VIRUSES AND HEMATOPOEIESIS

我们的实验室进行B19细小病毒的基础和临床研究， 细小病毒科中唯一对人类致病的病毒。 急性 感染导致第五种疾病，一种儿童皮疹疾病和一种 成人多关节痛综合征。 在基础疾病患者中， 溶血、急性感染导致短暂性再生障碍危象。 在 存在潜在免疫缺陷的患者，病毒感染持续存在， 引起慢性贫血;细小病毒感染是贫血的原因， 艾滋病患者。 该病毒嗜红系祖细胞 因为它使用了红细胞P抗原。 重组细小病毒 基于我们实验室开发的空衣壳的疫苗试剂， 进入I期试验;虽然没有毒性，但目前 剂量和常规佐剂，我们无法证明 志愿者血清中的中和抗体。 在临床研究中， 我们无法证实B19与 细小病毒和儿童暴发性肝炎。 的结构研究 使用冷冻电子显微镜观察B19细小病毒空衣壳， 确定了球糖苷受体的病毒结合位点为凹陷 存在于三重对称轴上， 突变体与中和抗体也映射。 在分子生物学中 研究，非结构蛋白，这是负责病毒 复制功能以及细胞毒性，已被证明含有 多个核定位信号并具有磷酸化和 非磷酸化形式。 除了B19细小病毒，我们还有 鉴定了几种其他相关但不同的西曼细小病毒， 在它们各自的宿主物种中引起相似的临床症状。 我们 已经将我们的细小病毒研究扩展到包括腺相关病毒 (AAV)as a vector载体for gene基因therapy治疗. 我们已经确认了一个 AAV-2的细胞受体，一种流行的载体骨架，作为150 kd 糖蛋白;然而，受体仅以低水平存在于 人类造血细胞 另一种人类腺相关病毒，AAV-3， 不与该受体结合。 绕过假定的块 受体介导的病毒进入宿主细胞，我们已经测序 并产生了AAV-3的感染性克隆。 最后，在研究 人类免疫缺陷病毒-1（HIV-1），我们已经产生了第一个 基于Epstein-Barr病毒的穿梭载体和最近稳定的 用于细胞内免疫逆转录病毒载体。 我们把目标对准了病毒 逆转录酶（RT）。 在病毒生命的这个阶段灭活 循环将阻止整合，此外， 来自不同物种的酶允许广泛的靶向。 细胞内 表达抗RT重链和重链与轻链的结合， 在体外实现并高度有效地预防甚至 解决细胞系的病毒感染。 目前的研究是针对 稳定地转导单链抗体的基因 分子进入淋巴细胞系和原代猿猴和人类 淋巴细胞 如果成功的话，非人类灵长类动物试验旨在预防 将进行猴免疫缺陷病毒感染。

Our laboratory performs basic and clinical studies of the B19 parvovirus, the only member of the Parvoviridae family pathogenic in humans. Acute infection causes fifth disease, a childhood rash illness and a polyarthralgia syndrome in adults. In patients with underlying hemolysis, acute infection results in transient aplastic crisis. In patients with underlying immunodeficiency, virus infection persists and causes chronic anemia; parvovirus infection is a cause of anemia in patients with AIDS. The virus is tropic for erythroid progenitor cells due to its use of erythrocyte P antigen. A recombinant parvovirus vaccine reagent, based on empty capsids developed in our laboratory, has entered phase I trials; although there was no toxicity, at the current dose and with conventional adjuvant, we were not able to demonstrate neutralizing antibodies in sera from volunteers. In clinical studies, we have been unable to confirm a single report of association between B19 parvovirus and fulminant hepatitis of childhood. Structural studies of B19 parvovirus empty capsids using cryo-electron microscopy have identified the viral binding site for globoside receptor as depressions present on the three-fold axes of symmetry, a region to which escape mutants to neutralizing antibodies also map. In molecular biologic studies, the nonstructural protein, which is responsible for viral replicative functions as well as cytotoxicity, has been shown to contain multiple nuclear localization signals and to have both phosphorylated and nonphosphorylated forms. In addition to B19 parvovirus, we have identified several other related but distinct siman parvoviruses which cause similar clinical syndromes in their respective host species. We have expanded our parvovirus studies to include adeno-associated virus (AAV) as a vector for gene therapy. We have identified a putative cellular receptor for AAV-2, a popular vector backbone, as a 150 kd glycoprotein; however, the receptor was present only at low levels on human hematopoietic cells. Another human adeno-associated virus, AAV-3, does not share binding to this receptor. To circumvent a putative block to receptor mediated entry of virus into host cells, we have sequenced and produced an infectious clone of AAV-3. Finally, in studies of the human immunodeficiency virus-1 (HIV-1), we have produced first an Epstein-Barr virus based shuttle vector and more recently a stable retroviral vector for intracellular immunization. We targeted the viral reverse transcriptase (RT). Inactivation at this stage of the virus life cycle would prevent integration and, in addition, high homology among enzymes from different species allows broad targeting. Intracellular expression of anti-RT heavy chain and heavy combined with light chain was achieved and was highly effective in vitro in preventing and even resolving viral infection of cell lines. Curent studies are directed towards stable transduction of the gene for a single antibody chain molecule into both lymphoid cell lines and primary simian and human lymphocytes. If successful, nonhuman primate trials aimed at preventing simian immunodeficiency virus infection will be conducted.