EAGER: Peptide affinity membranes for binding influenza viruses: H1N1 and H5H1

卡里姆智慧的优点：这个急切的提议是一个高风险、高收益的提议。最近H1N1流感的爆发和H5N1禽流感病毒的全球传播及其对人类的感染引发了全球对这些病毒导致大流行的担忧。根据世界卫生组织2009年5月20日的报告，41个国家已确认10243例H1N1流感病例，其中80人死亡。在美国，这一数字分别为5469和6。迫切需要针对H1N1和H5N1的治疗或快速疫苗接种过程。该提案在这一变革性的想法中解决了这个问题，即开发一种基于多肽的膜系统，以便可以从细胞培养液或从人类血液中捕获流感病毒。主要目标是开发一种基于细胞培养的疫苗生产方法，该方法使用基于多肽的纯化步骤。该多肽是利用有关流感病毒血凝素(HA)表面蛋白的可用基因组信息开发的。我们假设，对于给定的病毒，可以通过使用筛选工具来选择具有高特异性的亲和肽。此外，通过将亲和肽基团化学结合到膜表面，该亲和基质可用于重复结合和洗脱特定类型的病毒。我们进一步假设病毒表面蛋白在病毒结合过程中发挥重要作用。因此，根据特定的流感病毒株设计的亲和膜可用于结合与该病毒同源性较高的其他病毒。这项研究将有助于更好地了解病毒表面蛋白在亲和结合过程中的作用。“同质性”一词通常用来描述两种流感病毒株的相似程度。通过选择几种不同同质性的流感病毒，可以表征表面蛋白对病毒与亲和膜结合的影响。这项研究将回答关于同质性对病毒与给定亲和膜结合的影响有多敏感的问题。由于微孔亲和膜结合了微滤和树脂基亲和层析的优点，因此可以显著简化病毒的生产和纯化过程。本发明特别适用于以细胞培养为基础的流感病毒疫苗生产工艺。通过选择合适的膜孔径，可以一步实现细胞的去除和病毒的提纯。细胞培养物将通过微孔亲和膜的一侧飞行。在此期间，细胞和细胞碎片将被膜孔排斥，而病毒、蛋白质和/或DNA和其他小的生物成分将迁移到膜孔内。然后，病毒将被嫁接在膜孔表面的这些功能性多肽特异性结合，而蛋白质、DNA和其他生物成分将穿过膜孔。洗涤后可将高纯度的病毒从结合膜上洗脱出来。这种简化的纯化过程可能会加快H1N1和H5N1疫苗的开发、商业化和生产过程。广泛影响：拟议研究的完成将对H1N1和H5H1流感干预产生重大影响和可能的应用。所提出的新型多肽基膜将简化病毒纯化过程，并可能加快疫苗的开发、商业化和生产过程。所制备的膜还可用于病毒样本的快速采集和纯化，用于病毒诊断。此外，该方法还可以扩展到诊断其他病毒，如西尼罗河病毒。此外，可能会开发出一种可能的临床设备，它可以专门从人类血液中去除病毒。通过使人体血液循环通过含有针对特定流感病毒的亲和膜的膜装置，可以使这些病毒结合到膜表面，从而减少血液中的总病毒数量；然后可以将该装置与其他一些干预方法结合起来，如使用达菲。该项目将包括来自化学工程和TTU健康科学中心的多学科研究人员。该项目的教育目标之一是培养一名博士后和一名博士后，从事疫苗和多肽膜技术的前沿研究。本科生将从TTU广为人知的外展项目中挑选，例如工程外展中心和McNair学者项目。然后，UG的学生将被鼓励进入研究生院，并在生物相关领域从事职业。PI在吸引女性和少数族裔候选人加入他的研究小组方面拥有丰富的经验。与当地学校的接触将通过资助的项目进行，如科学是女孩的事情，大学预科工程，教师培训研讨会，和工程外展导师。

0943424KarimIntellectual Merit: This EAGER proposal is a high risk high gain proposal. Recent outbreak of H1N1 flu and the worldwide spread of H5N1 avian influenza virus and its infection to human beings has raised a global concern about these viruses to cause a pandemic. According to the World Health Organization report of May 20, 2009, 41 countries have confirmed 10,243 cases of H1N1 flu with 80 fatalities. In the US, these numbers are 5,469 and 6, respectively. A treatment or a rapid vaccination process for H1N1 and H5N1 is urgently needed. The proposal addresses this issue in this transformative idea to develop a peptide-based membrane system so that flu viruses can be captured from either a cell culture broth or from human blood. The main goal is to develop a cell culture-based vaccine production method, which uses a peptide-based purification step. The peptide is developed using the available genomic information regarding hemagglutinin (HA) surface protein of flu viruses. We hypothesize that for a given virus, an affinity peptide with a high specificity could be selected by using a screening tool. Moreover, with the chemical attachment of the affinity peptide group to a membrane surface, this affinity matrix could be used to repeatedly bind and elute the specific type of virus. We further hypothesize that the virus surface protein plays a significant role on the virus binding process. Therefore, an affinity membrane designed based on a specific strain of influenza virus may be used to bind other viruses which have high homogeneity to this virus. This research will be helpful in better understanding the role of the virus surface protein on the affinity binding process. A term of 'homogeneity' is usually used to describe how similar two influenza virus strains are. By choosing several influenza viruses with different homogeneity, the effect of the surface protein on the virus binding to an affinity membrane could be characterized. This research will answer the question regarding how sensitive the effect of the homogeneity is on the binding of a virus to a given affinity membrane.Since microporous affinity membrane combines both the advantages of the microfiltration and resin based affinity chromatography, the virus production and purification process could be significantly simplified. This is specifically suitable for the cell culture based influenza virus vaccine production process. By carefully choosing an appropriate membrane pore size, cells removal and virus purification could be achieved within one step. Cell cultures will be flown through one side of a microporous affinity membrane. During this period, cells and cell debris will be rejected by the membrane pores while viruses and proteins and/or DNA and other small biological components will migrate inside the membrane pores. Viruses will then be specifically bound by these functional peptide groups grafted on the membrane pore surface while protein, DNA and other biological components will pass through the membrane pores. After washing, viruses with high purity can be eluted from the bounded membranes. This simplification of the purification process may speed up the H1N1 and H5N1 vaccine development, commercialization and production process.Broader Impact: Completion of the proposed studies will have significant impact and possible applications in H1N1 and H5H1 flu intervention. The proposed new peptide-based membrane will simplify the virus purification process and it may speed up the vaccine development, commercialization and production process. The prepared membrane may also be used in the virus diagnosis by quick collection and purification of virus samples. Further, this method may be extended to diagnose other viruses, such as the West Nile virus. Further, a possible clinical device may be developed which can specifically remove viruses from human blood. By circulating human blood through a membrane device containing affinity membranes for a specific flu virus, these viruses can be made to bind to the membrane surfaces thus reducing the overall virus population in the blood; this device may be then combined with some other intervention methods such as using TAMIFLU. This project will involve multidisciplinary researchers from Chemical Engineering and TTU Health Sciences Center. One of the educational goals of the project is to train one post-doc and one PhD student in the cutting edge research of vaccine and peptide-based membrane technology. An undergraduate student will be selected from well publicized outreach programs at TTU, e.g. Engineering Outreach Center, and McNair Scholars Program. The UG student will then be encouraged to go to graduate school and have a career in bio related field. The PI has significant experience in attracting female and minority candidates to his research group. Outreach to local schools will be done through funded programs such as Science it's a Girl's Thing, Pre-college Engineering, Teacher Training Workshop, and Engineering Outreach Mentor.