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tonb依赖性整体外膜转运蛋白（TbpA）和表面暴露脂蛋白（TbpB）的表达。同源但不同的tonb依赖性转运蛋白由Ngo产生，使病原体能够利用其他先天免疫蛋白作为锌的唯一来源。与能够利用hTf的蛋白（TbpA和TbpB）一样，其他四种未充分表征的tonb依赖性转运蛋白（tdt）在奈瑟氏菌物种中保存良好，不受高频变异的影响，也不是由人类宿主或其他非奈瑟氏菌共生细菌产生的。目前的建议侧重于探索TdTs的疫苗潜力，最终目标是开发一种针对多种必需营养获取系统的多组分疫苗。总体假设是，合理设计的疫苗由工程TdTs与实验选择的佐剂相结合组成，将提供免疫交叉保护，防止定植和由多种非政府组织菌株引起的疾病。具体目标如下：目标1。tdt的结构、生物信息学和诱变分析。我们打算表征四种带配体和不带配体的tdt的结构，以指导突变和疫苗工作。我们将评估这些tdt在不同Ngo菌株中的存在性和可变性。我们将对tdt进行诱变以消除配体结合功能。目标2。优化佐剂和递送模式引发特异性免疫反应。佐剂、给药途径、剂量和时间表将在小鼠中测试最佳抗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。目标3。在新的人源感染模型中测试基于破伤风破伤风疫苗的保护效果。优化的抗原、佐剂、途径和时间表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进行保护试验。这些新的感染动物模型的免疫学相关因素和保护决定因素将被描述。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。将对优化的、合理设计的疫苗配方进行测试，以防范广泛的非政府组织菌株，包括耐抗生素的“超级细菌”菌株。保护的免疫学相关性将在所有人源化小鼠感染模型中与这些菌株进行验证。这些研究具有重要意义，因为它们可能导致开发针对对现有治疗方法产生耐药性的顽固病原体的有效疫苗。这些研究具有创新性，因为它们将采用结构引导的疫苗设计来开发配体结合不合格的疫苗抗原，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.