Novel antiviral strategy offering forward capability and reduced risk of escape

新颖的抗病毒策略提供前进能力并降低逃逸风险

• 批准号: 10593778
• 负责人: ANDREW HAYHURST
• 金额: $ 29.7万
• 依托单位: TEXAS BIOMEDICAL RESEARCH INSTITUTE
• 依托单位国家: 美国
• 财政年份: 2023
• 资助国家: 美国
• 起止时间: 2023-04-04 至 2025-03-31
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10593778
• 关键词: Affinity; Antibodies; Antibody Therapy; Antigen Targeting; Area; Avidity; Binding; Capsid; Cells; Compensation; Coronavirus; Crosslinker; DNA; Detection; Development; Diagnostic; Disease; Ebola virus; Engineering; Ensure; Epitopes; Escape Mutant; Escherichia coli; Evolution; Exhibits; Family; Felis catus; Foundations; Future; Generations; Genes; Glycosaminoglycans; Goals; Health; Human; Immune system; In Vitro; Individual; Infection; Influenza vaccination; Intervention; Intrabody; Mammalian Cell; Mediating; Modeling; Monitor; Mus; Mutation; Nature; Nucleoproteins; Parents; Penetration; Peptides; Pharmaceutical Preparations; Plasmids; Polymers; Predisposition; Process; Proteins; RNA Viruses; RNA vaccine; Reagent; Research; Resources; Risk; Seasons; Site; Small Interfering RNA; Specificity; Structural Protein; Surface; System; Technology; Testing; Therapeutic antibodies; Time; Transfection; Viral; Viral Genome; Viral Hemorrhagic Fevers; Viral Physiology; Viral Proteins; Viral Structural Proteins; Virion; Virus; Virus Assembly; Virus Cultivation; Virus Replication; Virus-like particle; Work; coronavirus pandemic; crosslink; design; dimer; env Gene Products; expression vector; human disease; interest; member; monomer; mutant; nanobodies; neutralizing antibody; nonhuman primate; novel; novel coronavirus; novel strategies; particle; pathogenic virus; preference; protein oligomer; stoichiometry; success; tissue culture; variants of concern

ABSTRACT The current coronavirus (CoV) pandemic, seasonal infections by other CoV and other “cold” viruses, plus the need for annual influenza vaccinations exemplify the challenges posed by viral antigenic drift and shift. Targeting landscapes of viral structural proteins displayed on the surfaces of virus particles and or the surfaces of infected cells has been the primary basis for developing antibody-based therapeutics. Although great advances have been made in trying to identify regions of these surface displayed proteins that are conserved and less prone to “escape” antibody binding, it appears to be a continual battle of cat and mouse as we are seeing with continual emergence of CoV “Variants of Concern”. In contrast, viral structural proteins that remain inside virus particles and cells, avoid the impact of cyclical antibody selection, and tend to be far more conserved. Oligomeric assemblies of these proteins can also blunt the impact of antiviral escape mutations owing to the mix of mutant and wild-type monomers present in the parent cell. Furthermore, the stoichiometry required of an antiviral to impede oligomer function need not be necessarily 1 antiviral to 1 monomer since, especially if the antiviral were a crosslinker, its impact would be relayed beyond the immediate contact to neighboring oligomers. Our long-term hypothesis is that affinity reagents capable of binding an internal oligomeric structural protein of all species of a viral genus uniformly will impede viral assembly when present as dimeric crosslinkers in a manner that is both forward capable and has much reduced susceptibility to viral escape. We will explore the antiviral potential of dimeric crosslinkers using viruses of the genus Ebolavirus as our model and a novel, rare nanobody manifesting uniform reactivity to nucleoprotein of all 6 species. Our two specific aims are: (1) we will engineer mammalian cell expression vectors encoding nanobody homodimers and assess antiviral activity using virus like particle surrogates at BSL-2 following plasmid transfection to drive intrabody expression, (2) we will engineer E. coli expression vectors encoding nanobody homodimers fused to cell penetrating peptides and glycosaminoglycan binding motifs and assess antiviral activity following protein transduction of virus infected cells at BSL-4. Success will demonstrate a novel antiviral strategy that can then be thoroughly explored for the propensity to select escape mutants relative to an existing neutralizing antibody regime to test whether the strategy is more “escape-proof”. The overall approach should be applicable to other human viral pathogens by carefully retuning the affinity reagent, with adequate time and resources, to maximize broad long-term impact in helping to safe guard human health.

摘要 目前的冠状病毒（CoV）大流行，其他CoV和其他“感冒”病毒的季节性感染， 再加上每年流感疫苗接种的需要，这就克服了病毒抗原漂移和转变带来的挑战。 病毒颗粒表面和/或表面展示的病毒结构蛋白的靶向景观 受感染细胞的生长一直是开发基于抗体的疗法的主要基础。虽然大 在试图鉴定这些表面展示蛋白的保守区域方面已经取得了进展 而且不太容易“逃脱”抗体结合，这似乎是一场猫和老鼠的持续战斗，就像我们一样。 看到冠状病毒“令人担忧的变体”的不断出现。相反，病毒结构蛋白， 在病毒颗粒和细胞内部，避免了周期性抗体选择的影响， 保守的这些蛋白质的寡聚体组装也可以减弱抗病毒逃逸突变的影响 这是由于亲本细胞中存在突变体和野生型单体的混合。此外，化学计量 阻止寡聚体功能所需的抗病毒剂不一定是1种抗病毒剂对1种单体， 特别是如果抗病毒药物是一种交联剂，其影响将超越直接接触， 相邻的低聚物。我们的长期假设是，能够结合内部 病毒属的所有物种的寡聚结构蛋白在存在时一致地将阻碍病毒装配 作为二聚体交联剂，其方式是既能够正向，又具有大大降低的对病毒的易感性， 逃跑我们将使用埃博拉病毒属的病毒探索二聚体交联剂的抗病毒潜力， 我们的模型和一个新的，罕见的纳米抗体表现出一致的反应性，所有6个物种的核蛋白。我们两 具体目标是：（1）设计编码纳米抗体同源二聚体的哺乳动物细胞表达载体 并在质粒转染后使用BSL-2的病毒样颗粒替代物评估抗病毒活性， 胞内抗体表达，（2）我们将工程化E.编码融合至 细胞穿透肽和糖胺聚糖结合基序，并评估蛋白质 在BSL-4处转导病毒感染的细胞。成功将证明一种新的抗病毒策略， 彻底探索相对于现有中和抗体选择逃逸突变体的倾向 这是一个新的制度，以测试该战略是否更“防逃”。整体方法应适用于其他 通过用足够的时间和资源仔细地重新调整亲和试剂， 最大限度地发挥广泛的长期影响，帮助保护人类健康。