DEVELOPMENT OF A VIRUS-FREE DNA VACCINE AGAINST SMALLPOX

开发针对天花的无病毒 DNA 疫苗

• 批准号: 8360153
• 负责人: Miguel Otero
• 金额: $ 10.26万
• 依托单位: UNIVERSITY OF PUERTO RICO
• 依托单位国家: 美国
• 财政年份: 2011
• 资助国家: 美国
• 起止时间: 2011-06-01 至 2012-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8360153
• 关键词: Adjuvant; Animals; Antigens; Biomedical Research; Categories; Cells; Core Protein; DNA Vaccines; Dendritic Cells; Development; Dose; Elderly; Enzyme-Linked Immunosorbent Assay; Epitopes; Event; Funding; Genes; Grant; Growth Factor; Histocompatibility Antigens Class II; Immune; Immune response; Immunocompromised Host; Immunology; Life; Macrophage Inflammatory Protein-1; Maps; Mediating; Memory; Modeling; Molecular; Mus; National Center for Research Resources; Population; Pregnant Women; Principal Investigator; Puerto Rico; Research; Research Infrastructure; Resources; Smallpox; Source; Structural Protein; System; Testing; Transplant Recipients; United States National Institutes of Health; Vaccinated; Vaccination; Vaccines; Vaccinia virus; Viral Load result; Viral Proteins; Virus; chemokine; cost; enzyme linked immunospot assay; hazard; interest; pathogen; plasmid DNA

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. Primary support for the subproject and the subproject's principal investigator may have been provided by other sources, including other NIH sources. The Total Cost listed for the subproject likely represents the estimated amount of Center infrastructure utilized by the subproject, not direct funding provided by the NCRR grant to the subproject or subproject staff. Smallpox is a threat in an event of a bioterrorist attack, therefore it is important to develop a safe vaccine against this virus. There is an important population of non-vaccinated immunocompromised people that could not be safely vaccinated with the current vaccine. Within these are patients of transplants, under immunosupressive therapy, pregnant women, and the elderly. We are interested in the development of a safer vaccine that protects the population against a possible attack. We hypothesize that the adjuvant-mediated enhancement of the molecular antigen immune response will control the viral burden on infected mice. However, as smallpox is only one of possible threats, this project will provide an excellent model to test the hypothesis of a molecular adjuvant that will help set up a system that could be applied to other Category A, B and C Priority Pathogens. Mice will be immunized with plasmid DNA encoding for the vaccinia virus proteins C7L and A55R genes that are non structural proteins known to generate a cellular immune response, and the recently discovered E6R and L3L core proteins. The immunology of those molecules has not been tested to the best of our knowledge. Animals will be vaccinated with each construct individually or combined with two molecular adjuvants, the chemokine MIP-1-alpha and another construct which encodes for the dendritic cell-specific growth factor Flt-3L, a combination that is known to increase the recruitment and expansion of dendritic cells. To the best of our knowledge, this combination of molecular adjuvants has never been tested in the context of smallpox vaccination. Cell- and humoral-mediated immune responses from each antigen will be determined by ELISPOT and ELISA, respectively. Molecular mapping of each antigen will be performed to identify dominant epitopes. Animals will be challenged with a lethal dose of vaccinia virus, and the viral load will be determined by qPCR analysis. Memory, activation and proliferation will be determined to detect the long-lasting effect of each antigen on the induction of an immune response. Strong cellular and humoral immune responses against vaccinia virus are expected so that protecting immune memory could be developed without the hazard of being exposed to live virus.

该子项目是利用资源的众多研究子项目之一 由 NIH/NCRR 资助的中心拨款提供。子项目的主要支持 并且子项目的首席研究员可能是由其他来源提供的， 包括其他 NIH 来源。 子项目可能列出的总成本 代表子项目使用的中心基础设施的估计数量， NCRR 赠款不直接向子项目或子项目工作人员提供资金。 天花在生物恐怖袭击中是一种威胁，因此开发针对这种病毒的安全疫苗非常重要。有一个重要的未接种疫苗的免疫功能低下人群无法安全地接种当前的疫苗。其中包括接受免疫抑制治疗的移植患者、孕妇和老年人。我们有兴趣开发一种更安全的疫苗来保护人们免受可能的攻击。我们假设佐剂介导的分子抗原免疫反应的增强将控制受感染小鼠的病毒负荷。然而，由于天花只是可能的威胁之一，该项目将提供一个出色的模型来测试分子佐剂的假设，这将有助于建立一个可应用于其他 A、B 和 C 类优先病原体的系统。小鼠将使用编码痘苗病毒蛋白 C7L 和 A55R 基因的质粒 DNA 进行免疫，这些基因是已知可产生细胞免疫反应的非结构蛋白，以及最近发现的 E6R 和 L3L 核心蛋白。据我们所知，这些分子的免疫学尚未经过测试。动物将单独接种每种构建体或与两种分子佐剂（趋化因子 MIP-1-α 和编码树突细胞特异性生长因子 Flt-3L 的另一种构建体）组合进行疫苗接种，这种组合已知可增加招募和扩增树突状细胞。据我们所知，这种分子佐剂组合从未在天花疫苗接种中进行过测试。每种抗原的细胞和体液介导的免疫反应将分别通过 ELISPOT 和 ELISA 测定。将进行每种抗原的分子图谱以确定主要表位。动物将受到致死剂量的牛痘病毒攻击，病毒载量将通过 qPCR 分析确定。将确定记忆、激活和增殖，以检测每种抗原对诱导免疫反应的长期影响。预计针对痘苗病毒的强烈细胞和体液免疫反应，以便可以在没有暴露于活病毒的危险的情况下形成保护免疫记忆。