Using gonococcal TonB-dependent transporters as vaccine antigens

人类病原体淋病奈瑟菌（Ngo）能够利用人类蛋白质作为必需金属如铁（Fe）和锌（Zn）的来源。采用人转铁蛋白（hTf）作为铁源的能力需要表达的TonB依赖性，完整的外膜转运蛋白（TbpA），和表面暴露的脂蛋白（TbpB）。Ngo产生同源但不同的TonB依赖性转运蛋白，使病原体能够利用其他先天免疫蛋白作为唯一的锌源。与能够使用hTf的蛋白质（TbpA和TbpB）一样，其他四种未充分表征的TonB依赖性转运蛋白（TdT）在奈瑟氏菌属物种中非常保守，不受高频变异的影响，也不由人类宿主或其他非奈瑟氏菌细菌产生。目前的提案集中于探索TDT的疫苗潜力，最终目标是针对多种必需营养素获取系统的多组分疫苗。总体假设是，由工程化TdT与实验选择的佐剂组合组成的合理设计的疫苗将提供针对由不同Ngo菌株引起的定殖和疾病的免疫交叉保护。具体目标如下：目标1。未充分表征的TdT的结构、生物信息学和诱变分析。我们打算表征四个TDT的结构，有和没有配体，以指导诱变和疫苗的努力。我们将评估不同Ngo菌株中这些TdT的存在和变异性。我们将诱变TdT以消除配体结合功能。目标2.优化佐剂和递送模式以引发特异性免疫应答。将测试佐剂、递送途径、剂量和时间表以获得小鼠中的最佳抗TdT应答。免疫应答将通过广泛测试体液和细胞免疫因子来定义。目标3.在新的人源化感染模型中测试基于TdT的疫苗的保护效力。将在女性下生殖道感染、盆腔炎、男性尿道炎和鼻咽感染的人源化小鼠模型中测试优化的抗原、佐剂、途径和时间表的保护作用。免疫学相关因素和决定因素的保护，在这些新的动物模型的感染将进行表征。目标4。评估合理设计的疫苗在所有感染模型中的交叉保护作用。将测试优化的、合理设计的疫苗制剂对广泛的Ngo菌株的保护作用，包括抗微生物耐药的“超级细菌”菌株。将在所有人源化小鼠感染模型中用这些菌株验证保护的免疫学相关性。这些研究是重要的，因为它们可能导致针对已对现有治疗方法产生抗性的寄生虫病原体的有效疫苗的开发。这些研究具有创新性，因为它们将采用结构指导的疫苗设计来开发配体结合缺陷的疫苗抗原，这些抗原将在新型人源化小鼠感染模型中进行保护测试。

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.