INDUCTION OF PERSISTENT IMMUNITY BY PARVOVIRAL VECTORS

描述：（改编自申请人的摘要）最佳HIV疫苗接种 战略将引发持久的，消毒，跨进化枝中和 抗病毒的抗体这一目标可能需要开发 病毒载体发挥主要作用的新型抗原递送策略。 在基于线性的向量构造中的改进， 自主复制细小病毒的单链DNA基因组 最近允许我们生产载体，以适合于体内使用的量， 没有复制能力的病毒。这些载体预计将分享 与其亲本病毒的一些独特特性。他们应该无法 在休眠细胞中启动复制，并神秘地持续到宿主 细胞进入S期响应外源信号，在这个时候，他们将 复制，表达它们编码的转基因，并杀死细胞。此外，本发明还 我们应该能够靶向它们在特定的细胞类型中表达， 包装的外套的属性。我们将使用矢量包装 在鼠嗜淋巴细胞细小病毒MVMi的外壳中， 抗原表达至宿主中非常小部分的T细胞。由于大多数 这些受感染的细胞将休息，很少的抗原应该是 在任何一个时间表达。然而，由于这些细胞可能会被激活， 在宿主生命的某个阶段， 将成为表达抗原的受感染、垂死细胞的持续来源， 从而引发对编码抗原的持续免疫应答。初步 结果显示用表达Borellia的MVM载体感染的小鼠 表面抗原OspA引起强烈保护性体液应答， 在单次静脉接种后150天仍然最大， 105个转导单位的载体。我们将使用矢量编码绿色荧光 蛋白质以测量载体感染静息鼠、人 和猕猴T细胞，并在激活后有条件地表达它们。另一 表达合成嵌合抗原的载体构建体将用于 监测这种独特病毒的几个方面：宿主相互作用和ELISA 用于分析免疫反应的生物学和寿命的测定 诱导。非结构性和非结构性突变对诱导免疫的影响 将检查载体的衣壳基因。C-末端截短形式的 将构建HIV-1 gp 160;有或没有糖基化修饰 进入矢量。由这些载体诱导的抗体应答，最初在 小鼠，将通过ELISA定量并分析中和活性， 首先使用携带gp 160的“替代”VSV载体，然后在交叉进化枝中 HIV中和试验。

DESCRIPTION: (Adapted from Applicant's Abstract) An optimal HIV vaccination strategy would elicit persistent, sterilizing, cross-clade neutralizing antibodies against the virus. This goal will likely require the development of novel antigen delivery strategies in which viral vectors play a major role. Improvements in the construction of vectors based on the linear, single-stranded DNA genomes of the autonomously replicating parvoviruses have recently allowed us to produce vectors, in amounts suitable for use in vivo, devoid of replication competent virus. These vectors are predicted to share several unique properties with their parental viruses. They should be unable to initiate replication in resting cells, and persist cryptically until the host cell enters S phase in response to exogenous signals, at which time they would replicate, express the transgene they encode, and kill the cell. Additionally, we should be able to target them to express in particular cell types as a property of the coat in which they are packaged. We will use vectors packaged in the coat of the murine lymphotropic parvovirus MVMi to target foreign antigen expression to a very small fraction of T-cells in the host. Since most of these infected cells will be resting, very little antigen should be expressed at any one time. However, since these cells will likely be activated for proliferation stochastically at some point in the life of the host, they will become a continual source of infected, dying cells expressing antigen, thus eliciting a persistent immune response to encoded antigen. Preliminary results show that mice infected with an MVM vector expressing the Borellia surface antigen OspA mount a vigorous, protective humoral response which is still maximal at 150 days after a single intravenous inoculation with as few as 105 transducing units of vector. We will use vectors encoding green fluorescent protein to measure the ability of the vector to infect resting murine, human and macaque T-cells, and express them conditionally upon activation. A further vector construct expressing a synthetic chimeric antigen will be used to monitor several aspects of this unique virus: host interaction, and ELISA assays developed to analyze the biology and longevity of the immune response induced. The effect on induced immunity of mutation of both non-structural and capsid genes of the vector will be examined. C-terminally truncated versions of HIV-1 gp160; with or without glycosylation modifications will be constructed into the vector. Antibody responses induced by these vectors, initially in mice, will be quantitated by ELISA and analyzed for neutralizing activity, firstly with a "surrogate" gp160-carrying VSV vector and then in cross-clade HIV neutralization assays.