The overall goal of this project is to develop novel, robust, high-throughput assays to evaluate the susceptibility of to inhibitors of HIV-1 Gag and protease proteins, with particular emphasis on virion assembly and maturation. These assays will be based on proven technologies underlying Monogram Bioscience's widely used PhenoSense HIVTM and GeneSeq HIVTM drug resistance tests, i.e. recombinant virus-based and nucleic acid sequencing-based assays first developed to evaluate inhibitors of HIV-1 protease and reverse transcriptase. Currently, several assembly/maturation inhibitors are undergoing evaluation in pre-clinical and clinical studies, e.g. bevirimat (Panacos), vivecon (Myriad Genetics). Consequently, reliable, high-throughput assays are needed to support the development efforts of this promising new class of antiretroviral drugs, and eventually, their use in routine clinical practice. Since their implementation in the Monogram Clinical Reference Laboratory and commercialization in 2000, the PhenoSense HIV and GeneSeq HIV assays have been used to characterize in excess of 150,000 patient samples. In addition, both assays have been used extensively to support phase III evaluations of antiretroviral drug candidates over the past eight years, both to characterize resistance to new agents and to guide the selection of optimized background regimens. In 2003, Monogram successfully adapted the PhenoSense and GeneSeq technologies to evaluate inhibitors of HIV-1 entry (e.g. enfuvirtide, maraviroc, vivcriviroc, ibalizumab, PRO-140), and this year we completed validations for two new assays to monitor resistance to integrase inhibitors (e.g. raltegravir, elvitegravir). This application describes a straightforward, tried and true strategy to develop PhenoSense and GeneSeq HIV drug resistance assay systems that are capable of evaluating inhibitors of HIV-1 Gag and protease proteins; specifically inhibitors that target virus assembly and maturation. Because these new assay systems will include the immuno- dominant Gag region, they arelikely to prove valuable to the study of cellular immune responses. Such studies may prove valuable to vaccine design and evaluation.
Public Health Relevance: Successful completion of the funding proposal will result in the development and validation of new genotypic and phenotypic assays that will enable the characterization of viruses that are resistant to HIV-1 assembly and maturation inhibitors and will help guide antiretroviral treatment decisions for HIV-1 infected patients. The assay will also greatly advance the genotypic and phenotypic characterization of gag-specific CTL escape variants and will prove valuable to the design and development of improved HIV vaccines and immune-modulating therapies.
Public Health Relevance Statement: PROJECT NARRATIVE Successful completion of the funding proposal will result in the development and validation of new genotypic and phenotypic assays that will enable the characterization of viruses that are resistant to HIV-1 assembly and maturation inhibitors and will help guide antiretroviral treatment decisions for HIV-1 infected patients. The assay will also greatly advance the genotypic and phenotypic characterization of gag-specific CTL escape variants and will prove valuable to the design and development of improved HIV vaccines and immune-modulating therapies.
NIH Spending Category: Biotechnology; Genetics
Project Terms: AIDS Virus; Acquired Immune Deficiency Syndrome Virus; Acquired Immunodeficiency Syndrome Virus; Amendment; Amino Acid Sequence; Anti-Retroviral Agents; Antigenic Determinants; Antiproteases; Antiretroviral Agents; Assay; Base Sequence; Binding Determinants; Bioassay; Biologic Assays; Biological Assay; Blood Plasma; Capsid; Cells; Chemistry; Clinic; Clinical; Clinical Research; Clinical Study; Clinical Trials; Clinical Trials, Phase I; Clinical Trials, Phase II; Clinical Trials, Unspecified; Cloning, Molecular; Closure by Ligation; Code; Coding System; Codon; Codon Nucleotides; Collection; Computer Software Tools; DNA; DNA Restriction Enzymes; DNA Sequence; Data; Data Reporting; Deoxynucleotide-triphosphate[{..