Difference: MinLu (1 vs. 5)
Revision 530 Oct 2009 - Main.DavidCowburn
My Links
Personal PreferencesUncomment preferences variables to activate them (remove the #-sign). Help and details on preferences variables are available in TWikiPreferences.
Related Topics
Public Information on Grants associated with NYSBC Grant Number: 5R01AI042382-09 Project Title: Structure and Function of the HIV-1 Envelope Protein PI Information: Name Email Title LU, MIN mlu@med.cornell.edu ASSOCIATE PROFESSOR Abstract: DESCRIPTION (provided by applicant): HIV-1 entry into cells is mediated by the envelope glycoprotein (Env) complex (gp120/gp41). Binding of gp120 to receptors on the target cell triggers a series of conformational changes in the native Env trimer that ultimately lead to formation of a six-helix bundle structure in gp41, the so-called fusogenic state of the protein. In this state, interactions between two helical regions (HR1 and HR2) in the gp41 ectodomain are coupled to membrane fusion. HR2-derived peptides, such as Fuzeon (T20), are thought to inhibit membrane fusion by preventing the HR regions from refolding into the six-helix bundle. Despite intensive efforts, high-resolution structural information about the native, trimeric Env complex is lacking. Recognizing this history, we propose to apply new protein dissection approaches to study the pre-fusion conformation(s) of gp41. The broad, longterm objective of this proposal is to define the structural transitions of gp41 by which the HR regions, initially sequestered in the native Env complex, are released and refolded in order to cause membrane fusion. The specific aims of the proposal are: (1) To determine the effects of the fusion inhibitor T20-resistant/dependent mutations in the HR regions on the equilibrium folding, thermodynamics, and conformation of the six-helix bundle, in order to understand the mechanism of action of T20 and how its antiviral effect is mitigated by the evolution of resistant viruses in vivo. (2) To characterize, at high resolution, the structural and dynamic properties of the conserved membraneproximal Trp-rich domain of gp41 in association with [3OG micelles and lipid vesicles, in order to provide structural insight into the mechanism of HIV-1 neutralization by mAbs 2F5 and 4E10. (3) To crystallize and determine the structure of a novel trimerization domain formed by the HR2 and Trp-rich membrane-proximal regions, and to utilize engineered envelope glycoproteins to define the roles of this trimeric structural domain in regulating the stabilization and controlled activation of the native Env complex. We wish to elucidate a mechanism for the precise switch between the metastable native and fusogenic forms of the protein. Public Health Relevance: This Public Health Relevance is not available. Thesaurus Terms: HIV envelope protein gp41, human immunodeficiency virus 1, protein structure function, virus infection mechanism conformation, gene mutation, neutralizing antibody X ray crystallography, circular dichroism, tissue /cell culture Institution: WEILL MEDICAL COLLEGE OF CORNELL UNIV 1300 YORK AVENUE NEW YORK, NY 10065 Fiscal Year: 2008 Department: BIOCHEMISTRY Project Start: 01-SEP-1999 Project End: 31-MAY-2009 ICD: NATIONAL INSTITUTE OF ALLERGY AND INFECTIOUS DISEASES IRG: AMCB Grant Number: 5R01AI068591-04 Project Title: Engineered gp41 Peptides as HIV-1 Topical Microbicides PI Information: Name Email Title LU, MIN mlu@med.cornell.edu ASSOCIATE PROFESSOR Abstract: This abstract is not available. Public Health Relevance: This Public Health Relevance is not available. Thesaurus Terms: Macaca, cell, infection, local antiinfective agent, model, peptide, topical drug application antiinfective agent, cell line, concept, gene mutation, glutamine, human, human immunodeficiency virus, immunodeficiency, irritation /irritant, lead, leucine, ligand, recombinant protein, recombinant virus, simian immunodeficiency virus, small molecule, solubility, tissue, vaccine, virus Institution: WEILL MEDICAL COLLEGE OF CORNELL UNIV 1300 YORK AVENUE NEW YORK, NY 10065 Fiscal Year: 2009 Department: BIOCHEMISTRY Project Start: 01-MAR-2006 Project End: 28-FEB-2010 ICD: NATIONAL INSTITUTE OF ALLERGY AND INFECTIOUS DISEASES IRG: ADDT Grant Number: 3R21AI071979-02S1 Project Title: Development of N-Peptides for Use in HIV-1 Topical Microbicides PI Information: Name Email Title LU, MIN ASSOCIATE PROFESSOR Abstract: DESCRIPTION (provided by applicant): HIV-1 continues to spread globally and no vaccine is available. Hence there is an urgent public health need for an effective microbicide to prevent sexual transmission of HIV-1. The topical application of potent combinations of viral fusion and entry inhibitors has been shown to exhibit microbicidal efficacy in the rhesus macaque vaginal transmission model. Peptides derived from the N- and C-terminal regions of the gp41 ectodomain (called N- and C-peptides, respectively) inhibit HIV-1 entry. N-peptides have generally proven far less potent than C-peptides. However, stabilization of a trimeric coiled-coil conformation of N peptides has been demonstrated to be a viable strategy to develop them as a new class of potent HIV-1 fusion inhibitors. We have recently identified and determined the crystal structure of an autonomously folded, N-peptide coiled-coil domain. The overall goal of this research plan is to gain a detailed understanding of the structural and thermodynamic properties of this novel coiled-coil domain, and to use this knowledge to design and produce a bacterially expressed N-peptide fusion inhibitor for inclusion in a HIV-1 topical microbicide. Our central hypothesis is that the synergistic inhibition of different stages of the viral fusion and entry process can offer a powerful benefit in formulating an efficacious and more economic microbicide product. Specific aims of this research are: (1) to use modern protein engineering methods to identify and develop stabilized variants of a trimeric coiled-coil domain that display potent inhibitory activity against HIV-1 membrane fusion. We will use isoleucine- and valine-scanning mutagenesis to identify and incorporate specific