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.

项目摘要/摘要 全世界每年约有8700万新发淋病病例。2020年，向CDC报告了677，769例病例， 自2009年的历史低点以来，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。头孢曲松耐药报告-目前唯一推荐的一线抗生素 几乎每个大陆的淋病都预示着一个无法治愈的淋病时代的到来。开发新的治疗方法 和预防Ng是全球公共卫生的优先事项。 我们的团队与Evaxion Biotech合作，已经确定了两种假定的细胞分裂蛋白（NGO 0265和 NGO 1549（FtsN））作为主要候选疫苗。NGO 0265和FtsN由所有Ng分离株表达，并且FtsN对于NGO 0265和FtsN的表达是必需的。 细菌活力用NGO 0265和FtsN免疫小鼠可显著增强杀菌抗体（Abs）， 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，其中它使用其表面LOS唾液酸转移酶（Lst）和宿主- 衍生的CMP-唾液酸。LOS唾液酸化使Ng能够逃避补体、阳离子抗菌肽（CAMP）和 通过接合宿主Siglec受体来下调炎症反应。淋球菌lst缺失突变体是 在小鼠中减毒。支持其在发病机制中的关键作用，迄今为止测序的所有淋球菌病分离株都具有 功能性Lst基因 骆驼科动物单域抗体称为VHH或纳米抗体，已被发现具有独特的性质， 巨大的通用性，以促进简单和经济的基于抗体的治疗的快速发展。VHH- 基于VHH的产品可以利用VHH组分的小尺寸和高稳定性，它们结合构象的倾向， 依赖性中和表位，以及它们作为异多聚体功能表达的易处理性。这些特点 使具有非靶向亲和力、广泛的天然变体 特异性和结合多个不同靶标的能力。 在这里，我们的目标是开发针对NGO 0265，FtsN和Lst的基于纳米抗体的治疗方法，用于连续治疗 也可以预防耐多药淋病。目的一：用纯化的重组蛋白免疫羊驼， 嵌合NGO 0265-NGO 1549和Lst，从免疫B细胞中产生纳米抗体展示噬菌体文库，并鉴定 以高亲和力识别其靶标上的非重叠表位的纳米抗体。纳米抗体的能力， 阻断酶功能或激活补体并在与IgG Fc连接时杀死Ng的能力将在目的2中进行评价。在目标3中， 我们将测试针对每种靶标的前导抗Ng纳米抗体清除淋球菌定植的能力， 良好建立的小鼠阴道定殖模型。 这项工作的成功完成将鉴定和表征具有抗多药耐药Ng活性的纳米抗体， 其然后可适于多种形式和药物递送平台。