes from sorted single plasma cells, coming from a subject immunized with the 2007 trivalent influenza vaccine. The crystal structure of a complex of the hemagglutinin (HA) from H1N1 strain A/Solomon Islands/3/2006 with the Fab of CH65 shows that the tip of the CH65 heavy-chain complementarity determining region 3 (CDR3) inserts into the receptor binding pocket on HA1, mimicking in many respects the interaction of the physiological receptor, sialic acid. CH65 neutralizes infectivity of 30 out of 36 H1N1 strains tested. The resistant strains have a single-residue insertion near the rim of the sialic-acid pocket. We conclude that broad neutralization of influenza virus can be achieved by antibodies with contacts that mimic those of the receptor.

  19. Broadly Neutralizing Antibody 8ANC195 Recognizes Closed and Open States of HIV-1 Env.

    Science.gov (United States)

    Scharf, Louise; Wang, Haoqing; Gao, Han; Chen, Songye; McDowall, Alasdair W; Bjorkman, Pamela J

    2015-09-10

    The HIV-1 envelope (Env) spike contains limited epitopes for broadly neutralizing antibodies (bNAbs); thus, most neutralizing antibodies are strain specific. The 8ANC195 epitope, defined by crystal and electron microscopy (EM) structures of bNAb 8ANC195 complexed with monomeric gp120 and trimeric Env, respectively, spans the gp120 and gp41 Env subunits. To investigate 8ANC195's gp41 epitope at higher resolution, we solved a 3.58 Å crystal structure of 8ANC195 complexed with fully glycosylated Env trimer, revealing 8ANC195 insertion into a glycan shield gap to contact gp120 and gp41 glycans and protein residues. To determine whether 8ANC195 recognizes the CD4-bound open Env conformation that leads to co-receptor binding and fusion, one of several known conformations of virion-associated Env, we solved EM structures of an Env/CD4/CD4-induced antibody/8ANC195 complex. 8ANC195 binding partially closed the CD4-bound trimer, confirming structural plasticity of Env by revealing a previously unseen conformation. 8ANC195's ability to bind different Env conformations suggests advantages for potential therapeutic applications.

  20. Pharmacokinetics and immunogenicity of broadly neutralizing HIV monoclonal antibodies in macaques.

    Science.gov (United States)

    Rosenberg, Yvonne; Sack, Markus; Montefiori, David; Labranche, Celia; Lewis, Mark; Urban, Lori; Mao, Lingjun; Fischer, Rainer; Jiang, Xiaoming

    2015-01-01

    The identification of highly potent broadly neutralizing antibodies (bnAbs) against HIV-1, and success in preventing SHIV infection following their passive administration, have increased the likelihood that immunotherapeutic strategies can be adopted to prevent and treat HIV-1 infection. However, while broad and potent neutralizing activity is an essential prerequisite, in vivo properties such as good circulatory stability and non-immunogenicity are equally critical for developing a human treatment. In the present study, glycoforms of the bnAbs 10-1074, NIH45-46G54W, 10E8, PGT121, PGT128, PGT145, PGT135, PG9, PG16, VRC01 and b12 were produced by Agrobacterium-mediated transient transfection of Nicotiana benthamiana and assessed following administration in rhesus macaques. The results indicate that (i) N-glycans within the VL domain impair plasma stability of plant-derived bnAbs and (ii) while PGT121 and b12 exhibit no immunogenicity in rhesus macaques after multiple injections, VRC01, 10-1074 and NIH45-46G54W elicit high titer anti-idiotypic antibodies following a second injection. These anti-idiotypic antibodies specifically bind the administered bnAb or a close family member, and inhibit the bnAb in neutralization assays. These findings suggest that specific mutations in certain bnAbs contribute to their immunogenicity and call attention to the prospect that these mutated bnAbs will be immunogenic in humans, potentially compromising their value for prophylaxis and therapy of HIV-1.

  1. Isolation of Human Antibodies Against Hepatitis E From Phage Display Library by Metal Affinity Chromatography

    Institute of Scientific and Technical Information of China (English)

    2007-01-01

    Objective To isolate human antibodies against hepatitis E virus from phage display library by a new method of panning phage antibody library based on immobilized metal affinity chromatography (IMAC). Methods Phage antibody library was allowed to mix with hex-His tagged expressed HEV specific antigen, NE2, in solution for adequate binding before affinity resin for hex-His was added. The non-specific phage antibodies were removed by extensive washing and the specific bound phage antibodies could then be eluted to infect TG1 or repeat the binding process for subsequent rounds of purification. The specificity of the selected human antibodies were tested by antigen competitive ELISA, human sera blocking ELISA, scFv expression, and sequence analysis. Results His-NE2 specific recombinant phages were successfully enriched after panning procedure. Two individual phage clones, 126 and 138, showed 50% inhibition in NE2 antigen competition ELISA and obvious blocking effect by HEV positive serum in blocking ELISA. Soluble scFv of 126, 138 bound to NE2 specifically. Conclusion Two specific human phage antibodies against hepatitis E virus (HEV) from phage display library were isolated by immobilized metal affinity chromatography. The immobilized metal affinity chromatography applied to phage antibody selection was a helpful supplement to the selection in solution.

  2. Raising an Antibody Specific to Breast Cancer Subpopulations Using Phage Display on Tissue Sections

    DEFF Research Database (Denmark)

    Larsen, Simon Asbjørn; Meldgaard, Theresa; Fridriksdottir, Agla Jael Rubner

    2016-01-01

    BACKGROUND/AIM: Primary tumors display a great level of intra-tumor heterogeneity in breast cancer. The current lack of prognostic and predictive biomarkers limits accurate stratification and the ability to predict response to therapy. The aim of the present study was to select recombinant antibody...... fragments specific against breast cancer subpopulations, aiding the discovery of novel biomarkers. MATERIALS AND METHODS: Recombinant antibody fragments were selected by phage display. A novel shadowstick technology enabled the direct selection using tissue sections of antibody fragments specific against...... small subpopulations of breast cancer cells. Selections were performed against a subpopulation of breast cancer cells expressing CD271(+), as these previously have been indicated to be potential breast cancer stem cells. The selected antibody fragments were screened by phage ELISA on both breast cancer...

  3. Use of bacteriophage particles displaying influenza virus hemagglutinin for the detection of hemagglutination-inhibition antibodies.

    Science.gov (United States)

    Domm, William; Brewer, Matthew; Baker, Steven F; Feng, Changyong; Martínez-Sobrido, Luis; Treanor, John; Dewhurst, Stephen

    2014-03-01

    Bacteriophage lambda capsids provide a flexible molecular scaffold that can be engineered to display a wide range of exogenous proteins, including full-length viral glycoproteins produced in eukaryotic cells. One application for such particles lies in the detection of virus-specific antibodies, since they may obviate the need to work with infectious stocks of highly pathogenic or emerging viruses that can pose significant biosafety and biocontainment challenges. Bacteriophage lambda capsids were produced that displayed an insect-cell derived, recombinant H5 influenza virus hemagglutinin (HA) on their surface. The particles agglutinated red blood cells efficiently, in a manner that could be blocked using H5 HA-specific monoclonal antibodies. The particles were then used to develop a modified hemagglutinination-inhibition (HAI) assay, which successfully identified human sera with H5 HA-specific HAI activity. These results demonstrate the utility of HA-displaying bacteriophage capsids for the detection of influenza virus-specific HAI antibodies.

  4. Toward Effective HIV Vaccination INDUCTION OF BINARY EPITOPE REACTIVE ANTIBODIES WITH BROAD HIV NEUTRALIZING ACTIVITY

    Energy Technology Data Exchange (ETDEWEB)

    Nishiyama, Yasuhiro; Planque, Stephanie; Mitsuda, Yukie; Nitti, Giovanni; Taguchi, Hiroaki; Jin, Lei; Symersky, Jindrich; Boivin, Stephane; Sienczyk, Marcin; Salas, Maria; Hanson, Carl V.; Paul, Sudhir; (Texas-MED); (Viral Rickettsial)

    2009-11-23

    We describe murine monoclonal antibodies (mAbs) raised by immunization with an electrophilic gp120 analog (E-gp120) expressing the rare ability to neutralize genetically heterologous human immunodeficiency virus (HIV) strains. Unlike gp120, E-gp120 formed covalent oligomers. The reactivity of gp120 and E-gp120 with mAbs to reference neutralizing epitopes was markedly different, indicating their divergent structures. Epitope mapping with synthetic peptides and electrophilic peptide analogs indicated binary recognition of two distinct gp120 regions by anti-E-gp120 mAbs, the 421-433 and 288-306 peptide regions. Univalent Fab and single chain Fv fragments expressed the ability to recognize both peptides. X-ray crystallography of an anti-E-gp120 Fab fragment revealed two neighboring cavities, the typical antigen-binding cavity formed by the complementarity determining regions (CDRs) and another cavity dominated by antibody heavy chain variable (VH) domain framework (FR) residues. Substitution of the FR cavity VH Lys-19 residue by an Ala residue resulted in attenuated binding of the 421-433 region peptide probe. The CDRs and VH FR replacement/silent mutation ratios exceeded the ratio for a random mutation process, suggesting adaptive development of both putative binding sites. All mAbs studied were derived from VH1 family genes, suggesting biased recruitment of the V gene germ line repertoire by E-gp120. The conserved 421-433 region of gp120 is essential for HIV binding to host CD4 receptors. This region is recognized weakly by the FR of antibodies produced without exposure to HIV, but it usually fails to induce adaptive synthesis of neutralizing antibodies. We present models accounting for improved CD4-binding site recognition and broad HIV neutralizing activity of the mAbs, long sought goals in HIV vaccine development.

  5. Broad-specificity immunoassays for sulfonamide detection: immunochemical strategy for generic antibodies and competitors.

    Science.gov (United States)

    Franek, Milan; Diblikova, Iva; Cernoch, Ivo; Vass, Maria; Hruska, Karel

    2006-03-01

    Development of antibodies with broad specificity recognition for sulfonamide drugs was found to be surprisingly difficult when conventional immunochemical strategies were applied to hapten design. To improve the cross-reactivity pattern of antibodies for the family of sulfonamide drugs, a novel strategy based on the single-ring (fragment-derived) hapten moieties with different spacer substituent lengths was employed for the preparation of immunogens, coating conjugates, and enzyme competitors. The rabbit antibodies raised against a common (one-ring) p-aminobenzenesulfonamide hapten moiety (attached to a carrier protein through the N-1 position) in combination with a homologous hapten-peroxidase tracer allowed the detection of 15 sulfonamide species at the maximum residue limit level using direct ELISA. The two-ring 6-(4-aminobenzensulfonylamino)hexanoic hapten mimics, previously reported in the literature as a weak generic antigen, generated surprisingly superior immune responses in rabbits. The antibodies raised against this two-ring hapten were capable of detecting at least 19 and 17 sulfonamides in a direct ELISA system at the regulatory level with sensitivities corresponding to 20 and 50% binding inhibition, respectively. A negligible cross-reaction with N4 metabolites makes it possible to measure responses of parent sulfonamides in the presence of their metabolized forms. In skimmed milk, the highest limit of detection (LOD) for sulfacetamide defined as 20% inhibition was 65.2 microg x L(-1) (IC20 value), whereas the additional 18 sulfonamides tested exhibited LODs in the range of 0.2-36.8 microg x L(-1). This sensitivity allows simple multisulfonamide tests to be established for use in the laboratory or on site.

  6. SINGLE CHAIN VARIABLE FRAGMENTS OF ANTIBODIES AGAINST DIPHTHERIA TOXIN B-SUBUNIT ISOLATED FROM PHAGE DISPLAY HUMAN ANTIBODY LIBRARY

    OpenAIRE

    Oliinyk O. S.; Kaberniuk A. A.; Kolibo D. V.; Komisarenko S. V.

    2014-01-01

    Diphtheria toxin is an exoantigen of Corynebacterium diphtheriae that inhibits protein synthesis and kills sensitive cells. The aim of this study was to obtain human recombinant single-chain variable fragment (scFv) antibodies against receptor-binding B subunit of diphtheria toxin. 12 specific clones were selected after three rounds of a phage display naїve (unimmunized) human antibody library against recombinant B-subunit. scFv DNA inserts from these 12 clones were digested with MvaI, an...

  7. Broadly Neutralizing Antibody Responses in a Large Longitudinal Sub-Saharan HIV Primary Infection Cohort.

    Science.gov (United States)

    Landais, Elise; Huang, Xiayu; Havenar-Daughton, Colin; Murrell, Ben; Price, Matt A; Wickramasinghe, Lalinda; Ramos, Alejandra; Bian, Charoan B; Simek, Melissa; Allen, Susan; Karita, Etienne; Kilembe, William; Lakhi, Shabir; Inambao, Mubiana; Kamali, Anatoli; Sanders, Eduard J; Anzala, Omu; Edward, Vinodh; Bekker, Linda-Gail; Tang, Jianming; Gilmour, Jill; Kosakovsky-Pond, Sergei L; Phung, Pham; Wrin, Terri; Crotty, Shane; Godzik, Adam; Poignard, Pascal

    2016-01-01

    Broadly neutralizing antibodies (bnAbs) are thought to be a critical component of a protective HIV vaccine. However, designing vaccines immunogens able to elicit bnAbs has proven unsuccessful to date. Understanding the correlates and immunological mechanisms leading to the development of bnAb responses during natural HIV infection is thus critical to the design of a protective vaccine. The IAVI Protocol C program investigates a large longitudinal cohort of primary HIV-1 infection in Eastern and South Africa. Development of neutralization was evaluated in 439 donors using a 6 cross-clade pseudo-virus panel predictive of neutralization breadth on larger panels. About 15% of individuals developed bnAb responses, essentially between year 2 and year 4 of infection. Statistical analyses revealed no influence of gender, age or geographical origin on the development of neutralization breadth. However, cross-clade neutralization strongly correlated with high viral load as well as with low CD4 T cell counts, subtype-C infection and HLA-A*03(-) genotype. A correlation with high overall plasma IgG levels and anti-Env IgG binding titers was also found. The latter appeared not associated with higher affinity, suggesting a greater diversity of the anti-Env responses in broad neutralizers. Broadly neutralizing activity targeting glycan-dependent epitopes, largely the N332-glycan epitope region, was detected in nearly half of the broad neutralizers while CD4bs and gp41-MPER bnAb responses were only detected in very few individuals. Together the findings suggest that both viral and host factors are critical for the development of bnAbs and that the HIV Env N332-glycan supersite may be a favorable target for vaccine design.

  8. Broadly Neutralizing Antibody Responses in a Large Longitudinal Sub-Saharan HIV Primary Infection Cohort.

    Directory of Open Access Journals (Sweden)

    Elise Landais

    2016-01-01

    Full Text Available Broadly neutralizing antibodies (bnAbs are thought to be a critical component of a protective HIV vaccine. However, designing vaccines immunogens able to elicit bnAbs has proven unsuccessful to date. Understanding the correlates and immunological mechanisms leading to the development of bnAb responses during natural HIV infection is thus critical to the design of a protective vaccine. The IAVI Protocol C program investigates a large longitudinal cohort of primary HIV-1 infection in Eastern and South Africa. Development of neutralization was evaluated in 439 donors using a 6 cross-clade pseudo-virus panel predictive of neutralization breadth on larger panels. About 15% of individuals developed bnAb responses, essentially between year 2 and year 4 of infection. Statistical analyses revealed no influence of gender, age or geographical origin on the development of neutralization breadth. However, cross-clade neutralization strongly correlated with high viral load as well as with low CD4 T cell counts, subtype-C infection and HLA-A*03(- genotype. A correlation with high overall plasma IgG levels and anti-Env IgG binding titers was also found. The latter appeared not associated with higher affinity, suggesting a greater diversity of the anti-Env responses in broad neutralizers. Broadly neutralizing activity targeting glycan-dependent epitopes, largely the N332-glycan epitope region, was detected in nearly half of the broad neutralizers while CD4bs and gp41-MPER bnAb responses were only detected in very few individuals. Together the findings suggest that both viral and host factors are critical for the development of bnAbs and that the HIV Env N332-glycan supersite may be a favorable target for vaccine design.

  9. Deep mutational scanning of an antibody against epidermal growth factor receptor using mammalian cell display and massively parallel pyrosequencing.

    Science.gov (United States)

    Forsyth, Charles M; Juan, Veronica; Akamatsu, Yoshiko; DuBridge, Robert B; Doan, Minhtam; Ivanov, Alexander V; Ma, Zhiyuan; Polakoff, Dixie; Razo, Jennifer; Wilson, Keith; Powers, David B

    2013-01-01

    We developed a method for deep mutational scanning of antibody complementarity-determining regions (CDRs) that can determine in parallel the effect of every possible single amino acid CDR substitution on antigen binding. The method uses libraries of full length IgGs containing more than 1000 CDR point mutations displayed on mammalian cells, sorted by flow cytometry into subpopulations based on antigen affinity and analyzed by massively parallel pyrosequencing. Higher, lower and neutral affinity mutations are identified by their enrichment or depletion in the FACS subpopulations. We applied this method to a humanized version of the anti-epidermal growth factor receptor antibody cetuximab, generated a near comprehensive data set for 1060 point mutations that recapitulates previously determined structural and mutational data for these CDRs and identified 67 point mutations that increase affinity. The large-scale, comprehensive sequence-function data sets generated by this method should have broad utility for engineering properties such as antibody affinity and specificity and may advance theoretical understanding of antibody-antigen recognition.

  10. Honing a harder-hitting hammerhead improves broadly neutralizing antibody breadth and potency.

    Science.gov (United States)

    Lewis, George K

    2015-06-01

    While current HIV-1 therapies have greatly improved the quality and duration of life for infected individuals, a vaccine to prevent transmission of the virus is lacking. Broadly neutralizing monoclonal antibodies (bnmAbs) with the capacity to neutralize multiple HIV-1 variants have been isolated from HIV-1-infected individuals, and there has been a great effort to investigate how these bnmAbs arise, due their potential for HIV-1 vaccination. In this issue of the JCI, Willis and colleagues apply a computational approach to design variants of the bnmAb PG9 in an attempt to enhance potency and neutralization breadth. One of these variants was able to target multiple PG9-resistant strains, as the result of stabilization of the long heavy chain complementarity determining region 3 (HCDR3). The results of this study provide important insight and a unique approach to optimizing HIV-1 bnmABs.

  11. Application of Current Hapten in the Production of Broad Specificity Antibodies Against Organophosphorus Pesticides

    Institute of Scientific and Technical Information of China (English)

    LIU Xian-jin; YAN Chun-rong; LIU Yuan; YU Xiang-yang; ZHANG Cun-zheng

    2008-01-01

    Diethylphosphono acetic acid (DPA) was used as a current hapten to generate broad specificity polycolonal antibodies against a group of organophosphorus pesticides. Six New Zealand white rabbits were immunized with immunogens synthesized by the active ester method (AEM) or 1-ethyl-3-(3-dimethylaminopropyl)-carbodimide method (EDC). The titers of antisera reached 25 600 by AEM and 6 400 by EDC, respectively. Polyclonal antibodies raised against DPA were screened and selected for the competitive indirect enzyme-linked immunosorbent assay (CI-ELISA). A CI-ELISA for DPA was developed with a detection limit of 3.536 ng mL-1 and an I50 value of 0.182 ug mL-1. The assay specificity was evaluated by obtaining competitive curves for several structurally related compounds as competitors. The antiserum showed high affinities to chlorpyrifos, diazinon, omethoate, parathion-ethyl and profenofos with I50 of 0.12, 0.15, 0.21, 0.88, 0.97 and 2.5 ug mL-1, respectively. The results indicate that the assay could be a screening tool for quantitation and semi-quantitation determination of the above former five organophosphorus pesticides.

  12. Immunologic characteristics of HIV-infected individuals who make broadly neutralizing antibodies.

    Science.gov (United States)

    Borrow, Persephone; Moody, M Anthony

    2017-01-01

    Induction of broadly neutralizing antibodies (bnAbs) capable of inhibiting infection with diverse variants of human immunodeficiency virus type 1 (HIV-1) is a key, as-yet-unachieved goal of prophylactic HIV-1 vaccine strategies. However, some HIV-infected individuals develop bnAbs after approximately 2-4 years of infection, enabling analysis of features of these antibodies and the immunological environment that enables their induction. Distinct subsets of CD4(+) T cells play opposing roles in the regulation of humoral responses: T follicular helper (Tfh) cells support germinal center formation and provide help for affinity maturation and the development of memory B cells and plasma cells, while regulatory CD4(+) (Treg) cells including T follicular regulatory (Tfr) cells inhibit the germinal center reaction to limit autoantibody production. BnAbs exhibit high somatic mutation frequencies, long third heavy-chain complementarity determining regions, and/or autoreactivity, suggesting that bnAb generation is likely to be highly dependent on the activity of CD4(+) Tfh cells, and may be constrained by host tolerance controls. This review discusses what is known about the immunological environment during HIV-1 infection, in particular alterations in CD4(+) Tfh, Treg, and Tfr populations and autoantibody generation, and how this is related to bnAb development, and considers the implications for HIV-1 vaccine design.

  13. Immunologic Basis for Long HCDR3s in Broadly Neutralizing Antibodies against HIV-1

    Directory of Open Access Journals (Sweden)

    Lei eYu

    2014-06-01

    Full Text Available A large panel of potent broadly neutralizing antibodies (bnAbs against HIV-1 has been reported in recent years, raising hope for the design of an effective vaccine based on epitopes recognized by these protective antibodies. However, many of these bnAbs contain the feature of long heavy chain complementarity-determining region 3 (HCDR3 sequences, which is viewed as an obstacle to the development of an HIV-1 vaccine targeting long HCDR3 bnAb responses. This mini-review summarizes the current literature and discusses the different potential Immunologic mechanisms for generating long HCDR3, including D-D fusion, VH replacement, long N region addition and skewed D-J genes usage, among which potential VH replacement products appear to be significant contributors. VH replacement occurs through RAG-mediated secondary recombination and contributes to the diversified naïve B cell repertoire. During VH replacement, a short stretch of nucleotides from previously rearranged VH genes is left within the newly formed HCDR3, thus elongating its length. Accumulating evidence suggests that long HCDR3s are present at significant numbers in the human mature naïve B cell repertoire and are primarily generated by recombination during B cell development. These new observations indicate that long HCDR3s, though in low frequency, are a normal feature of human antibody naïve repertoire and they appear to be selected to target conserved epitopes located in deep regions of the HIV-1 envelope trimer during HIV-1 infection. Therefore, the presence of long HCDR3 sequences should not necessarily be viewed as an obstacle to the development of an HIV-1 vaccine that promotes bnAb responses.

  14. Phage-display libraries of murine and human antibody Fab fragments

    DEFF Research Database (Denmark)

    Engberg, J; Andersen, P S; Nielsen, L K

    1996-01-01

    We provide efficient and detailed procedures for construction, expression, and screening of comprehensive libraries of murine or human antibody Fab fragments displayed on the surface of filamentous phage. In addition, protocols for producing and using ultra-electrocompetent cells, for producing Fab...

  15. Selection of a breast cancer subpopulation-specific antibody using phage display on tissue sections

    DEFF Research Database (Denmark)

    Larsen, Simon Asbjørn; Meldgaard, Theresa; Fridriksdottir, Agla J

    2015-01-01

    of breast cancer may provide crucial knowledge for the development of individualized therapy. However, this process is challenged by the availability of biomarkers able to identify subpopulations specifically. Here, we demonstrate an approach for phage display selection of recombinant antibody fragments...

  16. Monoclonal antibody proteomics: use of antibody mimotope displaying phages and the relevant synthetic peptides for mAb scouting.

    Science.gov (United States)

    Hajdú, István; Flachner, Beáta; Bognár, Melinda; Végh, Barbara M; Dobi, Krisztina; Lőrincz, Zsolt; Lázár, József; Cseh, Sándor; Takács, László; Kurucz, István

    2014-08-01

    Monoclonal antibody proteomics uses nascent libraries or cloned (Plasmascan™, QuantiPlasma™) libraries of mAbs that react with individual epitopes of proteins in the human plasma. At the initial phase of library creation, cognate protein antigen and the epitope interacting with the antibodies are not known. Scouting for monoclonal antibodies (mAbs) with the best binding characteristics is of high importance for mAb based biomarker assay development. However, in the absence of the identity of the cognate antigen the task represents a challenge. We combined phage display, and surface plasmon resonance (Biacore) experiments to test whether specific phages and the respective mimotope peptides obtained from large scale studies are applicable to determine key features of antibodies for scouting. We show here that mAb captured phage-mimotope heterogeneity that is the diversity of the selected peptide sequences, is inversely correlated with an important binding descriptor; the off-rate of the antibodies and that represents clues for driving the selection of useful mAbs for biomarker assay development. Carefully chosen synthetic mimotope peptides are suitable for specificity testing in competitive assays using the target proteome, in our case the human plasma.

  17. Variable epitope libraries: new vaccine immunogens capable of inducing broad human immunodeficiency virus type 1-neutralizing antibody response.

    Science.gov (United States)

    Charles-Niño, Claudia; Pedroza-Roldan, Cesar; Viveros, Monica; Gevorkian, Goar; Manoutcharian, Karen

    2011-07-18

    The extreme antigenic variability of human immunodeficiency virus (HIV) leads to immune escape of the virus, representing a major challenge in the design of effective vaccine. We have developed a novel concept for immunogen construction based on introduction of massive mutations within the epitopes targeting antigenically variable pathogens and diseases. Previously, we showed that these immunogens carrying large combinatorial libraries of mutated epitope variants, termed as variable epitope libraries (VELs), induce potent, broad and long lasting CD8+IFN-γ+ T-cell response. Moreover, we demonstrated that these T cells recognize more than 50% of heavily mutated variants (5 out of 10 amino acid positions were mutated in each epitope variant) of HIV-1 gp120 V3 loop-derived cytotoxic T lymphocyte epitope (RGPGRAFVTI) in mice. The constructed VELs had complexities of 10000 and 12500 individual members, generated as plasmid DNA or as M13 phage display combinatorial libraries, respectively, and with structural composition RGPGXAXXXX or XGXGXAXVXI, where X is any of 20 natural amino acids. Here, we demonstrated that sera from mice immunized with these VELs are capable of neutralizing 5 out of 10 viral isolates from Tier 2 reference panel of subtype B envelope clones, including HIV-1 isolates which are known to be resistant to neutralization by several potent monoclonal antibodies, described previously. These data indicate the feasibility of the application of immunogens based on VEL concept as an alternative approach for the development of molecular vaccines against antigenically variable pathogens.

  18. New broadly reactive neutralizing antibodies against hepatitis B virus surface antigen.

    Science.gov (United States)

    Kucinskaite-Kodze, Indre; Pleckaityte, Milda; Bremer, Corinna M; Seiz, Pia L; Zilnyte, Milda; Bulavaite, Aiste; Mickiene, Gitana; Zvirblis, Gintautas; Sasnauskas, Kestutis; Glebe, Dieter; Zvirbliene, Aurelija

    2016-01-01

    genotypes. Recombinant scFv consisting of immunoglobulin VH and VL regions joined by a 20 aa-long linker was generated by cloning the respective cDNA sequences from hybridoma HB1. The recombinant scFv generated in Escherichia coli recognized the same epitope as the parental MAb HB1. Cloning of HB1 VH and VL regions allowed determination of their primary structure and subsequent computer modeling of antibody-epitope interaction. The generated molecular models of HB1 variable region with its target peptides were in accordance with experimental data showing the importance of certain aa residues in antibody binding. In conclusion, the current study describes new HBsAg-specific antibodies with HBV-neutralizing potency and a broad cross-reactivity against different HBV strains. The generated MAb HB1 will be of great value in diagnostic and research settings, while the recombinant HB1-derived scFv represents a promising "building block" for producing anti-HBV tools with a potential biopharmaceutical application.

  19. Broadly neutralizing antibodies developed by an HIV-positive elite neutralizer exact a replication fitness cost on the contemporaneous virus.

    Science.gov (United States)

    Sather, D Noah; Carbonetti, Sara; Kehayia, Jenny; Kraft, Zane; Mikell, Iliyana; Scheid, Johannes F; Klein, Florian; Stamatatos, Leonidas

    2012-12-01

    Approximately 1% of those infected with HIV-1 develop broad and potent serum cross-neutralizing antibody activities. It is unknown whether or not the development of such immune responses affects the replication of the contemporaneous autologous virus. Here, we defined a pathway of autologous viral escape from contemporaneous potent and broad serum neutralizing antibodies developed by an elite HIV-1-positive (HIV-1(+)) neutralizer. These antibodies potently neutralize diverse isolates from different clades and target primarily the CD4-binding site (CD4-BS) of the viral envelope glycoprotein. Viral escape required mutations in the viral envelope glycoprotein which limited the accessibility of the CD4-binding site to the autologous broadly neutralizing anti-CD4-BS antibodies but which allowed the virus to infect cells by utilizing CD4 receptors on their surface. The acquisition of neutralization resistance, however, resulted in reduced cell entry potential and slower viral replication kinetics. Our results indicate that in vivo escape from autologous broadly neutralizing antibodies exacts fitness costs to HIV-1.

  20. Engineering HIV envelope protein to activate germline B cell receptors of broadly neutralizing anti-CD4 binding site antibodies.

    Science.gov (United States)

    McGuire, Andrew T; Hoot, Sam; Dreyer, Anita M; Lippy, Adriana; Stuart, Andrew; Cohen, Kristen W; Jardine, Joseph; Menis, Sergey; Scheid, Johannes F; West, Anthony P; Schief, William R; Stamatatos, Leonidas

    2013-04-08

    Broadly neutralizing antibodies (bnAbs) against HIV are believed to be a critical component of the protective responses elicited by an effective HIV vaccine. Neutralizing antibodies against the evolutionarily conserved CD4-binding site (CD4-BS) on the HIV envelope glycoprotein (Env) are capable of inhibiting infection of diverse HIV strains, and have been isolated from HIV-infected individuals. Despite the presence of anti-CD4-BS broadly neutralizing antibody (bnAb) epitopes on recombinant Env, Env immunization has so far failed to elicit such antibodies. Here, we show that Env immunogens fail to engage the germline-reverted forms of known bnAbs that target the CD4-BS. However, we found that the elimination of a conserved glycosylation site located in Loop D and two glycosylation sites located in variable region 5 of Env allows Env-binding to, and activation of, B cells expressing the germline-reverted BCRs of two potent broadly neutralizing antibodies, VRC01 and NIH45-46. Our results offer a possible explanation as to why Env immunogens have been ineffective in stimulating the production of such bNAbs. Importantly, they provide key information as to how such immunogens can be engineered to initiate the process of antibody-affinity maturation against one of the most conserved Env regions.

  1. Broadly Neutralizing Anti-Influenza Virus Antibodies: Enhancement of Neutralizing Potency in Polyclonal Mixtures and IgA Backbones

    Science.gov (United States)

    He, Wenqian; Mullarkey, Caitlin E.; Duty, J. Andrew; Moran, Thomas M.; Palese, Peter

    2015-01-01

    ABSTRACT Current influenza virus vaccines rely upon the accurate prediction of circulating virus strains months in advance of the actual influenza season in order to allow time for vaccine manufacture. Unfortunately, mismatches occur frequently, and even when perfect matches are achieved, suboptimal vaccine efficacy leaves several high-risk populations vulnerable to infection. However, the recent discovery of broadly neutralizing antibodies that target the hemagglutinin (HA) stalk domain has renewed hope that the development of “universal” influenza virus vaccines may be within reach. Here, we examine the functions of influenza A virus hemagglutinin stalk-binding antibodies in an endogenous setting, i.e., as polyclonal preparations isolated from human sera. Relative to monoclonal antibodies that bind to the HA head domain, the neutralization potency of monoclonal stalk-binding antibodies was vastly inferior in vitro but was enhanced by several orders of magnitude in the polyclonal context. Furthermore, we demonstrated a surprising enhancement in IgA-mediated HA stalk neutralization relative to that achieved by antibodies of IgG isotypes. Mechanistically, this could be explained in two ways. Identical variable regions consistently neutralized virus more potently when in an IgA backbone compared to an IgG backbone. In addition, HA-specific memory B cells isolated from human peripheral blood were more likely to be stalk specific when secreting antibodies of IgA isotypes compared to those secreting IgG. Taken together, our data provide strong evidence that HA stalk-binding antibodies perform optimally when in a polyclonal context and that the targeted elicitation of HA stalk-specific IgA should be an important consideration during “universal” influenza virus vaccine design. IMPORTANCE Influenza viruses remain one of the most worrisome global public health threats due to their capacity to cause pandemics. While seasonal vaccines fail to protect against the

  2. Broad and potent HIV-1 neutralization by a human antibody that binds the gp41-120 interface

    OpenAIRE

    Huang, Jinghe; Kang, Byong H; Pancera, Marie; Lee, Jeong Hyun; Tong, Tommy; Feng, Yu; Georgiev, Ivelin S.; Chuang, Gwo-Yu; Druz, Aliaksandr; Doria-Rose, Nicole A.; Laub, Leo; Sliepen, Kwinten; van Gils, Marit J.; de la Peña, Alba Torrents; Derking, Ronald

    2014-01-01

    The isolation of human monoclonal antibodies (mAbs) is providing important insights regarding the specificities that underlie broad neutralization of HIV-1 (reviewed in 1 ). Here we report a broad and extremely potent HIV-specific mAb, termed 35O22, which binds novel HIV-1 envelope glycoprotein (Env) epitope. 35O22 neutralized 62% of 181 pseudoviruses with an IC50

  3. NOVEL AMYLOID-BETA SPECIFIC scFv and VH ANTIBODY FRAGMENTS FROM HUMAN AND MOUSE PHAGE DISPLAY ANTIBODY LIBRARIES

    Science.gov (United States)

    Medecigo, M.; Manoutcharian, K.; Vasilevko, V.; Govezensky, T.; Munguia, M. E.; Becerril, B.; Luz-Madrigal, A.; Vaca, L.; Cribbs, D. H.; Gevorkian, G.

    2010-01-01

    Anti-amyloid immunotherapy has been proposed as an appropriate therapeutic approach for Alzheimer’s disease (AD). Significant efforts have been made towards the generation and assessment of antibody-based reagents capable of preventing and clearing amyloid aggregates as well as preventing their synaptotoxic effects. In this study, we selected a novel set of human anti-amyloid-beta peptide 1-42 (Aβ1-42) recombinant monoclonal antibodies in a single chain fragment variable (scFv) and a single domain (VH) formats. We demonstrated that these antibody fragments recognize in a specific manner amyloid beta deposits in APP/Tg mouse brains, inhibit toxicity of oligomeric Aβ1-42 in neuroblastoma cell cultures in a concentration-dependently manner and reduced amyloid deposits in APP/Tg2576 mice after intracranial administration. These antibody fragments recognize epitopes in the middle/C-terminus region of Aβ, which makes them strong therapeutic candidates due to the fact that most of the Aβ species found in the brains of AD patients display extensive N-terminus truncations/modifications. PMID:20451261

  4. Vaccine Elicitation of High Mannose-Dependent Neutralizing Antibodies against the V3-Glycan Broadly Neutralizing Epitope in Nonhuman Primates.

    Science.gov (United States)

    Saunders, Kevin O; Nicely, Nathan I; Wiehe, Kevin; Bonsignori, Mattia; Meyerhoff, R Ryan; Parks, Robert; Walkowicz, William E; Aussedat, Baptiste; Wu, Nelson R; Cai, Fangping; Vohra, Yusuf; Park, Peter K; Eaton, Amanda; Go, Eden P; Sutherland, Laura L; Scearce, Richard M; Barouch, Dan H; Zhang, Ruijun; Von Holle, Tarra; Overman, R Glenn; Anasti, Kara; Sanders, Rogier W; Moody, M Anthony; Kepler, Thomas B; Korber, Bette; Desaire, Heather; Santra, Sampa; Letvin, Norman L; Nabel, Gary J; Montefiori, David C; Tomaras, Georgia D; Liao, Hua-Xin; Alam, S Munir; Danishefsky, Samuel J; Haynes, Barton F

    2017-02-28

    Induction of broadly neutralizing antibodies (bnAbs) that target HIV-1 envelope (Env) is a goal of HIV-1 vaccine development. A bnAb target is the Env third variable loop (V3)-glycan site. To determine whether immunization could induce antibodies to the V3-glycan bnAb binding site, we repetitively immunized macaques over a 4-year period with an Env expressing V3-high mannose glycans. Env immunizations elicited plasma antibodies that neutralized HIV-1 expressing only high-mannose glycans-a characteristic shared by early bnAb B cell lineage members. A rhesus recombinant monoclonal antibody from a vaccinated macaque bound to the V3-glycan site at the same amino acids as broadly neutralizing antibodies. A structure of the antibody bound to glycan revealed that the three variable heavy-chain complementarity-determining regions formed a cavity into which glycan could insert and neutralized multiple HIV-1 isolates with high-mannose glycans. Thus, HIV-1 Env vaccination induced mannose-dependent antibodies with characteristics of V3-glycan bnAb precursors.

  5. All eyes on the next generation of HIV vaccines: strategies for inducing a broadly neutralizing antibody response.

    Science.gov (United States)

    Ahlers, Jeffrey D

    2014-04-01

    HIV-1 broadly neutralizing antibodies (BNAbs) develop after several years of infection through a recursive process of memory B cell adaptation and maturation against co-evolving virus quasispecies. Advances in single-cell sorting and memory B cell antibody cloning methods have identified many new HIV BNAbs targeting conserved epitopes on the HIV envelope (env) protein. 3D crystal structures and biophysical analyses of BNAbs bound to invariant virus structures expressed on monomeric gp120, epitope scaffolds, core structures, and native trimers have helped us to visualize unique binding interactions and paratope orientations that have been instrumental in guiding vaccine design. A paradigm shift in the approach to structure-based design of HIV-1 envelope immunogens came recently after several laboratories discovered that native viral envelopes or "env-structures" reverse-engineered to bind with high affinity to a handful of broadly neutralizing antibodies did not in fact bind the predicted germline precursors of these broadly neutralizing antibodies. A major challenge for HIV-1 B cell vaccine development moving forward is the design of new envelope immunogens that can trigger the selection and expansion of germline precursor and intermediate memory B cells to recapitulate B cell ontogenies associated with the maturation of a broadly neutralizing antibody response. Equally important for vaccine development is the identification of delivery systems, prime-boost strategies, and synergistic adjuvant combinations that can induce the magnitude and quality of antigen-specific T follicular helper (TFH) cell responses needed to drive somatic hypermutation (SHM) and B cell maturation against heterologous primary virus envelopes. Finding the combination of multi-protein envelope immunogens and immunization strategies that can evolve a potent broadly neutralizing antibody response portends to require a complex vaccine regimen that might be difficult to implement on any scale

  6. Phage display-based strategies for cloning and optimization of monoclonal antibodies directed against human pathogens.

    Science.gov (United States)

    Clementi, Nicola; Mancini, Nicasio; Solforosi, Laura; Castelli, Matteo; Clementi, Massimo; Burioni, Roberto

    2012-01-01

    In the last two decades, several phage display-selected monoclonal antibodies (mAbs) have been described in the literature and a few of them have managed to reach the clinics. Among these, the anti-respiratory syncytial virus (RSV) Palivizumab, a phage-display optimized mAb, is the only marketed mAb directed against microbial pathogens. Palivizumab is a clear example of the importance of choosing the most appropriate strategy when selecting or optimizing an anti-infectious mAb. From this perspective, the extreme versatility of phage-display technology makes it a useful tool when setting up different strategies for the selection of mAbs directed against human pathogens, especially when their possible clinical use is considered. In this paper, we review the principal phage display strategies used to select anti-infectious mAbs, with particular attention focused on those used against hypervariable pathogens, such as HCV and influenza viruses.

  7. Dose-response curve slope helps predict therapeutic potency and breadth of HIV broadly neutralizing antibodies.

    Science.gov (United States)

    Webb, Nicholas E; Montefiori, David C; Lee, Benhur

    2015-09-29

    A new generation of HIV broadly neutralizing antibodies (bnAbs) with remarkable potency, breadth and epitope diversity has rejuvenated interest in immunotherapeutic strategies. Potencies defined by in vitro IC50 and IC80 values (50 and 80% inhibitory concentrations) figure prominently into the selection of clinical candidates; however, much higher therapeutic levels will be required to reduce multiple logs of virus and impede escape. Here we predict bnAb potency at therapeutic levels by analysing dose-response curve slopes, and show that slope is independent of IC50/IC80 and specifically relates to bnAb epitope class. With few exceptions, CD4-binding site and V3-glycan bnAbs exhibit slopes >1, indicative of higher expected therapeutic effectiveness, whereas V2-glycan, gp41 membrane-proximal external region (MPER) and gp120-gp41 bnAbs exhibit less favourable slopes <1. Our results indicate that slope is one major predictor of both potency and breadth for bnAbs at clinically relevant concentrations, and may better coordinate the relationship between bnAb epitope structure and therapeutic expectations.

  8. HIV-1 broadly neutralizing antibody precursor B cells revealed by germline-targeting immunogen.

    Science.gov (United States)

    Jardine, Joseph G; Kulp, Daniel W; Havenar-Daughton, Colin; Sarkar, Anita; Briney, Bryan; Sok, Devin; Sesterhenn, Fabian; Ereño-Orbea, June; Kalyuzhniy, Oleksandr; Deresa, Isaiah; Hu, Xiaozhen; Spencer, Skye; Jones, Meaghan; Georgeson, Erik; Adachi, Yumiko; Kubitz, Michael; deCamp, Allan C; Julien, Jean-Philippe; Wilson, Ian A; Burton, Dennis R; Crotty, Shane; Schief, William R

    2016-03-25

    Induction of broadly neutralizing antibodies (bnAbs) is a major HIV vaccine goal. Germline-targeting immunogens aim to initiate bnAb induction by activating bnAb germline precursor B cells. Critical unmet challenges are to determine whether bnAb precursor naïve B cells bind germline-targeting immunogens and occur at sufficient frequency in humans for reliable vaccine responses. Using deep mutational scanning and multitarget optimization, we developed a germline-targeting immunogen (eOD-GT8) for diverse VRC01-class bnAbs. We then used the immunogen to isolate VRC01-class precursor naïve B cells from HIV-uninfected donors. Frequencies of true VRC01-class precursors, their structures, and their eOD-GT8 affinities support this immunogen as a candidate human vaccine prime. These methods could be applied to germline targeting for other classes of HIV bnAbs and for Abs to other pathogens.

  9. ß-defensin-2 in breast milk displays a broad antimicrobial activity against pathogenic bacteria

    Directory of Open Access Journals (Sweden)

    Joanna Baricelli

    2015-02-01

    Full Text Available OBJECTIVE: To describe the antimicrobial activity of ß-defensin-2 produced in the mammary gland and secreted in human breast milk. METHODS: The peptide production was performed by DNA cloning. ß-defensin-2 levels were quantified in 61 colostrum samples and 39 mature milk samples from healthy donors, by an indirect enzyme-linked immunosorbent assay (ELISA. Using halo inhibition assay, this study assessed activity against seven clinical isolates from diarrheal feces of children between 0 and 2 years of age. The activity of ß-defensin-2 against three opportunistic pathogens that can cause nosocomial infections was determined by microdilution test. RESULTS: The peptide levels were higher in colostrum (n = 61 than in mature milk samples (n = 39, as follows: median and range, 8.52 (2.6-16.3 µg/ml versus 0.97 (0.22-3.78, p < 0.0001; Mann-Whitney test. The recombinant peptide obtained showed high antimicrobial activity against a broad range of pathogenic bacteria. Its antibacterial activity was demonstrated in a disk containing between 1-4 µg, which produced inhibition zones ranging from 18 to 30 mm against three isolates of Salmonella spp. and four of E. coli. ß-defensin-2 showed minimum inhibitory concentrations (MICs of 0.25 µg/mL and 0.5 µg/mL for S. marcescen and P. aeruginosa, respectively, while a higher MIC (4 µg/mL was obtained against an isolated of multidrug-resistant strain of A. baumannii. CONCLUSIONS: To the authors' knowledge, this study is the first to report ß-defensin-2 levels in Latin American women. The production and the activity of ß-defensin-2 in breast milk prove its importance as a defense molecule for intestinal health in pediatric patients.

  10. Construction of Large Human Single-chain Antibody Phage Display Library

    Institute of Scientific and Technical Information of China (English)

    2002-01-01

    A large human naive single chain antibody (scFv) library is constructed from 60 healthy donors via phage display technique. During the period, some methods are employed to optimize the diversity, such as multi donors, different annealing temperature, half-nest PCR, and assembly by two-way fusion PCR. In this stud y, 78 electroporations resulted in 1010 library, diversity of which is assayed by enzyme fingerprint. The efficiency and diversity are all better than other rese arches.

  11. Identification of immunogenic proteins and generation of antibodies against Salmonella Typhimurium using phage display

    Directory of Open Access Journals (Sweden)

    Meyer Torsten

    2012-06-01

    Full Text Available Abstract Background Solely in Europoe, Salmonella Typhimurium causes more than 100,000 infections per year. Improved detection of livestock colonised with S. Typhimurium is necessary to prevent foodborne diseases. Currently, commercially available ELISA assays are based on a mixture of O-antigens (LPS or total cell lysate of Salmonella and are hampered by cross-reaction. The identification of novel immunogenic proteins would be useful to develop ELISA based diagnostic assays with a higher specificity. Results A phage display library of the entire Salmonella Typhimurium genome was constructed and 47 immunogenic oligopeptides were identified using a pool of convalescent sera from pigs infected with Salmonella Typhimurium. The corresponding complete genes of seven of the identified oligopeptids were cloned. Five of them were produced in E. coli. The immunogenic character of these antigens was validated with sera from pigs infeced with S. Tyhimurium and control sera from non-infected animals. Finally, human antibody fragments (scFv against these five antigens were selected using antibody phage display and characterised. Conclusion In this work, we identified novel immunogenic proteins of Salmonella Typhimurium and generated antibody fragments against these antigens completely based on phage display. Five immunogenic proteins were validated using a panel of positive and negative sera for prospective applications in diagnostics of Salmonela Typhimurium.

  12. Broad-spectrum inhibition of HIV-1 by a monoclonal antibody directed against a gp120-induced epitope of CD4.

    Directory of Open Access Journals (Sweden)

    Samuele E Burastero

    Full Text Available To penetrate susceptible cells, HIV-1 sequentially interacts with two highly conserved cellular receptors, CD4 and a chemokine receptor like CCR5 or CXCR4. Monoclonal antibodies (MAbs directed against such receptors are currently under clinical investigation as potential preventive or therapeutic agents. We immunized Balb/c mice with molecular complexes of the native, trimeric HIV-1 envelope (Env bound to a soluble form of the human CD4 receptor. Sera from immunized mice were found to contain gp120-CD4 complex-enhanced antibodies and showed broad-spectrum HIV-1-inhibitory activity. A proportion of MAbs derived from these mice preferentially recognized complex-enhanced epitopes. In particular, a CD4-specific MAb designated DB81 (IgG1Κ was found to preferentially bind to a complex-enhanced epitope on the D2 domain of human CD4. MAb DB81 also recognized chimpanzee CD4, but not baboon or macaque CD4, which exhibit sequence divergence in the D2 domain. Functionally, MAb DB81 displayed broad HIV-1-inhibitory activity, but it did not exert suppressive effects on T-cell activation in vitro. The variable regions of the heavy and light chains of MAb DB81 were sequenced. Due to its broad-spectrum anti-HIV-1 activity and lack of immunosuppressive effects, a humanized derivative of MAb DB81 could provide a useful complement to current preventive or therapeutic strategies against HIV-1.

  13. Multi-epitope Models Explain How Pre-existing Antibodies Affect the Generation of Broadly Protective Responses to Influenza.

    Directory of Open Access Journals (Sweden)

    Veronika I Zarnitsyna

    2016-06-01

    Full Text Available The development of next-generation influenza vaccines that elicit strain-transcendent immunity against both seasonal and pandemic viruses is a key public health goal. Targeting the evolutionarily conserved epitopes on the stem of influenza's major surface molecule, hemagglutinin, is an appealing prospect, and novel vaccine formulations show promising results in animal model systems. However, studies in humans indicate that natural infection and vaccination result in limited boosting of antibodies to the stem of HA, and the level of stem-specific antibody elicited is insufficient to provide broad strain-transcendent immunity. Here, we use mathematical models of the humoral immune response to explore how pre-existing immunity affects the ability of vaccines to boost antibodies to the head and stem of HA in humans, and, in particular, how it leads to the apparent lack of boosting of broadly cross-reactive antibodies to the stem epitopes. We consider hypotheses where binding of antibody to an epitope: (i results in more rapid clearance of the antigen; (ii leads to the formation of antigen-antibody complexes which inhibit B cell activation through Fcγ receptor-mediated mechanism; and (iii masks the epitope and prevents the stimulation and proliferation of specific B cells. We find that only epitope masking but not the former two mechanisms to be key in recapitulating patterns in data. We discuss the ramifications of our findings for the development of vaccines against both seasonal and pandemic influenza.

  14. Structure-guided alterations of the gp41-directed HIV-1 broadly neutralizing antibody 2F5 reveal new properties regarding its neutralizing function.

    Directory of Open Access Journals (Sweden)

    Javier Guenaga

    Full Text Available The broadly neutralizing HIV-1 antibody 2F5 recognizes an epitope in the gp41 membrane proximal external region (MPER. The MPER adopts a helical conformation as free peptide, as post-fusogenic forms of gp41, and when bound to the 4E10 monoclonal antibody (Mab. However, when bound to 2F5, the epitope is an extended-loop. The antibody-peptide structure reveals binding between the heavy and light chains with most the long, hydrophobic CDRH3 not contacting peptide. However, mutagenesis identifies this loop as critical for binding, neutralization and for putative hydrophobic membrane interactions. Here, we examined length requirements of the 2F5 CDRH3 and plasticity regarding binding and neutralization. We generated 2F5 variants possessing either longer or shorter CDRH3s and assessed function. The CDRH3 tolerated elongations and reductions up to four residues, displaying a range of binding affinities and retaining some neutralizing capacity. 2F5 antibody variants selective recognition of conformationally distinctive MPER probes suggests a new role for the CDRH3 loop in destabilizing the helical MPER. Binding and neutralization were enhanced by targeted tryptophan substitutions recapitulating fully the activities of the wild-type 2F5 antibody in a shorter CDRH3 variant. MPER alanine scanning revealed binding contacts of this variant downstream of the 2F5 core epitope, into the 4E10 epitope region. This variant displayed increased reactivity to cardiolipin-beta-2-glycoprotein. Tyrosine replacements maintained neutralization while eliminating cardiolipin-beta-2-glycoprotein interaction. The data suggest a new mechanism of action, important for vaccine design, in which the 2F5 CDRH3 contacts and destabilizes the MPER helix downstream of its core epitope to allow induction of the extended-loop conformation.

  15. Human Ig knockin mice to study the development and regulation of HIV-1 broadly neutralizing antibodies.

    Science.gov (United States)

    Verkoczy, Laurent; Alt, Frederick W; Tian, Ming

    2017-01-01

    A major challenge for HIV-1 vaccine research is developing a successful immunization approach for inducing broadly neutralizing antibodies (bnAbs). A key shortcoming in meeting this challenge has been the lack of animal models capable of identifying impediments limiting bnAb induction and ranking vaccine strategies for their ability to promote bnAb development. Since 2010, immunoglobulin knockin (KI) technology, involving inserting functional rearranged human variable exons into the mouse IgH and IgL loci has been used to express bnAbs in mice. This approach has allowed immune tolerance mechanisms limiting bnAb production to be elucidated and strategies to overcome such limitations to be evaluated. From these studies, along with the wealth of knowledge afforded by analyses of recombinant Ig-based bnAb structures, it became apparent that key functional features of bnAbs often are problematic for their elicitation in mice by classic vaccine paradigms, necessitating more iterative testing of new vaccine concepts. In this regard, bnAb KI models expressing deduced precursor V(D)J rearrangements of mature bnAbs or unrearranged germline V, D, J segments (that can be assembled into variable region exons that encode bnAb precursors), have been engineered to evaluate novel immunogens/regimens for effectiveness in driving bnAb responses. One promising approach emerging from such studies is the ability of sequentially administered, modified immunogens (designed to bind progressively more mature bnAb precursors) to initiate affinity maturation. Here, we review insights gained from bnAb KI studies regarding the regulation and induction of bnAbs, and discuss new Ig KI methodologies to manipulate the production and/or expression of bnAbs in vivo, to further facilitate vaccine-guided bnAb induction studies.

  16. Optimal Combinations of Broadly Neutralizing Antibodies for Prevention and Treatment of HIV-1 Clade C Infection.

    Science.gov (United States)

    Wagh, Kshitij; Bhattacharya, Tanmoy; Williamson, Carolyn; Robles, Alex; Bayne, Madeleine; Garrity, Jetta; Rist, Michael; Rademeyer, Cecilia; Yoon, Hyejin; Lapedes, Alan; Gao, Hongmei; Greene, Kelli; Louder, Mark K; Kong, Rui; Karim, Salim Abdool; Burton, Dennis R; Barouch, Dan H; Nussenzweig, Michel C; Mascola, John R; Morris, Lynn; Montefiori, David C; Korber, Bette; Seaman, Michael S

    2016-03-01

    The identification of a new generation of potent broadly neutralizing HIV-1 antibodies (bnAbs) has generated substantial interest in their potential use for the prevention and/or treatment of HIV-1 infection. While combinations of bnAbs targeting distinct epitopes on the viral envelope (Env) will likely be required to overcome the extraordinary diversity of HIV-1, a key outstanding question is which bnAbs, and how many, will be needed to achieve optimal clinical benefit. We assessed the neutralizing activity of 15 bnAbs targeting four distinct epitopes of Env, including the CD4-binding site (CD4bs), the V1/V2-glycan region, the V3-glycan region, and the gp41 membrane proximal external region (MPER), against a panel of 200 acute/early clade C HIV-1 Env pseudoviruses. A mathematical model was developed that predicted neutralization by a subset of experimentally evaluated bnAb combinations with high accuracy. Using this model, we performed a comprehensive and systematic comparison of the predicted neutralizing activity of over 1,600 possible double, triple, and quadruple bnAb combinations. The most promising bnAb combinations were identified based not only on breadth and potency of neutralization, but also other relevant measures, such as the extent of complete neutralization and instantaneous inhibitory potential (IIP). By this set of criteria, triple and quadruple combinations of bnAbs were identified that were significantly more effective than the best double combinations, and further improved the probability of having multiple bnAbs simultaneously active against a given virus, a requirement that may be critical for countering escape in vivo. These results provide a rationale for advancing bnAb combinations with the best in vitro predictors of success into clinical trials for both the prevention and treatment of HIV-1 infection.

  17. Hepatitis C virus resistance to broadly neutralizing antibodies measured using replication-competent virus and pseudoparticles.

    Science.gov (United States)

    Wasilewski, Lisa N; Ray, Stuart C; Bailey, Justin R

    2016-11-01

    A better understanding of natural variation in neutralization resistance and fitness of diverse hepatitis C virus (HCV) envelope (E1E2) variants will be critical to guide rational development of an HCV vaccine. This work has been hindered by inadequate genetic diversity in viral panels and by a lack of standardization of HCV entry assays. Neutralization assays generally use lentiviral pseudoparticles expressing HCV envelope proteins (HCVpp) or chimeric full-length viruses that are replication competent in cell culture (HCVcc). There have been few systematic comparisons of specific infectivities of E1E2-matched HCVcc and HCVpp, and to our knowledge, neutralization of E1E2-matched HCVpp and HCVcc has never been compared using a diverse panel of human broadly neutralizing monoclonal antibodies (bNAbs) targeting distinct epitopes. Here, we describe an efficient method for introduction of naturally occurring E1E2 genes into a full-length HCV genome, producing replication-competent chimeric HCVcc. We generated diverse panels of E1E2-matched HCVcc and HCVpp and measured the entry-mediating fitness of E1E2 variants using the two systems. We also compared neutralization of E1E2-matched HCVcc and HCVpp by a diverse panel of human bNAbs targeting epitopes across E1E2. We found no correlation between specific infectivities of E1E2-matched HCVcc versus HCVpp, but found a very strong positive correlation between relative neutralization resistance of these same E1E2-matched HCVcc and HCVpp variants. These results suggest that quantitative comparisons of neutralization resistance of E1E2 variants can be made with confidence using either HCVcc or HCVpp, allowing the use of either or both systems to maximize diversity of neutralization panels.

  18. Crystallographic Identification of Lipid as an Integral Component of the Epitope of HIV Broadly Neutralizing Antibody 4E10.

    Science.gov (United States)

    Irimia, Adriana; Sarkar, Anita; Stanfield, Robyn L; Wilson, Ian A

    2016-01-19

    Numerous studies of the anti-HIV-1 envelope glycoprotein 41 (gp41) broadly neutralizing antibody 4E10 suggest that 4E10 also interacts with membrane lipids, but the antibody regions contacting lipids and its orientation with respect to the viral membrane are unknown. Vaccine immunogens capable of re-eliciting these membrane proximal external region (MPER)-like antibodies may require a lipid component to be successful. We performed a systematic crystallographic study of lipid binding to 4E10 to identify lipids bound by the antibody and the lipid-interacting regions. We identified phosphatidic acid, phosphatidylglycerol, and glycerol phosphate as specific ligands for 4E10 in the crystal structures. 4E10 used its CDRH1 loop to bind the lipid head groups, while its CDRH3 interacted with the hydrophobic lipid tails. Identification of the lipid binding sites on 4E10 may aid design of immunogens for vaccines that include a lipid component in addition to the MPER on gp41 for generation of broadly neutralizing antibodies.

  19. Isolation by phage display and characterization of a singlechain antibody specific for O6-methyldeoxyguanosine

    Institute of Scientific and Technical Information of China (English)

    2001-01-01

    New approaches of making single chain Fv antibodies against O6-methyl-2′-deoxyguanosine (O6MdG) have been demonstrated by using the phage antibody display system. Using O6MdG as an antigen, 21 positive clones were identified by ELISA from this library, one of which, desig-nated H3, specifically binds to O6MdG with high affinity. The H3 scFv antibody has an affinity constant (Kaff) of 5.94×1011(mol/L)-1. H3 scFv has been successfully used to detect O6MdG in DNA hydrolyses from yeast or E. coli cells treated with a DNA methylating agent. To our knowledge, this is the first report of the selection of a specific scFv against DNA adducts. The results demonstrate the potential applications of the phage display technology for the detection of DNA lesions caused by mutagens and carcinogens.

  20. Epitope Mapping of Dengue-Virus-Enhancing Monoclonal-Antibody Using Phage Display Peptide Library

    Directory of Open Access Journals (Sweden)

    Chung-I Rai

    2008-01-01

    Full Text Available The Antibody-Dependent Enhancement (ADE hypothesis has been proposed to explain why more severe manifestations of Dengue Hemorrhagic Fever and Dengue Shock Syndrome (DHF/DSS occur predominantly during secondary infections of Dengue Virus (DV with different serotypes. However, the epitopes recognized by these enhancing antibodies are unclear. Recently, anti-pre-M monoclonal antibody (mAb 70-21, which recognized all DV serotypes without neutralizing activity, were generated and demonstrated as an enhancing antibody for DV infection. In the present study, the epitope recognized by mAb 70-21 was identified using a phage-displayed random-peptide library. After three rounds of biopanning, ELISA showed that immunopositive phage clones specifically bound to mAb 70-21 but not to serum or purified IgG from naive mice. DNA sequencing of these phage clones showed a consensus sequence, QNNLGPR. Like mAb70-21, these phage-induced antisera also enhanced the DV infection of cells. In addition, indirect fluorescent assays showed phage-induced antisera bound to human rhabdomyosarcoma or Vero cells. Western blotting and immunoprecipitation analysis showed that phage-induced antisera recognized hsp 60 in BHK cell lysate. Moreover, the sera levels of antibodies against the synthetic peptide QNNLGPR correlated with the disease severity of dengue patients. Taken together, these results suggest that antibodies which recognized epitopes shared by pre-M of DV and hsp 60 of host cells may enhance DV infection and be involved in the development of DHF or DSS.