residue substitutions that increase both trimer stability and antiviral potency. We will also construct chimeric N-peptides by using an isoleucine-zipper sequence, to stabilize the coiled-coil structure. Our emphasis is to generate potent N-peptide inhibitors suitable for development as an inexpensive component of a microbicide formulation. (2) To characterize the specificity, potency, and toxicity of improved N-peptide variants and their in vitro synergistic interactions with the virus-cell attachment inhibitors CMPD167 and BMS-378806, and the C-peptide fusion inhibitor C52L. We will conduct in vitro studies to determine inhibitory activity of select N-peptide variants against diverse primary HIV-1 isolates, and their toxic or inflammatory effects using the rabbit vaginal irritation model and in human cells. We will also study synergistic antiviral effects in vitro in order to make rational predictions for lead inhibitor combinations for formulation and efficacy testing in rhesus macaques. (3) To use the rhesus macaque high dose vaginal transmission model to assess the in vivo potency of an optimized N-peptide inhibitor alone and in combination with CMPD167, BMS- 378806, and C52L. We will evaluate the protection of macaques from vaginal challenge with both CCR5 and CXCR4 SHIVs by a vaginally delivered N-peptide inhibitor alone and in synergistic combination with CMPD167, BMS-378806, and C52L. Public Health Relevance: This Public Health Relevance is not available. Thesaurus Terms: There are no thesaurus terms on file for this project. Institution: WEILL MEDICAL COLLEGE OF CORNELL UNIV 1300 YORK AVENUE NEW YORK, NY 10065 Fiscal Year: 2008 Department: BIOCHEMISTRY Project Start: 15-SEP-2006 Project End: 31-AUG-2009 ICD: NATIONAL INSTITUTE OF ALLERGY AND INFECTIOUS DISEASES IRG: ZAI1 Grant Number: 5R21AI074469-02 Project Title: Stabilized gp41 Structural Motif for HIV-1 Vaccine PI Information: Name Email Title LU, MIN mlu@med.cornell.edu ASSOCIATE PROFESSOR Abstract: DESCRIPTION (provided by applicant): The development of immunogens capable of eliciting broadly neutralizing antibodies to HIV-1 remains one of the most difficult challenges confronting biomedical research today. Nonetheless, a small number of human monoclonal antibodies (mAbs) have been isolated from HIV-1-infected individuals that can neutralize primary isolates of HIV-1 from different genetic subtypes. Of these rare mAbs, two (2F5 and 4E10) recognize epitopes on the membrane-proximal region of the gp41 ectodomain. However, all attempts to elicit comparable anti-gp41 antibodies by vaccination with peptides or simple linear epitopes have thus far failed. Our approach to immunogen design is guided by our recent identification and structural determination of a novel two-stranded coiled-coil domain (C55) that includes the membrane-proximal epitope region of gp41. However, we have determined that the C55 dimer structure is too unstable to be suitable for immunogenicity studies. The overall goal of this research plan is to gain a detailed understanding of the structural and thermodynamic properties of the C55 coiled-coil domain, and to use this knowledge to design and produce stable immunogens for the elicitation of broadly neutralizing HIV-1 antibodies. Our central hypothesis is that presentation of the highly conserved sequences recognized by 2F5 and 4E10 in this new structural state opens a promising avenue for the discovery and development of novel immunogens to induce broadly reactive neutralizing antibodies. Specific aims of this research are: (1) To identify and develop stabilized versions of the C55 coiled-coil domain encompassing the 2F5 and 4E10 epitopes. We will identify and incorporate apolar side chain substitutions into the buried hydrophobic core of the dimer to stabilize the coiled-coil structure. We will also screen cysteine substitutions to introduce a disulfide bond between the two interacting helices. Our emphasis is to generate stable molecules that preserve both the dimer conformation and the critical surface-exposed residues of the membrane-proximal region. (2) To evaluate the immunological responses elicited by stabilized C55 coiled-coil variants in small animals. We will conduct immunogenicity studies in rabbits and guinea-pigs to determine whether the stabilized dimer structure can elicit neutralizing antibodies. These studies will involve immunization of the animals using soluble proteins. We will also evaluate the immunogenicity of the stable coiled-coil molecules captured onto nanometer-sized beads as particulate immunogens. Public Health Relevance: This Public Health Relevance is not available. Thesaurus Terms: antigen, dimer, vaccine Gastropoda, active immunization, antibody, conformation, cysteine, disulfide bond, genetics, guinea pig, human, immunization, membrane, monoclonal antibody, neutralizing antibody, peptide, protein Institution: WEILL MEDICAL COLLEGE OF CORNELL UNIV 1300 YORK AVENUE NEW YORK, NY 10065 Fiscal Year: 2008 Department: BIOCHEMISTRY Project Start: 01-MAY-2007 Project End: 30-APR-2010 ICD: NATIONAL INSTITUTE OF ALLERGY AND INFECTIOUS DISEASES IRG: VACC Grant Number: 5R21AI078466-02 Project Title: Presentation of Structural Determinants of the 2F5 Neutralization Epitope PI Information: Name Email Title LU, MIN mlu@med.cornell.edu ASSOCIATE PROFESSOR Abstract: DESCRIPTION (provided by applicant): Development of envelope protein immunogens capable of eliciting neutralizing antibodies against primary HIV-1 isolates from different genetic subtypes remains the most difficult challenge in the field of HIV-1 vaccinology. The conserved membrane-proximal external region of the gp41 ectodomain bears the epitopes of two broadly neutralizing human monoclonal antibodies, 2F5 and 4E10, and is therefore an important target of HIV-1 vaccine design. However, all attempts to elicit anti-gp41 antibodies comparable to these produced in infected humans by using unstructured peptide immunogens have thus far failed. Success will likely require a deeper understanding of the structural motifs of the gp120/gp41 complex that stabilize the mature envelope trimer on the virion surface. Current