}]DNA deoxynucleotidyltransferase (RNA-directed); Deoxyribonucleic Acid; Development; Digestion; Drug resistance; Drugs; EC 2.7.7.49; ENV; Early-Stage Clinical Trials; Endopeptidase Inhibitors; Engineering; Engineerings; Environment; Epitopes; Esteroproteases; Evaluation; Funding; Gagging; Gene Products, gag; Generations; Genes; Genes, Reporter; Genes, env; Genetic; Genetic Alteration; Genetic Change; Genetic defect; Genomics; Goals; Guidelines; HIV; HIV drug resistance; HIV drug resistant; HIV vaccine; HIV-1; HIV-1 Protease; HIV-1 Reverse Transcriptase; HIV-I; HIV/AIDS Vaccines; HIV1; HIV1 protease; HTLV-III; High Throughput Assay; Human Immunodeficiency Viruses; Human T-Cell Leukemia Virus Type III; Human T-Cell Lymphotropic Virus Type III; Human T-Lymphotropic Virus Type III; Human immunodeficiency virus 1; Immune; Immune response; Immunodeficiency Virus Type 1, Human; Immunologic, Luciferase; Infection; Integrase; Integrase Inhibitors; LAV-HTLV-III; Laboratories; Ligation; Luciferases; Lymphadenopathy-Associated Virus; Measurable; Measures; Medication; Minor; Molecular Biology, Nucleic Acid Sequencing; Molecular Cloning; Monitor; Mutagenesis, Site-Directed; Mutation; New Agents; Nucleic acid sequencing; Nucleocapsid; Nucleotide Sequence; Nucleotides; Oligo; Oligonucleotides; Parents; Patient Selection; Patients; Peptidase Inhibitors; Peptidases; Peptide Hydrolase Inhibitors; Peptide Hydrolases; Peptide Peptidohydrolase Inhibitors; Performance; Pharmaceutic Preparations; Pharmaceutical Preparations; Phase; Phase 1 Clinical Trials; Phase 2 Clinical Trials; Phase I Clinical Trials; Phase I Study; Phase II Clinical Trials; Plasma; Plasmids; Population; Predisposition; Preparation; Procedures; Process; Protease Antagonists; Protease Inhibitor; Protease, HIV-1; Proteases; Protein Structure, Primary; Proteinase Inhibitors; Proteinases; Proteins; Proteolytic Enzymes; Protocols, Treatment; Quality Control; RGM; RNA Transcriptase; RNA-Dependent DNA Polymerase; RNA-Directed DNA Polymerase; RT-PCR; RTPCR; Reaction; Recombinants; Reflex, Pharyngeal; Regimen; Reporter; Reporter Genes; Reporting; Reporting, Data; Reproducibility; Research; Resistance; Restriction Endonucleases; Reticuloendothelial System, Serum, Plasma; Retroviral Antigen gag Protein; Reverse Transcriptase; Reverse Transcriptase Polymerase Chain Reaction; Revertase; SEQ-AN; SP1; SP1 gene; Sampling; Science of Chemistry; Sensitivity and Specificity; Sequence Analyses; Sequence Analysis; Serum, Plasma; Site; Site-Directed Mutagenesis; Site-Specific Mutagenesis; Software Tools; Source; Specific qualifier value; Specificity; Specified; Staging; Susceptibility; System; System, LOINC Axis 4; T-20; T-20 cpd; Targeted DNA Modification; Targeted Modification; Technology; Testing; Tools, Software; Transcription Factor Sp1 Gene; Transfection; Treatment Protocols; Treatment Regimen; Treatment Schedule; Vaccine Design; Validation; Variant; Variation; Viral; Viral Burden; Viral Load; Viral Load result; Viral Vector; Viral gag Proteins; Virion; Virus; Virus Assembly; Virus Particle; Virus Replication; Virus-HIV; Viruses, General; anti-retroviral; antiretroviral; base; clinical investigation; clinical practice; coat (nonenveloped virus); commercialization; cost; design; designing; disease subtype; disorder subtype; drug candidate; drug resistant; drug/agent; enfuvirtide; env Genes; experience; experiment; experimental research; experimental study; fitness; gag Antigens; gag Gene Products; gag Polyproteins; gag Protein; gene product; genome mutation; group specific antigen; high throughput screening; host response; human T cell leukemia virus III; human T lymphotropic virus III; human immunodeficiency virus vaccine; immunoresponse; improved; inhibitor; inhibitor/antagonist; mutant; novel; nucleic acid sequence; pentafuside; phase 1 study; phase 1 trial; phase 2 study; phase 2 trial; phase I trial; phase II trial; pre-clinical; preclinical; prospective; protein sequence; protocol, phase I; protocol, phase II; public health relevance; recombinant virus; research study; resistance mutation; resistance to Drug; resistance to HIV drug; resistant; resistant to Drug; resistant to HIV drug; restriction enzyme; reverse transcriptase PCR; study, phase II; tool; validation studies; vector; virus multiplication
