Antibody (Ab) responses to a B-cell epitope can be produced by immunization with a peptide bearin... more Antibody (Ab) responses to a B-cell epitope can be produced by immunization with a peptide bearing the epitope of interest. Such peptides are often poor immunogens, requiring conjugation or fusion to carrier proteins that provide T-cell help. However, carrier proteins may also divert the Ab response away from the targeted peptide via its own B-cell epitopes. Thus, an ideal peptide carrier should provide T-cell help to drive B-cell responses but contain relatively few B-cell epitopes. The Ab response to filamentous phage is restricted to the 12 N-terminal residues of the major coat protein (pVIII) and the external domains of the minor coat protein, pIII. Previously, we reported that the Ab response against a synthetic peptide conjugated to phage was more focused on the peptide compared to ovalbumin as the carrier. In some cases, the anti-peptide Ab titer exceeded the anti-carrier Ab titer by > 20 fold. The reduced complexity of the phage surface compared to ovalbumin’s may be resp...
Despite the widespread use of immune repertoire profiling, there is currently no standardized for... more Despite the widespread use of immune repertoire profiling, there is currently no standardized format for output files from VDJ analysis. Researchers utilize software such as IgBlast and IMGT/High V-Quest to perform VDJ analysis and infer germline rearrangements. Each of these software tools produces results in a different file format, and can identify the same result using different labels. These differences make it challenging for users to perform additional analysis using the output file from one software to the next. We have addressed this problem by developing a standardized file format for representing results. The purpose of VDJML is to provide a common standardized format for different VDJ analysis applications and to facilitate downstream processing of the results in an application-agnostic manner. The VDJML file format is accompanied by a suite of analysis tools, which are accessible via command line and written in C++ and python. The VDJML suite will allow users to streaml...
High-throughput sequencing (HTS) of immunoglobulin (B-cell receptor, antibody) and T-cell recepto... more High-throughput sequencing (HTS) of immunoglobulin (B-cell receptor, antibody) and T-cell receptor repertoires has increased dramatically since the technique was introduced in 2009 (1-3). This experimental approach explores the maturation of the adaptive immune system and its response to antigens, pathogens, and disease conditions in exquisite detail. It holds significant promise for diagnostic and therapy-guiding applications. New technology often spreads rapidly, sometimes more rapidly than the understanding of how to make the products of that technology reliable, reproducible, or usable by others. As complex technologies have developed, scientific communities have come together to adopt common standards, protocols, and policies for generating and sharing data sets, such as the MIAME protocols developed for microarray experiments. The Adaptive Immune Receptor Repertoire (AIRR) Community formed in 2015 to address similar issues for HTS data of immune repertoires. The purpose of thi...
sharing of AIRR-seq data, and to address challenges involved in the management and analysis of th... more sharing of AIRR-seq data, and to address challenges involved in the management and analysis of these large data sets. To this end, three working groups were formed: (1) Common Repository, (2) Minimal Standards, and (3) Tools & Resources (Fig. 1). These working groups spent the next year prioritizing goals and defining recommendations that were discussed at the second AIRR Community meeting, in June 2016, which was held at the US National Institutes of Health in Rockville, Maryland. The resulting set of ratified recommendations, along with video recordings of the discussions, is available on the AIRR Community website (http://airr-community. org). The third AIRR Community meeting will take place in December 2017, also at the US National Institutes of Health. The three AIRR Community working groups meet regularly via teleconference, with many discussions continued online through the B-T.CR forum (https://b-t.cr/). A major goal of the Common Repository Working Group is to develop mechanisms to enable data sharing between multiple repositories that store AIRR-seq data, such as iReceptor (http://ireceptor.irmacs.sfu.ca/) and VDJServer (https://vdjserver.org/). This infrastructure will allow queries (such as a search for a particular CDR3 sequence) to span participating repositories. Along with technical hurdles, major issues involve legal constraints such as donor apy 4,6-8. With high-throughput technologies generating large, complex data sets, AIRR-seq has led to the development of a diverse set of sample-processing strategies 9 and bioinformatics data analysis tools 10,11. However, the ability to generate these data has outpaced the infrastructure available to manage it. Hundreds of studies are being published without common rules or standard procedures for the acquisition, storage, annotation, or sharing of the associated AIRR-seq data sets.
an alternative mechanism involving ring breaking has been proposed by C. B. Post and M. Karplus [... more an alternative mechanism involving ring breaking has been proposed by C. B. Post and M. Karplus [J. Am. Chem. Soc. 108, 1317 (1986)]. The lysozyme mechanism has been reviewed by A. J. Kirby [CRC Crit.
