The initiation of antiretroviral therapy (ART) suppresses HIV-1 replication below the limit of detection, reduces mortality and morbidity of HIV-infected patients  and reduces viral transmission . However, ART does not cure HIV-infected individuals. Indeed, upon cessation of ART the viral replication rebounds demonstrating that HIV persists despite conventional ART .
The main mechanisms that hinder HIV-1 eradication are the existence of long-lived latently HIV-1-infected cells and/or the existence of residual virus replication that replenishes the HIV latent cell reservoir [4,5]. Indeed, HIV-1 has been shown to persist within different compartments of the body, that is, blood , lymph nodes , brain , and other immune-privileged sites within different cell subsets [9,10]. One of the most described HIV-1 reservoirs is a small pool of latently infected long-lived memory CD4+ T cells which harbors limited virus transcription and thereby lacks the ability to produce viral proteins during treatment, limiting their elimination by conventional ART or by HIV-specific CD8+ T cells .
In this context, numerous strategies were/are currently being evaluated to target and kill HIV-1 infected cells to ultimately find a cure. One of these strategies, called the ‘shock and kill’ strategy, aims to induce HIV-1 replication from latently HIV-1-infected cells using pharmacological interventions . It has been hypothesized that HIV-1 replication may render HIV-1-infected cells susceptible to elimination either by HIV-specific CD8+ T cells or through virus-mediated cytopathicity . Other strategies focus on the elimination of HIV-1-infected blood and lymph node CD4+ T-cells using HIV-1-specific broad neutralizing antibodies  or antibodies directed against surface markers defining CD4+ T-cell populations enriched for HIV-1-infected cells containing replication competent virus [14–16,17▪]. The impact of such clinical interventions in HIV-infected patients were monitored using HIV viral load, total and integrated HIV DNA (Alu PCR), cell-associated RNA, quantitative viral outgrowth assay (Q-VOA), and time to rebound upon interruption of therapy [18–20], however, recent technical advances may offer new opportunities to comprehensively assess the impact of clinical interventions.
In this context, the objective of this review is to highlight the strengths and limits of standard in-vitro techniques currently being used in the assessment of HIV-1 reservoir in ART-treated patients and to put forward recent advances in the development of robust and highly scalable assays that may help to monitor the impact of clinical interventions in ART-treated patients.
MONITORING CELL-ASSOCIATED DNA AS A MARKER FOR HIV-1 PERSISTENCE
Total cell-associated HIV-1 DNA has been a standard method to estimate the size of the HIV reservoir in HIV-1-infected individuals . It includes the measurement of integrated, unintegrated and 2-long terminal repeat (2-LTR) forms of HIV by real-time PCR in latently and productively infected cells [22,23]. In general, this assay has been largely applied to whole blood, lymphocyte subsets, resting or purified CD4+ T cells in cell pellets and in tissue biopsies [7,16,21]. In blood, Chomont et al. showed using real-time PCR for the assessment of total HIV DNA that it was the central memory CD4+ T cells that contributed the most to the total pool of HIV-1-infected cells. Within tissues, quantification of total HIV-1 DNA further showed residual HIV infection in the gut and rectum of patients under ART . Recently, Strain et al. described and compared droplet digital PCR with conventional quantitative PCR to quantify HIV-1 DNA load in patients on ART and showed that droplet digital PCR indeed improved the signal to noise ratio and proved to be more robust in targeting HIV-1 sequence variations while providing the quantification in absolute values. The authors proposed a limit of detection of about three copies per million cells to limit false positive responses . Others have developed HIV DNA quantification assays based on the detection of highly conserved HIV-1 pol by PCR, with improved sensitivity of detection of up to three to five copies per million cells [25▪]. However, the aforementioned methods cannot discriminate between nonintegrative and integrative forms of HIV. To estimate the frequency of total HIV-infected cells, recent studies focused on Alu-gag PCR assay which allows the estimation of HIV proviruses using specific primers for detection of HIV-1 gag and ‘Alu’ repeat sequences in the human genome that are present approximately every 5000 base pairs [26–28]. The first step of the Alu-gag PCR has variable efficiency depending on the proximity of Alu repeats in human genome and the second step is a real-time PCR within LTR of HIV (Fig. 1) . This method allows exponential amplification of the integrated DNA whereas the unintegrated DNA is only linearly amplified by gag. Clinical trials monitoring the impact of cure interventions have thus focused on the assessment of HIV-integrated DNA [19,30]. In addition, the application of integrated DNA PCR assay in longitudinal assessments in clinical trials to detect changes over time in the size of the pool of infected cells is still under investigation.
