Philippe Andre Gallay
Scripps Research Institute
Project start date: 2010-01-15
Project end date: 2014-12-31
Sponsored Links Excellgen http://Excellgen.com
Grants awarded to Philippe Andre Gallay
C5A AS AN ANTI-HIV MICROBICIDAL CANDIDATE
Philippe Andre Gallay, Associate Professor
Scripps Research Institute, La Jolla, Ca 92037-1000
Grant 1R01AI087470-01A1 from National Institute Of Allergy And Infectious Diseases
Abstract: We identified a short peptide SWLRDIWDWICEVLSDFK called C5A, which represents a novel class of microbicidal candidates. C5A neutralizes HIV at an nM-M range without apparent cytotoxicity to human cells. C5A corresponds to a small (18 amino acids) N-terminal region (aa 3-20) of the hepatitis C virus (HCV) nonstructural protein 5A (NS5A) (477 amino acids). The sequence of C5A encompasses the region responsible for the anchoring of NS5A into the ER membrane. Importantly, in contrast to C5A (18 aa), full length NS5A (477 aa) does not inhibit HIV infection. We demonstrated that C5A disrupts the HIV membrane, but preserves the integrity of the cellular plasma membrane. The HIV membrane rupture is specific because C5A does not disturb the integrity of the plasma membrane of human cells even when used at high doses and because it does not inhibit the infection of other enveloped viruses such as influenza and vesicular stomatitis viruses. C5A possesses multiple attractive microbicidal properties it i) blocks HIV infection of primary targets including T cells, macrophages and dendritic cells; ii) exhibits a broad range of antiviral activity against primary HIV isolates, multi-drug resistant HIV isolates, SIV and SHIV; iii) interrupts an ongoing T cell infection; iv) prevents transmigration of HIV through primary human genital epithelial cells; v) blocks infection of dendritic and Langerhans cells ex vivo (skin tissues); vi) prevents HIV transfer from dendritic and Langerhans cells to T cells ex vivo; vii) is extremely efficacious since less than 15 min of exposure suffices for C5A to neutralize HIV; viii) is potent for a considerable length of time both prior to (at least 1 h) and after (at least 1 h) addition of HIV to cells; ix) is potent at a low pH; x) is soluble in water at inhibitory concentrations; xi) is not toxic to commensal Lactobacilli present in the vaginal tract; xii) exhibits minimal adverse changes, inflammation and toxicity in cervicovaginal tissue in vivo; xiii) is not immunogenic; xiv) does not affect cellular signaling pathways; xv) apparently does not allow viral development resistance; xvi) efficiently blocks HIV infectivity when diluted in genital fluids; and most importantly xvii) vaginal application of C5A offers complete protection against a vaginal viral challenge in the humanized BLT mouse HIV transmission model. Thus, C5A represents the prototype of a new generation of microbicidal agents that may have promise for HIV prevention. In this application, we would like to follow up on these exciting data by fully exploring the possibility that C5A represents a true microbicidal candidate. In the first aim of this application, we propose to conduct a series of experiments aimed at identifying the component of the viral membrane to which C5A binds because the C5A ligand, which resides in the membrane of HIV, represents a potential target for the development of a novel class of anti-HIV therapies with an unusual mechanism of antiviral action. Interestingly, we obtained several lines of evidence that the sphingolipid called dihydrosphingomyelin (DHSM) represents the main target of C5A in the HIV membrane i) DHSM, incorporated into HIV particles, is specifically pulled down by C5A beads; ii) C5A binds directly to adsorbed DHSM; iii) C5A ruptures liposomes constituted with DHSM; and most importantly iv) pre-incubation of C5A with soluble DHSM prevents HIV rupture by C5A and preserves HIV infectivity. The amphipathic property of C5A, the identity of DHSM as the C5A target in the HIV membrane, and the specific C5A rupture of DHSM-containing liposomes or HIV particles, provide the first hint for the antiviral mechanism of C5A action C5A, which encompasses the N-terminal region responsible for the anchoring of NS5A into the ER membrane, by binding to DHSM enriched within the HIV membrane, disturbs the integrity of the viral membrane due to its amphipathic nature. In the second aim of this application, we propose to optimize the in vitro potency and in vivo safety of C5A by creating a second generation of peptides using the parental C5A peptide as the archetype. All newly synthesized peptides will be tested in genital fluids for their in vitro microbicidal properties. The most potent compounds among the newly synthesized peptides will be selected. Remarkably, we found that acetylation, amidation and glycosylation of C5A greatly enhanced C5A anti-HIV activities in genital fluids. This is the proof-of-concept for the feasibility of identifying C5A derivates with enhanced anti-HIV activities. In the third aim, the most potent C5A derivates will be assessed for safety and efficacy in the HIV vaginal transmission BLT mouse model. Kinetic administration studies will be executed to determine how long before and/or after the viral challenge C5A precludes HIV transmission. In the fourth aim, safety and efficacy pilot studies will be conducted in another SIV/HIV vaginal transmission animal model the progesterone-treated macaque model. If similar protective results were obtained using the two transmission