INTRACELLULAR ANTIBODY-MEDIATED VIRUS NEUTRALIZATION

为许多病毒研制疫苗的最大障碍之一 病原体是病毒外壳蛋白的抗原性变异性。这个 一些病毒(最明显的是流感病毒)的快速变异率 和HIV)使病毒逃脱中和抗体反应 由抗病毒疫苗诱导。病毒的内部蛋白质，在 另一方面，它们通常高度保守，并没有进化到 迅速改变它们的抗原性。对这些内部的抗体反应 蛋白质，虽然对病毒的免疫反应是一致的特征， 几乎总是无法影响病毒的传染性，因为抗体 不能接触到他们的目标抗原，这些抗原位于 在病毒内部，或在病毒感染的细胞内。然而，如果， 抗体被引入细胞的胞浆中，它们确实有 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 当它们在胞浆中表达时，通过去除 它们的氨基末端ER插入序列。因此，单元格或 最终，表达这种胞浆抗体的转基因动物 应能抵抗病毒感染。利用电穿孔技术 将抗体引入胞浆，我们鉴定出能够 用流感病毒阻断细胞感染。在过去的一年里我们 从一株分泌重链和轻链的杂交瘤中克隆出重链和轻链基因 一种能够阻止感染任何人的抗体 甲型流感病毒亚型。在移除部分基因之后 编码ER插入序列，我们将这些基因插入到 构建了真核表达载体，并将其导入细胞 向量。我们正在对这一表达方式进行描述 这些胞浆抗体的折叠和抗体的抵抗力 表达细胞对甲型流感病毒的感染。我们的目标是创造 抗流感病毒感染的转基因动物。

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.