Viral genomes that fail to integrate within the cell might convert into circular episomes containing one or two copies of LTR . These circular genomes are useful markers of de-novo infection events . Because of the unique sequence junction that is formed upon end-to-end joining of linear genome, detection of 2-LTR by PCR is believed to be highly specific . However, the use of 2-LTR as a surrogate marker for recent cellular infection is controversial, primarily because of controversy regarding the half-life of episomes . Some studies have noted relative stability of the 2-LTR form of HIV-1 DNA in primary CD4+ T-cell cultures demonstrating that the presence of 2-LTR alone might not be sufficient to detect ongoing replication . Together with other assays, detection of 2-LTR circular form of HIV-1 was used to assess the impact of ART intensification with integrase inhibitor treatment of patients on ART upon the hypothesis that the intensification would increase the 2-LTR form if de novo infection was prevalent during ART . The study noted a transient increase in the 2-LTR form and concluded that at least in a few patients, there might be some residual ongoing HIV-1 replication that in part might contribute to HIV-1 persistence . Moreover, the 2-LTR form is the least abundant form of viral genome in infected patients on ART, and combining assessment with integrated form of the virus is more conclusive of the total burden of infection during treatment.
In summary, PCR-based assays detecting HIV-1 DNA are standardized, robust, and require fewer cells as compared with culture-based methods. However, the major disadvantage of such assays is that they fail to distinguish between defective and replication competent virus. Indeed, a recent characterization of full length proviruses have illustrated that more than 88% of proviruses were defective . Therefore PCR-based methods may on the one hand overestimate the size of the HIV reservoir by quantifying both cells containing replication competent and defective virus and on the other hand may underestimate the impact of clinical interventions aimed at reducing the size of the replication competent reservoir by also assessing the untargeted defective virus. However, till date the evaluation of the frequency of HIV-infected cells by integrated HIV-1 DNA remains important surrogate of the reservoir size.
VIRAL OUTGROWTH ASSAY FOR ASSESSMENT OF REPLICATION COMPETENT VIRUS DURING HIV-1 PERSISTENCE
To estimate the frequency of cells containing replication competent virus, Siliciano's group developed a limiting dilution coculture assay called the Q-VOA . This assay is usually performed on purified resting memory CD4+ T cells isolated from HIV-infected individuals and cocultured with allogeneic CD8+-depleted peripheral blood mononuclear cells (PBMCs) which allow virus amplification [36,37▪]. Using the conventional Q-VOA, cells are classically further stimulated with phytohemaglutinin to induce maximal T-cell activation and the virus production is classically assessed in VOA culture supernatants by conventional HIV P24 ELISA (Fig. 2a)[35,36]. The estimation of the frequency of cells containing replication competent virus is based on maximum likelihood methods and expressed as infectious unit per million (IUPM) (Fig. 2b) . Several studies then focused on the quantification of latently infected resting memory CD4+ T cells containing replication competent HIV-1 in blood of patients on ART and showed the frequency to be about one latently infected cell per million [7,39–42].
