Development of nanobody immunotherapeutics that prevent and treat gonorrhea

项目摘要/摘要 全球每年新增淋病病例约8700万例。2020年，向疾控中心报告的病例为677,769例， 与2009年的历史最低点相比，年发病率增加了111%。淋病奈瑟菌(Ng)对 几乎每一种临床使用的抗生素。对头孢曲松的耐药性报告--目前唯一推荐的一线治疗头孢曲松 来自几乎每个大洲的治疗预示着一个无法治愈的淋病时代。新型治疗方法的发展 预防Ng是全球公共卫生的优先事项。 我们的团队与Evaxion Biotech合作，已经确定了两种可能的细胞分裂蛋白(NGO0265和 NGO1549(FtsN))作为主要候选疫苗。NGO0265和FtsN在所有的Ng分离株中都有表达，并且FtsN是必需的 细菌生存能力。NGO0265和FtsN免疫小鼠可产生杀菌抗体 减少淋球菌在小鼠阴道定植的持续时间和负担。NG部署独一无二的免疫逃避 它的策略是利用其表面的脂寡糖(LOS)唾液酸转移酶(LST)和宿主- 衍生的CMP-唾液酸。LOS使Ng能够逃避补体、阳离子抗菌肽(CAMP)和 通过激活宿主的Siglec受体来下调炎症反应。淋球菌lst缺失突变株是 在小鼠身上减弱。到目前为止，所有已测序的淋球菌分离株都具有 功能性LST基因。 被称为VHH或纳米体的骆驼单域抗体已被发现具有独特的性质，可提供 巨大的通用性，促进了基于抗体的简单和经济疗法的快速发展。VHH- 基于产品可以利用VHH组件的小尺寸和高稳定性，它们结合构象的倾向- 依赖的中和表位，以及它们作为异源多聚体进行功能表达的可操作性。这些特点 使具有超高靶向亲和力、广泛的天然变体的商业上有利的抗体治疗药物成为可能 特殊性和绑定多个不同目标的能力。 在这里，我们的目标是开发针对NGO0265、FtsN和LST的纳米体疗法，作为辅助治疗 作为预防耐多药淋病的药物。在目标1中，我们将用纯化的重组羊驼免疫 NGO0265-NGO1549和LST嵌合，从免疫B细胞中创建纳米体展示噬菌体文库并鉴定 识别靶标上非重叠表位的纳米体具有高亲和力。纳米物体的能力 目标2将评估阻断酶功能或激活补体并在与Ig G Fc结合时杀死Ng。在目标3中， 我们将测试针对每个目标的先导抗Ng纳米抗体在一个 建立完善的小鼠阴道定植模型。 这项工作的成功完成将鉴定和表征具有抗多药耐药Ng活性的纳米体， 然后可以适应各种形式和药物输送平台。

PROJECT SUMMARY/ABSTRACT About 87 million new cases of gonorrhea occur worldwide annually. In 2020, 677,769 cases were reported to the CDC, a 111% increase in annual incidence since the historic low in 2009. Neisseria gonorrhoeae (Ng) has become resistant to almost every antibiotic in clinical use. Reports of resistance to ceftriaxone – the only currently recommended first-line of treatment – from almost every continent portends an era of untreatable gonorrhea. Development of novel treatments and preventives against Ng is a global public health priority. Our group, in collaboration with Evaxion Biotech, has identified two putative cell-division proteins (NGO0265 and NGO1549 (FtsN)) as lead vaccine candidates. NGO0265 and FtsN are expressed by all Ng isolates and FtsN is essential for bacterial viability. Immunization of mice with NGO0265 and FtsN elicits bactericidal antibodies (Abs) and significantly reduces the duration and burden of gonococcal colonization of mouse vaginas. Ng deploy a unique immune evasion strategy wherein it caps its lipooligosaccharide (LOS) with sialic acid using its surface LOS sialyltransferase (Lst) and host- derived CMP-sialic acid. LOS sialylation enables Ng to evade complement, cationic antimicrobial peptides (CAMPs) and down-regulate the inflammatory response by engaging host Siglec receptors. Gonococcal lst deletion mutants are attenuated in mice. Supporting its key role in pathogenesis, all gonococcal disease isolates sequenced thus far possess a functional Lst gene. Camelid single-domain antibodies called VHHs or nanobodies, have been found to possess unique properties that offer enormous versatility to facilitate the rapid development of simple and economical antibody-based therapeutics. VHH- based products can exploit the small size and high stability of VHH components, their propensity to bind conformation- dependent neutralizing epitopes, and their tractability for functional expression as heteromultimers. These characteristics make possible commercially favorable antibody therapeutic agents having ultrahigh target affinities, broad natural variant specificities and the ability to bind multiple different targets. Here, we aim to develop nanobody-based therapeutics against NGO0265, FtsN and Lst for use as an adjunctive treatment and as a preventive against multidrug-resistant gonorrhea. In Aim 1, we will immunize alpacas with purified recombinant chimeric NGO0265-NGO1549 and Lst, create a nanobody-display phage library from immune B cells and identify nanobodies that recognize non-overlapping epitopes on their targets with high-affinity. The ability of the nanobodies to block enzyme function or to activate complement and kill Ng when linked to IgG Fc will be evaluated in Aim 2. In Aim 3, we will test the ability of the lead anti-Ng nanobodies against each of the targets to clear gonococcal colonization in a well-established mouse vaginal colonization model. Successful completion of the work will identify and characterize nanobodies with activity against multidrug-resistant Ng, which can then be adapted to a variety of formats and drug delivery platforms.