The latent viral reservoir is a major barrier to eradicating HIV infection. Unfortunately, very few tools are available to quantify the reservoir and monitor the effectiveness of prospective therapies that aim to reduce it. The current gold standard assay, the quantitative viral outgrowth assay (QVOA), has enabled landmark scientific studies, but lacks the simplicity and robustness required for large-scale clinical implementation. Other options include a number of nucleic acid-based assays, but these are thought to overestimate the quantity of latent HIV that is actually replication-competent. Thus, there exists an unmet need for next-generation reservoir assay technologies that are capable of analyzing the HIV proviral reservoir in a straightforward and precise manner. The cornerstone of our proposed assay is a novel ?dual reporter? sensor cell (DRSC) line that contains two independent HIV infection-linked reporter systems, both of which must be triggered to designate a ?true positive? result, thereby enhancing specificity. Like QVOA, our DRSC requires successful spread of HIV infection between multiple cells to trigger a positive signal, thereby eliminating many of the false positive events seen with non-live cell assays (e.g., defective HIV DNA will be erroneously quantified in many PCR-based reservoir assay). Importantly, preliminary data suggests that the DRSC is significantly faster (2 vs. 7 days) and requires less sample volume when compared with the QVOA. In the proposed project, we further optimize the DRSC via development of two key concepts: First, we will evaluate and implement ?low convection? culture environments, which will accelerate assay kinetics (and improve sensitivity) via elimination of the convective dilution of secreted factors (including virions), resulting in the concentration of factors within the vicinity of producing cells. Second, we will continue to improve our automated imaging and image analysis techniques (including real-time imaging processes), facilitating the further optimization of assay performance (e.g., through better selection of assay timing) and streamlining the ultimate translation of this technology into broader research and clinical roles (e.g., via integration of ?image-based genotyping? into our readout). These two key developments enable a number of advantageous features, including: 1) Reduction in total assay time; 2) The spatial resolution of the DRSCs combined with diffusion- dominant fluid mechanics eliminates the need for limiting dilution endpoints (as with QVOA, which requires multiple replicates of multiple T cell dilutions to obtain a single endpoint), substantially reducing the amount of sample volume required; 3) an overall reduction in assay complexity, relative to the QVOA. Importantly, at the conclusion of this study, we will perform a pilot study comparing patient samples with expected high and low reservoir values using both the DRSC assay and QVOA, thus laying the foundation for larger future studies.
The latent viral reservoir is a major barrier to eradicating HIV infection. Unfortunately, very few tools are available to quantify the reservoir and monitor the effectiveness of prospective therapies that aim to reduce it. We propose to develop a novel assay, a ?dual reporter? sensor cell (DRSC) line that contains two independent HIV infection- linked reporter systems, both of which must be triggered to designate a ?true positive? result, thereby enhancing specificity.
