Powassan virus nanobodies from camelid monomeric variable antibody domains elicited by natural infection

自然感染引起的骆驼单体可变抗体域的波瓦桑病毒纳米抗体

• 批准号: 10043364
• 负责人: MARGARET R MACDONALD
• 金额: $ 25.43万
• 依托单位: ROCKEFELLER UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2020
• 资助国家: 美国
• 起止时间: 2020-06-15 至 2022-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10043364
• 关键词: Adopted; Affinity; Affinity Chromatography; Alpaca; Amino Acid Sequence; Animals; Antibodies; Antibody Response; Arboviruses; Area; Autoimmunity; Bacteria; Base Sequence; Binding; Biochemical; Biological; Biological Assay; Biological Products; Black-legged Tick; Bone Marrow; Camels; Canada; Cessation of life; Chemicals; Clinical; Clinical Trials; Communicable Diseases; Communities; Computer software; DNA Library; Data; Development; Diagnostic; Disease; E protein; Encephalitis; Environment; Enzyme-Linked Immunosorbent Assay; Epidemiology; Escherichia coli; Exposure to; Family; Fc domain; Flaviviridae; Flavivirus; Future; Goals; Great Lakes Region; High-Throughput Nucleotide Sequencing; Human; Immunity; Immunization; Immunize; In Vitro; Individual; Infection; Ixodes; Laboratories; Life; Llama; Longevity; Lymphocyte; Magic; Malignant Neoplasms; Mammals; Massachusetts; Medical; Medical Research; Methods; Modeling; Molecular; Monoclonal Antibodies; Monoclonal Antibody Therapy; Mus; Neurologic; Outcome; Penetrance; Peptides; Population; Powassan virus; Prevalence; Proteins; Publishing; RNA; Reagent; Recombinants; Recovery; Reporter; Research; Role; Russia; Safety; Series; Serological; Solubility; Specificity; Structure; Supportive care; System; Tertiary Protein Structure; Testing; Therapeutic; Therapeutic Agents; Therapeutic Uses; Ticks; Tissues; Translating; Treatment Efficacy; United States; Vaccination; Variant; Viral Antigens; Virion; Virus; Virus Diseases; base; cancer therapy; cost; cross reactivity; disorder control; env Gene Products; experimental study; immunogenicity; in silico; liquid chromatography mass spectrometry; medical attention; member; mosquito-borne pathogen; nanobodies; neutralizing antibody; neutralizing monoclonal antibodies; nonhuman primate; novel; novel virus; pathogen; programs; protein E; response; success; tick-borne; tool; virology; virus envelope

Powassan virus (POWV), a tick-borne flavivirus endemic to the USA, Canada, and Russia, has steadily increased in prevalence over the past decade. In a subset of cases, it leads to severe, often fatal, encephalitic disease. Furthermore, following recovery, long-term debilitating neurological sequelae occur in ~50% of individuals. No virus-specific therapeutic options currently exist, restricting treatment to supportive care. Monoclonal antibodies have been used clinically since 1986 for cancer, autoimmunity and infectious disease control and neutralizing monoclonal antibody therapy for POWV is one potential treatment approach. Nanobodies, or the variant domains (VHH) of heavy-chain only antibodies (HcAb) found in camelid species, have recently been adopted by the medical research community, with several currently in clinical trials for a variety of diseases and diagnostics. Nanobodies have low immunogenicity, high specificity, and exceptional stability and solubility, as well as demonstrated superior efficacy, safety, and utility, making them strong candidates for future therapeutics. Serological data suggests that many alpacas and llamas in the NE USA may be exposed to POWV during their lifespan, and therefore likely harbor neutralizing antibodies. This study combines virological, molecular biochemical, and epidemiological approaches to identify and characterize novel POWV nanobodies derived from naturally infected and immunized camelid species. In Aim 1, total sera from alpacas and llamas will be screened for POWV neutralization activity. Positive animals will be immunized with recombinant envelope protein in order to boost antibody responses. From animals whose sera show the highest neutralization potential, POWV- specific HcAb populations will be purified by affinity chromatography and the 15 kDa VHH domains isolated. Bone marrow will also be extracted from these same serologically positive animals and the VHH loci will be amplified by PCR. Utilizing a two-pronged identification strategy, VHH protein pools and VHH loci will be characterized by LC/MS and MiSeq high-throughput sequencing, respectively. Loci sequences will then be translated in silico and matched to the MS data using our program Llama-Magic, providing complete amino acid sequences for POWV-specific nanobodies. In Aim 2, recombinant nanobodies will be expressed in E. coli, purified, and tested in vitro for POWV binding and neutralization as well as cross reactivity to other flaviviruses. This will yield a panel of potent POWV-specific nanobodies that will be useful for research, diagnostic and therapeutic purposes. Future experiments could explore their therapeutic efficacy using murine and non-human primate models of POWV encephalitis.

鲍瓦桑病毒(POWV)是一种扁虱传播的黄病毒，流行于美国、加拿大和俄罗斯，在过去十年中流行率稳步上升。在一些病例中，它会导致严重的、往往是致命的脑炎疾病。此外，随着康复，约50%的人会出现长期的衰弱神经后遗症。目前没有针对病毒的治疗选择，将治疗限制在支持性护理上。自1986年以来，单抗已在临床上用于癌症、自身免疫和传染病控制，中和单抗治疗POWV是一种潜在的治疗方法。纳米抗体，或在骆驼物种中发现的重链抗体(HcAb)的变异域(VHH)，最近已被医学研究界采用，目前有几个正在进行各种疾病和诊断的临床试验。纳米抗体具有低免疫原性、高特异性、优异的稳定性和溶解性，并表现出优异的有效性、安全性和实用性，使其成为未来治疗的有力候选者。血清学数据表明，美国东北部的许多羊驼和骆驼在其生命周期内可能接触到POWV，因此可能含有中和抗体。这项研究结合了病毒学、分子生化和流行病学的方法来鉴定和表征来自自然感染和免疫的骆驼物种的新型POWV纳米抗体。在目标1中，将对羊驼和骆驼的总血清进行POWV中和活性的筛选。阳性动物将用重组包膜蛋白免疫，以增强抗体反应。从血清具有最高中和潜力的动物中，将通过亲和层析纯化POWV特异性HcAb群体，并分离出15 kDa的VHH结构域。还将从这些血清学阳性的动物身上提取骨髓，并通过聚合酶链式反应扩增VHH基因座。利用双管齐下的鉴定策略，VHH蛋白库和VHH基因座将分别通过LC/MS和MiSeq高通量测序进行表征。然后，基因座序列将被翻译成电子计算机，并使用我们的程序Llama-Magic与MS数据进行匹配，为POWV特定的纳米体提供完整的氨基酸序列。在目标2中，重组纳米体将在大肠杆菌中表达、纯化，并在体外测试POWV的结合和中和以及与其他黄病毒的交叉反应。这将产生一组有效的POWV特异性纳米体，将用于研究、诊断和治疗目的。未来的实验可能会利用POWV脑炎的小鼠和非人类灵长类动物模型来探索它们的治疗效果。