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

通过合成生物学构建 ETEC 抗腹泻减毒活疫苗

• 批准号: 8868927
• 负责人: John Robert Coleman
• 金额: $ 29.64万
• 依托单位: CODAGENIX, INC.
• 依托单位国家: 美国
• 财政年份: 2014
• 资助国家: 美国
• 起止时间: 2014-06-15 至 2017-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8868927
• 关键词: Adherence; Amino Acid Sequence; Animals; Antibodies; Antigens; Attenuated; Attenuated Live Virus Vaccine; Bacterial Vaccines; Child; Clinic; Code; Codon Nucleotides; Communicable Diseases; Derivation procedure; Development; Diagnostic; Diarrhea; Digestion; Disease; Ensure; Enteral; Enterotoxins; Enzyme-Linked Immunosorbent Assay; Epithelial Cells; Escherichia coli; Escherichia coli Infections; Escherichia coli Vaccines; Funding; Future; Gastrointestinal tract structure; Genes; Genome; Genomics; Health; Heating; Human; Human poliovirus; Immune; Immune response; Immunity; In Vitro; Individual; Infection; Influenza A virus; Intestinal Mucosa; Knock-out; Lethal Dose 50; Life; Lung; Methods; Mus; Phase; Pneumococcal vaccine; Polioviruses; Principal Investigator; Process; Production; Science; Serotyping; Small Business Technology Transfer Research; Software Design; Solutions; Streptococcus pneumoniae; Synthetic Genes; System; Targeted Toxins; Testing; Toxic effect; Toxin; Translation Process; Translations; Vaccination; Vaccines; Virulence; Virulence Factors; Virulent; acronyms; attenuation; base; combat; design; enteropathogenic Escherichia coli; enterotoxigenic Escherichia coli; field study; homologous recombination; immunogenicity; in vivo; killings; mouse model; novel; novel strategies; pathogen; programs; protein expression; prototype; research study; response; restriction enzyme; success; synthetic biology; vaccine candidate; vaccine development

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.

描述(由申请人提供)：鉴于产肠毒素大肠杆菌(ETEC)每年对全球2亿人的影响，开发对抗该病原体的疫苗至关重要；然而，需要一种全新的、非常规的方法。现场研究表明，对抗ETEC的惰性、基于抗原的疫苗“基本上没有效果”。因此，一种模拟自然感染的疫苗(即一种活的减毒株，但不是敲除株)将引起最强大的保护性反应。在首席研究人员最近利用合成生物学构建细菌疫苗的成功进展的基础上，基因设计软件将被用于对ETEC热不稳定(LT)和热稳定(ST)毒素进行“重新编码”，以显著降低但不能消除表达水平。这种“重新编码”的毒株未来可能成为一种减毒活疫苗，能够对ETEC免疫，ETEC是发展中国家最常见的腹泻原因。由PI开发的这种方法有望在ETEC中产生积极的结果，因为这种方法，特别是毒素定制，已经成功地用于开发一种新型的、活减毒肺炎链球菌(SP)候选疫苗(Coleman Jr等人)。JID 2011)。合成基因定制将实现构建一种能够对ETEC免疫的减毒活疫苗株，方法是将编码目标毒素的基因与合成基因交换，这些合成基因已被重新编码，以使用减慢翻译速度的密码子对。合成的重新设计将导致毒素表达减少，但不是完全消除(Coleman Jr，Science 2008)。以前的研究发现，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反应。PI发现，表达非破坏性水平毒素的合成细菌菌株的毒力甚至低于对照基因敲除菌株，这表明可能需要非常低水平的毒力因子产生才能诱导免疫，我们相信这一发现与ETEC疫苗的构建有关(Coleman Jr，JID 2011)。我们相信，类似的发现将出现在合成修饰的ETEC菌株中。ETEC的不耐热(LT)毒素需要附着和定植在肠粘膜上；因此，过去所有被杀死的和LT基因敲除菌株作为疫苗都是无效的(Johnson AM，J Bac。2009年；卡德里，F.疫苗，2004年)。此外，已知对第二次自然感染的保护主要归因于自然感染菌株的LT和ST毒素谱，而较少归因于该菌株的特定血清型(Johnson AM等人)。2009年)。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，这是已知的后续免疫所必需的。我们推测，合成修饰的ETEC产生这些毒素的低、亚致病水平将提供必要的宿主附着和诱导免疫反应，如SP所见。SAVE方法可以为ETEC疫苗领域几十年来一直寻求的问题提供解决方案--将LT和ST毒素包括在能够免疫刺激的水平，尽管水平足够低，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”的LT毒素的衍生品转化了ETEC H10407菌株，并在这里寻求资金，以支持更多菌株的构建，并在体外和体内对它们进行表征。该项目非常适合STTR项目，这是一项在第一阶段进行的明确实验，如果成功，将代表着一项重大进步，并将允许在第二阶段扩大规模。在第一阶段结束时，我们相信我们将拥有一种具有良好特性的减毒活疫苗株来对抗ETEC。在第二阶段，该菌株将在活体动物系统中进一步表征，该系统实际上模拟人类感染(小猪)，并可能在临床上使用。这项研究的结果 鉴于目前还没有针对ETEC的疫苗，可能会产生非常大的全球影响。我们寻求同时产生高影响的结果，同时扩大合理的基因设计对细菌病原体的适用性。