  1. Broad Anti-Hepatitis C Virus (HCV) Antibody Responses Are Associated with Improved Clinical Disease Parameters in Chronic HCV Infection

    Science.gov (United States)

    Swann, Rachael E.; Cowton, Vanessa M.; Robinson, Mark W.; Cole, Sarah J.; Barclay, Stephen T.; Mills, Peter R.; Thomson, Emma C.; McLauchlan, John

    2016-01-01

    ABSTRACT During hepatitis C virus (HCV) infection, broadly neutralizing antibody (bNAb) responses targeting E1E2 envelope glycoproteins are generated in many individuals. It is unclear if these antibodies play a protective or a pathogenic role during chronic infection. In this study, we investigated whether bNAb responses in individuals with chronic infection were associated with differences in clinical presentation. Patient-derived purified serum IgG was used to assess the breadth of HCV E1E2 binding and the neutralization activity of HCV pseudoparticles. The binding and neutralization activity results for two panels bearing viral envelope proteins representing either an intergenotype or an intragenotype 1 group were compared. We found that the HCV load was negatively associated with strong cross-genotypic E1E2 binding (P = 0.03). Overall, we observed only a modest correlation between total E1E2 binding and neutralization ability. The breadth of intergenotype neutralization did not correlate with any clinical parameters; however, analysis of individuals with genotype 1 (gt1) HCV infection (n = 20), using an intragenotype pseudoparticle panel, found a strong association between neutralization breadth and reduced liver fibrosis (P = 0.006). A broad bNAb response in our cohort with chronic infection was associated with a single nucleotide polymorphism (SNP) in the HLA-DQB1 gene (P = 0.038), as previously reported in a cohort with acute disease. Furthermore, the bNAbs in these individuals targeted more than one region of E2-neutralizing epitopes, as assessed through cross-competition of patient bNAbs with well-characterized E2 antibodies. We conclude that the bNAb responses in patients with chronic gt1 infection are associated with lower rates of fibrosis and host genetics may play a role in the ability to raise such responses. IMPORTANCE Globally, there are 130 million to 150 million people with chronic HCV infection. Typically, the disease is progressive and is a

  2. REAL-Select: full-length antibody display and library screening by surface capture on yeast cells.

    Science.gov (United States)

    Rhiel, Laura; Krah, Simon; Günther, Ralf; Becker, Stefan; Kolmar, Harald; Hock, Björn

    2014-01-01

    We describe a novel approach named REAL-Select for the non-covalent display of IgG-molecules on the surface of yeast cells for the purpose of antibody engineering and selection. It relies on the capture of secreted native full-length antibodies on the cell surface via binding to an externally immobilized ZZ domain, which tightly binds antibody Fc. It is beneficial for high-throughput screening of yeast-displayed IgG-libraries during antibody discovery and development. In a model experiment, antibody-displaying yeast cells were isolated from a 1:1,000,000 mixture with control cells confirming the maintenance of genotype-phenotype linkage. Antibodies with improved binding characteristics were obtained by affinity maturation using REAL-Select, demonstrating the ability of this system to display antibodies in their native form and to detect subtle changes in affinity by flow cytometry. The biotinylation of the cell surface followed by functionalization with a streptavidin-ZZ fusion protein is an approach that is independent of the genetic background of the antibody-producing host and therefore can be expected to be compatible with other eukaryotic expression hosts such as P. pastoris or mammalian cells.

  3. REAL-Select: full-length antibody display and library screening by surface capture on yeast cells.

    Directory of Open Access Journals (Sweden)

    Laura Rhiel

    Full Text Available We describe a novel approach named REAL-Select for the non-covalent display of IgG-molecules on the surface of yeast cells for the purpose of antibody engineering and selection. It relies on the capture of secreted native full-length antibodies on the cell surface via binding to an externally immobilized ZZ domain, which tightly binds antibody Fc. It is beneficial for high-throughput screening of yeast-displayed IgG-libraries during antibody discovery and development. In a model experiment, antibody-displaying yeast cells were isolated from a 1:1,000,000 mixture with control cells confirming the maintenance of genotype-phenotype linkage. Antibodies with improved binding characteristics were obtained by affinity maturation using REAL-Select, demonstrating the ability of this system to display antibodies in their native form and to detect subtle changes in affinity by flow cytometry. The biotinylation of the cell surface followed by functionalization with a streptavidin-ZZ fusion protein is an approach that is independent of the genetic background of the antibody-producing host and therefore can be expected to be compatible with other eukaryotic expression hosts such as P. pastoris or mammalian cells.

  4. Llama antibody fragments recognizing various epitopes of the CD4bs neutralize a broad range of HIV-1 subtypes A, B and C.

    Science.gov (United States)

    Strokappe, Nika; Szynol, Agnieszka; Aasa-Chapman, Marlèn; Gorlani, Andrea; Forsman Quigley, Anna; Hulsik, David Lutje; Chen, Lei; Weiss, Robin; de Haard, Hans; Verrips, Theo

    2012-01-01

    Many of the neutralising antibodies, isolated to date, display limited activities against the globally most prevalent HIV-1 subtypes A and C. Therefore, those subtypes are considered to be an important target for antibody-based therapy. Variable domains of llama heavy chain antibodies (VHH) have some superior properties compared with classical antibodies. Therefore we describe the application of trimeric forms of envelope proteins (Env), derived from HIV-1 of subtype A and B/C, for a prolonged immunization of two llamas. A panel of VHH, which interfere with CD4 binding to HIV-1 Env were selected with use of panning. The results of binding and competition assays to various Env, including a variant with a stabilized CD4-binding state (gp120(Ds2)), cross-competition experiments, maturation analysis and neutralisation assays, enabled us to classify the selected VHH into three groups. The VHH of group I were efficient mainly against viruses of subtype A, C and B'/C. The VHH of group II resemble the broadly neutralising antibody (bnmAb) b12, neutralizing mainly subtype B and C viruses, however some had a broader neutralisation profile. A representative of the third group, 2E7, had an even higher neutralization breadth, neutralizing 21 out of the 26 tested strains belonging to the A, A/G, B, B/C and C subtypes. To evaluate the contribution of certain amino acids to the potency of the VHH a small set of the mutants were constructed. Surprisingly this yielded one mutant with slightly improved neutralisation potency against 92UG37.A9 (subtype A) and 96ZM651.02 (subtype C). These findings and the well-known stability of VHH indicate the potential application of these VHH as anti-HIV-1 microbicides.

  5. Phage Display-based Strategies for Cloning and Optimization of Monoclonal Antibodies Directed against Human Pathogens

    Directory of Open Access Journals (Sweden)

    Roberto Burioni

    2012-07-01

    Full Text Available In the last two decades, several phage display-selected monoclonal antibodies (mAbs have been described in the literature and a few of them have managed to reach the clinics. Among these, the anti-respiratory syncytial virus (RSV Palivizumab, a phage-display optimized mAb, is the only marketed mAb directed against microbial pathogens. Palivizumab is a clear example of the importance of choosing the most appropriate strategy when selecting or optimizing an anti-infectious mAb. From this perspective, the extreme versatility of phage-display technology makes it a useful tool when setting up different strategies for the selection of mAbs directed against human pathogens, especially when their possible clinical use is considered. In this paper, we review the principal phage display strategies used to select anti-infectious mAbs, with particular attention focused on those used against hypervariable pathogens, such as HCV and influenza viruses.

  6. Administration of nucleoside-modified mRNA encoding broadly neutralizing antibody protects humanized mice from HIV-1 challenge

    Science.gov (United States)

    Pardi, Norbert; Secreto, Anthony J.; Shan, Xiaochuan; Debonera, Fotini; Glover, Joshua; Yi, Yanjie; Muramatsu, Hiromi; Ni, Houping; Mui, Barbara L.; Tam, Ying K.; Shaheen, Farida; Collman, Ronald G.; Karikó, Katalin; Danet-Desnoyers, Gwenn A.; Madden, Thomas D.; Hope, Michael J.; Weissman, Drew

    2017-01-01

    Monoclonal antibodies are one of the fastest growing classes of pharmaceutical products, however, their potential is limited by the high cost of development and manufacturing. Here we present a safe and cost-effective platform for in vivo expression of therapeutic antibodies using nucleoside-modified mRNA. To demonstrate feasibility and protective efficacy, nucleoside-modified mRNAs encoding the light and heavy chains of the broadly neutralizing anti-HIV-1 antibody VRC01 are generated and encapsulated into lipid nanoparticles. Systemic administration of 1.4 mg kg−1 of mRNA into mice results in ∼170 μg ml−1 VRC01 antibody concentrations in the plasma 24 h post injection. Weekly injections of 1 mg kg−1 of mRNA into immunodeficient mice maintain trough VRC01 levels above 40 μg ml−1. Most importantly, the translated antibody from a single injection of VRC01 mRNA protects humanized mice from intravenous HIV-1 challenge, demonstrating that nucleoside-modified mRNA represents a viable delivery platform for passive immunotherapy against HIV-1 with expansion to a variety of diseases. PMID:28251988

  7. Ontogeny of recognition specificity and functionality for the broadly neutralizing anti-HIV antibody 4E10.

    Directory of Open Access Journals (Sweden)

    Kathryn A K Finton

    2014-09-01

    Full Text Available The process of antibody ontogeny typically improves affinity, on-rate, and thermostability, narrows polyspecificity, and rigidifies the combining site to the conformer optimal for binding from the broader ensemble accessible to the precursor. However, many broadly-neutralizing anti-HIV antibodies incorporate unusual structural elements and recognition specificities or properties that often lead to autoreactivity. The ontogeny of 4E10, an autoreactive antibody with unexpected combining site flexibility, was delineated through structural and biophysical comparisons of the mature antibody with multiple potential precursors. 4E10 gained affinity primarily by off-rate enhancement through a small number of mutations to a highly conserved recognition surface. Controverting the conventional paradigm, the combining site gained flexibility and autoreactivity during ontogeny, while losing thermostability, though polyspecificity was unaffected. Details of the recognition mechanism, including inferred global effects due to 4E10 binding, suggest that neutralization by 4E10 may involve mechanisms beyond simply binding, also requiring the ability of the antibody to induce conformational changes distant from its binding site. 4E10 is, therefore, unlikely to be re-elicited by conventional vaccination strategies.

  8. Glycan shifting on hepatitis C virus (HCV) E2 glycoprotein is a mechanism for escape from broadly neutralizing antibodies.

    Science.gov (United States)

    Pantua, Homer; Diao, Jingyu; Ultsch, Mark; Hazen, Meredith; Mathieu, Mary; McCutcheon, Krista; Takeda, Kentaro; Date, Shailesh; Cheung, Tommy K; Phung, Qui; Hass, Phil; Arnott, David; Hongo, Jo-Anne; Matthews, David J; Brown, Alex; Patel, Arvind H; Kelley, Robert F; Eigenbrot, Charles; Kapadia, Sharookh B

    2013-06-12

    Hepatitis C virus (HCV) infection is a major cause of liver disease and hepatocellular carcinoma. Glycan shielding has been proposed to be a mechanism by which HCV masks broadly neutralizing epitopes on its viral glycoproteins. However, the role of altered glycosylation in HCV resistance to broadly neutralizing antibodies is not fully understood. Here, we have generated potent HCV neutralizing antibodies hu5B3.v3 and MRCT10.v362 that, similar to the previously described AP33 and HCV1, bind to a highly conserved linear epitope on E2. We utilize a combination of in vitro resistance selections using the cell culture infectious HCV and structural analyses to identify mechanisms of HCV resistance to hu5B3.v3 and MRCT10.v362. Ultra deep sequencing from in vitro HCV resistance selection studies identified resistance mutations at asparagine N417 (N417S, N417T and N417G) as early as 5days post treatment. Comparison of the glycosylation status of soluble versions of the E2 glycoprotein containing the respective resistance mutations revealed a glycosylation shift from N417 to N415 in the N417S and N417T E2 proteins. The N417G E2 variant was glycosylated neither at residue 415 nor at residue 417 and remained sensitive to MRCT10.v362. Structural analyses of the E2 epitope bound to hu5B3.v3 Fab and MRCT10.v362 Fab using X-ray crystallography confirmed that residue N415 is buried within the antibody-peptide interface. Thus, in addition to previously described mutations at N415 that abrogate the β-hairpin structure of this E2 linear epitope, we identify a second escape mechanism, termed glycan shifting, that decreases the efficacy of broadly neutralizing HCV antibodies.

  9. Construction and selection of the natural immune Fab antibody phage display library from patients with colorectal cancer

    Institute of Scientific and Technical Information of China (English)

    Bao-Ping Wu; Bing Xiao; Tian-Mo Wan; Ya-Li Zhang; Zhen-Shu Zhang; Dian-Yuan Zhou; Zhuo-Sheng Lai; Chun-Fang Gao

    2001-01-01

    AIM: To construct the natural immune Fab antibody phage display libraries of colorectal cancer and to select antibodies related with colorectal cancer. METHODS: Extract total RNA from tissue of local cancer metastasis lymph nodes of patients with colorectal cancer.RT-PCR was used to amplify the heavy chain Fd and light chain к and the amplification products were inserted successively into the vector pComb3 to construct the human libraries of Fab antibodies. They were then panned by phage display technology. By means of Dot immunoblotting and ELISA, the libraries were identified and the Fab phage antibodies binding with antigens of colorectal cancer were selected. RESULTS: The amplified fragments of Fd and к gained by RT-PCR were about 650bp. Fd and к PCR products were subsequently inserted into the vector pComb3, resulting in a recombination rate of 40% and the volume of Fab phage display library reached 1.48 x 106. The libraries were enriched about 120-fold by 3 cycles of adsorption-elution- multiplication (panning). Dot immunoblotting showed Fab expressions on the phage libraries and ELISA showed 5clones of Fab phage antibodies which had binding activities with antigens of colorectal cancer. CONCLUSION: The natural immune Fab antibody phage display libraries of colorectal cancer were constructed. They could be used to select the relative antibodies of colorectal cancer.

  10. Broadly neutralizing antibodies against the rapidly evolving porcine reproductive and respiratory syndrome virus.

    Science.gov (United States)

    Robinson, Sally R; Li, Juan; Nelson, Eric A; Murtaugh, Michael P

    2015-05-04

    Neutralizing antibodies are a critical part of the immune armory for defense against viruses, and the mechanism by which many effective vaccines work to protect against viral infections. However, infections by rapidly evolving and genetically diverse viruses are often characterized by ineffective neutralizing antibody responses. Porcine reproductive and respiratory syndrome virus (PRRSV) is a highly genetically diverse RNA virus that causes PRRS, the most significant disease of pigs worldwide. The prevailing view of immunity to PRRSV is characterized by delayed and ineffectual production of neutralizing antibodies lacking cross-reactivity that is necessary for vaccine efficacy. Using an ELISA-based neutralizing assay developed to analyze PRRSV growth in porcine alveolar macrophages, the naturally permissive cell of PRRSV, we showed that sera from previously infected commercial sows had high levels of neutralizing activity against diverse PRRSV strains, including across distinct genotypes of PRRSV. Fifty percent cross-neutralization titers in excess of 1/1024 were observed. Neutralizing activity was dose-dependent and was maintained in the immunoglobulin fraction. Presence of high-titer, anti-PRRSV antibody activity that cross-neutralizes diverse strains of virus has prompted reevaluation of the role of neutralizing antibodies for cross-protection against PRRSV under field conditions. Understanding conditions that favor development of cross-neutralizing activity will be crucial for improved strategies to enhance cross-protection against PRRSV. More detailed studies are expected to elucidate mechanisms of neutralizing antibody production and maturation and to investigate conserved epitope targets of cross-neutralization in this rapidly evolving virus.

  11. Broad and potent HIV-1 neutralization by a human antibody that binds the gp41-gp120 interface

    Energy Technology Data Exchange (ETDEWEB)

    Huang, Jinghe; Kang, Byong H.; Pancera, Marie; Lee, Jeong Hyun; Tong, Tommy; Feng, Yu; Imamichi, Hiromi; Georgiev, Ivelin S.; Chuang, Gwo-Yu; Druz, Aliaksandr; Doria-Rose, Nicole A.; Laub, Leo; Sliepen, Kwinten; van Gils, Marit J.; de la Peña, Alba Torrents; Derking, Ronald; Klasse, Per-Johan; Migueles, Stephen A.; Bailer, Robert T.; Alam, Munir; Pugach, Pavel; Haynes, Barton F.; Wyatt, Richard T.; Sanders, Rogier W.; Binley, James M.; Ward, Andrew B.; Mascola, John R.; Kwong, Peter D.; Connors, Mark [NIH

    2015-10-15

    The isolation of human monoclonal antibodies is providing important insights into the specificities that underlie broad neutralization of HIV-1 (reviewed in ref. 1). Here we report a broad and extremely potent HIV-specific monoclonal antibody, termed 35O22, which binds a novel HIV-1 envelope glycoprotein (Env) epitope. 35O22 neutralized 62% of 181 pseudoviruses with a half-maximum inhibitory concentration (IC50) <50 μg ml-1. The median IC50 of neutralized viruses was 0.033 μg ml-1, among the most potent thus far described. 35O22 did not bind monomeric forms of Env tested, but did bind the trimeric BG505 SOSIP.664. Mutagenesis and a reconstruction by negative-stain electron microscopy of the Fab in complex with trimer revealed that it bound to a conserved epitope, which stretched across gp120 and gp41. The specificity of 35O22 represents a novel site of vulnerability on HIV Env, which serum analysis indicates to be commonly elicited by natural infection. Binding to this new site of vulnerability may thus be an important complement to current monoclonal-antibody-based approaches to immunotherapies, prophylaxis and vaccine design.

  12. HIV-1 clade C escapes broadly neutralizing autologous antibodies with N332 glycan specificity by distinct mechanisms.

    Science.gov (United States)

    Deshpande, Suprit; Patil, Shilpa; Kumar, Rajesh; Hermanus, Tandile; Murugavel, Kailapuri G; Srikrishnan, Aylur K; Solomon, Suniti; Morris, Lynn; Bhattacharya, Jayanta

    2016-08-30

    The glycan supersite centered on N332 in the V3 base of the HIV-1 envelope (Env) is a target for broadly neutralizing antibodies (bnAbs) such as PGT121 and PGT128. In this study, we examined the basis of resistance of HIV-1 clade C Envs obtained from broadly cross neutralizing (BCN) plasma of an Indian donor with N332 specificity. Pseudotyped viruses expressing autologous envs were found to be resistant to autologous BCN plasma as well as to PGT121 and PGT128 mAbs despite the majority of Envs containing an intact N332 residue. While resistance of one of the Envs to neutralization by autologous plasma antibodies with shorter V1 loop length was found to be correlated with a N332S mutation, resistance to neutralization of rest of the Envs was found to be associated with longer V1 loop length and acquisition of protective N-glycans. In summary, we show evidence of escape of circulating HIV-1 clade C in an individual from autologous BCN antibodies by three distinct mechanisms.

  13. Czech ethanol-free propolis extract displays inhibitory activity against a broad spectrum of bacterial and fungal pathogens.

    Science.gov (United States)

    Netíková, Ladislava; Bogusch, Petr; Heneberg, Petr

    2013-09-01

    Propolis acts primarily as a biocide against invasive bacteria and fungi in the hive, suggesting its potential for industrial applications. In food application, propolis is considered as a chemical preservative in meat products, extending shelf life of frozen meat and other food. The mechanism of action is still unclear due to the synergy of multiple compounds contained in propolis and due to parallel targeting of multiple pathways within each affected organism. Here, we examined the antimicrobial properties of dimethylsulfoxide (DMSO) Czech propolis extract. Until recently, DMSO was only rarely used in the propolis studies, although the other solvents tested (mostly ethanol) may significantly affect the observed inhibitory effects, notwithstanding the antimicrobial effects of ethanol itself. Here, we provide results of zone inhibition tests against Aspergillus fumigatus, Microsporum gypseum, Microsporum canis, Candida albicans, Escherichia coli, Staphylococcus aureus, Listeria monocytogenes, and Enterococcus faecalis. Although we determined inhibitory effects against all the microorganisms tested, the dose-dependent response curves were not similar to each other. While inhibitory effects against C. albicans or S. aureus were strictly dose-dependent, responses of M. gypseum and E. faecalis displayed plateau across the broad range of concentrations tested. Interestingly, response of E. coli revealed the double-peak dose-dependent curve, and responses of M. canis and L. monocytogenes decreased at the highest concentrations tested. Suggested is evaluation of DMSO propolis extracts in experimental treatment of human and veterinary infections, preferably in multitherapy with antibiotics.

  14. Isolation of Osteosarcoma-Associated Human Antibodies from a Combinatorial Fab Phage Display Library

    Directory of Open Access Journals (Sweden)

    Carmela Dantas-Barbosa

    2009-01-01

    Full Text Available Osteosarcoma, a highly malignant disease, is the most common primary bone tumor and is frequently found in children and adolescents. In order to isolate antibodies against osteosarcoma antigens, a combinatorial osteosarcoma Fab library displayed on the surface of phages was used. After three rounds of selection on the surface of tumor cells, several osteosarcoma-reactive Fabs were detected. From these Fabs, five were better characterized, and despite having differences in their VH (heavy chain variable domain and Vκ (kappa chain variable domain regions, they all bound to a protein with the same molecular mass. Further analysis by cell ELISA and immunocytochemistry suggested that the Fabs recognize a membrane-associated tumor antigen expressed in higher amounts in neoplasic cells than in normal tissue. These results suggest that the human Fabs selected in this work are a valuable tool for the study of this neoplasia.

  15. Protection Against Clostridium difficile Infection With Broadly Neutralizing Antitoxin Monoclonal Antibodies

    OpenAIRE

    2012-01-01

    The spore-forming bacterium Clostridium difficile represents the principal cause of hospital-acquired diarrhea and pseudomembranous colitis worldwide. C. difficile infection (CDI) is mediated by 2 bacterial toxins, A and B; neutralizing these toxins with monoclonal antibodies (mAbs) provides a potential nonantibiotic strategy for combating the rising prevalence, severity, and recurrence of CDI. Novel antitoxin mAbs were generated in mice and were humanized. The humanized antitoxin A mAb PA-50...

  16. Development of a monoclonal antibody-based broad-specificity ELISA for fluroquinolone antibiotics in foods and molecular modeling studies of cross-reactive compounds

    Science.gov (United States)

    Development of a competitive indirect enzyme-linked immunosorbent assay (ciELISA) with monoclonal antibodies (Mabs) having broad specificity for fluoroquinolone (FQ) antibiotics is described. Four FQs, ciprofloxacin (CIP), norfloxacin (NOR), enrofloxacin (ENR) and ofloxacin (OFL) were conjugated to...

  17. Discovery of high affinity anti-ricin antibodies by B cell receptor sequencing and by yeast display of combinatorial VH:VL libraries from immunized animals.

    Science.gov (United States)

    Wang, Bo; Lee, Chang-Han; Johnson, Erik L; Kluwe, Christien A; Cunningham, Josephine C; Tanno, Hidetaka; Crooks, Richard M; Georgiou, George; Ellington, Andrew D

    2016-01-01

    Ricin is a toxin that could potentially be used as a bioweapon. We identified anti-ricin A chain antibodies by sequencing the antibody repertoire from immunized mice and by selecting high affinity antibodies using yeast surface display. These methods led to the isolation of multiple antibodies with high (sub-nanomolar) affinity. Interestingly, the antibodies identified by the 2 independent approaches are from the same clonal lineages, indicating for the first time that yeast surface display can identify native antibodies. The new antibodies represent well-characterized reagents for biodefense diagnostics and therapeutics development.

  18. Expression of HIV-1 broadly neutralizing antibodies mediated by recombinant adeno-associated virus 8 in vitro and in vivo.

    Science.gov (United States)

    Yu, Yongjiao; Fu, Lu; Jiang, Xiaoyu; Guan, Shanshan; Kuai, Ziyu; Kong, Wei; Shi, Yuhua; Shan, Yaming

    2016-12-01

    Despite unremitting efforts since the discovery of human immunodeficiency virus type 1 (HIV-1), an effective vaccine has not been generated. Viral vector-mediated transfer for expression of HIV-1 broadly neutralizing antibodies (BnAbs) is an attractive strategy. In this study, a recombinant adeno-associated virus 8 (rAAV8) vector was used to encode full-length antibodies against HIV-1 in 293T cells and Balb/c mice after gene transfer. The 10E8 or NIH45-46 BnAb was expressed from a single open reading frame by linking the heavy and light chains with a furin cleavage and a 2A self-processing peptide (F2A). The results showed that the BnAbs could be expressed in the 293T cell culture medium. A single intramuscular injection of rAAV8 led to long-term expression of BnAbs in Balb/c mice. The expressed antibodies in the supernatant of 293T cells and in Balb/c mice showed neutralization effects against HIV-1 pseudoviruses. Combined immunization of rAAV8 expressing 10E8 and rAAV8 expressing NIH45-46 in Balb/c mice could increase these neutralization effects on strains of HIV-1 sensitive to 10E8 or NIH45-46 antibody compared with a single injection of rAAV8 expressing either antibody alone. Therefore, the combined immunization may be a potential vaccine approach against HIV-1.

  19. Broadly neutralizing anti-influenza antibodies require Fc receptor engagement for in vivo protection.

    Science.gov (United States)

    DiLillo, David J; Palese, Peter; Wilson, Patrick C; Ravetch, Jeffrey V

    2016-02-01

    In vivo protection by antimicrobial neutralizing Abs can require the contribution of effector functions mediated by Fc-Fcγ receptor (Fc-FcγR) interactions for optimal efficacy. In influenza, broadly neutralizing anti-hemagglutinin (anti-HA) stalk mAbs require Fc-FcγR interactions to mediate in vivo protection, but strain-specific anti-HA head mAbs do not. Whether this rule applies only to anti-stalk Abs or is applicable to any broadly neutralizing Ab (bNAb) against influenza is unknown. Here, we characterized the contribution of Fc-FcγR interactions during in vivo protection for a panel of 13 anti-HA mAbs, including bNAbs and non-neutralizing Abs, against both the stalk and head domains. All classes of broadly binding anti-HA mAbs required Fc-FcγR interactions to provide protection in vivo, including those mAbs that bind the HA head and those that do not neutralize virus in vitro. Further, a broadly neutralizing anti-neuraminidase (anti-NA) mAb also required FcγRs to provide protection in vivo, but a strain-specific anti-NA mAb did not. Thus, these findings suggest that the breadth of reactivity of anti-influenza Abs, regardless of their epitope, necessitates interactions with FcγRs on effector cell populations to mediate in vivo protection. These findings will guide the design of antiviral Ab therapeutics and inform vaccine design to elicit Abs with optimal binding properties and effector functions.

  20. Naturally selected hepatitis C virus polymorphisms confer broad neutralizing antibody resistance

    Science.gov (United States)

    Bailey, Justin R.; Wasilewski, Lisa N.; Snider, Anna E.; El-Diwany, Ramy; Osburn, William O.; Keck, Zhenyong; Foung, Steven K.H.; Ray, Stuart C.

    2014-01-01

    For hepatitis C virus (HCV) and other highly variable viruses, broadly neutralizing mAbs are an important guide for vaccine development. The development of resistance to anti-HCV mAbs is poorly understood, in part due to a lack of neutralization testing against diverse, representative panels of HCV variants. Here, we developed a neutralization panel expressing diverse, naturally occurring HCV envelopes (E1E2s) and used this panel to characterize neutralizing breadth and resistance mechanisms of 18 previously described broadly neutralizing anti-HCV human mAbs. The observed mAb resistance could not be attributed to polymorphisms in E1E2 at known mAb-binding residues. Additionally, hierarchical clustering analysis of neutralization resistance patterns revealed relationships between mAbs that were not predicted by prior epitope mapping, identifying 3 distinct neutralization clusters. Using this clustering analysis and envelope sequence data, we identified polymorphisms in E2 that confer resistance to multiple broadly neutralizing mAbs. These polymorphisms, which are not at mAb contact residues, also conferred resistance to neutralization by plasma from HCV-infected subjects. Together, our method of neutralization clustering with sequence analysis reveals that polymorphisms at noncontact residues may be a major immune evasion mechanism for HCV, facilitating viral persistence and presenting a challenge for HCV vaccine development. PMID:25500884

  1. Baculovirus display of single chain antibody (scFv using a novel signal peptide

    Directory of Open Access Journals (Sweden)

    Gonzalez Gaëlle

    2010-11-01

    Full Text Available Abstract Background Cells permissive to virus can become refractory to viral replication upon intracellular expression of single chain fragment variable (scFv antibodies directed towards viral structural or regulatory proteins, or virus-coded enzymes. For example, an intrabody derived from MH-SVM33, a monoclonal antibody against a conserved C-terminal epitope of the HIV-1 matrix protein (MAp17, was found to exert an inhibitory effect on HIV-1 replication. Results Two versions of MH-SVM33-derived scFv were constructed in recombinant baculoviruses (BVs and expressed in BV-infected Sf9 cells, N-myristoylation-competent scFvG2/p17 and N-myristoylation-incompetent scFvE2/p17 protein, both carrying a C-terminal HA tag. ScFvG2/p17 expression resulted in an insoluble, membrane-associated protein, whereas scFvE2/p17 was recovered in both soluble and membrane-incorporated forms. When coexpressed with the HIV-1 Pr55Gag precursor, scFvG2/p17 and scFvE2/p17 did not show any detectable negative effect on virus-like particle (VLP assembly and egress, and both failed to be encapsidated in VLP. However, soluble scFvE2/p17 isolated from Sf9 cell lysates was capable of binding to its specific antigen, in the form of a synthetic p17 peptide or as Gag polyprotein-embedded epitope. Significant amounts of scFvE2/p17 were released in the extracellular medium of BV-infected cells in high-molecular weight, pelletable form. This particulate form corresponded to BV particles displaying scFvE2/p17 molecules, inserted into the BV envelope via the scFv N-terminal region. The BV-displayed scFvE2/p17 molecules were found to be immunologically functional, as they reacted with the C-terminal epitope of MAp17. Fusion of the N-terminal 18 amino acid residues from the scFvE2/p17 sequence (N18E2 to another scFv recognizing CD147 (scFv-M6-1B9 conferred the property of BV-display to the resulting chimeric scFv-N18E2/M6. Conclusion Expression of scFvE2/p17 in insect cells using a BV

  2. Low frequency of broadly neutralizing HIV antibodies during chronic infection even in quaternary epitope targeting antibodies containing large numbers of somatic mutations.

    Science.gov (United States)

    Hicar, Mark D; Chen, Xuemin; Kalams, Spyros A; Sojar, Hakimuddin; Landucci, Gary; Forthal, Donald N; Spearman, Paul; Crowe, James E

    2016-02-01

    Neutralizing antibodies (Abs) are thought to be a critical component of an appropriate HIV vaccine response. It has been proposed that Abs recognizing conformationally dependent quaternary epitopes on the HIV envelope (Env) trimer may be necessary to neutralize diverse HIV strains. A number of recently described broadly neutralizing monoclonal Abs (mAbs) recognize complex and quaternary epitopes. Generally, many such Abs exhibit extensive numbers of somatic mutations and unique structural characteristics. We sought to characterize the native antibody (Ab) response against circulating HIV focusing on such conformational responses, without a prior selection based on neutralization. Using a capture system based on VLPs incorporating cleaved envelope protein, we identified a selection of B cells that produce quaternary epitope targeting Abs (QtAbs). Similar to a number of broadly neutralizing Abs, the Ab genes encoding these QtAbs showed extensive numbers of somatic mutations. However, when expressed as recombinant molecules, these Abs failed to neutralize virus or mediate ADCVI activity. Molecular analysis showed unusually high numbers of mutations in the Ab heavy chain framework 3 region of the variable genes. The analysis suggests that large numbers of somatic mutations occur in Ab genes encoding HIV Abs in chronically infected individuals in a non-directed, stochastic, manner.

  3. Immune System Regulation in the Induction of Broadly Neutralizing HIV-1 Antibodies

    Directory of Open Access Journals (Sweden)

    Garnett Kelsoe

    2013-12-01

    Full Text Available In this brief review, we discuss immune tolerance as a factor that determines the magnitude and quality of serum antibody responses to HIV-1 infection and vaccination in the context of recent work. We propose that many conserved, neutralizing epitopes of HIV-1 are weakly immunogenic because they mimic host antigens. In consequence, B cells that strongly bind these determinants are removed by the physiological process of immune tolerance. This structural mimicry may represent a significant impediment to designing protective HIV-1 vaccines, but we note that several vaccine strategies may be able to mitigate this evolutionary adaptation of HIV and other microbial pathogens.

  4. Broadly neutralizing antibodies targeted to mucin-type carbohydrate epitopes of human immunodeficiency virus

    DEFF Research Database (Denmark)

    Hansen, J E; Nielsen, C; Arendrup, M

    1991-01-01

    The cancer-related mucin-type carbohydrate neoantigen Tn was found on gp160 and gp120 of human immunodeficiency virus (HIV). Immunoglobulin G (IgG) and IgM monoclonal antibodies (MAbs) against Tn neutralized infection with cell-free virus and blocked fusion between HIV-infected and uninfected cells......; this binding was inhibitable by pure Tn antigen, and indications were found that this inhibition occurred at a pre-entry step. Boosting the naturally occurring low-titer anti-Tn activity may be of prophylactic value, as suggested by the in vitro neutralization found in this study....

  5. Broadly neutralizing antibodies targeted to mucin-type carbohydrate epitopes of human immunodeficiency virus

    DEFF Research Database (Denmark)

    Hansen, J E; Nielsen, C; Arendrup, M;

    1991-01-01

    . This inhibition was found in infection of both lymphocytic cells and monocytoid cells. Viruses tested included six HIV-1 and five HIV-2 isolates propagated in different cells, as well as infectious plasma from AIDS patients. The antiviral effect of anti-Tn MAbs occurred by specific binding of the MAb to the virus......The cancer-related mucin-type carbohydrate neoantigen Tn was found on gp160 and gp120 of human immunodeficiency virus (HIV). Immunoglobulin G (IgG) and IgM monoclonal antibodies (MAbs) against Tn neutralized infection with cell-free virus and blocked fusion between HIV-infected and uninfected cells...

  6. Precisely Molded Nanoparticle Displaying DENV-E Proteins Induces Robust Serotype-Specific Neutralizing Antibody Responses

    Science.gov (United States)

    Hoekstra, Gabriel; Yi, Xianwen; Stone, Michelle; Horvath, Katie; Miley, Michael J.; DeSimone, Joseph; Luft, Chris J.; de Silva, Aravinda M.

    2016-01-01

    Dengue virus (DENV) is the causative agent of dengue fever and dengue hemorrhagic fever. The virus is endemic in over 120 countries, causing over 350 million infections per year. Dengue vaccine development is challenging because of the need to induce simultaneous protection against four antigenically distinct DENV serotypes and evidence that, under some conditions, vaccination can enhance disease due to specific immunity to the virus. While several live-attenuated tetravalent dengue virus vaccines display partial efficacy, it has been challenging to induce balanced protective immunity to all 4 serotypes. Instead of using whole-virus formulations, we are exploring the potentials for a particulate subunit vaccine, based on DENV E-protein displayed on nanoparticles that have been precisely molded using Particle Replication in Non-wetting Template (PRINT) technology. Here we describe immunization studies with a DENV2-nanoparticle vaccine candidate. The ectodomain of DENV2-E protein was expressed as a secreted recombinant protein (sRecE), purified and adsorbed to poly (lactic-co-glycolic acid) (PLGA) nanoparticles of different sizes and shape. We show that PRINT nanoparticle adsorbed sRecE without any adjuvant induces higher IgG titers and a more potent DENV2-specific neutralizing antibody response compared to the soluble sRecE protein alone. Antigen trafficking indicate that PRINT nanoparticle display of sRecE prolongs the bio-availability of the antigen in the draining lymph nodes by creating an antigen depot. Our results demonstrate that PRINT nanoparticles are a promising platform for delivering subunit vaccines against flaviviruses such as dengue and Zika. PMID:27764114

  7. The effects of somatic hypermutation on neutralization and binding in the PGT121 family of broadly neutralizing HIV antibodies.

    Directory of Open Access Journals (Sweden)

    Devin Sok

    Full Text Available Broadly neutralizing HIV antibodies (bnAbs are typically highly somatically mutated, raising doubts as to whether they can be elicited by vaccination. We used 454 sequencing and designed a novel phylogenetic method to model lineage evolution of the bnAbs PGT121-134 and found a positive correlation between the level of somatic hypermutation (SHM and the development of neutralization breadth and potency. Strikingly, putative intermediates were characterized that show approximately half the mutation level of PGT121-134 but were still capable of neutralizing roughly 40-80% of PGT121-134 sensitive viruses in a 74-virus panel at median titers between 15- and 3-fold higher than PGT121-134. Such antibodies with lower levels of SHM may be more amenable to elicitation through vaccination while still providing noteworthy coverage. Binding characterization indicated a preference of inferred intermediates for native Env binding over monomeric gp120, suggesting that the PGT121-134 lineage may have been selected for binding to native Env at some point during maturation. Analysis of glycan-dependent neutralization for inferred intermediates identified additional adjacent glycans that comprise the epitope and suggests changes in glycan dependency or recognition over the course of affinity maturation for this lineage. Finally, patterns of neutralization of inferred bnAb intermediates suggest hypotheses as to how SHM may lead to potent and broad HIV neutralization and provide important clues for immunogen design.

  8. Identification and Characterization of a Broadly Cross-Reactive HIV-1 Human Monoclonal Antibody That Binds to Both gp120 and gp41

    NARCIS (Netherlands)

    Zhang, Mei-Yun; Yuan, Tingting; Li, Jingjing; Borges, Andrew Rosa; Watkins, Jennifer D.; Guenaga, Javier; Yang, Zheng; Wang, Yanping; Wilson, Richard; Li, Yuxing; Polonis, Victoria R.; Pincus, Seth H.; Ruprecht, Ruth M.; Dimitrov, Dimiter S.

    2012-01-01

    Identification of broadly cross-reactive HIV-1-neutralizing antibodies (bnAbs) may assist vaccine immunogen design. Here we report a novel human monoclonal antibody (mAb), designated m43, which co-targets the gp120 and gp41 subunits of the HIV-1 envelope glycoprotein (Env). M43 bound to recombinant

  9. Broadly cross-reactive antibodies dominate the human B cell response against 2009 pandemic H1N1 influenza virus infection

    OpenAIRE

    2011-01-01

    The 2009 pandemic H1N1 influenza pandemic demonstrated the global health threat of reassortant influenza strains. Herein, we report a detailed analysis of plasmablast and monoclonal antibody responses induced by pandemic H1N1 infection in humans. Unlike antibodies elicited by annual influenza vaccinations, most neutralizing antibodies induced by pandemic H1N1 infection were broadly cross-reactive against epitopes in the hemagglutinin (HA) stalk and head domain of multiple influenza strains. T...

  10. Triosephosphate isomerase of Taenia solium (TTPI): phage display and antibodies as tools for finding target regions to inhibit catalytic activity.

    Science.gov (United States)

    Sanabria-Ayala, Víctor; Belmont, Iaraset; Abraham, Landa

    2015-01-01

    Previous studies demonstrated that antibodies against triosephosphate isomerase of Taenia solium (TTPI) can alter its enzymatic catalysis. In the present study, we used antibodies produced against the NH2-terminal region of TTPI (1/3NH2TTPI) and the phage display technology to find target regions to inhibit TTPI activity. As a first step, we obtained polyclonal antibodies against non-conserved regions from the 1/3NH2TTPI, which had an inhibitory effect of about 74 % on catalytic activity. Afterward, they were used to screen a library of phage-displayed dodecapeptides; as a result, 41 phage mimotope clones were isolated and grouped according to their amino acid sequence, finding the consensus A1 (VPTXPI), A2 (VPTXXI), B (LTPGQ), and D (DPLPR). Antibodies against selected phage mimotope clones were obtained by rabbit's immunization; these ones clearly recognized TTPI by both Western blot and ELISA. However, only the mimotope PDTS16 (DSVTPTSVMAVA) clone, which belongs to the VPTXXI consensus, raised antibodies capable of inhibiting the TTPI catalytic activity in 45 %. Anti-PDTS16 antibodies were confronted to several synthetic peptides that encompass the 1/3NH2TTPI, and they only recognized three, which share the motif FDTLQK belonging to the helix-α1 in TTPI. This suggests that this motif is the main part of the epitope recognized by anti-PDTS16 antibodies and revealed its importance for TTPI catalysis.

  11. Direct administration in the respiratory tract improves efficacy of broadly neutralizing anti-influenza virus monoclonal antibodies.

    Science.gov (United States)

    Leyva-Grado, Victor H; Tan, Gene S; Leon, Paul E; Yondola, Mark; Palese, Peter

    2015-07-01

    The emergence of influenza virus strains resistant to approved neuraminidase inhibitors and the time constrains after infection when these drugs can be effective constitute major drawbacks for this class of drugs. This highlights a critical need to discover new therapeutic agents that can be used for the treatment of influenza virus-infected patients. The use of broadly neutralizing anti-influenza monoclonal antibodies (MAbs) has been sought as an alternative immunotherapy against influenza infection. Here, we tested in mice previously characterized broadly neutralizing anti-hemagglutinin (HA) stalk MAbs prophylactically and therapeutically using different routes of administration. The efficacy of treatment against an influenza H1N1 pandemic virus challenge was compared between two systemic routes of administration, intraperitoneal (i.p.) and intravenous (i.v.), and two local routes, intranasal (i.n.) and aerosol (a.e.). The dose of MAb required for prophylactic protection was reduced by 10-fold in animals treated locally (i.n. or a.e.) compared with those treated systemically (i.p. or i.v.). Improved therapeutic protection was observed in animals treated i.n. on day 5 postinfection (60% survival) compared with those treated via the i.p. route (20% survival). An increase in therapeutic efficacy against other influenza virus subtypes (H5N1) was also observed when a local route of administration was used. Our findings demonstrate that local administration significantly decreases the amount of broadly neutralizing monoclonal antibody required for protection against influenza, which highlights the potential use of MAbs as a therapeutic agent for influenza-associated disease.

  12. A simplified procedure for antibody engineering by yeast surface display: Coupling display levels and target binding by ribosomal skipping.

    Science.gov (United States)

    Grzeschik, Julius; Hinz, Steffen C; Könning, Doreen; Pirzer, Thomas; Becker, Stefan; Zielonka, Stefan; Kolmar, Harald

    2017-02-01

    Yeast surface display is a valuable, widely used method for protein engineering. However, current yeast display applications rely on the staining of epitope tags in order to verify full-length presentation of the protein of interest on the cell surface. We aimed at developing a modified yeast display approach that relies on ribosomal skipping, thereby enabling the translation of two proteins from one open reading frame and, in that manner, generating an intracellular fluorescence signal. This improved setup is based on a 2A sequence that is encoded between the protein to be displayed and a gene for green fluorescent protein (GFP). The intracellular GFP fluorescence signal of yeast cells correlates with full-length protein presentation and omits the need for the immunofluorescence detection of epitope tags. For method validation, shark-derived IgNAR variable domains (vNAR) were subjected to affinity maturation using the 2A-GFP system. Yeast library screening of full-length vNAR variants which were detected via GFP expression yielded the same high-affinity binder that had previously been isolated by our group using the conventional epitope tag-based display format. The presented method obviates the need for additional immunofluorescence cell staining, offering an easy and cost-friendly alternative to conventional epitope tag detections.

  13. Production and radioimmunoimaging of novel fully human phage display recombinant antibodies and growth inhibition of lung adenocarcinoma cell line overexpressing Prx I.

    Science.gov (United States)

    Luo, Yi; Pang, Hua; Li, Shujie; Cao, Hui; Peng, Zhiping; Fan, Chunbo; Li, Shaolin

    2009-07-01

    The Peroxiredoxin I (Prx I) is a member of the Peroxiredoxin family, which is overexpressed in many diverse tumor types and is an anti-apoptosis protein for tumor cell proliferation and survival. Therapeutic strategies targeting the Prx I may therefore be effective broad-spectrum anticancer agents. We constructed a phage display single-chain variable fragment (scFv) antibody library and sieve out the fully human, lung adenocarcinoma-sepcific monoclonal antibodies. The selection on Prx I was performed using above-mentioned lung adenocarcinoma-sepcific monoclonal antibodies with high affinity to Prx I overexpressing lung adenocarcinoma cells. The candidate scFv sequences, based on enzyme-linked immunosorbent assay (ELISA) screening data, were chosen for soluble expression, and a 30 kDa band was observed on polyacrylamide gel electrophoresis as predicted. The purified antibodies were characterized by immunoblotting and showed high specificity to Prx I-overexpressing lung adenocarcinoma cells A549. Radioimmunoimaging was taken to evaluate specificity and distribution of antibodies in vivo. The radiolocalization index (RI) of tumor/serum and tumor/muscle gradually increased, reaching its peak (4.06 +/- 0.13 and 5.17 +/- 0.97, respectively) at 48 h postadministration. Single photon emission computed tomography (SPECT) imaging showed the radioactivity was aggregated in tumor locations and tumor imaging was clearly observed. The internalized scFv resulted in antibody-mediated cell apoptosis and downregulation of Prx I expression. These results demonstrate that the scFv possesses strong antitumor activity on lung adenocarcinoma and may therefore be an effective therapeutic candidate for the treatment of cancers that are dependent on Prx I for growth and survival.

  14. Protection against Clostridium difficile infection with broadly neutralizing antitoxin monoclonal antibodies.

    Science.gov (United States)

    Marozsan, Andre J; Ma, Dangshe; Nagashima, Kirsten A; Kennedy, Brian J; Kang, Yun Kenneth; Arrigale, Robert R; Donovan, Gerald P; Magargal, Wells W; Maddon, Paul J; Olson, William C

    2012-09-01

    The spore-forming bacterium Clostridium difficile represents the principal cause of hospital-acquired diarrhea and pseudomembranous colitis worldwide. C. difficile infection (CDI) is mediated by 2 bacterial toxins, A and B; neutralizing these toxins with monoclonal antibodies (mAbs) provides a potential nonantibiotic strategy for combating the rising prevalence, severity, and recurrence of CDI. Novel antitoxin mAbs were generated in mice and were humanized. The humanized antitoxin A mAb PA-50 and antitoxin B mAb PA-41 have picomolar potencies in vitro and bind to novel regions of the respective toxins. In a hamster model for CDI, 95% of animals treated with a combination of humanized PA-50 and PA-41 showed long-term survival relative to 0% survival of animals treated with standard antibiotics or comparator mAbs. These humanized mAbs provide insight into C. difficile intoxication and hold promise as potential nonantibiotic agents for improving clinical management of CDI.

  15. A broadly neutralizing human monoclonal antibody is effective against H7N9

    Science.gov (United States)

    Tharakaraman, Kannan; Subramanian, Vidya; Viswanathan, Karthik; Sloan, Susan; Yen, Hui-Ling; Barnard, Dale L.; Leung, Y. H. Connie; Szretter, Kristy J.; Koch, Tyree J.; Delaney, James C.; Babcock, Gregory J.; Wogan, Gerald N.; Sasisekharan, Ram; Shriver, Zachary

    2015-01-01

    Emerging strains of influenza represent a significant public health threat with potential pandemic consequences. Of particular concern are the recently emerged H7N9 strains which cause pneumonia with acute respiratory distress syndrome. Estimates are that nearly 80% of hospitalized patients with H7N9 have received intensive care unit support. VIS410, a human antibody, targets a unique conserved epitope on influenza A. We evaluated the efficacy of VIS410 for neutralization of group 2 influenza strains, including H3N2 and H7N9 strains in vitro and in vivo. VIS410, administered at 50 mg/kg, protected DBA mice infected with A/Anhui/2013 (H7N9), resulting in significant survival benefit upon single-dose (−24 h) or double-dose (−12 h, +48 h) administration (P H7N9) resulted in significant decreased lung viral load (P = 0.002) and decreased lung cytokine responses for nine of the 11 cytokines measured. Based on these results, we find that VIS410 may be effective either as monotherapy or combined with antivirals in treating H7N9 disease, as well as disease from other influenza strains. PMID:26283346

  16. Phage display used for gene cloning of human recombinant antibody against the erythrocyte surface antigen, rhesus D

    DEFF Research Database (Denmark)

    Dziegiel, M; Nielsen, L K; Andersen, P S;

    1995-01-01

    A novel phage display system has been developed for PCR amplification and cloning of the Fab fragments of human immunoglobulin genes. Using this system, we have cloned an antibody from a mouse-human hybridoma cell line directed against the erythrocyte antigen rhesus D. Intact erythrocytes were used...

  17. Phage display used for gene cloning of human recombinant antibody against the erythrocyte surface antigen, rhesus D

    DEFF Research Database (Denmark)

    Dziegiel, M; Nielsen, L K; Andersen, P S

    1995-01-01

    A novel phage display system has been developed for PCR amplification and cloning of the Fab fragments of human immunoglobulin genes. Using this system, we have cloned an antibody from a mouse-human hybridoma cell line directed against the erythrocyte antigen rhesus D. Intact erythrocytes were us...

  18. Broadly-Reactive Neutralizing and Non-neutralizing Antibodies Directed against the H7 Influenza Virus Hemagglutinin Reveal Divergent Mechanisms of Protection.

    Science.gov (United States)

    Tan, Gene S; Leon, Paul E; Albrecht, Randy A; Margine, Irina; Hirsh, Ariana; Bahl, Justin; Krammer, Florian

    2016-04-01

    In the early spring of 2013, Chinese health authorities reported several cases of H7N9 influenza virus infections in humans. Since then the virus has established itself at the human-animal interface in Eastern China and continues to cause several hundred infections annually. In order to characterize the antibody response to the H7N9 virus we generated several mouse monoclonal antibodies against the hemagglutinin of the A/Shanghai/1/13 (H7N9) virus. Of particular note are two monoclonal antibodies, 1B2 and 1H5, that show broad reactivity to divergent H7 hemagglutinins. Monoclonal antibody 1B2 binds to viruses of the Eurasian and North American H7 lineages and monoclonal antibody 1H5 reacts broadly to virus isolates of the Eurasian lineage. Interestingly, 1B2 shows broad hemagglutination inhibiting and neutralizing activity, while 1H5 fails to inhibit hemagglutination and demonstrates no neutralizing activity in vitro. However, both monoclonal antibodies were highly protective in an in vivo passive transfer challenge model in mice, even at low doses. Experiments using mutant antibodies that lack the ability for Fc/Fc-receptor and Fc/complement interactions suggest that the protection provided by mAb 1H5 is, at least in part, mediated by the Fc-fragment of the mAb. These findings highlight that a protective response to a pathogen may not only be due to neutralizing antibodies, but can also be the result of highly efficacious non-neutralizing antibodies not readily detected by classical in vitro neutralization or hemagglutination inhibition assays. This is of interest because H7 influenza virus vaccines induce only low hemagglutination inhibiting antibody titers while eliciting robust antibody titers as measured by ELISA. Our data suggest that these binding but non-neutralizing antibodies contribute to protection in vivo.

  19. Broadly-Reactive Neutralizing and Non-neutralizing Antibodies Directed against the H7 Influenza Virus Hemagglutinin Reveal Divergent Mechanisms of Protection.

    Directory of Open Access Journals (Sweden)

    Gene S Tan

    2016-04-01

    Full Text Available In the early spring of 2013, Chinese health authorities reported several cases of H7N9 influenza virus infections in humans. Since then the virus has established itself at the human-animal interface in Eastern China and continues to cause several hundred infections annually. In order to characterize the antibody response to the H7N9 virus we generated several mouse monoclonal antibodies against the hemagglutinin of the A/Shanghai/1/13 (H7N9 virus. Of particular note are two monoclonal antibodies, 1B2 and 1H5, that show broad reactivity to divergent H7 hemagglutinins. Monoclonal antibody 1B2 binds to viruses of the Eurasian and North American H7 lineages and monoclonal antibody 1H5 reacts broadly to virus isolates of the Eurasian lineage. Interestingly, 1B2 shows broad hemagglutination inhibiting and neutralizing activity, while 1H5 fails to inhibit hemagglutination and demonstrates no neutralizing activity in vitro. However, both monoclonal antibodies were highly protective in an in vivo passive transfer challenge model in mice, even at low doses. Experiments using mutant antibodies that lack the ability for Fc/Fc-receptor and Fc/complement interactions suggest that the protection provided by mAb 1H5 is, at least in part, mediated by the Fc-fragment of the mAb. These findings highlight that a protective response to a pathogen may not only be due to neutralizing antibodies, but can also be the result of highly efficacious non-neutralizing antibodies not readily detected by classical in vitro neutralization or hemagglutination inhibition assays. This is of interest because H7 influenza virus vaccines induce only low hemagglutination inhibiting antibody titers while eliciting robust antibody titers as measured by ELISA. Our data suggest that these binding but non-neutralizing antibodies contribute to protection in vivo.

  20. Generation of Potent Anti-Vascular Endothelial Growth Factor Neutralizing Antibodies from Mouse Phage Display Library for Cancer Therapy

    Directory of Open Access Journals (Sweden)

    Yan-Da Lai

    2016-02-01

    Full Text Available Vascular endothelial growth factor (VEGF is an important stimulator for angiogenesis in solid tumors. Blocking VEGF activity is an effective therapeutic strategy to inhibit tumor growth and metastasis. Avastin, a humanized monoclonal antibody recognizes VEGF, has been approved by the US Food and Drug Administration. To generate potential VEGF-recognizing antibodies with better tumor regression ability than that of Avastin, we have designed a systematic antibody selection plan. From mice immunized with recombinant human VEGF, we generated three phage display libraries, scFv-M13KO7, Fab-M13KO7, and scFv-Hyperphage, in single-chain Fv (scFv or Fab format, displayed using either M13KO7 helper phage or Hyperphage. Solid-phase and solution-phase selection strategies were then applied to each library, generating six panning combinations. A total of sixty-four antibodies recognizing VEGF were obtained. Based on the results of epitope mapping, binding affinity, and biological functions in tumor inhibition, eight antibodies were chosen to examine their abilities in tumor regression in a mouse xenograft model using human COLO 205 cancer cells. Three of them showed improvement in the inhibition of tumor growth (328%–347% tumor growth ratio (% of Day 0 tumor volume on Day 21 vs. 435% with Avastin. This finding suggests a potential use of these three antibodies for VEGF-targeted therapy.

  1. Broad-range neutralizing anti-influenza A human monoclonal antibodies: new perspectives in therapy and prophylaxis.

    Science.gov (United States)

    Clementi, Nicola; Criscuolo, Elena; Castelli, Matteo; Clementi, Massimo

    2012-10-01

    Broadly neutralizing monoclonal antibodies (mAbs) directed against different subtypes of influenza A viruses are novel tools for the potential development of effective anti-influenza prophylactic and therapeutic strategies. In both cases, the main candidates for passive transfer and new vaccine development are represented by protective mAbs directed against influenza hemagglutinin (HA). A large number of mAbs directed against influenza HA has been developed to date. However, even if they can be useful and contribute to develop new vaccinal strategies, only few of them can be a good candidate for human administration. In this review, we will describe the most relevant human mAb directed against influenza HA able to recognize highly divergent influenza isolates and possibly useful for human therapy and prophylaxis.

  2. Binding of inferred germline precursors of broadly neutralizing HIV-1 antibodies to native-like envelope trimers.

    Science.gov (United States)

    Sliepen, Kwinten; Medina-Ramírez, Max; Yasmeen, Anila; Moore, John P; Klasse, Per Johan; Sanders, Rogier W

    2015-12-01

    HIV-1 envelope glycoproteins (Env) and Env-based immunogens usually do not interact efficiently with the inferred germline precursors of known broadly neutralizing antibodies (bNAbs). This deficiency may be one reason why Env and Env-based immunogens are not efficient at inducing bNAbs. We evaluated the binding of 15 inferred germline precursors of bNAbs directed to different epitope clusters to three soluble native-like SOSIP.664 Env trimers. We found that native-like SOSIP.664 trimers bind to some inferred germline precursors of bNAbs, particularly ones involving the V1/V2 loops at the apex of the trimer. The data imply that native-like SOSIP.664 trimers will be an appropriate platform for structure-guided design improvements intended to create immunogens able to target the germline precursors of bNAbs.

  3. A Strategy for Generating a Broad-Spectrum Monoclonal Antibody and Soluble Single-Chain Variable Fragments against Plant Potyviruses.

    Science.gov (United States)

    Liu, Han-Lin; Lin, Wei-Fang; Hu, Wen-Chi; Lee, Yung-An; Chang, Ya-Chun

    2015-10-01

    Potyviruses are major pathogens that often cause mixed infection in calla lilies. To reduce the time and cost of virus indexing, a detection method for the simultaneous targeting of multiple potyviruses was developed by generating a broad-spectrum monoclonal antibody (MAb) for detecting the greatest possible number of potyviruses. The conserved 121-amino-acid core regions of the capsid proteins of Dasheen mosaic potyvirus (DsMV), Konjak mosaic potyvirus (KoMV), and Zantedeschia mild mosaic potyvirus (ZaMMV) were sequentially concatenated and expressed as a recombinant protein for immunization. After hybridoma cell fusion and selection, one stable cell line that secreted a group-specific antibody, named C4 MAb, was selected. In the reaction spectrum test, the C4 MAb detected at least 14 potyviruses by indirect enzyme-linked immunosorbent assay (I-ELISA) and Western blot analysis. Furthermore, the variable regions of the heavy (VH) and light (VL) chains of the C4 MAb were separately cloned and constructed as single-chain variable fragments (scFvs) for expression in Escherichia coli. Moreover, the pectate lyase E (PelE) signal peptide of Erwinia chrysanthemi S3-1 was added to promote the secretion of C4 scFvs into the medium. According to Western blot analysis and I-ELISA, the soluble C4 scFv (VL-VH) fragment showed a binding specificity similar to that of the C4 MAb. Our results demonstrate that a recombinant protein derived from fusion of the conserved regions of viral proteins has the potential to produce a broad-spectrum MAb against a large group of viruses and that the PelE signal peptide can improve the secretion of scFvs in E. coli.

  4. Safety, pharmacokinetics and neutralization of the broadly neutralizing HIV-1 human monoclonal antibody VRC01 in healthy adults.

    Science.gov (United States)

    Ledgerwood, J E; Coates, E E; Yamshchikov, G; Saunders, J G; Holman, L; Enama, M E; DeZure, A; Lynch, R M; Gordon, I; Plummer, S; Hendel, C S; Pegu, A; Conan-Cibotti, M; Sitar, S; Bailer, R T; Narpala, S; McDermott, A; Louder, M; O'Dell, S; Mohan, S; Pandey, J P; Schwartz, R M; Hu, Z; Koup, R A; Capparelli, E; Mascola, J R; Graham, B S

    2015-12-01

    VRC-HIVMAB060-00-AB (VRC01) is a broadly neutralizing HIV-1 monoclonal antibody (mAb) isolated from the B cells of an HIV-infected patient. It is directed against the HIV-1 CD4 binding site and is capable of potently neutralizing the majority of diverse HIV-1 strains. This Phase I dose-escalation study in healthy adults was conducted at the National Institutes of Health (NIH) Clinical Center (Bethesda, MD, USA). Primary objectives were the safety, tolerability and pharmacokinetics (PK) of VRC01 intravenous (i.v.) infusion at 5, 20 or 40 mg/kg, given either once (20 mg/kg) or twice 28 days apart (all doses), and of subcutaneous (s.c.) delivery at 5 mg/kg compared to s.c. placebo given twice, 28 days apart. Cumulatively, 28 subjects received 43 VRC01 and nine received placebo administrations. There were no serious adverse events or dose-limiting toxicities. Mean 28-day serum trough concentrations after the first infusion were 35 and 57 μg/ml for groups infused with 20 mg/kg (n = 8) and 40 mg/kg (n = 5) doses, respectively. Mean 28-day trough concentrations after the second infusion were 56 and 89 μg/ml for the same two doses. Over the 5-40 mg/kg i.v. dose range (n = 18), the clearance was 0.016 l/h and terminal half-life was 15 days. After infusion VRC01 retained expected neutralizing activity in serum, and anti-VRC01 antibody responses were not detected. The human monoclonal antibody (mAb) VRC01 was well tolerated when delivered i.v. or s.c. The mAb demonstrated expected half-life and pharmacokinetics for a human immunoglobulin G. The safety and PK results support and inform VRC01 dosing schedules for planning HIV-1 prevention efficacy studies.

  5. Antibody Complementarity-Determining Regions (CDRs) Can Display Differential Antimicrobial, Antiviral and Antitumor Activities

    Science.gov (United States)

    Polonelli, Luciano; Pontón, José; Elguezabal, Natalia; Moragues, María Dolores; Casoli, Claudio; Pilotti, Elisabetta; Ronzi, Paola; Dobroff, Andrey S.; Rodrigues, Elaine G.; Juliano, Maria A.; Maffei, Domenico Leonardo; Magliani, Walter; Conti, Stefania; Travassos, Luiz R.

