Development of nanobody immunotherapeutics that prevent and treat gonorrhea

开发预防和治疗淋病的纳米抗体免疫疗法

• 批准号: 10753164
• 负责人: SANJAY RAM
• 金额: $ 26.57万
• 依托单位: UNIV OF MASSACHUSETTS MED SCH WORCESTER
• 依托单位国家: 美国
• 财政年份: 2023
• 资助国家: 美国
• 起止时间: 2023-06-02 至 2025-05-31
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10753164
• 关键词: Affect; Affinity; Alpaca; Antibiotic Resistance; Antibiotics; Antibodies; Antibody Therapy; Antigens; Antimicrobial Cationic Peptides; Attenuated; B-Lymphocytes; Bacteria; Binding; Binding Proteins; Biotechnology; Ceftriaxone; Cell division; Cells; Characteristics; Chlamydia; Clinical; Collaborations; Complement; Complement Factor H; Complement Membrane Attack Complex; Cytidine Monophosphate N-Acetylneuraminic Acid; Development; Disease; Drug Delivery Systems; Drug-resistant Neisseria Gonorrhoeae; Ectopic Pregnancy; Enzymes; Epitopes; Flow Cytometry; Genes; Goals; Gonorrhea; HIV; Human; IgG1; Immune; Immune Evasion; Immunization; Immunize; Immunoglobulin G; Immunotherapeutic agent; In Vitro; Incidence; Individual; Infection; Infertility; Inflammatory Response; Lead; Libraries; Link; Mediating; Messenger RNA; Microbe; Modeling; Molecular Conformation; Multi-Drug Resistance; Mus; Mutation; Neisseria gonorrhoeae; Organism; Pathogenesis; Pathway interactions; Persons; Phage Display; Predisposition; Prevention; Preventive; Property; Proteins; Reaction; Recombinants; Recommendation; Reporting; Resistance; Sexually Transmitted Diseases; Sialic Acids; Sialyltransferases; Specificity; Surface; Technology; Testing; Therapeutic; Therapeutic Agents; Therapeutic antibodies; Topical application; United States; Vagina; Vaginal Ring; Variant; Woman; Work; antitoxin; bactericide; chronic pelvic pain; drug-resistant gonorrhea; efficacy testing; hyperimmunization; immune clearance; improved; in silico; in vivo; infection burden; inhibitor; lipooligosaccharide; mucosal site; mutant; nanobodies; novel; prevent; public health priorities; receptor; screening; sialic acid binding Ig-like lectin; sialylation; synergism; technology platform; transmission process; trend; vaccine candidate

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.

项目概要/摘要 2020 年，全球新增淋病病例约 8700 万例，美国疾病控制和预防中心 (CDC) 报告了 677,769 例病例。 自 2009 年历史最低点以来，年发病率增加了 111%。淋病奈瑟菌 (Ng) 已对 几乎所有临床使用的抗生素都有头孢曲松耐药的报告——头孢曲松是目前唯一推荐的一线药物。 治疗——几乎每个大陆都预示着一个无法治愈的淋病新疗法的开发时代。 预防 Ng 是全球公共卫生的优先事项。 我们的小组与 Evaxion Biotech 合作，鉴定了两种假定的细胞分裂蛋白（NGO0265 和 NGO1549 (FtsN)) 作为主要候选疫苗 NGO0265 和 FtsN 在所有 Ng 分离株中表达，并且 FtsN 对于 NGO0265 和 FtsN 至关重要。 用 NGO0265 和 FtsN 免疫小鼠可引发杀菌抗体 (Abs)，并显着提高细菌活力。 减少淋球菌在小鼠阴道定植的持续时间和负担，Ng 部署了独特的免疫逃避。 策略：它利用其表面 LOS 唾液酸转移酶 (Lst) 和宿主，用唾液酸对其脂寡糖 (LOS) 进行加帽 衍生的 CMP-唾液酸使 Ng 能够逃避补体、阳离子抗菌肽 (CAMP) 和 通过接合宿主淋球菌 lst 缺失突变体来下调炎症反应。 迄今为止已测序的所有淋球菌疾病分离株都具有在小鼠体内减弱的证据。 功能性 Lst 基因。 骆驼科单域抗体（称为 VHH 或纳米抗体）被发现具有独特的特性，可提供 巨大的多功能性有助于快速开发简单且经济的基于抗体的 VHH-疗法。 基于 VHH 的产品可以利用 VHH 成分的小尺寸和高稳定性，以及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易处理性。 使具有超高靶亲和力、广泛天然变体的商业上有利的抗体治疗剂成为可能 特异性和结合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 的基于纳米抗体的疗法，用作辅助治疗 在目标 1 中，我们将用纯化的重组体对羊驼进行免疫接种，以预防多重耐药性淋病。 嵌合 NGO0265-NGO1549 和 Lst，从免疫 B 细胞创建纳米抗体展示噬菌体库并鉴定 纳米抗体能够以高亲和力识别其靶标上的非重叠表位。 当与 IgG Fc 连接时阻断酶功能或激活补体并杀死 Ng 将在目标 2 中进行评估。在目标 3 中， 我们将测试先导抗 Ng 纳米抗体针对每个靶标清除淋球菌定植的能力 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重耐药性 Ng 活性的纳米抗体， 然后可以适应各种形式和药物输送平台。