models, it would further validate the use of these models for the screening of microbicidal candidates. In addition, protective results would provide proof-of-concept of the usefulness of topically applied microbicides, such as C5A, to prevent genital HIV transmission. The goal of this project is to conduct in vitro and in vivo studies designed to support the preclinical development of short peptides as novel anti-HIV microbicides. There is an urgent need for the development of safe, effective topical microbicides to prevent the sexual transmission of HIV and to allow the female partner to take personal responsibility for use and application. The development of a safe, effective acceptable topical microbicide to prevent the sexual transmission of HIV could play a major role in worldwide reduction of the over 14,000 new HIV infections per day, and potentially save millions of lives
Keywords: AIDS Virus; AIDS prevention; AIDS/HIV prevention; Acetylation; Acquired Immune Deficiency Syndrome Virus; Acquired Immunodeficiency Syndrome Virus; Active Follow-up; Address; Affect; Amino Acids; Animal Model; Animal Models and Related Studies; Anti-HIV Therapy; Antiviral Agents; Antiviral Drugs; Antivirals; Binding; Binding (Molecular Function); Body Tissues; Bone Marrow; C5 a; C5a; Cell membrane; Cells; Complement 5a; Complement C5a; Corpus Luteum Hormone; Cytoplasmic Membrane; DISSEC; Data; Delta4-pregnene-3, 20-dione; Dendritic Cells; Development; Dissection; Dose; Drug Administration, Topical; Drug Resistance, Multiple; Drug Resistant, Multiple; Epithelial Cells; Exhibits; Female; Generations; Genital; Genital System, Female, Vagina; Genital system; Goals; Grant; Grippe; HCV; HIV; HIV Infections; HIV Prevention; HIV/AIDS prevention; HTLV-III; HTLV-III Infections; HTLV-III-LAV Infections; Hepatitis C virus; Hepatitus C; Human; Human Immunodeficiency Viruses; Human T-Cell Leukemia Virus Type III; Human T-Cell Lymphotropic Virus Type III; Human T-Lymphotropic Virus Type III; Human, General; Hydrogen Oxide; IC50; INFLM; In Vitro; Infection; Inflammation; Influenza; Inhibitory Concentration 50; Kinetic; Kinetics; LAV-HTLV-III; Lactobacillus; Langerhans cell; Length; Life; Ligands; Liposomal; Liposomes; Liquid substance; Liver; Local Microbicides; Lymphadenopathy-Associated Virus; Lytotoxicity; Macaca; Macaque; Mammals, Mice; Man (Taxonomy); Man, Modern; Manuscripts; Membrane; Metabolic Glycosylation; Mice; Microbicides, Local; Modeling; Molecular Interaction; Multi-Drug Resistance; Multidrug Resistance; Murine; Mus; N-terminal; NH2-terminal; Nature; Nonstructural Protein; Peptides; Pilot Projects; Plasma Membrane; Play; Pregn-4-ene-3, 20-dione; Pregnenedione; Preparation; Progesterone; Property; Property, LOINC Axis 2; Publishing; Recombinant C5a; Research Design; Resistance development; Resistance to Multi-drug; Resistance to Multidrug; Resistance to Multiple Drug; Resistant development; Resistant to Multiple Drug; Resistant to multi-drug; Resistant to multidrug; Reticuloendothelial System, Bone Marrow; Reticuloendothelial System, Thymus; Role; Rupture; SHIV; SIV; Safety; Screening procedure; Series; Sexual Transmission; Signal Pathway; Simian Immunodeficiency Viruses; Skin Tissue; Sphingolipids; Study Section; Study Type; T-Cells; T-Lymphocyte; T-Lymphotropic Virus Type III Infections, Human; Testing; Therapeutic Progesterone; Thymus; Thymus Gland; Thymus Proper; Thymus-Dependent Lymphocytes; Time; Tissues; Titrations; Topical application; Toxic effect; Toxicities; Transmission; VSV; Vagina; Vaginal; Veiled Cells; Vesicular Stomatitis Virus; Vesicular stomatitis Indiana virus; Viral; Virus; Virus-HIV; Viruses, General; Water; Work; amidation; aminoacid; anti-HIV microbicide; body system, hepatic; comparative efficacy; cytotoxicity; developing resistance; experiment; experimental research; experimental study; flu infection; fluid; follow-up; glycosylation; immunogenic; improved; in vivo; in vivo Model; influenza infection; liquid; macrophage; membrane structure; microbicidal; microbicide; model organism; mouse model; multi-drug resistant; multidrug resistant; mutant; novel; organ system, hepatic; particle; pilot study; plasmalemma; pre-clinical; preclinical; preclinical evaluation; prevent; preventing; prototype; public health relevance; research study; screening; screenings; simian HIV; simian human immunodeficiency virus; social role; study design; tandem mass spectrometry; therapy, AIDS anti-HIV; thymus derived lymphocyte; topical administration; topical drug application; topical microbicide; topically applied; transmission process; urogenital system (genital part); vaginal transmission
Relevance: The goal of this project is to conduct in vitro and in vivo studies designed to support the preclinical development of short peptides as novel anti-HIV microbicides. There is an urgent need for the development of safe, effective topical microbicides to prevent the sexual transmission of HIV and to allow the female partner to take personal responsibility for use and application. The development of a safe, effective acceptable topical microbicide to prevent the sexual transmission of HIV could play a major role in worldwide reduction of the over 14,000 new HIV infections per day, and potentially save millions of lives
Project start date: 2010-06-01
Project end date: 2015-05-31
Budget start date: 1-JUN-2010
Budget end date: 31-MAY-2011
PFA/PA: PA-07-070
1R01AI087470-01A1 (2010): $474750
VIROCIDE AS AN ANTI-HIV MICROBICIDE CANDIDATE