    2008-01-01

    Background Complementarity-determining regions (CDRs) are immunoglobulin (Ig) hypervariable domains that determine specific antibody (Ab) binding. We have shown that synthetic CDR-related peptides and many decapeptides spanning the variable region of a recombinant yeast killer toxin-like antiidiotypic Ab are candidacidal in vitro. An alanine-substituted decapeptide from the variable region of this Ab displayed increased cytotoxicity in vitro and/or therapeutic effects in vivo against various bacteria, fungi, protozoa and viruses. The possibility that isolated CDRs, represented by short synthetic peptides, may display antimicrobial, antiviral and antitumor activities irrespective of Ab specificity for a given antigen is addressed here. Methodology/Principal Findings CDR-based synthetic peptides of murine and human monoclonal Abs directed to: a) a protein epitope of Candida albicans cell wall stress mannoprotein; b) a synthetic peptide containing well-characterized B-cell and T-cell epitopes; c) a carbohydrate blood group A substance, showed differential inhibitory activities in vitro, ex vivo and/or in vivo against C. albicans, HIV-1 and B16F10-Nex2 melanoma cells, conceivably involving different mechanisms of action. Antitumor activities involved peptide-induced caspase-dependent apoptosis. Engineered peptides, obtained by alanine substitution of Ig CDR sequences, and used as surrogates of natural point mutations, showed further differential increased/unaltered/decreased antimicrobial, antiviral and/or antitumor activities. The inhibitory effects observed were largely independent of the specificity of the native Ab and involved chiefly germline encoded CDR1 and CDR2 of light and heavy chains. Conclusions/Significance The high frequency of bioactive peptides based on CDRs suggests that Ig molecules are sources of an unlimited number of sequences potentially active against infectious agents and tumor cells. The easy production and low cost of small sized synthetic

  6. Antibody complementarity-determining regions (CDRs can display differential antimicrobial, antiviral and antitumor activities.

    Directory of Open Access Journals (Sweden)

    Luciano Polonelli

    Full Text Available BACKGROUND: Complementarity-determining regions (CDRs are immunoglobulin (Ig hypervariable domains that determine specific antibody (Ab binding. We have shown that synthetic CDR-related peptides and many decapeptides spanning the variable region of a recombinant yeast killer toxin-like antiidiotypic Ab are candidacidal in vitro. An alanine-substituted decapeptide from the variable region of this Ab displayed increased cytotoxicity in vitro and/or therapeutic effects in vivo against various bacteria, fungi, protozoa and viruses. The possibility that isolated CDRs, represented by short synthetic peptides, may display antimicrobial, antiviral and antitumor activities irrespective of Ab specificity for a given antigen is addressed here. METHODOLOGY/PRINCIPAL FINDINGS: CDR-based synthetic peptides of murine and human monoclonal Abs directed to: a a protein epitope of Candida albicans cell wall stress mannoprotein; b a synthetic peptide containing well-characterized B-cell and T-cell epitopes; c a carbohydrate blood group A substance, showed differential inhibitory activities in vitro, ex vivo and/or in vivo against C. albicans, HIV-1 and B16F10-Nex2 melanoma cells, conceivably involving different mechanisms of action. Antitumor activities involved peptide-induced caspase-dependent apoptosis. Engineered peptides, obtained by alanine substitution of Ig CDR sequences, and used as surrogates of natural point mutations, showed further differential increased/unaltered/decreased antimicrobial, antiviral and/or antitumor activities. The inhibitory effects observed were largely independent of the specificity of the native Ab and involved chiefly germline encoded CDR1 and CDR2 of light and heavy chains. CONCLUSIONS/SIGNIFICANCE: The high frequency of bioactive peptides based on CDRs suggests that Ig molecules are sources of an unlimited number of sequences potentially active against infectious agents and tumor cells. The easy production and low cost of small

  7. Isolation of anti-toxin single domain antibodies from a semi-synthetic spiny dogfish shark display library

    Directory of Open Access Journals (Sweden)

    Goldman Ellen R

    2007-11-01

    Full Text Available Abstract Background Shark heavy chain antibody, also called new antigen receptor (NAR, consists of one single Variable domain (VH, containing only two complementarity-determining regions (CDRs. The antigen binding affinity and specificity are mainly determined by these two CDRs. The good solubility, excellent thermal stability and complex sequence variation of small single domain antibodies (sdAbs make them attractive alternatives to conventional antibodies. In this report, we construct and characterize a diversity enhanced semi-synthetic NAR V display library based on naturally occurring NAR V sequences. Results A semi-synthetic shark sdAb display library with a complexity close to 1e9 was constructed. This was achieved by introducing size and sequence variations in CDR3 using randomized CDR3 primers of three different lengths. Binders against three toxins, staphylococcal enterotoxin B (SEB, ricin, and botulinum toxin A (BoNT/A complex toxoid, were isolated from panning the display library. Soluble sdAbs from selected binders were purified and evaluated using direct binding and thermal stability assays on the Luminex 100. In addition, sandwich assays using sdAb as the reporter element were developed to demonstrate their utility for future sensor applications. Conclusion We demonstrated the utility of a newly created hyper diversified shark NAR displayed library to serve as a source of thermal stable sdAbs against a variety of toxins.

  8. Affinity isolation of antigen-specific circulating B cells for generation of phage display-derived human monoclonal antibodies

    DEFF Research Database (Denmark)

    Ditzel, Henrik

    2009-01-01

    A method is described for affinity isolation of antigen-specific circulating B cells of interest for subsequent generation of immune antibody phage display libraries. This approach should overcome the problem of low yields of monoclonal antibodies of interest in the libraries generated from...... peripheral blood lymphocytes caused by the low abundance of antigen-specific B cells in the circulation. The preselection of B cells is based on the specificity of the surface Ig receptor and is accomplished using the antigen of interest conjugated to magnetic beads. This method should significantly increase...

  9. Epitope Mapping of Dengue-Virus-Enhancing Monoclonal-Antibody Using Phage Display Peptide Library

    OpenAIRE

    Chung-I Rai; Huan-Yao Lei; Yee-Shin Lin; Hsiao-Sheng Liu; Shun-Hua Chen; Lien-Cheng Chen; Trai-Ming Yeh

    2008-01-01

    The Antibody-Dependent Enhancement (ADE) hypothesis has been proposed to explain why more severe manifestations of Dengue Hemorrhagic Fever and Dengue Shock Syndrome (DHF/DSS) occur predominantly during secondary infections of Dengue Virus (DV) with different serotypes. However, the epitopes recognized by these enhancing antibodies are unclear. Recently, anti-pre-M monoclonal antibody (mAb 70-21), which recognized all DV serotypes without neutralizing activity, were generated and demonstrated...

  10. PRODUCTION OF PHAGE-DISPLAYED ANTI-IDIOTYPIC ANTIBODY SINGLE CHAIN VARIABLE FRAGMENTS TO MG7 MONOCLONAL ANTIBODY DIRECTED AGAINST GASTRIC CARCINOMA

    Institute of Scientific and Technical Information of China (English)

    何凤田; 聂勇战; 陈宝军; 乔太东; 韩者艺; 樊代明

    2002-01-01

    Objective. To generate phage-displayed anti-idiotypic antibody single chain variable fragments (anti -Id ScFv) to MG7 monoclonal antibody (McAb) directed against gastric carcinoma so as to lay a foundation for developing anti-Id ScFv vaccine of the cancer.Methods. Balb/c mice were immunized i. p. with MG7 McAb conjugated with keyhole limpet hemocyanin (KLH), and mRNA was isolated from the spleens of the immunized mice. Heavy and light chain (VH and VL)genes of antibody were amplified separately and assembled into ScFv genes with a linker DNA by PCR. The ScFv genes were ligated into the phagemid vector pCANTAB5E and the ligated sample was transformed into competent E. coli TG1. The transformants were infected with M13KO7 helper phage to yield recombinant phages displaying ScFv on the tips of M13 phage. After 4 rounds of panning with MG7, the MG7-positive clones were selected by ELISA from the enriched phages. Thetypesoftheanti-IdScFvdisplayedontheselectedphagecloneswerepreliminarily identified by competition ELISA.Results. The VH, VL and ScFv DNAs were about 340 bp, 320 bp and 750 bp respectively. Twenty-four MG7-positive clones were selected from 60 enriched phage clones, among which 5 displayed β or γtype anti-Id ScFv.Conclsion. The anti-Id ScFv to MG7 McAb can be successfully selected by recombinant phage antibody technique, which paves a way for the study of prevention and cure of gastric carcinoma by using anti-Id ScFv.

  11. Identification of the specificity of isolated phage display single-chain antibodies using yeast two-hybrid screens

    DEFF Research Database (Denmark)

    Rasmussen, Nicolaj; Ditzel, Henrik

    2009-01-01

    A method is described for the identification of the antigen recognised by an scFv isolated from an antibody phage display library using selection against a complex mixture of proteins (e.g. intact cells, purified cell surface membranes, and tissue sections). The method takes advantage of a yeast...... two-hybrid system that additionally allows for reorganization of post-translational modifications to the bait and target proteins. This technique is therefore especially useful for identifying surface-expressed antigens....

  12. 噬菌体抗体库筛选技术%Screening Isolates from Antibody Phage-display Libraries

    Institute of Scientific and Technical Information of China (English)

    高鹏; 胡立勇; 钱钰; 徐晨

    2012-01-01

    For almost 20 years, phage display antibody (Ab) libraries screening have been widely used in antibody screen, clinical diagnostic, treatment of disease and basic research areas. Rapid identification of the autofit monoclonal antibody, and reliable and efficient data management and analysis are very important in the application of this technology. This review described reported methods for high-throughput screening of antibody phage-display libraries and further more gave a brief introduction to data management.%在过去的20年中,噬菌体抗体库筛选技术被广泛的应用于抗体筛选、疾病治疗,临床诊断以及基础研究之中.在该技术的应用过程中,快速有效的筛选出最适合的单克隆抗体并进行可靠和高效的数据管理和分析是十分重要的.文中总结了近年来噬菌体抗体库的高通量筛选方法并且对数据管理做了简要介绍.

  13. Broadly Neutralizing Hemagglutinin Stalk-Specific Antibodies Induce Potent Phagocytosis of Immune Complexes by Neutrophils in an Fc-Dependent Manner

    Directory of Open Access Journals (Sweden)

    Caitlin E. Mullarkey

    2016-10-01

    Full Text Available Broadly neutralizing antibodies that recognize the conserved hemagglutinin (HA stalk have emerged as exciting new biotherapeutic tools to combat seasonal and pandemic influenza viruses. Our general understanding of the mechanisms by which stalk-specific antibodies achieve protection is rapidly evolving. It has recently been demonstrated that broadly neutralizing HA stalk-specific IgG antibodies require Fc-Fcγ receptor (FcγR interactions for optimal protection in vivo. Here we examine the neutrophil effector functions induced by stalk-specific antibodies. As the most abundant subset of blood leukocytes, neutrophils represent a critical innate effector cell population and serve an instrumental role in orchestrating downstream adaptive responses to influenza virus infection. Yet, the interplay of HA stalk-specific IgG, Fc-FcγR engagement, and neutrophils has remained largely uncharacterized. Using an in vitro assay to detect the production of reactive oxygen species (ROS, we show that human and mouse monoclonal HA stalk-specific IgG antibodies are able to induce the production of ROS by neutrophils, while HA head-specific antibodies do not. Furthermore, our results indicate that the production of ROS is dependent on Fc receptor (FcR engagement and phagocytosis. We went on to assess the ability of monoclonal HA stalk-specific IgA antibodies to induce ROS. Consistent with our findings for monoclonal IgGs, only HA stalk-specific IgA antibodies elicited ROS production by neutrophils. This induction is dependent on the engagement of FcαR1. Taken together, our findings describe a novel FcR-dependent effector function induced by HA stalk-specific IgG and IgA antibodies, and importantly, our studies shed light on the mechanisms by which HA stalk-specific antibodies achieve protection.

  14. Broadly Neutralizing Hemagglutinin Stalk-Specific Antibodies Induce Potent Phagocytosis of Immune Complexes by Neutrophils in an Fc-Dependent Manner.

    Science.gov (United States)

    Mullarkey, Caitlin E; Bailey, Mark J; Golubeva, Diana A; Tan, Gene S; Nachbagauer, Raffael; He, Wenqian; Novakowski, Kyle E; Bowdish, Dawn M; Miller, Matthew S; Palese, Peter

    2016-10-04

    Broadly neutralizing antibodies that recognize the conserved hemagglutinin (HA) stalk have emerged as exciting new biotherapeutic tools to combat seasonal and pandemic influenza viruses. Our general understanding of the mechanisms by which stalk-specific antibodies achieve protection is rapidly evolving. It has recently been demonstrated that broadly neutralizing HA stalk-specific IgG antibodies require Fc-Fcγ receptor (FcγR) interactions for optimal protection in vivo Here we examine the neutrophil effector functions induced by stalk-specific antibodies. As the most abundant subset of blood leukocytes, neutrophils represent a critical innate effector cell population and serve an instrumental role in orchestrating downstream adaptive responses to influenza virus infection. Yet, the interplay of HA stalk-specific IgG, Fc-FcγR engagement, and neutrophils has remained largely uncharacterized. Using an in vitro assay to detect the production of reactive oxygen species (ROS), we show that human and mouse monoclonal HA stalk-specific IgG antibodies are able to induce the production of ROS by neutrophils, while HA head-specific antibodies do not. Furthermore, our results indicate that the production of ROS is dependent on Fc receptor (FcR) engagement and phagocytosis. We went on to assess the ability of monoclonal HA stalk-specific IgA antibodies to induce ROS. Consistent with our findings for monoclonal IgGs, only HA stalk-specific IgA antibodies elicited ROS production by neutrophils. This induction is dependent on the engagement of FcαR1. Taken together, our findings describe a novel FcR-dependent effector function induced by HA stalk-specific IgG and IgA antibodies, and importantly, our studies shed light on the mechanisms by which HA stalk-specific antibodies achieve protection.

  15. A broadly neutralizing anti-influenza antibody reveals ongoing capacity of haemagglutinin-specific memory B cells to evolve

    Science.gov (United States)

    Fu, Ying; Zhang, Zhen; Sheehan, Jared; Avnir, Yuval; Ridenour, Callie; Sachnik, Thomas; Sun, Jiusong; Hossain, M. Jaber; Chen, Li-Mei; Zhu, Quan; Donis, Ruben O.; Marasco, Wayne A.

    2016-01-01

    Understanding the natural evolution and structural changes involved in broadly neutralizing antibody (bnAb) development holds great promise for improving the design of prophylactic influenza vaccines. Here we report an haemagglutinin (HA) stem-directed bnAb, 3I14, isolated from human memory B cells, that utilizes a heavy chain encoded by the IGHV3-30 germline gene. MAb 3I14 binds and neutralizes groups 1 and 2 influenza A viruses and protects mice from lethal challenge. Analysis of VH and VL germline back-mutants reveals binding to H3 and H1 but not H5, which supports the critical role of somatic hypermutation in broadening the bnAb response. Moreover, a single VLD94N mutation improves the affinity of 3I14 to H5 by nearly 10-fold. These data provide evidence that memory B cell evolution can expand the HA subtype specificity. Our results further suggest that establishing an optimized memory B cell pool should be an aim of ‘universal' influenza vaccine strategies. PMID:27619409

  16. Development of two murine antibodies against Neospora caninum using phage display technology and application on the detection of N. caninum.

    Directory of Open Access Journals (Sweden)

    Jinhua Dong

    Full Text Available Neosporosis, caused by an intracellular parasite, Neospora caninum, is an infectious disease primarily of cattle and dogs. It occurs worldwide and causes huge damages to dairy farms. In this study, we immunized mice with recombinant surface-associated protein 1 of N. caninum (rNcSAG1 and developed two novel monoclonal antibodies, A10 and H3, against NcSAG1 using phage-display technology. Both clones bound to purified rNcSAG1 and the half maximal inhibitory concentrations of A10 and H3 are 50 and 72 nM of rNcSAG1, respectively. In immunofluorescence assays, both A10 and H3 Fabs bound to N. caninum parasites. Direct detection of N. caninum parasites was developed firstly using an enzyme-linked immunosorbent assay (ELISA with A10 and H3. Binding of A10 and H3 antibodies to rNcSAG1 was also inhibited by some certain anti-N. caninum antibodies in the neosporosis-positive cattle sera, suggesting they might bind to the same epitopes of NcSAG1 with those anti-N. caninum antibodies of bovine. These antibodies were demonstrated to have a potential for monitoring the N. caninum parasites in a dairy farm, which may lead to protect livestock from parasite-infection.

  17. Development of an immunoaffinity column method using broad-specificity monoclonal antibodies for simultaneous extraction and cleanup of quinolone and sulfonamide antibiotics in animal muscle tissues

    Science.gov (United States)

    This paper describes a novel mixed-bed immunoaffinity column (IAC) method. The IAC was produced by coupling anti-fluoroquinolone and anti-sulfonamide broad-specificity monoclonal antibodies (Mabs) to Sepharose 4B for simultaneously isolating 13 fluoroquinolones (FQs) and 6 sulfonamides (SAs) from s...

  18. Rational Design and Characterization of the Novel, Broad and Potent Bispecific HIV-1 Neutralizing Antibody iMabm36

    Science.gov (United States)

    Sun, Ming; Pace, Craig S.; Yao, Xin; Yu, Faye; Padte, Neal N.; Huang, Yaoxing; Seaman, Michael S.; Li, Qihan; Ho, David D.

    2014-01-01

    While broadly neutralizing monoclonal antibodies (bNAbs) have always been considered potential therapeutic options for the prophylactic and treatment of HIV infection, their lack of breadth against all HIV variants has been one of the limiting factors. To provide sufficient neutralization breadth and potency against diverse viruses, including neutralization escape variants, strategies to combine different bNAbs have been explored recently. We rationally designed and engineered a novel bispecific HIV-1 neutralizing antibody (bibNAb), iMabm36, for high potency and breadth against HIV. iMabm36 is composed of the anti-CD4 Ab ibalizumab (iMab) linked to two copies of the single-domain Ab m36 which targets a highly conserved CD4-induced epitope. iMabm36 neutralizes a majority of a large, multi-clade panel of pseudoviruses (96%, n=118) at an IC50 concentration of less than 10 μg/mL, with 83% neutralized at an IC50 concentration of less than 0.1μg/ml. In addition, iMabm36 neutralizes six replication-competent transmitted-founder viruses to 100% inhibition at a concentration of less than 0.1μg/ml in a PBMC-based neutralizing assay. Mechanistically, improved antiviral activity of iMabm36 is dependent on both CD4 binding activity of iMab component and CD4i binding activity of the m36 component. After characterizing viral resistance to iMabm36 neutralization was due to mutations residing in the bridging sheet of gp120, an optimized m36 variant was engineered that, when fused to iMab, improved antiviral activity significantly. Together inter-dependency of this dual mechanism of action enables iMabm36 to potently inhibit HIV-1 entry. These results demonstrate that mechanistic-based design of bibNAbs could generate potential preventive and therapeutic candidates for HIV/AIDS. PMID:24853313

  19. Epimedium koreanum Nakai Displays Broad Spectrum of Antiviral Activity in Vitro and in Vivo by Inducing Cellular Antiviral State

    Directory of Open Access Journals (Sweden)

    Won-Kyung Cho

    2015-01-01

    Full Text Available Epimedium koreanum Nakai has been extensively used in traditional Korean and Chinese medicine to treat a variety of diseases. Despite the plant’s known immune modulatory potential and chemical make-up, scientific information on its antiviral properties and mode of action have not been completely investigated. In this study, the broad antiviral spectrum and mode of action of an aqueous extract from Epimedium koreanum Nakai was evaluated in vitro, and moreover, the protective effect against divergent influenza A subtypes was determined in BALB/c mice. An effective dose of Epimedium koreanum Nakai markedly reduced the replication of Influenza A Virus (PR8, Vesicular Stomatitis Virus (VSV, Herpes Simplex Virus (HSV and Newcastle Disease Virus (NDV in RAW264.7 and HEK293T cells. Mechanically, we found that an aqueous extract from Epimedium koreanum Nakai induced the secretion of type I IFN and pro-inflammatory cytokines and the subsequent stimulation of the antiviral state in cells. Among various components present in the extract, quercetin was confirmed to have striking antiviral properties. The oral administration of Epimedium koreanum Nakai exhibited preventive effects on BALB/c mice against lethal doses of highly pathogenic influenza A subtypes (H1N1, H5N2, H7N3 and H9N2. Therefore, an extract of Epimedium koreanum Nakai and its components play roles as immunomodulators in the innate immune response, and may be potential candidates for prophylactic or therapeutic treatments against diverse viruses in animal and humans.

  20. Functional characterization of a monoclonal antibody epitope using a lambda phage display-deep sequencing platform

    Science.gov (United States)

    Domina, Maria; Lanza Cariccio, Veronica; Benfatto, Salvatore; Venza, Mario; Venza, Isabella; Borgogni, Erica; Castellino, Flora; Midiri, Angelina; Galbo, Roberta; Romeo, Letizia; Biondo, Carmelo; Masignani, Vega; Teti, Giuseppe; Felici, Franco; Beninati, Concetta

    2016-01-01

    We have recently described a method, named PROFILER, for the identification of antigenic regions preferentially targeted by polyclonal antibody responses after vaccination. To test the ability of the technique to provide insights into the functional properties of monoclonal antibody (mAb) epitopes, we used here a well-characterized epitope of meningococcal factor H binding protein (fHbp), which is recognized by mAb 12C1. An fHbp library, engineered on a lambda phage vector enabling surface expression of polypeptides of widely different length, was subjected to massive parallel sequencing of the phage inserts after affinity selection with the 12C1 mAb. We detected dozens of unique antibody-selected sequences, the most enriched of which (designated as FrC) could largely recapitulate the ability of fHbp to bind mAb 12C1. Computational analysis of the cumulative enrichment of single amino acids in the antibody-selected fragments identified two overrepresented stretches of residues (H248-K254 and S140-G154), whose presence was subsequently found to be required for binding of FrC to mAb 12C1. Collectively, these results suggest that the PROFILER technology can rapidly and reliably identify, in the context of complex conformational epitopes, discrete “hot spots” with a crucial role in antigen-antibody interactions, thereby providing useful clues for the functional characterization of the epitope. PMID:27530334

  1. Human peripheral blood monocytes display surface antigens recognized by monoclonal antinuclear antibodies

    Energy Technology Data Exchange (ETDEWEB)

    Holers, V.M.; Kotzin, B.L.

    1985-09-01

    The authors used monoclonal anti-nuclear autoantibodies and indirect immunofluorescence to examine normal human peripheral blood mononuclear leukocytes for the presence of cell surface nuclear antigens. Only one monoclonal anti-histone antibody (MH-2) was found to bind to freshly isolated PBL, staining approximately 10% of large cells. However, after cells were placed into culture for 16-24 h, a high percentage (up to 60%) of large-sized cells were recognized by an anti-DNA (BWD-1) and several different antihistone monoclonal antibodies (BWH-1, MH-1, and MH-2). These antibodies recognize separate antigenic determinants on chromatin and histones extracted from chromatin. The histone antigen-positive cells were viable, and the monoclonal antibodies could be shown to be binding to the cell surface and not to the nucleus. Using monoclonal antibodies specific for monocytes and T cells, and complement-mediated cytotoxicity, the cells bearing histone antigens were shown to be primarily monocytes. The appearance of histone and DNA antigen-positive cells was nearly completely inhibited by the addition of low concentrations of cycloheximide at initiation of the cultures. In contrast, little effect on the percentage of positive cells was detected if cells were exposed to high doses of gamma irradiation before culture. These data further support the existence of cell surface nuclear antigens on selected cell subsets, which may provide insight into the immunopathogenesis of systemic lupus erythematosus and related autoimmune diseases.

  2. Modeling neutralization kinetics of HIV by broadly neutralizing monoclonal antibodies in genital secretions coating the cervicovaginal mucosa.

    Directory of Open Access Journals (Sweden)

    Scott A McKinley

    Full Text Available Eliciting broadly neutralizing antibodies (bnAb in cervicovaginal mucus (CVM represents a promising "first line of defense" strategy to reduce vaginal HIV transmission. However, it remains unclear what levels of bnAb must be present in CVM to effectively reduce infection. We approached this complex question by modeling the dynamic tally of bnAb coverage on HIV. This analysis introduces a critical, timescale-dependent competition: to protect, bnAb must accumulate at sufficient stoichiometry to neutralize HIV faster than virions penetrate CVM and reach target cells. We developed a model that incorporates concentrations and diffusivities of HIV and bnAb in semen and CVM, kinetic rates for binding (kon and unbinding (koff of select bnAb, and physiologically relevant thicknesses of CVM and semen layers. Comprehensive model simulations lead to robust conclusions about neutralization kinetics in CVM. First, due to the limited time virions in semen need to penetrate CVM, substantially greater bnAb concentrations than in vitro estimates must be present in CVM to neutralize HIV. Second, the model predicts that bnAb with more rapid kon, almost independent of koff, should offer greater neutralization potency in vivo. These findings suggest the fastest arriving virions at target cells present the greatest likelihood of infection. It also implies the marked improvements in in vitro neutralization potency of many recently discovered bnAb may not translate to comparable reduction in the bnAb dose needed to confer protection against initial vaginal infections. Our modeling framework offers a valuable tool to gaining quantitative insights into the dynamics of mucosal immunity against HIV and other infectious diseases.

  3. Schistosoma japonicum:construction of phage display antibody library and its application in the immunodiagnosis of infection

    Institute of Scientific and Technical Information of China (English)

    陈代雄; 何蔼; 詹希美; 俞慕华; 雷智刚; 孟锦绣; 李卓雅; 梁瑜; 张瑞琳

    2004-01-01

    Background A monoclonal antibody would be an effective tool for the detection of circulating antigens in the serum of patients with schistosomiasis, but the traditional way of producing monoclonal antibodies is not cost-effective. The objective of this study was to find a new method for the large-scale production of monoclonal antibodies against Schistosoma japonicum (Sj).Methods A phage display antibody library for Sj was constructed. To obtain a single-chain variable fragment antibody (scFv) against Sj, the library was screened with metabolic antigens from adult Sj worms (Sj-MAg) using enzyme-linked immunosorbent assay. The soluble scFvs selected were used to detect Sj antigens in the serum of acute and chronic schistosomiasis patients.Results Six positive clones with good reactivity to Sj-MAg were obtained from the phage display antibody library of about 1.07×106 individual clones. Only two of these six clones bound specifically to Sj-MAg and were chosen for further analysis. Specific soluble anti-Sj-MAg scFvs were produced by inducing the 2 clones with isopropyl-D-thiogalactopyranoside. The characteristics of the scFvs were then determined. The results of Western blot showed that these scFvs could bind to Sj-MAg specifically and had a molecular weight of about 31 kD. When testing serum from schistosomiasis patients with one of the two specific scFvs, its sensitivity was found to be 60% and 37% in acute and chronic patients, respectively, with a specificity of 90%. When the two specific scFvs were combined, their sensitivity was found to be 75% and 57% in acute and chronic patients, respectively, with a specificity of 85%.Conclusions The results indicate that the scFvs are potentially useful for the diagnosis of schistosomiasis. The library construction also provides a useful tool for the further screening of other antibodies for both diagnostic and immunotherapeutic applications and for epitope analysis and vaccine design.

  4. Phage display used for gene cloning of human recombinant antibody against the erythrocyte surface antigen, rhesus D

    DEFF Research Database (Denmark)

    Dziegiel, Morten Hanefeld; Nielsen, L K; Andersen, P S

    1995-01-01

    A novel phage display system has been developed for PCR amplification and cloning of the Fab fragments of human immunoglobulin genes. Using this system, we have cloned an antibody from a mouse-human hybridoma cell line directed against the erythrocyte antigen rhesus D. Intact erythrocytes were used...... for absorption of the Fab phages. Soluble Fab fragments produced from the cloned material showed identical performance to the parental antibody in agglutination assays. Gel filtration confirmed that the Fab fragment consists of a kappa-Fd heterodimer. The successful use of intact cells for selection of specific...... Fab phages demonstrates that it is possible to by-pass purification of the antigen of interest. Comparison with published germline sequences demonstrated that the immunoglobulin coding regions had the highest homology to the VH 1.9III and V kappa Hum kappa v325 germline genes, respectively....

  5. Application of phage display in selecting Tomato spotted wilt virus - specific single-chain antibodies (scFvs) for sensitive diagnosis in ELISA

    NARCIS (Netherlands)

    Griep, R.A.; Prins, M.; Twisk, van C.; Keller, H.J.H.G.; Kerschbaumer, R.J.; Kormelink, R.; Goldbach, R.W.; Schots, A.

    2000-01-01

    A panel of recombinant single-chain antibodies (scFvs) against structural proteins of Tomato spotted wilt virus (TSWV) was retrieved from a human combinatorial scFv antibody library using the novel phage display technique. After subcloning the encoding DNA sequences in the expression vector pSKAP/S,

  6. Multimeric scaffolds displaying the HIV-1 envelope MPER induce MPER-specific antibodies and cross-neutralizing antibodies when co-immunized with gp160 DNA.

    Directory of Open Access Journals (Sweden)

    Shelly J Krebs

    Full Text Available Developing a vaccine that overcomes the diversity of HIV-1 is likely to require a strategy that directs antibody (Ab responses toward conserved regions of the viral Envelope (Env. However, the generation of neutralizing Abs (NAbs targeting these regions through vaccination has proven to be difficult. One conserved region of particular interest is the membrane proximal external region (MPER of Env located within the gp41 ectodomain. In order to direct the immune response to this region, the MPER and gp41 ectodomain were expressed separately as N-terminal fusions to the E2 protein of Geobacillus stearothermophilus. The E2 protein acts as a scaffold by self-assembling into 60-mer particles, displaying up to 60 copies of the fused target on the surface. Rabbits were immunized with E2 particles displaying MPER and/or the gp41 ectodomain in conjunction with DNA encoding full-length gp160. Only vaccines including E2 particles displaying MPER elicited MPER-specific Ab responses. NAbs were elicited after two immunizations that largely targeted the V3 loop. To overcome V3 immunodominance in the DNA component, E2 particles displaying MPER were used in conjunction with gp160 DNA lacking hypervariable regions V2, V3, or combined V1V2V3. All rabbits had HIV binding Ab responses and NAbs following the second vaccination. Using HIV-2/HIV-1 MPER chimeric viruses as targets, NAbs were detected in 12/16 rabbits after three immunizations. Low levels of NAbs specific for Tier 1 and 2 viruses were observed in all groups. This study provides evidence that co-immunizing E2 particles displaying MPER and gp160 DNA can focus Ab responses toward conserved regions of Env.

  7. Antibody VH and VL recombination using phage and ribosome display technologies reveals distinct structural routes to affinity improvements with VH-VL interface residues providing important structural diversity.

    Science.gov (United States)

    Groves, Maria A T; Amanuel, Lily; Campbell, Jamie I; Rees, D Gareth; Sridharan, Sudharsan; Finch, Donna K; Lowe, David C; Vaughan, Tristan J

    2014-01-01

    In vitro selection technologies are an important means of affinity maturing antibodies to generate the optimal therapeutic profile for a particular disease target. Here, we describe the isolation of a parent antibody, KENB061 using phage display and solution phase selections with soluble biotinylated human IL-1R1. KENB061 was affinity matured using phage display and targeted mutagenesis of VH and VL CDR3 using NNS randomization. Affinity matured VHCDR3 and VLCDR3 library blocks were recombined and selected using phage and ribosome display protocol. A direct comparison of the phage and ribosome display antibodies generated was made to determine their functional characteristics.In our analyses, we observed distinct differences in the pattern of beneficial mutations in antibodies derived from phage and ribosome display selections, and discovered the lead antibody Jedi067 had a ~3700-fold improvement in KD over the parent KENB061. We constructed a homology model of the Fv region of Jedi067 to map the specific positions where mutations occurred in the CDR3 loops. For VL CDR3, positions 94 to 97 carry greater diversity in the ribosome display variants compared with the phage display. The positions 95a, 95b and 96 of VLCDR3 form part of the interface with VH in this model. The model shows that positions 96, 98, 100e, 100f, 100 g, 100h, 100i and 101 of the VHCDR3 include residues at the VH and VL interface. Importantly, Leu96 and Tyr98 are conserved at the interface positions in both phage and ribosome display indicating their importance in maintaining the VH-VL interface. For antibodies derived from ribosome display, there is significant diversity at residues 100a to 100f of the VH CDR3 compared with phage display. A unique deletion of isoleucine at position 102 of the lead candidate, Jedi067, also occurs in the VHCDR3.As anticipated, recombining the mutations via ribosome display led to a greater structural diversity, particularly in the heavy chain CDR3, which in turn

  8. Flow-Cytometric Isolation of Human Antibodies from a Nonimmune Saccharomyces cerevisiae Surface Display Library

    Energy Technology Data Exchange (ETDEWEB)

    Feldhaus, Michael (BATTELLE (PACIFIC NW LAB)); Siegel, Robert W.(BATTELLE (PACIFIC NW LAB)); Opresko, Lee (BATTELLE (PACIFIC NW LAB)); Coleman, James R.(BATTELLE (PACIFIC NW LAB)); Feldhaus, Jane M.(BATTELLE (PACIFIC NW LAB)); Yeung, Yik A.(Massachusetts Institute Of Tec); Cochran, Jennifer R.(Massachusetts Institute Of Tec); Heinzelman, Peter (Massachusetts Institute Of Tec); Colby, David (Massachusetts Institute Of Tec); Swers, Jeffrey (Massachusetts Institute Of Tec); Graff, Christilyn (Massachusetts Institute Of Tec); Wiley, H Steven (BATTELLE (PACIFIC NW LAB)); Wittrup, K D.(Massachusetts Institute Of Tec)

    2003-02-28

    A nonimmune library of 109 human antibody scFv fragments has been cloned and expressed on the surface of yeast, and nanomolar-affinity scFvs routinely obtained by magnetic bead screening and flow cytometric sorting. The yeast library can be amplified 1010-fold without measurable loss of clonal diversity, enabling effectively indefinite expansion of the library. The expression, stability, and antigen binding properties of more than 50 isolated scFv clones were assessed directly on the yeast cell surface by immunofluorescent labeling and flow cytometry, obviating separate subcloning, expression, and purification steps and thereby expediting the isolation of novel affinity reagents. The ability to use multiplex library screening demonstrates the utility of this approach for high throughput antibody isolation for proteomics applications.

  9. A constant threat for HIV: Fc-engineering to enhance broadly neutralizing antibody activity for immunotherapy of the acquired immunodeficiency syndrome.

    Science.gov (United States)

    Nimmerjahn, Falk

    2015-08-01

    Passive immunotherapy with polyclonal or hyperimmune serum immunoglobulin G (IgG) preparations provides an efficient means of protecting immunocompromised patients from microbial infections. More recently, the use of passive immunotherapy to prevent or to treat established infections with the human immunodeficiency virus (HIV) has gained much attention, due to promising preclinical data obtained in monkey and humanized mouse in vivo model systems, demonstrating that the transfer of HIV-specific antibodies can not only prevent HIV infection, but also diminish virus load during chronic infection. Furthermore, an array of broadly neutralizing HIV-specific antibodies has become available and the importance of the IgG constant region as a critical modulator of broadly neutralizing activity has been demonstrated. The aim of this review is to summarize the most recent findings with regard to the molecular and cellular mechanisms responsible for antibody-mediated clearance of HIV infection, and to discuss how this may help to improve HIV therapy via optimizing Fcγ-receptor-dependent activities of HIV-specific antibodies.

  10. Development of single chain variable fragment (scFv) antibodies against Xylella fastidiosa subsp. pauca by phage display.

    Science.gov (United States)

    Yuan, Qing; Jordan, Ramon; Brlansky, Ronald H; Istomina, Olga; Hartung, John

    2015-10-01

    Xylella fastidiosa is a member of the gamma proteobacteria. It is fastidious, insect-vectored and xylem-limited and causes a variety of diseases, some severe, on a wide range of economically important perennial crops, including grape and citrus. Antibody based detection assays are commercially available for X. fastidiosa, and are effective at the species, but not at the subspecies level. We have made a library of scFv antibody fragments directed against X. fastidiosa subsp. pauca strain 9a5c (citrus) by using phage display technology. Antibody gene repertoires were PCR-amplified using 23 primers for the heavy chain variable region (V(H)) and 21 primers for the light chain variable region (V(L)). The V(H) and V(L) were joined by overlap extension PCR, and then the genes of the scFv library were ligated into the phage vector pKM19. The library contained 1.2×10(7) independent clones with full-length scFv inserts. In each of 3cycles of affinity-selection with 9a5c, about 1.0×10(12) phage were used for panning with 4.1×10(6), 7.1×10(6), 2.1×10(7) phage recovered after the first, second and third cycles, respectively. Sixty-six percent of clones from the final library bound X. fastidiosa 9a5c in an ELISA. Some of these scFv antibodies recognized strain 9a5c and did not recognize X. fastidiosa strains that cause Pierce's disease of grapevine.

  11. Effective Optimization of Antibody Affinity by Phage Display Integrated with High-Throughput DNA Synthesis and Sequencing Technologies.

    Directory of Open Access Journals (Sweden)

    Dongmei Hu

    Full Text Available Phage display technology has been widely used for antibody affinity maturation for decades. The limited library sequence diversity together with excessive redundancy and labour-consuming procedure for candidate identification are two major obstacles to widespread adoption of this technology. We hereby describe a novel library generation and screening approach to address the problems. The approach started with the targeted diversification of multiple complementarity determining regions (CDRs of a humanized anti-ErbB2 antibody, HuA21, with a small perturbation mutagenesis strategy. A combination of three degenerate codons, NWG, NWC, and NSG, were chosen for amino acid saturation mutagenesis without introducing cysteine and stop residues. In total, 7,749 degenerate oligonucleotides were synthesized on two microchips and released to construct five single-chain antibody fragment (scFv gene libraries with 4 x 10(6 DNA sequences. Deep sequencing of the unselected and selected phage libraries using the Illumina platform allowed for an in-depth evaluation of the enrichment landscapes in CDR sequences and amino acid substitutions. Potent candidates were identified according to their high frequencies using NGS analysis, by-passing the need for the primary screening of target-binding clones. Furthermore, a subsequent library by recombination of the 10 most abundant variants from four CDRs was constructed and screened, and a mutant with 158-fold increased affinity (Kd = 25.5 pM was obtained. These results suggest the potential application of the developed methodology for optimizing the binding properties of other antibodies and biomolecules.

  12. Straightforward selection of broadly neutralizing single-domain antibodies targeting the conserved CD4 and coreceptor binding sites of HIV-1 gp120.

    Science.gov (United States)

    Matz, Julie; Kessler, Pascal; Bouchet, Jérôme; Combes, Olivier; Ramos, Oscar Henrique Pereira; Barin, Francis; Baty, Daniel; Martin, Loïc; Benichou, Serge; Chames, Patrick

    2013-01-01

    Few broadly neutralizing antibodies targeting determinants of the HIV-1 surface envelope glycoprotein (gp120) involved in sequential binding to host CD4 and chemokine receptors have been characterized. While these epitopes show low diversity among various isolates, HIV-1 employs many strategies to evade humoral immune response toward these sensitive sites, including a carbohydrate shield, low accessibility to these buried cavities, and conformational masking. Using trimeric gp140, free or bound to a CD4 mimic, as immunogens in llamas, we selected a panel of broadly neutralizing single-domain antibodies (sdAbs) that bind to either the CD4 or the coreceptor binding site (CD4BS and CoRBS, respectively). When analyzed as monomers or as homo- or heteromultimers, the best sdAb candidates could not only neutralize viruses carrying subtype B envelopes, corresponding to the Env molecule used for immunization and selection, but were also efficient in neutralizing a broad panel of envelopes from subtypes A, C, G, CRF01_AE, and CRF02_AG, including tier 3 viruses. Interestingly, sdAb multimers exhibited a broader neutralizing activity spectrum than the parental sdAb monomers. The extreme stability and high recombinant production yield combined with their broad neutralization capacity make these sdAbs new potential microbicide candidates for HIV-1 transmission prevention.

  13. Transfected Cell Microarrays for the Expression of Membrane-Displayed Single-Chain Antibodies

    Science.gov (United States)

    2011-01-01

    T4 DNA ligase (1 U/μL) and 5× ligation buffer (Invitro- gen) were stored at –20◦C for up to 6 months (see Note 6). 10. Ligation reaction buffer...the need to purify the dephosphory- lated vector. 6. T4 DNA ligase is unstable on ice for long periods of time. It is best to keep the enzyme at –20◦C...Delehanty 10. The digested, cleaned PCR products (7 μl) were ligated to 50 ng of pDisplay vector also digested with SfiI and SalI with 1U T4 DNA

  14. Broadly neutralizing antibody PGT121 allosterically modulates CD4 binding via recognition of the HIV-1 gp120 V3 base and multiple surrounding glycans.

    Directory of Open Access Journals (Sweden)

    Jean-Philippe Julien

    Full Text Available New broad and potent neutralizing HIV-1 antibodies have recently been described that are largely dependent on the gp120 N332 glycan for Env recognition. Members of the PGT121 family of antibodies, isolated from an African donor, neutralize ∼70% of circulating isolates with a median IC50 less than 0.05 µg ml(-1. Here, we show that three family members, PGT121, PGT122 and PGT123, have very similar crystal structures. A long 24-residue HCDR3 divides the antibody binding site into two functional surfaces, consisting of an open face, formed by the heavy chain CDRs, and an elongated face, formed by LCDR1, LCDR3 and the tip of the HCDR3. Alanine scanning mutagenesis of the antibody paratope reveals a crucial role in neutralization for residues on the elongated face, whereas the open face, which accommodates a complex biantennary glycan in the PGT121 structure, appears to play a more secondary role. Negative-stain EM reconstructions of an engineered recombinant Env gp140 trimer (SOSIP.664 reveal that PGT122 interacts with the gp120 outer domain at a more vertical angle with respect to the top surface of the spike than the previously characterized antibody PGT128, which is also dependent on the N332 glycan. We then used ITC and FACS to demonstrate that the PGT121 antibodies inhibit CD4 binding to gp120 despite the epitope being distal from the CD4 binding site. Together, these structural, functional and biophysical results suggest that the PGT121 antibodies may interfere with Env receptor engagement by an allosteric mechanism in which key structural elements, such as the V3 base, the N332 oligomannose glycan and surrounding glycans, including a putative V1/V2 complex biantennary glycan, are conformationally constrained.

  15. Broad antibody mediated cross-neutralization and preclinical immunogenicity of new codon-optimized HIV-1 clade CRF02_AG and G primary isolates.

    Directory of Open Access Journals (Sweden)

    Simon M Agwale

    Full Text Available Creation of an effective vaccine for HIV has been an elusive goal of the scientific community for almost 30 years. Neutralizing antibodies are assumed to be pivotal to the success of a prophylactic vaccine but previous attempts to make an immunogen capable of generating neutralizing antibodies to primary "street strain" isolates have resulted in responses of very limited breadth and potency. The objective of the study was to determine the breadth and strength of neutralizing antibodies against autologous and heterologous primary isolates in a cohort of HIV-1 infected Nigerians and to characterize envelopes from subjects with particularly broad or strong immune responses for possible use as vaccine candidates in regions predominated by HIV-1 CRF02_AG and G subtypes. Envelope vectors from a panel of primary Nigerian isolates were constructed and tested with plasma/sera from the same cohort using the PhenoSense HIV neutralizing antibody assay (Monogram Biosciences Inc, USA to assess the breadth and potency of neutralizing antibodies. The immediate goal of this study was realized by the recognition of three broadly cross-neutralizing sera: (NG2-clade CRF02_AG, NG3-clade CRF02_AG and NG9- clade G. Based on these findings, envelope gp140 sequences from NG2 and NG9, complemented with a gag sequence (Clade G and consensus tat (CRF02_AG and G antigens have been codon-optimized, synthesized, cloned and evaluated in BALB/c mice. The intramuscular administration of these plasmid DNA constructs, followed by two booster DNA immunizations, induced substantial specific humoral response against all constructs and strong cellular responses against the gag and tat constructs. These preclinical findings provide a framework for the design of candidate vaccine for use in regions where the HIV-1 epidemic is driven by clades CRF02_AG and G.

  16. Analysis of pectic epitopes recognised by hybridoma and phage display monoclonal antibodies using defined oligosaccharides, polysaccharides, and enzymatic degradation

    DEFF Research Database (Denmark)

    Willats, William George Tycho; Limberg, G.; Buchholt, H.C.;

    2000-01-01

    The structure of epitopes recognised by anti-pectin monoclonal antibodies (mAbs) has been investigated using a series of model lime-pectin samples with defined degrees and patterns of methyl esterification, a range of defined oligogalacturonides and enzymatic degradation of pectic polysaccharides....... In immuno-dot-assays, the anti-homogalacturonan (HG) mAbs JIM5 and JIM7 both bound to samples with a wide range of degrees of methyl esterification in preference to fully de-esterified samples. In contrast, the anti-HG phage display mAb PAM1 bound most effectively to fully de-esterified pectin...... occurs where specific but undefined methyl-esterification patterns are present on HG domains, although fully de-esterified HG samples contain sub-optimal JIM5 epitopes. The persistence of mAb binding to epitopes in pectic antigens, with 41% blockwise esterification (P41) and 43% random esterification (F...

  17. Antibody constant region peptides can display immunomodulatory activity through activation of the Dectin-1 signalling pathway.

    Directory of Open Access Journals (Sweden)

    Elena Gabrielli

    Full Text Available We previously reported that a synthetic peptide with sequence identical to a CDR of a mouse monoclonal antibody specific for difucosyl human blood group A exerted an immunomodulatory activity on murine macrophages. It was therapeutic against systemic candidiasis without possessing direct candidacidal properties. Here we demonstrate that a selected peptide, N10K, putatively deriving from the enzymatic cleavage of the constant region (Fc of human IgG(1, is able to induce IL-6 secretion and pIkB-α activation. More importantly, it causes an up-regulation of Dectin-1 expression. This leads to an increased activation of β-glucan-induced pSyk, CARD9 and pIkB-α, and an increase in the production of pro-inflammatory cytokines such as IL-6, IL-12, IL-1β and TNF-α. The increased activation of this pathway coincides with an augmented phagocytosis of non opsonized Candida albicans cells by monocytes. The findings suggest that some Fc-peptides, potentially deriving from the proteolysis of immunoglobulins, may cause an unexpected immunoregulation in a way reminiscent of innate immunity molecules.

  18. Insight into screening immunoglobulin gene combinatorial libraries in a phage display vector: a tale of two antibodies.

    Science.gov (United States)

    Kakinuma, A; Portolano, S; Chazenbalk, G; Rapoport, B; McLachlan, S M

    1997-01-01

    Combinatorial libraries of immunoglobulin genes in "phage display" vectors are a powerful tool for obtaining antigen-specific antibody fragments. To date, this approach has been used to isolate abundant, but not rare, human autoantibodies of IgG class. We have compared the relative efficiencies of panning pComb3 libraries made from intrathyroidal plasma cells for abundant human autoantibodies to thyroid peroxidase (TPO) and rare autoantibodies to the thyrotropin receptor (TSHR). TPO-specific Fab were readily obtained from a library using three different forms of recombinant antigen, (i) purified TPO, (ii) impure TPO in culture medium and, (iii) TPO expressed on the surface of CHO cells. In contrast, TSHR-specific Fab were not isolated. This was the case despite repeated pannings of six libraries from three optimal patients (IgG/kappa and IgG/lambda libraries for each patient). Both purified recombinant TSHR and CHO cells expressing TSHR on their surface were used. Library enrichment was observed on some screenings. However, Fab expressed by individual clones or from enriched libraries were not specific as determined by (i) binding to purified, radio-labeled antigen, (ii) FACS analysis of TSHR on intact CHO cells and, (iii) inhibition of radiolabeled TSH binding. Remarkably, in screening for both TPO- and TSHR-specific Fab, neither library enrichment nor the retention of cDNA inserts of the correct size correlated with obtaining Fab with the antigenic specificity sought. Indeed, excellent enrichment could be observed with conditioned medium from untransfected cells. Our data suggest that the key to isolating rare antibodies from phage display libraries is not the creation of vast libraries of greater diversity or even the development of more stable vectors. Rather, success in this endeavor appears to require reducing the "noise" of non-specific clones in a moderately sized library.

  19. In vitro neutralisation of rotavirus infection by two broadly specific recombinant monovalent llama-derived antibody fragments

    NARCIS (Netherlands)

    F. Aladin (Farah); A.W.C. Einerhand (Sandra); J. Bouma (Janneke); S. Bezemer (Sandra); P. Hermans (Pim); D. Wolvers (Danielle); K. Bellamy (Kate); L.G.J. Frenken (Leon); J. Gray (Jim); M. Iturriza-Gómara (Miren)

    2012-01-01

    textabstractRotavirus is the main cause of viral gastroenteritis in young children. Therefore, the development of inexpensive antiviral products for the prevention and/or treatment of rotavirus disease remains a priority. Previously we have shown that a recombinant monovalent antibody fragment (refe

  20. Structure of HIV-1 gp120 V1/V2 domain with broadly neutralizing antibody PG9

    Energy Technology Data Exchange (ETDEWEB)

    McLellan, Jason S.; Pancera, Marie; Carrico, Chris; Gorman, Jason; Julien, Jean-Philippe; Khayat, Reza; Louder, Robert; Pejchal, Robert; Sastry, Mallika; Dai, Kaifan; O’Dell, Sijy; Patel, Nikita; Shahzad-ul-Hussan, Syed; Yang, Yongping; Zhang, Baoshan; Zhou, Tongqing; Zhu, Jiang; Boyington, Jeffrey C.; Chuang, Gwo-Yu; Diwanji, Devan; Georgiev, Ivelin; Kwon, Young Do; Lee, Doyung; Louder, Mark K.; Moquin, Stephanie; Schmidt, Stephen D.; Yang, Zhi-Yong; Bonsignori, Mattia; Crump, John A.; Kapiga, Saidi H.; Sam, Noel E.; Haynes, Barton F.; Burton, Dennis R.; Koff, Wayne C.; Walker, Laura M.; Phogat, Sanjay; Wyatt, Richard; Orwenyo, Jared; Wang, Lai-Xi; Arthos, James; Bewley, Carole A.; Mascola, John R.; Nabel, Gary J.; Schief, William R.; Ward, Andrew B.; Wilson, Ian A.; Kwong, Peter D. (UWASH); (NIH); (Scripps); (Duke); (IAVI); (Maryland-MED)

    2012-12-13

    Variable regions 1 and 2 (V1/V2) of human immunodeficiency virus-1 (HIV-1) gp120 envelope glycoprotein are critical for viral evasion of antibody neutralization, and are themselves protected by extraordinary sequence diversity and N-linked glycosylation. Human antibodies such as PG9 nonetheless engage V1/V2 and neutralize 80% of HIV-1 isolates. Here we report the structure of V1/V2 in complex with PG9. V1/V2 forms a four-stranded {beta}-sheet domain, in which sequence diversity and glycosylation are largely segregated to strand-connecting loops. PG9 recognition involves electrostatic, sequence-independent and glycan interactions: the latter account for over half the interactive surface but are of sufficiently weak affinity to avoid autoreactivity. The structures of V1/V2-directed antibodies CH04 and PGT145 indicate that they share a common mode of glycan penetration by extended anionic loops. In addition to structurally defining V1/V2, the results thus identify a paradigm of antibody recognition for highly glycosylated antigens, which - with PG9 - involves a site of vulnerability comprising just two glycans and a strand.

  1. Isolation and characterization of anti ROR1 single chain fragment variable antibodies using phage display technique.

    Science.gov (United States)

    Aghebati-Maleki, Leili; Younesi, Vahid; Jadidi-Niaragh, Farhad; Baradaran, Behzad; Majidi, Jafar; Yousefi, Mehdi

    2017-01-01

    Receptor tyrosine kinase-like orphan receptor (ROR1) belongs to one of the families of receptor tyrosine kinases (RTKs). RTKs are involved in the various physiologic cellular functions including proliferation, migration, survival, signaling and differentiation. Several RTKs are deregulated in various cancers implying the targeting potential of these molecules in cancer therapy. ROR1 has recently been shown to be expressed in various types of cancer cells but not in normal adult cells. Hence a molecular inhibitor of extracellular domain of ROR1 that inhibits ROR1-cell surface interaction is of great therapeutic importance. In an attempt to develop molecular inhibitors of ROR1, we screened single chain variable fragment (scFv) phage display libraries, Tomlinson I + J, against one specific synthetic oligopeptide from extracellular domain of ROR1 and selected scFvs were characterized using various immunological techniques. Several ROR1 specific scFvs were selected following five rounds of panning procedure. The scFvs showed specific binding to ROR1 using immunological techniques. Our results demonstrate successful isolation and characterization of specific ROR1 scFvs that may have great therapeutic potential in cancer immunotherapy.

  2. High affinity antibodies against Lex and sialyl Lex from a phage display library.

    Science.gov (United States)

    Dinh, Q; Weng, N P; Kiso, M; Ishida, H; Hasegawa, A; Marcus, D M

    1996-07-15

    Our previous studies of seven murine mAbs against the carbohydrate Lex Ag demonstrated that they were all encoded by VH441 and V kappa 24B. To obtain higher affinity Abs, and to ascertain whether their L chains could be encoded by other genes, we constructed a phage display library in a modified pComb 8 vector. The library contained random L chains, and Fd segments enriched in VH domains encoded by the VHX24 gene family. We selected phage with an Lex-BSA Ag, and obtained two Fab mAbs, clones 23 and 24, whose affinities were more than 100-fold higher than hybridoma mAb PM81. Both new mAbs were encoded by VH441, and their L chains were encoded by genes of the V kappa Ox1 and V kappa 9 families. In contrast to hybridoma mAb PM81, which binds only Lex, clones 23 and 24 bound sialyl Lex (SLex) as well as Lex, and clone 23 also binds the backbone carbohydrate structure nLacCer. Analysis of the binding of these three mAbs to synthetic glycolipids that contained structural modifications indicated that they recognize different aspects of the Lex structure, and suggested that they bind to limited regions of the oligosaccharide.

  3. Ability to develop broadly neutralizing HIV-1 antibodies is not restricted by the germline Ig gene repertoire.

    Science.gov (United States)

    Scheepers, Cathrine; Shrestha, Ram K; Lambson, Bronwen E; Jackson, Katherine J L; Wright, Imogen A; Naicker, Dshanta; Goosen, Mark; Berrie, Leigh; Ismail, Arshad; Garrett, Nigel; Abdool Karim, Quarraisha; Abdool Karim, Salim S; Moore, Penny L; Travers, Simon A; Morris, Lynn

    2015-05-01

    The human Ig repertoire is vast, producing billions of unique Abs from a limited number of germline Ig genes. The IgH V region (IGHV) is central to Ag binding and consists of 48 functional genes. In this study, we analyzed whether HIV-1-infected individuals who develop broadly neutralizing Abs show a distinctive germline IGHV profile. Using both 454 and Illumina technologies, we sequenced the IGHV repertoire of 28 HIV-infected South African women from the Centre for the AIDS Programme of Research in South Africa (CAPRISA) 002 and 004 cohorts, 13 of whom developed broadly neutralizing Abs. Of the 259 IGHV alleles identified in this study, approximately half were not found in the International Immunogenetics Database (IMGT). This included 85 entirely novel alleles and 38 alleles that matched rearranged sequences in non-IMGT databases. Analysis of the rearranged H chain V region genes of mAbs isolated from seven of these women, as well as previously isolated broadly neutralizing Abs from other donors, provided evidence that at least eight novel or non-IMGT alleles contributed to functional Abs. Importantly, we found that, despite a wide range in the number of IGHV alleles in each individual, including alleles used by known broadly neutralizing Abs, there were no significant differences in germline IGHV repertoires between individuals who do and do not develop broadly neutralizing Abs. This study reports novel IGHV repertoires and highlights the importance of a fully comprehensive Ig database for germline gene usage prediction. Furthermore, these data suggest a lack of genetic bias in broadly neutralizing Ab development in HIV-1 infection, with positive implications for HIV vaccine design.

  4. Lipid interactions and angle of approach to the HIV-1 viral membrane of broadly neutralizing antibody 10E8: Insights for vaccine and therapeutic design

    Science.gov (United States)

    Irimia, Adriana; Sarkar, Anita; Schiffner, Torben; Tingle, Ryan; Adachi, Yumiko; Deller, Marc C.; Burton, Dennis R.

    2017-01-01

    Among broadly neutralizing antibodies to HIV, 10E8 exhibits greater neutralizing breadth than most. Consequently, this antibody is the focus of prophylactic/therapeutic development. The 10E8 epitope has been identified as the conserved membrane proximal external region (MPER) of gp41 subunit of the envelope (Env) viral glycoprotein and is a major vaccine target. However, the MPER is proximal to the viral membrane and may be laterally inserted into the membrane in the Env prefusion form. Nevertheless, 10E8 has not been reported to have significant lipid-binding reactivity. Here we report x-ray structures of lipid complexes with 10E8 and a scaffolded MPER construct and mutagenesis studies that provide evidence that the 10E8 epitope is composed of both MPER and lipid. 10E8 engages lipids through a specific lipid head group interaction site and a basic and polar surface on the light chain. In the model that we constructed, the MPER would then be essentially perpendicular to the virion membrane during 10E8 neutralization of HIV-1. As the viral membrane likely also plays a role in selecting for the germline antibody as well as size and residue composition of MPER antibody complementarity determining regions, the identification of lipid interaction sites and the MPER orientation with regard to the viral membrane surface during 10E8 engagement can be of great utility for immunogen and therapeutic design. PMID:28225819

  5. Structural basis for penetration of the glycan shield of hepatitis C virus E2 glycoprotein by a broadly neutralizing human antibody.

    Science.gov (United States)

    Li, Yili; Pierce, Brian G; Wang, Qian; Keck, Zhen-Yong; Fuerst, Thomas R; Foung, Steven K H; Mariuzza, Roy A

    2015-04-17

    Hepatitis C virus (HCV) is a major cause of liver cirrhosis and hepatocellular carcinoma. A challenge for HCV vaccine development is to identify conserved epitopes able to elicit protective antibodies against this highly diverse virus. Glycan shielding is a mechanism by which HCV masks such epitopes on its E2 envelope glycoprotein. Antibodies to the E2 region comprising residues 412-423 (E2(412-423)) have broadly neutralizing activities. However, an adaptive mutation in this linear epitope, N417S, is associated with a glycosylation shift from Asn-417 to Asn-415 that enables HCV to escape neutralization by mAbs such as HCV1 and AP33. By contrast, the human mAb HC33.1 can neutralize virus bearing the N417S mutation. To understand how HC33.1 penetrates the glycan shield created by the glycosylation shift to Asn-415, we determined the structure of this broadly neutralizing mAb in complex with its E2(412-423) epitope to 2.0 Å resolution. The conformation of E2(412-423) bound to HC33.1 is distinct from the β-hairpin conformation of this peptide bound to HCV1 or AP33, because of disruption of the β-hairpin through interactions with the unusually long complementarity-determining region 3 of the HC33.1 heavy chain. Whereas Asn-415 is buried by HCV1 and AP33, it is solvent-exposed in the HC33.1-E2(412-423) complex, such that glycosylation of Asn-415 would not prevent antibody binding. Furthermore, our results highlight the structural flexibility of the E2(412-423) epitope, which may serve as an immune evasion strategy to impede induction of antibodies targeting this site by reducing its antigenicity.

  6. Antibody

    Science.gov (United States)

    An antibody is a protein produced by the body's immune system when it detects harmful substances, called antigens. Examples ... microorganisms (bacteria, fungi, parasites, and viruses) and chemicals. Antibodies may be produced when the immune system mistakenly ...

  7. Complement-Dependent Lysis of Influenza A Virus-Infected Cells by Broadly Cross-Reactive Human Monoclonal Antibodies

    Science.gov (United States)

    Terajima, Masanori; Cruz, John; Co, Mary Dawn T.; Lee, Jane-Hwei; Kaur, Kaval; Wilson, Patrick C.; Ennis, Francis A.

    2011-01-01

    We characterized human monoclonal antibodies (MAbs) cloned from influenza virus-infected patients and from influenza vaccine recipients by complement-dependent lysis (CDL) assay. Most MAbs active in CDL were neutralizing, but not all neutralizing MAbs can mediate CDL. Two of the three stalk-specific neutralizing MAbs tested were able to mediate CDL and were more cross-reactive to temporally distant H1N1 strains than the conventional hemagglutination-inhibiting and neutralizing MAbs. One of the stalk-specific MAbs was subtype cross-reactive to H1 and H2 hemagglutinins, suggesting a role for stalk-specific antibodies in protection against influenza illness, especially by a novel viral subtype which can cause pandemics. PMID:21994454

  8. Isolation and characterization of broadly neutralizing human monoclonal antibodies to the e1 glycoprotein of hepatitis C virus

    DEFF Research Database (Denmark)

    Meunier, Jean-Christophe; Russell, Rodney S.; Goossens, Vera

    2008-01-01

    The relative importance of humoral and cellular immunity in the prevention or clearance of hepatitis C virus (HCV) infection is poorly understood. However, there is considerable evidence that neutralizing antibodies are involved in disease control. Here we describe the detailed analysis of human...... monoclonal antibodies (MAbs) directed against HCV glycoprotein E1, which may have the potential to control HCV infection. We have identified two MAbs that can strongly neutralize HCV-pseudotyped particles (HCVpp) bearing the envelope glycoproteins of genotypes 1a, 1b, 4a, 5a, and 6a and less strongly...... neutralize HCVpp bearing the envelope glycoproteins of genotype 2a. Genotype 3a was not neutralized. The epitopes for both MAbs were mapped to the region encompassing amino acids 313 to 327. In addition, robust neutralization was also observed against cell culture-adapted viruses of genotypes 1a and 2a...

