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

基本信息

批准号：
10192656
负责人：
MARGARET R MACDONALD
金额：
$ 21.19万
依托单位：
ROCKEFELLER UNIVERSITY
依托单位国家：
美国
项目类别：
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/10192656
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 Ixodidae 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 Serology 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 emerging pathogen 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 programs protein E response success tick-borne tick-borne flavivirus tick-borne virus tool virology virus envelope

项目摘要

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.

波瓦桑病毒 (POWV) 是一种蜱传黄病毒，在美国、加拿大和俄罗斯流行，在过去十年中流行率稳步上升。在一部分病例中，它会导致严重的、通常是致命的脑炎疾病。此外，恢复后，约 50% 的人会出现长期衰弱的神经系统后遗症。目前不存在针对病毒的治疗方案，治疗仅限于支持性护理。单克隆抗体自 1986 年以来已在临床上用于癌症、自身免疫和传染病控制，针对 POWV 的中和单克隆抗体疗法是一种潜在的治疗方法。纳米抗体，即在骆驼科动物中发现的纯重链抗体 (HcAb) 的变体结构域 (VHH)，最近已被医学研究界采用，其中几种纳米抗体目前正处于针对多种疾病和诊断的临床试验中。纳米抗体具有低免疫原性、高特异性、出色的稳定性和溶解度，并表现出卓越的功效、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美洲驼在其一生中可能接触过 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方法来鉴定和表征源自自然感染和免疫的骆驼科动物物种的新型 POWV 纳米抗体。在目标 1 中，将对羊驼和美洲驼的总血清进行 POWV 中和活性筛查。阳性动物将用重组包膜蛋白进行免疫，以增强抗体反应。通过亲和层析从血清显示最高中和潜力的动物中纯化 POWV 特异性 HcAb 群体并分离 15 kDa VHH 结构域。还将从这些相同的血清学阳性动物中提取骨髓，并通过 PCR 扩增 VHH 基因座。利用双管齐下的鉴定策略，VHH 蛋白池和 VHH 位点将分别通过 LC/MS 和 MiSeq 高通量测序进行表征。然后使用我们的 Llama-Magic 程序将基因座序列进行计算机翻译并与 MS 数据进行匹配，从而为 POWV 特异性纳米抗体提供完整的氨基酸序列。在目标 2 中，重组纳米抗体将在大肠杆菌中表达、纯化，并在体外测试 POWV 结合和中和以及与其他黄病毒的交叉反应性。这将产生一组有效的 POWV 特异性纳米抗体，可用于研究、诊断和治疗目的。未来的实验可以使用小鼠和非人类灵长类 POWV 脑炎模型来探索其治疗效果。