Philippe Andre Gallay, Associate Professor
Scripps Research Institute, La Jolla, Ca 92037-1000
Grant 3R21AI078791-02S1 from National Institute Of Allergy And Infectious Diseases
Abstract: Topical microbicides are defined as vaginally applied products that prevent male-to-female or female-to-male HIV transmission. The high error rate of the HIV reverse transcriptase (RT) drives the development of resistance and genomic diversity in HIV. The current pan-resistance to all classes of HIV inhibitors, such as those found in O group virus for non-nucleoside RT and protease (PR) inhibitors, raises an important question with regard to the development of microbicides targeting RT or PR. These concerns may be extended to inhibitors of HIV entry due to rapid emergence of mutations in the envelope (Env) glycoprotein under selective pressure. There is thus an urgent need to identify new anti-HIV compounds, which target viral components other than PR, RT and Env, and which may be developed as safe and effective microbicides. We identified a short linear peptide called Virocide, which neutralizes HIV at an nM range. Several lines of evidence s cells even when applied twice daily to cells at a concentration 20-200-fold superior to that which blocks HIV infection; iii) Virocide does not harm mice injected i.v. twice daily with high peptide concentrations (0.5 mg) for a period of 3 days; and iv) Virocide apparently does not create lesions in the vaginal epithelium of humanized mice since the peptide, rather than promoting transmission, completely blocks intravaginal transmission. Virocide represents an attractive microbicide candidate for several reasons i) Virocide inhibits infection of a broad range of primary isolates in various primary human cell types; ii) it interferes with the three mechanisms involved in HIV transmission genital epithelial transmigration, dendritic cell-mediated transmission, and infection of mucosal target cells; iii) Virocide is extremely efficacious since less than 15 min of exposure suffices to neutralize HIV; iv) it is potent for two hours both prior to and after addition of HIV to cells, suggze the anti-HIV efficacy of Virocide in vitro as well as its toxicity and immunogenicity in vivo. If these studies unambiguously demonstrate that Virocide represents an attractive anti-HIV microbicide candidate, we will be in a position to test its efficacy as a topical microbicide in vivo
Keywords: AIDS Virus; Acquired Immune Deficiency Syndrome Virus; Acquired Immunodeficiency Syndrome Virus; Administration, Intravaginal; Administration, Vaginal; Amino Acids; Antiproteases; Antiviral Agents; Antiviral Drugs; Antivirals; Assay; Associated Viruses; Binding; Binding (Molecular Function); Bioassay; Biologic Assays; Biological Assay; Cell Growth in Number; Cell Multiplication; Cell Proliferation; Cell membrane; Cells; Cellular Membrane; Cellular Proliferation; Chimp; Chimpanzee; Coitus; Cytokines, Chemotactic; Cytoplasmic Membrane; Dendritic Cells; Deoxynucleotide-triphosphate[{..}]DNA deoxynucleotidyltransferase (RNA-directed); Development; Dose; Douching; Drug resistance; EC 2.7.7.49; Endopeptidase Inhibitors; Entry Inhibitors, HIV; Environment; Epithelial; Epithelium; Esteroproteases; Exhibits; Female; Figs; Figs - dietary; Generations; Genetic Alteration; Genetic Change; Genetic defect; Genital; Genital System, Female, Vagina; Genital system; Genomics; Glycoproteins; Goals; HCV; HIV; HIV Entry Inhibitors; HIV Infections; HIV-1; HIV-2; HIV-I; HIV-II; HIV1; HIV2; HSV; HTLV-III; HTLV-III Infections; HTLV-III-LAV Infections; HTLV-IV; Hepatitis C virus; Hepatitus C; Herpes Simplex; Herpes Simplex Infections; Herpes Simplex Virus; Herpes labialis Virus; Herpes simplex disease; Herpesvirus hominis; Herpesvirus hominis disease; Homologous Chemotactic Cytokines; Hour; Human; Human Immunodeficiency Viruses; Human Sexual Intercourse; Human T-Cell Leukemia Virus Type III; Human T-Cell Lymphotropic Virus Type III; Human T-Lymphotropic Virus Type III; Human T-Lymphotropic Virus Type IV; Human immunodeficiency virus 1; Human immunodeficiency virus 2; Human, General; IFN; INFLM; Immune; Immune response; Immunodeficiency Virus Type 1, Human; Immunodeficiency Virus Type 2, Human; In Vitro; Infection; Inflammation; Inflammatory; Intercrines; Interferons; Intravaginal Administration; LAV-2; LAV-HTLV-III; Length; Lesion; Life; Liquid substance; Local Microbicides; Lymphadenopathy-Associated Virus; Lytotoxicity; Macaca; Macaque; Mammals, Mice; Mammals, Rabbits; Man (Taxonomy); Man, Modern; Mediating; Membrane; Mice; Microbicides, Local; Modeling; Molecular Interaction; Mucosa; Mucosal Tissue; Mucous Membrane; Murine; Mus; Mutation; N-terminal; NH2-terminal; Nonstructural Protein; Nucleosides; Oryctolagus cuniculus; Pan; Pan Genus; Pan Species; Pathway interactions; Peptidase Inhibitors; Peptidases; Peptide Hydrolase Inhibitors; Peptide Hydrolases; Peptide Peptidohydrolase Inhibitors; Peptide antibodies; Peptides; Plasma Membrane; Plasma, Seminal; Play; Position; Positioning Attribute; Pressure; Pressure- physical agent; Property; Property, LOINC Axis 2; Protease Antagonists; Protease Inhibitor; Proteases; Protein, Nonstructural; Proteinase Inhibitors; Proteinases; Proteolytic Enzymes; RNA Transcriptase; RNA-Dependent DNA Polymerase; RNA-Directed DNA Polymerase; Rabbit, Domestic; Rabbits; Reporting; Research Design; Resistance; Resistance development; Resistant development; Reverse Transcriptase; Revertase; Role; SHIV; SIS cytokines; Safety; Satellite