  9. Structure and function of broadly reactive antibody PG16 reveal an H3 subdomain that mediates potent neutralization of HIV-1

    Energy Technology Data Exchange (ETDEWEB)

    Pejchal, Robert; Walker, Laura M.; Stanfield, Robyn L.; Phogat, Sanjay K.; Koff, Wayne C.; Poignard, Pascal; Burton, Dennis R.; Wilson, Ian A. (Scripps); (IAVI)

    2010-11-15

    Development of an effective vaccine against HIV-1 will likely require elicitation of broad and potent neutralizing antibodies against the trimeric surface envelope glycoprotein (Env). Monoclonal antibodies (mAbs) PG9 and PG16 neutralize {approx}80% of HIV-1 isolates across all clades with extraordinary potency and target novel epitopes preferentially expressed on Env trimers. As these neutralization properties are ideal for a vaccine-elicited antibody response to HIV-1, their structural basis was investigated. The crystal structure of the antigen-binding fragment (Fab) of PG16 at 2.5 {angstrom} resolution revealed its unusually long, 28-residue, complementarity determining region (CDR) H3 forms a unique, stable subdomain that towers above the antibody surface. A 7-residue 'specificity loop' on the 'hammerhead' subdomain was identified that, when transplanted from PG16 to PG9 and vice versa, accounted for differences in the fine specificity and neutralization of these two mAbs. The PG16 electron density maps also revealed that a CDR H3 tyrosine was sulfated, which was confirmed for both PG9 (doubly) and PG16 (singly) by mass spectral analysis. We further showed that tyrosine sulfation plays a role in binding and neutralization. An N-linked glycan modification is observed in the variable light chain, but not required for antigen recognition. Further, the crystal structure of the PG9 light chain at 3.0 {angstrom} facilitated homology modeling to support the presence of these unusual features in PG9. Thus, PG9 and PG16 use unique structural features to mediate potent neutralization of HIV-1 that may be of utility in antibody engineering and for high-affinity recognition of a variety of therapeutic targets.

  10. Antibody-based PET of uPA/uPAR signaling with broad applicability for cancer imaging

    DEFF Research Database (Denmark)

    Yang, Dongzhi; Severin, Gregory; Dougherty, Casey A.;

    2016-01-01

    Mounting evidence suggests that the urokinase plasminogen activator (uPA) and its receptor (uPAR) play a central role in tumor progression. The goal of this study was to develop an 89Zr-labeled, antibody-based positron emission tomography (PET) tracer for quantitative imaging of the uPA/uPAR system....... An anti-uPA monoclonal antibody (ATN-291) was conjugated with a deferoxamine (Df) derivative and subsequently labeled with 89Zr. Flow cytometry, microscopy studies, and competitive binding assays were conducted to validate the binding specificity of Df-ATN-291 against uPA. PET imaging with 89Zr-Df-ATN-291...... was achieved in good radiochemical yield and high specific activity. Serial PET imaging demonstrated that, in most tumors studied (except uPA- LNCaP), the uptake of 89Zr-Df-ATN-291 was higher compared to major organs at 120 h post-injection, providing excellent tumor contrast. The tumor-to-muscle ratio of 89Zr...

  11. Development of Broadly Neutralizing Antibodies and Their Mapping by Monomeric gp120 in Human Immunodeficiency Virus Type 1-Infected Humans and Simian-Human Immunodeficiency Virus SHIVSF162P3N-Infected Macaques

    Science.gov (United States)

    Jia, Manxue; Lu, Hong; Markowitz, Martin; Cheng-Mayer, Cecilia

    2016-01-01

    ABSTRACT To improve our understanding of the similarities and differences between neutralizing antibodies elicited by simian-human immunodeficiency virus (SHIV)-infected rhesus macaques and human immunodeficiency virus type 1 (HIV-1)-infected humans, we examined the plasma of 13 viremic macaques infected with SHIVSF162P3N and 85 HIV-1-infected humans with known times of infection. We identified 5 macaques (38%) from 1 to 2 years postinfection (p.i.) with broadly neutralizing antibodies (bnAbs) against tier 2 HIV-1. In comparison, only 2 out of 42 (5%) human plasma samples collected in a similar time frame of 1 to 3 years p.i. exhibited comparable neutralizing breadths and potencies, with the number increasing to 7 out of 21 (30%) after 3 years p.i. Plasma mapping with monomeric gp120 identified only 2 out of 9 humans and 2 out of 4 macaques that contained gp120-reactive neutralizing antibodies, indicating distinct specificities in these plasma samples, with most of them recognizing the envelope trimer (including gp41) rather than the gp120 monomer. Indeed, a total of 20 gp120-directed monoclonal antibodies (MAbs) isolated from a human subject (AD358) and a Chinese rhesus macaque (GB40) displayed no or limited neutralizing activity against tier 2 strains. These isolated MAbs, mapped to the CD4-binding site, the V3 loop, the inner domain, and the C5 region of gp120, revealed genetic similarity between the human and macaque immunoglobulin genes used to encode some V3-directed MAbs. These results also support the use of envelope trimer probes for efficient isolation of HIV-1 bnAbs. IMPORTANCE HIV-1 vaccine research can benefit from understanding the development of broadly neutralizing antibodies (bnAbs) in rhesus macaques, commonly used to assess vaccine immunogenicity and efficacy. Here, we examined 85 HIV-1-infected humans and 13 SHIVSF162P3N-infected macaques for bnAbs and found that, similar to HIV-1-infected humans, bnAbs in SHIV-infected macaques are also rare

  12. DNA prime and virus-like particle boost from a single H5N1 strain elicits broadly neutralizing antibody responses against head region of H5 hemagglutinin.

    Science.gov (United States)

    Wang, Guiqin; Zhou, Fan; Buchy, Philippe; Zuo, Teng; Hu, Hongxing; Liu, Jingjing; Song, Yufeng; Ding, Heng; Tsai, Cheguo; Chen, Ze; Zhang, Linqi; Deubel, Vincent; Zhou, Paul

    2014-03-01

    Since 1996, highly pathogenic avian influenza (HPAI) H5N1 virus has presented a persistent threat to public health. Its high degree of genetic diversity also poses enormous challenges in developing effective vaccines. To search for vaccine regimens that could elicit broadly neutralizing antibody responses against diverse HPAI H5N1 strains, in the present study we tested H5 hemagglutinin (HA) from an A/Thailand/1(KAN)-1/2004 strain in a heterologous prime-boost vaccination. We demonstrated that priming mice with DNA and boosting with virus-like particle induced antibody responses that cross-neutralize all reported clades and subclades of HPAI H5N1 viruses and protect mice from high lethal dose HPAI H5N1 challenge in both active and passive immunizations. Unexpectedly, cross-divergent H5 neutralizing antibodies are directed to the HA head and block both attachment and postattachment of virus entry. Thus, we conclude that as a promising pan-H5 vaccine candidate this prime-boost regimen could be further developed in ferrets and in humans.

  13. Limited naturally occurring escape in broadly neutralizing antibody epitopes in hepatitis C glycoprotein E2 and constrained sequence usage in acute infection.

    Science.gov (United States)

    Rodrigo, Chaturaka; Walker, Melanie R; Leung, Preston; Eltahla, Auda A; Grebely, Jason; Dore, Gregory J; Applegate, Tanya; Page, Kimberly; Dwivedi, Sunita; Bruneau, Julie; Morris, Meghan D; Cox, Andrea L; Osburn, William; Kim, Arthur Y; Schinkel, Janke; Shoukry, Naglaa H; Lauer, Georg M; Maher, Lisa; Hellard, Margaret; Prins, Maria; Luciani, Fabio; Lloyd, Andrew R; Bull, Rowena A

    2017-04-01

    Broadly neutralizing antibodies have been associated with spontaneous clearance of the hepatitis C infection as well as viral persistence by immune escape. Further study of neutralizing antibody epitopes is needed to unravel pathways of resistance to virus neutralization, and to identify conserved regions for vaccine design. All reported broadly neutralizing antibody (BNAb) epitopes in the HCV Envelope (E2) glycoprotein were identified. The critical contact residues of these epitopes were mapped onto the linear E2 sequence. All publicly available E2 sequences were then downloaded and the contact residues within the BNAb epitopes were assessed for the level of conservation, as well as the frequency of occurrence of experimentally-proven resistance mutations. Epitopes were also compared between two sequence datasets obtained from samples collected at well-defined time points from acute (180days) infections, to identify any significant differences in residue usage. The contact residues for all BNAbs were contained within 3 linear regions of the E2 protein sequence. An analysis of 1749 full length E2 sequences from public databases showed that only 10 out of 29 experimentally-proven resistance mutations were present at a frequency >5%. Comparison of subtype 1a viral sequences obtained from samples collected during acute or chronic infection revealed significant differences at positions 610 and 655 with changes in residue (p<0.05), and at position 422 (p<0.001) with a significant difference in variability (entropy). The majority of experimentally-described escape variants do not occur frequently in nature. The observed differences between acute and chronically isolated sequences suggest constraints on residue usage early in infection.

  14. A phage-displayed chicken single-chain antibody fused to alkaline phosphatase detects Fusarium pathogens and their presence in cereal grains

    Energy Technology Data Exchange (ETDEWEB)

    Hu, Zu-Quan [Molecular Biotechnology Laboratory of Triticeae Crops, Huazhong Agricultural University, Wuhan 430070 (China); Li, He-Ping [Molecular Biotechnology Laboratory of Triticeae Crops, Huazhong Agricultural University, Wuhan 430070 (China); College of Life Science and Technology, Huazhong Agricultural University, Wuhan 430070 (China); Zhang, Jing-Bo [Molecular Biotechnology Laboratory of Triticeae Crops, Huazhong Agricultural University, Wuhan 430070 (China); College of Plant Science and Technology, Huazhong Agricultural University, Wuhan 430070 (China); Huang, Tao [Molecular Biotechnology Laboratory of Triticeae Crops, Huazhong Agricultural University, Wuhan 430070 (China); College of Life Science and Technology, Huazhong Agricultural University, Wuhan 430070 (China); Liu, Jin-Long; Xue, Sheng [Molecular Biotechnology Laboratory of Triticeae Crops, Huazhong Agricultural University, Wuhan 430070 (China); Wu, Ai-Bo [Institute for Agri-food Standards and Testing Technology, Laboratory of Quality and Safety Risk Assessment for Agro-products, Ministry of Agriculture, Shanghai Academy of Agricultural Sciences, 1000 Jinqi Road, Shanghai 201403 (China); Liao, Yu-Cai, E-mail: ycliao06@yahoo.com.cn [Molecular Biotechnology Laboratory of Triticeae Crops, Huazhong Agricultural University, Wuhan 430070 (China); College of Plant Science and Technology, Huazhong Agricultural University, Wuhan 430070 (China); National Center of Plant Gene Research, Wuhan 430070 (China)

    2013-02-18

    Graphical abstract: A phage-displayed chicken scFv antibody, FvSG7, binds on the surface antigen of conidiospores and the mycelia of F. verticillioides. Its fusion with alkaline phosphatase (AP) through a 218 linker displayed a 4-fold higher affinity compared with the parent scFv antibody and efficiently detected toxigenic Fusarium pathogens in cereal grains. Highlights: ► Generation of a highly reactive scFv antibody against F. verticillioides. ► Localization of the antibody binding to the surface target of F. verticillioides. ► Expression of the antibody–alkaline phosphatase (AP) fusion linked by a 218 linker. ► The antibody–AP fusion has a higher affinity than the parental antibody. ► The antibody–AP fusion detects toxigenic Fusarium pathogens in cereal grains. -- Abstract: Fusarium and its poisonous mycotoxins are distributed worldwide and are of particular interest in agriculture and food safety. A simple analytical method to detect pathogens is essential for forecasting diseases and controlling mycotoxins. This article describes a proposed method for convenient and sensitive detection of Fusarium pathogens that uses the fusion of single-chain variable fragment (scFv) and alkaline phosphatase (AP). A highly reactive scFv antibody specific to soluble cell wall-bound proteins (SCWPs) of F. verticillioides was selected from an immunized chicken phagemid library by phage display. The antibody was verified to bind on the surface of ungerminated conidiospores and mycelia of F. verticillioides. The scFv–AP fusion was constructed, and soluble expression in bacteria was confirmed. Both the antibody properties and enzymatic activity were retained, and the antigen-binding capacity of the fusion was enhanced by the addition of a linker. Surface plasmon resonance measurements confirmed that the fusion displayed 4-fold higher affinity compared with the fusion's parental scFv antibody. Immunoblot analyses showed that the fusion had good binding

  15. A glycoconjugate antigen based on the recognition motif of a broadly neutralizing human immunodeficiency virus antibody, 2G12, is immunogenic but elicits antibodies unable to bind to the self glycans of gp120

    DEFF Research Database (Denmark)

    Astronomo, Rena D; Lee, Hing-Ken; Scanlan, Christopher N

    2008-01-01

    The glycan shield of human immunodeficiency virus type 1 (HIV-1) gp120 contributes to viral evasion from humoral immune responses. However, the shield is recognized by the HIV-1 broadly neutralizing antibody (Ab), 2G12, at a relatively conserved cluster of oligomannose glycans. The discovery of 2G......12 raises the possibility that a carbohydrate immunogen may be developed that could elicit 2G12-like neutralizing Abs and contribute to an AIDS vaccine. We have previously dissected the fine specificity of 2G12 and reported that the synthetic tetramannoside (Man(4)) that corresponds to the D1 arm...

  16. Antibody binding site mapping of SARS-CoV spike protein receptor-binding domain by a combination of yeast surface display and phage peptide library screening.

    Science.gov (United States)

    Zhang, Xiaoping; Wang, Jingxue; Wen, Kun; Mou, Zhirong; Zou, Liyun; Che, Xiaoyan; Ni, Bing; Wu, Yuzhang

    2009-12-01

    The receptor-binding domain (RBD) of severe acute respiratory syndrome coronavirus (SARS-CoV) spike (S) protein plays an important role in viral infection, and is a potential major neutralizing determinant. In this study, three hybridoma cell lines secreting specific monoclonal antibodies against the RBD of the S protein were generated and their exact binding sites were identified. Using yeast surface display, the binding sites of these antibodies were defined to two linear regions on the RBD: S(337-360) and S(380-399). Using these monoclonal antibodies in phage peptide library screening identified 10 distinct mimotopes 12 amino acids in length. Sequence comparison between native epitopes and these mimotopes further confirmed the binding sites, and revealed key amino acid residues involved in antibody binding. None of these antibodies could neutralize the murine leukemia virus pseudotyped expressing the SARS-CoV spike protein (MLV/SARS-CoV). However, these mAbs could be useful in the diagnosis of SARS-CoV due to their exclusive reactivity with SARS-CoV. Furthermore, this study established a feasible platform for epitope mapping. Yeast surface display combined with phage peptide library screening provides a convenient strategy for the identification of epitope peptides from certain antigenic proteins.

  17. A novel method for in Situ detection of hydrolyzable casein fragments in a cheese matrix by antibody phage display technique and CLSM

    DEFF Research Database (Denmark)

    Duan, Zhi; Brüggemann, Dagmar Adeline; Siegumfeldt, Henrik

    2009-01-01

    was successfully developed for the detection of scFvs binding to different alpha(s1)-casein fragments inside a cheese matrix by CLSM. To our knowledge, this is the first demonstrated immunofluorescent labeling method for in situ analysis of proteolysis phenomena in the cheese matrix. Additionally, this technique......A novel method to monitor in situ hydrolyzable casein fragments during cheese ripening by using immunofluorescent labeling and confocal laser scanning microscopy (CLSM) was developed. Monoclonal single chain variable fragments of antibody (scFvs) were generated by antibody phage display toward...

  18. A next-generation cleaved, soluble HIV-1 Env trimer, BG505 SOSIP.664 gp140, expresses multiple epitopes for broadly neutralizing but not non-neutralizing antibodies.

    Directory of Open Access Journals (Sweden)

    Rogier W Sanders

    2013-09-01

    Full Text Available A desirable but as yet unachieved property of a human immunodeficiency virus type 1 (HIV-1 vaccine candidate is the ability to induce broadly neutralizing antibodies (bNAbs. One approach to the problem is to create trimeric mimics of the native envelope glycoprotein (Env spike that expose as many bNAb epitopes as possible, while occluding those for non-neutralizing antibodies (non-NAbs. Here, we describe the design and properties of soluble, cleaved SOSIP.664 gp140 trimers based on the subtype A transmitted/founder strain, BG505. These trimers are highly stable, more so even than the corresponding gp120 monomer, as judged by differential scanning calorimetry. They are also homogenous and closely resemble native virus spikes when visualized by negative stain electron microscopy (EM. We used several techniques, including ELISA and surface plasmon resonance (SPR, to determine the relationship between the ability of monoclonal antibodies (MAbs to bind the soluble trimers and neutralize the corresponding virus. In general, the concordance was excellent, in that virtually all bNAbs against multiple neutralizing epitopes on HIV-1 Env were highly reactive with the BG505 SOSIP.664 gp140 trimers, including quaternary epitopes (CH01, PG9, PG16 and PGT145. Conversely, non-NAbs to the CD4-binding site, CD4-induced epitopes or gp41ECTO did not react with the trimers, even when their epitopes were present on simpler forms of Env (e.g. gp120 monomers or dissociated gp41 subunits. Three non-neutralizing MAbs to V3 epitopes did, however, react strongly with the trimers but only by ELISA, and not at all by SPR and to only a limited extent by EM. These new soluble trimers are useful for structural studies and are being assessed for their performance as immunogens.

  19. Identification and characterization of a broadly cross-reactive HIV-1 human monoclonal antibody that binds to both gp120 and gp41.

    Directory of Open Access Journals (Sweden)

    Mei-Yun Zhang

    Full Text Available Identification of broadly cross-reactive HIV-1-neutralizing antibodies (bnAbs may assist vaccine immunogen design. Here we report a novel human monoclonal antibody (mAb, designated m43, which co-targets the gp120 and gp41 subunits of the HIV-1 envelope glycoprotein (Env. M43 bound to recombinant gp140 s from various primary isolates, to membrane-associated Envs on transfected cells and HIV-1 infected cells, as well as to recombinant gp120 s and gp41 fusion intermediate structures containing N-trimer structure, but did not bind to denatured recombinant gp140 s and the CD4 binding site (CD4bs mutant, gp120 D368R, suggesting that the m43 epitope is conformational and overlaps the CD4bs on gp120 and the N-trimer structure on gp41. M43 neutralized 34% of the HIV-1 primary isolates from different clades and all the SHIVs tested in assays based on infection of peripheral blood mononuclear cells (PBMCs by replication-competent virus, but was less potent in cell line-based pseudovirus assays. In contrast to CD4, m43 did not induce Env conformational changes upon binding leading to exposure of the coreceptor binding site, enhanced binding of mAbs 2F5 and 4E10 specific for the membrane proximal external region (MPER of gp41 Envs, or increased gp120 shedding. The overall modest neutralization activity of m43 is likely due to the limited binding of m43 to functional Envs which could be increased by antibody engineering if needed. M43 may represent a new class of bnAbs targeting conformational epitopes overlapping structures on both gp120 and gp41. Its novel epitope and possibly new mechanism(s of neutralization could helpdesign improved vaccine immunogens and candidate therapeutics.

  20. A combinatorial mutagenesis approach for functional epitope mapping on phage-displayed target antigen: application to antibodies against epidermal growth factor.

    Science.gov (United States)

    Infante, Yanelys Cabrera; Pupo, Amaury; Rojas, Gertrudis

    2014-01-01

    Although multiple different procedures to characterize the epitopes recognized by antibodies have been developed, site-directed mutagenesis remains the method of choice to define the energetic contribution of antigen residues to binding. These studies are useful to identify critical residues and to delineate functional maps of the epitopes. However, they tend to underestimate the roles of residues that are not critical for binding on their own, but contribute to the formation of the target epitope in an additive, or even cooperative, way. Mapping antigenic determinants with a diffuse energetic landscape, which establish multiple individually weak interactions with the antibody paratope, resulting in high affinity and specificity recognition of the epitope as a whole, is thus technically challenging. The current work was aimed at developing a combinatorial strategy to overcome the limitations of site-directed mutagenesis, relying on comprehensive randomization of discrete antigenic regions within phage-displayed antigen libraries. Two model antibodies recognizing epidermal growth factor were used to validate the mapping platform. Abrogation of antibody recognition due to the introduction of simultaneous replacements was able to show the involvement of particular amino acid clusters in epitope formation. The abundance of some of the original residues (or functionally equivalent amino acids sharing their physicochemical properties) among the set of mutated antigen variants selected on a given antibody highlighted their contributions and allowed delineation of a detailed functional map of the corresponding epitope. The use of the combinatorial approach could be expanded to map the interactions between other antigens/antibodies.

  1. Improved Binding Activity of Antibodies against Major Histocompatibility Complex Class I Chain-Related Gene A by Phage Display Technology for Cancer-Targeted Therapy

    Directory of Open Access Journals (Sweden)

    Achara Phumyen

    2012-01-01

    Full Text Available Major histocompatibility complex class I chain-related gene A (MICA is an NKG2D ligand that is over-expressed under cellular stress including cancer transformation and viral infection. High expression of MICA in cancer tissues or patients' sera is useful for prognostic or follow-up markers in cancer patients. In this study, phage display technology was employed to improve antigen-binding activities of anti-MICA monoclonal antibodies (WW2G8, WW6B7, and WW9B8. The 12 amino acid residues in the complementarity determining regions (CDRs on the V domain of the heavy chain CDR3 (HCDR3 of these anti-MICA antibodies were modified by PCR-random mutagenesis, and phages displaying mutated anti-MICA Fab were constructed. After seven rounds of panning, five clones of phages displaying mutant anti-MICA Fab which exhibited 3–7-folds higher antigen-binding activities were isolated. Two clones of the mutants (phage-displayed mutant Fab WW9B8.1 and phage-displayed mutant Fab WW9B8.21 were confirmed to have antigen-binding specificity for cell surface MICA proteins by flow cytometry. These phage clones are able to recognize MICA in a native form according to positive results obtained by indirect ELISA and flow cytometry. Thus, these phage particles could be potentially used for further development of nanomedicine specifically targeting cancer cells expressing MICA proteins.

  2. Re-engineering of the PAM1 phage display monoclonal antibody to produce a soluble, versatile anti-homogalacturonan scFv

    DEFF Research Database (Denmark)

    Manfield, I. W.; Bernal Giraldo, Adriana Jimena; Møller, I.;

    2006-01-01

    Antibody phage display is an increasingly important alternative method for the production of monoclonal antibodies (mAbs) and involves the expression of antibody fragments (scFvs) at the surface of bacteriophage particles. We have previously used this technique to generate a phage mAb (PAM1phage...... of the PAM1 mAb, we describe here the production of a phage-free, soluble scFv version of the PAM1 mAb (PAM1scFv). Using the new PAM1scFv probe, the occurrence of the HG epitope recognized can now be localized with high resolution within micro-domains of plant cell walls....

  3. Rapid Selection of Phage Se-scFv with GPX Activity via Combination of Phage Display Antibody Library with Chemical Modification

    Institute of Scientific and Technical Information of China (English)

    2007-01-01

    Glutathione peroxidase (GPX) plays an important role in scavenging reactive oxygen species. A series of catalytic antibodies with GPX activity have been generated by the authors of this study. To obtain humanized catalytic antibodies, the phage-displayed human antibody library was used to select novel antibodies by repetitive screening. Phage antibodies, scFv-B8 and scFv-H6 with the GSH-binding site, were obtained from the library by enzyme-linked immunosorbent assay(ELISA) analysis with 4 rounds of selection against their respective haptens, S-2,4-dinitriphenyl t-butyl ester(GSH-s-DNP-Bu) and S-2,4-dinitriphenyl t-hexyl ester(GSH-s-DNP-He). Nevertheless, several studies need to be conducted to determine whether scFv-B8 and scFv-H6 possess GPX activity. To enhance the speed of the selection, selenocysteine(Sec, the catalytic group of GPX) was incorporated directly into the phages, scFv-B8 and scFv-H6, by chemical mutation to form the phages Se-scFv-B8 and Se-scFv-H6. The GPX activities were found to be 3012 units/μmol and 2102 units/μmol, respectively. To improve the GPX activity of the phage Se-scFv-B8, DNA shuffling was used to construct a secondary library and another positive phage antibody scFv-B9 was screened out by another panning against GSH-s-DNP-Bu. When Sec was incorporated via chemical mutation into the phage antibody scFv-B9, its GPX activity reached 3560 units/μmol, which is 1.17-fold higher than the phage antibody Se-scFv-B8and almost approached the order of magnitude of native GPX. The rapid selection is the prerequisite for generating humanized Se-scFv with GPX activity.

  4. A hepatitis C virus (HCV) vaccine comprising envelope glycoproteins gpE1/gpE2 derived from a single isolate elicits broad cross-genotype neutralizing antibodies in humans

    DEFF Research Database (Denmark)

    Law, John Lok Man; Chen, Chao; Wong, Jason

    2013-01-01

    , an effective HCV vaccine which could elicit broadly cross-neutralizing antibodies has represented a major challenge. In this study, we tested for the presence of cross-neutralizing antibodies in human volunteers who were immunized with recombinant glycoproteins gpE1/gpE2 derived from a single HCV strain (HCV1...... of genotype 1a). Cross neutralization was tested in Huh-7.5 human hepatoma cell cultures using infectious recombinant HCV (HCVcc) expressing structural proteins of heterologous HCV strains from all known major genotypes, 1-7. Vaccination induced significant neutralizing antibodies against heterologous HCV...... genotype 1a virus which represents the most common genotype in North America. Of the 16 vaccinees tested, 3 were selected on the basis of strong 1a virus neutralization for testing of broad cross-neutralizing responses. At least 1 vaccinee was shown to elicit broad cross-neutralization against all HCV...

  5. Screening of scFvs against cTnI from Phage Display Antibody Library and Their Expression in E.coli Rosetta

    Institute of Scientific and Technical Information of China (English)

    WEI Jing-yan; ZHANG Han-qi; JIN Qin-han; LI Wei; LUO Gui-min; LI Shan-yu; MU Ying; ZHU Xue-jun; LIU Lei; GAO Li-zeng; SONG Da-qian; SUN Zhi-wei; YAN Gang-lin

    2005-01-01

    The single chain variable fragments of antibodies(scFvs) against eTnI were screened from the phage display antibody library by using cTnI as the target antigen. After four rounds of panning, four clones(H2, G5,A9, B9) from the phage display antibody library were verified to show higher binding affinity for cTnI by ELISA and to contain the variable region genes of the light and heavy chains of scFvs by sequencing. The variable region genes of scFvs H2 and G5 were successfully amplified by polymerase chain reactions (PCR)and cloned into expression vector pPELB and expressed as a soluble protein in E. coli Rosetta, whose expression yield was about 2% of total proteins. The expressed proteins were purified by nickel(Ni) affinity chromatography and a single band is shown in the position of 28 kDa on SDS-PAGE. The western blot analysis result verifies that the expressed scFv proteins are capable of binding with monoclonal antibodies against hexa-histidine, indicating that they are hexa-histidin-tagged aim proteins. The immunoassay demonstrates that the expressed scFv proteins are able to specifically react with cTnI molecules. The association constant (KA)values range from 1.2×104 to 1.7×105 L/mol that are correspondent to the affinities of polyclonal antibodies against cTnI from rabbits. These antibodies can be valuable reagents for the immunoassay of cTnI.

  6. Identification of Broad-Genotype HPV L2 Neutralization Site for Pan-HPV Vaccine Development by a Cross-Neutralizing Antibody.

    Directory of Open Access Journals (Sweden)

    Daning Wang

    Full Text Available Human Papillomavirus (HPV, a non-enveloped, double-stranded DNA virus, is responsible for 5% of human cancers. The HPV capsid consists of major and minor structural proteins, L1 and L2. L1 proteins form an icosahedral shell with building blocks of the pentameric capsomere, and one L2 molecule extends outward from the central hole of the capsid. Thus, L2 is concealed within L1 and only becomes exposed when the capsid interacts with host cells. The low antigenic variation of L2 means that this protein could offer a target for the development of a pan-HPV vaccine. Toward this goal, here we describe an anti-L2 monoclonal antibody, 14H6, which broadly neutralizes at least 11 types of HPV, covering types 6, 11, 16, 18, 31, 33, 35, 45, 52, 58 and 59, in pseudovirion--based cell neutralization assay. The mAb 14H6 recognizes a minimal linear epitope located on amino acids 21 to 30 of the L2 protein. Alanine scanning mutagenesis and sequence alignment identified several conserved residues (Cys22, Lys23, Thr27, Cys28 and Pro29 that are involved in the 14H6 binding with L2. The epitope was grafted to several scaffolding proteins, including HPV16 L1 virus-like particles, HBV 149 core antigen and CRM197. The resultant chimeric constructs were expressed in Escherichia coli and purified with high efficiency. Immunization with these pan-HPV vaccine candidates elicited high titers of the L2-specific antibody in mice and conferred robust (3-log titers of cross-genotype neutralization, including against HPV11, 16, 18, 45, 52, 58 and 59. These findings will help in the development of an L2-based, pan-HPV vaccine.

  7. Extra-epitopic hepatitis C virus polymorphisms confer resistance to broadly neutralizing antibodies by modulating binding to scavenger receptor B1

    Science.gov (United States)

    El-Diwany, Ramy; Mankowski, Madeleine C.; Wasilewski, Lisa N.; Brady, Jillian K.; Snider, Anna E.; Osburn, William O.; Murrell, Ben; Ray, Stuart C.

    2017-01-01

    Broadly-neutralizing monoclonal antibodies (bNAbs) may guide vaccine development for highly variable viruses including hepatitis C virus (HCV), since they target conserved viral epitopes that could serve as vaccine antigens. However, HCV resistance to bNAbs could reduce the efficacy of a vaccine. HC33.4 and AR4A are two of the most potent anti-HCV human bNAbs characterized to date, binding to highly conserved epitopes near the amino- and carboxy-terminus of HCV envelope (E2) protein, respectively. Given their distinct epitopes, it was surprising that these bNAbs showed similar neutralization profiles across a panel of natural HCV isolates, suggesting that some viral polymorphisms may confer resistance to both bNAbs. To investigate this resistance, we developed a large, diverse panel of natural HCV envelope variants and a novel computational method to identify bNAb resistance polymorphisms in envelope proteins (E1 and E2). By measuring neutralization of a panel of HCV pseudoparticles by 10 μg/mL of each bNAb, we identified E1E2 variants with resistance to one or both bNAbs, despite 100% conservation of the AR4A binding epitope across the panel. We discovered polymorphisms outside of either binding epitope that modulate resistance to both bNAbs by altering E2 binding to the HCV co-receptor, scavenger receptor B1 (SR-B1). This study is focused on a mode of neutralization escape not addressed by conventional analysis of epitope conservation, highlighting the contribution of extra-epitopic polymorphisms to bNAb resistance and presenting a novel mechanism by which HCV might persist even in the face of an antibody response targeting multiple conserved epitopes. PMID:28235087

  8. Extra-epitopic hepatitis C virus polymorphisms confer resistance to broadly neutralizing antibodies by modulating binding to scavenger receptor B1.

    Science.gov (United States)

    El-Diwany, Ramy; Cohen, Valerie J; Mankowski, Madeleine C; Wasilewski, Lisa N; Brady, Jillian K; Snider, Anna E; Osburn, William O; Murrell, Ben; Ray, Stuart C; Bailey, Justin R

    2017-02-01

    Broadly-neutralizing monoclonal antibodies (bNAbs) may guide vaccine development for highly variable viruses including hepatitis C virus (HCV), since they target conserved viral epitopes that could serve as vaccine antigens. However, HCV resistance to bNAbs could reduce the efficacy of a vaccine. HC33.4 and AR4A are two of the most potent anti-HCV human bNAbs characterized to date, binding to highly conserved epitopes near the amino- and carboxy-terminus of HCV envelope (E2) protein, respectively. Given their distinct epitopes, it was surprising that these bNAbs showed similar neutralization profiles across a panel of natural HCV isolates, suggesting that some viral polymorphisms may confer resistance to both bNAbs. To investigate this resistance, we developed a large, diverse panel of natural HCV envelope variants and a novel computational method to identify bNAb resistance polymorphisms in envelope proteins (E1 and E2). By measuring neutralization of a panel of HCV pseudoparticles by 10 μg/mL of each bNAb, we identified E1E2 variants with resistance to one or both bNAbs, despite 100% conservation of the AR4A binding epitope across the panel. We discovered polymorphisms outside of either binding epitope that modulate resistance to both bNAbs by altering E2 binding to the HCV co-receptor, scavenger receptor B1 (SR-B1). This study is focused on a mode of neutralization escape not addressed by conventional analysis of epitope conservation, highlighting the contribution of extra-epitopic polymorphisms to bNAb resistance and presenting a novel mechanism by which HCV might persist even in the face of an antibody response targeting multiple conserved epitopes.

  9. Evolution of an interloop disulfide bond in high-affinity antibody mimics based on fibronectin type III domain and selected by yeast surface display: molecular convergence with single-domain camelid and shark antibodies.

    Science.gov (United States)

    Lipovsek, Dasa; Lippow, Shaun M; Hackel, Benjamin J; Gregson, Melissa W; Cheng, Paul; Kapila, Atul; Wittrup, K Dane

    2007-05-11

    The 10th human fibronectin type III domain ((10)Fn3) is one of several protein scaffolds used to design and select families of proteins that bind with high affinity and specificity to macromolecular targets. To date, the highest affinity (10)Fn3 variants have been selected by mRNA display of libraries generated by randomizing all three complementarity-determining region -like loops of the (10)Fn3 scaffold. The sub-nanomolar affinities of such antibody mimics have been attributed to the extremely large size of the library accessible by mRNA display (10(12) unique sequences). Here we describe the selection and affinity maturation of (10)Fn3-based antibody mimics with dissociation constants as low as 350 pM selected from significantly smaller libraries (10(7)-10(9) different sequences), which were constructed by randomizing only 14 (10)Fn3 residues. The finding that two adjacent loops in human (10)Fn3 provide a large enough variable surface area to select high-affinity antibody mimics is significant because a smaller deviation from wild-type (10)Fn3 sequence is associated with a higher stability of selected antibody mimics. Our results also demonstrate the utility of an affinity-maturation strategy that led to a 340-fold improvement in affinity by maximizing sampling of sequence space close to the original selected antibody mimic. A striking feature of the highest affinity antibody mimics selected against lysozyme is a pair of cysteines on adjacent loops, in positions 28 and 77, which are critical for the affinity of the (10)Fn3 variant for its target and are close enough to form a disulfide bond. The selection of this cysteine pair is structurally analogous to the natural evolution of disulfide bonds found in new antigen receptors of cartilaginous fish and in camelid heavy-chain variable domains. We propose that future library designs incorporating such an interloop disulfide will further facilitate the selection of high-affinity, highly stable antibody mimics from

  10. Evolution of the Humoral Response during HCV Infection: Theories on the Origin of Broadly Neutralizing Antibodies and Implications for Vaccine Design.

    Science.gov (United States)

    Murira, Armstrong; Lapierre, Pascal; Lamarre, Alain

    2016-01-01

    Similar to human immunodeficiency virus (HIV)-1, vaccine-induced elicitation of broadly neutralizing (bNt) antibodies (Abs) is gaining traction as a key goal toward the eradication of the hepatitis C virus (HCV) pandemic. Previously, the significance of the Ab response against HCV was underappreciated given the prevailing evidence advancing the role of the cellular immune response in clearance and overall control of the infection. However, recent findings have driven growing interest in the humoral arm of the immune response and in particular the role of bNt responses due to their ability to confer protective immunity upon passive transfer in animal models. Nevertheless, the origin and development of bNt Abs is poorly understood and their occurrence is rare as well as delayed with emergence only observed in the chronic phase of infection. In this review, we characterize the interplay between the host immune response and HCV as it progresses from the acute to chronic phase of infection. In addition, we place these events in the context of current hypotheses on the origin of bNt Abs against the HIV-1, whose humoral immune response is better characterized. Based on the increasing significance of the humoral immune response against HCV, characterization of these events may be critical in understanding the development of the bNt responses and, thus, provide strategies toward effective vaccine design.

  11. Selection of single domain antibodies from immune libraries displayed on the surface of E. coli cells with two β-domains of opposite topologies.

    Directory of Open Access Journals (Sweden)

    Valencio Salema

    Full Text Available Screening of antibody (Ab libraries by direct display on the surface of E. coli cells is hampered by the presence of the outer membrane (OM. In this work we demonstrate that the native β-domains of EhaA autotransporter and intimin, two proteins from enterohemorrhagic E. coli O157:H7 (EHEC with opposite topologies in the OM, are effective systems for the display of immune libraries of single domain Abs (sdAbs from camelids (nanobodies or VHH on the surface of E. coli K-12 cells and for the selection of high affinity sdAbs using magnetic cell sorting (MACS. We analyzed the capacity of EhaA and intimin β-domains to display individual sdAbs and sdAb libraries obtained after immunization with the extracellular domain of the translocated intimin receptor from EHEC (TirM(EHEC. We demonstrated that both systems displayed functional sdAbs on the surface of E. coli cells with little proteolysis and cellular toxicity, although E. coli cells displaying sdAbs with the β-domain of intimin showed higher antigen-binding capacity. Both E. coli display libraries were screened for TirM(EHEC binding clones by MACS. High affinity binders were selected by both display systems, although more efficiently with the intimin β-domain. The specificity of the selected clones against TirM(EHEC was demonstrated by flow cytometry of E. coli cells, along with ELISA and surface plasmon resonance with purified sdAbs. Finally, we employed the E. coli cell display systems to provide an estimation of the affinity of the selected sdAb by flow cytometry analysis under equilibrium conditions.

  12. Crystal structure of a human rhinovirus that displays part of the HIV-1 V3 loop and induces neutralizing antibodies against HIV-1.

    Science.gov (United States)

    Ding, Jianping; Smith, Allen D; Geisler, Sheila C; Ma, Xuejun; Arnold, Gail Ferstandig; Arnold, Eddy

    2002-07-01

    We report the 2.7 A resolution structure of a chimeric rhinovirus, MN-III-2, that displays part of the HIV-1 gp120 V3 loop and elicits HIV-neutralizing antibodies. The V3 loop insert is dominated by two type I beta turns. The structures of two adjacent tripeptides resemble those of analogous segments in three Fab/V3 loop peptide complexes. Although two of the three corresponding antibodies bind and neutralize MN-III-2 well, only one of the three can bind without significant rearrangement. These results suggest that the V3 loop insert: (1) can share some local conformational similarity to V3 loop sequences presented on different structural frameworks; (2) must be able to adopt multiple conformations, even in a relatively constrained environment; and (3) may mimic the conformational variability of the epitope on HIV-1, increasing the likelihood of eliciting appropriate neutralizing immune responses.

  13. Broad blockade antibody responses in human volunteers after immunization with a multivalent norovirus VLP candidate vaccine: immunological analyses from a phase I clinical trial.

    Directory of Open Access Journals (Sweden)

    Lisa C Lindesmith

    2015-03-01

    Full Text Available Human noroviruses (NoVs are the primary cause of acute gastroenteritis and are characterized by antigenic variation between genogroups and genotypes and antigenic drift of strains within the predominant GII.4 genotype. In the context of this diversity, an effective NoV vaccine must elicit broadly protective immunity. We used an antibody (Ab binding blockade assay to measure the potential cross-strain protection provided by a multivalent NoV virus-like particle (VLP candidate vaccine in human volunteers.Sera from ten human volunteers immunized with a multivalent NoV VLP vaccine (genotypes GI.1/GII.4 were analyzed for IgG and Ab blockade of VLP interaction with carbohydrate ligand, a potential correlate of protective immunity to NoV infection and illness. Immunization resulted in rapid rises in IgG and blockade Ab titers against both vaccine components and additional VLPs representing diverse strains and genotypes not represented in the vaccine. Importantly, vaccination induced blockade Ab to two novel GII.4 strains not in circulation at the time of vaccination or sample collection. GII.4 cross-reactive blockade Ab titers were more potent than responses against non-GII.4 VLPs, suggesting that previous exposure history to this dominant circulating genotype may impact the vaccine Ab response. Further, antigenic cartography indicated that vaccination preferentially activated preexisting Ab responses to epitopes associated with GII.4.1997. Study interpretations may be limited by the relevance of the surrogate neutralization assay and the number of immunized participants evaluated.Vaccination with a multivalent NoV VLP vaccine induces a broadly blocking Ab response to multiple epitopes within vaccine and non-vaccine NoV strains and to novel antigenic variants not yet circulating at the time of vaccination. These data reveal new information about complex NoV immune responses to both natural exposure and to vaccination, and support the potential

  14. The epitope and neutralization mechanism of AVFluIgG01, a broad-reactive human monoclonal antibody against H5N1 influenza virus.

    Directory of Open Access Journals (Sweden)

    Zhiliang Cao

    Full Text Available The continued spread of highly pathogenic avian influenza (HPAI H5N1 virus underscores the importance of effective antiviral approaches. AVFluIgG01 is a potent and broad-reactive H5N1-neutralizing human monoclonal antibody (mAb showing great potential for use either for therapeutic purposes or as a basis of vaccine development, but its antigenic epitope and neutralization mechanism have not been finely characterized. In this study, we first demonstrated that AVFluIgG01 targets a novel conformation-dependent epitope in the globular head region of H5N1 hemagglutinin (HA. By selecting mimotopes from a random peptide library in combination with computational algorithms and site-directed mutagenesis, the epitope was mapped to three conserved discontinuous sites (I-III that are located closely at the three-dimensional structure of HA. Further, we found that this HA1-specific human mAb can efficiently block both virus-receptor binding and post-attachment steps, while its Fab fragment exerts the post-attachment inhibition only. Consistently, AVFluIgG01 could inhibit HA-mediated cell-cell membrane fusion at a dose-dependent manner and block the acquisition of pH-induced protease sensitivity. These results suggest a neutralization mechanism of AVFluIgG01 by simultaneously blocking viral attachment to the receptors on host cells and interfering with HA conformational rearrangements associated with membrane fusion. The presented data provide critical information for developing novel antiviral therapeutics and vaccines against HPAI H5N1 virus.

  15. 人免疫缺陷病毒1型广谱中和抗体研究进展%Research Progress on HIV-1 Broadly Neutralizing Antibodies

    Institute of Scientific and Technical Information of China (English)

    范燕峰; 赵娟

    2015-01-01

    人类免疫缺陷病毒1型(HIV-1 )是获得性免疫缺陷综合征的主要致病因素. 目前为止依然缺乏能快速诱导出HIV特异性广谱中和抗体的有效疫苗. 一旦HIV-1潜伏在细胞内,则很难被彻底消除,如何活化潜伏感染的细胞进而进行药物治疗是摆在全世界科学家面前的又一难题. 而用药物控制HIV-1传播策略的贯彻落实也遇到了诸如经济、政策和社会等各方面的困难. 因此,新的预防策略的研究迫在眉睫.%Human immunodeficiency virus type 1 ( HIV-1 ) is the major pathogenic factor of acquired immunodeficiency disease ( AIDS ) .So far, there still has no effective vaccine developed that can rapidly induce broadly neutralizing antibodies.How to activate latently infected cells and then apply drug treatment is another problem faced by scientists around the world,because once the latent HIV-1 is in the cell,it is diffi-cult to be completely eliminated .Besides,the implementation of the strategy of controlling HIV-1 infection by anti-HIV drugs has encountered economic,policy,social and other difficulties.Therefore,researches on new prevention strategies are at urgent needs.

  16. Retargeted oncolytic adenovirus displaying a single variable domain of camelid heavy-chain-only antibody in a fiber protein.

    Science.gov (United States)

    van Erp, Elisabeth A; Kaliberova, Lyudmila N; Kaliberov, Sergey A; Curiel, David T

    2015-01-01

    Conditionally replicative adenoviruses are promising agents for oncolytic virotherapy. Various approaches have been attempted to retarget adenoviruses to tumor-specific antigens to circumvent deficiency of receptor for adenoviral binding and to provide an additional level of tumor specificity. Functional incorporation of highly specific targeting molecules into the viral capsid can potentially retarget adenoviral infection. However, conventional antibodies are not compatible with the cytoplasmic adenovirus capsid synthesis. The goal of this study was to evaluate the utility of single variable domains derived from heavy chain camelid antibodies for retargeting of adenovirus infection. We have combined transcriptional targeting using a tumor-specific promoter with transductional targeting through viral capsid incorporation of antihuman carcinoembryonic antigen single variable domains. Obtained data demonstrated that employment of a single variable domain genetically incorporated into an adenovirus fiber increased specificity of infection and efficacy of replication of single variable domain-targeted oncolytic adenovirus. The double targeting, both transcriptional through the C-X-C chemokine receptor type 4 promoter and transductional using the single variable domain, is a promising means to improve the therapeutic index for these advanced generation conditionally replicative adenoviruses. A successful strategy to transductional retargeting of oncolytic adenovirus infection has not been shown before and therefore we believe this is the first employment of transductional targeting using single variable domains derived from heavy chain camelid antibodies to enhance specificity of conditionally replicative adenoviruses.

  17. Antibody Engineering for Pursuing a Healthier Future

    Science.gov (United States)

    Saeed, Abdullah F. U. H.; Wang, Rongzhi; Ling, Sumei; Wang, Shihua

    2017-01-01

    Since the development of antibody-production techniques, a number of immunoglobulins have been developed on a large scale using conventional methods. Hybridoma technology opened a new horizon in the production of antibodies against target antigens of infectious pathogens, malignant diseases including autoimmune disorders, and numerous potent toxins. However, these clinical humanized or chimeric murine antibodies have several limitations and complexities. Therefore, to overcome these difficulties, recent advances in genetic engineering techniques and phage display technique have allowed the production of highly specific recombinant antibodies. These engineered antibodies have been constructed in the hunt for novel therapeutic drugs equipped with enhanced immunoprotective abilities, such as engaging immune effector functions, effective development of fusion proteins, efficient tumor and tissue penetration, and high-affinity antibodies directed against conserved targets. Advanced antibody engineering techniques have extensive applications in the fields of immunology, biotechnology, diagnostics, and therapeutic medicines. However, there is limited knowledge regarding dynamic antibody development approaches. Therefore, this review extends beyond our understanding of conventional polyclonal and monoclonal antibodies. Furthermore, recent advances in antibody engineering techniques together with antibody fragments, display technologies, immunomodulation, and broad applications of antibodies are discussed to enhance innovative antibody production in pursuit of a healthier future for humans.

  18. Transplastomic Nicotiana benthamiana plants expressing multiple defence genes encoding protease inhibitors and chitinase display broad-spectrum resistance against insects, pathogens and abiotic stresses.

    Science.gov (United States)

    Chen, Peng-Jen; Senthilkumar, Rajendran; Jane, Wann-Neng; He, Yong; Tian, Zhihong; Yeh, Kai-Wun

    2014-05-01

    Plastid engineering provides several advantages for the next generation of transgenic technology, including the convenient use of transgene stacking and the generation of high expression levels of foreign proteins. With the goal of generating transplastomic plants with multiresistance against both phytopathogens and insects, a construct containing a monocistronic patterned gene stack was transformed into Nicotiana benthamiana plastids harbouring sweet potato sporamin, taro cystatin and chitinase from Paecilomyces javanicus. Transplastomic lines were screened and characterized by Southern/Northern/Western blot analysis for the confirmation of transgene integration and respective expression level. Immunogold localization analyses confirmed the high level of accumulation proteins that were specifically expressed in leaf and root plastids. Subsequent functional bioassays confirmed that the gene stacks conferred a high level of resistance against both insects and phytopathogens. Specifically, larva of Spodoptera litura and Spodoptera exigua either died or exhibited growth retardation after ingesting transplastomic plant leaves. In addition, the inhibitory effects on both leaf spot diseases caused by Alternaria alternata and soft rot disease caused by Pectobacterium carotovorum subsp. carotovorum were markedly observed. Moreover, tolerance to abiotic stresses such as salt/osmotic stress was highly enhanced. The results confirmed that the simultaneous expression of sporamin, cystatin and chitinase conferred a broad spectrum of resistance. Conversely, the expression of single transgenes was not capable of conferring such resistance. To the best of our knowledge, this is the first study to demonstrate an efficacious stacked combination of plastid-expressed defence genes which resulted in an engineered tolerance to various abiotic and biotic stresses.

  19. Variable domain antibodies specific for viral hemorrhagic septicemia virus (VHSV) selected from a randomized IgNAR phage display library.

    Science.gov (United States)

    Ohtani, Maki; Hikima, Jun-ichi; Jung, Tae-Sung; Kondo, Hidehiro; Hirono, Ikuo; Takeyama, Haruko; Aoki, Takashi

    2013-02-01

    Phage display libraries are used to screen for nucleotide sequences that encode immunoglobulin variable (V) regions that are specific for a target antigen. We previously constructed an immunoglobulin new antigen receptor (IgNAR) phage display library. Here we used this library to obtain an IgNAR V region that is specific for viral hemorrhagic septicemia virus (VHSV). A phage clone (clone 653) was found to be specific for VHSV by the biopanning method. The V region of clone 653 was used to construct a 6 × His tagged recombinant IgNAR-653 V protein (rIgNAR-653) using the Escherichia coli pET system. The rIgNAR-653 protein bound specifically to VHSV, confirming its activity.

  20. An Exopolysaccharide-Deficient Mutant of Lactobacillus rhamnosus GG Efficiently Displays a Protective Llama Antibody Fragment against Rotavirus on Its Surface.

    Science.gov (United States)

    Álvarez, Beatriz; Krogh-Andersen, Kasper; Tellgren-Roth, Christian; Martínez, Noelia; Günaydın, Gökçe; Lin, Yin; Martín, M Cruz; Álvarez, Miguel A; Hammarström, Lennart; Marcotte, Harold

    2015-09-01

    Rotavirus is the leading cause of infantile diarrhea in developing countries, where it causes a high number of deaths among infants. Two vaccines are available, being highly effective in developed countries although markedly less efficient in developing countries. As a complementary treatment to the vaccines, a Lactobacillus strain producing an anti-rotavirus antibody fragment in the gastrointestinal tract could potentially be used. In order to develop such an alternative therapy, the effectiveness of Lactobacillus rhamnosus GG to produce and display a VHH antibody fragment (referred to as anti-rotavirus protein 1 [ARP1]) on the surface was investigated. L. rhamnosus GG is one of the best-characterized probiotic bacteria and has intrinsic antirotavirus activity. Among four L. rhamnosus GG strains [GG (CMC), GG (ATCC 53103), GG (NCC 3003), and GG (UT)] originating from different sources, only GG (UT) was able to display ARP1 on the bacterial surface. The genomic analysis of strain GG (UT) showed that the genes welE and welF of the EPS cluster are inactivated, which causes a defect in exopolysaccharide (EPS) production, allowing efficient display of ARP1 on its surface. Finally, GG (UT) seemed to confer a level of protection against rotavirus-induced diarrhea similar to that of wild-type GG (NCC 3003) in a mouse pup model, indicating that the EPS may not be involved in the intrinsic antirotavirus activity. Most important, GG (EM233), a derivative of GG (UT) producing ARP1, was significantly more protective than the control strain L. casei BL23.

  1. DNA immunization combined with scFv phage display identifies antagonistic GCGR specific antibodies and reveals new epitopes on the small extracellular loops.

    Science.gov (United States)

    van der Woning, Bas; De Boeck, Gitte; Blanchetot, Christophe; Bobkov, Vladimir; Klarenbeek, Alex; Saunders, Michael; Waelbroeck, Magali; Laeremans, Toon; Steyaert, Jan; Hultberg, Anna; De Haard, Hans

    2016-01-01

    The identification of functional monoclonal antibodies directed against G-protein coupled receptors (GPCRs) is challenging because of the membrane-embedded topology of these molecules. Here, we report the successful combination of llama DNA immunization with scFv-phage display and selections using virus-like particles (VLP) and the recombinant extracellular domain of the GPCR glucagon receptor (GCGR), resulting in glucagon receptor-specific antagonistic antibodies. By immunizing outbred llamas with plasmid DNA containing the human GCGR gene, we sought to provoke their immune system, which generated a high IgG1 response. Phage selections on VLPs allowed the identification of mAbs against the extracellular loop regions (ECL) of GCGR, in addition to multiple VH families interacting with the extracellular domain (ECD) of GCGR. Identifying mAbs binding to the ECL regions of GCGR is challenging because the large ECD covers the small ECLs in the energetically most favorable 'closed conformation' of GCGR. Comparison of Fab with scFv-phage display demonstrated that the multivalent nature of scFv display is essential for the identification of GCGR specific clones by selections on VLPs because of avid interaction. Ten different VH families that bound 5 different epitopes on the ECD of GCGR were derived from only 2 DNA-immunized llamas. Seven VH families demonstrated interference with glucagon-mediated cAMP increase. This combination of technologies proved applicable in identifying multiple functional binders in the class B GPCR context, suggesting it is a robust approach for tackling difficult membrane proteins.

  2. Establishment of bacteria display technology for Fab antibody library screening%筛选Fab抗体库的细菌展示技术的建立

    Institute of Scientific and Technical Information of China (English)

    徐黎明; 尹成凯; 任桂萍; 田辉; 王雪苯; 丁良君; 李德山

    2011-01-01

    目的:利用NlpA蛋白的前六个氨基酸(CDQSSS)将抗体锚定在细菌内膜建立筛选Fabs抗体库的展示技术,为今后抗体的研发工作奠定基础.方法:从pNAD质粒中克隆出NlpA leader(含有CDQSSS序列)基因序列,利用相应的酶切位点将该序列插入pComb3表达载体中构建成用于展示Fab的重组质粒pBFD.将从pEAI质粒克隆得到的anti-human IL-1β抗体的重链Fab和全长轻链分别插入到NlpA leader和pelB leader( pComb3载体自带的果胶酶基因前导肽)的下游.将pBFD-Fab转入到E.coli DH5α中诱导表达,原生质球制备后,采用梯度浓度的抗原进行孵育,最后经流式细胞术(FCM)检测抗体展示情况并且分选阳性群体,利用质粒提取的方法来替代PCR方法拯救阳性基因,转化E.coli DH5α,利用FCM再次检测该群体展示的抗体与抗原结合情况.结果:所展示的anti-hlL-1β Fab抗体依次与抗原和FITC标记的抗原特异性抗体孵育后,用FCM实时检测,结果显示出很强的荧光信号并且表现出抗原浓度依赖性.拯救出的pBFD-Fab-原生质球的FCM检测结果与首次展示的FCM结果一致,该系统能够稳定的展示抗体.结论:经过该细菌展示系统展示的Fab抗体能够有效的折叠,与相应的抗原具有很好的特异性结合能力.成功改进该展示技术的基因拯救方法,避免了基因突变和链置换的发生.此外还证明了该展示技术具有很好的稳定性.本实验成功构建了筛选Fab抗体库的细菌展技术.%AIM:To establish bacterial display technology for the purpose of Fab antibody library screening, by u-sing six amino acids (CDQSSS) of the amino termimus of NlpA protein to anchore antibodies to the periplasmic side of the bacterial inner membrane. METHODS: The NlpA Leader sequences (encoding CDQSSS) was amplified from pNAD plasmid. The PCR product was subcloned into pComb3 expression vector to generate Fab display vector pBFD. The heavy chains of the Fab gene

  3. Env-2dCD4 S60C complexes act as super immunogens and elicit potent, broadly neutralizing antibodies against clinically relevant human immunodeficiency virus type 1 (HIV-1).

    Science.gov (United States)

    Killick, Mark A; Grant, Michelle L; Cerutti, Nichole M; Capovilla, Alexio; Papathanasopoulos, Maria A

    2015-11-17

    The ability to induce a broadly neutralizing antibody (bNAb) response following vaccination is regarded as a crucial aspect in developing an effective vaccine against human immunodeficiency virus type 1 (HIV-1). The bNAbs target the HIV-1 envelope glycoprotein (Env) which is exposed on the virus surface, thereby preventing cell entry. To date, conventional vaccine approaches such as the use of Env-based immunogens have been unsuccessful. We expressed, purified, characterized and evaluated the immunogenicity of several unique HIV-1 subtype C Env immunogens in small animals. Here we report that vaccine immunogens based on Env liganded to a two domain CD4 variant, 2dCD4(S60C) are capable of consistently eliciting potent, broadly neutralizing antibody responses in New Zealand white rabbits against a panel of clinically relevant HIV-1 pseudoviruses. This was irrespective of the Env protein subtype and context. Importantly, depletion of the anti-CD4 antibodies appeared to abrogate the neutralization activity in the rabbit sera. Taken together, this data suggests that the Env-2dCD4(S60C) complexes described here are "super" immunogens, and potentially immunofocus antibody responses to a unique epitope spanning the 2dCD4(60C). Recent data from the two available anti-CD4 monoclonal antibodies, Ibalizumab and CD4-Ig (and bispecific variants thereof) have highlighted that the use of these broad and potent entry inhibitors could circumvent the need for a conventional vaccine targeting HIV-1. Overall, the ability of the unique Env-2dCD4(S60C) complexes to elicit potent bNAb responses has not been described previously, reinforcing that further investigation for their utility in preventing and controlling HIV-1/SIV infection is warranted.

  4. Human monoclonal antibodies derived from a patient infected with 2009 pandemic influenza A virus broadly cross-neutralize group 1 influenza viruses

    Energy Technology Data Exchange (ETDEWEB)

    Pan, Yang [Research Institute for Microbial Diseases, Osaka University, Suita, Osaka (Japan); Sasaki, Tadahiro [Research Institute for Microbial Diseases, Osaka University, Suita, Osaka (Japan); JST/JICA, Science and Technology Research Partnership for Sustainable Development (SATREPS), Tokyo (Japan); Kubota-Koketsu, Ritsuko [Research Institute for Microbial Diseases, Osaka University, Suita, Osaka (Japan); Kanonji Institute, The Research Foundation for Microbial Diseases of Osaka University, Kanonji, Kagawa (Japan); JST/JICA, Science and Technology Research Partnership for Sustainable Development (SATREPS), Tokyo (Japan); Inoue, Yuji [Research Institute for Microbial Diseases, Osaka University, Suita, Osaka (Japan); JST/JICA, Science and Technology Research Partnership for Sustainable Development (SATREPS), Tokyo (Japan); Yasugi, Mayo [Research Institute for Microbial Diseases, Osaka University, Suita, Osaka (Japan); Graduate School of Life and Environmental Sciences, Osaka Prefecture University, Izumisano, Osaka (Japan); JST/JICA, Science and Technology Research Partnership for Sustainable Development (SATREPS), Tokyo (Japan); Yamashita, Akifumi; Ramadhany, Ririn; Arai, Yasuha [Research Institute for Microbial Diseases, Osaka University, Suita, Osaka (Japan); Du, Anariwa [Research Institute for Microbial Diseases, Osaka University, Suita, Osaka (Japan); JST/JICA, Science and Technology Research Partnership for Sustainable Development (SATREPS), Tokyo (Japan); Boonsathorn, Naphatsawan [Research Institute for Microbial Diseases, Osaka University, Suita, Osaka (Japan); Department of Medical Sciences, Ministry of Public Health, Muang, Nonthaburi (Thailand); JST/JICA, Science and Technology Research Partnership for Sustainable Development (SATREPS), Tokyo (Japan); Ibrahim, Madiha S. [Research Institute for Microbial Diseases, Osaka University, Suita, Osaka (Japan); Department of Microbiology and Immunology, Faculty of Veterinary Medicine, Damanhour University, Damanhour (Egypt); and others

    2014-07-18

    Highlights: • Influenza infection can elicit heterosubtypic antibodies to group 1 influenza virus. • Three human monoclonal antibodies were generated from an H1N1-infected patient. • The antibodies predominantly recognized α-helical stem of viral hemagglutinin (HA). • The antibodies inhibited HA structural activation during the fusion process. • The antibodies are potential candidates for future antibody therapy to influenza. - Abstract: Influenza viruses are a continuous threat to human public health because of their ability to evolve rapidly through genetic drift and reassortment. Three human monoclonal antibodies (HuMAbs) were generated in this study, 1H11, 2H5 and 5G2, and they cross-neutralize a diverse range of group 1 influenza A viruses, including seasonal H1N1, 2009 pandemic H1N1 (H1N1pdm) and avian H5N1 and H9N2. The three HuMAbs were prepared by fusing peripheral blood lymphocytes from an H1N1pdm-infected patient with a newly developed fusion partner cell line, SPYMEG. All the HuMAbs had little hemagglutination inhibition activity but had strong membrane-fusion inhibition activity against influenza viruses. A protease digestion assay showed the HuMAbs targeted commonly a short α-helix region in the stalk of the hemagglutinin. Furthermore, Ile45Phe and Glu47Gly double substitutions in the α-helix region made the HA unrecognizable by the HuMAbs. These two amino acid residues are highly conserved in the HAs of H1N1, H5N1 and H9N2 viruses. The HuMAbs reported here may be potential candidates for the development of therapeutic antibodies against group 1 influenza viruses.

