Using gonococcal TonB-dependent transporters as vaccine antigens

人类病原体淋病奈瑟氏菌(NGO)能够利用人类蛋白质作为铁(Fe)和锌(Zn)等必需金属的来源。利用人转铁蛋白(HTF)作为铁源的能力需要表达依赖于TonB的完整外膜转运蛋白(TbpA)和表面暴露的脂蛋白(TbpB)。NGO产生同源但不同的TonB依赖转运蛋白，使病原菌能够利用其他天然免疫蛋白作为唯一的锌来源。与允许使用HTF的蛋白质(TbpA和TbpB)一样，其他四种功能不足的TonB依赖转运蛋白(TdT)在奈瑟菌物种中保存良好，不受高频变异的影响，也不由人类宿主或其他非共生奈瑟菌细菌产生。目前的提案侧重于探索TDTs的疫苗潜力，最终目标是开发一种针对多种基本营养获取系统的多组分疫苗。最重要的假设是，由工程TdT和实验选择的佐剂组成的合理设计的疫苗将提供免疫交叉保护，防止不同的NGO毒株引起的定植和疾病。具体目标如下：目的1.特征不充分的TdT的结构、生物信息学和诱变分析。我们打算表征四个带有和不带有配体的TdT的结构，以指导突变和疫苗努力。我们将评估这些TdT在不同的NGO毒株中的存在和变异性。我们将对TdT进行突变，以消除配体结合功能。目的2.优化佐剂和给药方式以诱导特异性免疫应答。将对佐剂、给药路线、剂量和时间表进行测试，以获得最佳的小鼠抗TDT反应。免疫反应将通过广泛测试体液和细胞免疫因子来定义。目的3.检测TDT疫苗在新的人源化感染模型中的保护效果。优化的抗原、佐剂、路线和时间表将在人源化小鼠模型中进行测试，以保护下女性生殖道感染、盆腔炎、男性尿道炎和鼻咽感染。这些新的感染动物模型中的免疫学相关性和保护决定因素将被描述。目的4.评价合理设计的疫苗在所有感染模式中的交叉保护作用。将对优化、合理设计的疫苗配方进行测试，以防止广泛的NGO毒株，包括抗菌素耐药的“超级细菌”毒株。在所有人源化的小鼠感染模型中，将用这些菌株验证保护的免疫学相关性。这些研究意义重大，因为它们可能导致开发一种有效的疫苗，以对抗一种对现有治疗方法产生抗药性的顽固病原体。这些研究具有创新性，因为他们将使用结构引导的疫苗设计来开发与配体结合的无能疫苗抗原，并将在新的人源化小鼠感染模型中测试其保护作用。

The human pathogen, Neisseria gonorrhoeae (Ngo) is capable of utilizing human proteins as sources of necessary metals such as iron (Fe) and zinc (Zn). The ability to employ human transferrin (hTf) as an Fe source requires expression of a TonB-dependent, integral outer membrane transporter (TbpA), and a surface-exposed lipoprotein (TbpB). Homologous but distinct TonB-dependent transporters are produced by Ngo that enable the pathogen to utilize other innate immunity proteins as sole Zn sources. Like the proteins that enable use of hTf (TbpA and TbpB), the other four under-characterized TonB-dependent transporters (TdTs) are well-conserved among Neisseria species, are not subject to high-frequency variation and are not produced by the human host or other non-Neisseria commensal bacteria. The current proposal is focused on exploring the vaccine potential of the TdTs with the ultimate goal being a multi-component vaccine that targets multiple essential nutrient acquisition systems. The overarching hypothesis is that a rationally-designed vaccine consisting of engineered TdTs combined with experimentally-selected adjuvants will provide immunologic cross-protection against colonization and disease caused by diverse Ngo strains. The specific aims are as follows: Aim 1. Structural, bioinformatics and mutagenic analysis of under-characterized TdTs. We intend to characterize the structures of four TdTs with and without ligand to guide mutagenesis and vaccine efforts. We will assess the presence and variability of these TdTs among diverse Ngo strains. And we will mutagenize the TdTs to eliminate ligand-binding functions. Aim 2. Optimizing adjuvants and delivery modes for elicitation of specific immune responses. Adjuvants, delivery routes, doses and schedules will be tested for optimal anti-TdT responses in mice. Immunological responses will be defined by broadly testing humoral and cellular immunity factors. Aim 3. Test for protective efficacy of TdT-based vaccines in new humanized infection models. Optimized antigens, adjuvants, routes and schedules will be tested for protection in humanized mouse models of lower female genital tract infection, pelvic inflammatory disease, male urethritis and nasopharyngeal infection. Immunological correlates and determinants of protection in these new animal models of infection will be characterized. Aim 4. Evaluate rationally designed vaccines for cross-protection in all models of infection. Optimized, rationally-designed vaccine formulations will be tested for protection against a broad group of Ngo strains, including antimicrobial resistant “superbug” strains. Immunological correlates of protection will be validated with these strains in all of the humanized mouse models of infection. These studies are significant since they may lead to the development of an efficacious vaccine against a recalcitrant pathogen that has developed resistance to existing therapeutic methods. These studies are innovative because they will employ structure-guided vaccine design to develop ligand-binding incompetent vaccine antigens, which will be tested for protection in novel humanized mouse models of infection.