Notably, this assay was recently modified to estimate the efficiency of latency reversing agents (LRAs) to reactivate HIV replication in vitro in resting memory CD4+ T cells isolated from long-term-treated aviremic HIV infected patients [37▪] and further improved by detecting HIV P24 by electrochemiluminescence (ECL) (Fig. 2c) [37▪]. Of note, to increase the sensitivity of the Q-VOA, several groups focused on the detection of HIV-1 RNA in VOA culture supernatants and expressed the frequency of cells producing HIV RNA as RNA-unit per million (RUPM) [37▪,43]. To limit background reactivation potentially induced by mixed leukocyte reaction in the assessment of LRA efficiency ex vivo by Q-VOA, this assay was recently further modified by replacing allogeneic CD8+-depleted PBMCs by transformed CD4+ T-cell line (MOLT-4/CCR5) which allows amplification of both X4 and R5 tropic viruses (Fig. 2d) . Using this experimental strategy, Bullen et al. showed that histone deacetylase inhibitors (HDACis) may have limited effectiveness in the reactivation of replication competent HIV-1 in primary resting memory CD4+ T cells, unless combination of mechanistically distinct LRAs was used .
The frequencies of HIV-infected cells estimated by culture-based methods have been, however, shown to be about 300-fold lower than the one estimated by Alu PCR methods . Providing insight into this discrepancy, Ho et al. demonstrated using characterization of full length proviruses that only 10–12% of provirus were inducible, whereas only a fraction of it were indeed induced following VOA. Therefore, these data demonstrate that conventional VOA provides a minimal estimate of the frequency of cells containing replication competent virus. In this context, Cillo et al., proposed to compare the efficiency of LRAs to reactivate HIV replication with HIV replication induced following maximal T-cell activation. The fraction of provirus induced by LRA was then expressed as fractional provirus expression .
Taken together, culture-based methods such as Q-VOA are robust, reproducible and well standardized methods used to estimate the frequency of cells containing replication competent virus. Newly developed and ultrasensitive HIV-1 P24 detection method might further enhance the sensitivity of the Q-VOA [46▪]. However, as Q-VOA requires large number of cells, it may be difficult to apply on lymph node biopsy samples where the cells are limited. In addition, single round of activation used in these methods might not induce all competent proviruses and thus may underestimate the frequency of cells containing replication competent virus . Therefore, these findings highlight the need to develop high throughput and highly scalable assays that could robustly and comprehensively characterize the size of the pool of infected cells containing potentially replication competent virus.
DETECTION OF CELL-ASSOCIATED HIV-1 RNA IN HIV-1 PERSISTENCE
Assessment of cell-associated HIV RNA has been extensively used to evaluate residual virus replication in patients on ART [16,47,48]. However, it has become increasingly clear that the detection of RNA transcripts within the cell does not demonstrate new infection events . Rather, attempts have been made to evaluate viral evolution under ART  and the impact of ART intensification on the production of cell-associated RNA transcripts to demonstrate ongoing virus replication . Moreover, HIV-cure clinical trials evaluating the effectiveness of novel HDACi in purging latently infected cells have evaluated induction of cell-associated RNA upon administration [18,19,51]. Especially with the quantification up to single cell, this method provides a highly sensitive approach to measure HIV-1 persistence. Recently, Procopio et al.[52▪▪], developed a novel assay, the tat/rev-induced limiting dilution assay (TILDA), which quantifies the frequency of cells harboring viral genomes that produce tat/rev multiply spliced HIV-1 RNA upon maximal stimulation. This assay combines the ultrasensitive detection of multiply spliced RNA upon maximal activation of CD4+ T cells with a limiting dilution format to assess the frequency of cells capable of being induced to produce viral RNA transcripts [52▪▪]. The assay is based on the concept that the tat/rev multiply spliced HIV-RNA is essential and sufficient for viral production and can be used as a surrogate marker to detect productively infected cells [52▪▪]. The assay was developed for high throughput studies, requires low numbers of CD4+ T cells, is reproducible (coefficient of variation = 0.2) and covers a wide dynamic range of reservoir size (over 3 logs) [37▪]. However, as the quantification is based on detection of induced tat/rev, this assay still may overestimate the size of the pool of infected cells as mutations in other parts of viral genomes might still render the virus defective. To address this point, pilot clinical trials evaluating the efficacy of the HDACi Romidepsin in reversing HIV-1 latency have used TILDA to measure induced HIV-1 RNA upon drug administration and further studies might reveal the reproducibility and usefulness of TILDA in the evaluation of HIV replication [30,53].