thinking postulates that the receptor-mediated activation of gp41 involves a regulated sequence of structural transitions with one or more on-pathway intermediate(s) that may reveal cryptic neutralization epitopes. Our recent identification of a four-stranded coiled-coil structure (C43) encompassing the 2F5 epitope suggests that structural aspects of the 2F5 and 4E10 epitopes may be transiently exposed and required for stimulating neutralizing antibody responses. The broad, long-term objective of this research plan is to use protein chemistry and structural biology approaches to engineer and produce stable forms of the C43 coiled-coil motif for the induction of broadly reactive neutralizing antibodies. The premise of the proposed research is that definition of the structural requirements for optimal presentation of the 2F5 neutralization epitope can lead to the identification of stable immunogen products to generate effective anti-HIV-1 immunity. The Specific Aims of the proposed research are: (1) To design and develop stabilized versions of the C43 coiled-coil domain bearing the 2F5 epitope for immunogenicity studies. We will generate a single-chain analog of the tetraplex coiled coil in which the four mutagenesis to identify specific residue substitutions that favorably influence inter-helical packing interactions in the tetramer in order to stabilize an ordered helical peptide structure of the 2F5 epitope that preserves surface-exposed side chains. (2) To evaluate the immunological responses elicited by stabilized C43 coiled-coil variants in small animals. We will conduct immunogenicity studies in rabbits and guinea-pigs to determine whether the stabilized involve immunization of the animals using the stabilized C43 peptide proteins. We will also evaluate the immunogenicity of the stable coiled-coil molecules captured onto nanometer-sized beads as particulate immunogens. The research and public health communities concur that a preventive vaccine is the obvious long-term solution to bring the global HIV-1 epidemic under control (1-8). Unfortunately, this goal has proven elusive and no such vaccine is available. Overcoming this important biomedical problem will require new and imaginative design strategies to bring us closer to the goal of a successful AIDS vaccine. Public Health Relevance: This Public Health Relevance is not available. Thesaurus Terms: There are no thesaurus terms on file for this project. Institution: WEILL MEDICAL COLLEGE OF CORNELL UNIV 1300 YORK AVENUE NEW YORK, NY 10065 Fiscal Year: 2009 Department: BIOCHEMISTRY Project Start: 15-APR-2008 Project End: 31-MAR-2010 ICD: NATIONAL INSTITUTE OF ALLERGY AND INFECTIOUS DISEASES IRG: ZAI1
| |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Revision 408 Apr 2009 - Main.SherryllJones
My Links
Personal PreferencesUncomment preferences variables to activate them (remove the #-sign). Help and details on preferences variables are available in TWikiPreferences.
Related Topics
Public Information on Grants associated with NYSBC Grant Number: 5R01AI042382-09 Project Title: Structure and Function of the HIV-1 Envelope Protein PI Information: Name Email Title LU, MIN mlu@med.cornell.edu ASSOCIATE PROFESSOR Abstract: DESCRIPTION (provided by applicant): HIV-1 entry into cells is mediated by the envelope glycoprotein (Env) complex (gp120/gp41). Binding of gp120 to receptors on the target cell triggers a series of conformational changes in the native Env trimer that ultimately lead to formation of a six-helix bundle structure in gp41, the so-called fusogenic state of the protein. In this state, interactions between two helical regions (HR1 and HR2) in the gp41 ectodomain are coupled to membrane fusion. HR2-derived peptides, such as Fuzeon (T20), are thought to inhibit membrane fusion by preventing the HR regions from refolding into the six-helix bundle. Despite intensive efforts, high-resolution structural information about the native, trimeric Env complex is lacking. Recognizing this history, we propose to apply new protein dissection approaches to study the pre-fusion conformation(s) of gp41. The broad, longterm objective of this proposal is to define the structural transitions of gp41 by which the HR regions, initially sequestered in the native Env complex, are released and refolded in order to cause membrane fusion. The specific aims of the proposal are: (1) To determine the effects of the fusion inhibitor T20-resistant/dependent mutations in the HR regions on the equilibrium folding, thermodynamics, and conformation of the six-helix bundle, in order to understand the mechanism of action of T20 and how its antiviral effect is mitigated by the evolution of resistant viruses in vivo. (2) To characterize, at high resolution, the structural and dynamic properties of the conserved membraneproximal Trp-rich domain of gp41 in association with [3OG micelles and lipid vesicles, in order to provide structural insight into the mechanism of HIV-1 neutralization by mAbs 2F5 and 4E10. (3) To crystallize and determine the structure of a novel trimerization domain formed by the HR2 and Trp-rich membrane-proximal regions, and to utilize engineered envelope glycoproteins to define the roles of this trimeric structural domain in regulating the stabilization and controlled activation of the native Env complex. We wish to elucidate a mechanism for the precise switch between the metastable native and fusogenic forms of the protein. Public Health Relevance: This Public Health Relevance is not available. Thesaurus Terms: HIV envelope protein gp41, human immunodeficiency virus 1, protein structure function, virus infection mechanism conformation, gene mutation, neutralizing antibody X ray crystallography, circular dichroism, tissue /cell culture Institution: WEILL MEDICAL COLLEGE OF CORNELL UNIV 1300 YORK AVENUE NEW YORK, NY 10065 Fiscal Year: 2008 Department: BIOCHEMISTRY Project Start: 01-SEP-1999 Project End: 31-MAY-2009 ICD: NATIONAL INSTITUTE OF ALLERGY AND INFECTIOUS DISEASES IRG: AMCB Grant Number: 5R01AI068591-04 Project Title: Engineered gp41 Peptides as HIV-1 Topical Microbicides PI Information: Name Email Title LU, MIN