VIRUSES AND HEMATOPOEIESIS

我们的实验室对B19细小病毒进行基础和临床研究，

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.