Strategies to eradicate HIV-1 infection in individuals with suppressed or undetectable viral loads are currently being explored in clinical trials. HIV+ individuals with suppressed 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 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 regimen of anti-retroviral drugs. The virus elimination strategy is predicated on the assumption that multiple rounds of treatment with latency reversing drugs will decrease the reservoir over time leading to the eventual eradication of infection. The quantitative viral outgrowth assay (VOA) is considered the best method currently available to measure the level of latently infected cells. However the VOA is expensive, time consuming and labor intensive. And recent research shows that even the VOA may not be able to accurately quantitate the size of the latent reservoir as reactivation of latent functional viruses may involve poorly understood and/or stochastic mechanisms. By examining both the genomic RNA sequences of the reactivated viruses generated in the VOA and the archived proviral DNA sequences in the resting CD4+ T cells we should be able to obtain a greater understanding of the extent of latent infections. Developing a robust and sensitive method to amplify and sequence whole HIV-1 genomes found in early VOA supernatants would potentially save assay turnaround time and give access to a wealth of sequence information on these activated viruses. Additionally, the sensitive and reliable amplification of full-length HIV templates from enriched populations of CD4+ resting memory cells prior to activation, should enable direct comparisons of virus variants that appear after latent reservoir activation to those found in the cellular archive. Recently developed digital PCR platforms can be used to quantitate the number of copies of HIV DNA found in a sample and sequencing full length HIV proviral genomes would allow the estimation of what percentage of those copies appear to encode functional viruses. We propose developing robust and sensitive methods of amplifying HIV genomes from VOA supernatants as well as memory T cells and sequencing those templates using both conventional and next generation sequencing methods. This study proposal addresses several specific objectives of research interest 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 viruses 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).
Due to advances in anti-retroviral treatment, millions of people worldwide are infected by HIV but have no evidence of virus replication. However, HIV still resides in an inactive state in long lived immune system cells. This project would advance our ability to detect the extent and viability of this latent reservoir of HIV and asses the effectiveness of therapies aimed at reducing or eliminating this hidden reservoir of HIV infection.