mlu@med.cornell.edu ASSOCIATE PROFESSOR Abstract: This abstract is not available. Public Health Relevance: This Public Health Relevance is not available. Thesaurus Terms: Macaca, cell, infection, local antiinfective agent, model, peptide, topical drug application antiinfective agent, cell line, concept, gene mutation, glutamine, human, human immunodeficiency virus, immunodeficiency, irritation /irritant, lead, leucine, ligand, recombinant protein, recombinant virus, simian immunodeficiency virus, small molecule, solubility, tissue, vaccine, virus Institution: WEILL MEDICAL COLLEGE OF CORNELL UNIV 1300 YORK AVENUE NEW YORK, NY 10065 Fiscal Year: 2009 Department: BIOCHEMISTRY Project Start: 01-MAR-2006 Project End: 28-FEB-2010 ICD: NATIONAL INSTITUTE OF ALLERGY AND INFECTIOUS DISEASES IRG: ADDT Grant Number: 3R21AI071979-02S1 Project Title: Development of N-Peptides for Use in HIV-1 Topical Microbicides PI Information: Name Email Title LU, MIN ASSOCIATE PROFESSOR Abstract: DESCRIPTION (provided by applicant): HIV-1 continues to spread globally and no vaccine is available. Hence there is an urgent public health need for an effective microbicide to prevent sexual transmission of HIV-1. The topical application of potent combinations of viral fusion and entry inhibitors has been shown to exhibit microbicidal efficacy in the rhesus macaque vaginal transmission model. Peptides derived from the N- and C-terminal regions of the gp41 ectodomain (called N- and C-peptides, respectively) inhibit HIV-1 entry. N-peptides have generally proven far less potent than C-peptides. However, stabilization of a trimeric coiled-coil conformation of N peptides has been demonstrated to be a viable strategy to develop them as a new class of potent HIV-1 fusion inhibitors. We have recently identified and determined the crystal structure of an autonomously folded, N-peptide coiled-coil domain. The overall goal of this research plan is to gain a detailed understanding of the structural and thermodynamic properties of this novel coiled-coil domain, and to use this knowledge to design and produce a bacterially expressed N-peptide fusion inhibitor for inclusion in a HIV-1 topical microbicide. Our central hypothesis is that the synergistic inhibition of different stages of the viral fusion and entry process can offer a powerful benefit in formulating an efficacious and more economic microbicide product. Specific aims of this research are: (1) to use modern protein engineering methods to identify and develop stabilized variants of a trimeric coiled-coil domain that display potent inhibitory activity against HIV-1 membrane fusion. We will use isoleucine- and valine-scanning mutagenesis to identify and incorporate specific residue substitutions that increase both trimer stability and antiviral potency. We will also construct chimeric N-peptides by using an isoleucine-zipper sequence, to stabilize the coiled-coil structure. Our emphasis is to generate potent N-peptide inhibitors suitable for development as an inexpensive component of a microbicide formulation. (2) To characterize the specificity, potency, and toxicity of improved N-peptide variants and their in vitro synergistic interactions with the virus-cell attachment inhibitors CMPD167 and BMS-378806, and the C-peptide fusion inhibitor C52L. We will conduct in vitro studies to determine inhibitory activity of select N-peptide variants against diverse primary HIV-1 isolates, and their toxic or inflammatory effects using the rabbit vaginal irritation model and in human cells. We will also study synergistic antiviral effects in vitro in order to make rational predictions for lead inhibitor combinations for formulation and efficacy testing in rhesus macaques. (3) To use the rhesus macaque high dose vaginal transmission model to assess the in vivo potency of an optimized N-peptide inhibitor alone and in combination with CMPD167, BMS- 378806, and C52L. We will evaluate the protection of macaques from vaginal challenge with both CCR5 and CXCR4 SHIVs by a vaginally delivered N-peptide inhibitor alone and in synergistic combination with CMPD167, BMS-378806, and C52L. Public Health Relevance: This Public Health Relevance is not available. Thesaurus Terms: There are no thesaurus terms on file for this project. Institution: WEILL MEDICAL COLLEGE OF CORNELL UNIV 1300 YORK AVENUE NEW YORK, NY 10065 Fiscal Year: 2008 Department: BIOCHEMISTRY Project Start: 15-SEP-2006 Project End: 31-AUG-2009 ICD: NATIONAL INSTITUTE OF ALLERGY AND INFECTIOUS DISEASES IRG: ZAI1 Grant Number: 5R21AI074469-02 Project Title: Stabilized gp41 Structural Motif for HIV-1 Vaccine PI Information: Name Email Title LU, MIN mlu@med.cornell.edu ASSOCIATE PROFESSOR Abstract: DESCRIPTION (provided by applicant): The development of immunogens capable of eliciting broadly neutralizing antibodies to HIV-1 remains one of the most difficult challenges confronting biomedical research today. Nonetheless, a small number of human monoclonal antibodies (mAbs) have been isolated from HIV-1-infected individuals that can neutralize primary isolates of HIV-1 from different genetic subtypes. Of these rare mAbs, two (2F5 and 4E10) recognize epitopes on the membrane-proximal region of the gp41 ectodomain. However, all attempts to elicit comparable anti-gp41 antibodies by vaccination with peptides or simple linear epitopes have thus far failed. Our approach to immunogen design is guided by our recent identification and structural determination of a novel two-stranded coiled-coil domain (C55) that includes the membrane-proximal epitope region of gp41. However, we have determined that the C55 dimer structure is too unstable to be suitable for immunogenicity studies. The overall goal of this research plan is to gain a detailed understanding of the structural and thermodynamic properties of the C55 coiled-coil domain, and to use this knowledge to design and produce stable immunogens for the elicitation of broadly neutralizing HIV-1 antibodies. Our central hypothesis is that presentation of the highly conserved sequences recognized by 2F5 and 4E10 in this new structural state opens a promising avenue for the discovery and development