Viruses; Seminal Plasma; Sexual Intercourse; Sexual Transmission; Signal Pathway; Simplexvirus; Specificity; Stomatitis; Study Type; Surface; T-Cells; T-Lymphocyte; T-Lymphotropic Virus Type III Infections, Human; TLR protein; Testing; Thymus-Dependent Lymphocytes; Time; Toll-like receptors; Toxic effect; Toxicities; Transmission; Transmission, Sexual; VSV; Vagina; Vaginal; Vaginal Douching; Vaginal Irrigation; Vaginal Lavage; Veiled Cells; Vesicular Stomatitis Virus; Vesicular stomatitis Indiana virus; Viral; Viral Gene Products; Viral Gene Proteins; Viral Proteins; Virus; Virus-HIV; Viruses, General; Work; aminoacid; anti-HIV microbicide; cell type; chemoattractant cytokine; chemokine; cytokine; cytotoxicity; developing resistance; drug resistant; experiment; experimental research; experimental study; fluid; genome mutation; herpesvirus; host response; human T cell leukemia virus III; human T lymphotropic virus III; human herpesvirus 1 group; immunogenic; immunogenicity; immunoresponse; improved; in vitro testing; in vivo; inhibitor; inhibitor/antagonist; liquid; male; membrane structure; microbicidal; microbicide; mouse model; novel; particle; pathway; plasmalemma; pre-clinical; preclinical; pressure; prevent; preventing; public health relevance; research study; resistance to Drug; resistant; resistant to Drug; response; sedimentation velocity; simian HIV; simian human immunodeficiency virus; social role; study design; thymus derived lymphocyte; topical microbicide; transmission process; urogenital system (genital part); vaginal transmission; virus protein
Relevance: The goal of this project is to conduct in vitro and in vivo studies designed to support the preclinical development of a short peptide as a novel anti-HIV microbicide candidate. There is an urgent need for the development of safe, effective topical microbicides to prevent the sexual transmission of HIV and to allow the female partner to take personal responsibility for use and application. The development of a safe, effective acceptable topical microbicide to prevent the sexual transmission of HIV-1 could play a major role in worldwide reduction of the over 14,000 new HIV-1 infections per day, and potentially save millions of lives
Project start date: 2009-07-22
Project end date: 2010-06-30
Budget start date: 22-JUL-2009
Budget end date: 30-JUN-2010
PFA/PA: PA-06-181
3R21AI078791-02S1 (2009): $451431
ANTI-HIV MICROBICIDAL PEPTIDES
Philippe Andre Gallay, Associate Professor
Scripps Research Institute, La Jolla, Ca 92037-1000
Grant 5R21AI079782-02 from National Institute Of Allergy And Infectious Diseases
Abstract: Topical microbicides are defined as vaginally applied products that prevent male-to-female or female-to-male HIV transmission. The high error rate of the HIV reverse transcriptase (RT) drives the development of resistance and genomic diversity in HIV. The current pan-resistance to all classes of HIV inhibitors such as that found in O group virus for non-nucleoside RT and protease (PR) inhibitors, raises an important question with regard to the development of microbicides targeting RT or PR. These concerns may be extended to inhibitors of HIV entry due to rapid emergence of mutations in the envelope (Env) under selective pressure. There is thus an urgent need to identify new anti-HIV compounds, which target viral components other than PR, RT and Env, and which may be developed as safe and effective microbicides. We identified a short linear peptide, which neutralizes HIV at an nM range. The peptide blocks HIV entry into cells in an Env-independent manner. Several lines of evidence suggest that the peptide destabilizes the HIV membrane, liberating the Env glycoprotein. The peptide-mediated HIV membrane destabilization is specific because i) the peptide does not disturb the membrane of other enveloped viruses (i.e., HSV and VSV); ii) it does not destabilize the membrane of human cervical cells even when applied twice daily to cells at a concentration 20-200-fold superior to that which blocks HIV infection; iii) the peptide does not harm mice injected i.v. twice daily with high peptide concentrations (0.5 mg) for a period of three days; and iv) the peptide apparently does not create lesions in the vaginal epithelium of humanized mice since the peptide, rather than promoting transmission, completely blocks intravaginal transmission The peptide represents an attractive microbicide candidate for several reasons i) the peptide inhibits infection of a broad range of primary isolates in various primary human cell types; ii) it interferes with the three mechanisms involved in HIV transmission genital epithelial transmigration, dendritic cell-mediated transmission, and infection of mucosal target cells; iii) the peptide is extremely efficacious since less than 15 minutes of exposure suffices to neutralize HIV; iv) it is potent for two hours both prior to and after addition of HIV to cells, suggesting that it should be active for an acceptable length of time both prior to and after sexual intercourse; v) the peptide retains its antiviral properties at a low pH; vi) it is not toxic or immunogenic in mice, even after repeated i.v. administrations of high peptide doses; and vii) most importantly, the peptide totally blocks the intravaginal transmission of HIV in humanized mice. In this application, we propose to fully characterize the anti-HIV efficacy of the peptide and derivatives in vitro as well as their toxicity and immunogenicity in vivo (R21 phase). If these studies unambiguously demonstrate that the peptides represent attractive anti-HIV microbicide candidates, they will then be tested as topical microbicides in vivo (R33 phase). The goal of this project is to conduct in vitro and in vivo studies designed to support the preclinical development of short peptides as novel anti-HIV microbicides. There is an urgent need for the development of safe, effective topical microbicides to prevent the sexual transmission of HIV and to allow the female partner to take personal responsibility for use and application. The development of a safe, effective acceptable topical microbicide to prevent the sexual transmission of HIV-1 could play a major role in worldwide reduction of the over 14,000 new HIV-1 infections per day, and potentially save millions of lives
