A novel and effective nanobody to prevent and treat Zika virus infection

摘要 寨卡病毒(Zika Virus，ZIKV)是一种最新出现的引起世界关注的传染病病毒。寨卡病毒感染已导致 严重疾病，包括神经和先天性寨卡病毒综合征，表现为小头畸形症和 胎儿损伤、胎儿和幼崽死亡、大脑异常和其他畸形。寨卡病毒可能会感染孕妇 胎儿并持续存在于神经元和生殖组织中数月，导致严重的组织 损坏。目前，还没有可用的治疗剂来预防和治疗寨卡病毒引起的严重 人类的疾病，呼吁开发人类使用的新型抗寨卡病毒疗法。ZIKV信封 (E)蛋白质是关键的治疗和疫苗目标。ZIKV E特异性中和单抗(MAbs) 正在进行临床前和/或临床测试，但其中一些未能完全停止垂直 ZIKV的传播。此外，常规的单抗通常体积较大、结构复杂。 结构不稳定、行为不稳定和/或组织渗透性差。相反，纳米体(Nb)是单一结构域 骆驼重链抗体的可变区的抗体，它表现出如下独特的性质 稳定性和特异性强，结合亲和力高，基于其体积小(~15)具有良好的组织渗透性 KDA)，使其成为预防和治疗人类疾病的一种有吸引力的治疗工具。我们之前的研究已经 为设计和开发新型、高效的NBS奠定了技术平台 东方呼吸综合征冠状病毒(MERS-CoV)。与常规单抗相比，这些抗MERS冠状病毒 NBS表现出显著增强的稳定性和半衰期。我们还展示了ZIKV E的能力 在我们已建立的动物模型中，以蛋白质为基础的疫苗和治疗药物可以预防ZIKV感染。在……里面 拟议的研究，我们假设，ZIKV E特定的NBS，经过适当的工程，将提出 半衰期延长，稳定性极佳，组织渗透性好，无毒性，改善了对 寨卡病毒感染。具体目标是(1)设计ZIKV E专用NBS，以生成人性化的NBS 具有延长的半衰期和增强的疗效，(2)表征这些NBS的稳定性和广泛的组织 无毒通透性；(3)体外交叉中和活性和体内疗效评价。 抗ZIKV感染的特异性NBS。在这笔赠款的有效期内，总的目标是迅速产生 新型高效抗ZIKV NB，半衰期长，稳定性强，组织渗透性好， 提高预防寨卡病毒感染的效力。即使ZIKV会导致严重的胎儿损伤和死亡，而且 在神经元和生殖组织中持续长达数月，严重损害，属性和 最终和充分发展的NB的特性预计将有助于其完全 防止寨卡病毒的垂直传播并将其从感染组织中清除，导致显著减少 与寨卡病毒相关的小头畸形、脑畸形、其他畸形和组织损伤。 好了！

Abstract Zika virus (ZIKV) is the latest emerging infectious virus raising worldwide attention. ZIKV infection has led to severe diseases, including neurological and congenital Zika syndromes, as represented by microcephaly with fetal damage, fetal and pup death, brain abnormalities, and other malformations. ZIKV may infect pregnant fetuses and persistently exist in neuronal and reproductive tissues for months, resulting in severe tissue damage. Currently, there are no therapeutic agents available to prevent and treat ZIKV-caused severe diseases in humans, calling for the development of novel anti-ZIKV therapeutics for human use. ZIKV envelope (E) protein is a key therapeutic and vaccine target. ZIKV E-specific neutralizing monoclonal antibodies (mAbs) are being developed preclinically and/or tested clinically, but some of them have failed to fully stop vertical transmission of ZIKV. Moreover, the conventional mAbs usually have relatively large size, complicated structure, unstable behavior, and/or poor tissue penetration. In contrast, a nanobody (Nb) is a single-domain antibody of the variable domain of Camelid heavy-chain antibody, and it presents such unique properties as strong stability and specificity, high binding affinity, and good tissue penetration based on its small size (~15 kDa), making it an attractive therapeutic tool to prevent and treat human diseases. Our previous studies have established the technological platform for designing and developing novel and effective Nbs against Middle East respiratory syndrome coronavirus (MERS-CoV). Compared to conventional mAbs, these anti-MERS-CoV Nbs showed significantly enhanced stability and half-life. We have also demonstrated the ability of ZIKV E protein-based vaccines and therapeutics to protect against ZIKV infection in our established animal models. In the proposed study, we hypothesize that ZIKV E-specific Nbs, after appropriate engineering, will present extended half-life, excellent stability, good tissue penetration without toxicity, and improved efficacy against ZIKV infection. The specific aims are to (1) engineer ZIKV E-specific Nbs for generation of humanized Nbs with extended half-life and enhanced efficacy, (2) characterize these Nbs for stability and broad tissue permeability without toxicity, and (3) evaluate in vitro cross-neutralizing activity and in vivo efficacy of ZIKV E- specific Nbs against ZIKV infection. Within the tenure of this grant, the overall goal is to rapidly generate a novel and effective anti-ZIKV Nb with extended half-life, strong stability, excellent tissue penetration, and increased efficacy to prevent ZIKV infection. Even though ZIKV causes severe fetal damage and demise, and persists in neuronal and reproductive tissues for up to months with severe damage, the properties and characteristics of the final and fully developed Nb is expected to contribute to its efficacy in completely preventing the vertical transmission of ZIKV and clear it from infected tissues, leading to a significant reduction of ZIKV-associated microcephaly, brain abnormalities, other malformations and tissue damage. !