Background: The genes that produce antibodies and the immune receptors expressed on lymphocytes a... more Background: The genes that produce antibodies and the immune receptors expressed on lymphocytes are not germline encoded; rather, they are somatically generated in each developing lymphocyte by a process called V(D)J recombination, which assembles specific, independent gene segments into mature composite genes. The full set of composite genes in an individual at a single point in time is referred to as the immune repertoire. V(D)J recombination is the distinguishing feature of adaptive immunity and enables effective immune responses against an essentially infinite array of antigens. Characterization of immune repertoires is critical in both basic research and clinical contexts. Recent technological advances in repertoire profiling via high-throughput sequencing have resulted in an explosion of research activity in the field. This has been accompanied by a proliferation of software tools for analysis of repertoire sequencing data. Despite the widespread use of immune repertoire profiling and analysis software, there is currently no standardized format for output files from V(D)J analysis. Researchers utilize software such as IgBLAST and IMGT/High V-QUEST to perform V(D)J analysis and infer the structure of germline rearrangements. However, each of these software tools produces results in a different file format, and can annotate the same result using different labels. These differences make it challenging for users to perform additional downstream analyses. Results: To help address this problem, we propose a standardized file format for representing V(D)J analysis results. The proposed format, VDJML, provides a common standardized format for different V(D)J analysis applications to facilitate downstream processing of the results in an application-agnostic manner. The VDJML file format specification is accompanied by a support library, written in C++ and Python, for reading and writing the VDJML file format. Conclusions: The VDJML suite will allow users to streamline their V(D)J analysis and facilitate the sharing of scientific knowledge within the community. The VDJML suite and documentation are available from https://vdjserver.org/vdjml/. We welcome participation from the community in developing the file format standard, as well as code contributions.
Antibody Engineering & Therapeutics, the largest meeting devoted to antibody science and technolo... more Antibody Engineering & Therapeutics, the largest meeting devoted to antibody science and technology and the annual meeting of The Antibody Society, will be held in San Diego, CA on December 11-15, 2016. Each of 14 sessions will include six presentations by leading industry and academic experts. In this meeting preview, the session chairs discuss the relevance of their topics to current and future antibody therapeutics development. Session topics include bispecifics and designer polyclonal antibodies; antibodies for neurodegenerative diseases; the interface between passive and active immunotherapy; antibodies for non-cancer indications; novel antibody display, selection and screening technologies; novel checkpoint modulators / immuno-oncology; engineering antibodies for T-cell therapy; novel engineering strategies to enhance antibody functions; and the biological Impact of Fc receptor engagement. The meeting will open with keynote speakers Dennis R. Burton (The Scripps Research Insti...