  5. Selection and characterization of naturally occurring single-domain (IgNAR) antibody fragments from immunized sharks by phage display.

    Science.gov (United States)

    Dooley, Helen; Flajnik, Martin F; Porter, Andrew J

    2003-09-01

    The novel immunoglobulin isotype novel antigen receptor (IgNAR) is found in cartilaginous fish and is composed of a heavy-chain homodimer that does not associate with light chains. The variable regions of IgNAR function as independent domains similar to those found in the heavy-chain immunoglobulins of Camelids. Here, we describe the successful cloning and generation of a phage-displayed, single-domain library based upon the variable domain of IgNAR. Selection of such a library generated from nurse sharks (Ginglymostoma cirratum) immunized with the model antigen hen egg-white lysozyme (HEL) enabled the successful isolation of intact antigen-specific binders matured in vivo. The selected variable domains were shown to be functionally expressed in Escherichia coli, extremely stable, and bind to antigen specifically with an affinity in the nanomolar range. This approach can therefore be considered as an alternative route for the isolation of minimal antigen-binding fragments with favorable characteristics.

  6. Fully human broadly neutralizing monoclonal antibodies against influenza A viruses generated from the memory B cells of a 2009 pandemic H1N1 influenza vaccine recipient

    Energy Technology Data Exchange (ETDEWEB)

    Hu, Weibin [Molecular Virus Unit, Key Laboratory of Molecular Virology and Immunology, Institut Pasteur of Shanghai, Chinese Academy of Sciences, Shanghai 200025 (China); Chen, Aizhong [Key Laboratory of Molecular Cell Biology, Institute of Biochemistry and Cell Biology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, Shanghai 200031 (China); Miao, Yi [Shanghai Xuhui Central Hospital, Shanghai 200031 (China); Xia, Shengli [Center for Disease Control and Prevention of Henan Province, Zhengzhou 450016 (China); Ling, Zhiyang; Xu, Ke; Wang, Tongyan [Molecular Virus Unit, Key Laboratory of Molecular Virology and Immunology, Institut Pasteur of Shanghai, Chinese Academy of Sciences, Shanghai 200025 (China); Xu, Ying; Cui, Jun; Wu, Hongqiang; Hu, Guiyu; Tian, Lin; Wang, Lingling [Key Laboratory of Molecular Cell Biology, Institute of Biochemistry and Cell Biology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, Shanghai 200031 (China); Shu, Yuelong [Chinese Center for Disease Control and Prevention, Beijing 102206 (China); Ma, Xiaowei [Hualan Biological Bacterin Company, Xinxiang 453003 (China); Xu, Bianli; Zhang, Jin [Center for Disease Control and Prevention of Henan Province, Zhengzhou 450016 (China); Lin, Xiaojun, E-mail: linxiaojun@hualan.com [Hualan Biological Bacterin Company, Xinxiang 453003 (China); Bian, Chao, E-mail: cbian@sibs.ac.cn [Key Laboratory of Molecular Cell Biology, Institute of Biochemistry and Cell Biology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, Shanghai 200031 (China); Sun, Bing, E-mail: bsun@sibs.ac.cn [Molecular Virus Unit, Key Laboratory of Molecular Virology and Immunology, Institut Pasteur of Shanghai, Chinese Academy of Sciences, Shanghai 200025 (China); Key Laboratory of Molecular Cell Biology, Institute of Biochemistry and Cell Biology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, Shanghai 200031 (China)

    2013-01-20

    Whether the 2009 pandemic H1N1 influenza vaccine can induce heterosubtypic cross-protective anti-hemagglutinin (HA) neutralizing antibodies is an important issue. We obtained a panel of fully human monoclonal antibodies from the memory B cells of a 2009 pandemic H1N1 influenza vaccine recipient. Most of the monoclonal antibodies targeted the HA protein but not the HA1 fragment. Among the analyzed antibodies, seven mAbs exhibited neutralizing activity against several influenza A viruses of different subtypes. The conserved linear epitope targeted by the neutralizing mAbs (FIEGGWTGMVDGWYGYHH) is part of the fusion peptide on HA2. Our work suggests that a heterosubtypic neutralizing antibody response primarily targeting the HA stem region exists in recipients of the 2009 pandemic H1N1 influenza vaccine. The HA stem region contains various conserved neutralizing epitopes with the fusion peptide as an important one. This work may aid in the design of a universal influenza A virus vaccine.

  7. A strategy for the generation of specific human antibodies by directed evolution and phage display. An example of a single-chain antibody fragment that neutralizes a major component of scorpion venom.

    Science.gov (United States)

    Riaño-Umbarila, Lidia; Juárez-González, Victor Rivelino; Olamendi-Portugal, Timoteo; Ortíz-León, Mauricio; Possani, Lourival Domingos; Becerril, Baltazar

    2005-05-01

    This study describes the construction of a library of single-chain antibody fragments (scFvs) from a single human donor by individual amplification of all heavy and light variable domains (1.1 x 10(8) recombinants). The library was panned using the phage display technique, which allowed selection of specific scFvs (3F and C1) capable of recognizing Cn2, the major toxic component of Centruroides noxius scorpion venom. The scFv 3F was matured in vitro by three cycles of directed evolution. The use of stringent conditions in the third cycle allowed the selection of several improved clones. The best scFv obtained (6009F) was improved in terms of its affinity by 446-fold, from 183 nm (3F) to 410 pm. This scFv 6009F was able to neutralize 2 LD(50) of Cn2 toxin when a 1 : 10 molar ratio of toxin-to-antibody fragment was used. It was also able to neutralize 2 LD(50) of the whole venom. These results pave the way for the future generation of recombinant human antivenoms.

  8. Complete epitopes for vaccine design derived from a crystal structure of the broadly neutralizing antibodies PGT128 and 8ANC195 in complex with an HIV-1 Env trimer.

    Science.gov (United States)

    Kong, Leopold; Torrents de la Peña, Alba; Deller, Marc C; Garces, Fernando; Sliepen, Kwinten; Hua, Yuanzi; Stanfield, Robyn L; Sanders, Rogier W; Wilson, Ian A

    2015-10-01

    The HIV-1 envelope gp160 glycoprotein (Env) is a trimer of gp120 and gp41 heterodimers that mediates cell entry and is the primary target of the humoral immune response. Broadly neutralizing antibodies (bNAbs) to HIV-1 have revealed multiple epitopes or sites of vulnerability, but mapping of most of these sites is incomplete owing to a paucity of structural information on the full epitope in the context of the Env trimer. Here, a crystal structure of the soluble BG505 SOSIP gp140 trimer at 4.6 Å resolution with the bNAbs 8ANC195 and PGT128 reveals additional interactions in comparison to previous antibody-gp120 structures. For 8ANC195, in addition to previously documented interactions with gp120, a substantial interface with gp41 is now elucidated that includes extensive interactions with the N637 glycan. Surprisingly, removal of the N637 glycan did not impact 8ANC195 affinity, suggesting that the antibody has evolved to accommodate this glycan without loss of binding energy. PGT128 indirectly affects the N262 glycan by a domino effect, in which PGT128 binds to the N301 glycan, which in turn interacts with and repositions the N262 glycan, thereby illustrating the important role of neighboring glycans on epitope conformation and stability. Comparisons with other Env trimer and gp120 structures support an induced conformation for glycan N262, suggesting that the glycan shield is allosterically modified upon PGT128 binding. These complete epitopes of two broadly neutralizing antibodies on the Env trimer can now be exploited for HIV-1 vaccine design.

  9. Drift of the HIV-1 envelope glycoprotein gp120 toward increased neutralization resistance over the course of the epidemic: a comprehensive study using the most potent and broadly neutralizing monoclonal antibodies.

    Science.gov (United States)

    Bouvin-Pley, M; Morgand, M; Meyer, L; Goujard, C; Moreau, A; Mouquet, H; Nussenzweig, M; Pace, C; Ho, D; Bjorkman, P J; Baty, D; Chames, P; Pancera, M; Kwong, P D; Poignard, P; Barin, F; Braibant, M

    2014-12-01

    Extending our previous analyses to the most recently described monoclonal broadly neutralizing antibodies (bNAbs), we confirmed a drift of HIV-1 clade B variants over 2 decades toward higher resistance to bNAbs targeting almost all the identified gp120-neutralizing epitopes. In contrast, the sensitivity to bNAbs targeting the gp41 membrane-proximal external region remained stable, suggesting a selective pressure on gp120 preferentially. Despite this evolution, selected combinations of bNAbs remain capable of neutralizing efficiently most of the circulating variants.

  10. Influenza virus antigenicity and broadly neutralizing epitopes.

    Science.gov (United States)

    Air, Gillian M

    2015-04-01

    A vaccine formulation that would be effective against all strains of influenza virus has long been a goal of vaccine developers, but antibodies after infection or vaccination were seen to be strain specific and there was little evidence of cross-reactive antibodies that neutralized across subtypes. Recently a number of broadly neutralizing monoclonal antibodies have been characterized. This review describes the different classes of broadly neutralizing antibodies and discusses the potential of their therapeutic use or for design of immunogens that induce a high proportion of broadly neutralizing antibodies.

  11. Non-random escape pathways from a broadly neutralizing human monoclonal antibody map to a highly conserved region on the hepatitis C virus E2 glycoprotein encompassing amino acids 412-423.

    Directory of Open Access Journals (Sweden)

    Zhen-yong Keck

    2014-08-01

    Full Text Available A challenge for hepatitis C virus (HCV vaccine development is to define epitopes that are able to elicit protective antibodies against this highly diverse virus. The E2 glycoprotein region located at residues 412-423 is conserved and antibodies to 412-423 have broadly neutralizing activities. However, an adaptive mutation, N417S, is associated with a glycan shift in a variant that cannot be neutralized by a murine but by human monoclonal antibodies (HMAbs against 412-423. To determine whether HCV escapes from these antibodies, we analyzed variants that emerged when cell culture infectious HCV virions (HCVcc were passaged under increasing concentrations of a specific HMAb, HC33.1. Multiple nonrandom escape pathways were identified. Two pathways occurred in the context of an N-glycan shift mutation at N417T. At low antibody concentrations, substitutions of two residues outside of the epitope, N434D and K610R, led to variants having improved in vitro viral fitness and reduced sensitivity to HC33.1 binding and neutralization. At moderate concentrations, a S419N mutation occurred within 412-423 in escape variants that have greatly reduced sensitivity to HC33.1 but compromised viral fitness. Importantly, the variants generated from these pathways differed in their stability. N434D and K610R-associated variants were stable and became dominant as the virions were passaged. The S419N mutation reverted back to N419S when immune pressure was reduced by removing HC33.1. At high antibody concentrations, a mutation at L413I was observed in variants that were resistant to HC33.1 neutralization. Collectively, the combination of multiple escape pathways enabled the virus to persist under a wide range of antibody concentrations. Moreover, these findings pose a different challenge to vaccine development beyond the identification of highly conserved epitopes. It will be necessary for a vaccine to induce high potency antibodies that prevent the formation of escape

  12. Novel Clostridium difficile Anti-Toxin (TcdA and TcdB Humanized Monoclonal Antibodies Demonstrate In Vitro Neutralization across a Broad Spectrum of Clinical Strains and In Vivo Potency in a Hamster Spore Challenge Model.

    Directory of Open Access Journals (Sweden)

    Hongyu Qiu

    Full Text Available Clostridium difficile (C. difficile infection (CDI is the main cause of nosocomial antibiotic-associated colitis and increased incidence of community-associated diarrhea in industrialized countries. At present, the primary treatment of CDI is antibiotic administration, which is effective but often associated with recurrence, especially in the elderly. Pathogenic strains produce enterotoxin, toxin A (TcdA, and cytotoxin, toxin B (TcdB, which are necessary for C. difficile induced diarrhea and gut pathological changes. Administration of anti-toxin antibodies provides an alternative approach to treat CDI, and has shown promising results in preclinical and clinical studies. In the current study, several humanized anti-TcdA and anti-TcdB monoclonal antibodies were generated and their protective potency was characterized in a hamster infection model. The humanized anti-TcdA (CANmAbA4 and anti-TcdB (CANmAbB4 and CANmAbB1 antibodies showed broad spectrum in vitro neutralization of toxins from clinical strains and neutralization in a mouse toxin challenge model. Moreover, co-administration of humanized antibodies (CANmAbA4 and CANmAbB4 cocktail provided a high level of protection in a dose dependent manner (85% versus 57% survival at day 22 for 50 mg/kg and 20 mg/kg doses, respectively in a hamster gastrointestinal infection (GI model. This study describes the protective effects conferred by novel neutralizing anti-toxin monoclonal antibodies against C. difficile toxins and their potential as therapeutic agents in treating CDI.

  13. Why Does the Molecular Structure of Broadly Neutralizing Monoclonal Antibodies Isolated from Individuals Infected with HIV-1 not Inform the Rational Design of an HIV-1 Vaccine?

    Directory of Open Access Journals (Sweden)

    Marc H V Van Regenmortel

    2015-05-01

    Full Text Available It is commonly assumed that neutralizing Mabs that bind to the HIV-1 Env glycoprotein are more specific reagents than anti-HIV-1 polyclonal antisera and that knowledge of the structure of these Mabs facilitates the rational design of effective HIV-1 vaccine immunogens. However, after more than ten years of unsuccessful experimentation using the structure-based reverse vaccinology approach, it is now evident that it is not possible to infer from the structure of neutralizing Mabs which HIV immunogens induced their formation nor which vaccine immunogens will elicit similar Abs in an immunized host. The use of Mabs for developing an HIV-1 vaccine was counterproductive because it overlooked the fact that the apparent specificity of a Mab very much depends on the selection procedure used to obtain it and also did not take into account that an antibody is never monospecific for a single epitope but is always polyspecific for many epitopes. When the rationale of the proponents of the unsuccessful rational design strategy is analyzed, it appears that investigators who claim they are designing a vaccine immunogen are only improving the binding reactivity of a single epitope-paratope pair and are not actually designing an immunogen able to generate protective antibodies. The task of a designer consists in imagining what type of immunogen is likely to elicit a protective immune response but in the absence of knowledge regarding which features of the immune system are responsible for producing a functional neutralizing activity in antibodies, it is not feasible to intentionally optimize a potential immunogen candidate in order to obtain the desired outcome. The only available option is actually to test possible solutions by trial-and-error experiments until the preset goal is perhaps attained. Rational design and empirical approaches in HIV vaccine research should thus not be opposed as alternative options since empirical testing is an integral part of a so

  14. Human monoclonal antibodies derived from a patient infected with 2009 pandemic influenza A virus broadly cross-neutralize group 1 influenza viruses.

    Science.gov (United States)

    Pan, Yang; Sasaki, Tadahiro; Kubota-Koketsu, Ritsuko; Inoue, Yuji; Yasugi, Mayo; Yamashita, Akifumi; Ramadhany, Ririn; Arai, Yasuha; Du, Anariwa; Boonsathorn, Naphatsawan; Ibrahim, Madiha S; Daidoji, Tomo; Nakaya, Takaaki; Ono, Ken-ichiro; Okuno, Yoshinobu; Ikuta, Kazuyoshi; Watanabe, Yohei

    2014-07-18

    Influenza viruses are a continuous threat to human public health because of their ability to evolve rapidly through genetic drift and reassortment. Three human monoclonal antibodies (HuMAbs) were generated in this study, 1H11, 2H5 and 5G2, and they cross-neutralize a diverse range of group 1 influenza A viruses, including seasonal H1N1, 2009 pandemic H1N1 (H1N1pdm) and avian H5N1 and H9N2. The three HuMAbs were prepared by fusing peripheral blood lymphocytes from an H1N1pdm-infected patient with a newly developed fusion partner cell line, SPYMEG. All the HuMAbs had little hemagglutination inhibition activity but had strong membrane-fusion inhibition activity against influenza viruses. A protease digestion assay showed the HuMAbs targeted commonly a short α-helix region in the stalk of the hemagglutinin. Furthermore, Ile45Phe and Glu47Gly double substitutions in the α-helix region made the HA unrecognizable by the HuMAbs. These two amino acid residues are highly conserved in the HAs of H1N1, H5N1 and H9N2 viruses. The HuMAbs reported here may be potential candidates for the development of therapeutic antibodies against group 1 influenza viruses.

  15. B cells from knock-in mice expressing broadly neutralizing HIV antibody b12 carry an innocuous B cell receptor responsive to HIV vaccine candidates.

    Science.gov (United States)

    Ota, Takayuki; Doyle-Cooper, Colleen; Cooper, Anthony B; Doores, Katherine J; Aoki-Ota, Miyo; Le, Khoa; Schief, William R; Wyatt, Richard T; Burton, Dennis R; Nemazee, David

    2013-09-15

    Broadly neutralizing Abs against HIV protect from infection, but their routine elicitation by vaccination has not been achieved. To generate small animal models to test vaccine candidates, we have generated targeted transgenic ("knock-in") mice expressing, in the physiological Ig H and L chain loci, two well-studied broadly neutralizing Abs: 4E10, which interacts with the membrane proximal external region of gp41, and b12, which binds to the CD4 binding site on gp120. 4E10HL mice are described in the companion article (Doyle-Cooper et al., J. Immunol. 191: 3186-3191). In this article, we describe b12 mice. B cells in b12HL mice, in contrast to the case in 4E10 mice, were abundant and essentially monoclonal, retaining the b12 specificity. In cell culture, b12HL B cells responded avidly to HIV envelope gp140 trimers and to BCR ligands. Upon transfer to wild-type recipients, b12HL B cells responded robustly to vaccination with gp140 trimers. Vaccinated b12H mice, although generating abundant precursors and Abs with affinity for Env, were unable to rapidly generate neutralizing Abs, highlighting the importance of developing Ag forms that better focus responses to neutralizing epitopes. The b12HL and b12H mice should be useful in optimizing HIV vaccine candidates to elicit a neutralizing response while avoiding nonprotective specificities.

  16. Antithyroglobulin antibody

    Science.gov (United States)

    Thyroglobulin antibody; Thyroiditis - thyroglobulin antibody; Hypothyroidism - thyroglobulin antibody; Thyroiditis - thyroglobulin antibody; Graves disease - thyroglobulin antibody; Underactive thyroid - thyroglobulin antibody

  17. Amplification and polyclonal antibody preparation of M13 phage display library%M13噬菌体肽库扩增及兔抗血清制备

    Institute of Scientific and Technical Information of China (English)

    任晓峰; 马晓微

    2013-01-01

    为制备M13噬菌体多克隆抗体,将噬菌体十二肽原始文库进行大量扩增,作为免疫原制备兔抗M13的多克隆抗体血清并进行间接ELISA鉴定.结果表明,该多抗效价达1∶1 048 576,说明此多抗可与扩增噬菌体文库发生很好的抗原抗体反应.将制备的噬菌体M13多克隆抗体与商业化M13抗体水平比较,制备多抗与商业化M13抗体效果相当.本研究成功制备兔抗M13噬菌体多克隆抗体,为深入研究噬菌体展示技术提供了材料.%In order to generate polyclonal antibodies of the M13 Phage,one M13 of Ph..D.-12TM Phage Display Peptide Library was amplified and used to immunize a rabbit to generate polyclonal antibody.Indirect ELISA analysis showed that the titer of the polyclonal antibody was approximately 1∶1 048 576,showing that the anti-M13 antibody had a high titer.Compared this polyclonal antibody with a Rabbit polyclonal antibody (Rb pAb) to M13 Bacteriophage Coat Proteins (commercialization),the binding activity of the produced polyclonal antibody to the identical target was as good as that of the Rb pAb to M13 Bacteriophage Coat Proteins.In the study,polyclonal antibody to this M13 of phage library was successfully generated and such phage polyclonal antibody is important material for functional analysis with phage.

  18. Detection of antibodies to a pathogenic mycoplasma in American alligators (Alligator mississippiensis), broad-nosed Caimans (Caiman latirostris), and Siamese crocodiles (Crocodylus siamensis).

    Science.gov (United States)

    Brown, D R; Schumacher, I M; Nogueira, M F; Richey, L J; Zacher, L A; Schoeb, T R; Vliet, K A; Bennett, R A; Jacobson, E R; Brown, M B

    2001-01-01

    An epidemic of pneumonia with fibrinous polyserositis and multifocal arthritis emerged in captive American alligators (Alligator mississippiensis) in Florida, United States, in 1995. Mycoplasma alligatoris sp. nov. was cultured from multiple organs, peripheral blood, synovial fluid, and cerebrospinal fluid of affected alligators. In a subsequent experimental inoculation study, the Henle-Koch-Evans postulates were fulfilled for M. alligatoris as the etiological agent of fatal mycoplasmosis of alligators. That finding was remarkable because mycoplasmal disease is rarely fatal in animals. An enzyme-linked immunosorbent assay (ELISA) for the detection of antibodies produced by alligators in response to M. alligatoris exposure was developed by using plasma obtained from naturally infected alligators during the original epidemic. The assay was validated by using plasma obtained during an experimental dose-response study and applied to analyze plasma obtained from captive and wild crocodilian species. The ELISA reliably detected alligator seroconversion (P crocodile Crocodylus siamensis following experimental inoculation with M. alligatoris. The ELISA may be used to monitor exposure to the lethal pathogen M. alligatoris among captive, repatriated, and wild crocodilian species.

  19. Host-Primed Ebola Virus GP Exposes a Hydrophobic NPC1 Receptor-Binding Pocket, Revealing a Target for Broadly Neutralizing Antibodies

    Directory of Open Access Journals (Sweden)

    Zachary A. Bornholdt

    2016-02-01

    Full Text Available The filovirus surface glycoprotein (GP mediates viral entry into host cells. Following viral internalization into endosomes, GP is cleaved by host cysteine proteases to expose a receptor-binding site (RBS that is otherwise hidden from immune surveillance. Here, we present the crystal structure of proteolytically cleaved Ebola virus GP to a resolution of 3.3 Å. We use this structure in conjunction with functional analysis of a large panel of pseudotyped viruses bearing mutant GP proteins to map the Ebola virus GP endosomal RBS at molecular resolution. Our studies indicate that binding of GP to its endosomal receptor Niemann-Pick C1 occurs in two distinct stages: the initial electrostatic interactions are followed by specific interactions with a hydrophobic trough that is exposed on the endosomally cleaved GP1 subunit. Finally, we demonstrate that monoclonal antibodies targeting the filovirus RBS neutralize all known filovirus GPs, making this conserved pocket a promising target for the development of panfilovirus therapeutics.

  20. The Broad Anti-AML Activity of the CD33/CD3 BiTE Antibody Construct, AMG 330, Is Impacted by Disease Stage and Risk.

    Science.gov (United States)

    Harrington, Kimberly H; Gudgeon, Chelsea J; Laszlo, George S; Newhall, Kathryn J; Sinclair, Angus M; Frankel, Stanley R; Kischel, Roman; Chen, Guang; Walter, Roland B

    2015-01-01

    The CD33/CD3-bispecific T-cell engaging (BiTE) antibody construct, AMG 330, potently lyses CD33+ leukemic cells in vitro. Using specimens from 41 patients with acute myeloid leukemia (AML), we studied the factors that might contribute to clinical response or resistance. For this purpose, thawed aliquots of primary AML samples were immunophenotypically characterized and subjected to various doses of AMG 330 in the presence or absence of healthy donor T-cells. After 48 hours, drug-specific cytotoxicity was quantified and correlated with CD33 expression levels, amounts of T-cells present, and other disease characteristics. AMG 330 caused modest cytotoxicity that was correlated with the amount of autologous T-cells (P = 0.0001) but not CD33 expression, as AMG 330 exerted marked cytotoxic effects in several specimens with minimal CD33 expression. With healthy donor T-cells added, AMG 330 cytotoxicity depended on the drug dose and effector:target (E:T) cell ratio. High cytotoxic activity was observed even with minimal CD33 expression, and AMG 330 cytotoxicity and CD33 expression correlated only at high E:T cell ratio and high AMG 330 doses (Pspectrum, while suggesting the presence of yet undefined, CD33-independent, relative resistance mechanisms in specific patient subsets.

  1. The Broad Anti-AML Activity of the CD33/CD3 BiTE Antibody Construct, AMG 330, Is Impacted by Disease Stage and Risk.

    Directory of Open Access Journals (Sweden)

    Kimberly H Harrington

    Full Text Available The CD33/CD3-bispecific T-cell engaging (BiTE antibody construct, AMG 330, potently lyses CD33+ leukemic cells in vitro. Using specimens from 41 patients with acute myeloid leukemia (AML, we studied the factors that might contribute to clinical response or resistance. For this purpose, thawed aliquots of primary AML samples were immunophenotypically characterized and subjected to various doses of AMG 330 in the presence or absence of healthy donor T-cells. After 48 hours, drug-specific cytotoxicity was quantified and correlated with CD33 expression levels, amounts of T-cells present, and other disease characteristics. AMG 330 caused modest cytotoxicity that was correlated with the amount of autologous T-cells (P = 0.0001 but not CD33 expression, as AMG 330 exerted marked cytotoxic effects in several specimens with minimal CD33 expression. With healthy donor T-cells added, AMG 330 cytotoxicity depended on the drug dose and effector:target (E:T cell ratio. High cytotoxic activity was observed even with minimal CD33 expression, and AMG 330 cytotoxicity and CD33 expression correlated only at high E:T cell ratio and high AMG 330 doses (P<0.003. AMG 330 resulted in significantly higher cytotoxicity in specimens from patients with newly diagnosed AML than those with relapsed/refractory disease despite similar levels of CD33 on myeloblasts. AMG 330 cytotoxicity also appeared greater in specimens from patients with favorable-risk disease as compared to other specimens. Together, our data demonstrate that AMG 330 is highly active in primary AML specimens across the entire disease spectrum, while suggesting the presence of yet undefined, CD33-independent, relative resistance mechanisms in specific patient subsets.

  2. The isolation of novel phage display-derived human recombinant antibodies against CCR5, the major co-receptor of HIV.

    Science.gov (United States)

    Shimoni, Moria; Herschhorn, Alon; Britan-Rosich, Yelena; Kotler, Moshe; Benhar, Itai; Hizi, Amnon

    2013-08-01

    Selecting for antibodies against specific cell-surface proteins is a difficult task due to many unrelated proteins that are expressed on the cell surface. Here, we describe a method to screen antibody-presenting phage libraries against native cell-surface proteins. We applied this method to isolate antibodies that selectively recognize CCR5, which is the major co-receptor for HIV entry (consequently, playing a pivotal role in HIV transmission and pathogenesis). We employed a phage screening strategy by using cells that co-express GFP and CCR5, along with an excess of control cells that do not express these proteins (and are otherwise identical to the CCR5-expressing cells). These control cells are intended to remove most of the phages that bind the cells nonspecifically; thus leading to an enrichment of the phages presenting anti-CCR5-specific antibodies. Subsequently, the CCR5-presenting cells were quantitatively sorted by flow cytometry, and the bound phages were eluted, amplified, and used for further successive selection rounds. Several different clones of human single-chain Fv antibodies that interact with CCR5-expressing cells were identified. The most specific monoclonal antibody was converted to a full-length IgG and bound the second extracellular loop of CCR5. The experimental approach presented herein for screening for CCR5-specific antibodies can be applicable to screen antibody-presenting phage libraries against any cell-surface expressed protein of interest.

  3. Phage display derived human monoclonal antibodies isolated by binding to the surface of live primary breast cancer cells recognize GRP78

    DEFF Research Database (Denmark)

    Jakobsen, Charlotte G; Rasmussen, Nicolaj; Laenkholm, Anne-Vibeke

    2007-01-01

    Clinical trials using monoclonal antibodies (mAb) against cell-surface markers have yielded encouraging therapeutic results in several cancer types. Generally, however, anticancer antibodies are only efficient against a subpopulation of cancers, and there is a strong need for identification of no...... therapies including mAb-based immunotherapy. Our results suggest that the human antibody Ab39 may be a useful starting point for further genetic optimization that could render it a useful diagnostic and therapeutic reagent for a variety of cancers...

  4. Broadly neutralizing antibodies against human immunodeficiency virus type 1 (HIV-1) and rational design of HIV-1 vaccines%抗人类免疫缺陷病毒广谱中和抗体与疫苗设计

    Institute of Scientific and Technical Information of China (English)

    王若珂; 郭建影; 张林琦

    2013-01-01

    自发现人类免疫缺陷病毒1型(HIV-1)30年来,科学家们不断探索有效的 HIV-1疫苗,但至今效果不理想。由于“精英患者”的广谱中和血清能起到有效的抗病毒作用,研究人员希望通过对体液保护免疫机制的研究,推动疫苗的设计和优化,为尽早研发成功 HIV疫苗提供关键理论和技术支撑。近几年来,由于技术的突破和改进,从HIV感染者中分离获得广谱中和抗体的概率和数量大大提高。本文针对近几年来分离的有代表性的广谱中和抗体,根据其不同的识别位点分为四大类进行详细介绍,并总结了从研究广谱中和抗体中所获得的疫苗设计创新思路与启示。%Antibody response is a crucial host defense against human immunodeficiency virus type 1 (HIV-1) infection .In recent years , tremendous progress has been made in isolating and characterizing of broadly neutralizing monoclonal antibodies (bnmAbs ) from “elite neutralizers” who remain healthy despite of prolonged infection . These bnmAbs were found to target four major vulnerable sites on the envelope glycoprotein .Structural and functional characterization of these bnmAbs has provided critical foundation for our better understanding of protective antibody response in vivo .In the current review ,we summarize the characteristics of these bnmAbs and discuss on their implications for rational design of novel HIV-1 vaccines capable of inducing antibodies similar to those bnmAbs .

  5. Modulation of the CD40-CD40 ligand interaction using human anti-CD40 single-chain antibody fragments obtained from the n-CoDeR phage display library.

    Science.gov (United States)

    Ellmark, Peter; Ottosson, Camilla; Borrebaeck, Carl A K; Malmborg Hager, Ann-Christin; Furebring, Christina

    2002-08-01

    CD40 plays a central regulatory role in the immune system and antibodies able to modulate CD40 signalling may consequently have a potential in immunotherapy, in particular for treatment of lymphomas and autoimmune disease like multiple sclerosis. As a first step to achieve this goal, we describe the selection and characterization of a novel set of fully human anti-CD40 antibody fragments (scFv) from a phage display library (n-CoDeR). In order to determine their biological potential, these antibody fragments have been analysed for their ability to promote B-cell activation, rescue from apoptosis and to block the CD40-CD40 ligand (CD40L) interaction. The selected cohort of human scFv could be subcategorized, each expressing a distinct functional signature. Thus scFv were generated that induced B-cell proliferation, rescued B cells from apoptosis and blocked the CD40-CD40L interaction to different extents. In particular, one of the scFv clones (F33) had the ability to abrogate completely this interaction. The epitope recognition patterns as well as individual rate constants were also determined and the affinity was shown to vary from low to high nanomolar range. In conclusion, this panel of human anti-CD40 scFv fragments displays a number of distinct properties, which may constitute a valuable source when evaluating candidates for in vivo trials.

  6. Phage Display Approaches for the Isolation of Monoclonal Antibodies Against Dengue Virus Envelope Domain III from Human and Mouse Derived Libraries

    Directory of Open Access Journals (Sweden)

    Subhash G. Vasudevan

    2012-02-01

    Full Text Available Domain III of the dengue virus envelope protein (EDIII, aa295-395 has an immunoglobulin fold and is the proposed receptor-binding domain of the virus. Previous studies have shown that monoclonal antibodies against EDIII can be neutralizing and have therapeutic potential. Here, cloned Fab-phage libraries of human and mouse origin were screened for DENV specific antibodies. Firstly, bacterially expressed EDIII or whole virus particles were used as bait in biopanning against a large naïve human Fab-phage library ( > 10 billion independent clones. Multiple panning strategies were employed, and in excess of 1000 clones were screened, but all of the antibodies identified bound the envelope in regions outside EDIII suggesting EDIII antibodies are virtually absent from the naïve human repertoire. Next, a chimeric Fab-phage library was constructed from a panel of EDIII specific mouse hybridomas by pooling the VH and VL chain sequences from the hybridomas and cloning these into the pComb3X phagemid vector with human CH and CL encoding sequences. Biopanning against EDIII identified a unique antibody (C9 that cross-reacts with EDIII from DENV1-3 and, in the IgG format, binds and neutralizes DENV2 in cell-based assays. Sequence analysis and saturation mutagenesis of complementary determining regions (CDR in the C9 light chain suggest an antigen recognition model in which the LCDR3 is a key determinant of EDIII specificity, while modifications in LCDR1 and LCDR2 affect DENV serotype cross-reactivity. Overall, this study supports the current prevailing opinion that neutralizing anti-EDIII monoclonal antibodies can be readily generated in murine systems, but in humans the anti-DENV immune response is directed away from domain III.

  7. Generation of a phage-display library of single-domain camelid VH H antibodies directed against Chlamydomonas reinhardtii antigens, and characterization of VH Hs binding cell-surface antigens.

    Science.gov (United States)

    Jiang, Wenzhi; Rosenberg, Julian N; Wauchope, Akelia D; Tremblay, Jacqueline M; Shoemaker, Charles B; Weeks, Donald P; Oyler, George A

    2013-11-01

    Single-domain antibodies (sdAbs) are powerful tools for the detection, quantification, purification and subcellular localization of proteins of interest in biological research. We have generated camelid (Lama pacos) heavy chain-only variable VH domain (VH H) libraries against antigens in total cell lysates from Chlamydomonas reinhardtii. The sdAbs in the sera from immunized animals and VH H antibody domains isolated from the library show specificity to C. reinhardtii and lack of reactivity to antigens from four other algae: Chlorella variabilis, Coccomyxa subellipsoidea, Nannochloropsis oceanica and Thalassiosira pseudonana. Antibodies were produced against a diverse representation of antigens as evidenced by sera ELISA and protein-blot analyses. A phage-display library consisting of the VH H region contained at least 10(6) individual transformants, and thus should represent a wide range of C. reinhardtii antigens. The utility of the phage library was demonstrated by using live C. reinhardtii cells to pan for VH H clones with specific recognition of cell-surface epitopes. The lead candidate VH H clones (designated B11 and H10) bound to C. reinhardtii with EC50 values ≤ 0.5 nm. Treatment of cells with VH H B11 fused to the mCherry or green fluorescent proteins allowed brilliant and specific staining of the C. reinhardtii cell wall and analysis of cell-wall genesis during cell division. Such high-complexity VH H antibody libraries for algae will be valuable tools for algal researchers and biotechnologists.

  8. Two ScFv antibody libraries derived from identical VL-VH framework with different binding site designs display distinct binding profiles.

    Science.gov (United States)

    Huovinen, Tuomas; Syrjänpää, Markku; Sanmark, Hanna; Brockmann, Eeva-Christine; Azhayev, Alex; Wang, Qi; Vehniäinen, Markus; Lamminmäki, Urpo

    2013-10-01

    In directed evolution experiments, a single randomization scheme of an antibody gene does not provide optimal diversity for recognition of all sizes of antigens. In this study, we have expanded the recognition potential of our universal library, termed ScFvP, with a second distinct diversification scheme. In the second library, termed ScFvM, diversity was designed closer to the center of the antigen binding site in the same antibody framework as earlier. Also, the CDR-H3 loop structures were redesigned to be shorter, 5-12 aa and mostly without the canonical salt bridge between Arg106H and Asp116H to increase the flexibility of the loop and to allow more space in the center of the paratope for binding smaller targets. Antibodies were selected from the two libraries against various antigens separately and as a mixture. The origin and characteristics of the retrieved antibodies indicate that complementary diversity results in complementary functionality widening the spectrum of targets amenable for selection.

  9. Construction and characterization of a non-immune Llama variable heavy chain phage display antibody library%羊驼非免疫重链单域抗体库的构建和鉴定

    Institute of Scientific and Technical Information of China (English)

    吴标; 王树军; 夏立亮; 季萍; 葛海良; 赵国屏; 王颖

    2011-01-01

    本研究旨在通过构建羊驼非免疫重链单域抗体库,完成抗体库多样性的鉴定,为进一步筛选抗原特异性重链抗体奠定基础.我们从未经免疫的羊驼外周血中分离外周血单个核细胞(PBMC),抽提RNA后,用RT-PCR方法特异性扩增羊驼重链抗体可变区(VHH)片段;并采用两步连接方法将重链抗体可变区片段与噬菌粒载体pCANTAB5E连接获得重组子,多次电转感受态大肠杆菌TG1后获得VHH抗体基因库;并采用稀释计数法测定抗体库库容量,随机挑取克隆测序验证抗体库多样性.结果显示,我们所构建的羊驼非免疫重链单域抗体库的库容量为1.5×109,随机克隆测序验证多样性良好,独立克隆所占比例为80%,并显示出和人源抗体较高的同源性.上述结果表明,我们已经成功构建获得大容量的羊驼非免疫重链单域抗体库,为进一步筛选抗原特异性重链抗体奠定基础.%To construct a non-immune Llama variable domain of heavy chain antibody(VHH) phage display antibody library (VHH antibody library). Llama peripheral blood mononuclear lymphocytes were isolated from whole blood by Ficoll-hypaque density gradient centrifugation. Total RNA was extracted from PBMCs and reverse-transcripted into cDNA by using specific primers. VHH were amplified by nested PCR. PCR products of VHH fragments were then purified and ligated with phagemid vector pCANTAB5E by a modified two-step ligation method. Recombinant pCANTAB5E-VHH vectors were electroporated into competent TGI E.coli cells to obtain the primary VHH antibody library. The library capacity was titrated through limited dilution. Recombination efficacy and diversity of VHH antibody library was determined by sequencing analysis. Alignment a-nalysis was performed to compare the homology between Llama VHH domain and human/mouse variable regions of heavy chains. By using a modified strategy, we have constructed a non-immune Llama VHH antibody library with 1. 5

  10. 流感病毒广谱中和抗体与广谱流感病毒疫苗%Broadly neutralizing antibodies against influenza virus andb roader influenza vaccine

    Institute of Scientific and Technical Information of China (English)

    于永利

    2013-01-01

    A series of breakthroughs have recently been made in discovering broadly neutralizing antibodies ( bnAb) against infleu nza virus, and the discoveries have spawned the research on developing broader influenza vaccine ( BIV) .The bnAb and BIV are being developed as new biologics for the prevention or treatment of infection caused by different types of influ-enza virues s that continuously undergo antigenic evolution in various hosts .%近年来,在流感病毒的广谱中和抗体( bnAb )研究领域中有了突破性的进展,这些进展力推了广谱流感疫苗( BIV)的研制。bnAb 和BIV可能成为有效预防并治疗多型、易变、宿主众多的流感病毒感染的新型生物制品。

  11. Isolation of ScFv antibodies of rP27Kip1 from phage display libraries constructed from immunized and non-immunized repertoires

    Institute of Scientific and Technical Information of China (English)

    曹跃琼; 乔守怡; 袁有忠; 黄建生; 赵寿元

    1999-01-01

    Through mRNA extract, RT and a series of PCR, phage antibody libraries were constructed from rP27Kiplimmunized and non-immunized mice. After only one round of selection with rP27Kipl, clones from each library were chosen randomly and digested by Taq I and Hinf I. 11 of 64 clones from the immunized animal had consistent restriction pattern, while none of the 64 clones from the non-immunized animal had, except that one had the same fragments pattern as that of the 11 clones. The 12 fragments were expressed in E. coli BL21(DE3)/pET-28b(+) system. ELISA showed that some of the fragments could bind to rP27Kipl specifically. All these results implied that specific antibody can be obtained by genetic engineering without hybridoma or many rounds of growth and panning selection.

  12. Identification of Hitherto Undefined B-Cell Epitopes by Antibodies in the Sera of Vitiligo Patients Using Phage-Display Peptide Library

    Directory of Open Access Journals (Sweden)

    Zohreh Jadali

    2003-12-01

    Full Text Available A random 12 mers phage library was used to screen a pool of immunoglo¬bulin fractions obtained from vitiligo patients. Subsequent to panning experiments, a panel of affinity selected phage from vitiligo patients were obtained. This panel was tested using an ELIS A for their reactivity with pooled sera from patients and normal controls. Among the 16 randomly selected clones, two of clones showed distinct positive reactivity with the patient's sera compared with controls. The peptides displayed by these phages expressed the following amino acid sequences: SHMPLANQYQWA and NHVQAWEQFWDS. Thus, screening with phage-displayed random peptide library of vitiligo sera can reveal peptide sequences that mimic vitiligo-related self-antigen.

  13. In vitro improvement of a shark IgNAR antibody by Qbeta replicase mutation and ribosome display mimics in vivo affinity maturation.

    Science.gov (United States)

    Kopsidas, George; Roberts, Anthony S; Coia, Gregory; Streltsov, Victor A; Nuttall, Stewart D

    2006-11-15

    We have employed a novel mutagenesis system, which utilizes an error-prone RNA dependent RNA polymerase from Qbeta bacteriophage, to create a diverse library of single domain antibody fragments based on the shark IgNAR antibody isotype. Coupling of these randomly mutated mRNA templates directly to the translating ribosome allowed in vitro selection of affinity matured variants showing enhanced binding to target, the apical membrane antigen 1 (AMA1) from Plasmodium falciparum. One mutation mapping to the IgNAR CDR1 loop was not readily additive to other changes, a result explained by structural analysis of aromatic interactions linking the CDR1, CDR3, and Ig framework regions. This combination appeared also to be counter-selected in experiments, suggesting that in vitro affinity maturation is additionally capable of discriminating against incorrectly produced protein variants. Interestingly, a further mutation was directed to a position in the IgNAR heavy loop 4 which is also specifically targeted during the in vivo shark response to antigen, providing a correlation between natural processes and laboratory-based affinity maturation systems.

  14. 人源Fab抗体库的构建和抗hPRLR抗体的筛选鉴定%Screening and identification of human Fab antibody against hPRLR from large phage-display library originated from breast cancer patients

    Institute of Scientific and Technical Information of China (English)

    屈芫; 魏钦俊; 姚俊; 鲁雅洁; 王天明; 曹新; 冯振卿

    2012-01-01

    cWe aim to get specific Fab antibody against human prolactin receptor (hPRLR) from the human Fab antibody library constructed by phage display technology. Human lymphocytes were collected from peripheral blood of 24 breast cancer patients. And then the total RNA was extracted and reversely transcribed to cDNA. Genes of light and heavy chains of human antibody were amplified by RT-PCR to construct anti-hPRLR immunized human antibody library. After three rounds of panning and one round of crossed-panning with against his-hPRLR fusion protein, BSA-polypeptide (epitopes of hPRLR) and GST-hPRLR fusion protein, positive clones were chosen by Phage-ELISA and DNA sequencing. Then the positive clones were transformed into E. coli Top 10' and induced to express antibody protein. The results indicated that the human Fab phage-display library consisting of l.0×l09 clones were successfully constructed, and six clones were selected after four rounds. One of them named FabG2 expressed protein correctly. ELISA and Western blot analysis showed that FabG2 could bind hPRLR specifically. We concluded that the hPRLR specific Fab antibody selected from large phage-display library could be used as candidates for therapy agent of breast cancer which over-expresses hPRLR.%目的 构建人源Fab噬菌体抗体库,筛选抗hPRLR抗体片段并进行初步鉴定.方法 从乳腺癌患者外周血提取总RNA,通过RT-PCR扩增人抗体轻链和重链基因,构建抗hPRLR人源Fab抗体库.分别以His-hPRLR融合蛋白、BSA-hPRLR表位多肽融合蛋白和GST-hPRLR融合蛋白作为抗原包板,经过3轮循环的吸附一洗脱一扩增的筛选及1轮交叉筛选,挑单克隆用Phage-ELISA、DNA测序筛选阳性克隆,将筛选到的阳性克隆Fabc2转化至Top10’受体菌,诱导表达可溶性Fab抗体,通过Western blot和ELISA进行特异性的鉴定.结果 构建的人源Fab库容为1.0×109,4轮的筛选,获得6株能与hPRLR结合的人源抗体克隆.选取的Fabc2能够进行

  15. Phage displaying peptides mimic schistosoma antigenic epitopes selected by rat natural antibodies and protective immunity induced by their immunization in mice

    Institute of Scientific and Technical Information of China (English)

    Min Wang; Xin-Yuan Yi; Xian-Ping Li; Dong-Ming Zhou; McReynolds Larry; Xian-Fang Zeng

    2005-01-01

    AIM: To obtain the short peptides mimic antigenic epitopes selected by rat natural antibodies to schistosomes, and to explore their immunoprotection against schistosomiasis in mice.METHODS: Adults worm antigens (AWA) were analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and enzyme-linked transferred immunoblotting methods with normal SD rat sera (NRS). The killing effects on schistosomula with fresh and heat-inactivated sera from SD rats were observed. Then the purified IgG from sera of SD rats was used to biopan a phage random peptide library and 20 randomly selected positive clones were detected by ELISA and 2 of them were sequenced.Sixty female mice were immunized thrice with positive phage clones (0, 2nd, 4th wk). Each mouse was challenged with 40 cercariae, and all mice were killed 42 d after challenge. The worms and the liver eggs were counted. RESULTS: NRS could specifically react to the molecules of 75 000, 47 000, 34 500 and 23 000 of AWA. Sera from SD rats showed that the mortality rate of schistosomula was 76.2%, and when the sera were heat-inactivated in vitro, the mortality rate was decreased to 41.0% after being cultured for 48 h. The specific phages bound to IgG were enriched about 300-folds after three rounds of biopanning. Twenty clones were detected by ELISA, 19 of them bound to the specific IgG of rat sera. Immunization with these epitopes was carried out in mice. Compared with the control groups, the mixture of two mimic peptides could induce 34.9% (P = 0.000) worm reduction and 67.6% (P = 0.000) total liver egg reduction in mice. Two different mimic peptides could respectively induce 31.0% (P = 0.001), 14.5% (P = 0.074) worm reduction and 61.2% (P = 0.000), 35.7% (P = 0.000) total liver egg reduction. The specific antibody could be induced by immunization of the mimic peptides, and the antibody titer in immunized mice reached more than 1:6 400 as detected by ELISA.CONCLUSION: Specific peptides mimic antigenic

  16. 抗呼吸道合胞病毒Fab噬菌体抗体库的构建及初步筛选%Construction and preliminary panning of Fab phage display antibody library against respiratory syncytial virus

    Institute of Scientific and Technical Information of China (English)

    汪治华; 张国成; 李安茂; 周南; 陈一; 李小青; 苏字飞; 邓阳彬; 王治静

    2008-01-01

    Objective To construct a human phage display antibody library,which will help to develop new drugs and vaccines against respiratory syncytial virus (RSV) and solve many of the issues that have limited the progression and application of murine monoclonal antibodies (McAbs) in the clinic.This can provide a platform for human antibody preparation and diagnosis,prophylaxis and therapy of RSV infection in children.Methods Peripheral blood lymphocytes were isolated from 52 children with RSV infection,cDNA was synthesized from the total RNA of lymphocytes.The light and heavy chain Fd (VH-CH1)fragments of immunoglobulin gene were amplified by RT-PCR.The amplified products were cloned into phagemid vector pComb3x and the clone samples were electrotransformed into competent E.coli XL1-Blue.The transformed cells were then infected with M13K07 helper phage to yield recombinant phage antibody of Fabs.The plasmids extracted from amplified E.coil were digested with restriction endonucleases Sac 1,Xba I,Spe I and Xho I to monitor the insertion of the light or heavy chain Fd genes.RSV virions were utilized as antigens to screen Fab antibodies.Results By recombination of light and heavy chain genes,an immune Fab phage display antibody library against RSV containing 2.08×107 different clones was constructed,in which 70% clones had light chains and heavy chain Fd genes.The capacity of Fab phage antibody gene library was 1.46×107 and the titre of the original Fab antibody library was about 1.06 x 1012 pfu/mL The antibody library gained an enrichment in different degrees after the preliminary panning.Condusions Utilizing the technology of phage display,an immune Fab phage display antibody library against RSV was successfully constructed in this study,which laid a valuable experimental foundation for further study and created favorable conditions for preparing human McAbs. This may also contribute to the improvement in the diagnosis,therapy and prophylaxis of RSV infection in children

  17. Magnetic Affinity Immunoassay Based Enzyme-Labeled Phage Displayed Antibody%基于酶标噬菌体抗体的磁分离免疫分析方法

    Institute of Scientific and Technical Information of China (English)

    穆晞惠; 童朝阳; 黄启斌; 刘冰; 刘志伟; 郝兰群; 张金平

    2014-01-01

    A new magnetic affinity immunoassay (MAIA) strategy based on enzyme-labeled phage displayed antibody was developed. The assay consisted of a sandwich format in which immobilized polyclonal antibody (pcAb) on magnetic microparticle was used for capture probe, and enzyme-labeled phage displayed antibody for specific detection probe to increase enzyme amount and enhance detection signal. By the proposed method,β-bungarotoxin (β-BGT) was successfully detected. A linear relationship between absorbance value and the concentration of β-BGT in the range of 0. 016-62. 5 μg / L was obtained. The linear regression equation was Y=0. 641X+1. 355 (R =0. 9925, n = 13, p<0. 0001) with a detection limit of 0. 016 μg / L. In comparison with the traditional ELISA, this method gave a 10-fold better sensitivity in β-BGT detection. This strategy also gave a 4-fold better sensitivity comparing with the MAIA based on enzyme labeled monoclonal antibody (mcAb). Due to low detection limit, acceptable reproducibility and high specificity, this method holds great promise in toxin trace detection.%以磁微粒偶联多抗为磁性捕获探针,酶标噬菌体抗体为特异信号检测探针,采用“磁性捕获探针-待测物-酶标噬菌体抗体探针”的检测模式,成功建立了一种基于酶标噬菌体抗体的磁分离免疫分析方法。本方法检测β-银环蛇毒素线性范围为0.016~62.5μg/ L,回归方程为 Y =0.641X+1.355( R =0.9925,n =13, p<0.0001),检出限为0.016μg/ L。本方法比传统 ELISA 法检测灵敏度提高了10倍,与采用酶标单抗复合物探针的双抗体夹心磁分离免疫分析法相比,检测灵敏度提高4倍。本方法灵敏度高,具有较好重现性与特异性,在毒素的痕量检测方面具有广阔的应用前景。

  18. Targeting the active site of the placental isozyme of alkaline phosphatase by phage-displayed scFv antibodies selected by a specific uncompetitive inhibitor

    Directory of Open Access Journals (Sweden)

    Kala Mrinalini

    2005-12-01

    . Conclusion The results demonstrate the biochemical modulation of scFv binding. Also, the scFvs bound to the active site and denied the access to the substrate. The selection strategy could generate specific anti-enzyme antibodies to PLAP that can potentially be used for targeting, for modulating enzyme activity in in vitro and in vivo and as probes for the active site. This strategy also has a general application in selecting antibodies from combinatorial libraries to closely related molecules and conformations.

  19. Development of a surface display ELISA to detect anti-IgG antibodies against bovine αS1-casein in human sera.

    Science.gov (United States)

    Saenger, Thorsten; Braukmann, Achim; Vordenbäumen, Stefan; Altendorfer, Irina; Bleck, Ellen; Hochwallner, Heidrun; Valenta, Rudolf; Schneider, Matthias; Jose, Joachim

    2014-08-01

    The aim of the present study was to develop a surface display ELISA (SD-ELISA) for IgG-serum reaction against bovine casein αS1 (CSN1S1). In a SD-ELISA, the antigen is displayed on the surface of Escherichia coli using the autodisplay technology and whole cells of E. coli are used to coat the microplates for serum testing. After establishing the setup of the SD-ELISA with polyclonal rabbit antiserum against bovine CSN1S1, the SD-ELISA was validated with 20 human sera, of which 10 sera were proven to have an IgG-mediated reaction against bovine CSN1S1 and 10 sera were shown to be negative for this reaction. Receiver operating characteristics (ROC) analysis revealed sensitivity of 100% and a specificity of 100% at a cut-off value of 0.133. Furthermore, human serum of 48 patients with known reactivity against human CSN1S1 (31 positive and 17 negative) was examined by the newly developed SD-ELISA to exclude cross-reactivity. Twenty human sera showed an IgG-mediated reaction against bovine CSN1S1. Eleven of these sera were positive for the reactivity against human CSN1S1, and nine were negative. In conclusion it was demonstrated that the performance of SD-ELISA is comparable to established ELISA without loss in sensitivity or specificity. Based on the advantages of this method - in particular no need for time-consuming and expensive antigen production and purification - the SD-ELISA is a potent alternative to convenient methods for identification and especially high-throughput screening of new antigens in the field of food allergies.

  20. 人源性抗乳腺癌单链抗体库的筛选与初步鉴定%Isolation and identification of single chain Fv antibodies against breast cancer from a human phage display library

    Institute of Scientific and Technical Information of China (English)

    王净; 王慧; 王超; 袁媛; 崔岩; 叶菁; 李青

    2012-01-01

    [目的]本研究旨在已构建的大容量人源性抗乳腺癌噬菌体单链抗体库的基础上,筛选出高亲和力的特异性单链抗体(scFv)并对抗体基本特性进行初步鉴定.[方法]以人乳腺癌细胞系MCF-7为靶标,经过4轮淘洗,筛选出高亲和力的特异性抗乳腺癌scFv,并对其结构序列进行分析;通过ELISA和Western blot方法,鉴定scFv的亲和力和特异性,以及其蛋白的基本表达情况.[结果]成功构建具有高亲和力的抗乳腺癌单链抗体库,获得scFv的长度约为750 bp,ELISA证实所得抗体对乳腺癌细胞具有良好的亲和力和高度的特异性,IPTG诱导表达及Western blot结果显示,scFv为相对分子质量(Mr)30 000的可溶性蛋白.[结论]本研究在已构建的大容量抗乳腺癌单链抗体库的基础上,筛选获得了高亲和力的抗乳腺癌单链抗体库.研究结果为进一步获得可应用于临床诊断和治疗的乳腺癌靶向性抗体奠定了良好的基础.%AIM: To Isolate and identify single chain Fv (scFv) antibodies against breast cancer from a constructed human phage display library. METHODS: Recombinant phages specific for breast cancer cells were enriched after four-round screening with MCF-7 cells. We selected the antigen-positive ones from the enriched clones by phage ELISA. The positive clones were used to infect E. coli HB2151 to express soluble scFv antibody. The antigen binding activity of the soluble antibodies was detected by West-em blotting. RESULTS; The specific antibodies against MCF-7 cells were enriched after four rounds of affinity selection. SDS-PAGE and Western blotting showed a band at relative molecular mass 30 000 Da, which indicated soluble antibodies were present. ELISA analysis revealed that soluble antibodies had the affinity to a human breast cancer cell line MCF-7 but not to other cancer cell line, which demonstrated scFv could react specifically with breast cancer cells. CONCLUSION; We constructed a scFv phage

  1. Structural and Functional Bases for Broad-Spectrum Neutralization of Avian and Human Influenza A Viruses

    OpenAIRE

    Sui, Jianhua; Hwang, William C; Perez, Sandra; Wei, Ge; Aird, Daniel; Chen, Li-Mei; Santelli, Eugenio; Stec, Boguslaw; Cadwell, Greg; Ali, Maryam; Wan, Hongquan; Murakami, Akikazu; Yammanuru, Anuradha; Han, Thomas; Cox, Nancy J

    2009-01-01

    Influenza virus remains a constant public health threat, owing to its ability to evade immune surveillance through rapid genetic drift and reassortment. Monoclonal antibody (mAb)-based immunotherapy is a promising strategy for disease control. Here we use a human Ab phage display library and H5 hemagglutinin (HA) ectodomain to select ten neutralizing mAbs (nAbs) with a remarkably broad range among Group 1 influenza viruses, including the H5N1 “bird flu” and the H1N1 “Spanish flu” strains. Not...

  2. Microbial transglutaminase displays broad acyl-acceptor substrate specificity

    DEFF Research Database (Denmark)

    T. Gundersen, Maria; Keillor, Jeffrey W.; Pelletier, Joelle N.

    2013-01-01

    The great importance of amide bonds in industrial synthesis has encouraged the search for efficient catalysts of amide bond formation. Microbial transglutaminase (MTG) is heavily utilized in crosslinking proteins in the food and textile industries, where the side chain of a glutamine reacts...

  3. Antibody affinity maturation

    DEFF Research Database (Denmark)

    Skjødt, Mette Louise

    surface expression of various antibody formats in the generated knockout strain. Functional scFv and scFab fragments were efficiently displayed on yeast whereas impaired chain assembly and heavy chain degradation was observed for display of full-length IgG molecules. To identify the optimal polypeptide...... linker for yeast surface display of scFv and scFab fragments, we compared a series of different Gly-Ser-based linkers in display and antigen binding proficiency. We show that these formats of the model antibody can accommodate linkers of different lengths and that introduction of alanine or glutamate...... fragments by in vivo homologous recombination large combinatorial antibody libraries can easily be generated. We have optimized ordered assembly of three CDR fragments into a gapped vector and observed increased transformation efficiency in a yeast strain carrying a deletion of the SGS1 helicase...

  4. Exosome Display technology: applications to the development of new diagnostics and therapeutics.

    Science.gov (United States)

    Delcayre, Alain; Estelles, Angeles; Sperinde, Jeffrey; Roulon, Thibaut; Paz, Pedro; Aguilar, Barbara; Villanueva, Janeth; Khine, SuSu; Le Pecq, Jean-Bernard

    2005-01-01

    Exosome Display is a novel methodology enabling the manipulation of exosome protein content. This technology stems from the identification of addressing domains that mediate the specific distribution of proteins on exosomes. More particularly, Lactadherin expressed in non-mammary gland tissue has been found to localize to exosomes via binding of its C1C2 domain to exosome lipids. Exosome Display of soluble antigens and extracellular domains of membrane proteins that are not naturally found on exosomes occurs upon fusion of proteins with the Lactadherin C1C2 domain. Exosome Display of native full-length membrane proteins can also be achieved by non-restricted expression or sampling of membrane proteins on exosomes. These novel findings enable us to manipulate exosome composition and tailor exosomes with new desirable properties. The Exosome Display technology is very versatile since soluble, membrane-bound, trans-membrane or multimeric antigens that are not naturally found on exosomes can now be efficiently expressed at their surface in a native conformation. The technology was applied to the generation of antibodies against tumor biomarkers such as HLA/peptide complex. This antibody method called ExoMAb can be used to generate antibodies against any drug target candidates, notably including G-protein coupled receptors. The potential of Exosome Display technology for developing a broad range of novel diagnostics and therapeutics is discussed.

  5. Heterologous prime-boost vaccination with MF59-adjuvanted H5 vaccines promotes antibody affinity maturation towards the hemagglutinin HA1 domain and broad H5N1 cross-clade neutralization.

    Directory of Open Access Journals (Sweden)

    Surender Khurana

    Full Text Available In an open label clinical study (2007, MF59-adjuvanted hemagglutinin (HA vaccine from H5N1-A/Vietnam/1194/2004 (clade 1 was administered to subjects previously vaccinated (primed with clade 0 H5N3 (A/duck/Singapore/97 vaccine at least 6 years earlier (in 1999 or 2001. The primed individuals responded rapidly and generated high neutralizing antibody titers against the H5N1-Vietnam strain within 7 days of a single booster vaccination. Furthermore, significant cross-neutralization titers were measured against H5N1 clade 0, 1, and 2 viruses. In the current study, the impact of MF59 adjuvant during heterologous priming on the quality of humoral polyclonal immune response in different vaccine arms were further evaluated using real time kinetics assay by surface plasmon resonance (SPR. Total anti-H5N1 HA1 polyclonal sera antibody binding from the heterologous prime-boost groups after a single MF59-H5N1 boost was significantly higher compared with sera from unprimed individuals that received two MF59-H5N1 vaccinations. The antigen-antibody complex dissociation rates (surrogate for antibody affinity of the polyclonal sera against HA1 of H5N1-A/Vietnam/1194/2004 from the MF59-H5N3 primed groups were significantly higher compared to sera from unadjuvanted primed groups or unprimed individuals that received two MF59-H5N1 vaccines. Furthermore, strong inverse correlations were observed between the antibody dissociation off-rates of the immune sera against HA1 (but not HA2 and the virus neutralization titers against H5 vaccine strains and heterologous H5N1 strains. These findings supports the use of oil-in-water-adjuvanted pandemic influenza vaccines to elicit long term memory B cells with high affinity BCR capable of responding to potential variant pandemic viruses likely to emerge and adapt to human transmissions.

