Strategies to eradicate HIV-1 infection in individuals with suppressed or undetectable viral replication are being actively explored in clinical trials. HIV+ individuals with suppressed viral replication are treated with agents that remodel chromatin, e.g. histone deacetylase inhibitors (HDACIs) or other T cell stimulators in efforts to reactivate expression of latent HIV resulting in de novo virus production, which will subsequently results in the death of latent infected cells through a variety of postulated mechanisms, including programmed cell death (apoptosis) and/or immune activation. New virions produced by activated T cells are prevented from infecting new cells by the ongoing treatment of the individual with a fully suppressive anti-retroviral drug regimen. Such virus elimination strategies are predicated on the assumption that multiple rounds of treatment with latency reversing drugs will incrementally diminish the latent vial reservoir over time leading to an eventual eradication of infection. A major challenge in evaluating the efficacy of eradication protocols in clinical trils is the lack of a standardized assay for detecting latent virus depletion as a measure of reservoir eradication. The current recognized gold-standard assay (Q-VOA) is based on quantitative measures of viral outgrowth using limit dilutions of primary resting T cells isolated following exposure to candidate therapeutic T cell stimulators and potent control compounds. These assays require large cell numbers and require days, if not weeks to complete. Virus production is quantitated from cell or cell culture media fractions using various methods that include ELISA and PCR. The use of various assays and detection methods has led to inconsistent results from the various performing laboratories and hinders inter-laboratory comparisons. Previously, the introduction of standardized neutralizing antibody assays for the evaluation of HIV vaccine candidates enabled fine distinctions in efficacy when evaluating different immunogens and vaccination strategies. In much the same way, the implementation of a reliable, standardized Q-VOA assay should provide the ability to discern subtle differences in latent reservoir size during eradication studies. Monogram Biosciences (www.monogrambio.com) has more than 15 years of experience developing high throughput cell based viral assays that evaluate HIV-1 therapeutics. We intend to leverage our established expertise to pursue and complete the proposed study objectives. Monogram will explore different Q-VOA approaches to select and develop a method that generates a robust, reproducible, rapid and affordable method to accurately measure the HIV-1 latent reservoir in clinical samples. This study proposal addresses several specific objectives as specified in the funding opportunity announcement (PA-12-162): (a) Development of new assays (including but not limited to development of new quantitative assays for sensitive detection of HIV-1 in tissue, a simple method for detecting replication-competent virus in latently infected cells, assays to measure diversification of viruse in reservoirs, assays to accurately discriminate and measure vDNA in integrated and unintegrated forms. (b) Technology advancement (including but not limited to methods to standardize isolation and quantification of replication competent vRNA and viral DNA (vDNA) from cells and tissues, and nanotechnology).
Clinical investigators are attempting to cure HIV infection in cases where successful antiretroviral treatment has resulted in long term suppression of viral replication. In such cases, latent virus is believed to persist in a quiescent state in certain lymphocytes and possibly other cells. Curative strategies are designed to re-activate virus expression in latently infected cells prompting the subsequent death of the infected cell. It is hoped that repeated treatment over time will eventually rid the body of all latently infected cells and so affect a cure. The aim of this project is to develop and standardize a sensitive, accurate and reproducible assay to more conveniently and reliably assess the size of the latent reservoir and the effectiveness of curative treatments.