of novel immunogens to induce broadly reactive neutralizing antibodies. Specific aims of this research are: (1) To identify and develop stabilized versions of the C55 coiled-coil domain encompassing the 2F5 and 4E10 epitopes. We will identify and incorporate apolar side chain substitutions into the buried hydrophobic core of the dimer to stabilize the coiled-coil structure. We will also screen cysteine substitutions to introduce a disulfide bond between the two interacting helices. Our emphasis is to generate stable molecules that preserve both the dimer conformation and the critical surface-exposed residues of the membrane-proximal region. (2) To evaluate the immunological responses elicited by stabilized C55 coiled-coil variants in small animals. We will conduct immunogenicity studies in rabbits and guinea-pigs to determine whether the stabilized dimer structure can elicit neutralizing antibodies. These studies will involve immunization of the animals using soluble proteins. We will also evaluate the immunogenicity of the stable coiled-coil molecules captured onto nanometer-sized beads as particulate immunogens. Public Health Relevance: This Public Health Relevance is not available. Thesaurus Terms: antigen, dimer, vaccine Gastropoda, active immunization, antibody, conformation, cysteine, disulfide bond, genetics, guinea pig, human, immunization, membrane, monoclonal antibody, neutralizing antibody, peptide, protein Institution: WEILL MEDICAL COLLEGE OF CORNELL UNIV 1300 YORK AVENUE NEW YORK, NY 10065 Fiscal Year: 2008 Department: BIOCHEMISTRY Project Start: 01-MAY-2007 Project End: 30-APR-2010 ICD: NATIONAL INSTITUTE OF ALLERGY AND INFECTIOUS DISEASES IRG: VACC Grant Number: 5R21AI078466-02 Project Title: Presentation of Structural Determinants of the 2F5 Neutralization Epitope PI Information: Name Email Title LU, MIN mlu@med.cornell.edu ASSOCIATE PROFESSOR Abstract: DESCRIPTION (provided by applicant): Development of envelope protein immunogens capable of eliciting neutralizing antibodies against primary HIV-1 isolates from different genetic subtypes remains the most difficult challenge in the field of HIV-1 vaccinology. The conserved membrane-proximal external region of the gp41 ectodomain bears the epitopes of two broadly neutralizing human monoclonal antibodies, 2F5 and 4E10, and is therefore an important target of HIV-1 vaccine design. However, all attempts to elicit anti-gp41 antibodies comparable to these produced in infected humans by using unstructured peptide immunogens have thus far failed. Success will likely require a deeper understanding of the structural motifs of the gp120/gp41 complex that stabilize the mature envelope trimer on the virion surface. Current thinking postulates that the receptor-mediated activation of gp41 involves a regulated sequence of structural transitions with one or more on-pathway intermediate(s) that may reveal cryptic neutralization epitopes. Our recent identification of a four-stranded coiled-coil structure (C43) encompassing the 2F5 epitope suggests that structural aspects of the 2F5 and 4E10 epitopes may be transiently exposed and required for stimulating neutralizing antibody responses. The broad, long-term objective of this research plan is to use protein chemistry and structural biology approaches to engineer and produce stable forms of the C43 coiled-coil motif for the induction of broadly reactive neutralizing antibodies. The premise of the proposed research is that definition of the structural requirements for optimal presentation of the 2F5 neutralization epitope can lead to the identification of stable immunogen products to generate effective anti-HIV-1 immunity. The Specific Aims of the proposed research are: (1) To design and develop stabilized versions of the C43 coiled-coil domain bearing the 2F5 epitope for immunogenicity studies. We will generate a single-chain analog of the tetraplex coiled coil in which the four mutagenesis to identify specific residue substitutions that favorably influence inter-helical packing interactions in the tetramer in order to stabilize an ordered helical peptide structure of the 2F5 epitope that preserves surface-exposed side chains. (2) To evaluate the immunological responses elicited by stabilized C43 coiled-coil variants in small animals. We will conduct immunogenicity studies in rabbits and guinea-pigs to determine whether the stabilized involve immunization of the animals using the stabilized C43 peptide proteins. We will also evaluate the immunogenicity of the stable coiled-coil molecules captured onto nanometer-sized beads as particulate immunogens. The research and public health communities concur that a preventive vaccine is the obvious long-term solution to bring the global HIV-1 epidemic under control (1-8). Unfortunately, this goal has proven elusive and no such vaccine is available. Overcoming this important biomedical problem will require new and imaginative design strategies to bring us closer to the goal of a successful AIDS vaccine. Public Health Relevance: This Public Health Relevance is not available. Thesaurus Terms: There are no thesaurus terms on file for this project. Institution: WEILL MEDICAL COLLEGE OF CORNELL UNIV 1300 YORK AVENUE NEW YORK, NY 10065 Fiscal Year: 2009 Department: BIOCHEMISTRY Project Start: 15-APR-2008 Project End: 31-MAR-2010 ICD: NATIONAL INSTITUTE OF ALLERGY AND INFECTIOUS DISEASES IRG: ZAI1
| ||||||||||||||||||||||||||||||||
| Changed: | ||||||||||||||||||||||||||||||||
| < < |
| |||||||||||||||||||||||||||||||
| > > |
| |||||||||||||||||||||||||||||||
| ||||||||||||||||||||||||||||||||
| Changed: | ||||||||||||||||||||||||||||||||
| < < |
| |||||||||||||||||||||||||||||||
| > > |
| |||||||||||||||||||||||||||||||
| ||||||||||||||||||||||||||||||||
Revision 302 Apr 2009 - Main.SherryllJones
| ||||||||||||||||||||||||||||||||||||||||||||||||
| Deleted: | ||||||||||||||||||||||||||||||||||||||||||||||||
| < < | ||||||||||||||||||||||||||||||||||||||||||||||||
My Links
Personal PreferencesUncomment preferences variables to activate them (remove the #-sign). Help and details on preferences variables are available in TWikiPreferences.