Keywords: AIDS Drugs; AIDS Virus; Acquired Immune Deficiency Syndrome Virus; Acquired Immunodeficiency Syndrome Virus; Amino Acids; Animal Model; Animal Models and Related Studies; Anti-AIDS Agents; Anti-AIDS Drugs; Anti-HIV Agents; Anti-HIV Drugs; Anti-Human Immunodeficiency Virus Agents; Antiproteases; Antiviral Agents; Antiviral Drugs; Antivirals; Assay; Associated Viruses; Binding; Binding (Molecular Function); Bioassay; Biologic Assays; Biological Assay; Cell Growth in Number; Cell Multiplication; Cell Proliferation; Cell membrane; Cells; Cellular Proliferation; Cervical; Coitus; Corpus Luteum Hormone; Cytoplasmic Membrane; Delta4-pregnene-3, 20-dione; Dendritic Cells; Deoxynucleotide-triphosphate[{..}]DNA deoxynucleotidyltransferase (RNA-directed); Development; Dose; Drug Resistance, Multiple; Drug Resistant, Multiple; EC 2.7.7.49; Endopeptidase Inhibitors; Environment; Epithelial; Epithelium; Esteroproteases; Exhibits; Female; Figs; Figs - dietary; Generations; Genetic Alteration; Genetic Change; Genetic defect; Genital; Genital System, Female, Vagina; Genital system; Genomics; Glycoproteins; Goals; HCV; HIV; HIV Entry Inhibitors; HIV Infections; HIV-1; HIV-2; HIV-I; HIV-II; HIV1; HIV2; HSV; HTLV-III; HTLV-III Infections; HTLV-III-LAV Infections; HTLV-IV; Hepatitis C virus; Hepatitus C; Herpes Simplex; Herpes Simplex Infections; Herpes Simplex Virus; Herpes labialis Virus; Herpes simplex disease; Herpesvirus hominis; Herpesvirus hominis disease; Hour; Human; Human Immunodeficiency Viruses; Human Sexual Intercourse; Human T-Cell Leukemia Virus Type III; Human T-Cell Lymphotropic Virus Type III; Human T-Lymphotropic Virus Type III; Human T-Lymphotropic Virus Type IV; Human immunodeficiency virus 1; Human immunodeficiency virus 2; Human, General; IFN; INFLM; Immune; Immune response; Immunodeficiency Virus Type 1, Human; Immunodeficiency Virus Type 2, Human; In Vitro; Infection; Inflammation; Interferons; LAV-2; LAV-HTLV-III; Length; Lesion; Life; Liquid substance; Local Microbicides; Lymphadenopathy-Associated Virus; Lytotoxicity; Macaca; Macaque; Mammals, Mice; Mammals, Rabbits; Man (Taxonomy); Man, Modern; Mediating; Membrane; Mice; Microbicides, Local; Modeling; Molecular Interaction; Multi-Drug Resistance; Multidrug Resistance; Murine; Mus; Mutate; Mutation; N-terminal; NH2-terminal; Nonstructural Protein; Nucleosides; Oryctolagus cuniculus; Pathway interactions; Peptidase Inhibitors; Peptidases; Peptide Hydrolase Inhibitors; Peptide Hydrolases; Peptide Peptidohydrolase Inhibitors; Peptide antibodies; Peptides; Phase; Plasma Membrane; Play; Position; Positioning Attribute; Pregn-4-ene-3, 20-dione; Pregnenedione; Pressure; Pressure- physical agent; Progesterone; Property; Property, LOINC Axis 2; Protease Antagonists; Protease Inhibitor; Proteases; Proteinase Inhibitors; Proteinases; Proteolytic Enzymes; RNA Transcriptase; RNA-Dependent DNA Polymerase; RNA-Directed DNA Polymerase; Rabbit, Domestic; Rabbits; Research Design; Resistance; Resistance development; Resistance to Multi-drug; Resistance to Multidrug; Resistance to Multiple Drug; Resistant development; Resistant to Multiple Drug; Resistant to multi-drug; Resistant to multidrug; Reverse Transcriptase; Revertase; Role; SHIV; Safety; Satellite Viruses; Seminal Plasma; Sexual Intercourse; Sexual Transmission; Signal Pathway; Simplexvirus; Specificity; Stomatitis; Study Type; Surface; T-Lymphotropic Virus Type III Infections, Human; TLR protein; Testing; Therapeutic Progesterone; Time; Toll-like receptors; Toxic effect; Toxicities; Transmission; VSV; Vagina; Vaginal; Veiled Cells; Vesicular Stomatitis Virus; Vesicular stomatitis Indiana virus; Viral; Viral Gene Products; Viral Gene Proteins; Viral Proteins; Virus; Virus-HIV; Viruses, General; Work; aminoacid; anti-HIV microbicide; antiAIDS agent; cell type; cytotoxicity; developing resistance; experiment; experimental research; experimental study; fluid; genome mutation; herpesvirus; host response; human T cell leukemia virus III; human T lymphotropic virus III; human herpesvirus 1 group; immunogenic; immunogenicity; immunoresponse; improved; in vitro testing; in vivo; inhibitor; inhibitor/antagonist; liquid; male; membrane structure; microbicidal; microbicide; model organism; mouse model; multi-drug resistant; multidrug resistant; novel; oncogene protein pp60(v-src) (137-157); particle; pathway; peptide A; plasmalemma; pre-clinical; preclinical; pressure; prevent; preventing; research study; resistant; response; sedimentation velocity; simian HIV; simian human immunodeficiency virus; social role; study design; topical microbicide; transmission process; urogenital system (genital part); vaginal transmission; virus protein
