Harvard Catalyst Profiles

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The overall goals of this project ar to determine if a single chain antibody that recognizes the CD4+ T cells for AIDS gene therapy. The single chain antibody that will be used is derived from the broadly neutralizing F105 human MAb and is comprised of an immunoglobulin heavy chain leader sequence and heavy and light chain variable regions that are joined by an interchain linker. We have shown that this antibody is stably expressed and retained in the endoplasmic reticulum and is not toxic in stably transformed cells. Cell morphology and replication rates of the transduced cells are normal. The antibody binds to the envelope protein within the cell and inhibits the maturation of this critical virus protein and markedly inhibits syncytia formation. The infectivity of HIV-1 particles produced by cells that express the single chain antibody is also substantially reduced. In stably transformed CD4+ SupT cells after HIV-1 infection, surface gp120 expression is substantially reduced, surface CD4 expression is restored to normal and syncytia formation is greatly inhibited. Thus this novel approach may have a role in AIDS gene therapy. In this proposal we will first determine through the use of plasmid based eukaryotic expression vectors the optimal promoter, both constitutive and inducible, to efficiency express the sFv105 in CD4+ SupT and Jurkat T cells and to determine the range of laboratory strains of HIV-1 that are inhibited. Inhibition of syncytia formation, single cell lysis and production of infectious HIV-1 virions will be tested. Long term cultures will be followed for the development of HIV-1 escape mutants. To transduce cells at a high level of efficiency for AIDS gene therapy, two different gene transfer systems will be used. For retroviral mediated gene transfer, we will also use the N2 retroviral shuttle vector to transduce the sFv105 at a high level of efficiency into uninfected CD4+ Sup T and Jurkat T cells. We will transfer either the entire promotor-sFv105 cassette (that gave maximal inhibitory activity) or the sFv105 gene alone into the N2 retroviral shuttle vector. In the latter, the 5' LTR promotor of the vector will be used to drive transcription. Following stable transection of the sFv105 containing N2 vector into the amphotropic packaging cell line PA317, supernatant will be harvested and used to infect CD4+ SupT and Jurkat T cell lines. We will also study the effect of the stably transduced sFv105 gene in chronically HIV-1 infected cell lines. For these experiments, the latently infected U1 promonocytic cell line and the T-lymphocytic cell line ACH-2 will be used. Both cell lines contain integrated HIV-1 proviral DNA and can be induced to unregulated HIV-1 replication by the addition of PDB or TNF-alpha. We will also transduce the sFv105 into uninfected and HIV-1 infected CD4+ primary lymphocytes using the N2 shuttle vector. For HIV-1 infected CD4+ primary lymphocytes, sCD4 and/or F105 MAb will be used to prevent HIV-1 spread following cell activation. We will test these transduced lymphocytes for their ability to inhibit viral infectivity in primary cultures using different laboratory strains of HIV-1 and several primary HIV-1 isolates. We will also establish that the basal function of the transduced T cells are intact. For non-retroviral mediated gene transfer of sFv105, and adeno-associated virus (AAV) vector will be used. Similar gene transfer experiments as detailed for the N2 vector will be performed on CD4+ SupT and Jurkat T cells, and on uninfected and HIV-1 infected CD4+ primary lymphocytes. These studies will form the basis for determining whether intracellular immunization using an engineered human antibody is a viable strategy for AIDS gene therapy.

Funded by the NIH National Center for Advancing Translational Sciences through its Clinical and Translational Science Awards Program, grant number UL1TR002541.