Related Topics
| ||||||||||||||||||||||||||||||||||||||||||||||||
| Added: | ||||||||||||||||||||||||||||||||||||||||||||||||
| > > | Public Information on Grants associated with NYSBC | |||||||||||||||||||||||||||||||||||||||||||||||
| Added: | ||||||||||||||||||||||||||||||||||||||||||||||||
| > > | Grant Number: 5R01AI042382-09 Project Title: Structure and Function of the HIV-1 Envelope Protein PI Information: Name Email Title LU, MIN mlu@med.cornell.edu ASSOCIATE PROFESSOR | |||||||||||||||||||||||||||||||||||||||||||||||
| Added: | ||||||||||||||||||||||||||||||||||||||||||||||||
| > > | Abstract: DESCRIPTION (provided by applicant): HIV-1 entry into cells is mediated by the envelope glycoprotein (Env) complex (gp120/gp41). Binding of gp120 to receptors on the target cell triggers a series of conformational changes in the native Env trimer that ultimately lead to formation of a six-helix bundle structure in gp41, the so-called fusogenic state of the protein. In this state, interactions between two helical regions (HR1 and HR2) in the gp41 ectodomain are coupled to membrane fusion. HR2-derived peptides, such as Fuzeon (T20), are thought to inhibit membrane fusion by preventing the HR regions from refolding into the six-helix bundle. Despite intensive efforts, high-resolution structural information about the native, trimeric Env complex is lacking. Recognizing this history, we propose to apply new protein dissection approaches to study the pre-fusion conformation(s) of gp41. The broad, longterm objective of this proposal is to define the structural transitions of gp41 by which the HR regions, initially sequestered in the native Env complex, are released and refolded in order to cause membrane fusion. The specific aims of the proposal are: (1) To determine the effects of the fusion inhibitor T20-resistant/dependent mutations in the HR regions on the equilibrium folding, thermodynamics, and conformation of the six-helix bundle, in order to understand the mechanism of action of T20 and how its antiviral effect is mitigated by the evolution of resistant viruses in vivo. (2) To characterize, at high resolution, the structural and dynamic properties of the conserved membraneproximal Trp-rich domain of gp41 in association with [3OG micelles and lipid vesicles, in order to provide structural insight into the mechanism of HIV-1 neutralization by mAbs 2F5 and 4E10. (3) To crystallize and determine the structure of a novel trimerization domain formed by the HR2 and Trp-rich membrane-proximal regions, and to utilize engineered envelope glycoproteins to define the roles of this trimeric structural domain in regulating the stabilization and controlled activation of the native Env complex. We wish to elucidate a mechanism for the precise switch between the metastable native and fusogenic forms of the protein.
Public Health Relevance:
This Public Health Relevance is not available.
Thesaurus Terms:
HIV envelope protein gp41, human immunodeficiency virus 1, protein structure function, virus infection mechanism
conformation, gene mutation, neutralizing antibody
X ray crystallography, circular dichroism, tissue /cell culture
Institution: WEILL MEDICAL COLLEGE OF CORNELL UNIV
1300 YORK AVENUE
NEW YORK, NY 10065
Fiscal Year: 2008
Department: BIOCHEMISTRY
Project Start: 01-SEP-1999
Project End: 31-MAY-2009
ICD: NATIONAL INSTITUTE OF ALLERGY AND INFECTIOUS DISEASES
IRG: AMCB
Grant Number: 5R01AI068591-04 Project Title: Engineered gp41 Peptides as HIV-1 Topical Microbicides PI Information: Name Email Title LU, MIN mlu@med.cornell.edu ASSOCIATE PROFESSOR Abstract: This abstract is not available. Public Health Relevance: This Public Health Relevance is not available. Thesaurus Terms: Macaca, cell, infection, local antiinfective agent, model, peptide, topical drug application antiinfective agent, cell line, concept, gene mutation, glutamine, human, human immunodeficiency virus, immunodeficiency, irritation /irritant, lead, leucine, ligand, recombinant protein, recombinant virus, simian immunodeficiency virus, small molecule, solubility, tissue, vaccine, virus Institution: WEILL MEDICAL COLLEGE OF CORNELL UNIV 1300 YORK AVENUE NEW YORK, NY 10065 Fiscal Year: 2009 Department: BIOCHEMISTRY Project Start: 01-MAR-2006 Project End: 28-FEB-2010 ICD: NATIONAL INSTITUTE OF ALLERGY AND INFECTIOUS DISEASES IRG: ADDT Grant Number: 3R21AI071979-02S1 Project Title: Development of N-Peptides for Use in HIV-1 Topical Microbicides PI Information: Name Email Title LU, MIN ASSOCIATE PROFESSOR Abstract: DESCRIPTION (provided by applicant): HIV-1 continues to spread globally and no vaccine is available. Hence there is an urgent public health need for an effective microbicide to prevent sexual transmission of HIV-1. The topical application of potent combinations of viral fusion and entry inhibitors has been shown to exhibit microbicidal efficacy in the rhesus macaque vaginal transmission model. Peptides derived from the N- and C-terminal regions of the gp41 ectodomain (called N- and C-peptides, respectively) inhibit HIV-1 entry. N-peptides have generally proven far less potent than C-peptides. However, stabilization of a trimeric coiled-coil conformation of N peptides has been demonstrated to be a viable strategy to develop them as a new class of potent HIV-1 fusion inhibitors. We have recently identified and determined the crystal structure of an autonomously folded, N-peptide coiled-coil domain. The overall goal of this research plan is to gain a detailed understanding of the structural and thermodynamic properties of this novel coiled-coil domain, and to use this knowledge