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: 8781197
• 负责人: John Robert Coleman
• 金额: $ 30万
• 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 2016-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8781197
• 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; 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; public health relevance; 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）：鉴于该病原体每年影响全球2亿人，开发对抗产肠毒素大肠杆菌（ETEC）的疫苗至关重要；然而，需要一种全新的、非传统的方法。实地研究表明，用于对抗ETEC的惰性、基于抗原的疫苗“基本上没有效力”。因此，模仿自然感染的疫苗（即减毒活株，但不是敲除毒株）将引起最强烈的保护性反应。在首席研究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（但不是消除）表达水平。这种“重新编码”的菌株将来可能作为一种减毒活疫苗，能够免疫ETEC，这是发展中国家最常见的腹泻原因。PI开发的这种方法有望在ETEC中产生积极的结果，因为该方法，特别是毒素定制，已成功用于开发一种新型减毒肺炎链球菌（SP）候选疫苗（Coleman JR等）。JID 2011)。合成基因定制将实现构建能够免疫ETEC的减毒活疫苗株，方法是将编码目标毒素的基因与已被“重新编码”的合成基因交换，使用减缓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）。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反应。PI发现，表达非破坏性毒素水平的合成菌株的毒性甚至低于对照敲除菌株，这表明诱导免疫可能需要非常低水平的毒力因子产生，我们认为这一发现与ETEC疫苗构建相关（Coleman 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）毒素需要在肠粘膜上附着和定植；因此，过去所有灭活株和LT敲除株作为疫苗都是无效的（Johnson AM, J Bac. 2009; Qadri， F. Vaccine 2004）。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，而较少归因于菌株的特定血清型（Johnson AM, et al. 2009）。因此，通过利用