  6. The 2010 Broad Prize

    Science.gov (United States)

    Education Digest: Essential Readings Condensed for Quick Review, 2011

    2011-01-01

    A new data analysis, based on data collected as part of The Broad Prize process, provides insights into which large urban school districts in the United States are doing the best job of educating traditionally disadvantaged groups: African-American, Hispanics, and low-income students. Since 2002, The Eli and Edythe Broad Foundation has awarded The…

  7. Learning from the 2009 H1N1 pandemic: prospects for more broadly effective influenza vaccines

    Institute of Scientific and Technical Information of China (English)

    Ethan C. Settembre; Philip R. Dormitzer; Rino Rappuoli

    2011-01-01

    Calls to develop a universal influenza vaccine have increased in the wake of the 2009 H1 N1 influenza pandemic. This demand comes at a time when analyses of the human antibody repertoire, informed by structures of complexes between broadly neutralizing antibodies and influenza hemagglutinin, have revealed the target of a class of broadly neutralizing antibodies. Recent studies suggest a path forward to more broadly protective influenza vaccines.%@@ Calls to develop a universal influenza vaccine have increased in the wake of the 2009 H1 N1 influenza pandemic.This demand comes at a time when analyses of the human antibody repertoire, informed by structures of complexes between broadly neutralizing antibodies and influenza hemagglutinin, have revealed the target of a class of broadly neutralizing antibodies.Recent studies suggest a path forward to more broadly protective influenza vaccines.

  8. Human monoclonal antibodies to a novel cluster of conformational epitopes on HCV E2 with resistance to neutralization escape in a genotype 2a isolate

    DEFF Research Database (Denmark)

    Keck, Zhen-yong; Xia, Jinming; Wang, Yong

    2012-01-01

    The majority of broadly neutralizing antibodies to hepatitis C virus (HCV) are against conformational epitopes on the E2 glycoprotein. Many of them recognize overlapping epitopes in a cluster, designated as antigenic domain B, that contains residues G530 and D535. To gain information on other...... regions that will be relevant for vaccine design, we employed yeast surface display of antibodies that bound to genotype 1a H77C E2 mutant proteins containing a substitution either at Y632A (to avoid selecting non-neutralizing antibodies) or D535A. A panel of nine human monoclonal antibodies (HMAbs......) was isolated and designated as HC-84-related antibodies. Each HMAb neutralized cell culture infectious HCV (HCVcc) with genotypes 1-6 envelope proteins with varying profiles, and each inhibited E2 binding to the viral receptor CD81. Five of these antibodies neutralized representative genotypes 1-6 HCVcc...

  9. 一种特异性识别斑马鱼卵黄蛋白原的噬菌体展示单链抗体的可溶性表达方案%Strategy for soluble expression of phage-displayed scFv antibody specific for zebrafish vitellogenin

    Institute of Scientific and Technical Information of China (English)

    饶瑜; 钟利桥; 张凤; 张晓华; 戴和平

    2011-01-01

    We developed a new method for soluble expression of phage-displayed scFv antibody specific for zebrafish vitellogenin. The scFv antibody F5 could bind zebrafish vitellogenin specifically in phage-displayed form but not soluble form. The gene of scFv antibody F5 was cloned into vector pET 32a and transferred into Escherichia coli ori DE3. With inducible expression, soluble scFv antibody 32a-F5 was obtained successfully and could also specifically bind to zebrafish vitellogenin. The insoluble expression of phage-displayed scFv antibody was a common problem in the practical use of phage display. This study offered a feasible way to express soluble scFv antibodies with biological activity.%为了实现特异性识别斑马鱼卵黄蛋白原的噬菌体展示单链抗体的可溶性表达,将不能以可溶性蛋白形式表达的、只能以噬菌体展示形式特异性识别斑马鱼卵黄蛋白原的单链抗体F5的基因,克隆到pET 32a载体并转化入大肠杆菌ori DE3中.结果表明,通过诱导表达,可获得可溶性的并且仍特异性识别斑马鱼卵黄蛋白原的单链抗体32a-F5.噬菌体展示单链抗体不能可溶性表达是噬菌体展示技术应用中常见的问题,该方法提供了一种表达可溶性单链抗体的可行性方案.

  10. Structure of a novel shoulder-to-shoulder p24 dimer in complex with the broad-spectrum antibody A10F9 and its implication in capsid assembly.

    Directory of Open Access Journals (Sweden)

    Ying Gu

    Full Text Available Mature HIV-1 viral particles assemble as a fullerene configuration comprising p24 capsid hexamers, pentamers and dimers. In this paper, we report the X-ray crystal structures of the p24 protein from natural HIV-1 strain (BMJ4 in complex with Fab A10F9, which recognizes a conserved epitope in the C-terminal domain of the BMJ4 p24 protein. Our structures reveal a novel shoulder-to-shoulder p24 dimerization mode that is mediated by an S-S bridge at C177. Consistent with these structures, the shoulder-to-shoulder dimer that was obtained from the BMJ4 strain was also observed in p24 proteins from other strains by the introduction of a cysteine residue at position 177. The potential biological significance was further validated by the introduction of a C177A mutation in the BMJ4 strain, which then displays a low infectivity. Our data suggest that this novel shoulder-to-shoulder dimer interface trapped by this unique S-S bridge could represent a physiologically relevant mode of HIV-1 capsid assembly during virus maturation, although Cys residue itself may not be critical for HIV-I replication.

  11. Antimitochondrial antibody

    Science.gov (United States)

    ... page: //medlineplus.gov/ency/article/003529.htm Antimitochondrial antibody To use the sharing features on this page, please enable JavaScript. Antimitochondrial antibodies (AMA) are substances ( antibodies ) that form against mitochondria. ...

  12. Universal Numeric Segmented Display

    CERN Document Server

    Azad, Md Abul kalam; Kamruzzaman, S M

    2010-01-01

    Segmentation display plays a vital role to display numerals. But in today's world matrix display is also used in displaying numerals. Because numerals has lots of curve edges which is better supported by matrix display. But as matrix display is costly and complex to implement and also needs more memory, segment display is generally used to display numerals. But as there is yet no proposed compact display architecture to display multiple language numerals at a time, this paper proposes uniform display architecture to display multiple language digits and general mathematical expressions with higher accuracy and simplicity by using a 18-segment display, which is an improvement over the 16 segment display.

  13. Antibody-targeted NY-ESO-1 to mannose receptor or DEC-205 in vitro elicits dual human CD8+ and CD4+ T cell responses with broad antigen specificity.

    Science.gov (United States)

    Tsuji, Takemasa; Matsuzaki, Junko; Kelly, Marcus P; Ramakrishna, Venky; Vitale, Laura; He, Li-Zhen; Keler, Tibor; Odunsi, Kunle; Old, Lloyd J; Ritter, Gerd; Gnjatic, Sacha

    2011-01-15

    Immunization of cancer patients with vaccines containing full-length tumor Ags aims to elicit specific Abs and both CD4(+) and CD8(+) T cells. Vaccination with protein Ags, however, often elicits only CD4(+) T cell responses without inducing Ag-specific CD8(+) T cells, as exogenous protein is primarily presented to CD4(+) T cells. Recent data revealed that Ab-mediated targeting of protein Ags to cell surface receptors on dendritic cells could enhance the induction of both CD4(+) and CD8(+) T cells. We investigated in this study if these observations were applicable to NY-ESO-1, a cancer-testis Ag widely used in clinical cancer vaccine trials. We generated two novel targeting proteins consisting of the full-length NY-ESO-1 fused to the C terminus of two human mAbs against the human mannose receptor and DEC-205, both internalizing molecules expressed on APC. These targeting proteins were evaluated for their ability to activate NY-ESO-1-specific human CD4(+) and CD8(+) T cells in vitro. Both targeted NY-ESO-1 proteins rapidly bound to their respective targets on APC. Whereas nontargeted and Ab-targeted NY-ESO-1 proteins similarly activated CD4(+) T cells, cross-presentation to CD8(+) T cells was only efficiently induced by targeted NY-ESO-1. In addition, both mannose receptor and DEC-205 targeting elicited specific CD4(+) and CD8(+) T cells from PBLs of cancer patients. Receptor-specific delivery of NY-ESO-1 to APC appears to be a promising vaccination strategy to efficiently generate integrated and broad Ag-specific immune responses against NY-ESO-1 in cancer patients.

  14. The Broad Foundations, 2006

    Science.gov (United States)

    Broad Foundation, 2006

    2006-01-01

    The mission of the Broad Foundations is to transform K-12 urban public education through better governance, management, labor relations and competition; make significant contributions to advance major scientific and medical research; foster public appreciation of contemporary art by increasing access for audiences worldwide; and lead and…

  15. Expression of recombinant antibodies.

    Science.gov (United States)

    Frenzel, André; Hust, Michael; Schirrmann, Thomas

    2013-01-01

    Recombinant antibodies are highly specific detection probes in research, diagnostics, and have emerged over the last two decades as the fastest growing class of therapeutic proteins. Antibody generation has been dramatically accelerated by in vitro selection systems, particularly phage display. An increasing variety of recombinant production systems have been developed, ranging from Gram-negative and positive bacteria, yeasts and filamentous fungi, insect cell lines, mammalian cells to transgenic plants and animals. Currently, almost all therapeutic antibodies are still produced in mammalian cell lines in order to reduce the risk of immunogenicity due to altered, non-human glycosylation patterns. However, recent developments of glycosylation-engineered yeast, insect cell lines, and transgenic plants are promising to obtain antibodies with "human-like" post-translational modifications. Furthermore, smaller antibody fragments including bispecific antibodies without any glycosylation are successfully produced in bacteria and have advanced to clinical testing. The first therapeutic antibody products from a non-mammalian source can be expected in coming next years. In this review, we focus on current antibody production systems including their usability for different applications.

  16. Recombinant renewable polyclonal antibodies.

    Science.gov (United States)

    Ferrara, Fortunato; D'Angelo, Sara; Gaiotto, Tiziano; Naranjo, Leslie; Tian, Hongzhao; Gräslund, Susanne; Dobrovetsky, Elena; Hraber, Peter; Lund-Johansen, Fridtjof; Saragozza, Silvia; Sblattero, Daniele; Kiss, Csaba; Bradbury, Andrew R M

    2015-01-01

    Only a small fraction of the antibodies in a traditional polyclonal antibody mixture recognize the target of interest, frequently resulting in undesirable polyreactivity. Here, we show that high-quality recombinant polyclonals, in which hundreds of different antibodies are all directed toward a target of interest, can be easily generated in vitro by combining phage and yeast display. We show that, unlike traditional polyclonals, which are limited resources, recombinant polyclonal antibodies can be amplified over one hundred million-fold without losing representation or functionality. Our protocol was tested on 9 different targets to demonstrate how the strategy allows the selective amplification of antibodies directed toward desirable target specific epitopes, such as those found in one protein but not a closely related one, and the elimination of antibodies recognizing common epitopes, without significant loss of diversity. These recombinant renewable polyclonal antibodies are usable in different assays, and can be generated in high throughput. This approach could potentially be used to develop highly specific recombinant renewable antibodies against all human gene products.

  17. Construction and Identification of Antibodies 2F5 and 4E10 Target Gene Vectors with the Neutralization of Anti-HIV Broad Spectrum%含抗HIV广谱中和抗体2F5、4E10靶基因载体的构建及鉴定

    Institute of Scientific and Technical Information of China (English)

    王晶妍; 张煜; 王珂; 王建英

    2012-01-01

    目的:构建含HIV gp120,gp41序列中广谱中和抗体2F5,4E10作用靶基因的载体并进行鉴定,为后期重组载体表达产物诱导产生中和抗体及抗HIV亚单位疫苗的研究奠定基础.方法:根据NCBI中HIV gp120,gp41基因序列中可与2F5、4E10结合的区域设计引物并进行PCR反应,将PCR得到的目的片段插入到载体pET28a中,对重组载体进行PCR鉴定、酶切鉴定及DNA测序.结果:PCR鉴定、酶切鉴定及DNA测序结果证实重组载体构建成功.结论:成功构建了含HIV gp120,gp41序列中广谱中和抗体2F5,4E10作用靶基因的载体.%Objective Antibodies 2F5 and 4E10 target gene vectors with the neutralization of anti-HIV broad spectrum were constructed and identified. This will certainly lay the foundation for future research of neutralizing antibodies from recom-binanl vector products in expression and induction and anti-HIV subunit vaccine. Method Primers were designed and PCR was conducted according to the binding region of the HIV gp120, gp41 gene sequences in NCBI and the 2F5 , 4E10. Then the target fragments from PCR were inserted into the vector pET28a. In the end. PCR, restriction enzyme digestion and DNA sequencing were performed for the recombinant vectors. The results; PCR, restriction enzyme digestion and DNA sequencing confirmed that the recombinant vector were successfully constructed. The conclusion; The construction of vectors of broad-spectrum neutralizing antibodies 2F5, 4E10 target genes containing HIV gp120. gp41 gene sequences was successfully conducted in this paper.

  18. Antibody engineering and therapeutics, The Annual Meeting of the Antibody Society: December 8-12, 2013, Huntington Beach, CA.

    Science.gov (United States)

    Almagro, Juan Carlos; Gilliland, Gary L; Breden, Felix; Scott, Jamie K; Sok, Devin; Pauthner, Matthias; Reichert, Janice M; Helguera, Gustavo; Andrabi, Raiees; Mabry, Robert; Bléry, Mathieu; Voss, James E; Laurén, Juha; Abuqayyas, Lubna; Barghorn, Stefan; Ben-Jacob, Eshel; Crowe, James E; Huston, James S; Johnston, Stephen Albert; Krauland, Eric; Lund-Johansen, Fridtjof; Marasco, Wayne A; Parren, Paul W H I; Xu, Kai Y

    2014-01-01

    The 24th Antibody Engineering & Therapeutics meeting brought together a broad range of participants who were updated on the latest advances in antibody research and development. Organized by IBC Life Sciences, the gathering is the annual meeting of The Antibody Society, which serves as the scientific sponsor. Preconference workshops on 3D modeling and delineation of clonal lineages were featured, and the conference included sessions on a wide variety of topics relevant to researchers, including systems biology; antibody deep sequencing and repertoires; the effects of antibody gene variation and usage on antibody response; directed evolution; knowledge-based design; antibodies in a complex environment; polyreactive antibodies and polyspecificity; the interface between antibody therapy and cellular immunity in cancer; antibodies in cardiometabolic medicine; antibody pharmacokinetics, distribution and off-target toxicity; optimizing antibody formats for immunotherapy; polyclonals, oligoclonals and bispecifics; antibody discovery platforms; and antibody-drug conjugates.

  19. Recombinant baculovirus displayed vaccine

    Science.gov (United States)

    Prabakaran, Mookkan; Kwang, Jimmy

    2014-01-01

    The rapid evolution of new sublineages of H5N1 influenza in Asia poses the greatest challenge in vaccine development for pre-pandemic preparedness. To overcome the antigenic diversity of H5N1 strains, multiple vaccine strains can be designed based on the distribution of neutralizing epitopes in the globular head of H5 hemagglutinin (HA). Recently, we selected two different HAs of H5N1 strains based on the neutralizing epitopes and reactivity with different neutralizing antibodies. The HAs of selected vaccine strains were individually expressed on the baculovirus envelope (bivalent-BacHA) with its native antigenic configuration. Further, oral delivery of live bivalent-BacHA elicited broadly reactive humoral, mucosal and cell-mediated immune responses and showed complete protection against antigenically distinct H5N1 strains in mice. The strategy for the vaccine strain selection, vaccine design and route of administration will provide an idea for development of a widely protective vaccine against highly pathogenic H5N1 for pre-pandemic preparedness. PMID:23941989

  20. Classification of influenza A virus and its value for research of universal influenza vaccine and broad-spectrum neutralization antibody%甲型流感病毒分类与通用流感疫苗及广谱中和活性抗体的研究

    Institute of Scientific and Technical Information of China (English)

    蒋露芳; 居丽雯; 姜庆五

    2011-01-01

    Influenza is a great harm contagious disease for human. Vaccination is considered to be the most effective means to prevent influenza. For the problems of existing vaccine on timeliness and effectiveness,universal influenza vaccine,which can preclude all the influenza virus and induce duration protective immunity, has been a hot research. Meanwhile, the broad-spectrum neutralization antibody against all influenza A viruses is also the current focus. All these have made the request to explore applicable classification of influenza A virus.%流感是对人类危害极大的传染病,疫苗接种被认为是预防流感的最有效手段.为解决流感病毒变异导致现有疫卣时效性与有效性的问题,研制能够预防所有流感病毒毒株、可诱导持久保护性免疫的通用流感疫苗一直是流感研究的热点,同时,能中和所有甲型流感病毒的广谱中和活性抗体的研究也已成为目前关注的重点,因此对甲型流感病毒分类研究也提出了相应的要求.

  1. Autologous antibodies that bind neuroblastoma cells.

    Science.gov (United States)

    Sun, Yujing; Sholler, Giselle S; Shukla, Girja S; Pero, Stephanie C; Carman, Chelsea L; Zhao, Ping; Krag, David N

    2015-11-01

    Antibody therapy of neuroblastoma is promising and our goal is to derive antibodies from patients with neuroblastoma for developing new therapeutic antibodies. The feasibility of using residual bone marrow obtained for clinical indications as a source of tumor cells and a source of antibodies was assessed. From marrow samples, neuroblastoma cells were recovered, grown in cell culture and also implanted into mice to create xenografts. Mononuclear cells from the marrow were used as a source to generate phage display antibody libraries and also hybridomas. Growth of neuroblastoma patient cells was possible both in vitro and as xenografts. Antibodies from the phage libraries and from the monoclonal hybridomas bound autologous neuroblastoma cells with some selectivity. It appears feasible to recover neuroblastoma cells from residual marrow specimens and to generate human antibodies that bind autologous neuroblastoma cells. Expansion of this approach is underway to collect more specimens, optimize methods to generate antibodies, and to evaluate the bioactivity of neuroblastoma-binding antibodies.

  2. Displaying gray shades in liquid crystal displays

    Indian Academy of Sciences (India)

    T N Ruckmongathan

    2003-08-01

    Quality of image in a display depends on the contrast, colour, resolution and the number of gray shades. A large number of gray shades is necessary to display images without any contour lines. These contours are due to limited number of gray shades in the display causing abrupt changes in grayness of the image, while the original image has a gradual change in brightness. Amplitude modulation has the capability to display a large number of gray shades with minimum number of time intervals [1,2]. This paper will cover the underlying principle of amplitude modulation, some variants and its extension to multi-line addressing. Other techniques for displaying gray shades in passive matrix displays are reviewed for the sake of comparison.

  3. Sensitivity of HIV-1 to neutralization by antibodies against O-linked carbohydrate epitopes despite deletion of O-glycosylation signals in the V3 loop

    DEFF Research Database (Denmark)

    Hansen, J E; Jansson, B; Gram, G J

    1996-01-01

    It has been suggested that threonine or serine residues in the V3 loop of HIV-1 gp120 are glycosylated with the short-chain O-linked oligosaccharides Tn or sialosyl-Tn that function as epitopes for broadly neutralizing carbohydrate specific antibodies. In this study we examined whether mutation...... with deletions of O-glycosylation signals in the V3 loop displayed any decrease in sensitivity to anti-Tn or anti-sialosyl-Tn antibody. This indicates that these broadly specific neutralization epitopes are located outside the V3 loop of gp 120........ Additionally, one of these T-A mutants (T308A) also abrogated the signal for N-glycosylation at N306 inside the V3-loop. The mutant clones were compared with the wild type virus as to sensitivity to neutralization with monoclonal and polyclonal antibodies specific for the tip of the V3 loop of BRU or for the O...

  4. Selection of peptide mimics of HIV-1 epitope recognized by neutralizing antibody VRC01.

    Directory of Open Access Journals (Sweden)

    Anton N Chikaev

    Full Text Available The ability to induce anti-HIV-1 antibodies that can neutralize a broad spectrum of viral isolates from different subtypes seems to be a key requirement for development of an effective HIV-1 vaccine. The epitopes recognized by the most potent broadly neutralizing antibodies that have been characterized are largely discontinuous. Mimetics of such conformational epitopes could be potentially used as components of a synthetic immunogen that can elicit neutralizing antibodies. Here we used phage display technology to identify peptide motifs that mimic the epitope recognized by monoclonal antibody VRC01, which is able to neutralize up to 91% of circulating primary isolates. Three rounds of biopanning were performed against 2 different phage peptide libraries for this purpose. The binding specificity of selected phage clones to monoclonal antibody VRC01 was estimated using dot blot analysis. The putative peptide mimics exposed on the surface of selected phages were analyzed for conformational and linear homology to the surface of HIV-1 gp120 fragment using computational analysis. Corresponding peptides were synthesized and checked for their ability to interfere with neutralization activity of VRC01 in a competitive inhibition assay. One of the most common peptides selected from 12-mer phage library was found to partially mimic a CD4-binding loop fragment, whereas none of the circular C7C-mer peptides was able to mimic any HIV-1 domains. However, peptides identified from both the 12-mer and C7C-mer peptide libraries showed rescue of HIV-1 infectivity in the competitive inhibition assay. The identification of epitope mimics may lead to novel immunogens capable of inducing broadly reactive neutralizing antibodies.

  5. Conceptual Design of Industrial Process Displays

    DEFF Research Database (Denmark)

    Pedersen, C.R.; Lind, Morten

    1999-01-01

    by a simple example from a plant with batch processes. Later the method is applied to develop a supervisory display for a condenser system in a nuclear power plant. The differences between the continuous plant domain of power production and the batch processes from the example are analysed and broad...... categories of display types are proposed. The problems involved in specification and invention of a supervisory display are analysed and conclusions from these problems are made. It is concluded that the design method proposed provides a framework for the progress of the display design and is useful in pin......-pointing the actual problems. The method was useful in reducing the number of existing displays that could fulfil the requirements of the supervision task. The method provided at the same time a framework for dealing with the problems involved in inventing new displays based on structured analysis. However...

  6. Production of High Affinity Human Single-chain Antibody Against PreS1 of Hepatitis B Virus:Comparison of Large Na(i)ve and In vitro Immune Phage Displayed Antibody Library%高亲和力抗乙型肝炎病毒PreS1的人源单链抗体的获得:天然及免疫抗体库的对比研究

    Institute of Scientific and Technical Information of China (English)

    张志超; 胡学军; 包永明; 杨青; 张红梅; 安利佳

    2002-01-01

    A large nave phage displayed human single-chain variable fragments antibody (scFv) library and an in vitro immune library were constructed in parallel conditions, based on the PBLs from healthy and sero-negative blood donors, part of which were in vitro immunized by peptide PreS1 conjugated to BSA. After 3 rounds of panning against PreS1, measurement of antibody-antigen reaction revealed: a scFv specific to PreS1 from the immune library was obtained, which affinity (k=10-7~10-8 M) was higher than that from the nave one (k=10-6~10-7 M). Sequencing of the two scFv showed they were human antibodies, which may be of interest in therapy of Hepatitis B. This investigation also illustrated that the method of in vitro immunization results in antibody library more satisfied even than the large nave one.%以健康人的外周血淋巴细胞为来源,以偶联BSA的乙型肝炎病毒PreS1肽体外免疫.分别从免疫和未经免疫的淋巴细胞提取RNA,扩增抗体基因,构建大容量天然单链抗体(scFv)噬菌体展示文库和体外免疫scFv抗体库.以PreS1肽进行3轮淘选后,抗原抗体反应结果显示,从免疫库中获得了亲和力10-7~10-8 M的抗乙型肝炎病毒PreS1的单链抗体,高于天然库的结果(10-6~10-7 M).测序结果表明两株抗体均为人抗体.为基因工程抗体用于临床治疗乙型肝炎奠定基础.同时证明淋巴细胞体外免疫方法构建的免疫抗体库优于大容量天然抗体库.

  7. Construction and selection of human Fab antibody phage display library of extracellular domain of HER 2%人源性抗HER2胞外段Fab噬菌体抗体库的构建及筛选

    Institute of Scientific and Technical Information of China (English)

    张为家; 刘孝荣; 李官成; 贺智敏

    2011-01-01

    目的:构建全人源抗HER2胞外段(HER7. ECD)噬菌体Fab抗体库,从中筛选出特异性的抗体,并对其进行鉴定.方法:体外致敏并用EB病毒(EBV)转化HER2高表达乳腺癌患者的外周血单核细胞(PBMC),用PCR分别扩增重链Fd和轻链k/λ基因.经Sac I/Xba I和Xho/SpeI双酶切,依次克隆入噬菌体载体pComb3中,并电转化大肠杆菌XL1Blue,以辅助噬菌体VCSM13进行超感染,构建抗HER2 ECD人源化Fab噬菌体抗体库.以纯化HER2 ECD蛋白为抗原进行3轮固相淘选,富集抗HER2 ECD的抗体,并随机挑选克隆进行ELISA,获得的阳性克隆进一步以Western blot鉴定其抗原结合活性,对其中活性最高的克隆进行DNA测序.结果:构建了容量为2. 5 X 107的抗HER2 ECD的Fab噬菌体抗体库,并筛选获得了4株与HE2 ECD特异性结合的阳性克隆,Western blot分析显示其与HER?-ECD能较好的结合.选取结合活性最高的阳性克隆进行DNA序列分析,结果显示其重链可变区、轻链可变区分别与人胚系免疫球蛋白基因有高度的同源性.结论:成功构建了全人源抗HER2 ECD噬菌体抗体库,并筛选出抗HER2 ECD特异性较强的噬菌体克隆,为获得新的有临床应用价值的HER2 ECD抗体提供了实验基础.%AIM: To construct the fully humanized antiextracellular domain (ECD) of HER2.Fab fragment phage library, select antibodies against HER2 ECD specifically and identify its characteristics.METHODS: Peripheral blood monouclear cells (PBMCs) of breast cancer patients with HER2-overexpressing were immunized in vitro with purification protein of recombinant HEF2 ECD and were then transformed by Epstein-Barr virus (EBV).After total RNA was extracted, the heavy chain Fd and k/λ light chain were am plified by RT-PCR.Following restrictive digestion with Sac I/ Xba I and Xho I/Spe I, the light chain κ/λ genes and heavy chain genes Fd were inserted into the phagemid vector pComb3 successively and then electroporated into E.coil XL1-Blue

  8. Application of broad-spectrum humanized neutralizing monoclonal antibodies to influenza A virus in prevention and treatment of influenza%广谱全人源化甲型流感病毒中和抗体在流感预防与治疗中的应用

    Institute of Scientific and Technical Information of China (English)

    高灵茜; 杨利

    2013-01-01

    针对多个亚型甲型流感病毒的广谱中和性单克隆抗体(monoclonalantibody,mAb)是极具潜力的新型而有效的流感防治手段.抗流感病毒血凝素(hemagglutinin,HA)的保护性mAb,无论通过主动免疫还是被动免疫,均具有很好的效果.迄今,已有不少甲型流感病毒广谱中和性mAb被开发出来.尽管这些mAb具备临床应用潜能以及作为研制新型疫苗(抗独特型抗体疫苗)的可能,但仅有几种HA mAb对人类流感防治真正有效.此文主要对能高效识别多个亚型HA的人源化甲型流感病毒mAb做一综述.%Broad-spectrum neutralizing monoclonal antibodies (mAbs) against different subtypes of influenza A viruses are potential tools to prevent and treat influenza.Protective mAbs to influenza hemagglutinin (HA) have good results by passive and active immunization.So far,a number of mAbs against influenza HA have been developed.But only few of them are effective for influenza prevention and treatment.In this article,the human mAbs which can recognize divergent influenza isolates are reviewed.

  9. H5N1型禽流感病毒广谱中和单克隆抗体的筛选及其中和机制初步研究%Broad-spectrum Neutralizing Monoclonal Antibodies Against H5N1 Avian Influenza A Viruses and Primary Research on The Mechanism

    Institute of Scientific and Technical Information of China (English)

    张晓; 曾晓燕; 刘哲; 金秋; 徐言; 冯振卿; 焦永军

    2011-01-01

    Avian influenza is a highly contagious disease of birds caused by A influenza viruses. The circulation in humans by the highly pathogenic H5N1 avian flu in the past few years have caused most pandemics and have heightened fear that the next influenza pandemic is due. Antibodies could be used as an efficient anti-virus agent in clinical therapy. The full-length HA of the A/Jiangsu/1/2007(H5N 1) about 1.7 kb was amplified, subcloned to the pFastBac vector and recombinant bacmid DNA was selected. The recombinant HA was expressed and purified HA about 70 ku was used as the antigen to immunize Balb/c mice. The whole H5N1 virus was used to select 5 mono-antibodies (mAbs), and all of them were tested using microneutralization assays. 8G10D7, one of the antibodies, had broad neutralizing effect against clade 2 and clade 9 H5N1 avian influenza A viruses, and the IC50 was from 1 : 256 to I ' 64. When detected with 8G10D7, all 4 viruses showed 70 ku and 43 ku protein band,which confirms that the binding site of the scFv antibodies were located at the HA1 domain. The nucleuses of MDCK cells infected by 4 viruses were colored purple, and red around the nucleus. 8Gl 0D7 showed HI activity to the 4 viruses, the HI has a positive correlation with neutralization concentration IC50, which also further confirms that the binding site of the scFv antibodies were located at the HA1 domain. When the mAb 8Gl 0D7 was used for the study of prophylaxis and therapeutic effect on influenza A viruses infection in an embryonated chicken eggs model. It had a complete 100% protection effect on the H5N1 viruses in avian host in the prophylactic and therapeutic groups. The 100% preventive protection effect could be reached when challenged with H5N1 avian influenza A viruse in human host in the prophylactic groups, and there is also a 87.5% protection effect with H5N1 viruses in human host in the therapeutic groups. Thereby, the study suggests that the mAb 8G10D7 could be used in therapies to

  10. Handbook of display technology

    CERN Document Server

    Castellano, Joseph A

    1992-01-01

    This book presents a comprehensive review of technical and commercial aspects of display technology. It provides design engineers with the information needed to select proper technology for new products. The book focuses on flat, thin displays such as light-emitting diodes, plasma display panels, and liquid crystal displays, but it also includes material on cathode ray tubes. Displays include a large number of products from televisions, auto dashboards, radios, and household appliances, to gasoline pumps, heart monitors, microwave ovens, and more.For more information on display tech

  11. Rapid, Quantitative Mapping of Anti-HIV Type 1 Envelope Serum Antibody Specificities

    OpenAIRE

    Powell, Rebecca L.R.; Lindsay, Ross W.B.; Wilson, Aaron; Carpov, Alexei; Rabinovich, Svetlana; Hoffenberg, Simon; Caulfield, Michael J.

    2013-01-01

    A new generation of extremely broad and potent neutralizing antibodies (bNAbs) has been isolated from HIV-infected subjects. This has refocused interest in the sites of vulnerability targeted by these bNAbs and in the potential for designing Envelope (Env) immunogens that display these sites. Standard methods for evaluating HIV-1 vaccine candidates do not enable epitope mapping on the HIV Env spike, the target for NAbs. To meet the need for rapid analysis of Ab specificity, we designed a mult...

  12. A Compressive Superresolution Display

    KAUST Repository

    Heide, Felix

    2014-06-22

    In this paper, we introduce a new compressive display architecture for superresolution image presentation that exploits co-design of the optical device configuration and compressive computation. Our display allows for superresolution, HDR, or glasses-free 3D presentation.

  13. Lunar Sample Display Locations

    Data.gov (United States)

    National Aeronautics and Space Administration — NASA provides a number of lunar samples for display at museums, planetariums, and scientific expositions around the world. Lunar displays are open to the public....

  14. Targeting N-Glycan Cryptic Sugar Moieties for Broad-Spectrum Virus Neutralization: Progress in Identifying Conserved Molecular Targets in Viruses of Distinct Phylogenetic Origins

    Directory of Open Access Journals (Sweden)

    Denong Wang

    2015-03-01

    Full Text Available Identifying molecular targets for eliciting broadly virus-neutralizing antibodies is one of the key steps toward development of vaccines against emerging viral pathogens. Owing to genomic and somatic diversities among viral species, identifying protein targets for broad-spectrum virus neutralization is highly challenging even for the same virus, such as HIV-1. However, viruses rely on host glycosylation machineries to synthesize and express glycans and, thereby, may display common carbohydrate moieties. Thus, exploring glycan-binding profiles of broad-spectrum virus-neutralizing agents may provide key information to uncover the carbohydrate-based virus-neutralizing epitopes. In this study, we characterized two broadly HIV-neutralizing agents, human monoclonal antibody 2G12 and Galanthus nivalis lectin (GNA, for their viral targeting activities. Although these agents were known to be specific for oligomannosyl antigens, they differ strikingly in virus-binding activities. The former is HIV-1 specific; the latter is broadly reactive and is able to neutralize viruses of distinct phylogenetic origins, such as HIV-1, severe acute respiratory syndrome coronavirus (SARS-CoV, and human cytomegalovirus (HCMV. In carbohydrate microarray analyses, we explored the molecular basis underlying the striking differences in the spectrum of anti-virus activities of the two probes. Unlike 2G12, which is strictly specific for the high-density Man9GlcNAc2Asn (Man9-clusters, GNA recognizes a number of N-glycan cryptic sugar moieties. These include not only the known oligomannosyl antigens but also previously unrecognized tri-antennary or multi-valent GlcNAc-terminating N-glycan epitopes (Tri/m-Gn. These findings highlight the potential of N-glycan cryptic sugar moieties as conserved targets for broad-spectrum virus neutralization and suggest the GNA-model of glycan-binding warrants focused investigation.

  15. STUDIES ON THE HIV-1 GP41 DISPLAY ON YEAST SURFACE FOR ANTIBODY DETECTION IN SERA%酵母细胞表面展示HIV-1 gp41用于血清中抗体的检测的研究

    Institute of Scientific and Technical Information of China (English)

    向柱方; 赵树进; 杜红丽; 林影

    2008-01-01

    以Hexa-His和Flag为标签,利用酵母表面展示技术将HIV-1囊膜蛋白gp41展示于酿酒酵母(Saccharomycws cerevisiase)细胞MT8-1表面.流式细胞测定结果表明His标签成功展示于重组酵母细胞MT8-1/pICAS-His和MT8-1/pICAS-His-gp41表面;活性的gp41成功展于重组酵母MT8-1/pICAS-His-gp41表面,荧光显微镜观测结果表明了这一点;Flag标签可以在TM8-1/pICAS-Flag表面检测到,但Flag和gp41均不能在MT8-1/pICAS-Flag-gp41表面检测到活性.利用展示在酵母表面的gp41蛋白建立酵母细胞CbELISA体系,可成功用于检测单克隆抗体,其浓度达到了10 ng/mL,此体系可用于检测血清中的抗体.%The envelope glycoprotein gp41 of HIV-1 has been successfully displayed on the cell surface of Saccharomyces cerevisiase MT8-1 by cell-surface engineering using Hexa-His and Flag tags for detection, respectively. Flow cytograms showed that His tag was displayed on the surface of MT8-1/pICAS-His and MT8-1/pICAS-His-gp41, the active gp41 could be detected on the cell surface of MT8-1/pICAS-His-gp41 and was confirmed by fluorescence microscopy;Flag tag was detected on the surface of MT8-1/pICAS-Flag, but Flag tag and gp41 were not detected on the surface of MT8-1/pICAS-Flag-gp41 separately. Further, a yeast CbELISA was developed by using the yeast cells displaying gp41 on the cell surface. It was able to detect the monoclonal antibody at the concentration of 10 ng/mL , and also capable of detecting the antibody in the sera.

  16. Volumetric Three-Dimensional Display Systems

    Science.gov (United States)

    Blundell, Barry G.; Schwarz, Adam J.

    2000-03-01

    A comprehensive study of approaches to three-dimensional visualization by volumetric display systems This groundbreaking volume provides an unbiased and in-depth discussion on a broad range of volumetric three-dimensional display systems. It examines the history, development, design, and future of these displays, and considers their potential for application to key areas in which visualization plays a major role. Drawing substantially on material that was previously unpublished or available only in patent form, the authors establish the first comprehensive technical and mathematical formalization of the field, and examine a number of different volumetric architectures. System level design strategies are presented, from which proposals for the next generation of high-definition predictable volumetric systems are developed. To ensure that researchers will benefit from work already completed, they provide: * Descriptions of several recent volumetric display systems prepared from material supplied by the teams that created them * An abstract volumetric display system design paradigm * An historical summary of 90 years of development in volumetric display system technology * An assessment of the strengths and weaknesses of many of the systems proposed to date * A unified presentation of the underlying principles of volumetric display systems * A comprehensive bibliography Beautifully supplemented with 17 color plates that illustrate volumetric images and prototype displays, Volumetric Three-Dimensional Display Systems is an indispensable resource for professionals in imaging systems development, scientific visualization, medical imaging, computer graphics, aerospace, military planning, and CAD/CAE.

  17. Thyroid Antibodies

    Science.gov (United States)

    ... e.g., at regular intervals after thyroid cancer treatment) Thyroid stimulating hormone receptor antibody, Thyroid Stimulating Immunoglobulin TRAb, TSHR Ab, TSI Graves disease When a person has symptoms of hyperthyroidism If a pregnant woman has a known autoimmune ...

  18. Construction and screening of phage display single chain antibody library against histidine-rich protein Ⅱ of Plasmodium falciparum%抗恶性疟原虫富含组氨酸蛋白Ⅱ单链抗体库的构建及筛选

    Institute of Scientific and Technical Information of China (English)

    侯云霞; 董文其; 徐伟文; 王萍; 陈白虹; 李明

    2001-01-01

    Objective To construct phage display single-chain antibody fragments (scFvs) library against histidine-rich protein Ⅱ (HRP-Ⅱ) of Plasmodium falciparum and select specific scFvs of anti- HRP-Ⅱ for the purpose of malaria diagnosis. Method The genes of variable fragments of heavy chain (VH) and light chain (VL) were gained from the spleen cells of BALB/c mice immunized with HRP-Ⅱ protein. The VH and VL genes were then assembled by the method of splicing overlapping extension and cloned into phagemid vector pCANTAB 5E. The scFv phage antibodies were expressed at the surface of the phage after the rescue by helper phage M13K07. HRP- Ⅱ protein was used as antigenic reagent for panning and screening. Results The total RNA from the spleen cells was isolated, and cDNA obtained and VH and VL gene regions amplified using PCR. The VH and VL gene regions were combined with a flexible linker ligated into the pCANTAB 5E phagemid vector, and transformed into TG1 Escherichia coli. The repertoire of the phage antibody was about 106. After panning and screening, 8 positive clones expressed scFv antibodies which were specific for HPR-Ⅱ as demonstrated by ELISA. Conclusion Phage display technology can be used as a powerful tool in making scFv antibodies which have the potential to be used as reagents in the diagnosis and therapy of malaria.%目的构建抗恶性疟原虫富含组氨酸蛋白Ⅱ(Histidine- rich protein II, HRP-II)单链抗体库,并筛选出阳性克隆。方法用噬菌体抗体库技术构建抗恶性疟原虫HRP-Ⅱ单链抗体库,并以HRP-Ⅱ为靶抗原对该库进行了三轮"亲和吸附-援救-感染扩增"的富集,挑取单菌落筛选并鉴定阳性克隆。结果获得目的基因并成功构建抗恶性疟原虫HRP-Ⅱ的单链抗体库,库容为106,并从中筛选出8株阳性克隆。结论噬菌体抗体库技术具有高效的筛选性能,抗 HRP-Ⅱ单链抗体的制备为其在恶性疟的免疫快速诊断方法中的应用奠定了基础。

  19. Broad-spectrum antiviral agents

    Directory of Open Access Journals (Sweden)

    Jun-Da eZhu

    2015-05-01

    Full Text Available Development of highly effective, broad-spectrum antiviral agents is the major objective shared by the fields of virology and pharmaceutics. Antiviral drug development has focused on targeting viral entry and replication, as well as modulating cellular defense system. High throughput screening of molecules, genetic engineering of peptides, and functional screening of agents have identified promising candidates for development of optimal broad-spectrum antiviral agents to intervene in viral infection and control viral epidemics. This review discusses current knowledge, prospective applications, opportunities, and challenges in the development of broad-spectrum antiviral agents.

  20. Pitfalls to avoid when using phage display for snake toxins.

    Science.gov (United States)

    Laustsen, Andreas Hougaard; Lauridsen, Line Præst; Lomonte, Bruno; Andersen, Mikael Rørdam; Lohse, Brian

    2017-02-01

    Antivenoms against bites and stings from snakes, spiders, and scorpions are associated with immunological side effects and high cost of production, since these therapies are still derived from the serum of hyper-immunized production animals. Biotechnological innovations within envenoming therapies are thus warranted, and phage display technology may be a promising avenue for bringing antivenoms into the modern era of biologics. Although phage display technology represents a robust and high-throughput approach for the discovery of antibody-based antitoxins, several pitfalls may present themselves when animal toxins are used as targets for phage display selection. Here, we report selected critical challenges from our own phage display experiments associated with biotinylation of antigens, clone picking, and the presence of amber codons within antibody fragment structures in some phage display libraries. These challenges may be detrimental to the outcome of phage display experiments, and we aim to help other researchers avoiding these pitfalls by presenting their solutions.

  1. Broad resonances in transport theory

    CERN Document Server

    Leupold, S

    2003-01-01

    The extension of the transport theoretical framework to include states with a broad mass distribution is discussed. The proper life-time and cross sections for a state with an arbitrarily given invariant mass is discussed in detail. (author)

  2. Invisible Display in Aluminum

    DEFF Research Database (Denmark)

    Prichystal, Jan Phuklin; Hansen, Hans Nørgaard; Bladt, Henrik Henriksen

    2005-01-01

    Bang & Olufsen a/s has been working with ideas for invisible integration of displays in metal surfaces. Invisible integration of information displays traditionally has been possible by placing displays behind transparent or semitransparent materials such as plastic or glass. The wish for an integ...... be obtained by shining light from the backside of the workpiece. When there is no light from the backside, the front surface seems totally untouched. This was achieved by laser ablation with ultra-short pulses.......Bang & Olufsen a/s has been working with ideas for invisible integration of displays in metal surfaces. Invisible integration of information displays traditionally has been possible by placing displays behind transparent or semitransparent materials such as plastic or glass. The wish...... for an integrated display in a metal surface is often ruled by design and functionality of a product. The integration of displays in metal surfaces requires metal removal in order to clear the area of the display to some extent. The idea behind an invisible display in Aluminum concerns the processing of a metal...

  3. Polyplanar optic display

    Energy Technology Data Exchange (ETDEWEB)

    Veligdan, J.; Biscardi, C.; Brewster, C.; DeSanto, L. [Brookhaven National Lab., Upton, NY (United States). Dept. of Advanced Technology; Beiser, L. [Leo Beiser Inc., Flushing, NY (United States)

    1997-07-01

    The Polyplanar Optical Display (POD) is a unique display screen which can be used with any projection source. This display screen is 2 inches thick and has a matte black face which allows for high contrast images. The prototype being developed is a form, fit and functional replacement display for the B-52 aircraft which uses a monochrome ten-inch display. The new display uses a 100 milliwatt green solid state laser (532 nm) as its optical source. In order to produce real-time video, the laser light is being modulated by a Digital Light Processing (DLP{trademark}) chip manufactured by Texas Instruments, Inc. A variable astigmatic focusing system is used to produce a stigmatic image on the viewing face of the POD. In addition to the optical design, the authors discuss the electronic interfacing to the DLP{trademark} chip, the opto-mechanical design and viewing angle characteristics.

  4. OLED displays and lighting

    CERN Document Server

    Koden, Mitsuhiro

    2017-01-01

    Organic light-emitting diodes (OLEDs) have emerged as the leading technology for the new display and lighting market. OLEDs are solid-state devices composed of thin films of organic molecules that create light with the application of electricity. OLEDs can provide brighter, crisper displays on electronic devices and use less power than conventional light-emitting diodes (LEDs) or liquid crystal displays (LCDs) used today. This book covers both the fundamentals and practical applications of flat and flexible OLEDs.

  5. Scalable Resolution Display Walls

    KAUST Repository

    Leigh, Jason

    2013-01-01

    This article will describe the progress since 2000 on research and development in 2-D and 3-D scalable resolution display walls that are built from tiling individual lower resolution flat panel displays. The article will describe approaches and trends in display hardware construction, middleware architecture, and user-interaction design. The article will also highlight examples of use cases and the benefits the technology has brought to their respective disciplines. © 1963-2012 IEEE.

  6. JAVA Stereo Display Toolkit

    Science.gov (United States)

    Edmonds, Karina

    2008-01-01

    This toolkit provides a common interface for displaying graphical user interface (GUI) components in stereo using either specialized stereo display hardware (e.g., liquid crystal shutter or polarized glasses) or anaglyph display (red/blue glasses) on standard workstation displays. An application using this toolkit will work without modification in either environment, allowing stereo software to reach a wider audience without sacrificing high-quality display on dedicated hardware. The toolkit is written in Java for use with the Swing GUI Toolkit and has cross-platform compatibility. It hooks into the graphics system, allowing any standard Swing component to be displayed in stereo. It uses the OpenGL graphics library to control the stereo hardware and to perform the rendering. It also supports anaglyph and special stereo hardware using the same API (application-program interface), and has the ability to simulate color stereo in anaglyph mode by combining the red band of the left image with the green/blue bands of the right image. This is a low-level toolkit that accomplishes simply the display of components (including the JadeDisplay image display component). It does not include higher-level functions such as disparity adjustment, 3D cursor, or overlays all of which can be built using this toolkit.

  7. Elicitation of structure-specific antibodies by epitope scaffolds

    OpenAIRE

    2010-01-01

    Elicitation of antibodies against targets that are immunorecessive, cryptic, or transient in their native context has been a challenge for vaccine design. Here we demonstrate the elicitation of structure-specific antibodies against the HIV-1 gp41 epitope of the broadly neutralizing antibody 2F5. This conformationally flexible region of gp41 assumes mostly helical conformations but adopts a kinked, extended structure when bound by antibody 2F5. Computational techniques were employed to transpl...

  8. Helmet-Mounted Displays (HMD)

    Data.gov (United States)

    Federal Laboratory Consortium — The Helmet-Mounted Display labis responsible for monocular HMD day display evaluations; monocular HMD night vision performance processes; binocular HMD day display...

  9. Standardizing visual display quality

    NARCIS (Netherlands)

    Besuijen, Ko; Spenkelink, Gerd P.J.

    1998-01-01

    The current ISO 9241–3 standard for visual display quality and the proposed user performance tests are reviewed. The standard is found to be more engineering than ergonomic and problems with system configuration, software applications, display settings, user behaviour, wear and physical environment

  10. Polyplanar optical display electronics

    Energy Technology Data Exchange (ETDEWEB)

    DeSanto, L.; Biscardi, C. [Brookhaven National Lab., Upton, NY (United States). Dept. of Advanced Technology

    1997-07-01

    The Polyplanar Optical Display (POD) is a unique display screen which can be used with any projection source. The prototype ten inch display is two inches thick and has a matte black face which allows for high contrast images. The prototype being developed is a form, fit and functional replacement display for the B-52 aircraft which uses a monochrome ten-inch display. In order to achieve a long lifetime, the new display uses a 100 milliwatt green solid-state laser (10,000 hr. life) at 532 nm as its light source. To produce real-time video, the laser light is being modulated by a Digital Light Processing (DLP{trademark}) chip manufactured by Texas Instruments. In order to use the solid-state laser as the light source and also fit within the constraints of the B-52 display, the Digital Micromirror Device (DMD{trademark}) circuit board is removed from the Texas Instruments DLP light engine assembly. Due to the compact architecture of the projection system within the display chassis, the DMD{trademark} chip is operated remotely from the Texas Instruments circuit board. The authors discuss the operation of the DMD{trademark} divorced from the light engine and the interfacing of the DMD{trademark} board with various video formats (CVBS, Y/C or S-video and RGB) including the format specific to the B-52 aircraft. A brief discussion of the electronics required to drive the laser is also presented.

  11. Visual merchandising window display

    Directory of Open Access Journals (Sweden)

    Opris (Cas. Stanila M.

    2013-12-01

    Full Text Available Window display plays a major part in the selling strategies; it does not only include the simple display of goods, nowadays it is a form of art, also having the purpose of sustaining the brand image. This article wants to reveal the tools that are essential in creating a fabulous window display. Being a window designer is not an easy job, you have to always think ahead trends, to have a sense of colour, to know how to use light to attract customers in the store after only one glance at the window. The big store window displays are theatre scenes: with expensive backgrounds, special effects and high fashion mannequins. The final role of the displays is to convince customers to enter the store and trigger the purchasing act which is the final goal of the retail activity.

  12. Yeast mating for combinatorial Fab library generation and surface display

    Energy Technology Data Exchange (ETDEWEB)

    Feldhaus, Jane M.; Lou, Jianlong; Coleman, James R.; Siegel, Robert W.; Marks, James D.; Feldhaus, Michael

    2004-04-23

    Yeast display of antibody fragments has proven to be an efficient and productive means for directed evolution of single chain Fv (scFv) antibodies for increased affinity and thermal stability, and more recently for the display and screening of a non-immune library. In this paper, we describe an elegant and simple method for constructing large combinatorial Fab libraries for display on the surface of Saccharomyces cerevisiae, from modestly sized, and easily constructed, heavy and light chain libraries. To this end, we have constructed a set of yeast strains and a two vector system for heavy chain and light chain surface display of Fab fragments with free native amino termini. Through yeast mating of the haploid libraries, a very large heterodimeric immune Fab library was displayed on the diploids and high affinity antigen specific Fabs were isolated from the library.

  13. The Broad Autism Phenotype Questionnaire

    Science.gov (United States)

    Hurley, Robert S. E.; Losh, Molly; Parlier, Morgan; Reznick, J. Steven; Piven, Joseph

    2007-01-01

    The broad autism phenotype (BAP) is a set of personality and language characteristics that reflect the phenotypic expression of the genetic liability to autism, in non-autistic relatives of autistic individuals. These characteristics are milder but qualitatively similar to the defining features of autism. A new instrument designed to measure the…

  14. Panoramic projection avionics displays

    Science.gov (United States)

    Kalmanash, Michael H.

    2003-09-01

    Avionics projection displays are entering production in advanced tactical aircraft. Early adopters of this technology in the avionics community used projection displays to replace or upgrade earlier units incorporating direct-view CRT or AMLCD devices. Typical motivation for these upgrades were the alleviation of performance, cost and display device availability concerns. In these systems, the upgraded (projection) displays were one-for-one form / fit replacements for the earlier units. As projection technology has matured, this situation has begun to evolve. The Lockheed-Martin F-35 is the first program in which the cockpit has been specifically designed to take advantage of one of the more unique capabilities of rear projection display technology, namely the ability to replace multiple small screens with a single large conformal viewing surface in the form of a panoramic display. Other programs are expected to follow, since the panoramic formats enable increased mission effectiveness, reduced cost and greater information transfer to the pilot. Some of the advantages and technical challenges associated with panoramic projection displays for avionics applications are described below.

  15. Antiparietal cell antibody test

    Science.gov (United States)

    APCA; Anti-gastric parietal cell antibody; Atrophic gastritis - anti-gastric parietal cell antibody; Gastric ulcer - anti-gastric parietal cell antibody; Pernicious anemia - anti-gastric parietal cell antibody; ...

  16. Phage display-derived human antibodies against specific glycosaminoglycan epitopes.

    NARCIS (Netherlands)

    Smits, N.C.; Lensen, J.F.M.; Wijnhoven, T.J.M.; Dam, G.B. ten; Jenniskens, G.J.; Kuppevelt, A.H.M.S.M. van

    2006-01-01

    Glycosaminoglycans (GAGs) are long unbranched polysaccharides, most of which are linked to a core protein to form proteoglycans. Depending on the nature of their backbone, one can discern galactosaminoglycans (chondroitin sulfate [CS] and dermatan sulfate [DS]) and glucosaminoglycans (heparan sulfat

  17. "Unconventional" Neutralizing Activity of Antibodies Against HIV

    Institute of Scientific and Technical Information of China (English)

    2007-01-01

    Neutralizing antibodies are recognized to be one of the essential elements of the adaptive immune response that must be induced by an effective vaccine against HIV. However, only a limited number of antibodies have been identified to neutralize a broad range of primary isolates of HIV-1 and attempts to induce such antibodies by immunization were unsuccessful. The difficulties to generate such antibodies are mainly due to intrinsic properties of HIV-1 envelope spikes, such as high sequence diversity, heavy glycosylation, and inducible and transient nature of certain epitopes. In vitro neutralizing antibodies are identified using "conventional" neutralization assay which uses phytohemagglutinin (PHA)-stimulated human PBMCs as target cells. Thus, in essence the assay evaluates HIV-1 replication in CD4+ T cells. Recently, several laboratories including us demonstrated that some monoclonal antibodies and HIV-1-specific polyclonal IgG purified from patient sera, although they do not have neutralizing activity when tested by the "conventional" neutralization assay, do exhibit potent and broad neutralizing activity in "unconventional" ways. The neutralizing activity of these antibodies and IgG fractions is acquired through post-translational modifications, through opsonization of virus particles into macrophages and immature dendritic cells (iDCs), or through expression of antibodies on the surface of HIV-1-susceptible cells. This review will focus on recent findings of this area and point out their potential applications in the development of preventive strategies against HIV.

  18. Small - Display Cartography

    DEFF Research Database (Denmark)

    Nissen, Flemming; Hvas, Anders; Münster-Swendsen, Jørgen

    This report comprises the work carried out in the work-package of small display cartography. The work-package has aimed at creating a general framework for the small-display cartography. A solid framework facilitates an increased use of spatial data in mobile devices - thus enabling, together...... with the rapidly evolving positioning techniques, a new category of position-dependent, map-based services to be introduced. The report consists of the following parts: Part I: Categorization of handheld devices, Part II: Cartographic design for small-display devices, Part III: Study on the GiMoDig Client ? Portal...... Appendix E consisting of confidential material and Appendix F with examples.   The work has resulted in a small device categorisation, a cartographic design specification for a small-display device for a specified navigation task as well as a comparison between OpenLS and an in house developed protocol...

  19. Flexible displays, rigid designs?

    DEFF Research Database (Denmark)

    Hornbæk, Kasper

    2015-01-01

    Rapid technological progress has enabled a wide range of flexible displays for computing devices, but the user experience--which we're only beginning to understand--will be the key driver for successful designs....

  20. Military display performance parameters

    Science.gov (United States)

    Desjardins, Daniel D.; Meyer, Frederick

    2012-06-01

    The military display market is analyzed in terms of four of its segments: avionics, vetronics, dismounted soldier, and command and control. Requirements are summarized for a number of technology-driving parameters, to include luminance, night vision imaging system compatibility, gray levels, resolution, dimming range, viewing angle, video capability, altitude, temperature, shock and vibration, etc., for direct-view and virtual-view displays in cockpits and crew stations. Technical specifications are discussed for selected programs.

  1. Liquid Crystal Airborne Display

    Science.gov (United States)

    1977-08-01

    81/2X 11- 10 -9 .8 display using a large advertising alphanimeric ( TCI ) has been added to the front of the optical box used in the F-4 aircraft for HUD...properties over a wide range of tempera - tures, including normal room temperature. What are Liquid Crystals? Liquid crystals have been classified in three...natic fanctions and to present data needed for the semi- automatic and manual control of system functions. Existing aircraft using CRT display

  2. Broad Diphotons from Narrow States

    CERN Document Server

    An, Haipeng; Zhang, Yue

    2015-01-01

    ATLAS and CMS have each reported a modest diphoton excess consistent with the decay of a broad resonance at ~ 750 GeV. We show how this signal can arise in a weakly coupled theory comprised solely of narrow width particles. In particular, if the decaying particle is produced off-shell, then the associated diphoton resonance will have a broad, adjustable width. We present simplified models which explain the diphoton excess through the three-body decay of a scalar or fermion. Our minimal ultraviolet completion is a weakly coupled and renormalizable theory of a singlet scalar plus a heavy vector-like quark and lepton. The smoking gun of this mechanism is an asymmetric diphoton peak recoiling against missing transverse energy, jets, or leptons.

  3. Raster graphics display library

    Science.gov (United States)

    Grimsrud, Anders; Stephenson, Michael B.

    1987-01-01

    The Raster Graphics Display Library (RGDL) is a high level subroutine package that give the advanced raster graphics display capabilities needed. The RGDL uses FORTRAN source code routines to build subroutines modular enough to use as stand-alone routines in a black box type of environment. Six examples are presented which will teach the use of RGDL in the fastest, most complete way possible. Routines within the display library that are used to produce raster graphics are presented in alphabetical order, each on a separate page. Each user-callable routine is described by function and calling parameters. All common blocks that are used in the display library are listed and the use of each variable within each common block is discussed. A reference on the include files that are necessary to compile the display library is contained. Each include file and its purpose are listed. The link map for MOVIE.BYU version 6, a general purpose computer graphics display system that uses RGDL software, is also contained.

  4. Engineering Recombinant Reoviruses To Display gp41 Membrane-Proximal External-Region Epitopes from HIV-1

    Science.gov (United States)

    Boehme, Karl W.; Ikizler, Mine'; Iskarpatyoti, Jason A.; Wetzel, J. Denise; Willis, Jordan; Crowe, James E.; LaBranche, Celia C.; Montefiori, David C.

    2016-01-01

    ABSTRACT The gp41 membrane-proximal external region (MPER) is a target for broadly neutralizing antibody responses against human immunodeficiency virus type 1 (HIV-1). However, replication-defective virus vaccines currently under evaluation in clinical trials do not efficiently elicit MPER-specific antibodies. Structural modeling suggests that the MPER forms an α-helical coiled coil that is required for function and immunogenicity. To maintain the native MPER conformation, we used reverse genetics to engineer replication-competent reovirus vectors that displayed MPER sequences in the α-helical coiled-coil tail domain of viral attachment protein σ1. Sequences in reovirus strain type 1 Lang (T1L) σ1 were exchanged with sequences encoding HIV-1 strain Ba-L MPER epitope 2F5 or the entire MPER. Individual 2F5 or MPER substitutions were introduced at virion-proximal or virion-distal sites in the σ1 tail. Recombinant reoviruses containing heterologous HIV-1 sequences were viable and produced progeny yields comparable to those with wild-type virus. HIV-1 sequences were retained following 10 serial passages in cell culture, indicating that the substitutions were genetically stable. Recombinant viruses engineered to display the 2F5 epitope or full-length MPER in σ1 were recognized by purified 2F5 antibody. Inoculation of mice with 2F5-containing vectors or rabbits with 2F5- or MPER-containing vectors elicited anti-reovirus antibodies, but HIV-1-specific antibodies were not detected. Together, these findings indicate that heterologous sequences that form α-helices can functionally replace native sequences in the α-helical tail domain of reovirus attachment protein σ1. However, although these vectors retain native antigenicity, they were not immunogenic, illustrating the difficulty of experimentally inducing immune responses to this essential region of HIV-1. IMPORTANCE Vaccines to protect against HIV-1, the causative agent of AIDS, are not approved for use

  5. Discovery of diverse and functional antibodies from large human repertoire antibody libraries.

    Science.gov (United States)

    Schwimmer, Lauren J; Huang, Betty; Giang, Hoa; Cotter, Robyn L; Chemla-Vogel, David S; Dy, Francis V; Tam, Eric M; Zhang, Fangjiu; Toy, Pamela; Bohmann, David J; Watson, Susan R; Beaber, John W; Reddy, Nithin; Kuan, Hua-Feng; Bedinger, Daniel H; Rondon, Isaac J

    2013-05-31

    Phage display antibody libraries have a proven track record for the discovery of therapeutic human antibodies, increasing the demand for large and diverse phage antibody libraries for the discovery of new therapeutics. We have constructed naïve antibody phage display libraries in both Fab and scFv formats, with each library having more than 250 billion clones that encompass the human antibody repertoire. These libraries show high fidelity in open reading frame and expression percentages, and their V-gene family distribution, VH-CDR3 length and amino acid usage mirror the natural diversity of human antibodies. Both the Fab and scFv libraries show robust sequence diversity in target-specific binders and differential V-gene usage for each target tested, supporting the use of libraries that utilize multiple display formats and V-gene utilization to maximize antibody-binding diversity. For each of the targets, clones with picomolar affinities were identified from at least one of the libraries and for the two targets assessed for activity, functional antibodies were identified from both libraries.

  6. The Ultimate Display

    CERN Document Server

    Fluke, C J

    2016-01-01

    Astronomical images and datasets are increasingly high-resolution and multi-dimensional. The vast majority of astronomers perform all of their visualisation and analysis tasks on low-resolution, two-dimensional desktop monitors. If there were no technological barriers to designing the ultimate stereoscopic display for astronomy, what would it look like? What capabilities would we require of our compute hardware to drive it? And are existing technologies even close to providing a true 3D experience that is compatible with the depth resolution of human stereoscopic vision? We consider the CAVE2 (an 80 Megapixel, hybrid 2D and 3D virtual reality environment directly integrated with a 100 Tflop/s GPU-powered supercomputer) and the Oculus Rift (a low- cost, head-mounted display) as examples at opposite financial ends of the immersive display spectrum.