to design and produce a bacterially expressed N-peptide fusion inhibitor for inclusion in a HIV-1 topical microbicide. Our central hypothesis is that the synergistic inhibition of different stages of the viral fusion and entry process can offer a powerful benefit in formulating an efficacious and more economic microbicide product. Specific aims of this research are: (1) to use modern protein engineering methods to identify and develop stabilized variants of a trimeric coiled-coil domain that display potent inhibitory activity against HIV-1 membrane fusion. We will use isoleucine- and valine-scanning mutagenesis to identify and incorporate specific residue substitutions that increase both trimer stability and antiviral potency. We will also construct chimeric N-peptides by using an isoleucine-zipper sequence, to stabilize the coiled-coil structure. Our emphasis is to generate potent N-peptide inhibitors suitable for development as an inexpensive component of a microbicide formulation. (2) To characterize the specificity, potency, and toxicity of improved N-peptide variants and their in vitro synergistic interactions with the virus-cell attachment inhibitors CMPD167 and BMS-378806, and the C-peptide fusion inhibitor C52L. We will conduct in vitro studies to determine inhibitory activity of select N-peptide variants against diverse primary HIV-1 isolates, and their toxic or inflammatory effects using the rabbit vaginal irritation model and in human cells. We will also study synergistic antiviral effects in vitro in order to make rational predictions for lead inhibitor combinations for formulation and efficacy testing in rhesus macaques. (3) To use the rhesus macaque high dose vaginal transmission model to assess the in vivo potency of an optimized N-peptide inhibitor alone and in combination with CMPD167, BMS- 378806, and C52L. We will evaluate the protection of macaques from vaginal challenge with both CCR5 and CXCR4 SHIVs by a vaginally delivered N-peptide inhibitor alone and in synergistic combination with CMPD167, BMS-378806, and C52L. Public Health Relevance: This Public Health Relevance is not available. Thesaurus Terms: There are no thesaurus terms on file for this project. Institution: WEILL MEDICAL COLLEGE OF CORNELL UNIV 1300 YORK AVENUE NEW YORK, NY 10065 Fiscal Year: 2008 Department: BIOCHEMISTRY Project Start: 15-SEP-2006 Project End: 31-AUG-2009 ICD: NATIONAL INSTITUTE OF ALLERGY AND INFECTIOUS DISEASES IRG: ZAI1 Grant Number: 5R21AI074469-02 Project Title: Stabilized gp41 Structural Motif for HIV-1 Vaccine PI Information: Name Email Title LU, MIN mlu@med.cornell.edu ASSOCIATE PROFESSOR Abstract: DESCRIPTION (provided by applicant): The development of immunogens capable of eliciting broadly neutralizing antibodies to HIV-1 remains one of the most difficult challenges confronting biomedical research today. Nonetheless, a small number of human monoclonal antibodies (mAbs) have been isolated from HIV-1-infected individuals that can neutralize primary isolates of HIV-1 from different genetic subtypes. Of these rare mAbs, two (2F5 and 4E10) recognize epitopes on the membrane-proximal region of the gp41 ectodomain. However, all attempts to elicit comparable anti-gp41 antibodies by vaccination with peptides or simple linear epitopes have thus far failed. Our approach to immunogen design is guided by our recent identification and structural determination of a novel two-stranded coiled-coil domain (C55) that includes the membrane-proximal epitope region of gp41. However, we have determined that the C55 dimer structure is too unstable to be suitable for immunogenicity studies. The overall goal of this research plan is to gain a detailed understanding of the structural and thermodynamic properties of the C55 coiled-coil domain, and to use this knowledge to design and produce stable immunogens for the elicitation of broadly neutralizing HIV-1 antibodies. Our central hypothesis is that presentation of the highly conserved sequences recognized by 2F5 and 4E10 in this new structural state opens a promising avenue for the discovery and development of novel immunogens to induce broadly reactive neutralizing antibodies. Specific aims of this research are: (1) To identify and develop stabilized versions of the C55 coiled-coil domain encompassing the 2F5 and 4E10 epitopes. We will identify and incorporate apolar side chain substitutions into the buried hydrophobic core of the dimer to stabilize the coiled-coil structure. We will also screen cysteine substitutions to introduce a disulfide bond between the two interacting helices. Our emphasis is to generate stable molecules that preserve both the dimer conformation and the critical surface-exposed residues of the membrane-proximal region. (2) To evaluate the immunological responses elicited by stabilized C55 coiled-coil variants in small animals. We will conduct immunogenicity studies in rabbits and guinea-pigs to determine whether the stabilized dimer structure can elicit neutralizing antibodies. These studies will involve immunization of the animals using soluble proteins. We will also evaluate the immunogenicity of the stable coiled-coil molecules captured onto nanometer-sized beads as particulate immunogens. Public Health Relevance: This Public Health Relevance is not available. Thesaurus Terms: antigen, dimer, vaccine Gastropoda, active immunization, antibody, conformation, cysteine, disulfide bond, genetics, guinea pig, human, immunization, membrane, monoclonal antibody, neutralizing antibody, peptide, protein Institution: WEILL MEDICAL COLLEGE OF CORNELL UNIV 1300 YORK AVENUE NEW