We are pleased to present peer reviewed forum proceedings of the 2 nd synchronicity forum of GHRI... more We are pleased to present peer reviewed forum proceedings of the 2 nd synchronicity forum of GHRI/CHVIfunded Canadian and African HIV prevention and vaccine teams Forum objectives GHRI-funded capacity building and HIV prevention research teams presented highlights of achievements Teams discussed how to jointly build on achievements for sustainability Provided an opportunity for inter-team collaboration, synchronize best approach to capacity building, mentoring of new researchers and building leadership Provided opportunities for informal discussions and networking among the teams. Teams learnt about recent advances in the area of African regulatory and ethics review process The forum proceedings was a special supplement in an openaccess journal was produced Forum partners
For the past 25 years, phage display technology has been an invaluable tool for studies of protei... more For the past 25 years, phage display technology has been an invaluable tool for studies of protein-protein interactions. However, the inherent biological, biochemical, and biophysical properties of filamentous bacteriophage, as well as the ease of its genetic manipulation, also make it an attractive platform outside the traditional phage display canon. This review will focus on the unique properties of the filamentous bacteriophage and highlight its diverse applications in current research. Particular emphases are placed on: (i) the advantages of the phage as a vaccine carrier, including its high immunogenicity, relative antigenic simplicity and ability to activate a range of immune responses, (ii) the phage's potential as a prophylactic and therapeutic agent for infectious and chronic diseases, (iii) the regularity of the virion major coat protein lattice, which enables a variety of bioconjugation and surface chemistry applications, particularly in nanomaterials, and (iv) the p...
Antibody Engineering & Therapeutics, the annual meeting of The Antibody Society, will be held in ... more Antibody Engineering & Therapeutics, the annual meeting of The Antibody Society, will be held in San Diego, CA in early December 2015. In this meeting preview, the chairs provide their thoughts on the importance of their session topics, which include antibody effector functions, reproducibility of research and diagnostic antibodies, new developments in antibody-drug conjugates (ADCs), preclinical and clinical ADC data, new technologies and applications for bispecific antibodies, antibody therapeutics for non-cancer and orphan indications, antibodies to harness the cellular immune system, overcoming resistance to clinical immunotherapy, and building comprehensive IGVH-gene repertoires through discovering, confirming and cataloging new germline IGVH genes. The Antibody Society's special session will focus on "Antibodies to watch" in 2016, which are a subset of the nearly 50 antibodies currently in Phase 3 clinical studies. Featuring over 100 speakers in total, the meetin...
The 4E10 antibody recognizes the membrane-proximal external region (MPER) of the HIV-1 Env glycop... more The 4E10 antibody recognizes the membrane-proximal external region (MPER) of the HIV-1 Env glycoprotein gp41 transmembrane subunit, exhibiting one of the broadest neutralizing activities known to date. The neutralizing activity of 4E10 requires solvent-exposed hydrophobic residues at the apex of the complementarity-determining region (CDR) H3 loop, but the molecular basis for this requirement has not been clarified. Here, we report the cocrystal structures and the energetic parameters of binding of a peptide bearing the 4E10-epitope sequence (4E10ep) to nonneutralizing versions of the 4E10 Fab. Nonneutralizing Fabs were obtained by shortening and decreasing the hydrophobicity of the CDR-H3 loop (termed ΔLoop) or by substituting the two tryptophan residues of the CDR-H3 apex with Asp residues (termed WDWD), which also decreases hydrophobicity but preserves the length of the loop. The analysis was complemented by the first crystal structure of the 4E10 Fab in its ligand-free state. Co...
G Protein Pathways, Part B: G Proteins and their Regulators, 2002
Heterotrimeric G protein βγ subunits bind to many different effector proteins. 1 The detailed pro... more Heterotrimeric G protein βγ subunits bind to many different effector proteins. 1 The detailed protein-protein interactions involved in βγ binding to these targets are not well understood. One proposed model for interaction between βγ subunits and effectors suggests that each effector may have shared and unique binding sites on the βγ surface. Evidence in favor of this hypothesis is that activation of all effectors is blocked by GDP bound α subunits, yet specific site-directed mutants can be found that block activation of some effectors while having little effect on others. 2-3 That unique binding interactions may exist for various effectors suggests that specific ligands could be identified that bind to some of these unique interaction sites and thus specifically inhibit the activation of some effectors by βγ subunits. One method for defining sequences in effectors that bind to βγ subunits has been to use peptides derived from the effectors in competition-based assays of effector activation. 4,5 Since βγ subunits bind to peptides derived from various effectors, we reasoned that combinatorial phage-displayed peptide library screening could identify novel ligands for binding to G protein βγ subunits at both common and unique effector interaction sites. One such screen in our hands yielded a family of peptides that further defines the protein requirements for interaction at specific sites on βγ subunits. 