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.

抽象的 当前的冠状病毒（COV）大流行，其他COV和其他“冷”病毒的季节性感染， 再加上年度影响疫苗的需求体现了病毒抗原漂移和转移带来的挑战。 在病毒颗粒表面上显示的病毒结构蛋白的靶向景观 感染细胞的主要基础是开发基于抗体的治疗。虽然很棒 试图确定这些表面显示的蛋白质的区域已取得进步 而且不太容易“逃脱”抗体约束，这似乎是一场连续的猫和老鼠战斗 随着COV的持续出现“关注的变体”。相反，保留的病毒结构蛋白 内部病毒颗粒和细胞，避免选择周期性抗体的影响，并且往往更大 保守。这些蛋白质的寡聚组件也可以钝化抗病毒逃生突变的影响 由于存在于母细胞中存在的突变体和野生型单体的混合物。此外，静态测定法 抗病毒需要阻碍低聚物功能的必要 特别是如果抗病毒是一个交联，其影响将被直接接触到 相邻的低聚物。我们的长期假设是能够结合内部的亲和力试剂 当存在时，病毒属的所有种类的寡聚结构蛋白会均匀地妨碍病毒组装 作为二聚体交联的方式，既具有前进且对病毒的敏感性都大大降低 逃脱。我们将使用埃博拉病毒属的病毒作为二聚体交联的抗病毒潜力 我们的模型和一种新颖的罕见纳米病，表现出对所有6种的核蛋白的均匀反应性。我们的两个 具体目的是：（1）我们将设计编码纳米型均二聚体的哺乳动物细胞表达向量 并在质粒转染后使用病毒（如粒子替代物）等病毒评估抗病毒活性 内部表达，（2）我们将设计编码纳米型同型二聚体的大肠杆菌表达载体 细胞穿透性宠物和糖胺聚糖结合基序以及蛋白质后的评估抗病毒活性 在BSL-4处转导病毒感染细胞。成功将展示一种新颖的抗病毒策略，然后 要彻底探索，以便选择相对于现有中和抗体选择逃生突变体 测试该策略是否更“防逃生”的政权。总体方法应适用于其他方法 人类病毒病原体通过仔细用足够的时间和资源来重新调整亲和力试剂 最大程度地提高长期影响，以帮助保护人类健康。