TZA: a novel assay for measuring the latent HIV-1 reservoir

Combination antiretroviral therapy (cART) has significantly reduced mortality and morbidity of those infected with HIV-1 [1,2]. During cART, the plasma virus levels become undetectable by standard methods of detection [3–5]. Investigators have determined that the major cellular reservoir of latent HIV-1 proviral DNA is contained primarily within the central memory (TCM) and transitional memory (TTM) compartments of resting CD4+ T cells (rCD4) [6–8]. However, further analysis indicates that replication-competent virus is more readily detected in TCM than in TTM [9]. It is thought that the establishment of the latent reservoir occurs when activated CD4+ T cells become infected as they return to a resting memory state [2]. While the frequency of latently infected cells is extremely low, estimated to be 0.1–10 infectious units per million (IUPM) rCD4 in most patients on long-term therapy [7,10,11] during cessation of cART, these long-lasting rCD4 are believed to be the source of emerging virus [12,13]. Therefore, to cure those infected with HIV-1, these latently infected rCD4 must be either eliminated or suppressed in their capacity to produce infectious HIV-1 upon therapy interruption [1,14,15]. However, due to the low frequency of the latently infected cells, lack of cell surface markers specific for latently infected cells, and presence of a replication defective viral genome in latently infected cells [16], it is extremely challenging to accurately quantify the size of the replication-competent reservoir, and hence, to determine the efficacy of any eradication strategy designed to eliminate the infected cells. There have been a number of reports that have identified associations with a few cell surface markers expressed on the CD4+ lymphocytes harboring latent HIV including the immune checkpoint molecules PD-1, LAG-3, TIGIT, and more recently CD32a [17]. Although all of these are important findings, it is unclear if the expression of these particular markers can be directly correlated with or are useful toward measuring of the size of the viral reservoir. Quantification of the latent HIV-1 viral reservoir requires a highly sensitive assay capable of measuring the low frequency of rCD4 carrying functional provirus. Preferably, such an assay should be cost-effective and adaptable for utilization under highthroughput conditions and large-scale clinical applications. To date, the standard method for determining the size of the reservoir utilizes a quantitative viral outgrowth assay (Q-VOA), in which limiting dilutions of rCD4 are cultured with Phytohaemagglutinin (PHA)-stimulated CD4+ cells, and virus production is determined by measuring HIV-1 protein or RNA via ELISA or quantitative polymerase chain reaction (QPCR), respectively [10,11,18,19]. The frequency of the latently infected cells has been estimated using either Poisson Statistics or by maximum likelihood analysis [20,21]. However, the original Q-VOA method is labor-intensive, time-consuming, expensive, and requires the frequent addition of activated CD4+ T cells. Recently, Laird et al. [16] reported a modified version of the original Q-VOA assay that implements the use of the MOLT-4/ CCR5 cell line for viral expansion instead of activated CD4+ T cells, followed by viral RNA measurement. However, this modified procedure is still time-consuming and difficult to perform in a high-throughput setting. The primary limitation of theQ-VOA is its inability to detect all of the latently infected cells containing replication-competent virus, and therefore only provides a minimal definitive estimate of the size of the latent HIV-1 replicationcompetent pool [22–24]. Underestimating the size of this reservoir may give a false impression that an infected individual is ‘cured’ when in fact they may still have latently infected cells capable of producing virus, as has been documented for the Boston patients [25] and the Mississippi baby [26]. While polymerase chain reaction (PCR)-based assays are commonly used to quantify proviral DNA or intracellular RNA transcripts [27–30], they cannot distinguish between replication-competent and defective proviruses [31]. This is important when one considers that the majority of provirus is not replication competent [16]. A failure to distinguish between the two would make it impossible to know when the latent reservoir has been successfully cleared, and when or if patients can safely interrupt therapy. Therefore, there is an urgent need for a fast and sensitive assay that can be used to quantify replication-competent HIV-1 in blood as well as in tissues. Such an assay would be helpful toward the identification and development of novel latency reversing agents (LRAs), and useful as a tool for monitoring HIV-1 clinical trials.