Quick and Accurate Measurements of HIV Broadly Neutralizing Antibody Susceptibility

快速准确地测量 HIV 广泛中和抗体敏感性

• 批准号: 10676422
• 负责人: Zizhang Sheng
• 金额: $ 100.24万
• 依托单位: COLUMBIA UNIVERSITY HEALTH SCIENCES
• 依托单位国家: 美国
• 财政年份: 2023
• 资助国家: 美国
• 起止时间: 2023-04-01 至 2026-03-31
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10676422
• 关键词: AIDS clinical trial group; Address; Affect; Algorithms; Antibodies; Area Under Curve; Bar Codes; Biological Assay; Clinical; Clinical Trials; Cloning; Computer Models; Consumption; DNA; DNA analysis; Data; Detection; Development; Dideoxy Chain Termination DNA Sequencing; Enrollment; Exposure to; Genetic; Genome; Genomic DNA; Genotype; Goals; HIV; HIV Envelope Protein gp120; HIV Infections; HIV Vaccine Trials Network; HIV envelope protein; Human; IgG1; Individual; Infection; Knowledge; Laboratories; Length; Libraries; Link; Literature; Luciferases; Measurement; Measures; Medical center; Minor; Minority; Neutralization Tests; Participant; Patients; Performance; Peripheral Blood Mononuclear Cell; Persons; Phenotype; Predisposition; Procedures; Property; Provirus Integration; Research Personnel; Resistance; Running; Sampling; Site; Societies; System; Technology; Testing; Third Generation Sequencing; Time; Training; Universities; Validation; Variant; Viral; Viral reservoir; Virion; Virus; antiretroviral therapy; assay development; biobank; clinical development; clinical trial enrollment; early phase clinical trial; env Genes; experimental study; improved; machine learning model; multiplex assay; nanopore; neutralizing antibody; novel; participant enrollment; point of care; portability; prediction algorithm; prevent; public health relevance; research clinical testing; viral RNA

PROJECT SUMMARY HIV-infected individuals harbor viral quasispecies that differ in genetic sequences and susceptibility to broadly neutralizing antibodies (bNAbs) targeting the HIV envelope (Env). Due to difficulties with current assays in confirming viral sensitivity to bNAbs, most clinical trials enrolled participants without knowledge of their viral susceptibility. Thus, an accurate and sensitive assay with quick turnaround (1-2 weeks) is needed. We propose to approach this aim with two prongs: Aim 1, a phenotypic (Env function) assay for bulk Env neutralization assessment and Aim 2, a genotypic (Env sequence) algorithm for high-throughput prediction of neutralization susceptibility. We propose 10 HIV bNAbs targeting 5 distinct gp120 sites for assay development, with 2 non-HIV human IgG1 antibodies as negative controls. Current assays are difficult because of two labor-intensive and time-consuming procedures: Env single-genome amplification (SGA) and individual Env cloning. Aim 1 will address the latter procedure required because the current TZM-bl neutralization assay cannot tease out bulk Env clones that differ in bNAb susceptibility. Aim 1 will apply DNA barcoding to simultaneously track bNAb sensitivity of hundreds of Env variants in a high-throughput manner. This will be achieved by a novel Env- expressing lentiviral system in which each Env is linked to a unique barcode sequence. Aim 2 will address Env sequencing, for which SGA is necessary because Sanger sequencing cannot sequence bulk templates. We propose in Aim 2 to apply a third-generation sequencing technology, Nanopore sequencing, for effective bulk template sequencing and develop machine learning models for a fast and reliable prediction algorithm. Combining with the ample bNAb neutralization data from literature and Aim 1 of this project, trained computational models can be fast, accurate, and high-throughput to predict bNAb susceptibility. Since the detection of a low yield of functional Env sequences and minority variants from participant’s HIV DNA reservoir is essential, we propose in Aim 3 to investigate whether a virion capture assay could be applied to enrich functional Envs. We will also apply a DNA analysis to enrich minor variants from the viral reservoir. We have biospecimens stored onsite and access to a wide distribution of HIV subtypes from the AIDS Clinical Trials Group (ACTG) and HIV Vaccine Trials Network (HVTN) biorepositories for assay development and validation. All proposed R61 Aims have clear milestones defined and the R61 assays will be developed with streamlined and standard operating procedure (SOP) for adaptation to clinical labs. The Columbia Clinical Trials Support Laboratory is fully equipped and staffed to perform the SOPs and provide input and validation on assay performance. Successful development of an accurate and sensitive bNAb susceptibility assay with quick turnaround will alter the enrollment for bNAb trials and greatly improve clinical development of bNAb products.

项目摘要 HIV感染者携带病毒准种，这些准种在基因序列和对广泛的 靶向HIV包膜（Env）的中和抗体（bNAb）。由于目前的测定方法在 确认病毒对bNAb的敏感性，大多数临床试验招募的参与者不知道他们的病毒 易感性因此，需要具有快速周转（1-2周）的准确且灵敏的测定。我们提出 为了实现这一目标，我们从两个方面着手：目标1，用于大量Env中和的表型（Env功能）测定 评估和Aim 2，用于中和的高通量预测的基因型（Env序列）算法 易感性我们提出了10种针对5个不同gp 120位点的HIV bNAb，用于检测开发，其中2种为非HIV bNAb。 人IgG 1抗体作为阴性对照。目前的分析是困难的，因为两个劳动密集型和 耗时的程序：Env单基因组扩增（SGA）和单个Env克隆。目标1将 解决后一个程序所需的，因为目前的TZM-bl中和试验不能梳理出散装 bNAb敏感性不同的Env克隆。目标1将应用DNA条形码同时跟踪bNAb 以高通量方式检测数百种Env变体的灵敏度。这将通过一个新的环境来实现- 本发明涉及表达慢病毒系统，其中每个Env连接至独特的条形码序列。目标2将解决环境问题 因为桑格测序不能对大量模板进行测序，所以SGA是必需的。我们 在目标2中建议应用第三代测序技术，纳米孔测序， 模板测序和开发机器学习模型，以实现快速可靠的预测算法。 结合文献中丰富的bNAb中和数据和本项目的目标1， 计算模型可以快速、准确和高通量地预测bNAb易感性。以来 从参与者的HIV DNA库中检测到低产量的功能性Env序列和少数变异体 是必不可少的，我们在目标3中提出研究是否可以应用病毒体捕获测定来富集 功能环境我们还将应用DNA分析来富集病毒库中的微小变异。我们有 现场储存的生物标本，以及从艾滋病临床试验组获得广泛分布的艾滋病毒亚型 （ACTG）和HIV疫苗试验网络（HVTN）生物储存库，用于试验开发和验证。所有 拟议的R61目标有明确的里程碑定义，R61检测试剂盒将以简化和 适用于临床实验室的标准操作规程（SOP）。哥伦比亚临床试验支持 实验室设备齐全，人员配备齐全，可执行SOP并提供试验输入和验证 性能成功开发了一种准确灵敏的bNAb药敏试验， 这一转变将改变bNAb试验的招募情况，并大大改善bNAb产品的临床开发。