INTRACELLULAR ANTIBODY-MEDIATED VIRUS NEUTRALIZATION

细胞内抗体介导的病毒中和

• 批准号: 3790852
• 负责人: J W YEWDELL
• 金额: --
• 依托单位: NATIONAL INSTITUTE OF ALLERGY AND INFECTIOUS DISEASES
• 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/3790852
• 关键词: antigen antibody reaction; hybridomas; microorganism immunology; neutralizing antibody; nucleic acid sequence; protein folding; tissue /cell culture; transfection; virus antigen

One of the greatest hurdles in creating vaccines for a number of viral pathogens is the antigenic variability of viral coat proteins. The rapid mutation rate of a number of viruses (most notably influenza virus and HIV) allows the virus to escape the neutralizing antibody response induced by anti-viral vaccines. The internal proteins of the virus, on the other hand, are generally highly conserved, and have not evolved to rapidly alter their antigenicity. Antibody responses to these internal proteins, while a consistent feature of immune responses to viruses, almost always fail to influence viral infectivity, since the antibodies do not have access to their target antigens, which are located either inside the virus, or inside the virus infected cells. If however, antibodies are introduced into the cytosol of cells, they do have the ability to prevent viral infection. Recent advances in understanding antibody folding indicate that antibodies have the ability to properly fold and bind antigen when they are expressed in the cytosol by removing their amino terminal ER insertion sequences. Therefore, cells, or eventually, transgenic animals expressing such cytosolic antibodies should be resistant to virus infection. Using electroporation to introduce antibodies into the cytosol, we identified antibodies able to block infection of cells with influenza viruses. In the past year we have cloned the heavy and light chain genes from a hybridoma secreting an antibody that is able to block infection with any of the human influenza A virus subtypes. After removing the portion of the genes encoding the ER insertion sequence, we have inserted these gene into eukaryotic expression vectors, and have transfected cells with the vectors. We are in the process of characterizing the expression and folding of these cytosolic antibodies, and the resistance of antibody expressing cells to influenza A virus infection. Our goal is to create transgenic animals resistant to influenza virus infection.

在为许多病毒制造疫苗的过程中， 病原体是病毒外壳蛋白的抗原变异性。 的 许多病毒（最显著的是流感病毒）的快速突变率 和HIV）使病毒逃避中和抗体反应 由抗病毒疫苗引起。 病毒的内部蛋白质， 另一方面，通常是高度保守的，并没有进化到 迅速改变其抗原性。 抗体对这些内部 蛋白质，虽然是对病毒的免疫反应的一致特征， 几乎总是不能影响病毒的传染性，因为抗体 不能接触到它们的靶抗原，这些抗原位于 或者病毒感染的细胞内。 但是，如果， 抗体被引入细胞的胞质溶胶中，它们确实具有 预防病毒感染的能力。 认识的最新进展 抗体折叠表明抗体有能力正确地 当它们在胞质溶胶中表达时， 其氨基末端ER插入序列。 因此，细胞，或 最终，表达这种胞质抗体的转基因动物 应该能抵抗病毒感染。 使用电穿孔来 将抗体引入胞质溶胶，我们发现抗体能够 阻止流感病毒感染细胞。 在过去的一年里，我们 已经从一个杂交瘤中克隆了重链和轻链基因， 一种抗体，能够阻止感染任何人类 甲型流感病毒亚型。 在去除了一部分基因后 编码ER插入序列，我们将这些基因插入到 真核表达载体，并已转染细胞与 向量。 我们正在描述这种表达的特征， 这些胞质抗体的折叠，以及抗体的抗性 表达细胞对甲型流感病毒感染的耐受性。 我们的目标是创造 抗流感病毒感染的转基因动物。