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| Figure 6: Comparison of sscFvPRO140-mediated inhibition of HIV infection with other protein-based entry inhibitors. (a) U373-MAGI-CCR5E cells were transduced with vector particles encoding sscFvPRO140 (U373-MAGI-CCR5E/ sscFvPRO140) or sCD4 (U373-MAGI-CCR5E/sCD4) at a low multiplicity of infection to generate a mixed population of gene-modified and unmodified target cells (level of gene-modification, ~50%) as described in Materials and Methods. Cells transduced with LVX vector particles served as a control. Single-round infection assays were performed with R5 HIVJRFL Env-pseudotyped reporter vector particles expressing dsRed. The number of infected cells in the gene-modified population (dsRed+ZsGreen1+) and in the unmodified population (dsRed+ZsGreen1-) was quantified by flow cytometry. (b) 1×106 genemodified 293T cells expressing sscFvPRO140 or sCD4 were cultured for 2 days in 2 ml of culture medium. The culture supernatant was filtered and used in single-round infection assays with R5 HIVJRFL Env-pseudotyped reporter vector particles expressing dsRed. A relative infection of 100% corresponded to 15% of infected cells; n=2. (c) Single-round infection assays were performed in the presence of purified sscFvPRO140, sCD4, or FDA-approved FIT20 at the indicated concentrations. A relative infection of 100% corresponded to 15% of infected cells; n=2. |
