Collaborative Research: Cell-free glycoprotein synthesis technology for point-of-care vaccine biomanufacturing

耐药细菌对人类健康的威胁越来越大。到2050年，每年可能有多达1000万人的生命处于危险之中。需要新的战略来应对这一威胁。已经开发出了安全有效地预防危险细菌感染的疫苗。该项目寻求解决目前疫苗生产中的限制。将开发可轻松扩大规模的疫苗生产的无细胞技术。这可能导致便携式、按需疫苗的开发和生产。该项目将推进结合疫苗的生物制造。这将改善资源贫乏社区的可获得性。同时，将开发实践学习模块，并将其提供给人数不足的高中生、本科生和研究生。细菌细胞表面装饰着复杂的糖结构。这些物质包括胶囊多糖(CPS)和O-多糖(O-PS)。这些结构是重要的毒力因子、黏附介质和免疫调节剂。CPS和O-PS结构通常不存在于宿主细胞表面。因此，这些多糖可以作为疫苗亚单位，用于保护人类免受危及生命的细菌感染。结合疫苗是一种亚单位疫苗，将多糖抗原连接到蛋白质载体上。它们是诱导对致病菌产生免疫力的最安全和最有效的方法之一。目前的生产技术复杂，依赖活细胞。冷藏在分销链上的每一步都是必要的。因此，制造业是集中的、资本密集型的，需要高技能的劳动力。该项目将创造一种可扩展的、无细胞的生物合成技术，用于产生结合疫苗候选。实验和计算方法将结合起来，开发和优化一锅无细胞糖蛋白合成系统。这些糖偶联物可以冷冻干燥的形式储存，只需加水即可重组。一项补充技术，鸟枪扫描糖突变，将能够全面评估糖基化效率作为连接部位和抗原负载密度的函数，这将与疫苗免疫原性相关。该奖项反映了NSF的法定使命，并通过使用基金会的智力优势和更广泛的影响审查标准进行评估，被认为值得支持。

Drug-resistant bacteria are a growing threat to human health. By the year 2050, up to 10 million lives per year could be at risk. New strategies will be needed to counter this threat. Vaccines have been developed to safely and effectively prevent dangerous bacterial infections. This project seeks to address current limitations in vaccine production. Cell-free technology for vaccine production that can be easily scaled up will be developed. This could lead to portable, on-demand vaccine development and production. The project will advance the biomanufacturing of conjugate vaccines. This would improve their availability to resource-poor communities. In parallel, hands-on learning modules will be developed and delivered to underrepresented high school students, and to undergraduate and graduate students. The bacterial cell surface is decorated with complex sugar structures. These include capsular polysaccharides (CPS) and O-polysaccharides (O-PS). These structures are important virulence factors, adhesion mediators, and immunomodulators. CPS and O-PS structures are typically absent from the surfaces of host cells. These polysaccharides can, therefore, be formulated as vaccine subunits and used to protect humans against life-threatening bacterial infections. Conjugate vaccines are a type of subunit vaccine whereby polysaccharide antigens are linked to a protein carrier. They are among the safest and most effective methods for inducing immunity against pathogenic bacteria. Current production technology is technically complex and relies on living cells. Refrigeration is necessary at every step along the distribution chain. As a result, manufacturing is centralized, capital intensive, and requires highly skilled labor. This project will create a scalable, cell-free biosynthesis technology for generating conjugate vaccine candidates. Experimental and computational approaches will be combined to develop and optimize a one-pot cell-free glycoprotein synthesis system. These glycoconjugates can be stored in freeze-dried formats and reconstituted simply by adding water. A complementary technology, shotgun scanning glycomutagenesis, will enable comprehensive evaluation of glycosylation efficiency as a function of conjugation site and antigen loading density, which will be correlated with vaccine immunogenicity.This award reflects NSF's statutory mission and has been deemed worthy of support through evaluation using the Foundation's intellectual merit and broader impacts review criteria.