Project start date: 2009-07-17
Project end date: 2011-06-30
Budget start date: 1-JUL-2010
Budget end date: 30-JUN-2011
PFA/PA: RFA-AI-07-034
5R21AI079782-02 (2010): $237375
1R21AI079782-01 (2009): $284850
INHIBITORS OF HIV-DENDRITIC CELL INTERACTIONS AS MICROBICIDES
Philippe Andre Gallay
Scripps Research Institute, La Jolla, Ca 92037-1000
Grant 5R33AI076005-04 from National Institute Of Allergy And Infectious Diseases
Abstract: The development of a safe, effective acceptable topical microbicide to prevent the sexual transmission of HIV could play a major role in worldwide reduction of the over 14,000 new HIV infections per day, and potentially save millions of lives. Given that cell-free virus ineffectively crosses the genital epithelium in the absence of lesions, it is likely that HIV hijacks host cells as Trojan horses to cross the normally impermeable genital epithelium. It has been postulated that HIV exploits Langerhans (LC) and dendritic cells (DC) to facilitate its safe passage through the genital epithelium. In this application, we propose to develop compounds that prevent HIV-LC and HIV-DC interactions in vitro (R21 phase) and to test them as topical microbicides in vivo (R33 phase). We demonstrated that HIV uses three specific receptors to mediate its initial contact with vaginal LC and DC. We propose to generate reagents that target each of these receptors. These receptor antagonists will then be tested for their capacities to prevent HIV-LC and -DC interactions. The goal is to identify reagents, which are the most potent at blocking in vitro HIV hijacking of vaginal LC and DC. These LC and DC receptor inhibitors will serve as microbicide candidates for subsequent in vivo studies proposed in the R33 phase. We demonstrated that gp120 mediates the contact between HIV and vaginal LC and DC. We propose to generate reagents, which prevent gp120-LC and -DC interactions. Specifically, we propose to generate soluble reagents that mimic the receptors that HIV exploits to interact with LC and DC. These receptor mimics will serve as decoys to prevent gp120 contact with vaginal LC and DC. The goal is to identify receptor mimics, which are the most potent at blocking in vitro HIV hijacking of vaginal LC and DC. These receptor mimics will serve as microbicide candidates for in vivo studies proposed in the R33 phase. One advantage of using compounds that prevent HIV-LC and -DC interactions is that many sexually transmitted pathogens also exploit LC and DC for host colonization, and thus the ability to block HIV-LC and -DC interactions may have the additional benefit of preventing other sexually transmitted pathogens. This approach may not only provide valuable novel microbicides, but it will also allow us to assess the contribution of LC and DC to HIV transmission
Keywords: AIDS Virus; AIDS Virus Receptors; Acquired Immune Deficiency Syndrome Virus; Acquired Immunodeficiency Syndrome Virus; Cell Communication; Cell Interaction; Cell-to-Cell Interaction; Cells; Corpus Luteum Hormone; Delta4-pregnene-3, 20-dione; Dendritic Cells; Development; Drugs; Envelope Glycoprotein gp120, HIV; Epidemic; Epithelium; Equine; Equine Species; Equus caballus; Equus przewalskii; Genital; Genital System, Female, Vagina; Genital system; Goals; HIV; HIV Envelope Protein gp120; HIV Infections; HIV Receptors; HTLV-III; HTLV-III Infections; HTLV-III Receptors; HTLV-III gp120; HTLV-III-LAV Infections; Horse, Domestic; Horses; Human Immunodeficiency Viruses; Human T-Cell Leukemia Virus Type III; Human T-Cell Lymphotropic Virus Type III; Human T-Lymphotropic Virus Type III; In Vitro; Infection; LAV-HTLV-III; Lesion; Life; Local Microbicides; Lymphadenopathy-Associated Virus; Macaca; Macaque; Mediating; Medication; Microbicides, Local; Modeling; Pharmaceutic Preparations; Pharmaceutical Preparations; Phase; Play; Pregn-4-ene-3, 20-dione; Pregnenedione; Progesterone; Reagent; Receptor Protein; Receptors, HIV; Role; SHIV; Sexual Transmission; T-Lymphotropic Virus Type III Infections, Human; Testing; Therapeutic Progesterone; Transmission; Vaccines; Vagina; Vaginal; Veiled Cells; Virus; Virus-HIV; Viruses, General; Work; drug/agent; env Protein gp120, HIV; gp120; gp120 ENV Glycoprotein; gp120(HIV); in vivo; inhibitor; inhibitor/antagonist; microbicidal; microbicide; novel; pathogen; prevent; preventing; receptor; simian HIV; simian human immunodeficiency virus; social role; topical microbicide; transmission process; urogenital system (genital part)
Relevance: Sexual transmission is the most common mode of infection in the global HIV epidemic. In the absence of an effective vaccine, there is an urgent need for additional strategies to prevent new HIV infections. Mucosal Langerhans and dendritic cells are the first cells that HIV encounters and infects. In this application, we propose to develop drugs, which specifically and completely prevent the contact between the virus and these cells, in order to use them as novel microbicide candidates
Project start date: 2007-09-30
Project end date: 2012-08-31
Budget start date: 1-SEP-2010
Budget end date: 31-AUG-2011
PFA/PA: RFA-AI-06-042
5R33AI076005-04 (2010): $527027
CYCLOPHILINS, CYCLOPHILIN INHIBITORS AND HEPATITIS C
Philippe Andre Gallay, Associate Professor
Scripps Research Institute, La Jolla, Ca 92037-1000
Grant 1R01AI087746-01 from National Institute Of Allergy And Infectious Diseases
