Live-attenuated ETEC Anti-Diarrhea Vaccine Construction via Synthetic Biology

描述（由申请人提供）：开发疫苗以对抗肠致病性大肠杆菌。大肠杆菌（ETEC）是至关重要的，因为病原体的全球影响，每年有2亿人;然而，需要一个全新的，非传统的方法。实地研究表明，对抗ETEC的惰性抗原疫苗“基本上没有效力”。因此，模拟自然感染的疫苗（即减毒活病毒株，但不是敲除病毒株）将引起最强的保护性应答。基于主要研究者最近成功开发的使用合成生物学构建细菌疫苗的进展，基因设计软件将用于“重新编码”ETEC热不稳定（LT）和热稳定（ST）毒素，以显着降低但不消除表达水平。这种“重新编码”的菌株在未来可以作为一种减毒活疫苗，能够免疫ETEC，这是发展中国家腹泻的最常见原因。PI开发的这种方法预期在ETEC中产生阳性结果，因为该方法（特别是毒素定制）已成功用于开发新型减毒活链球菌（SP）候选疫苗（科尔曼JR等人JID 2011）。合成基因定制将通过将编码靶毒素的基因与合成基因交换以使用减缓翻译速率的密码子对来实现能够对ETEC免疫的减毒活疫苗株的构建。合成重新设计将导致毒素表达减少，但不完全消除（科尔曼JR，Science 2008）。先前的研究发现，使用表达低水平合成毒力因子的SP菌株的毒力明显低于野生型，但可以诱导保护性免疫应答。PI发现，表达非损伤水平毒素的合成细菌菌株的毒力甚至低于对照敲除菌株，表明诱导免疫可能需要极低水平的毒力因子产生，我们认为该发现与ETEC疫苗构建相关（科尔曼JR，JID 2011）。我们相信在合成修饰的ETEC菌株中也会出现类似的发现。ETEC的热不稳定（LT）毒素是在肠粘膜上附着和定殖所必需的;因此，过去所有杀死的和LT敲除的菌株作为疫苗都是无效的（约翰逊AM，J Bac. 2009; Qadri，F. Vaccine 2004）。此外，已知针对第二次自然感染的保护作用主要归因于自然感染菌株的LT和ST毒素谱，而与菌株的特定血清型关系较小（约翰逊AM等人，2009）。因此，通过利用 除了LT和ST毒素基因的"重新编码"，人们可以构建分泌低水平野生型毒素的疫苗株，这是随后免疫所必需的。我们假设，合成修饰的ETEC产生低的亚致病水平的这些毒素将提供必要的宿主附着和诱导免疫应答，如SP中所见。SAVE方法可以为ETEC疫苗领域数十年来一直寻求的问题提供解决方案-以能够免疫刺激的水平包含LT和ST毒素，但足够低以避免毒性（腹泻）（Steinsland H. J Clin Microbiol 2004）。我们已经进行了初步的中试实验，并用一种"去优化" LT毒素的衍生物转化了ETEC H10407菌株，并在此寻求资金支持构建其他菌株并对其进行体外和体内表征。该项目非常适合STTR计划-第一阶段的定义实验，如果成功，将代表一个重大进展，并将允许在第二阶段扩展。在第一阶段结束时，我们相信我们将有一个良好的特点，减毒活疫苗株，以打击ETEC。在II期研究中，将在实际模拟人类感染的动物系统（仔猪）中以及可能在临床中进一步对该菌株进行体内表征。本研究结果 由于目前还没有针对ETEC的疫苗，因此可能会产生非常大的全球影响。我们寻求同时产生高影响力的结果，同时扩大合理的基因设计的细菌病原体的适用性。

DESCRIPTION (provided by applicant): The development of vaccine to combat Enterotoxigenic E. coli (ETEC) is vital given the pathogen's global impact on 200 million individuals annually; however, an entirely new, non-conventional approach is needed. Field studies have suggested that inert, antigen-based vaccines to combat ETEC have "essentially no efficacy". Therefore, a vaccine that mimics a natural infection (i.e. a live-attenuated, but not knockout strain) will elicit the most robust, protective response. Building on a recently successfu advance developed by the principal investigator that uses synthetic biology to construct bacterial vaccines, gene design software will be used to 're-code' the ETEC heat-labile (LT) and heat-stable (ST) toxins to have significantly decreased, but not eliminated, levels of expression. This 're-coded' strain could in the future serve as a live-attenuated vaccine capable of immunizing against ETEC, the most common cause of diarrhea in the developing world. This approach developed by the PI is expected to yield positive results in ETEC because the approach, specifically toxin customization, has been successfully utilized for the development of a novel, live-attenuated Streptococcus Pneumoniae (SP) vaccine candidate (Coleman JR et al. JID 2011). Synthetic gene customization will achieve the construction of a live-attenuated vaccine-strain capable of immunizing against ETEC by swapping the genes encoding the target toxin with synthetic genes that have been 're-coded' to use codon-pairs that slow the rate of translation. The synthetic re-design will result in decreased, but not entirely eliminated, toxin expression (Coleman JR, Science 2008). Previous findings using SP strains expressing low levels of synthetic virulence factors were significantly less virulent than the wild type, yet coul induce a protective immune response. The PI found that synthetic bacterial strains expressing non-damaging levels of toxin were even less virulent than a control knockout strain, suggesting very low levels of virulence factor production may be needed to induce immunity and we believe this finding is pertinent for ETEC vaccine construction (Coleman JR, JID 2011). We believe a similar finding will occur in synthetically modified ETEC strains. The heat-labile (LT) toxin of ETEC is required for attachment and colonization on the intestinal mucosa; thus all killed and LT knockout strains in the past have been ineffective as vaccines (Johnson AM, J Bac. 2009; Qadri, F. Vaccine 2004). Furthermore, it is known that protection against a second natural infection was attributed mostly to the LT and ST toxin profile of the naturally infecting strain an much less to the specific serotype of the strain (Johnson AM, et al. 2009). Therefore, by utilizing SAVE 're-coding' of the LT and ST toxin genes, one could construct vaccine strains that secrete low-levels of the wild type toxins, which are a known necessity for subsequent immunity. We hypothesize that synthetically modified ETEC producing low, sub-pathogenic levels of these toxins will provide the necessary host attachment and induction of the immune response as seen in SP. The SAVE approach could provide the solution to what has been sought by the ETEC vaccine field for decades - inclusion of the LT and ST toxins at levels capable of immune stimulation however low enough to avoid toxicity (diarrhea) (Steinsland H. J Clin Microbiol 2004). We have performed an initial pilot experiment and have transformed the ETEC H10407 strain with one derivation of a 'de-optimized' LT toxin and seek funds here to support the construction of additional strains and characterize them in vitro and in vivo. This project is ideally suited for the STTR program - a defined experiment in Phase I, which if successful represents a significant advance and will allow for expansion in Phase II. At the conclusion of this Phase I, we believe we will have a well-characterized live-attenuated vaccine strain to combat ETEC. In Phase II this strain will be further characterized in vivo in an animal system that actually mimics human infections (piglets) and possibly in the clinic. Results from this study could have a very large global impact given that currently there is no vaccine against ETEC. We seek to simultaneously yield high impact results while expanding the applicability of rational gene design to bacterial pathogens.