5a The peptides have a relatively high affinity for βγ subunits and selectively interfere with certain βγ-mediated pathways. The phage bearing the peptides provide a convenient tool for comparing the βγ binding sites of various effectors to determine if they are shared or unique. Ultimately such ligands could lead to the development of specific small-molecule inhibitors of βγ subunit mediated pathways. Many phage display methods have been published elsewhere. 6,7 Here we describe many of these methods as they specifically relate to analysis of G protein βγ subunits. These methods also have the potential to be directed toward defining ligands for other signal transduction molecules. General Phage and Bacterial Methods f88-4 Phage Display System The phage display system for these studies uses the phage f88-4, which is a derivative of fdtet which itself is in the M13 family of filamentous phage. 8 In this system individual peptides or peptide libraries are fused with the N terminus of the pVIII coat protein. Each phage particle contains 2000-2500 copies of the pVIII coat protein. The DNA sequences for the peptides to be displayed are inserted at the 5′ end of a second copy of gene VIII in the
ABSTRACT Immunoassays have become a standard in secretome analysis in clinical and research analy... more ABSTRACT Immunoassays have become a standard in secretome analysis in clinical and research analysis. In this field there is a need for a high throughput method that uses low sample volumes. Microfluidics and nanofluidics have been developed for this purpose. Our lab has developed a nanofluidic bioarray (NBA) chip with the goal being a high throughput system that assays low sample volumes against multiple probes. A combination of horizontal and vertical channels are produced to create an array antigens on the surface of the NBA chip in one dimension that is probed by flowing in the other dimension antibodies from biological fluids. We have tested the NBA chip by immobilizing streptavidin and then biotinylated peptide to detect the presence of a mouse monoclonal antibody (MAb) that is specific for the peptide. Bound antibody is detected by an AlexaFluor 647 labeled goat (anti-mouse IgG) polyclonal antibody. Using the NBA chip, we have successfully detected peptide binding by small-volume (0.5 μl) samples containing 50 attomoles (100 pM) MAb.
INTRODUCTIONThere are several ways to quantitate solutions of nucleic acids. If the solution is p... more INTRODUCTIONThere are several ways to quantitate solutions of nucleic acids. If the solution is pure, one can use a spectrophotometer to measure the amount of ultraviolet radiation absorbed by the bases. DNA can also be quantified by measuring the UV-induced emission of fluorescence from intercalated ethidium bromide. This method is useful if there is not enough DNA to quantify with a spectrophotometer, or if the DNA solution is contaminated. Strategies for accurately quantifying nucleic acids using these approaches are discussed here.
F ilamentous bacteriophage are commonly used as immunogenic carriers for peptides and proteins di... more F ilamentous bacteriophage are commonly used as immunogenic carriers for peptides and proteins displayed on the phage surface. Previously, we showed that immunization with phage to which peptides had been chemically conjugated can elicit a focused anti-peptide antibody response compared with traditional carrier molecules bearing the same peptide, perhaps due to the low surface complexity of the phage. The regularity of its surface also gives the phage other advantages as a carrier, including immunological simplicity and thousands of well-defined sites for chemical conjugation. More recently, we showed that focusing of antibody responses against 'target' peptides was enhanced when the phage's molecular surface was simplified by removal of immunodominant B-cell epitopes present on the minor coat protein, pIII. The pIII-truncated variant elicits an antibody response that is largely restricted to the exposed N-terminus of the major coat protein, pVIII, and to phage-associated bacterial lipopolysaccharide, and a significant fraction of this response crossreacts with a 12-residue peptide covering the surface-exposed region of pVIII. This allows one to track antibody responses against the phage (and any associated haptens) as they develop over time, and characterize them using a combination of serological, flow cytometric, cellular and immunogenetic assays. The filamentous phage thus provides an excellent model system for studying various aspects of the antibody response, all with the goal of targeting antibody production against weakly immunogenic peptides, proteins and carbohydrates. Developing strategies to enhance and focus humoral immune responses using filamentous phage as a model antigen
Antibody (Ab) responses to a B-cell epitope can be produced by immunization with a peptide bearin... more Antibody (Ab) responses to a B-cell epitope can be produced by immunization with a peptide bearing the epitope of interest. Such peptides are often poor immunogens, requiring conjugation or fusion to carrier proteins that provide T-cell help. However, carrier proteins may also divert the Ab response away from the targeted peptide via its own B-cell epitopes. Thus, an ideal peptide carrier should provide T-cell help to drive B-cell responses but contain relatively few B-cell epitopes. The Ab response to filamentous phage is restricted to the 12 N-terminal residues of the major coat protein (pVIII) and the external domains of the minor coat protein, pIII. Previously, we reported that the Ab response against a synthetic peptide conjugated to phage was more focused on the peptide compared to ovalbumin as the carrier. In some cases, the anti-peptide Ab titer exceeded the anti-carrier Ab titer by > 20 fold. The reduced complexity of the phage surface compared to ovalbumin’s may be resp...