Abstract: Current therapy for patients with chronic HCV infection includes high doses of IFN in combination with ribavirin. However, success rates remain low (around 50% of treated patients depending on genotype). Furthermore, the IFN/ribavirin treatment carries a significant risk of serious side effects. Thus, the development of alternative treatments is essential. The immunosuppressive drug cyclosporine A (CsA) was reported to be clinically effective against HCV infection. Moreover, recent studies provided evidence that non- immunosuppressive CsA analogs such as NIM811, Debio 025 and SCY-635 inhibit HCV RNA replication both in vitro and in vivo in an IFN-independent manner. A growing body of evidence suggests that cyclophilins (Cyps) are important for the HCV life cycle, suggesting that Cyp inhibitors (CsA, Debio 025, NIM811 or SCY- 635), by acting on intracellular Cyps, block HCV replication. Three independent studies recently demonstrated that HCV highly relies on cyclophilin A (CypA) to replicate in human hepatocytes. We showed that CypA, but not CypB, CypC and CypD, is critical for HCV replication. We demonstrated that the hydrophobic pocket of CypA, where Cyp inhibitors bind, and which control the isomerase activity of CypA, is critical for HCV replication. We obtained several lines of evidence that a population of CypA molecules resides in a protease- resistant membrane compartment similar to that where HCV replicates. We also found that the association of CypA with this compartment does not depend on the presence of HCV proteins. Importantly, we showed that Cyp inhibitors deplete this membrane compartment of CypA. The depletion of CypA from the protected ER spherules where HCV replication occurs, may provide the first hint for the mechanism of antiviral action of Cyp inhibitors. However, the molecular requirements for CypA in HCV replication as well as the antiviral mechanisms of action of this novel class of potent anti-HCV agents - the Cyp inhibitors - are completely obscure. By selecting Cyp inhibitor-resistant HCV mutant´s in vitro, studies mapped mutations into two nonstructural (NS) proteins - NS5A and NS5B. Altogether these data suggest the existence of a relationship between CypA, NS5A and NS5B that is critical for HCV replication. To test this hypothesis, we propose in this application to conduct a set of experiments aimed at fully characterizing CypA-NS5A-NS5B interactions at a molecular level; analyzing the development of HCV resistance to Cyp inhibitors; and investigating the effect of Cyp inhibitors on the composition and polymerase activities of HCV replication complexes. The ultimate goal of this application is to improve our understanding of the role of CypA in the HCV life cycle and of the in vitro and in vivo block mediated by this novel class of potent anti-HCV agents - the Cyp inhibitors. An estimated 170 million people worldwide are chronically infected with Hepatitis C virus. 10-20% of these will develop cirrhosis and 1-5% will develop hepatocellular carcinoma. The only currently approved therapy is weekly injection with pegylated interferon and daily oral ribavirin for 6-12 months. The therapy is associated with severe side effects and results in sustained viral clearance in only 50% of all patients. There is therefore a high need to develop new therapies with better efficacy and tolerability. By demonstrating that cyclophilin (Cyp) inhibitors have potent efficacy against HCV, our recent clinical study opens a new line of opportunity to eradicate this prime human threat. The current proposal outlines a comprehensive series of experiments aimed at determining the mechanism of action of Cyp inhibitors that should lead to the identification of new targets for inhibition of HCV. The study of the roles of Cyp - the intracellular targets of the Cyp inhibitors - in HCV replication is novel and should lead to the identification of new host and viral targets for the development of innovative anti-HCV therapies
Keywords: 1-Beta-D-ribofuranosyl-1, 2, 4-triazolo-3-carboxamide; 1-Beta-D-ribofuranosyl-1H-1, 2, 4-triazole-3-carboxamide; Adverse effects; Antiviral Agents; Antiviral Drugs; Antivirals; Assay; Binding; Binding (Molecular Function); Bioassay; Biologic Assays; Biological Assay; Biosynthetic Proteins; Carcinoma of the Liver Cells; Cells; Chronic Hepatitis C; Chronic type C viral hepatitis; Chronic viral hepatitis C; Ciclosporin; Cirrhosis; Clinical Research; Clinical Study; Complex; CsA; Cyclophilin A; Cyclophilins; Cyclosporin A; Cyclosporin-Binding Proteins; Cyclosporine; Cyclosporine A; DNA-Dependent RNA Polymerases; DNA-Directed RNA Polymerase; Data; Dependence; Development; Dose; Drug Industry; Drugs; EC 2.7.7.48; EC 2.7.7.6; Esteroproteases; Genetic Alteration; Genetic Change; Genetic defect; Genotype; Goals; HCC; HCV; HCV Chronic Infection; HCV infection; Hepatic Cells; Hepatic Parenchymal Cell; Hepatitis C; Hepatitis C virus; Hepatitis C virus infection; Hepatitis C, Chronic; Hepatitis, Viral, Non-A, Non-B, Parenterally-Transmitted; Hepatitus C; Hepatocellular Carcinoma; Hepatocellular cancer; Hepatocyte; Hepatoma; Human; Human, General; IFN; Immunosuppressants; Immunosuppressive Agents; In Vitro; Industry, Pharmaceutic; Injection of therapeutic agent; Injections; Interferons; Isomerase; Lead; Life Cycle; Life Cycle Stages; Link; Liver Cells; Man (Taxonomy); Man, Modern; Maps; Mediating; Medication; Membrane; Molecular; Molecular Interaction; Mutate; Mutation; NANBH; Nonstructural Protein; Nucleoside-triphosphate[{..}]RNA nucleotidyltransferase (DNA-directed); Nucleoside-triphosphate[{..