Evaluation of Memory Responses and Biomarkers from a Phase IEnterotoxigenic Escherichia coli (ETEC) Intramuscular SubunitVaccine with dmLT Adjuvant

产肠毒素大肠大肠杆菌（ETEC）是儿童、旅行者和部署人员细菌感染性腹泻的主要原因 危险地区的军事人员。因此，疫苗的开发将有利于公共卫生。 一种策略是使用定殖因子的亚基与不耐热毒素（LT）的类毒素组合。最近， 进行首次人体安全性和免疫原性1期疫苗试验（NCT 03404674）。剂量- 与LT-R192 G/L211 A（dmLT）组合的CS6-亚基抗原CssBA的递增肌内递送。没有 报道了严重的不良事件并且我们在几个组群中观察到了强的体液免疫原性， 与dmLT剂量显著相关。然而，临床试验样本的完整分析，包括体液和细胞 记忆是缺乏的。正如该疫苗试验所表明的，dmLT不仅是LT类毒素，而且是一种有效的佐剂， 刺激对共同递送的抗原的免疫;然而，在我们对分子生物学的理解中存在差距。 负责启动与dmLT共同递送的抗原的疫苗接种结果的机制。 本提案的目的是扩大对ETEC I期临床研究中血清和PBMC样本的分析 试验，并确定指导疫苗结果的关键生物标志物和分子机制。拟议 我们的研究旨在探索（1）疫苗接种剂量如何调节记忆，寿命和多样性的发展 （2）疫苗接种如何改变持久细胞免疫的发展;（3）早期是否 信号事件可以作为免疫的生物标志物;（4）免疫过程中的分子机制 影响疫苗接种效果。 为此，我们将分析我们储存的临床试验样本，并进行一些复杂的分析， 包括使用来自相关ETEC 2b期疫苗的样本进行转录和代谢组学分析- 有效性试验，以确定疫苗诱导保护的特征，然后我们将使用I期试验进行验证 样品此外，我们将通过细胞分析和小鼠模型验证研究结果。这些发现将有助于 随着通过胃肠外途径用于人用的ETEC疫苗的开发， 深入了解指导疫苗接种结果的关键事件。

Enterotoxigenic E. coli (ETEC) is a major cause of bacterial infectious diarrhea in children, travelers and deployed military personnel in risk areas. As such, development of a vaccine would be advantageous for public health. One strategy is to use subunits of colonization factors combined with toxoids of heat-labile toxin (LT). Recently, a first-in-humans safety and immunogenicity Phase 1 vaccine trial (NCT03404674) was conducted. with dose- escalating intramuscular delivery of CS6-subunit antigen CssBA combined with LT-R192G/L211A (dmLT). No serious adverse events were reported and we observed strong humoral immunogenicity in several cohorts, notably related to dmLT dose. Yet a complete analysis of clinical trial samples, including humoral and cellular memory is lacking. As this vaccine trial indicates, dmLT is not only an LT toxoid but also a potent adjuvant that stimulates immunity to co-delivered antigens; however, there is a gap in our understanding of molecular mechanisms responsible for initiating vaccination outcomes with antigens co-delivered with dmLT. The objective of this proposal is to expand analysis on serum and PBMC samples from an ETEC Phase 1 clinical trial and to define the key biomarkers and molecular mechanisms directing vaccine outcomes. In the proposed studies we aim to explore (1) how vaccination doses modulated development of memory, longevity and diversity of the humoral response; (2) how vaccination altered development of durable cellular immunity; (3) whether early signaling events can serve as biomarkers of immunity; and (4) what molecular mechanisms during immunization shape vaccination outcomes. To do so we will analyze our stored clinical trial samples and perform a number of sophisticated analyses, including transcriptional and metabolomics assays using samples from a related ETEC Phase 2b vaccine- efficacy trail to define signatures of vaccine induced protection which we will then validate using the Phase 1 trial samples. In addition, we will validate findings with cellular analyses and mouse models. These findings will help with the development of an ETEC vaccine for human use by parenteral route and also provide mechanistic insight into key events directing vaccination outcomes.