Evaluation of Memory Responses and Biomarkers from a Phase 1 Enterotoxigenic Escherichia coli (ETEC) Intramuscular Subunit Vaccine with dmLT Adjuvant

产肠毒素大肠大肠杆菌（ETEC）是儿童、旅行者和 在风险地区部署军事人员。因此，疫苗的开发将有利于公众 健康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，进行了首次人体安全性和免疫原性1期疫苗试验（NCT 03404674）。 与LT-R192G/L211A组合的CS6亚基抗原CssBA的剂量递增肌内递送 （dmLT）。未报告严重不良事件，我们在几个队列中观察到强免疫原性， 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。 本提案的目的是完成对来自ETEC I期的血清和PBMC样本的分析 临床试验，并确定指导疫苗结果的关键生物标志物和分子机制。在 建议的研究，我们的目标是探索（1）疫苗剂量如何调节记忆，寿命的发展 （2）疫苗接种如何改变持久细胞免疫的发展; (3)在免疫细胞中由CssBA + dmLT刺激引发的早期信号传导事件是否可以作为免疫细胞的免疫应答。 免疫生物标志物;以及（4）免疫制剂的关键分子机制是什么， 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， 包括转录和代谢组学分析。此外，我们还将通过细胞分析来验证研究结果， 小鼠模型和来自相关ETEC 2b期疫苗试验的样品。这些发现将有助于 通过肠外途径开发人用ETEC疫苗，并提供了对以下机制的深入了解 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 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 complete 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 initiated by CssBA+dmLT stimulation in immune cells can serve as biomarkers of immunity; and (4) what are the key molecular mechanisms of the immunization formulation that 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. In addition, we will validate findings with cellular analyses, mouse models, and samples from a related ETEC Phase 2b vaccine trial. 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.