MECHANISMS AND EVALUATION OF VACCINE NEUROVIRULENCE IN VIVO AND IN VITRO

疫苗体内外神经毒力的机制和评价

• 批准号: 2456627
• 负责人: K. M CARBONE
• 金额: --
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/2456627
• 关键词: brain disorders; developmental neurobiology; disease /disorder model; genetic strain; microorganism growth; mumps virus; neurotropic virus; tissue /cell culture; viral vaccines; virulence; virus replication

Brain damage due to intrauterine/perinatal virus infection is a significant congenital health problem. In addition, the CNS continues to develop during the first year of postnatal life, providing for continued susceptibility of the infant CNS to damage by viral infection after birth. Since the developing nervous system is uniquely sensitive to damage following virus infection, administering neurovirulent vaccines to infants also places the child's CNS at increased risk for injury. However, little is known about specific cellular components that support preferential replication of neurovirulent wild type and vaccine viruses in neuronal cells. Wild type mumps virus, and some strains of mumps vaccine virus (Urabe Am9, Leningrad 3), are among the most neurotropic of the early childhood viruses, and new MMR combinations continue to be proposed that include new strains of mumps vaccine virus. Thus, identification of the specific cellular characteristics that lead to enhanced susceptibility to damage by neurovirulent viruses will lead to the development of molecular biological, in vitro and/or small animal models of neurovirulence. Development of these non-simian models will lead to cost saving and improved predictability of neurovirulence testing. Identification of intracellular and cell surface molecules responsible for susceptibility to neurovirulent wild type and vaccine viruses will also provide information to help genetically engineer non-neurovirulent vaccine virus strains. Notably, information obtained in these studies about mumps virus vaccines will likely be useful in generalizing to other potentially neurovirulent vaccines (e.g., measles, Japanese encephalitis). Previous studies comparing virus growth in neuronal and non neuronal cell lines have suffered from problems related to the comparison of virus replication in cells of different lineage, strain and, sometimes, species. We have a human cell line (NT2) that can be differentiated from fibroblastic to neuronal cell type using retinoic acid. By comparing virus replication in these cells before and after differentiation to neuronal cell type, we can identify specific cell features important in neurovirulence using only one human cell line. Before and after differentiation, NT2 cells will be infected with parent or neurovirulent variants of non-neurovirulent (e.g., Jeryl Lynn vaccine), or parent or non-neurovirulent variants of neurovirulent (e.g., Kilham wild type) vaccine and wild type mumps virus. Differences in virus replication (e.g., binding of virus, titer of infectious virions, and viral proteins and RNA) will be compared between wild type and vaccine viruses and their variants before (fibroblastic) and after (neuronal) differentiation. Cell surface analysis, cell fractionation experiments and gel shift analysis will be used to identfy specific cellular components that interact with neurovirulent viruses before and after neuronal differentiation of cells. Once specific neuron-linked cell components that support neurovirulent virus replication are identified, we will identify the viral sequences responsible for this interaction. Using this information, virus vaccines can be developed with altered neurovirulence sequences to generate safer vaccines.

宫内/围产期病毒感染引起的脑损伤是一种 严重先天性健康问题。 此外，CNS继续 在出生后第一年发育， 婴儿CNS对病毒感染损伤的持续易感性 出生后 因为发育中的神经系统对 病毒感染后，给予神经毒性疫苗， 也会增加儿童中枢神经系统损伤的风险。 然而，很少有人知道特定的细胞成分， 神经毒性野生型和疫苗病毒的优先复制 在神经元细胞中。 野生型腮腺炎病毒和一些腮腺炎病毒株 疫苗病毒（Urabe Am 9，Leningrad 3）是最嗜神经的病毒之一。 早期儿童病毒，和新的MMR组合继续 包括新的腮腺炎疫苗病毒株。 因此，在本发明中， 识别导致 对神经毒性病毒损伤的敏感性增强将导致 分子生物学、体外和/或小动物 神经毒性模型。 这些非猿类模型的开发将 从而节省成本并提高神经毒力可预测性 试验. 细胞内和细胞表面分子的鉴定 负责对神经毒性野生型和疫苗的易感性 病毒还将提供信息， 非神经毒性疫苗病毒株。 值得注意的是， 在这些关于腮腺炎病毒疫苗的研究中， 推广到其它潜在的神经毒性疫苗（例如，麻疹， 日本脑炎）。 以前的研究比较了 神经元和非神经元细胞系都存在与 对于不同谱系细胞中病毒复制的比较， 品种，有时也是物种。 我们有一个人类细胞系（NT 2）， 使用视黄酸将成纤维细胞类型分化为神经元细胞类型 酸 通过比较病毒在这些细胞中的复制前后 分化为神经元细胞类型，我们可以识别特定的细胞 仅使用一种人类细胞系的神经毒力的重要特征。 在分化前后，NT 2细胞将被亲本感染， 或非神经毒性的神经毒性变体（例如，杰里尔林恩 疫苗），或神经毒性的亲本或非神经毒性变体（例如， Kilham野生型）疫苗和野生型腮腺炎病毒。 差异 病毒复制（例如，病毒结合，感染性病毒粒子滴度， 以及病毒蛋白和RNA）将在野生型和 疫苗病毒及其变体之前（成纤维细胞）和之后 （神经元）分化。 细胞表面分析，细胞分级分离 实验和凝胶位移分析将用于鉴定特定的 与神经毒性病毒相互作用的细胞成分， 在细胞的神经元分化之后。 一旦特定的神经元连接 支持神经毒性病毒复制的细胞成分是 我们将确定导致这种情况的病毒序列 互动 利用这些信息，可以研制出病毒疫苗 用改变的神经毒力序列来生产更安全的疫苗。