Despite the widespread use of immune repertoire profiling, there is currently no standardized for... more Despite the widespread use of immune repertoire profiling, there is currently no standardized format for output files from VDJ analysis. Researchers utilize software such as IgBlast and IMGT/High V-Quest to perform VDJ analysis and infer germline rearrangements. Each of these software tools produces results in a different file format, and can identify the same result using different labels. These differences make it challenging for users to perform additional analysis using the output file from one software to the next. We have addressed this problem by developing a standardized file format for representing results. The purpose of VDJML is to provide a common standardized format for different VDJ analysis applications and to facilitate downstream processing of the results in an application-agnostic manner. The VDJML file format is accompanied by a suite of analysis tools, which are accessible via command line and written in C++ and python. The VDJML suite will allow users to streaml...
High-throughput sequencing (HTS) of immunoglobulin (B-cell receptor, antibody) and T-cell recepto... more High-throughput sequencing (HTS) of immunoglobulin (B-cell receptor, antibody) and T-cell receptor repertoires has increased dramatically since the technique was introduced in 2009 (1-3). This experimental approach explores the maturation of the adaptive immune system and its response to antigens, pathogens, and disease conditions in exquisite detail. It holds significant promise for diagnostic and therapy-guiding applications. New technology often spreads rapidly, sometimes more rapidly than the understanding of how to make the products of that technology reliable, reproducible, or usable by others. As complex technologies have developed, scientific communities have come together to adopt common standards, protocols, and policies for generating and sharing data sets, such as the MIAME protocols developed for microarray experiments. The Adaptive Immune Receptor Repertoire (AIRR) Community formed in 2015 to address similar issues for HTS data of immune repertoires. The purpose of thi...
sharing of AIRR-seq data, and to address challenges involved in the management and analysis of th... more sharing of AIRR-seq data, and to address challenges involved in the management and analysis of these large data sets. To this end, three working groups were formed: (1) Common Repository, (2) Minimal Standards, and (3) Tools & Resources (Fig. 1). These working groups spent the next year prioritizing goals and defining recommendations that were discussed at the second AIRR Community meeting, in June 2016, which was held at the US National Institutes of Health in Rockville, Maryland. The resulting set of ratified recommendations, along with video recordings of the discussions, is available on the AIRR Community website (http://airr-community. org). The third AIRR Community meeting will take place in December 2017, also at the US National Institutes of Health. The three AIRR Community working groups meet regularly via teleconference, with many discussions continued online through the B-T.CR forum (https://b-t.cr/). A major goal of the Common Repository Working Group is to develop mechanisms to enable data sharing between multiple repositories that store AIRR-seq data, such as iReceptor (http://ireceptor.irmacs.sfu.ca/) and VDJServer (https://vdjserver.org/). This infrastructure will allow queries (such as a search for a particular CDR3 sequence) to span participating repositories. Along with technical hurdles, major issues involve legal constraints such as donor apy 4,6-8. With high-throughput technologies generating large, complex data sets, AIRR-seq has led to the development of a diverse set of sample-processing strategies 9 and bioinformatics data analysis tools 10,11. However, the ability to generate these data has outpaced the infrastructure available to manage it. Hundreds of studies are being published without common rules or standard procedures for the acquisition, storage, annotation, or sharing of the associated AIRR-seq data sets.
an alternative mechanism involving ring breaking has been proposed by C. B. Post and M. Karplus [... more an alternative mechanism involving ring breaking has been proposed by C. B. Post and M. Karplus [J. Am. Chem. Soc. 108, 1317 (1986)]. The lysozyme mechanism has been reviewed by A. J. Kirby [CRC Crit.