}]RNA nucleotidyltransferase (RNA-directed); Oral; PT-NANBH; Parenterally-Transmitted Non-A, Non-B Hepatitis; Patients; Pb element; Peptidases; Peptide Hydrolases; Peptidyl-Prolyl cis-trans Isomerase A; Pharmaceutic Preparations; Pharmaceutical Industry; Pharmaceutical Preparations; Phosphorylation; Polymerase; Population; Primary carcinoma of the liver cells; Proteases; Protein Phosphorylation; Protein, Nonstructural; Proteinases; Proteins; Proteolytic Enzymes; RNA Polymerases; RNA Replicase; RNA replication; RNA, Viral; RNA-Dependent RNA Polymerase; RNA-Directed RNA Polymerase; RTCA; Recombinant Proteins; Replication Unit; Replicon; Reporting; Research; Resistance; Ribavirin; Ribovirin; Risk; Role; Sandimmun; SangCya; Series; Source; Specificity; System; System, LOINC Axis 4; Testing; Treatment Side Effects; Tribavirin; Two Hybrid; Variant; Variation; Viral; Viral Gene Products; Viral Gene Proteins; Viral Proteins; Virus; Viruses, General; Work; Yeast One Hybrid System; Yeast One/Two-Hybrid System; alternative treatment; analog; base; domain mapping; drug/agent; experiment; experimental research; experimental study; gene product; genome mutation; heavy metal Pb; heavy metal lead; hepatitis non A non B; hepatitis nonA nonB; immunosuppressive; improved; in vitro Assay; in vivo; inhibitor; inhibitor/antagonist; innovate; innovation; innovative; life course; membrane structure; mutant; neoral; non A non B hepatitis; non A, non B hepatitis; non-A non-B hepatitis; non-A, non-B hepatitis; novel; public health relevance; research study; resistance mechanism; resistant; resistant mechanism; sandimmune; side effect; social role; success; therapy adverse effect; treatment adverse effect; viral RNA; virus RNA; virus protein; yeast two hybrid system
Relevance: An estimated 170 million people worldwide are chronically infected with Hepatitis C virus. 10-20% of these will develop cirrhosis and 1-5% will develop hepatocellular carcinoma. The only currently approved therapy is weekly injection with pegylated interferon and daily oral ribavirin for 6-12 months. The therapy is associated with severe side effects and results in sustained viral clearance in only 50% of all patients. There is therefore a high need to develop new therapies with better efficacy and tolerability. By demonstrating that cyclophilin (Cyp) inhibitors have potent efficacy against HCV, our recent clinical study opens a new line of opportunity to eradicate this prime human threat. The current proposal outlines a comprehensive series of experiments aimed at determining the mechanism of action of Cyp inhibitors that should lead to the identification of new targets for inhibition of HCV. The study of the roles of Cyp - the intracellular targets of the Cyp inhibitors - in HCV replication is novel and should lead to the identification of new host and viral targets for the development of innovative anti-HCV therapies
Project start date: 2010-01-15
Project end date: 2014-12-31
Budget start date: 15-JAN-2010
Budget end date: 31-DEC-2010
PFA/PA: PA-07-070
1R01AI087746-01 (2010): $474750
SYNDECAN AGONISTS AND ANTAGONISTS AS MICROBICIDES
Philippe Andre Gallay, Associate Professor
Scripps Research Institute, La Jolla, Ca 92037-1000
Grant 5R33AI071952-04 from Office Of The Director, National Institutes Of Health
Keywords: AIDS Virus; Agonist; Arginine; Arginine, L-Isomer; Binding; Binding (Molecular Function); Blood - brain barrier anatomy; Blood-Brain Barrier; Brain; C-C CKR-5 Gene; C-C Chemokine Receptor Type 5 Gene; CC-CKR-5 Gene; CCCKR5 Gene; CCL5; CCR-5 Gene; CCR5; CCR5 gene; CD195 Antigen Gene; CHEMR13 Gene; CKR-5 Gene; CKR5 Gene; CMKBR5 Gene; Cells; Cervical; Charge; Chemokine (C-C Motif) Ligand 5; Chemokine (C-C) Receptor 5 Gene; D17S136E; Data; Dendritic Cells; Drugs; Encephalon; Encephalons; Endothelial Cells; Endothelium; Envelope Glycoprotein gp120, HIV; Epithelial; Epithelial Cells; Epithelium; Genital; Genital System, Female, Vagina; Genital system; Goals; HB-GAM receptor; HIV Envelope Protein gp120; HIV-1; HIV-1 Fusion Co-Receptor Gene; HIV-I; HIV1; HSPG; HTLV-III gp120; HeLa; Hela Cells; Hemato-Encephalic Barrier; Heparan Sulfate; Heparan Sulfate Proteoglycan; Heparitin Sulfate; Human immunodeficiency virus 1; Immunity; Immunodeficiency Virus Type 1, Human; Immunoglobulin V; Immunoglobulin Variable Region; In Vitro; L-Arginine; MGC17164; Macaca; Macaque; Mediating; Medication; Microbe; Molecular Interaction; N-syndecan; Nervous System, Brain; Pathogenesis; Pharmaceutic Preparations; Pharmaceutical Preparations; Phase; Play; Pressure; Pressure- physical agent; Proteoheparan Sulfate; Publishing; RANTES; RANTES Protein, T-Cell; Reagent; Receptor Protein; Resistance; Role; SCYA5; SIS delta; SIS-delta; SISd; Small Inducible Cytokine A5; Surface; T-Cell Specific Protein p288; TCP228; Testing; Time; Vagina; Vaginal; Variable Region; Variable Region, Ig; Veiled Cells; Virus; Viruses, General; Work; base; drug/agent; env Protein gp120, HIV; fibroglycan; gp120; gp120 ENV Glycoprotein; gp120(HIV); heparin-binding growth associated molecule receptor; human T cell leukemia virus III; human T lymphotropic virus III; in vivo; macrophage; microbicidal; microbicide; monolayer; polyanion; pressure; prevent; preventing; receptor; resistant; social role; sulfation; syndecan; syndecan 3; syndecan-2; transcytosis; urogenital system (genital part)
Project start date: 2006-09-01
Project end date: 2011-08-31
Budget start date: 1-SEP-2009
Budget end date: 31-AUG-2010
5R33AI071952-04 (2009): $531750