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

波瓦桑病毒（POWV）是一种美国、加拿大和俄罗斯特有的蜱传黄病毒，在过去十年中流行率稳步上升。在一小部分病例中，它会导致严重的、通常是致命的脑病。此外，在康复后，约50%的个体会出现长期衰弱的神经系统后遗症。目前尚无针对病毒的治疗方案，因此治疗仅限于支持性护理。自1986年以来，单克隆抗体已在临床上用于癌症、自身免疫和传染病控制，中和单克隆抗体治疗是一种潜在的治疗方法。纳米体，或在骆驼物种中发现的仅重链抗体（HcAb）的变异结构域（VHH），最近已被医学研究界采用，其中一些目前正在进行临床试验，用于各种疾病和诊断。纳米体具有低免疫原性、高特异性、卓越的稳定性和溶解度，以及卓越的疗效、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大羊驼在其一生中可能暴露于POWV，因此可能含有中和抗体。本研究结合病毒学、分子生化和流行病学方法，鉴定和表征来自自然感染和免疫的骆驼物种的新型POWV纳米体。在目标1中，将筛选羊驼和美洲驼的总血清，以检测POWV中和活性。阳性动物将用重组包膜蛋白免疫，以增强抗体反应。从具有最高中和潜力的动物血清中，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中提取骨髓，并通过PCR扩增VHH位点。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。然后将位点序列在计算机上翻译，并使用我们的程序lama- magic与MS数据匹配，为powv特异性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特异性纳米体，可用于研究、诊断和治疗目的。未来的实验可以在小鼠和非人灵长类动物的POWV脑炎模型上探索其治疗效果。

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.