  7. Broad-spectrum antiviral therapeutics.

    Directory of Open Access Journals (Sweden)

    Todd H Rider

    Full Text Available Currently there are relatively few antiviral therapeutics, and most which do exist are highly pathogen-specific or have other disadvantages. We have developed a new broad-spectrum antiviral approach, dubbed Double-stranded RNA (dsRNA Activated Caspase Oligomerizer (DRACO that selectively induces apoptosis in cells containing viral dsRNA, rapidly killing infected cells without harming uninfected cells. We have created DRACOs and shown that they are nontoxic in 11 mammalian cell types and effective against 15 different viruses, including dengue flavivirus, Amapari and Tacaribe arenaviruses, Guama bunyavirus, and H1N1 influenza. We have also demonstrated that DRACOs can rescue mice challenged with H1N1 influenza. DRACOs have the potential to be effective therapeutics or prophylactics for numerous clinical and priority viruses, due to the broad-spectrum sensitivity of the dsRNA detection domain, the potent activity of the apoptosis induction domain, and the novel direct linkage between the two which viruses have never encountered.

  8. Sublingual immunization with M2-based vaccine induces broad protective immunity against influenza.

    Directory of Open Access Journals (Sweden)

    Byoung-Shik Shim

    Full Text Available BACKGROUND: The ectodomain of matrix protein 2 (M2e of influenza A virus is a rationale target antigen candidate for the development of a universal vaccine against influenza as M2e undergoes little sequence variation amongst human influenza A strains. Vaccine-induced M2e-specific antibodies (Abs have been shown to display significant cross-protective activity in animal models. M2e-based vaccine constructs have been shown to be more protective when administered by the intranasal (i.n. route than after parenteral injection. However, i.n. administration of vaccines poses rare but serious safety issues associated with retrograde passage of inhaled antigens and adjuvants through the olfactory epithelium. In this study, we examined whether the sublingual (s.l. route could serve as a safe and effective alternative mucosal delivery route for administering a prototype M2e-based vaccine. The mechanism whereby s.l. immunization with M2e vaccine candidate induces broad protection against infection with different influenza virus subtypes was explored. METHODS AND RESULTS: A recombinant M2 protein with three tandem copies of the M2e (3M2eC was expressed in Escherichia coli. Parenteral immunizations of mice with 3M2eC induced high levels of M2e-specific serum Abs but failed to provide complete protection against lethal challenge with influenza virus. In contrast, s.l. immunization with 3M2eC was superior for inducing protection in mice. In the latter animals, protection was associated with specific Ab responses in the lungs. CONCLUSIONS: The results demonstrate that s.l. immunization with 3M2eC vaccine induced airway mucosal immune responses along with broad cross-protective immunity to influenza. These findings may contribute to the understanding of the M2-based vaccine approach to control epidemic and pandemic influenza infections.

  9. Virtual Auditory Displays

    Science.gov (United States)

    2000-01-01

    timbre , intensity, distance, room modeling, radio communication Virtual Environments Handbook Chapter 4 Virtual Auditory Displays Russell D... musical note “A” as a pure sinusoid, there will be 440 condensations and rarefactions per second. The distance between two adjacent condensations or...and complexity are pitch, loudness, and timbre respectively. This distinction between physical and perceptual measures of sound properties is an

  10. Refreshing Refreshable Braille Displays.

    Science.gov (United States)

    Russomanno, Alexander; O'Modhrain, Sile; Gillespie, R Brent; Rodger, Matthew W M

    2015-01-01

    The increased access to books afforded to blind people via e-publishing has given them long-sought independence for both recreational and educational reading. In most cases, blind readers access materials using speech output. For some content such as highly technical texts, music, and graphics, speech is not an appropriate access modality as it does not promote deep understanding. Therefore blind braille readers often prefer electronic braille displays. But, these are prohibitively expensive. The search is on, therefore, for a low-cost refreshable display that would go beyond current technologies and deliver graphical content as well as text. And many solutions have been proposed, some of which reduce costs by restricting the number of characters that can be displayed, even down to a single braille cell. In this paper, we demonstrate that restricting tactile cues during braille reading leads to poorer performance in a letter recognition task. In particular, we show that lack of sliding contact between the fingertip and the braille reading surface results in more errors and that the number of errors increases as a function of presentation speed. These findings suggest that single cell displays which do not incorporate sliding contact are likely to be less effective for braille reading.

  11. Developing recombinant antibodies for biomarker detection

    Energy Technology Data Exchange (ETDEWEB)

    Baird, Cheryl L.; Fischer, Christopher J.; Pefaur, Noah B.; Miller, Keith D.; Kagen, Jacob; Srivastava, Sudhir; Rodland, Karin D.

    2010-10-01

    Monoclonal antibodies (mAbs) have an essential role in biomarker validation and diagnostic assays. A barrier to pursuing these applications is the reliance on immunization and hybridomas to produce mAbs, which is time-consuming and may not yield the desired mAb. We recommend a process flow for affinity reagent production that utilizes combinatorial protein display systems (eg, yeast surface display or phage display) rather than hybridomas. These systems link a selectable phenotype-binding conferred by an antibody fragment-with a means for recovering the encoding gene. Recombinant libraries obtained from immunizations can produce high-affinity antibodies (<10 nM) more quickly than other methods. Non-immune libraries provide an alternate route when immunizations are not possible, or when suitable mAbs are not recovered from an immune library. Directed molecular evolution (DME) is an integral part of optimizing mAbs obtained from combinatorial protein display, but can also be used on hybridoma-derived mAbs. Variants can easily be obtained and screened to increase the affinity of the parent mAb (affinity maturation). We discuss examples where DME has been used to tailor affinity reagents to specific applications. Combinatorial protein display also provides an accessible method for identifying antibody pairs, which are necessary for sandwich-type diagnostic assays.

  12. Use of antibody gene library for the isolation of specific single chain antibodies by ampicillin-antigen conjugates.

    Science.gov (United States)

    Neumann-Schaal, Meina; Messerschmidt, Katrin; Grenz, Nicole; Heilmann, Katja

    2013-03-01

    Isolation of recombinant antibodies from antibody libraries is commonly performed by different molecular display formats including phage display and ribosome display or different cell-surface display formats. We describe a new method which allows the selection of Escherichia coli cells producing the required single chain antibody by cultivation in presence of ampicillin conjugated to the antigen of interest. The method utilizes the neutralization of the conjugate by the produced single chain antibody which is secreted to the periplasm. Therefore, a new expression system based on the pET26b vector was designed and a library was constructed. The method was successfully established first for the selection of E. coli BL21 Star (DE3) cells expressing a model single chain antibody (anti-fluorescein) by a simple selection assay on LB-agar plates. Using this selection assay, we could identify a new single chain antibody binding biotin by growing E. coli BL21 Star (DE3) containing the library in presence of a biotin-ampicillin conjugate. In contrast to methods as molecular or cell surface display our selection system applies the soluble single chain antibody molecule and thereby avoids undesired effects, e.g. by the phage particle or the yeast fusion protein. By selecting directly in an expression strain, production and characterization of the selected single chain antibody is possible without any further cloning or transformation steps.

  13. Virtual acoustic displays

    Science.gov (United States)

    Wenzel, Elizabeth M.

    1991-01-01

    A 3D auditory display can potentially enhance information transfer by combining directional and iconic information in a quite naturalistic representation of dynamic objects in the interface. Another aspect of auditory spatial clues is that, in conjunction with other modalities, it can act as a potentiator of information in the display. For example, visual and auditory cues together can reinforce the information content of the display and provide a greater sense of presence or realism in a manner not readily achievable by either modality alone. This phenomenon will be particularly useful in telepresence applications, such as advanced teleconferencing environments, shared electronic workspaces, and monitoring telerobotic activities in remote or hazardous situations. Thus, the combination of direct spatial cues with good principles of iconic design could provide an extremely powerful and information-rich display which is also quite easy to use. An alternative approach, recently developed at ARC, generates externalized, 3D sound cues over headphones in realtime using digital signal processing. Here, the synthesis technique involves the digital generation of stimuli using Head-Related Transfer Functions (HRTF's) measured in the two ear-canals of individual subjects. Other similar approaches include an analog system developed by Loomis, et. al., (1990) and digital systems which make use of transforms derived from normative mannikins and simulations of room acoustics. Such an interface also requires the careful psychophysical evaluation of listener's ability to accurately localize the virtual or synthetic sound sources. From an applied standpoint, measurement of each potential listener's HRTF's may not be possible in practice. For experienced listeners, localization performance was only slightly degraded compared to a subject's inherent ability. Alternatively, even inexperienced listeners may be able to adapt to a particular set of HRTF's as long as they provide adequate

  14. Combination effect on HIV infection in vitro of soluble CD4 and HIV-neutralizing antibodies

    DEFF Research Database (Denmark)

    Hansen, J E; Sørensen, A M; Olofsson, S;

    1994-01-01

    In combination with HIV gp120 V3-loop antibody, two carbohydrate specific neutralizing antibodies (83D4 and 2G12) had a synergistic neutralizing effect on HIV infection. However, sCD4 and an antibody which blocks gp 120/CD4 binding (1B1) both displayed antagonism.......In combination with HIV gp120 V3-loop antibody, two carbohydrate specific neutralizing antibodies (83D4 and 2G12) had a synergistic neutralizing effect on HIV infection. However, sCD4 and an antibody which blocks gp 120/CD4 binding (1B1) both displayed antagonism....

  15. Ghost imaging with broad distance

    Institute of Scientific and Technical Information of China (English)

    段德洋; 张路; 杜少将; 夏云杰

    2015-01-01

    We present a scheme that is able to achieve the ghost imaging with broad distance. The physical nature of our scheme is that the different wavelength beams are separated in free space by an optical media according to the slow light or dispersion principle. Meanwhile, the equality of the optical distance of the two light arms is not violated. The photon correlation is achieved by the rotating ground glass plate (RGGP) and spatial light modulator (SLM), respectively. Our work shows that a monochromic ghost image can be obtained in the case of RGGP. More importantly, the position (or distance) of the object can be ascertained by the color of the image. Thus, the imaging and ranging processes are combined as one process for the first time to the best of our knowledge. In the case of SLM, we can obtain a colored image regardless of where the object is.

  16. Antibody function in neutralization and protection against HIV-1

    NARCIS (Netherlands)

    Hessell, A.J.

    2009-01-01

    The ability to induce neutralizing antibodies is generally thought to be of great importance for vaccine efficacy. In HIV-1 research this quality has been elusive as the HIV-1 virus has evolved multiple mechanisms to evade neutralizing antibodies. This thesis traces studies with four broadly neutral

  17. Immunologically driven chemical engineering of antibodies for catalytic activity.

    Science.gov (United States)

    Dias, Sonia; Jovic, Florence; Renard, Pierre-Yves; Taran, Fréderic; Créminon, Christophe; Mioskowski, Charles; Grassi, Jacques

    2002-11-01

    We describe a new strategy for the preparation of catalytic antibodies based on a two-step procedure. Firstly, monoclonal antibodies are selected only if displaying the following binding features: binding both the substrate and a reactive group in such a way that the two groups are in a reactive position towards each other. Secondly, the selected monoclonal antibodies (mAbs) are chemically engineered by covalently binding the reactive group into the binding pocket of the antibody. Using previously isolated monoclonal antibodies, we have focused our studies on the control of this second step.

  18. Pulmonary manifestations of the antiphospholipid antibody syndrome.

    Science.gov (United States)

    Ford, H James; Roubey, Robert A S

    2010-09-01

    A broad spectrum of pulmonary disease may occur in antiphospholipid antibody syndrome. The most common pulmonary manifestations are pulmonary thromboembolism and pulmonary hypertension. In this article the authors review these manifestations, as well as less common findings including acute respiratory distress syndrome, alveolar hemorrhage, and pulmonary capillaritis.

  19. Refrigerated display cabinets; Butikskyla

    Energy Technology Data Exchange (ETDEWEB)

    Fahlen, Per

    2000-07-01

    This report summarizes experience from SP research and assignments regarding refrigerated transport and storage of food, mainly in the retail sector. It presents the fundamentals of heat and mass transfer in display cabinets with special focus on indirect systems and secondary refrigerants. Moreover, the report includes a brief account of basic food hygiene and the related regulations. The material has been compiled for educational purposes in the Masters program at Chalmers Technical University.

  20. Phage display library screening for identification of interacting protein partners.

    Science.gov (United States)

    Addepalli, Balasubrahmanyam; Rao, Suryadevara; Hunt, Arthur G

    2015-01-01

    Phage display is a versatile high-throughput screening method employed to understand and improve the chemical biology, be it production of human monoclonal antibodies or identification of interacting protein partners. A majority of cell proteins operate in a concerted fashion either by stable or transient interactions. Such interactions can be mediated by recognition of small amino acid sequence motifs on the protein surface. Phage display can play a crucial role in identification of such motifs. This report describes the use of phage display for the identification of high affinity sequence motifs that could be responsible for interactions with a target (bait) protein.

  1. Broad Leaves in Strong Flow

    CERN Document Server

    Miller, Laura

    2013-01-01

    Flexible broad leaves are thought to reconfigure in the wind and water to reduce the drag forces that act upon them. Simple mathematical models of a flexible beam immersed in a two-dimensional flow will also exhibit this behavior. What is less understood is how the mechanical properties of a leaf in a three-dimensional flow will passively allow roll up into a cone shape and reduce both drag and vortex induced oscillations. In this fluid dynamics video, the flows around the leaves are compared with those of simplified sheets using 3D numerical simulations and physical models. For some reconfiguration shapes, large forces and oscillations due to strong vortex shedding are produced. In the actual leaf, a stable recirculation zone is formed within the wake of the reconfigured cone. In physical and numerical models that reconfigure into cones, a similar recirculation zone is observed with both rigid and flexible tethers. These results suggest that the three-dimensional cone structure in addition to flexibility is ...

  2. Oligopeptide M13 Phage Display in Pathogen Research

    Directory of Open Access Journals (Sweden)

    Michael Hust

    2013-10-01

    Full Text Available Phage display has become an established, widely used method for selection of peptides, antibodies or alternative scaffolds. The use of phage display for the selection of antigens from genomic or cDNA libraries of pathogens which is an alternative to the classical way of identifying immunogenic proteins is not well-known. In recent years several new applications for oligopeptide phage display in disease related fields have been developed which has led to the identification of various new antigens. These novel identified immunogenic proteins provide new insights into host pathogen interactions and can be used for the development of new diagnostic tests and vaccines. In this review we focus on the M13 oligopeptide phage display system for pathogen research but will also give examples for lambda phage display and for applications in other disease related fields. In addition, a detailed technical work flow for the identification of immunogenic oligopeptides using the pHORF system is given. The described identification of immunogenic proteins of pathogens using oligopeptide phage display can be linked to antibody phage display resulting in a vaccine pipeline.

  3. [Antinuclear antibodies].

    Science.gov (United States)

    Cabiedes, Javier; Núñez-Álvarez, Carlos A

    2010-01-01

    Anti-nuclear antibodies (ANA) are immunoglobulin directed against autologous cell nuclear and cytoplasmic components. Besides the autoimmune ANA there are other ANA that can be detected in circulation, like natural and infectious ANA. Because of its high sensibility, detection of the ANA must be done by indirect immunofluorescence (IIF) as screening test and all of those positive samples are convenient to confirm its specificity by ELISA, western blot or other techniques. Positive ANA detected by IIF must be evaluated taking in to account the pattern and titer. The following recommended step is the specificity characterization (reactivity against extractable nuclear antigens [ENA], dsDNA, etc.) which is useful for the diagnosis and follow up of patients with autoimmune diseases, and by such reasoning, its detection must be performed in an orderly and reasonable way using guides or strategies focused to the good use and interpretation of the autoantibodies. The objective of this review is to present a compilation of the literature and our experience in the detection and study of the ANA.

  4. Book Display as Adult Service

    Directory of Open Access Journals (Sweden)

    Matthew S. Moore

    1997-03-01

    Full Text Available 無Book display as an adult service is defined as choosing and positioning adult books from the collection to increase their circulation. The author contrasts bookstore arrangement for sales versus library arrangement for access. The paper considers the library-as-a-whole as a display, examines the right size for an in-library display, and discusses mass displays, end-caps, on-shelf displays, and the Tiffany approach. The author proposes that an effective display depends on an imaginative, unifying theme, and that book displays are part of the joy of libraries.

  5. Handbook of Visual Display Technology

    CERN Document Server

    Cranton, Wayne; Fihn, Mark

    2012-01-01

    The Handbook of Visual Display Technology is a unique work offering a comprehensive description of the science, technology, economic and human interface factors associated with the displays industry. An invaluable compilation of information, the Handbook will serve as a single reference source with expert contributions from over 150 international display professionals and academic researchers. All classes of display device are covered including LCDs, reflective displays, flexible solutions and emissive devices such as OLEDs and plasma displays, with discussion of established principles, emergent technologies, and particular areas of application. The wide-ranging content also encompasses the fundamental science of light and vision, image manipulation, core materials and processing techniques, display driving and metrology.

  6. Antibody-Mediated Internalization of Infectious HIV-1 Virions Differs among Antibody Isotypes and Subclasses.

    Science.gov (United States)

    Tay, Matthew Zirui; Liu, Pinghuang; Williams, LaTonya D; McRaven, Michael D; Sawant, Sheetal; Gurley, Thaddeus C; Xu, Thomas T; Dennison, S Moses; Liao, Hua-Xin; Chenine, Agnès-Laurence; Alam, S Munir; Moody, M Anthony; Hope, Thomas J; Haynes, Barton F; Tomaras, Georgia D

    2016-08-01

    Emerging data support a role for antibody Fc-mediated antiviral activity in vaccine efficacy and in the control of HIV-1 replication by broadly neutralizing antibodies. Antibody-mediated virus internalization is an Fc-mediated function that may act at the portal of entry whereby effector cells may be triggered by pre-existing antibodies to prevent HIV-1 acquisition. Understanding the capacity of HIV-1 antibodies in mediating internalization of HIV-1 virions by primary monocytes is critical to understanding their full antiviral potency. Antibody isotypes/subclasses differ in functional profile, with consequences for their antiviral activity. For instance, in the RV144 vaccine trial that achieved partial efficacy, Env IgA correlated with increased risk of HIV-1 infection (i.e. decreased vaccine efficacy), whereas V1-V2 IgG3 correlated with decreased risk of HIV-1 infection (i.e. increased vaccine efficacy). Thus, understanding the different functional attributes of HIV-1 specific IgG1, IgG3 and IgA antibodies will help define the mechanisms of immune protection. Here, we utilized an in vitro flow cytometric method utilizing primary monocytes as phagocytes and infectious HIV-1 virions as targets to determine the capacity of Env IgA (IgA1, IgA2), IgG1 and IgG3 antibodies to mediate HIV-1 infectious virion internalization. Importantly, both broadly neutralizing antibodies (i.e. PG9, 2G12, CH31, VRC01 IgG) and non-broadly neutralizing antibodies (i.e. 7B2 mAb, mucosal HIV-1+ IgG) mediated internalization of HIV-1 virions. Furthermore, we found that Env IgG3 of multiple specificities (i.e. CD4bs, V1-V2 and gp41) mediated increased infectious virion internalization over Env IgG1 of the same specificity, while Env IgA mediated decreased infectious virion internalization compared to IgG1. These data demonstrate that antibody-mediated internalization of HIV-1 virions depends on antibody specificity and isotype. Evaluation of the phagocytic potency of vaccine

  7. Functionally fused antibodies--a novel adjuvant fusion system

    DEFF Research Database (Denmark)

    Larsen, Martin; Jensen, Kim Bak; Christensen, Peter Astrup

    2008-01-01

    Antibodies capable of recognizing key molecular targets isolated e.g. by phage display technology have been used in the pursuit of new and improved therapies for prevalent human diseases. These approaches often take advantage of non-immunogenic antibody fragments to achieve specific toxin-, radio...

  8. HIV-1 resistance to neutralizing antibodies: Determination of antibody concentrations leading to escape mutant evolution.

    Science.gov (United States)

    Magnus, Carsten; Reh, Lucia; Trkola, Alexandra

    2016-06-15

    Broadly neutralizing antibodies against human immunodeficiency virus type 1 (HIV-1) are considered vital components of novel therapeutics and blueprints for vaccine research. Yet escape to even the most potent of these antibodies is imminent in natural infection. Measures to define antibody efficacy and prevent mutant selection are thus urgently needed. Here, we derive a mathematical framework to predict the concentration ranges for which antibody escape variants can outcompete their viral ancestors, referred to as mutant selection window (MSW). When determining the MSW, we focus on the differential efficacy of neutralizing antibodies against HIV-1 in two canonical infection routes, free-virus infection and cell-cell transmission. The latter has proven highly effective in vitro suggesting its importance for both in vivo spread as well as for escaping targeted intervention strategies. We observed a range of MSW patterns that highlight the potential of mutants to arise in both transmission pathways and over wide concentration ranges. Most importantly, we found that only when the arising mutant has both, residual sensitivity to the neutralizing antibody and reduced infectivity compared to the parental virus, antibody dosing outside of the MSW to restrict mutant selection is possible. Emergence of mutants that provide complete escape and have no considerable fitness loss cannot be prevented by adjusting antibody doses. The latter may in part explain the ubiquitous resistance to neutralizing antibodies observed in natural infection and antibody treatment. Based on our findings, combinations of antibodies targeting different epitopes should be favored for antibody-based interventions as this may render complete resistance less likely to occur and also increase chances that multiple escapes result in severe fitness loss of the virus making longer-term antibody treatment more feasible.

  9. 76 FR 34087 - Broad Stakeholder Survey

    Science.gov (United States)

    2011-06-10

    ... SECURITY Broad Stakeholder Survey AGENCY: National Protection and Programs Directorate, DHS. ACTION: 60-day... comments concerning the Broad Stakeholder Survey. DATES: Comments are encouraged and will be accepted until.... The Broad Stakeholder Survey is designed to gather stakeholder feedback on the effectiveness of...

  10. 78 FR 20119 - Broad Stakeholder Survey

    Science.gov (United States)

    2013-04-03

    ... SECURITY Broad Stakeholder Survey AGENCY: National Protection and Programs Directorate, DHS. ACTION: 30-day... soliciting comments concerning the Broad Stakeholder Survey. DHS previously published this ICR in the Federal... responders across the Nation. The Broad Stakeholder Survey is designed to gather stakeholder feedback on...

  11. 77 FR 50144 - Broad Stakeholder Survey

    Science.gov (United States)

    2012-08-20

    ... SECURITY Broad Stakeholder Survey AGENCY: National Protection and Programs Directorate, DHS. ACTION: 60-day... comments concerning the Broad Stakeholder Survey. DATES: Comments are encouraged and will be accepted until... across the Nation. The Broad Stakeholder Survey is designed to gather stakeholder feedback on...

  12. Selection of antibodies from synthetic antibody libraries.

    Science.gov (United States)

    Harel Inbar, Noa; Benhar, Itai

    2012-10-15

    More than 2 dozen years had passed since the field of antibody engineering was established, with the first reports of bacterial [1-3] and mammalian cells [4] expression of recombinant antibody fragments, and in that time a lot of effort was dedicated to the development of efficient technological means, intended to assist in the creation of therapeutic monoclonal antibodies (mAbs). Research focus was given to two intertwined technological aspects: the selection platform and the recombinant antibody repertoires. In accordance with these areas of interest, it is the goal of this chapter to describe the various selection tools and antibody libraries existing, with emphasis on the later, and their applications. This chapter gives a far from exhaustive, subjective "historic account" of the field, describing the selection platforms, the different formats of antibody repertoires and the applications of both for selecting recombinant antibodies. Several excellent books provide detailed protocols for constructing antibody libraries and selecting antibodies from those libraries [5-13]. Such books may guide a newcomer to the field in the fine details of antibody engineering. We would like to offer advice to the novice: although seemingly simple, effective library construction and antibody isolation provide best benefits in the hands of professionals. It is an art as much as it is science.

  13. A critical question for HIV vaccine development: Which antibodies to induce?

    OpenAIRE

    Zolla-Pazner, Susan

    2014-01-01

    A vaccine against HIV-1 must prevent infection against genetically diverse virus strains. Two approaches are currently being pursued to elicit antibody-mediated protection: vaccines that induce potent and broadly reactive neutralizing antibodies (bnAbs) or vaccines that induce “conventional antibodies,” which are less potent and broadly neutralizing in comparison. Although bnAbs may provide the greatest level of protection, their structural and genetic characteristics make their elicitation t...

  14. A nanobody:GFP bacterial platform that enables functional enzyme display and easy quantification of display capacity

    DEFF Research Database (Denmark)

    Wendel, Sofie; Christian Fischer, Emil; Martinez, Virginia;

    2016-01-01

    Background: Bacterial surface display is an attractive technique for the production of cell-anchored, functional proteins and engineering of whole-cell catalysts. Although various outer membrane proteins have been used for surface display, an easy and versatile high-throughput-compatible assay...... for evaluating and developing surface display systems is missing.Results: Using a single domain antibody (also called nanobody) with high affinity for green fluorescent protein (GFP), we constructed a system that allows for fast, fluorescence-based detection of displayed proteins. The outer membrane hybrid...... protein LppOmpA and the autotransporter C-IgAP exposed the nanobody on the surface of Escherichia coli with very different efficiency. Both anchors were capable of functionally displaying the enzyme Chitinase A as a fusion with the nanobody, and this considerably increased expression levels compared...

  15. Latest development of display technologies

    Science.gov (United States)

    Gao, Hong-Yue; Yao, Qiu-Xiang; Liu, Pan; Zheng, Zhi-Qiang; Liu, Ji-Cheng; Zheng, Hua-Dong; Zeng, Chao; Yu, Ying-Jie; Sun, Tao; Zeng, Zhen-Xiang

    2016-09-01

    In this review we will focus on recent progress in the field of two-dimensional (2D) and three-dimensional (3D) display technologies. We present the current display materials and their applications, including organic light-emitting diodes (OLEDs), flexible OLEDs quantum dot light emitting diodes (QLEDs), active-matrix organic light emitting diodes (AMOLEDs), electronic paper (E-paper), curved displays, stereoscopic 3D displays, volumetric 3D displays, light field 3D displays, and holographic 3D displays. Conventional 2D display devices, such as liquid crystal devices (LCDs) often result in ambiguity in high-dimensional data images because of lacking true depth information. This review thus provides a detailed description of 3D display technologies.

  16. Cross-reactivity between immunoglobulin G antibodies of whales and dolphins correlates with evolutionary distance.

    Science.gov (United States)

    Nollens, Hendrik H; Ruiz, Carolina; Walsh, Michael T; Gulland, Frances M D; Bossart, Gregory; Jensen, Eric D; McBain, James F; Wellehan, James F X

    2008-10-01

    Growing morphological and molecular evidence indicates that the porpoises, dolphins, and whales evolved within the even-toed ungulates, formerly known as Artiodactyla. These animals are now grouped in the Cetartiodactyla. We evaluated the antigenic similarity of the immunoglobulin G (IgG) molecules of 15 cetacean species and the domestic cow. The similarity was scored using three distinct antibodies raised against bottlenose dolphin (Tursiops truncatus) IgG in a Western blot, an indirect enzyme-linked immunosorbent assay (ELISA), and a competitive ELISA format. A score was generated for the genetic distance between each species and T. truncatus using the cytochrome b sequence. Each antibody displayed a distinct pattern of reactivity with the IgG antibodies of the various species. The monoclonal antibody (MAb) specific for the gamma heavy chain of T. truncatus was reactive with all monodontids, delphinids, and phocoenids. The light-chain-specific MAb reacted with IgG of delphinoid and phocoenid species and one of the two mysticete species tested. The polyclonal antibody was broadly cross-reactive across all cetaceans and the domestic cow. Using the MAb specific for the gamma heavy chain, the degree of IgG cross-reactivity ranged from less than 17% for the mysticetes to 106% for killer whale Orcinus orca. The IgG in beaked whale and baleen whale sera was significantly less cross-reactive with bottlenose dolphin IgG than sera from other toothed whales. A strong negative correlation was demonstrated between antigenic cross-reactivity of IgG molecules and the genetic distance of their hosts. The data generated will be useful for the development of clinical serodiagnostics in diverse cetacean species.

  17. A fully synthetic human Fab antibody library based on fixed VH/VL framework pairings with favorable biophysical properties.

    Science.gov (United States)

    Tiller, Thomas; Schuster, Ingrid; Deppe, Dorothée; Siegers, Katja; Strohner, Ralf; Herrmann, Tanja; Berenguer, Marion; Poujol, Dominique; Stehle, Jennifer; Stark, Yvonne; Heßling, Martin; Daubert, Daniela; Felderer, Karin; Kaden, Stefan; Kölln, Johanna; Enzelberger, Markus; Urlinger, Stefanie

    2013-01-01

    This report describes the design, generation and testing of Ylanthia, a fully synthetic human Fab antibody library with 1.3E+11 clones. Ylanthia comprises 36 fixed immunoglobulin (Ig) variable heavy (VH)/variable light (VL) chain pairs, which cover a broad range of canonical complementarity-determining region (CDR) structures. The variable Ig heavy and Ig light (VH/VL) chain pairs were selected for biophysical characteristics favorable to manufacturing and development. The selection process included multiple parameters, e.g., assessment of protein expression yield, thermal stability and aggregation propensity in fragment antigen binding (Fab) and IgG1 formats, and relative Fab display rate on phage. The framework regions are fixed and the diversified CDRs were designed based on a systematic analysis of a large set of rearranged human antibody sequences. Care was taken to minimize the occurrence of potential posttranslational modification sites within the CDRs. Phage selection was performed against various antigens and unique antibodies with excellent biophysical properties were isolated. Our results confirm that quality can be built into an antibody library by prudent selection of unmodified, fully human VH/VL pairs as scaffolds.

  18. Organic transistors in optical displays and microelectronic applications.

    Science.gov (United States)

    Gelinck, Gerwin; Heremans, Paul; Nomoto, Kazumasa; Anthopoulos, Thomas D

    2010-09-08

    Organic thin-film transistors (OTFTs) offer unprecedented opportunities for implementation in a broad range of technological applications spanning from large-volume microelectronics and optical displays to chemical and biological sensors. In this Progress Report, we review the application of organic transistors in the fields of flexible optical displays and microelectronics. The advantages associated with the use of OTFT technology are discussed with primary emphasis on the latest developments in the area of active-matrix electrophoretic and organic light-emitting diode displays based on OTFT backplanes and on the application of organic transistors in microelectronics including digital and analog circuits.

  19. Organic transistors in optical displays and microelectronic applications

    NARCIS (Netherlands)

    Gelinck, G.H.; Heremans, P.; Nomoto, K.; Anthopoulos, T.D.

    2010-01-01

    Organic thin-film transistors (OTFTs) offer unprecedented opportunities for implementation in a broad range of technological applications spanning from large-volume microelectronics and optical displays to chemical and biological sensors. In this Progress Report, we review the application of organic

  20. Mining gut microbiome oligopeptides by functional metaproteome display

    Science.gov (United States)

    Zantow, Jonas; Just, Sarah; Lagkouvardos, Ilias; Kisling, Sigrid; Dübel, Stefan; Lepage, Patricia; Clavel, Thomas; Hust, Michael

    2016-01-01

    Pathogen infections, autoimmune diseases, and chronic inflammatory disorders are associated with systemic antibody responses from the host immune system. Disease-specific antibodies can be important serum biomarkers, but the identification of antigens associated with specific immune reactions is challenging, in particular if complex communities of microorganisms are involved in the disease progression. Despite promising new diagnostic opportunities, the discovery of these serological markers becomes more difficult with increasing complexity of microbial communities. In the present work, we used a metagenomic M13 phage display approach to select immunogenic oligopeptides from the gut microbiome of transgenic mice suffering from chronic ileitis. We constructed three individual metaproteome phage display libraries with a library size of approximately 107 clones each. Using serum antibodies, we selected and validated three oligopeptides that induced specific antibody responses in the mouse model. This proof-of-concept study provides the first successful application of functional metaproteome display for the study of protein-protein interactions and the discovery of potential disease biomarkers. PMID:27703179

  1.  De novo isolation of antibodies with pH-dependent binding properties

    OpenAIRE

    Bonvin, Pauline; Venet, Sophie; Fontaine, Gaëlle; Ravn, Ulla; Gueneau, Franck; Kosco-Vilbois, Marie; Proudfoot, Amanda; Fischer, Nicolas

    2015-01-01

    pH-dependent antibodies are engineered to release their target at a slightly acidic pH, a property making them suitable for clinical as well as biotechnological applications. Such antibodies were previously obtained by histidine scanning of pre-existing antibodies, a labor-intensive strategy resulting in antibodies that displayed residual binding to their target at pH 6.0. We report here the de novo isolation of pH-dependent antibodies selected by phage display from libraries enriched in hist...

  2. Interaction Analysis through Proteomic Phage Display

    Directory of Open Access Journals (Sweden)

    Gustav N. Sundell

    2014-01-01

    Full Text Available Phage display is a powerful technique for profiling specificities of peptide binding domains. The method is suited for the identification of high-affinity ligands with inhibitor potential when using highly diverse combinatorial peptide phage libraries. Such experiments further provide consensus motifs for genome-wide scanning of ligands of potential biological relevance. A complementary but considerably less explored approach is to display expression products of genomic DNA, cDNA, open reading frames (ORFs, or oligonucleotide libraries designed to encode defined regions of a target proteome on phage particles. One of the main applications of such proteomic libraries has been the elucidation of antibody epitopes. This review is focused on the use of proteomic phage display to uncover protein-protein interactions of potential relevance for cellular function. The method is particularly suited for the discovery of interactions between peptide binding domains and their targets. We discuss the largely unexplored potential of this method in the discovery of domain-motif interactions of potential biological relevance.

  3. A Strategy for Screening Monoclonal Antibodies for Arabidopsis Flowers

    Science.gov (United States)

    Shi, Qian; Zhou, Lian; Wang, Yingxiang; Ma, Hong

    2017-01-01

    The flower is one of the most complex structures of angiosperms and is essential for sexual reproduction. Current studies using molecular genetic tools have made great advances in understanding flower development. Due to the lack of available antibodies, studies investigating the localization of proteins required for flower development have been restricted to use commercial antibodies against known antigens such as GFP, YFP, and FLAG. Thus, knowledge about cellular structures in the floral organs is limited due to the scarcity of antibodies that can label cellular components. To generate monoclonal antibodies that can facilitate molecular studies of the flower, we constructed a library of monoclonal antibodies against antigenic proteins from Arabidopsis inflorescences and identified 61 monoclonal antibodies. Twenty-four of these monoclonal antibodies displayed a unique band in a western blot assay in at least one of the examined tissues. Distinct cellular distribution patterns of epitopes were detected by these 24 antibodies by immunofluorescence microscopy in a flower section. Subsequently, a combination of immunoprecipitation and mass spectrometry analysis identified potential targets for three of these antibodies. These results provide evidence for the generation of an antibody library using the total plant proteins as antigens. Using this method, the present study identified 61 monoclonal antibodies and 24 of them were efficiently detecting epitopes in both western blot experiments and immunofluorescence microscopy. These antibodies can be applied as informative cellular markers to study the biological mechanisms underlying floral development in plants. PMID:28293248

  4. Yeast surface display for screening combinatorial polypeptide libraries.

    Science.gov (United States)

    Boder, E T; Wittrup, K D

    1997-06-01

    Display on the yeast cell wall is well suited for engineering mammalian cell-surface and secreted proteins (e.g., antibodies, receptors, cytokines) that require endoplasmic reticulum-specific post-translational processing for efficient folding and activity. C-terminal fusion to the Aga2p mating adhesion receptor of Saccharomyces cerevisiae has been used for the selection of scFv antibody fragments with threefold decreased antigen dissociation rate from a randomly mutated library. A eukaryotic host should alleviate expression biases present in bacterially propagated combinatorial libraries. Quantitative flow cytometric analysis enables fine discrimination of kinetic parameters for protein binding to soluble ligands.

  5. Colorimetry for CRT displays.

    Science.gov (United States)

    Golz, Jürgen; MacLeod, Donald I A

    2003-05-01

    We analyze the sources of error in specifying color in CRT displays. These include errors inherent in the use of the color matching functions of the CIE 1931 standard observer when only colorimetric, not radiometric, calibrations are available. We provide transformation coefficients that prove to correct the deficiencies of this observer very well. We consider four different candidate sets of cone sensitivities. Some of these differ substantially; variation among candidate cone sensitivities exceeds the variation among phosphors. Finally, the effects of the recognized forms of observer variation on the visual responses (cone excitations or cone contrasts) generated by CRT stimuli are investigated and quantitatively specified. Cone pigment polymorphism gives rise to variation of a few per cent in relative excitation by the different phosphors--a variation larger than the errors ensuing from the adoption of the CIE standard observer, though smaller than the differences between some candidate cone sensitivities. Macular pigmentation has a larger influence, affecting mainly responses to the blue phosphor. The estimated combined effect of all sources of observer variation is comparable in magnitude with the largest differences between competing cone sensitivity estimates but is not enough to disrupt very seriously the relation between the L and M cone weights and the isoluminance settings of individual observers. It is also comparable with typical instrumental colorimetric errors, but we discuss these only briefly.

  6. LHCb Event display

    CERN Document Server

    Trisovic, Ana

    2014-01-01

    The LHCb Event Display was made for educational purposes at the European Organization for Nuclear Research, CERN in Geneva, Switzerland. The project was implemented as a stand-alone application using C++ and ROOT, a framework developed by CERN for data analysis. This paper outlines the development and architecture of the application in detail, as well as the motivation for the development and the goals of the exercise. The application focuses on the visualization of events recorded by the LHCb detector, where an event represents a set of charged particle tracks in one proton-proton collision. Every particle track is coloured by its type and can be selected to see its essential information such as mass and momentum. The application allows students to save this information and calculate the invariant mass for any pair of particles. Furthermore, the students can use additional calculating tools in the application and build up a histogram of these invariant masses. The goal for the students is to find a $D^0$ par...

  7. Data Display in Qualitative Research

    Directory of Open Access Journals (Sweden)

    Susana Verdinelli PsyD

    2013-02-01

    Full Text Available Visual displays help in the presentation of inferences and conclusions and represent ways of organizing, summarizing, simplifying, or transforming data. Data displays such as matrices and networks are often utilized to enhance data analysis and are more commonly seen in quantitative than in qualitative studies. This study reviewed the data displays used by three prestigious qualitative research journals within a period of three years. The findings include the types of displays used in these qualitative journals, the frequency of use, and the purposes for using visual displays as opposed to presenting data in text.

  8. Unique interactive projection display screen

    Energy Technology Data Exchange (ETDEWEB)

    Veligdan, J.T.

    1997-11-01

    Projection systems continue to be the best method to produce large (1 meter and larger) displays. However, in order to produce a large display, considerable volume is typically required. The Polyplanar Optic Display (POD) is a novel type of projection display screen, which for the first time, makes it possible to produce a large projection system that is self-contained and only inches thick. In addition, this display screen is matte black in appearance allowing it to be used in high ambient light conditions. This screen is also interactive and can be remotely controlled via an infrared optical pointer resulting in mouse-like control of the display. Furthermore, this display need not be flat since it can be made curved to wrap around a viewer as well as being flexible.

  9. The antibody mining toolbox: an open source tool for the rapid analysis of antibody repertoires.

    Science.gov (United States)

    D'Angelo, Sara; Glanville, Jacob; Ferrara, Fortunato; Naranjo, Leslie; Gleasner, Cheryl D; Shen, Xiaohong; Bradbury, Andrew R M; Kiss, Csaba

    2014-01-01

    In vitro selection has been an essential tool in the development of recombinant antibodies against various antigen targets. Deep sequencing has recently been gaining ground as an alternative and valuable method to analyze such antibody selections. The analysis provides a novel and extremely detailed view of selected antibody populations, and allows the identification of specific antibodies using only sequencing data, potentially eliminating the need for expensive and laborious low-throughput screening methods such as enzyme-linked immunosorbant assay. The high cost and the need for bioinformatics experts and powerful computer clusters, however, have limited the general use of deep sequencing in antibody selections. Here, we describe the AbMining ToolBox, an open source software package for the straightforward analysis of antibody libraries sequenced by the three main next generation sequencing platforms (454, Ion Torrent, MiSeq). The ToolBox is able to identify heavy chain CDR3s as effectively as more computationally intense software, and can be easily adapted to analyze other portions of antibody variable genes, as well as the selection outputs of libraries based on different scaffolds. The software runs on all common operating systems (Microsoft Windows, Mac OS X, Linux), on standard personal computers, and sequence analysis of 1-2 million reads can be accomplished in 10-15 min, a fraction of the time of competing software. Use of the ToolBox will allow the average researcher to incorporate deep sequence analysis into routine selections from antibody display libraries.

  10. Generation and characterization of novel stromal specific antibodies

    Institute of Scientific and Technical Information of China (English)

    2005-01-01

    Rheumatoid synovial fibroblasts were used as an immunogen to produce monoclonal antibodies selected for their reactivity with stromal cell antigens. Mice were immunised with low passage whole cell preparations and the subsequent hybridomas were screened by immunohistochemistry on rheumatoid synovium and tonsil sections. The aim was to identify those antibodies that recognised antigens that were restricted to stromal cells and were not expressed on CD45 positive leucocytes. A significant number of antibodies detected antigen that identified endothelial cells. These antibodies were further characterised to determine whether the vessels identified by these antibodies were vascular or lymphatic.From five fusions clones were identified with predominant reactivity with: 1) fibroblasts and endothelial cells; or 2)broad stromal elements (fibroblast, endothelium, epithelium, follicular dendritic cells). A fibroblast-specific antibody that did not also identify vessels was not generated. Examples of each reactivity pattern are discussed.

  11. Antibody engineering: facing new challenges in cancer therapy

    Institute of Scientific and Technical Information of China (English)

    Laura SANZ; (A)ngel M CUESTA; Marta COMPTE; Luis (A)LVAREZ-VALLINA

    2005-01-01

    Antibody-based therapeutics are beginning to realize the promise enclosed in their early denomination as "magic bullets". Initial disappointment has turned into clinical and commercial success, and engineered antibodies currently represent over 30% of biopharmaceuticals in clinical trials. Recent structural and functional data have allowed the design of a new generation of therapeutic antibodies, with strategies ranging from complement-mediated and antibody-dependant cellular cytotoxicity enhancement to improved cytotoxic payloads using toxins, drugs,radionucleids and viral delivery. This review considers the structure of different types of recombinant antibodies, their mechanism of action and how their efficacy has been increased using a broad array of approaches. We will also focus on the additional benefits offered by the use of gene therapy methods for the in vivo production of therapeutic antibodies.

  12. Broad Prize: Do the Successes Spread?

    Science.gov (United States)

    Samuels, Christina A.

    2011-01-01

    When the Broad Prize for Urban Education was created in 2002, billionaire philanthropist Eli Broad said he hoped the awards, in addition to rewarding high-performing school districts, would foster healthy competition; boost the prestige of urban education, long viewed as dysfunctional; and showcase best practices. Over the 10 years the prize has…

  13. Augmenting digital displays with computation

    Science.gov (United States)

    Liu, Jing

    As we inevitably step deeper and deeper into a world connected via the Internet, more and more information will be exchanged digitally. Displays are the interface between digital information and each individual. Naturally, one fundamental goal of displays is to reproduce information as realistically as possible since humans still care a lot about what happens in the real world. Human eyes are the receiving end of such information exchange; therefore it is impossible to study displays without studying the human visual system. In fact, the design of displays is rather closely coupled with what human eyes are capable of perceiving. For example, we are less interested in building displays that emit light in the invisible spectrum. This dissertation explores how we can augment displays with computation, which takes both display hardware and the human visual system into consideration. Four novel projects on display technologies are included in this dissertation: First, we propose a software-based approach to driving multiview autostereoscopic displays. Our display algorithm can dynamically assign views to hardware display zones based on multiple observers' current head positions, substantially reducing crosstalk and stereo inversion. Second, we present a dense projector array that creates a seamless 3D viewing experience for multiple viewers. We smoothly interpolate the set of viewer heights and distances on a per-vertex basis across the arrays field of view, reducing image distortion, crosstalk, and artifacts from tracking errors. Third, we propose a method for high dynamic range display calibration that takes into account the variation of the chrominance error over luminance. We propose a data structure for enabling efficient representation and querying of the calibration function, which also allows user-guided balancing between memory consumption and the amount of computation. Fourth, we present user studies that demonstrate that the ˜ 60 Hz critical flicker fusion

  14. Acetylcholine receptor antibody

    Science.gov (United States)

    ... page: //medlineplus.gov/ency/article/003576.htm Acetylcholine receptor antibody To use the sharing features on this page, please enable JavaScript. Acetylcholine receptor antibody is a protein found in the blood ...

  15. Antinuclear antibody panel

    Science.gov (United States)

    ... page: //medlineplus.gov/ency/article/003535.htm Antinuclear antibody panel To use the sharing features on this page, please enable JavaScript. The antinuclear antibody panel is a blood test that looks at ...

  16. Lyme disease antibody

    Science.gov (United States)

    ... JavaScript. The Lyme disease blood test looks for antibodies in the blood to the bacteria that causes ... needed. A laboratory specialist looks for Lyme disease antibodies in the blood sample using the ELISA test . ...

  17. The antibody mining toolbox

    OpenAIRE

    D'Angelo, Sara; Glanville, Jacob; Ferrara, Fortunato; Naranjo, Leslie; Gleasner, Cheryl D.; Shen, Xiaohong; Bradbury, Andrew RM; Kiss, Csaba

    2013-01-01

    In vitro selection has been an essential tool in the development of recombinant antibodies against various antigen targets. Deep sequencing has recently been gaining ground as an alternative and valuable method to analyze such antibody selections. The analysis provides a novel and extremely detailed view of selected antibody populations, and allows the identification of specific antibodies using only sequencing data, potentially eliminating the need for expensive and laborious low-throughput ...

  18. Differential Antibody Responses to Conserved HIV-1 Neutralizing Epitopes in the Context of Multivalent Scaffolds and Native-Like gp140 Trimers

    Science.gov (United States)

    Morris, Charles D.; Azadnia, Parisa; de Val, Natalia; Vora, Nemil; Honda, Andrew; Giang, Erick; Saye-Francisco, Karen; Cheng, Yushao; Lin, Xiaohe; Mann, Colin J.; Tang, Jeffrey; Sok, Devin; Burton, Dennis R.; Law, Mansun; Ward, Andrew B.

    2017-01-01

    ABSTRACT Broadly neutralizing antibodies (bNAbs) have provided valuable insights into the humoral immune response to HIV-1. While rationally designed epitope scaffolds and well-folded gp140 trimers have been proposed as vaccine antigens, a comparative understanding of their antibody responses has not yet been established. In this study, we probed antibody responses to the N332 supersite and the membrane-proximal external region (MPER) in the context of heterologous protein scaffolds and native-like gp140 trimers. Ferritin nanoparticles and fragment crystallizable (Fc) regions were utilized as multivalent carriers to display scaffold antigens with grafted N332 and MPER epitopes, respectively. Trimeric scaffolds were also identified to stabilize the MPER-containing BG505 gp140.681 trimer in a native-like conformation. Following structural and antigenic evaluation, a subset of scaffold and trimer antigens was selected for immunization in BALB/c mice. Serum binding revealed distinct patterns of antibody responses to these two bNAb targets presented in different structural contexts. For example, the N332 nanoparticles elicited glycan epitope-specific antibody responses that could also recognize the native trimer, while a scaffolded BG505 gp140.681 trimer generated a stronger and more rapid antibody response to the trimer apex than its parent gp140.664 trimer. Furthermore, next-generation sequencing (NGS) of mouse splenic B cells revealed expansion of antibody lineages with long heavy-chain complementarity-determining region 3 (HCDR3) loops upon activation by MPER scaffolds, in contrast to the steady repertoires primed by N332 nanoparticles and a soluble gp140.664 trimer. These findings will facilitate the future development of a coherent vaccination strategy that combines both epitope-focused and trimer-based approaches. PMID:28246356

  19. Cross-Reactive Human IgM-Derived Monoclonal Antibodies that Bind to HIV-1 Envelope Glycoproteins

    Directory of Open Access Journals (Sweden)

    Barton F. Haynes

    2010-02-01

    Full Text Available Elicitation of antibodies with potent and broad neutralizing activity against HIV by immunization remains a challenge. Several monoclonal antibodies (mAbs isolated from humans with HIV-1 infection exhibit such activity but vaccine immunogens based on structures containing their epitopes have not been successful for their elicitation. All known broadly neutralizing mAbs (bnmAbs are immunoglobulin (Ig Gs (IgGs and highly somatically hypermutated which could impede their elicitation. Ig Ms (IgMs are on average significantly less divergent from germline antibodies and are relevant for the development of vaccine immunogens but are underexplored compared to IgGs. Here we describe the identification and characterization of several human IgM-derived mAbs against HIV-1 which were selected from a large phage-displayed naive human antibody library constructed from blood, lymph nodes and spleens of 59 healthy donors. These antibodies bound with high affinity to recombinant envelope glycoproteins (gp140s, Envs of HIV-1 isolates from different clades. They enhanced or did not neutralize infection by some of the HIV-1 primary isolates using CCR5 as a coreceptor but neutralized all CXCR4 isolates tested although weakly. One of these antibodies with relatively low degree of somatic hypermutation was more extensively characterized. It bound to a highly conserved region partially overlapping with the coreceptor binding site and close to but not overlapping with the CD4 binding site. These results suggest the existence of conserved structures that could direct the immune response to non-neutralizing or even enhancing antibodies which may represent a strategy used by the virus to escape neutralizing immune responses. Further studies will show whether such a strategy plays a role in HIV infection of humans, how important that role could be, and what the mechanisms of infection enhancement are. The newly identified mAbs could be used as reagents to further

  20. Immune history profoundly affects broadly protective B cell responses to influenza

    Science.gov (United States)

    Andrews, Sarah F.; Huang, Yunping; Kaur, Kaval; Popova, Lyubov I.; Ho, Irvin Y.; Pauli, Noel T.; Dunand, Carole J. Henry; Taylor, William M; Lim, Samuel; Huang, Min; Qu, Xinyan; Lee, Jane-Hwei; Salgado-Ferrer, Marlene; Krammer, Florian; Palese, Peter; Wrammert, Jens; Ahmed, Rafi; Wilson, Patrick C.

    2016-01-01

    Generating a broadly protective influenza vaccine is critical to global health. Understanding how immune memory influences influenza immunity is central to this goal. We undertook an in-depth study of the B cell response to the pandemic 2009 H1N1 vaccine over consecutive years. Analysis of monoclonal Abs generated from vaccine-induced plasmablasts demonstrated that individuals with low preexisting serological titers to the vaccinating strain generated a broadly reactive, HA stalk-biased, response. Higher preexisting serum antibody levels correlated with a strain-specific HA head-dominated response. We demonstrate that this HA head immunodominance encompasses poor accessibility of the HA stalk epitopes. Further, we show polyreactivity of HA stalk-reactive antibodies that could cause counterselection of these cells. Thus, preexisting memory against HA head epitopes predominate, inhibiting a broadly protective response against the HA stalk upon revaccination with similar strains. Consideration of influenza exposure history is critical for new vaccine strategies designed to elicit broadly neutralizing antibodies. PMID:26631631

  1. X-1 on display

    Science.gov (United States)

    1949-01-01

    A Bell Aircraft Corporation X-1 series aircraft on display at an Open House at NACA Muroc Flight Test Unit or High-Speed Flight Research Station hangar on South Base of Edwards Air Force Base, California. (The precise date of the photo is uncertain, but it is probably before 1948.) The instrumentation that was carried aboard the aircraft to gather data is on display. The aircraft data was recorded on oscillograph film that was read, calibrated, and converted into meaningful parameters for the engineers to evaluate from each research flight. In the background of the photo are several early U.S. jets. These include several Lockheed P-80 Shooting Stars, which were used as chase planes on X-1 flights; two Bell P-59 Airacomets, the first U.S. jet pursuit aircraft (fighter in later parlance); and a prototype Republic XP-84 Thunderjet. There were five versions of the Bell X-1 rocket-powered research aircraft that flew at the NACA High-Speed Flight Research Station, Edwards, California. The bullet-shaped X-1 aircraft were built by Bell Aircraft Corporation, Buffalo, N.Y. for the U.S. Army Air Forces (after 1947, U.S. Air Force) and the National Advisory Committee for Aeronautics (NACA). The X-1 Program was originally designated the XS-1 for eXperimental Sonic. The X-1's mission was to investigate the transonic speed range (speeds from just below to just above the speed of sound) and, if possible, to break the 'sound barrier.' Three different X-1s were built and designated: X-1-1, X-1-2 (later modified to become the X-1E), and X-1-3. The basic X-1 aircraft were flown by a large number of different pilots from 1946 to 1951. The X-1 Program not only proved that humans could go beyond the speed of sound, it reinforced the understanding that technological barriers could be overcome. The X-1s pioneered many structural and aerodynamic advances including extremely thin, yet extremely strong wing sections; supersonic fuselage configurations; control system requirements; powerplant

  2. Laser illuminated flat panel display

    Energy Technology Data Exchange (ETDEWEB)

    Veligdan, J.T.

    1995-12-31

    A 10 inch laser illuminated flat panel Planar Optic Display (POD) screen has been constructed and tested. This POD screen technology is an entirely new concept in display technology. Although the initial display is flat and made of glass, this technology lends itself to applications where a plastic display might be wrapped around the viewer. The display screen is comprised of hundreds of planar optical waveguides where each glass waveguide represents a vertical line of resolution. A black cladding layer, having a lower index of refraction, is placed between each waveguide layer. Since the cladding makes the screen surface black, the contrast is high. The prototype display is 9 inches wide by 5 inches high and approximately I inch thick. A 3 milliwatt HeNe laser is used as the illumination source and a vector scanning technique is employed.

  3. Antibody-mediated Prevention of Fusarium Mycotoxins in the Field

    Directory of Open Access Journals (Sweden)

    Yu-Cai Liao

    2008-10-01

    Full Text Available Fusarium mycotoxins directly accumulated in grains during the infection of wheat and other cereal crops by Fusarium head blight (FHB pathogens are detrimental to humans and domesticated animals. Prevention of the mycotoxins via the development of FHB-resistant varieties has been a challenge due to the scarcity of natural resistance against FHB pathogens. Various antibodies specific to Fusarium fungi and mycotoxins are widely used in immunoassays and antibody-mediated resistance in planta against Fusarium pathogens has been demonstrated. Antibodies fused to antifungal proteins have been shown to confer a very significantly enhanced Fusarium resistance in transgenic plants. Thus, antibody fusions hold great promise as an effective tool for the prevention of mycotoxin contaminations in cereal grains. This review highlights the utilization of protective antibodies derived from phage display to increase endogenous resistance of wheat to FHB pathogens and consequently to reduce mycotoxins in field. The role played by Fusarium-specific antibody in the resistance is also discussed.

  4. The advances and application prospects of monoclonal antibody preparation technology%单克隆抗体制备技术的最新进展及应用前景

    Institute of Scientific and Technical Information of China (English)

    孔君; 刘箐; 韩跃武; 王天昌; 刘金芝

    2011-01-01

    单克隆抗体技术是现代生命科学研究的重要工具,其在基因和蛋白质的结构与功能研究方面有着不可或缺的作用,在人类和动植物的免疫学诊断方面至今仍有着无可代替的重要作用.本文综述了单克隆抗体的制备技术,包括嵌合抗体、噬菌体展示技术、核糖体展示技术、基因工程抗体等,及其在临床医学和疾病的诊断与治疗等领域的广阔应用前景.%Monoclonal antibody technique, as an important tool in modem life science research, plays an indispensable role in the study of gene structure and protein function, as well as in the immunological diagnosis of humans, animals and plants. This review gives an overview on the preparation of monoclonal antibody, such as chimeric antibody, phage display, ribosome display, genetically engineered antibody, etc. and its broad application prospects in clinical medicine, diagnosis and therapy of diseases.

  5. Miniature information displays: primary applications

    Science.gov (United States)

    Alvelda, Phillip; Lewis, Nancy D.

    1998-04-01

    Positioned to replace current liquid crystal display technology in many applications, miniature information displays have evolved to provide several truly portable platforms for the world's growing personal computing and communication needs. The technology and functionality of handheld computer and communicator systems has finally surpassed many of the standards that were originally established for desktop systems. In these new consumer electronics, performance, display size, packaging, power consumption, and cost have always been limiting factors for fabricating genuinely portable devices. The rapidly growing miniature information display manufacturing industry is making it possible to bring a wide range of highly anticipated new products to new markets.

  6. Maintenance Procedure Display: Head Mounted Display (HMD) Evaluations

    Science.gov (United States)

    Whitmore, Milrian; Litaker, Harry L., Jr.; Solem, Jody A.; Holden, Kritina L.; Hoffman, Ronald R.

    2007-01-01

    A viewgraph presentation describing maintenance procedures for head mounted displays is shown. The topics include: 1) Study Goals; 2) Near Eye Displays (HMDs); 3) Design; 4) Phase I-Evaluation Methods; 5) Phase 1 Results; 6) Improved HMD Mounting; 7) Phase 2 -Evaluation Methods; 8) Phase 2 Preliminary Results; and 9) Next Steps.

  7. Focused Evolution of HIV-1 Neutralizing Antibodies Revealed by Structures and Deep Sequencing

    Energy Technology Data Exchange (ETDEWEB)

    Wu, Xueling; Zhou, Tongqing; Zhu, Jiang; Zhang, Baoshan; Georgiev, Ivelin; Wang, Charlene; Chen, Xuejun; Longo, Nancy S.; Louder, Mark; McKee, Krisha; O’Dell, Sijy; Perfetto, Stephen; Schmidt, Stephen D.; Shi, Wei; Wu, Lan; Yang, Yongping; Yang, Zhi-Yong; Yang, Zhongjia; Zhang, Zhenhai; Bonsignori, Mattia; Crump, John A.; Kapiga, Saidi H.; Sam, Noel E.; Haynes, Barton F.; Simek, Melissa; Burton, Dennis R.; Koff, Wayne C.; Doria-Rose, Nicole A.; Connors, Mark; Mullikin, James C.; Nabel, Gary J.; Roederer, Mario; Shapiro, Lawrence; Kwong, Peter D.; Mascola, John R. (Tumaini); (NIH); (Duke); (Kilimanjaro Repro.); (IAVI)

    2013-03-04

    Antibody VRC01 is a human immunoglobulin that neutralizes about 90% of HIV-1 isolates. To understand how such broadly neutralizing antibodies develop, we used x-ray crystallography and 454 pyrosequencing to characterize additional VRC01-like antibodies from HIV-1-infected individuals. Crystal structures revealed a convergent mode of binding for diverse antibodies to the same CD4-binding-site epitope. A functional genomics analysis of expressed heavy and light chains revealed common pathways of antibody-heavy chain maturation, confined to the IGHV1-2*02 lineage, involving dozens of somatic changes, and capable of pairing with different light chains. Broadly neutralizing HIV-1 immunity associated with VRC01-like antibodies thus involves the evolution of antibodies to a highly affinity-matured state required to recognize an invariant viral structure, with lineages defined from thousands of sequences providing a genetic roadmap of their development.

  8. Discovery of functional antibodies targeting ion channels.

    Science.gov (United States)

    Wilkinson, Trevor C I; Gardener, Matthew J; Williams, Wendy A

    2015-04-01

    Ion channels play critical roles in physiology and disease by modulation of cellular functions such as electrical excitability, secretion, cell migration, and gene transcription. Ion channels represent an important target class for drug discovery that has been largely addressed, to date, using small-molecule approaches. A significant opportunity exists to target these channels with antibodies and alternative formats of biologics. Antibodies display high specificity and affinity for their target antigen, and they have the potential to target ion channels very selectively. Nevertheless, isolating antibodies to this target class is challenging due to the difficulties in expression and purification of ion channels in a format suitable for antibody drug discovery in addition to the complexity of screening for function. In this article, we will review the current state of ion channel biologics discovery and the progress that has been made. We will also highlight the challenges in isolating functional antibodies to these targets and how these challenges may be addressed. Finally, we also illustrate successful approaches to isolating functional monoclonal antibodies targeting ion channels by way of a number of case studies drawn from recent publications.

  9. [VGKC-complex antibodies].

    Science.gov (United States)

    Watanabe, Osamu

    2013-04-01

    Various antibodies are associated with voltage-gated potassium channels (VGKCs). Representative antibodies to VGKCs were first identified by radioimmunoassays using radioisotope-labeled alpha-dendrotoxin-VGKCs solubilized from rabbit brain. These antibodies were detected only in a proportion of patients with acquired neuromyotonia (Isaacs' syndrome). VGKC antibodies were also detected in patients with Morvan's syndrome and in those with a form of autoimmune limbic encephalitis. Recent studies indicated that the "VGKC" antibodies are mainly directed toward associated proteins (for example LGI-1 and CASPR-2) that complex with the VGKCs themselves. The "VGKC" antibodies are now commonly known as VGKC-complex antibodies. In general, LGI-1 antibodies are most commonly detected in patients with limbic encephalitis with syndrome of inappropriate secretion of antidiuretic hormone. CASPR-2 antibodies are present in the majority of patients with Morvan's syndrome. These patients develop combinations of CNS symptoms, autonomic dysfunction, and peripheral nerve hyperexcitability. Furthermore, VGKC-complex antibodies are tightly associated with chronic idiopathic pain. Hyperexcitability of nociceptive pathways has also been implicated. These antibodies may be detected in sera of some patients with neurodegenerative diseases (for example, amyotrophic lateral sclerosis and Creutzfeldt-Jakob disease).

