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

产肠毒素大肠杆菌(ETEC)是引起儿童、旅行者和儿童细菌性感染性腹泻的主要原因 在危险地区部署军事人员。因此，疫苗的开发对公众是有利的 健康。一种策略是使用定居因子的亚基与不耐热毒素(LT)的类毒素相结合。 最近，首次进行了人类安全和免疫原性第一阶段疫苗试验(NCT03404674)。 CS6亚单位抗原CSSBA与LT-R192G/L211A联合肌肉注射的剂量递增 (Dmlt)。没有报告严重的不良反应，我们在几个队列中观察到了很强的免疫原性， 与dmLT剂量显著相关。然而，对临床试验样本的完整分析，包括体液和细胞 记忆力不足。正如本疫苗试验表明的那样，dmLT不仅是一种LT类毒素，而且是一种有效的佐剂， 激发对联合递送抗原的免疫力；然而，我们对分子的理解存在差距 与dmLT共同传递的抗原启动疫苗接种结果的机制。 这项建议的目标是完成对ETEC第一阶段的血清和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 我们提出的研究旨在探索(1)疫苗剂量如何调节记忆力、寿命的发育 和体液反应的多样性；(2)疫苗接种如何改变持久细胞免疫的发展； (3)CSSBA+dmLT刺激在免疫细胞中启动的早期信号事件是否可以作为 免疫的生物标志物；以及(4)免疫制剂的关键分子机制是什么 塑造疫苗接种结果。 为此，我们将分析我们存储的临床试验样本并执行一些复杂的分析， 包括转录和代谢组学分析。此外，我们将用细胞分析来验证研究结果， 小鼠模型，以及相关ETEC 2b期疫苗试验的样本。这些发现将有助于 通过肠外途径开发人类使用的ETEC疫苗，并提供对 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.