Transduction and Expansion of HIV+ CD4 T Cells with an HIV-1 Based Lentiviral Vector and Immobilized CD3/CD28 Antibodies Maintains the Diversity of the TCR Vβ Repertoire.

Recently, we initiated the first ex vivo HIV-based gene therapy trial in humans with HIV⁺ CD4⁺ T cells. In this protocol, a modified lentiviral vector carrying an anti-HIV payload is used to modify CD4⁺ T cells isolated from HIV-infected patients by apheresis and CD8 negative selection. The T cells are activated in the presence of vector and expanded using immobilized CD3/CD28 antibodies, and then infused back into the patient. T cell receptor (TCR) repertoire analysis has value for safety monitoring of adoptive T cell transfers in the detection of aberrant clonal expansions or deletions. In this study, the TCR Vβ repertoire was assessed using a flow cytometry based assay at various time points in the selection/transduction/expansion process of CD4⁺ T cells. PBMC isolated from whole blood of HIV⁺ patients were CD4-selected using a CD8 negative selection, followed by enrichment by CD3 antibody. CD4⁺ purified cells were transduced with the lentiviral vector, VRX496, in the presence of retronectin, and then co-cultured with CD3/CD28 coated M450 Dynabeads for ten days. The TCR Vβ repertoire was assessed in throughout the process using a FACS-based assay that employs a panel of 20 monoclonal antibodies recognizing most of the 24 Vβ families in PBMC and CD4+ T cells. Repertoires from subjects with normal polyclonal TCR profiles were conserved, as shown by the absence of any significant change in any Vβ family. Moreover, the transduction/expansion of CD4⁺ T cells from a patient with a previously skewed TCR profile allowed the improvement of the TCR Vβ repertoire. Finally, no significant difference was observed in the repertoire of cells transduced with VRX496 versus mock-transduced cells. These data demonstrate stability of the repertoire diversity and thus provide important support information in favor of the safety of a gene therapy approach involving lentiviral vector mediated modification and expansion of CD4⁺ T-cells.