Background: The genes that produce antibodies and the immune receptors expressed on lymphocytes a... more Background: The genes that produce antibodies and the immune receptors expressed on lymphocytes are not germline encoded; rather, they are somatically generated in each developing lymphocyte by a process called V(D)J recombination, which assembles specific, independent gene segments into mature composite genes. The full set of composite genes in an individual at a single point in time is referred to as the immune repertoire. V(D)J recombination is the distinguishing feature of adaptive immunity and enables effective immune responses against an essentially infinite array of antigens. Characterization of immune repertoires is critical in both basic research and clinical contexts. Recent technological advances in repertoire profiling via high-throughput sequencing have resulted in an explosion of research activity in the field. This has been accompanied by a proliferation of software tools for analysis of repertoire sequencing data. Despite the widespread use of immune repertoire profiling and analysis software, there is currently no standardized format for output files from V(D)J analysis. Researchers utilize software such as IgBLAST and IMGT/High V-QUEST to perform V(D)J analysis and infer the structure of germline rearrangements. However, each of these software tools produces results in a different file format, and can annotate the same result using different labels. These differences make it challenging for users to perform additional downstream analyses. Results: To help address this problem, we propose a standardized file format for representing V(D)J analysis results. The proposed format, VDJML, provides a common standardized format for different V(D)J analysis applications to facilitate downstream processing of the results in an application-agnostic manner. The VDJML file format specification is accompanied by a support library, written in C++ and Python, for reading and writing the VDJML file format. Conclusions: The VDJML suite will allow users to streamline their V(D)J analysis and facilitate the sharing of scientific knowledge within the community. The VDJML suite and documentation are available from https://vdjserver.org/vdjml/. We welcome participation from the community in developing the file format standard, as well as code contributions.
Antibody Engineering & Therapeutics, the largest meeting devoted to antibody science and technolo... more Antibody Engineering & Therapeutics, the largest meeting devoted to antibody science and technology and the annual meeting of The Antibody Society, will be held in San Diego, CA on December 11-15, 2016. Each of 14 sessions will include six presentations by leading industry and academic experts. In this meeting preview, the session chairs discuss the relevance of their topics to current and future antibody therapeutics development. Session topics include bispecifics and designer polyclonal antibodies; antibodies for neurodegenerative diseases; the interface between passive and active immunotherapy; antibodies for non-cancer indications; novel antibody display, selection and screening technologies; novel checkpoint modulators / immuno-oncology; engineering antibodies for T-cell therapy; novel engineering strategies to enhance antibody functions; and the biological Impact of Fc receptor engagement. The meeting will open with keynote speakers Dennis R. Burton (The Scripps Research Insti...
We are pleased to present peer reviewed forum proceedings of the 2 nd synchronicity forum of GHRI... more We are pleased to present peer reviewed forum proceedings of the 2 nd synchronicity forum of GHRI/CHVIfunded Canadian and African HIV prevention and vaccine teams Forum objectives GHRI-funded capacity building and HIV prevention research teams presented highlights of achievements Teams discussed how to jointly build on achievements for sustainability Provided an opportunity for inter-team collaboration, synchronize best approach to capacity building, mentoring of new researchers and building leadership Provided opportunities for informal discussions and networking among the teams. Teams learnt about recent advances in the area of African regulatory and ethics review process The forum proceedings was a special supplement in an openaccess journal was produced Forum partners
For the past 25 years, phage display technology has been an invaluable tool for studies of protei... more For the past 25 years, phage display technology has been an invaluable tool for studies of protein-protein interactions. However, the inherent biological, biochemical, and biophysical properties of filamentous bacteriophage, as well as the ease of its genetic manipulation, also make it an attractive platform outside the traditional phage display canon. This review will focus on the unique properties of the filamentous bacteriophage and highlight its diverse applications in current research. Particular emphases are placed on: (i) the advantages of the phage as a vaccine carrier, including its high immunogenicity, relative antigenic simplicity and ability to activate a range of immune responses, (ii) the phage's potential as a prophylactic and therapeutic agent for infectious and chronic diseases, (iii) the regularity of the virion major coat protein lattice, which enables a variety of bioconjugation and surface chemistry applications, particularly in nanomaterials, and (iv) the p...