SEQUENCING OF HIV-1 GENOMES TO ASSESS HIV-1 PERSISTENCE
Sequencing of HIV genome has been instrumental in the assessment of low-level HIV replication and molecular evolution during long-term therapy . To reveal the nature of the rebound virus, sequencing can be applied to cell pellets of CD4+ T-cell populations and/or to the virus clones obtained from VOA supernatants and compared with the virus on-ART versus virus obtained upon cessation of ART . In this context, extensive sequencing on the virus env obtained from plasma of treated patients with detectable low-level viremia were compared and aligned to the DNA recovered from blood resting memory CD4+ T cells, revealing that indeed the predominant virus clone in plasma was underrepresented in DNA but was identical to the replicating clone cultured in the virus outgrowth assay of resting memory CD4+ T cells . Phylogenetic analysis of longitudinally obtained sequences from blood CD4+ T cells of treated HIV-1-infected individuals has been crucial in the assessment of genetic evolution of HIV during therapy and has revealed no genetic diversity in the virus sequences when compared with plasma virus obtained from pre-ART [54,56] and confirmed previous findings . Rather, upon initiation of therapy, an accumulation of clonally expanded cells with defective provirus was noted by other studies [57,58]. Recently, deep sequencing was applied to blood and lymph node HIV DNA+ cells and using phylogenetic analysis and mathematical modelling, was able to shed insight into the evolutionary and infection dynamics of HIV-1 within the host, revealing that HIV-1 can continue to replicate in tissues and replenish the viral reservoir despite potent ART .
Taken together, while assessment of HIV-integrated DNA overestimates and culture-based methods underestimate the size of HIV reservoir, the application of sequencing technology might be one of the assays that may provide an accurate frequency of replication competent virus in HIV-infected patients on ART. In addition, the study by Ho et al. showed that direct sequencing and genome synthesis technology might be crucial to delineate induced provirus versus noninduced replication competent or defective virus. However, sequencing of viral clones requires a large number of cells and may be difficult because of large and diverse HIV populations within distinct integration sites and various cell subsets  (Table 1).
In the past few years, a large amount of effort has been made to compare, standardize, develop and further improve currently available assays to quantitatively and qualitatively monitor the pool of HIV-1-infected cells in HIV-infected patients (Table 1). However, the recent interventional HIV-1-cure clinical trials have highlighted the difficulty to comprehensively monitor the impact of such interventions on HIV reservoirs using currently available assays. Indeed, assessing time to viral rebound upon analytical treatment interruption remains the most reliable assay to evaluate the effectiveness of HIV-1 cure interventions in reducing the size of the reservoir. In fact, persistent HIV-1 infection may be confounded by the presence of multiple affected compartments, cell types, status of the virus, and state of the infected cell, whereas currently available assays quantify HIV-1 persistence mainly within peripheral blood using either PCR-based (quantifying DNA and RNA) or culture-based methods (quantifying replication competent virus). Therefore, there is still an important need to develop a reliable and sensitive assay that could be performed in cell populations isolated from blood and tissues that may effectively predict the time to viral rebound upon treatment interruption.
We would like to thank Aaron Weddle for his assistance in figure preparation.
Financial support and sponsorship
The work was supported by an educational grant of Bristol Myer Squibb to M.P.
Conflicts of interest
There are no conflicts of interest.
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