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  • Disclosure br Acknowledgments br A critical knowledge gap in

    2018-11-13

    Disclosure
    Acknowledgments
    A critical knowledge gap in the field of HIV cure research is how best to measure the persistence of replication-competent HIV in the form of intact but latent proviruses. One approach to curing HIV is to eradicate all replication-competent HIV from an individual. It is unlikely that any method, now or in the future, will be able to declare with certainty that all intact proviruses have been eliminated from an individual\'s body. The difficulty in declaring that all intact proviruses have been eradicated is best illustrated by the two Boston patients who underwent allogeneic, hematopoietic stem cell transplantation for uncontrolled malignancy. After successful transplantation and several years of suppressive antiretroviral therapy (ART), HIV DNA or RNA could not be detected in large volumes of blood K03861 Supplier assayed many times from either patient, yet viremia rebound occurred 7–32weeks after cessation of ART (). Similarly, it has been difficult to conclude that all HIV has been eliminated even from the Berlin patient who was transplanted with HIV-resistant cells and in whom HIV has not been detected in blood for the past decade off of ART (). As such, laboratory assays to exclude the persistence of intact proviruses in a person are no more likely to be found than is a therapy that eradicates all HIV from a person. The more realistic goal of HIV cure research is to reduce HIV reservoirs and enhance immune control of HIV to undetectable levels in the blood after cessation of ART, also termed, HIV remission. Researchers studying interventions aimed at HIV remission struggle to understand how much their interventions are affecting intact proviruses because of the shortcomings of existing methods to measure them. What matters for the goal of HIV cure is how much HIV can replicate. Polymerase chain reaction (PCR)-based methods are used to quantify the amount of HIV DNA and HIV RNA in mononuclear cells from blood and lymphoid tissues. Having detectable HIV DNA, however, does not tell us how much HIV could replicate as most of the DNA is defective (). Cell-associated HIV RNA has been used as a proxy for “active” reservoirs since RNA is short-lived and its presence implies recent transcription (). Measuring low level HIV RNA in blood or other body fluids is another marker for ongoing viral production (). However, all these methods are not direct measures of the ability to infect other cells, replicate and K03861 Supplier produce infectious progeny. The current “gold standard” of such test is the viral outgrowth assay (VOA), a co-culture assay that stimulates resting CD4+ T cells from HIV-infected patients to produce virus capable of infecting HIV-uninfected cells from another donor. However, this method underestimates the amount of replication competent virus by 60-fold compared to detection of intact proviruses by sequence analysis. This underestimation can be reduced by sequential stimulation and co-culture of patient and donors cells to activate a greater fraction of proviruses (), but this approach is impractical because the VOA, even with a single round of stimulation, requires a large blood volume, weeks to complete, and costs more than $1000 per assay for reagents and labor. In this issue of , Procopio and Chomont et al., proposed the Tal/rev Induced Limiting Dilution Assay or TILDA, a cell-associated HIV RNA PCR that is based on the VOA concept in that it relies on stimulating patient\'s CD4+ T cells, but instead of the cumbersome co-culture methods, an early product of viral transcription, the tat/rev multi-spliced RNA, is measured (). When HIV RNA enters the cytoplasm, it reverse transcribes into DNA that penetrates the nuclease to integrate into the host genome. When cells with integrated DNA are activated, they transcribe RNA that is multiply spliced for translation of tat and rev proteins that accelerate transcription and permit genomic viral RNA to exit the nucleus, respectively, which is translated into the structural proteins required for assembly and release of virions from the cell (). Because TILDA uses small blood volume and is relatively easy to perform without the expense and labor intensiveness of VOA, it is an attractive option for assessing HIV persistence.