YORK, NY 10065 Fiscal Year: 2008 Department: BIOCHEMISTRY Project Start: 01-MAY-2007 Project End: 30-APR-2010 ICD: NATIONAL INSTITUTE OF ALLERGY AND INFECTIOUS DISEASES IRG: VACC Grant Number: 5R21AI078466-02 Project Title: Presentation of Structural Determinants of the 2F5 Neutralization Epitope PI Information: Name Email Title LU, MIN mlu@med.cornell.edu ASSOCIATE PROFESSOR Abstract: DESCRIPTION (provided by applicant): Development of envelope protein immunogens capable of eliciting neutralizing antibodies against primary HIV-1 isolates from different genetic subtypes remains the most difficult challenge in the field of HIV-1 vaccinology. The conserved membrane-proximal external region of the gp41 ectodomain bears the epitopes of two broadly neutralizing human monoclonal antibodies, 2F5 and 4E10, and is therefore an important target of HIV-1 vaccine design. However, all attempts to elicit anti-gp41 antibodies comparable to these produced in infected humans by using unstructured peptide immunogens have thus far failed. Success will likely require a deeper understanding of the structural motifs of the gp120/gp41 complex that stabilize the mature envelope trimer on the virion surface. Current thinking postulates that the receptor-mediated activation of gp41 involves a regulated sequence of structural transitions with one or more on-pathway intermediate(s) that may reveal cryptic neutralization epitopes. Our recent identification of a four-stranded coiled-coil structure (C43) encompassing the 2F5 epitope suggests that structural aspects of the 2F5 and 4E10 epitopes may be transiently exposed and required for stimulating neutralizing antibody responses. The broad, long-term objective of this research plan is to use protein chemistry and structural biology approaches to engineer and produce stable forms of the C43 coiled-coil motif for the induction of broadly reactive neutralizing antibodies. The premise of the proposed research is that definition of the structural requirements for optimal presentation of the 2F5 neutralization epitope can lead to the identification of stable immunogen products to generate effective anti-HIV-1 immunity. The Specific Aims of the proposed research are: (1) To design and develop stabilized versions of the C43 coiled-coil domain bearing the 2F5 epitope for immunogenicity studies. We will generate a single-chain analog of the tetraplex coiled coil in which the four mutagenesis to identify specific residue substitutions that favorably influence inter-helical packing interactions in the tetramer in order to stabilize an ordered helical peptide structure of the 2F5 epitope that preserves surface-exposed side chains. (2) To evaluate the immunological responses elicited by stabilized C43 coiled-coil variants in small animals. We will conduct immunogenicity studies in rabbits and guinea-pigs to determine whether the stabilized involve immunization of the animals using the stabilized C43 peptide proteins. We will also evaluate the immunogenicity of the stable coiled-coil molecules captured onto nanometer-sized beads as particulate immunogens. The research and public health communities concur that a preventive vaccine is the obvious long-term solution to bring the global HIV-1 epidemic under control (1-8). Unfortunately, this goal has proven elusive and no such vaccine is available. Overcoming this important biomedical problem will require new and imaginative design strategies to bring us closer to the goal of a successful AIDS vaccine. Public Health Relevance: This Public Health Relevance is not available. Thesaurus Terms: There are no thesaurus terms on file for this project. Institution: WEILL MEDICAL COLLEGE OF CORNELL UNIV 1300 YORK AVENUE NEW YORK, NY 10065 Fiscal Year: 2009 Department: BIOCHEMISTRY Project Start: 15-APR-2008 Project End: 31-MAR-2010 ICD: NATIONAL INSTITUTE OF ALLERGY AND INFECTIOUS DISEASES IRG: ZAI1 | |||||||||||||||||||||||||||||||||||||||||||||||
| ||||||||||||||||||||||||||||||||||||||||||||||||
| Added: | ||||||||||||||||||||||||||||||||||||||||||||||||
| > > |
| |||||||||||||||||||||||||||||||||||||||||||||||
| ||||||||||||||||||||||||||||||||||||||||||||||||
Revision 201 Apr 2008 - Main.DavidCowburn
| ||||||||||||||||||
| Deleted: | ||||||||||||||||||
| < < | ||||||||||||||||||
My Links
Personal PreferencesUncomment preferences variables to activate them (remove the #-sign). Help and details on preferences variables are available in TWikiPreferences.
Related Topics
| ||||||||||||||||||
| Changed: | ||||||||||||||||||
| < < |
| |||||||||||||||||
| > > |
| |||||||||||||||||
| ||||||||||||||||||
| Changed: | ||||||||||||||||||
| < < |
| |||||||||||||||||
| > > |
| |||||||||||||||||
| ||||||||||||||||||
| Changed: | ||||||||||||||||||
| < < |
| |||||||||||||||||
| > > |
| |||||||||||||||||
| ||||||||||||||||||
| Changed: | ||||||||||||||||||
| < < |
| |||||||||||||||||
| > > |
| |||||||||||||||||
| Deleted: | ||||||||||||||||||
| < < |
| |||||||||||||||||
| ||||||||||||||||||
Revision 101 Apr 2008 - Main.TWikiRegistrationAgent
My Links
Personal PreferencesUncomment preferences variables to activate them (remove the #-sign). Help and details on preferences variables are available in TWikiPreferences.
Related Topics
| |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
View topic
| History: r5 < r4 < r3 < r2
| More topic actions...
Copyright © by the contributing authors. All material on this collaboration platform is the property of the contributing authors.
Ideas, requests, problems regarding this intranet, Send feedback
Ideas, requests, problems regarding this intranet, Send feedback