Antibody Engineering & Therapeutics, the annual meeting of The Antibody Society, will be held in ... more Antibody Engineering & Therapeutics, the annual meeting of The Antibody Society, will be held in San Diego, CA in early December 2015. In this meeting preview, the chairs provide their thoughts on the importance of their session topics, which include antibody effector functions, reproducibility of research and diagnostic antibodies, new developments in antibody-drug conjugates (ADCs), preclinical and clinical ADC data, new technologies and applications for bispecific antibodies, antibody therapeutics for non-cancer and orphan indications, antibodies to harness the cellular immune system, overcoming resistance to clinical immunotherapy, and building comprehensive IGVH-gene repertoires through discovering, confirming and cataloging new germline IGVH genes. The Antibody Society's special session will focus on "Antibodies to watch" in 2016, which are a subset of the nearly 50 antibodies currently in Phase 3 clinical studies. Featuring over 100 speakers in total, the meetin...
The 4E10 antibody recognizes the membrane-proximal external region (MPER) of the HIV-1 Env glycop... more The 4E10 antibody recognizes the membrane-proximal external region (MPER) of the HIV-1 Env glycoprotein gp41 transmembrane subunit, exhibiting one of the broadest neutralizing activities known to date. The neutralizing activity of 4E10 requires solvent-exposed hydrophobic residues at the apex of the complementarity-determining region (CDR) H3 loop, but the molecular basis for this requirement has not been clarified. Here, we report the cocrystal structures and the energetic parameters of binding of a peptide bearing the 4E10-epitope sequence (4E10ep) to nonneutralizing versions of the 4E10 Fab. Nonneutralizing Fabs were obtained by shortening and decreasing the hydrophobicity of the CDR-H3 loop (termed ΔLoop) or by substituting the two tryptophan residues of the CDR-H3 apex with Asp residues (termed WDWD), which also decreases hydrophobicity but preserves the length of the loop. The analysis was complemented by the first crystal structure of the 4E10 Fab in its ligand-free state. Co...
G Protein Pathways, Part B: G Proteins and their Regulators, 2002
Heterotrimeric G protein βγ subunits bind to many different effector proteins. 1 The detailed pro... more Heterotrimeric G protein βγ subunits bind to many different effector proteins. 1 The detailed protein-protein interactions involved in βγ binding to these targets are not well understood. One proposed model for interaction between βγ subunits and effectors suggests that each effector may have shared and unique binding sites on the βγ surface. Evidence in favor of this hypothesis is that activation of all effectors is blocked by GDP bound α subunits, yet specific site-directed mutants can be found that block activation of some effectors while having little effect on others. 2-3 That unique binding interactions may exist for various effectors suggests that specific ligands could be identified that bind to some of these unique interaction sites and thus specifically inhibit the activation of some effectors by βγ subunits. One method for defining sequences in effectors that bind to βγ subunits has been to use peptides derived from the effectors in competition-based assays of effector activation. 4,5 Since βγ subunits bind to peptides derived from various effectors, we reasoned that combinatorial phage-displayed peptide library screening could identify novel ligands for binding to G protein βγ subunits at both common and unique effector interaction sites. One such screen in our hands yielded a family of peptides that further defines the protein requirements for interaction at specific sites on βγ subunits. 5a The peptides have a relatively high affinity for βγ subunits and selectively interfere with certain βγ-mediated pathways. The phage bearing the peptides provide a convenient tool for comparing the βγ binding sites of various effectors to determine if they are shared or unique. Ultimately such ligands could lead to the development of specific small-molecule inhibitors of βγ subunit mediated pathways. Many phage display methods have been published elsewhere. 