  10. IBC's 21st Annual Antibody Engineering and 8th Annual Antibody Therapeutics International Conferences and 2010 Annual Meeting of the Antibody Society. December 5-9, 2010, San Diego, CA USA.

    Science.gov (United States)

    Arnett, Samantha O; Teillaud, Jean-Luc; Wurch, Theirry; Reichert, Janice M; Dunlop, Cameron; Huber, Michael

    2011-01-01

    The 21st Annual Antibody Engineering and 8th Annual Antibody Therapeutics international conferences, and the 2010 Annual Meeting of The Antibody Society, organized by IBC Life Sciences with contributions from The Antibody Society and two Scientific Advisory Boards, were held December 5-9, 2010 in San Diego, CA. The conferences were organized with a focus on antibody engineering only on the first day and a joint engineering/therapeutics session on the last day. Delegates could select from presentations that occurred in two simultaneous sessions on days 2 and 3. Day 1 included presentations on neutralizing antibodies and the identification of vaccine targets, as well as a historical overview of 20 years of phage display utilization. Topics presented in the Antibody Engineering sessions on day 2 and 3 included antibody biosynthesis, structure and stability; antibodies in a complex environment; antibody half-life; and targeted nanoparticle therapeutics. In the Antibody Therapeutics sessions on days 2 and 3, preclinical and early stage development and clinical updates of antibody therapeutics, including TRX518, SYM004, MM111, PRO140, CVX-241, ASG-5ME, U3-1287 (AMG888), R1507 and trastuzumab emtansine, were discussed, and perspectives were provided on the development of biosimilar and biobetter antibodies, including coverage of regulatory and intellectual property issues. The joint engineering/therapeutics session on the last day focused on bispecific and next-generation antibodies.

  11. Recent Progress toward Engineering HIV-1-Specific Neutralizing Monoclonal Antibodies

    OpenAIRE

    Ming Sun; Yue Li; Huiwen Zheng; Yiming Shao

    2016-01-01

    The recent discoveries of broadly potent neutralizing human monoclonal antibodies represent a new generation of antiretrovirals for the treatment and prophylaxis. Antibodies are generally considered more effective and safer and have been proved to provide passive protection against mucosal challenge in humanized mice and macaques. Several neutralizing Abs could protect animals against HIV-1 but are not effective when used in an established infected model for therapy. In order to overcome the ...

  12. Measuring Prevention More Broadly, An Empirical...

    Data.gov (United States)

    U.S. Department of Health & Human Services — Measuring Prevention More Broadly, An Empirical Assessment of CHIPRA Core Measures Differences in CHIP design and structure, across states and over time, may limit...

  13. Flexible Bistable Cholesteric Reflective Displays

    Science.gov (United States)

    Yang, Deng-Ke

    2006-03-01

    Cholesteric liquid crystals (ChLCs) exhibit two stable states at zero field condition-the reflecting planar state and the nonreflecting focal conic state. ChLCs are an excellent candidate for inexpensive and rugged electronic books and papers. This paper will review the display cell structure,materials and drive schemes for flexible bistable cholesteric (Ch) reflective displays.

  14. Multivalent dendritic molecules as broad spectrum bacteria agglutination agents.

    Science.gov (United States)

    Xiao, Shuzhang; Abu-Esba, Lica; Turkyilmaz, Serhan; White, Alexander G; Smith, Bradley D

    2013-01-01

    This study reports the first set of synthetic molecules that act as broad spectrum agglutination agents and thus are complementary to the specific targeting of antibodies. The molecules have dendritic architecture and contain multiple copies of zinc(II)-dipicolylamine (ZnDPA) units that have selective affinity for the bacterial cell envelope. A series of molecular structures were evaluated, with the number of appended ZnDPA units ranging from four to thirty-two. Agglutination assays showed that the multivalent probes rapidly cross-linked ten different strains of bacteria, regardless of Gram-type and cell morphology. Fluorescence microscopy studies using probes with four ZnDPA units indicated a high selectivity for bacteria agglutination in the presence of mammalian cells and no measurable effect on the health of the cells. The high bacterial selectivity was confirmed by conducting in vivo optical imaging studies of a mouse leg infection model. The results suggest that multivalent ZnDPA molecular probes with dendritic structures have great promise as selective, broad spectrum bacterial agglutination agents for infection imaging and theranostic applications.

  15. Three-dimensional display technologies.

    Science.gov (United States)

    Geng, Jason

    2013-01-01

    The physical world around us is three-dimensional (3D), yet traditional display devices can show only two-dimensional (2D) flat images that lack depth (i.e., the third dimension) information. This fundamental restriction greatly limits our ability to perceive and to understand the complexity of real-world objects. Nearly 50% of the capability of the human brain is devoted to processing visual information [Human Anatomy & Physiology (Pearson, 2012)]. Flat images and 2D displays do not harness the brain's power effectively. With rapid advances in the electronics, optics, laser, and photonics fields, true 3D display technologies are making their way into the marketplace. 3D movies, 3D TV, 3D mobile devices, and 3D games have increasingly demanded true 3D display with no eyeglasses (autostereoscopic). Therefore, it would be very beneficial to readers of this journal to have a systematic review of state-of-the-art 3D display technologies.

  16. Flat panel display - Impurity doping technology for flat panel displays

    Energy Technology Data Exchange (ETDEWEB)

    Suzuki, Toshiharu [Advanced Technology Planning, Sumitomo Eaton Nova Corporation, SBS Tower 9F, 10-1, Yoga 4-chome, Setagaya-ku, 158-0097 Tokyo (Japan)]. E-mail: suzuki_tsh@senova.co.jp

    2005-08-01

    Features of the flat panel displays (FPDs) such as liquid crystal display (LCD) and organic light emitting diode (OLED) display, etc. using low temperature poly-Si (LTPS) thin film transistors (TFTs) are briefly reviewed comparing with other FPDs. The requirements for fabricating TFTs used for high performance FPDs and system on glass (SoG) are addressed. This paper focuses on the impurity doping technology, which is one of the key technologies together with crystallization by laser annealing, formation of high quality gate insulator and gate-insulator/poly-Si interface. The issues to be solved in impurity doping technology for state of the art and future TFTs are clarified.

  17. Evolution of the HIV-1 Envelope Glycoprotein Genes and Neutralizing Antibody Response in an Individual with Broadly Cross Neutralizing Antibodies

    Science.gov (United States)

    2010-08-31

    global mY/AIDS epidemic .... .. ... ..... ...... ... ..... ........ ...... ...... .... ....... ........... . 4 my classification and genetic ...Pneumocystis carinni pneumonia (PCP) among previously healthy homosexual men in Los Angeles, CA. At the time of publication, two of the five men had...healthy homosexual men across the United States. Disturbingly, severe and aggressive cases of Kaposi’s sarcoma, normally a slowly progressive cancer

  18. Key gp120 Glycans Pose Roadblocks to the Rapid Development of VRC01-Class Antibodies in an HIV-1-Infected Chinese Donor.

    Science.gov (United States)

    Kong, Leopold; Ju, Bin; Chen, Yajing; He, Linling; Ren, Li; Liu, Jiandong; Hong, Kunxue; Su, Bin; Wang, Zheng; Ozorowski, Gabriel; Ji, Xiaolin; Hua, Yuanzi; Chen, Yanli; Deller, Marc C; Hao, Yanling; Feng, Yi; Garces, Fernando; Wilson, Richard; Dai, Kaifan; O'Dell, Sijy; McKee, Krisha; Mascola, John R; Ward, Andrew B; Wyatt, Richard T; Li, Yuxing; Wilson, Ian A; Zhu, Jiang; Shao, Yiming

    2016-04-19

    VRC01-class antibodies neutralize diverse HIV-1 strains by targeting the conserved CD4-binding site. Despite extensive investigations, crucial events in the early stage of VRC01 development remain elusive. We demonstrated how VRC01-class antibodies emerged in a Chinese donor by antigen-specific single B cell sorting, structural and functional studies, and longitudinal antibody and virus repertoire analyses. A monoclonal antibody DRVIA7 with modest neutralizing breadth was isolated that displayed a subset of VRC01 signatures. X-ray and EM structures revealed a VRC01-like angle of approach, but less favorable interactions between the DRVIA7 light-chain CDR1 and the N terminus with N276 and V5 glycans of gp120. Although the DRVIA7 lineage was unable to acquire broad neutralization, longitudinal analysis revealed a repertoire-encoded VRC01 light-chain CDR3 signature and VRC01-like neutralizing heavy-chain precursors that rapidly matured within 2 years. Thus, light chain accommodation of the glycan shield should be taken into account in vaccine design targeting this conserved site of vulnerability.

  19. Identification of anti-CD98 antibody mimotopes for inducing antibodies with antitumor activity by mimotope immunization.

    Science.gov (United States)

    Saito, Misa; Kondo, Masahiro; Ohshima, Motohiro; Deguchi, Kazuki; Hayashi, Hideki; Inoue, Kazuyuki; Tsuji, Daiki; Masuko, Takashi; Itoh, Kunihiko

    2014-04-01

    A mimotope is an antibody-epitope-mimicking peptide retrieved from a phage display random peptide library. Immunization with antitumor antibody-derived mimotopes is promising for inducing antitumor immunity in hosts. In this study, we isolated linear and constrained mimotopes from HBJ127, a tumor-suppressing anti-CD98 heavy chain mAb, and determined their abilities for induction of antitumor activity equal to that of the parent antibody. We detected elevated levels of antipeptide responses, but failed to detect reactivity against native CD98-expressing HeLa cells in sera of immunized mice. Phage display panning and selection of mimotope-immunized mouse spleen-derived antibody Fab library showed that HeLa cell-reactive Fabs were successfully retrieved from the library. This finding indicates that native antigen-reactive Fab clones represented an undetectable minor population in mimotope-induced antibody repertoire. Functional and structural analysis of retrieved Fab clones revealed that they were almost identical to the parent antibody. From these results, we confirmed that mimotope immunization was promising for retrieving antitumor antibodies equivalent to the parent antibody, although the co-administration of adjuvant compounds such as T-cell epitope peptides and Toll-like receptor 4 agonist peptides is likely to be necessary for inducing stronger antitumor immunity than mimotope injection alone.

  20. Tone compatibility between HDR displays

    Science.gov (United States)

    Bist, Cambodge; Cozot, Rémi; Madec, Gérard; Ducloux, Xavier

    2016-09-01

    High Dynamic Range (HDR) is the latest trend in television technology and we expect an in ux of HDR capable consumer TVs in the market. Initial HDR consumer displays will operate on a peak brightness of about 500-1000 nits while in the coming years display peak brightness is expected to go beyond 1000 nits. However, professionally graded HDR content can range from 1000 to 4000 nits. As with Standard Dynamic Range (SDR) content, we can expect HDR content to be available in variety of lighting styles such as low key, medium key and high key video. This raises concerns over tone-compatibility between HDR displays especially when adapting to various lighting styles. It is expected that dynamic range adaptation between HDR displays uses similar techniques as found with tone mapping and tone expansion operators. In this paper, we survey simple tone mapping methods of 4000 nits color-graded HDR content for 1000 nits HDR displays. We also investigate tone expansion strategies when HDR content graded in 1000 nits is displayed on 4000 nits HDR monitors. We conclude that the best tone reproduction technique between HDR displays strongly depends on the lighting style of the content.

  1. A strategy for eliciting antibodies against cryptic, conserved, conformationally dependent epitopes of HIV envelope glycoprotein.

    Directory of Open Access Journals (Sweden)

    Hanna C Kelker

    Full Text Available BACKGROUND: Novel strategies are needed for the elicitation of broadly neutralizing antibodies to the HIV envelope glycoprotein, gp120. Experimental evidence suggests that combinations of antibodies that are broadly neutralizing in vitro may protect against challenge with HIV in nonhuman primates, and a small number of these antibodies have been selected by repertoire sampling of B cells and by the fractionation of antiserum from some patients with prolonged disease. Yet no additional strategies for identifying conserved epitopes, eliciting antibodies to these epitopes, and determining whether these epitopes are accessible to antibodies have been successful to date. The defining of additional conserved, accessible epitopes against which one can elicit antibodies will increase the probability that some may be the targets of broadly neutralizing antibodies. METHODOLOGY/PRINCIPAL FINDINGS: We postulate that additional cryptic epitopes of gp120 are present, against which neutralizing antibodies might be elicited even though these antibodies are not elicited by gp120, and that many of these epitopes may be accessible to antibodies should they be formed. We demonstrate a strategy for eliciting antibodies in mice against selected cryptic, conformationally dependent conserved epitopes of gp120 by immunizing with multiple identical copies of covalently linked peptides (MCPs. This has been achieved with MCPs representing 3 different domains of gp120. We show that some cryptic epitopes on gp120 are accessible to the elicited antibodies, and some epitopes in the CD4 binding region are not accessible. The antibodies bind to gp120 with relatively high affinity, and bind to oligomeric gp120 on the surface of infected cells. CONCLUSIONS/SIGNIFICANCE: Immunization with MCPs comprised of selected peptides of HIV gp120 is able to elicit antibodies against conserved, conformationally dependent epitopes of gp120 that are not immunogenic when presented as gp120. Some

  2. Anti-aquaporin-4 antibody-positive dorsal midbrain syndrome.

    Science.gov (United States)

    Lee, Juyoun; Jeong, Seong-Hae; Park, Sang Min; Sohn, Eun Hee; Lee, Ae Young; Kim, Jae-Moon; Jo, Hyun-Jin; Lee, Yeon-Hee; Kim, Ji-Soo

    2015-04-01

    Neuromyelitis optica spectrum disorders (NMOSD) can cause various ocular motor disorders in addition to optic neuritis. Ocular motor findings associated with NMOSD include spontaneous vertical and gaze-evoked nystagmus, wall-eyed bilateral internuclear ophthalmoplegia, and trochlear nerve palsy. The association between dorsal midbrain syndrome and anti-aquaporin-4 antibody seropositivity has not been reported. Here, we report a patient displaying typical dorsal midbrain syndrome and anti-aquaporin-4 antibody seropositivity.

  3. Candidate antibody-based therapeutics against HIV-1.

    Science.gov (United States)

    Gong, Rui; Chen, Weizao; Dimitrov, Dimiter S

    2012-06-01

    Antibody-based therapeutics have been successfully used for the treatment of various diseases and as research tools. Several well characterized, broadly neutralizing monoclonal antibodies (bnmAbs) targeting HIV-1 envelope glycoproteins or related host cell surface proteins show sterilizing protection of animals, but they are not effective when used for therapy of an established infection in humans. Recently, a number of novel bnmAbs, engineered antibody domains (eAds), and multifunctional fusion proteins have been reported which exhibit exceptionally potent and broad neutralizing activity against a wide range of HIV-1 isolates from diverse genetic subtypes. eAds could be more effective in vivo than conventional full-size antibodies generated by the human immune system. Because of their small size (12∼15 kD), they can better access sterically restricted epitopes and penetrate densely packed tissue where HIV-1 replicates than the larger full-size antibodies. HIV-1 possesses a number of mechanisms to escape neutralization by full-size antibodies but could be less likely to develop resistance to eAds. Here, we review the in vitro and in vivo antiviral efficacies of existing HIV-1 bnmAbs, summarize the development of eAds and multispecific fusion proteins as novel types of HIV-1 inhibitors, and discuss possible strategies to generate more potent antibody-based candidate therapeutics against HIV-1, including some that could be used to eradicate the virus.

  4. Antibody Conjugates: From Heterogeneous Populations to Defined Reagents

    Directory of Open Access Journals (Sweden)

    Patrick Dennler

    2015-08-01

    Full Text Available Monoclonal antibodies (mAbs and their derivatives are currently the fastest growing class of therapeutics. Even if naked antibodies have proven their value as successful biopharmaceuticals, they suffer from some limitations. To overcome suboptimal therapeutic efficacy, immunoglobulins are conjugated with toxic payloads to form antibody drug conjugates (ADCs and with chelating systems bearing therapeutic radioisotopes to form radioimmunoconjugates (RICs. Besides their therapeutic applications, antibody conjugates are also extensively used for many in vitro assays. A broad variety of methods to functionalize antibodies with various payloads are currently available. The decision as to which conjugation method to use strongly depends on the final purpose of the antibody conjugate. Classical conjugation via amino acid residues is still the most common method to produce antibody conjugates and is suitable for most in vitro applications. In recent years, however, it has become evident that antibody conjugates, which are generated via site-specific conjugation techniques, possess distinct advantages with regard to in vivo properties. Here, we give a comprehensive overview on existing and emerging strategies for the production of covalent and non-covalent antibody conjugates.

  5. Antibody-Based Strategies to Prevent and Treat Influenza

    Directory of Open Access Journals (Sweden)

    Ram eSasisekharan

    2015-07-01

    Full Text Available Passive immunization using antibodies has been suggested to offer several benefits in comparison to other antiviral treatment options. The potential for seasonal protection arising from a single injection of antibodies is appealing and has been pursued for a number of infectious agents. However, until recently, antibody-based strategies to combat infectious agents has been hampered due to the fact that typical antibodies have been found to be strain-specific, with the virus evolving resistance in many cases. The discovery of broadly neutralizing antibodies (bNAbs in, for example, influenza, dengue virus, and HIV, which bind to multiple, structurally-diverse strains has provided renewed interest in this area. This review will focus on new technologies that enable the discovery of bNAbs, the challenges and opportunities of immunotherapies as an important addition to existing antiviral therapy, and the role of antibody discovery in informing rational vaccine discovery – with agents targeting influenza specifically addressed. Multiple agents have entered the clinic and raise the possibility that a single antibody or small combination of antibodies can effectively neutralize a wide variety of strains. However, challenges remain - including combating escape variants, pharmacodynamics of antibody distribution, and development of efficacy biomarkers beyond virologic endpoints.

  6. Ultraminiature, Micropower Multipurpose Display Project

    Data.gov (United States)

    National Aeronautics and Space Administration — High information content electronic displays remain the most difficult element of the human-machine interface to effectively miniaturize. Mobile applications need a...

  7. Color speckle in laser displays

    Science.gov (United States)

    Kuroda, Kazuo

    2015-07-01

    At the beginning of this century, lighting technology has been shifted from discharge lamps, fluorescent lamps and electric bulbs to solid-state lighting. Current solid-state lighting is based on the light emitting diodes (LED) technology, but the laser lighting technology is developing rapidly, such as, laser cinema projectors, laser TVs, laser head-up displays, laser head mounted displays, and laser headlamps for motor vehicles. One of the main issues of laser displays is the reduction of speckle noise1). For the monochromatic laser light, speckle is random interference pattern on the image plane (retina for human observer). For laser displays, RGB (red-green-blue) lasers form speckle patterns independently, which results in random distribution of chromaticity, called color speckle2).

  8. ENERGY STAR Certified Displays - Deprecated

    Data.gov (United States)

    U.S. Environmental Protection Agency — This dataset is up-to-date but newer better data can be retrieved at: https://data.energystar.gov/dataset/ENERGY-STAR-Certified-Displays/xsyb-v8gs Certified models...

  9. Ten inch Planar Optic Display

    Energy Technology Data Exchange (ETDEWEB)

    Beiser, L. [Beiser (Leo) Inc., Flushing, NY (United States); Veligdan, J. [Brookhaven National Lab., Upton, NY (United States)

    1996-04-01

    A Planar Optic Display (POD) is being built and tested for suitability as a high brightness replacement for the cathode ray tube, (CRT). The POD display technology utilizes a laminated optical waveguide structure which allows a projection type of display to be constructed in a thin (I to 2 inch) housing. Inherent in the optical waveguide is a black cladding matrix which gives the display a black appearance leading to very high contrast. A Digital Micromirror Device, (DMD) from Texas Instruments is used to create video images in conjunction with a 100 milliwatt green solid state laser. An anamorphic optical system is used to inject light into the POD to form a stigmatic image. In addition to the design of the POD screen, we discuss: image formation, image projection, and optical design constraints.

  10. Fluorescent labeling of antibody fragments using split GFP.

    Directory of Open Access Journals (Sweden)

    Fortunato Ferrara

    Full Text Available Antibody fragments are easily isolated from in vitro selection systems, such as phage and yeast display. Lacking the Fc portion of the antibody, they are usually labeled using small peptide tags recognized by antibodies. In this paper we present an efficient method to fluorescently label single chain Fvs (scFvs using the split green fluorescent protein (GFP system. A 13 amino acid tag, derived from the last beta strand of GFP (termed GFP11, is fused to the C terminus of the scFv. This tag has been engineered to be non-perturbing, and we were able to show that it exerted no effect on scFv expression or functionality when compared to a scFv without the GFP11 tag. Effective functional fluorescent labeling is demonstrated in a number of different assays, including fluorescence linked immunosorbant assays, flow cytometry and yeast display. Furthermore, we were able to show that this split GFP system can be used to determine the concentration of scFv in crude samples, as well an estimate of antibody affinity, without the need for antibody purification. We anticipate this system will be of widespread interest in antibody engineering and in vitro display systems.

  11. Production and characterization of human anti-V3 monoclonal antibodies from the cells of HIV-1 infected Indian donors

    Directory of Open Access Journals (Sweden)

    Andrabi Raiees

    2012-09-01

    Full Text Available Abstract Background Analysis of human monoclonal antibodies (mAbs developed from HIV-1 infected donors have enormously contributed to the identification of neutralization sensitive epitopes on the HIV-1 envelope glycoprotein. The third variable region (V3 is a crucial target on gp120, primarily due to its involvement in co-receptor (CXCR4 or CCR5 binding and presence of epitopes recognized by broadly neutralizing antibodies. Methods Thirty-three HIV-1 seropositive drug naive patients (18 males and 15 females within the age range of 20–57 years (median = 33 years were recruited in this study for mAb production. The mAbs were selected from EBV transformed cultures with conformationally constrained Cholera-toxin-B containing V3C (V3C-CTB fusion protein. We tested the mAbs for their binding with HIV-1 derived proteins and peptides by ELISA and for neutralization against HIV-1 viruses by TZM-bl assays. Results We isolated three anti-V3 mAbs, 277, 903 and 904 from the cells of different individuals. The ELISA binding revealed a subtype-C and subtype-A specific binding of antibody 277 and 903 while mAb 904 exhibited cross reactivity also with subtype-B V3. Epitope mapping of mAbs with overlapping V3 peptides showed exclusive binding to V3 crown. The antibodies displayed high and low neutralizing activity against 2/5 tier 1 and 1/6 tier 2 viruses respectively. Overall, we observed a resistance of the tier 2 viruses to neutralization by the anti-V3 mAbs, despite the exposure of the epitopes recognized by these antibodies on two representative native viruses (Du156.12 and JRFL, suggesting that the affinity of mAb might equally be crucial for neutralization, as the epitope recognition. Conclusions Our study suggests that the anti-V3 antibodies derived from subtype-C infected Indian patients display neutralization potential against tier 1 viruses while such activity may be limited against more resistant tier 2 viruses. Defining the fine epitope

  12. High throughput functional epitope mapping: revisiting phage display platform to scan target antigen surface.

    Science.gov (United States)

    Rojas, Gertrudis; Tundidor, Yaima; Infante, Yanelys Cabrera

    2014-01-01

    Antibody engineering must be accompanied by mapping strategies focused on identifying the epitope recognized by each antibody to define its unique functional identity. High throughput fine specificity determination remains technically challenging. We review recent experiences aimed at revisiting the oldest and most extended display technology to develop a robust epitope mapping platform, based on the ability to manipulate target-derived molecules (ranging from the whole native antigen to antigen domains and smaller fragments) on filamentous phages. Single, multiple and combinatorial mutagenesis allowed comprehensive scanning of phage-displayed antigen surface that resulted in the identification of clusters of residues contributing to epitope formation. Functional pictures of the epitope(s) were thus delineated in the natural context. Successful mapping of antibodies against interleukin-2, epidermal growth factor and its receptor, and vascular endothelial growth factor showed the versatility of these procedures, which combine the accuracy of site-directed mutagenesis with the high throughput potential of phage display.

  13. Giant Broad Line Regions in Dwarf Seyferts

    CERN Document Server

    Devereux, Nick

    2015-01-01

    High angular resolution spectroscopy obtained with the Hubble Space Telescope (HST) has revealed a remarkable population of galaxies hosting dwarf Seyfert nuclei with an unusually large broad-line region (BLR). These objects are remarkable for two reasons. Firstly, the size of the BLR can, in some cases, rival those seen in the most luminous quasars. Secondly, the size of the BLR is not correlated with the central continuum luminosity, an observation that distinguishes them from their reverberating counterparts. Collectively, these early results suggest that non-reverberating dwarf Seyferts are a heterogeneous group and not simply scaled versions of each other. Careful inspection reveals broad H Balmer emission lines with single peaks, double peaks, and a combination of the two, suggesting that the broad emission lines are produced in kinematically distinct regions centered on the black hole (BH). Because the gravitational field strength is already known for these objects, by virtue of knowing their BH mass, ...

  14. Syngeneic anti-idiotypic monoclonal antibodies to an anti-NeuGc-containing ganglioside monoclonal antibody.

    Science.gov (United States)

    Vázquez, A M; Pérez, A; Hernández, A M; Macías, A; Alfonso, M; Bombino, G; Pérez, R

    1998-12-01

    An IgM monoclonal antibody (MAb), named P3, has the characteristic to react specifically with a broad battery of N-glycolyl containing-gangliosides and with antigens expressed on breast tumors. When this MAb was administered alone in syngeneic mice, an specific IgG anti-idiotypic antibody (Ab2) response was induced, this Ab2 response was increased when P3 MAb was injected coupled to a carrier protein and in the presence of Freund's adjuvant. Spleen cells from these mice were used in somatic-cell hybridization experiments, using the murine myeloma cell line P3-X63-Ag8.653 as fusion partner. Five Ab2 MAbs specific to P3 MAb were selected. These IgG1 Ab2 MAbs were able to block the binding of P3 MAb to GM3(NeuGc) ganglioside and to a human breast carcinoma cell line. Cross-blocking experiments demonstrated that these Ab2 MAbs are recognizing the same or very close sites on the Abl MAb. The five Ab2 MAbs were injected into syngeneic mice and four of them produced strong anti-anti-idiotypic antibody (Ab3) response. While these Ab2 MAbs were unable to generate Ab3 antibodies with the same antigenic specificity than P3 MAb, three of them induced antibodies bearing P3 MAb idiotopes (Ag-Id+ Ab3). These results demonstrated that these Ab2 MAbs are not "internal image" antibodies, but they could define "regulatory idiotopes."

  15. Monoclonal antibody disulfide reduction during manufacturing

    Science.gov (United States)

    Hutterer, Katariina M.; Hong, Robert W.; Lull, Jonathon; Zhao, Xiaoyang; Wang, Tian; Pei, Rex; Le, M. Eleanor; Borisov, Oleg; Piper, Rob; Liu, Yaoqing Diana; Petty, Krista; Apostol, Izydor; Flynn, Gregory C.

    2013-01-01

    Manufacturing-induced disulfide reduction has recently been reported for monoclonal human immunoglobulin gamma (IgG) antibodies, a widely used modality in the biopharmaceutical industry. This effect has been tied to components of the intracellular thioredoxin reduction system that are released upon cell breakage. Here, we describe the effect of process parameters and intrinsic molecule properties on the extent of reduction. Material taken from cell cultures at the end of production displayed large variations in the extent of antibody reduction between different products, including no reduction, when subjected to the same reduction-promoting harvest conditions. Additionally, in a reconstituted model in which process variables could be isolated from product properties, we found that antibody reduction was dependent on the cell line (clone) and cell culture process. A bench-scale model using a thioredoxin/thioredoxin reductase regeneration system revealed that reduction susceptibility depended on not only antibody class but also light chain type; the model further demonstrates that the trend in reducibility was identical to DTT reduction sensitivity following the order IgG1λ > IgG1κ > IgG2λ > IgG2κ. Thus, both product attributes and process parameters contribute to the extent of antibody reduction during production. PMID:23751615

  16. Modelling Demand for Broad Money in Australia

    OpenAIRE

    Abbas Valadkhani

    2002-01-01

    The existence of a stable demand for money is very important for the conduct of monetary policy. It is argued that previous work on the demand for money in Australia has not been very satisfactory in a number of ways. This paper examines the long- and short-run determinants of the demand for broad money employing the Johansen cointegration technique and a short-run dynamic model. Using quarterly data for the period 1976:3-2002:2, this paper finds, inter alia, that the demand for broad money i...

  17. Yeast surface display of a noncovalent MHC class II heterodimer complexed with antigenic peptide.

    Science.gov (United States)

    Boder, Eric T; Bill, Jerome R; Nields, Andrew W; Marrack, Philippa C; Kappler, John W

    2005-11-20

    Microbial protein display technologies have enabled directed molecular evolution of binding and stability properties in numerous protein systems. In particular, dramatic improvements to antibody binding affinity and kinetics have been accomplished using these tools in recent years. Examples of successful application of display technologies to other immunological proteins have been limited to date. Herein, we describe the expression of human class II major histocompatibility complex allele (MHCII) HLA-DR4 on the surface of Saccharomyces cerevisiae as a noncovalently associated heterodimer. The yeast-displayed MHCII is fully native as assessed by binding of conformationally specific monoclonal antibodies; failure of antibodies specific for empty HLA-DR4 to bind yeast-displayed protein indicates antigenic peptide is bound. This report represents the first example of a noncovalent protein dimer displayed on yeast and of successful display of wild-type MHCII. Results further point to the potential for using yeast surface display for engineering and analyzing the antigen binding properties of MHCII.

  18. Shigella IpaB and IpaD displayed on L. lactis bacterium-like particles induce protective immunity in adult and infant mice.

    Science.gov (United States)

    Heine, Shannon J; Franco-Mahecha, Olga L; Chen, Xiaotong; Choudhari, Shyamal; Blackwelder, William C; van Roosmalen, Maarten L; Leenhouts, Kees; Picking, Wendy L; Pasetti, Marcela F

    2015-08-01

    Shigella spp. are among the enteric pathogens with the highest attributable incidence of moderate-to-severe diarrhea in children under 5 years of age living in endemic areas. There are no vaccines available to prevent this disease. In this work, we investigated a new Shigella vaccine concept consisting of nonliving, self-adjuvanted, Lactococcus lactis bacterium-like particles (BLP) displaying Shigella invasion plasmid antigen (Ipa) B and IpaD and examined its immunogenicity and protective efficacy in adult and newborn/infant mice immunized via the nasal route. Unique advantages of this approach include the potential for broad protection due to the highly conserved structure of the Ipas and the safety and practicality of a probiotic-based mucosal/adjuvant delivery platform. Immunization of adult mice with BLP-IpaB and BLP-IpaD (BLP-IpaB/D) induced high levels of Ipa-specific serum IgG and stool IgA in a dose-dependent manner. Immune responses and protection were enhanced by BLP delivery. Vaccine-induced serum antibodies exhibited opsonophagocytic and cytotoxic neutralizing activity, and IpaB/D IgG titers correlated with increased survival post-challenge. Ipa-specific antibody secreting cells were detected in nasal tissue and lungs, as well as IgG in bronchoalveolar lavage. Bone marrow cells produced IpaB/D-specific antibodies and contributed to protection after adoptive transfer. The BLP-IpaB/D vaccine conferred 90% and 80% protection against S. flexneri and S. sonnei, respectively. Mice immunized with BLP-IpaB/D as newborns also developed IpaB and IpaD serum antibodies; 90% were protected against S. flexneri and 44% against S. sonnei. The BLP-IpaB/D vaccine is a promising candidate for safe, practical and potentially effective immunization of children against shigellosis.

  19. Defining Key Features of the Broad Autism Phenotype

    Science.gov (United States)

    Losh, Molly; Childress, Debra; Lam, Kristen; Piven, Joseph

    2009-01-01

    This study examined the frequency of personality, language, and social-behavioral characteristics believed to comprise the broad autism phenotype (BAP), across families differing in genetic liability to autism. We hypothesized that within this unique sample comprised of multiple-incidence autism families (MIAF), single-incidence autism families (SIAF), and control Down syndrome families (DWNS), a graded expression would be observed for the principal characteristics conferring genetic susceptibility to autism, in which such features would express most profoundly among parents from MIAFs, less strongly among SIAFs, and least of all among comparison parents from DWNS families, who should display population base rates. Analyses detected linear expression of traits in line with hypotheses, and further suggested differential intrafamilial expression across family types. In the vast majority of MIAFs both parents displayed BAP characteristics, whereas within SIAFs, it was equally likely that one, both, or neither parent show BAP features. The significance of these findings is discussed in relation to etiologic mechanisms in autism and relevance to molecular genetic studies. PMID:17948871

  20. Automated Detection of Conformational Epitopes Using Phage Display Peptide Sequences

    Directory of Open Access Journals (Sweden)

    Surendra S Negi

    2009-01-01

    Full Text Available Background: Precise determination of conformational epitopes of neutralizing antibodies represents a key step in the rational design of novel vaccines. A powerful experimental method to gain insights on the physical chemical nature of conformational epitopes is the selection of linear peptides that bind with high affinities to a monoclonal antibody of interest by phage display technology. However, the structural characterization of conformational epitopes from these mimotopes is not straightforward, and in the past the interpretation of peptide sequences from phage display experiments focused on linear sequence analysis to find a consensus sequence or common sequence motifs.Results: We present a fully automated search method, EpiSearch that predicts the possible location of conformational epitopes on the surface of an antigen. The algorithm uses peptide sequences from phage display experiments as input, and ranks all surface exposed patches according to the frequency distribution of similar residues in the peptides and in the patch. We have tested the performance of the EpiSearch algorithm for six experimental data sets of phage display experiments, the human epidermal growth factor receptor-2 (HER-2/neu, the antibody mAb Bo2C11 targeting the C2 domain of FVIII, antibodies mAb 17b and mAb b12 of the HIV envelope protein gp120, mAb 13b5 targeting HIV-1 capsid protein and 80R of the SARS coronavirus spike protein. In all these examples the conformational epitopes as determined by the X-ray crystal structures of the antibody-antigen complexes, were found within the highest scoring patches of EpiSearch, covering in most cases more than 50% residues of experimental observed conformational epitopes. Input options of the program include mapping of a single peptide or a set of peptides on the antigen structure, and the results of the calculation can be visualized on our interactive web server.Availability: Users can access the EpiSearch from our web

  1. Measuring Single-Domain Antibody Interactions with Epitopes in Jet Fuel Using Microscale Thermophoresis

    Science.gov (United States)

    2015-01-01

    J. G. Renisio, J. J. Prompers, C. J. van Platerink, C. Cambillau, H. Darbonand, and L. G. Frenken. 2001. Thermal unfolding of a llama antibody...K. Srivastava, W. Haasnoot, et al. 2011. A broad set of different llama antibodies specific for a 16 kDa heat shock protein of Mycobacterium

  2. Interaction Tasks and Controls for Public Display Applications

    Directory of Open Access Journals (Sweden)

    Jorge C. S. Cardoso

    2014-01-01

    Full Text Available Public displays are becoming increasingly interactive and a broad range of interaction mechanisms can now be used to create multiple forms of interaction. However, the lack of interaction abstractions forces each developer to create specific approaches for dealing with interaction, preventing users from building consistent expectations on how to interact across different display systems. There is a clear analogy with the early days of the graphical user interface, when a similar problem was addressed with the emergence of high-level interaction abstractions that provided consistent interaction experiences to users and shielded developers from low-level details. This work takes a first step in that same direction by uncovering interaction abstractions that may lead to the emergence of interaction controls for applications in public displays. We identify a new set of interaction tasks focused on the specificities of public displays; we characterise interaction controls that may enable those interaction tasks to be integrated into applications; we create a mapping between the high-level abstractions provided by the interaction tasks and the concrete interaction mechanisms that can be implemented by those displays. Together, these contributions constitute a step towards the emergence of programming toolkits with widgets that developers could incorporate into their public display applications.

  3. Therapeutic Recombinant Monoclonal Antibodies

    Science.gov (United States)

    Bakhtiar, Ray

    2012-01-01

    During the last two decades, the rapid growth of biotechnology-derived techniques has led to a myriad of therapeutic recombinant monoclonal antibodies with significant clinical benefits. Recombinant monoclonal antibodies can be obtained from a number of natural sources such as animal cell cultures using recombinant DNA engineering. In contrast to…

  4. Teaching the Broad, Interdisciplinary Impact of Evolution

    Science.gov (United States)

    Benson, David; Atlas, Pierre; Haberski, Raymond; Higgs, Jamie; Kiley, Patrick; Maxwell, Michael, Jr.; Mirola, William; Norton, Jamey

    2009-01-01

    As perhaps the most encompassing idea in biology, evolution has impacted not only science, but other academic disciplines as well. The broad, interdisciplinary impact of evolution was the theme of a course taught at Marian College, Indianapolis, Indiana in 2002, 2004, and 2006. Using a strategy that could be readily adopted at other institutions,…

  5. The GREGOR Broad-Band Imager

    Science.gov (United States)

    von der Lühe, O.; Volkmer, R.; Kentischer, T. J.; Geißler, R.

    2012-11-01

    The design and characteristics of the Broad-Band Imager (BBI) of GREGOR are described. BBI covers the visible spectral range with two cameras simultaneously for a large field and with critical sampling at 390 nm, and it includes a mode for observing the pupil in a Foucault configuration. Samples of first-light observations are shown.

  6. Phosphors for flat panel emissive displays

    Energy Technology Data Exchange (ETDEWEB)

    Anderson, M.T.; Walko, R.J.; Phillips, M.L.F.

    1995-07-01

    An overview of emissive display technologies is presented. Display types briefly described include: cathode ray tubes (CRTs), field emission displays (FEDs), electroluminescent displays (ELDs), and plasma display panels (PDPs). The critical role of phosphors in further development of the latter three flat panel emissive display technologies is outlined. The need for stable, efficient red, green, and blue phosphors for RGB fall color displays is emphasized.

  7. Human monoclonal antibody HCV1 effectively prevents and treats HCV infection in chimpanzees.

    Directory of Open Access Journals (Sweden)

    Trevor J Morin

    Full Text Available Hepatitis C virus (HCV infection is a leading cause of liver transplantation and there is an urgent need to develop therapies to reduce rates of HCV infection of transplanted livers. Approved therapeutics for HCV are poorly tolerated and are of limited efficacy in this patient population. Human monoclonal antibody HCV1 recognizes a highly-conserved linear epitope of the HCV E2 envelope glycoprotein (amino acids 412-423 and neutralizes a broad range of HCV genotypes. In a chimpanzee model, a single dose of 250 mg/kg HCV1 delivered 30 minutes prior to infusion with genotype 1a H77 HCV provided complete protection from HCV infection, whereas a dose of 50 mg/kg HCV1 did not protect. In addition, an acutely-infected chimpanzee given 250 mg/kg HCV1 42 days following exposure to virus had a rapid reduction in viral load to below the limit of detection before rebounding 14 days later. The emergent virus displayed an E2 mutation (N415K/D conferring resistance to HCV1 neutralization. Finally, three chronically HCV-infected chimpanzees were treated with a single dose of 40 mg/kg HCV1 and viral load was reduced to below the limit of detection for 21 days in one chimpanzee with rebounding virus displaying a resistance mutation (N417S. The other two chimpanzees had 0.5-1.0 log(10 reductions in viral load without evidence of viral resistance to HCV1. In vitro testing using HCV pseudovirus (HCVpp demonstrated that the sera from the poorly-responding chimpanzees inhibited the ability of HCV1 to neutralize HCVpp. Measurement of antibody responses in the chronically-infected chimpanzees implicated endogenous antibody to E2 and interference with HCV1 neutralization although other factors may also be responsible. These data suggest that human monoclonal antibody HCV1 may be an effective therapeutic for the prevention of graft infection in HCV-infected patients undergoing liver transplantation.

  8. Selection of functional T cell receptor mutants from a yeast surface-display library.

    Science.gov (United States)

    Kieke, M C; Shusta, E V; Boder, E T; Teyton, L; Wittrup, K D; Kranz, D M

    1999-05-11

    The heterodimeric alphabeta T cell receptor (TCR) for antigen is the key determinant of T cell specificity. The structure of the TCR is very similar to that of antibodies, but the engineering of TCRs by directed evolution with combinatorial display libraries has not been accomplished to date. Here, we report that yeast surface display of a TCR was achieved only after the mutation of specific variable region residues. These residues are located in two regions of the TCR, at the interface of the alpha- and beta-chains and in the beta-chain framework region that is thought to be in proximity to the CD3 signal-transduction complex. The mutations are encoded naturally in many antibody variable regions, indicating specific functional differences that have not been appreciated between TCRs and antibodies. The identification of these residues provides an explanation for the inherent difficulties in the display of wild-type TCRs compared with antibodies. Yeast-displayed mutant TCRs bind specifically to the peptide/MHC antigen, enabling engineering of soluble T cell receptors as specific T cell antagonists. This strategy of random mutagenesis followed by selection for surface expression may be of general use in the directed evolution of other eukaryotic proteins that are refractory to display.

  9. Anti-insulin antibody test

    Science.gov (United States)

    Insulin antibodies - serum; Insulin Ab test; Insulin resistance - insulin antibodies; Diabetes - insulin antibodies ... You appear to have an allergic response to insulin Insulin no longer seems to control your diabetes

  10. BES Monitoring & Displaying System

    Institute of Scientific and Technical Information of China (English)

    MengWANG; BingyunZHANG; 等

    2001-01-01

    BES1 Monitoring & Displaying System(BESMDS)is projected to monitor and display the running status of DAQ and Slow Control systems of BES through the Web for worldwide accessing.It provides a real-time remote means of monitoring as well as an approach to study the environmental influence upon physical data taking.The system collects real-time data separately from BES online subsystems by network sockets and stores the data into a database.People can access the system through its web site.which retrieves data on request from the database and can display results in dynamically created images.Its web address in http:// besmds,ihep.ac.cn/

  11. PROGRAMMABLE DISPLAY PUSHBUTTON LEGEND EDITOR

    Science.gov (United States)

    Busquets, A. M.

    1994-01-01

    The Programmable Display Pushbutton (PDP) is a pushbutton device available from Micro Switch which has a programmable 16 x 35 matrix of LEDs on the pushbutton surface. Any desired legends can be displayed on the PDPs, producing user-friendly applications which greatly reduce the need for dedicated manual controls. Because the PDP can interact with the operator, it can call for the correct response before transmitting its next message. It is both a simple manual control and a sophisticated programmable link between the operator and the host system. The Programmable Display Pushbutton Legend Editor, PDPE, is used to create the LED displays for the pushbuttons. PDPE encodes PDP control commands and legend data into message byte strings sent to a Logic Refresh and Control Unit (LRCU). The LRCU serves as the driver for a set of four PDPs. The legend editor (PDPE) transmits to the LRCU user specified commands that control what is displayed on the LED face of the individual pushbuttons. Upon receiving a command, the LRCU transmits an acknowledgement that the message was received and executed successfully. The user then observes the effect of the command on the PDP displays and decides whether or not to send the byte code of the message to a data file so that it may be called by an applications program. The PDPE program is written in FORTRAN for interactive execution. It was developed on a DEC VAX 11/780 under VMS. It has a central memory requirement of approximately 12800 bytes. It requires four Micro Switch PDPs and two RS-232 VAX 11/780 terminal ports. The PDPE program was developed in 1985.

  12. Immunoproteomic Profiling of Bordetella pertussis Outer Membrane Vesicle Vaccine Reveals Broad and Balanced Humoral Immunogenicity.

    Science.gov (United States)

    Raeven, René H M; van der Maas, Larissa; Tilstra, Wichard; Uittenbogaard, Joost P; Bindels, Tim H E; Kuipers, Betsy; van der Ark, Arno; Pennings, Jeroen L A; van Riet, Elly; Jiskoot, Wim; Kersten, Gideon F A; Metz, Bernard

    2015-07-01

    The current resurgence of whooping cough is alarming, and improved pertussis vaccines are thought to offer a solution. Outer membrane vesicle vaccines (omvPV) are potential vaccine candidates, but omvPV-induced humoral responses have not yet been characterized in detail. The purpose of this study was to determine the antigen composition of omvPV and to elucidate the immunogenicity of the individual antigens. Quantitative proteome analysis revealed the complex composition of omvPV. The omvPV immunogenicity profile in mice was compared to those of classic whole cell vaccine (wPV), acellular vaccine (aPV), and pertussis infection. Pertussis-specific antibody levels, antibody isotypes, IgG subclasses, and antigen specificity were determined after vaccination or infection by using a combination of multiplex immunoassays, two-dimensional immunoblotting, and mass spectrometry. The vaccines and infection raised strong antibody responses, but large quantitative and qualitative differences were measured. The highest antibody levels were obtained by omvPV. All IgG subclasses (IgG1/IgG2a/IgG2b/IgG3) were elicited by omvPV and in a lower magnitude by wPV, but not by aPV (IgG1) or infection (IgG2a/b). The majority of omvPV-induced antibodies were directed against Vag8, BrkA, and LPS. The broad and balanced humoral response makes omvPV a promising pertussis vaccine candidate.

  13. Recombinant lipidated dengue-3 envelope protein domain III stimulates broad immune responses in mice.

    Science.gov (United States)

    Chiang, Chen-Yi; Liu, Shih-Jen; Hsieh, Chun-Hsiang; Chen, Mei-Yu; Tsai, Jy-Ping; Liu, Hsueh-Hung; Chen, I-Hua; Chong, Pele; Leng, Chih-Hsiang; Chen, Hsin-Wei

    2016-02-17

    The linkage of an immunogen with a toll-like receptor ligand has great potential to induce highly potent immune responses with the initial features of antigen-presenting cell activation. In the current study, we expressed recombinant dengue-3 envelope protein domain III (D3ED III) in lipidated form using an Escherichia coli-based system. The recombinant lipidated dengue-3 envelope protein domain III (LD3ED III) augments the expression levels of IL-12 family cytokines. LD3ED III-immunized mice enhance wide ranges of T cell responses as indicated by IFN-γ, IL-17, IL-21 production. Additionally, LD3ED III-immunized mice increase the frequencies of anti-D3ED III antibody producing cells. The boosted antibody titers cover various IgG isotypes, including IgG1, IgG2a, IgG2b, and IgG3. Importantly, LD3ED III-immunized mice induce neutralizing antibody capacity associated with a reduction of viremia levels after challenges. In contrast, mice that are immunized with D3ED III formulated with aluminum phosphate (D3ED III/Alum) only enhance Th2 responses and boost IgG1 antibody titers. Neither neutralizing antibody responses nor the inhibition of viremia levels after challenge is observed in mice that are immunized with D3ED III/Alum. These results suggest that LD3ED III can induce broad profiles of cellular and humoral immune responses.

  14. Putative bioactive motif of tritrpticin revealed by an antibody with biological receptor-like properties.

    Directory of Open Access Journals (Sweden)

    Raghava Sharma

    Full Text Available Antimicrobial peptides represent one of the most promising future strategies for combating infections and microbial drug resistance. Tritrpticin is a 13mer tryptophan-rich cationic antimicrobial peptide with a broad spectrum of activity whose application in antimicrobial therapy has been hampered by ambiguity about its biological target and consequently the molecular interactions necessary for its antimicrobial activity. The present study provides clues about the mechanism of action of tritripticin by using a unique monoclonal antibody (mAb as a 'physiological' structural scaffold. A pool of mAbs were generated against tritrpticin and based on its high affinity and ability to bind tritrpticin analogs, mAb 6C6D7 was selected and characterized further. In a screening of phage displayed random peptides, this antibody was able to identify a novel antimicrobial peptide with low sequence homology to tritrpticin, suggesting that the mAb possessed the physico-chemical characteristics mimicking the natural receptor. Subsequently, thermodynamics and molecular modeling identified a core group of hydrophobic residues in tritrpticin arranged in a distorted's' shaped conformation as critical for antibody binding. Comparison of the mAb induced conformation with the micelle bound structure of tritrpticin reveals how a common motif may be able to interact with multiple classes of biomolecules thus extending the target range of this innate immune peptide. Based on the concurrence between thermodynamic and structural data our results reveal a template that can be used to design novel antimicrobial pharmacophores while simultaneously demonstrating at a more fundamental level the potential of mAbs to act as receptor surrogates.

  15. Computational multi-projection display.

    Science.gov (United States)

    Moon, Seokil; Park, Soon-Gi; Lee, Chang-Kun; Cho, Jaebum; Lee, Seungjae; Lee, Byoungho

    2016-04-18

    A computational multi-projection display is proposed by employing a multi-projection system combining with compressive light field displays. By modulating the intensity of light rays from a spatial light modulator inside a single projector, the proposed system can offer several compact views to observer. Since light rays are spread to all directions, the system can provide flexible positioning of viewpoints without stacking projectors in vertical direction. Also, if the system is constructed properly, it is possible to generate view images with inter-pupillary gap and satisfy the super multi-view condition. We explain the principle of the proposed system and verify its feasibility with simulations and experimental results.

  16. Display standards for commercial flight decks

    Science.gov (United States)

    Lamberth, Larry S.; Penn, Cecil W.

    1994-06-01

    SAE display standards are used as guidelines for certifying commercial airborne electronic displays. The SAE document generation structure and approval process is described. The SAE committees that generate display standards are described. Three SAE documents covering flat panel displays (AS-8034, ARP-4256, and ARP-4260) are discussed with their current status. Head-Up Display documents are also in work.

  17. The germinal center antibody response in health and disease.

    Science.gov (United States)

    DeFranco, Anthony L

    2016-01-01

    The germinal center response is the delayed but sustained phase of the antibody response that is responsible for producing high-affinity antibodies of the IgG, IgA and/or IgE isotypes. B cells in the germinal center undergo re-iterative cycles of somatic hypermutation of immunoglobulin gene variable regions, clonal expansion, and Darwinian selection for cells expressing higher-affinity antibody variants. Alternatively, selected B cells can terminally differentiate into long-lived plasma cells or into a broad diversity of mutated memory B cells; the former secrete the improved antibodies to fight an infection and to provide continuing protection from re-infection, whereas the latter may jumpstart immune responses to subsequent infections with related but distinct infecting agents. Our understanding of the molecules involved in the germinal center reaction has been informed by studies of human immunodeficiency patients with selective defects in the production of antibodies. Recent studies have begun to reveal how innate immune recognition via Toll-like receptors can enhance the magnitude and selective properties of the germinal center, leading to more effective control of infection by a subset of viruses. Just as early insights into the nature of the germinal center found application in the development of the highly successful conjugate vaccines, more recent insights may find application in the current efforts to develop new generations of vaccines, including vaccines that can induce broadly protective neutralizing antibodies against influenza virus or HIV-1.

  18. Monoclonal antibody "gold rush".

    Science.gov (United States)

    Maggon, Krishan

    2007-01-01

    The market, sales and regulatory approval of new human medicines, during the past few years, indicates increasing number and share of new biologics and emergence of new multibillion dollar molecules. The global sale of monoclonal antibodies in 2006 were $20.6 billion. Remicade had annual sales gain of $1 billion during the past 3 years and five brands had similar increase in 2006. Rituxan with 2006 sales of $4.7 billion was the best selling monoclonal antibody and biological product and the 6th among the top selling medicinal brand. It may be the first biologic and monoclonal antibody to reach $10 billion annual sales in the near future. The strong demand from cancer and arthritis patients has surpassed almost all commercial market research reports and sales forecast. Seven monoclonal antibody brands in 2006 had sales exceeding $1 billion. Humanized or fully human monoclonal antibodies with low immunogenicity, enhanced antigen binding and reduced cellular toxicity provide better clinical efficacy. The higher technical and clinical success rate, overcoming of technical hurdles in large scale manufacturing, low cost of market entry and IND filing, use of fully human and humanized monoclonal antibodies has attracted funds and resources towards R&D. Review of industry research pipeline and sales data during the past 3 years indicate a real paradigm shift in industrial R&D from pharmaceutical to biologics and monoclonal antibodies. The antibody bandwagon has been joined by 200 companies with hundreds of new projects and targets and has attracted billions of dollars in R&D investment, acquisitions and licensing deals leading to the current Monoclonal Antibody Gold Rush.

  19. Antiphospholipid antibody syndrome.

    Science.gov (United States)

    Kutteh, William H; Hinote, Candace D

    2014-03-01

    Antiphospholipid antibodies (aPLs) are acquired antibodies directed against negatively charged phospholipids. Obstetric antiphospholipid antibody syndrome (APS) is diagnosed in the presence of certain clinical features in conjunction with positive laboratory findings. Obstetric APS is one of the most commonly identified causes of recurrent pregnancy loss. Thus, obstetric APS is distinguished from APS in other organ systems where the most common manifestation is thrombosis. Several pathophysiologic mechanisms of action of aPLs have been described. This article discusses the diagnostic and obstetric challenges of obstetric APS, proposed pathophysiologic mechanisms of APS during pregnancy, and the management of women during and after pregnancy.

  20. Multiple and broad frequency response Gunn diodes

    Science.gov (United States)

    Pilgrim, N. J.; Macpherson, R. F.; Khalid, A.; Dunn, G. M.; Cumming, D. R. S.

    2009-10-01

    Gunn diodes, operating in transit time mode, are usually thought of as incapable of generating power at multiple frequencies or over a broad frequency range. In this paper, we report experimental results showing that these diodes can generate power at several frequencies and, using Monte Carlo simulations of both planar and vertical devices, we offer an explanation of how this unusual behaviour may come into being and suggest possible applications for this novel device.

  1. Development of recombinant antibody technology for application in plant pathogen diagnosis

    NARCIS (Netherlands)

    Griep, R.A.

    1999-01-01

    This thesis describes the applicability of the novel phage display technique to select plant-pathogen-specific monoclonal antibodies (MAbs) from combinatorial antibody libraries. The retrieved MAbs are so specific that they can be used as diagnostic tools in sensitive immunoassays for the detection

  2. Human single chain antibodies against heparin: selection, characterization, and effect on coagulation.

    NARCIS (Netherlands)

    Westerlo, E.M.A. van de; Smetsers, T.F.; Dennissen, M.A.B.A.; Linhardt, R.J.; Veerkamp, J.H.; Muijen, G.N.P. van; Kuppevelt, A.H.M.S.M. van

    2002-01-01

    Heparin, located in mast cells and basophilic granulocytes, is widely used as an anticoagulant. It belongs to a class of linear polysaccharides called glycosaminoglycans (GAGs). Using phage display technology, we have selected 19 unique human antiheparin antibodies. Some antibodies react almost excl

  3. An improved single-chain Fab platform for efficient display and recombinant expression.

    Science.gov (United States)

    Koerber, James T; Hornsby, Michael J; Wells, James A

    2015-01-30

    Antibody phage display libraries combined with high-throughput selections have recently demonstrated tremendous promise to create the next generation of renewable, recombinant antibodies to study proteins and their many post-translational modification states; however, many challenges still remain, such as optimized antibody scaffolds. Recently, a single-chain fragment antigen binding (Fab) (scFab) format, in which the carboxy-terminus of the light chain is linked to the amino-terminus of the heavy chain, was described to potentially combine the high display levels of a single-chain fragment variable with the high stability of purified Fabs. However, this format required removal of the interchain disulfide bond to achieve modest display levels and subsequent bacterial expression resulted in high levels of aggregated scFab, hindering further use of scFabs. Here, we developed an improved scFab format that retains the interchain disulfide bond by increasing the linker length between the light and heavy chains to improve display and bacterial expression levels to 1-3 mg/L. Furthermore, rerouting of the scFab to the co-translational signal recognition particle pathway combined with reengineering of the signal peptide sequence results in display levels 24-fold above the original scFab format and 3-fold above parent Fab levels. This optimized scFab scaffold can be easily reformatted in a single step for expression in a bacterial or mammalian host to produce stable (Tm of 81 °C), predominantly monomeric (>90%) antibodies at a high yield. Ultimately, this new scFab format will advance high-throughput antibody generation platforms to discover the next generation of research and therapeutic antibodies.

  4. Autostereoscopic display with eye tracking

    Science.gov (United States)

    Tomono, Takao; Hoon, Kyung; Ha, Yong Soo; Kim, Sung-Sik; Son, Jung-Young

    2002-05-01

    Auto-stereoscopic 21-inch display with eye tracking having wide viewing zone and bright image was fabricated. The image of display is projected to retinal through several optical components. We calculated optical system for wider viewing zone by using Inverse-Ray Trace Method. The viewing zone of first model is 155mm (theoretical value: 161mm). We could widen viewing zone by controlling paraxial radius of curvature of spherical mirror, the distance between lenses and so on. The viewing zone of second model is 208mm. We used two spherical mirrors to obtain twice brightness. We applied eye-tracking system to the display system. Eye recognition is based on neural network card based on ZICS technology. We fabricated Auto-stereoscopic 21-inch display with eye tracking. We measured viewing zone based on illumination area. The viewing zone was 206mm, which was close to theoretical value. We could get twice brightness also. We could see 3D image according to position without headgear.

  5. Graphics Display of Foreign Scripts.

    Science.gov (United States)

    Abercrombie, John R.

    1987-01-01

    Describes Graphics Project for Foreign Language Learning at the University of Pennsylvania, which has developed ways of displaying foreign scripts on microcomputers. Character design on computer screens is explained; software for graphics, printing, and language instruction is discussed; and a text editor is described that corrects optically…

  6. Real Time Sonic Boom Display

    Science.gov (United States)

    Haering, Ed

    2014-01-01

    This presentation will provide general information about sonic boom mitigation technology to the public in order to supply information to potential partners and licensees. The technology is a combination of flight data, atmospheric data and terrain information implemented into a control room real time display for flight planning. This research is currently being performed and as such, any results and conclusions are ongoing.

  7. Verbal Modification via Visual Display

    Science.gov (United States)

    Richmond, Edmun B.; Wallace-Childers, La Donna

    1977-01-01

    The inability of foreign language students to produce acceptable approximations of new vowel sounds initiated a study to devise a real-time visual display system whereby the students could match vowel production to a visual pedagogical model. The system used amateur radio equipment and a standard oscilloscope. (CHK)

  8. Information retrieval and display system

    Science.gov (United States)

    Groover, J. L.; King, W. L.

    1977-01-01

    Versatile command-driven data management system offers users, through simplified command language, a means of storing and searching data files, sorting data files into specified orders, performing simple or complex computations, effecting file updates, and printing or displaying output data. Commands are simple to use and flexible enough to meet most data management requirements.

  9. Colour displays for categorical images

    NARCIS (Netherlands)

    Glasbey, C.; Heijden, van der G.W.A.M.; Toh, V.F.K.; Gray, A.J.

    2007-01-01

    We propose a method for identifying a set of colours for displaying 2D and 3D categorical images when the categories are unordered labels. The principle is to find maximally distinct sets of colours. We either generate colours sequentially, to maximize the dissimilarity or distance between a new col

  10. Book Display as Adult Service.

    Science.gov (United States)

    Moore, Matthew S.

    1997-01-01

    Defines book display as an adult service as choosing and positioning adult books from the library collection to increase their circulation. The author contrasts bookstore arrangement for sales versus library arrangement for access, including contrasting missions, genre grouping, weeding, problems, and dimensions. (Author/LRW)

  11. Memory B cell antibodies to HIV-1 gp140 cloned from individuals infected with clade A and B viruses.

    Directory of Open Access Journals (Sweden)

    Hugo Mouquet

    Full Text Available Understanding the antibody response to HIV-1 in humans that show broad neutralizing serologic activity is a crucial step in trying to reproduce such responses by vaccination. Investigating antibodies with cross clade reactivity is particularly important as these antibodies may target conserved epitopes on the HIV envelope gp160 protein. To this end we have used a clade B YU-2 gp140 trimeric antigen and single-cell antibody cloning methods to obtain 189 new anti-gp140 antibodies representing 51 independent B cell clones from the IgG memory B cells of 3 patients infected with HIV-1 clade A or B viruses and exhibiting broad neutralizing serologic activity. Our results support previous findings showing a diverse antibody response to HIV gp140 envelope protein, characterized by differentially expanded B-cell clones producing highly hypermutated antibodies with heterogenous gp140-specificity and neutralizing activity. In addition to their high-affinity binding to the HIV spike, the vast majority of the new anti-gp140 antibodies are also polyreactive. Although none of the new antibodies are as broad or potent as VRC01 or PG9, two clonally-related antibodies isolated from a clade A HIV-1 infected donor, directed against the gp120 variable loop 3, rank in the top 5% of the neutralizers identified in our large collection of 185 unique gp140-specific antibodies in terms of breadth and potency.