6,7 Here we describe many of these methods as they specifically relate to analysis of G protein βγ subunits. These methods also have the potential to be directed toward defining ligands for other signal transduction molecules. General Phage and Bacterial Methods f88-4 Phage Display System The phage display system for these studies uses the phage f88-4, which is a derivative of fdtet which itself is in the M13 family of filamentous phage. 8 In this system individual peptides or peptide libraries are fused with the N terminus of the pVIII coat protein. Each phage particle contains 2000-2500 copies of the pVIII coat protein. The DNA sequences for the peptides to be displayed are inserted at the 5′ end of a second copy of gene VIII in the
ABSTRACT Immunoassays have become a standard in secretome analysis in clinical and research analy... more ABSTRACT Immunoassays have become a standard in secretome analysis in clinical and research analysis. In this field there is a need for a high throughput method that uses low sample volumes. Microfluidics and nanofluidics have been developed for this purpose. Our lab has developed a nanofluidic bioarray (NBA) chip with the goal being a high throughput system that assays low sample volumes against multiple probes. A combination of horizontal and vertical channels are produced to create an array antigens on the surface of the NBA chip in one dimension that is probed by flowing in the other dimension antibodies from biological fluids. We have tested the NBA chip by immobilizing streptavidin and then biotinylated peptide to detect the presence of a mouse monoclonal antibody (MAb) that is specific for the peptide. Bound antibody is detected by an AlexaFluor 647 labeled goat (anti-mouse IgG) polyclonal antibody. Using the NBA chip, we have successfully detected peptide binding by small-volume (0.5 μl) samples containing 50 attomoles (100 pM) MAb.
INTRODUCTIONThere are several ways to quantitate solutions of nucleic acids. If the solution is p... more INTRODUCTIONThere are several ways to quantitate solutions of nucleic acids. If the solution is pure, one can use a spectrophotometer to measure the amount of ultraviolet radiation absorbed by the bases. DNA can also be quantified by measuring the UV-induced emission of fluorescence from intercalated ethidium bromide. This method is useful if there is not enough DNA to quantify with a spectrophotometer, or if the DNA solution is contaminated. Strategies for accurately quantifying nucleic acids using these approaches are discussed here.
F ilamentous bacteriophage are commonly used as immunogenic carriers for peptides and proteins di... more F ilamentous bacteriophage are commonly used as immunogenic carriers for peptides and proteins displayed on the phage surface. Previously, we showed that immunization with phage to which peptides had been chemically conjugated can elicit a focused anti-peptide antibody response compared with traditional carrier molecules bearing the same peptide, perhaps due to the low surface complexity of the phage. The regularity of its surface also gives the phage other advantages as a carrier, including immunological simplicity and thousands of well-defined sites for chemical conjugation. More recently, we showed that focusing of antibody responses against 'target' peptides was enhanced when the phage's molecular surface was simplified by removal of immunodominant B-cell epitopes present on the minor coat protein, pIII. The pIII-truncated variant elicits an antibody response that is largely restricted to the exposed N-terminus of the major coat protein, pVIII, and to phage-associated bacterial lipopolysaccharide, and a significant fraction of this response crossreacts with a 12-residue peptide covering the surface-exposed region of pVIII. This allows one to track antibody responses against the phage (and any associated haptens) as they develop over time, and characterize them using a combination of serological, flow cytometric, cellular and immunogenetic assays. The filamentous phage thus provides an excellent model system for studying various aspects of the antibody response, all with the goal of targeting antibody production against weakly immunogenic peptides, proteins and carbohydrates. Developing strategies to enhance and focus humoral immune responses using filamentous phage as a model antigen
Uploads
Papers by jamie scott