Current approaches to assess HIV-1 persistence

Banga, Riddhima; Procopio, Francesco A.; Perreau, Matthieu

Current Opinion in HIV & AIDS:
doi: 10.1097/COH.0000000000000282
STRATEGIES FOR TARGETING RESIDUAL HIV INFECTION: Edited by Matthieu Perreau and Nicolas Chomont
Editor's Choice
Abstract

Purpose of review: The persistence of HIV within long-lived HIV-infected CD4+ T cells is the primary obstacle towards HIV eradication and numerous strategies are currently being evaluated to target and kill HIV-infected cells to ultimately find a cure. HIV reservoirs are classically quantified by standard methods such as integrated HIV DNA (Alu PCR) and/or quantitative viral outgrowth assay; however, recent technical advances may offer new opportunities to comprehensively assess the impact of clinical interventions.

Recent findings: Digital droplet PCR, tat/rev-induced limiting dilution analysis, enhanced quantitative viral outgrowth assay, and whole genome sequencing technologies offer increased precision and/or higher sensitivity to quantify and characterize HIV reservoirs in antiretroviral therapy-treated HIV-infected patients.

Summary: The objective of this review is to highlight the characteristics and limits of recent technical advances that may help to monitor the impact of clinical interventions in antiretroviral therapy-treated patients.

Author Information

Division of Immunology and Allergy, Lausanne University Hospital, University of Lausanne, Lausanne, Switzerland

Correspondence to Matthieu Perreau, PhD, Division of Immunology and Allergy, Lausanne University Hospital, 1011 Lausanne, Switzerland. Tel: +41213141073; e-mail: Matthieu.Perreau@chuv.ch

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INTRODUCTION

The initiation of antiretroviral therapy (ART) suppresses HIV-1 replication below the limit of detection, reduces mortality and morbidity of HIV-infected patients [1] and reduces viral transmission [2]. However, ART does not cure HIV-infected individuals. Indeed, upon cessation of ART the viral replication rebounds demonstrating that HIV persists despite conventional ART [3].

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 [6], lymph nodes [7], brain [8], 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 [11].

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 [12]. 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 [12]. Other strategies focus on the elimination of HIV-1-infected blood and lymph node CD4+ T-cells using HIV-1-specific broad neutralizing antibodies [13] 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.

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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 [21]. 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.[6] 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 [16]. Recently, Strain et al.[24] 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 [24]. 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) [29]. This method allows exponential amplification of the integrated DNA whereas the unintegrated DNA is only linearly amplified by gag[29]. 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 [31]. These circular genomes are useful markers of de-novo infection events [31]. 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 [31]. 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 [31]. 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 [32]. 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 [33]. 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 [33]. 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 [34]. 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.

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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 [35]. 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) [38]. 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) [44]. Using this experimental strategy, Bullen et al.[38] 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 [45].

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 [27]. Providing insight into this discrepancy, Ho et al.[34] 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.[43], 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 [43].

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 [34]. 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.

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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 [47]. Rather, attempts have been made to evaluate viral evolution under ART [49] and the impact of ART intensification on the production of cell-associated RNA transcripts to demonstrate ongoing virus replication [50]. 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].

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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 [54]. 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 [55]. 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 [55]. 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 [56]. 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 [59].

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.[34] 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 [60] (Table 1).

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CONCLUSION

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.

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Acknowledgements

We would like to thank Aaron Weddle for his assistance in figure preparation.

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Financial support and sponsorship

The work was supported by an educational grant of Bristol Myer Squibb to M.P.

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Conflicts of interest

There are no conflicts of interest.

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REFERENCES AND RECOMMENDED READING

Papers of particular interest, published within the annual period of review, have been highlighted as:

▪ of special interest

▪▪ of outstanding interest

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REFERENCES

1. Palella FJ, Armon C, Buchacz K, et al. Factors associated with mortality among persistently viraemic triple-antiretroviral-class-experienced patients receiving antiretroviral therapy in the HIV Outpatient Study (HOPS). J Antimicrob Chemother 2014; 69:2826–2834.
2. Williams G, Lima V, Gouws E. Modelling the impact of antiretroviral therapy on the epidemic of HIV. Curr HIV Res 2011; 9:367–382.
3. Ruiz L, Martinez-Picado J, Romeu J, et al. Structured treatment interruption in chronically HIV-1 infected patients after long-term viral suppression. AIDS 2000; 14:397–403.
4. Chun T-W, Nickle DC, Justement JS, et al. HIV-infected individuals receiving effective antiviral therapy for extended periods of time continually replenish their viral reservoir. J Clin Invest 2005; 115:3250–3255.
5. Chun T-W, Stuyver L, Mizell SB, et al. Presence of an inducible HIV-1 latent reservoir during highly active antiretroviral therapy. Proc Natl Acad Sci U S A 1997; 94:13193–13197.
6. Chomont N, El-Far M, Ancuta P, et al. HIV reservoir size and persistence are driven by T cell survival and homeostatic proliferation. Nat Med 2009; 15:893–900.
7. Wong JK, Hezareh M, Günthard HF, et al. Recovery of replication-competent HIV despite prolonged suppression of plasma viremia. Science 1997; 278:1291–1295.
8. Heaton R, Clifford D, Franklin D, et al. HIV-associated neurocognitive disorders persist in the era of potent antiretroviral therapy CHARTER Study. Neurology 2010; 75:2087–2096.
9. Muciaccia B, Filippini A, Ziparo E, et al. Testicular germ cells of HIV-seropositive asymptomatic men are infected by the virus. J Reprod Immunol 1998; 41:81–93.
10. Eisele E, Siliciano RF. Redefining the viral reservoirs that prevent HIV-1 eradication. Immunity 2012; 37:377–388.
11. Van Lint C, Bouchat S, Marcello A. HIV-1 transcription and latency: an update. Retrovirology 2013; 10:1.
12. Deeks SG, Autran B, Berkhout B, et al. Towards an HIV cure: a global scientific strategy. Nat Rev Immunol 2012; 12:607–614.
13. Pegu A, Asokan M, Wu L, et al. Activation and lysis of human CD4 cells latently infected with HIV-1. Nat Commun 2015; 6:8447.
14. Perreau M, Savoye A-L, De Crignis E, et al. Follicular helper T cells serve as the major CD4 T cell compartment for HIV-1 infection, replication, and production. J Exp Med 2013; 210:143–156.
15. Gosselin A, Monteiro P, Chomont N, et al. Peripheral blood CCR4+ CCR6+ and CXCR3+ CCR6+ CD4+ T cells are highly permissive to HIV-1 infection. J Immunol 2010; 184:1604–1616.
16. Yukl SA, Shergill AK, Ho T, et al. The distribution of HIV DNA and RNA in cell subsets differs in gut and blood of HIV-positive patients on ART: implications for viral persistence. J Infect Dis 2013; 208:1212–1220.
17▪. Buzon MJ, Sun H, Li C, et al. HIV-1 persistence in CD4+ T cells with stem cell-like properties. Nat Med 2014; 20:139–142.

The study showed a transient increase in 2-LTR circles in a few patients when suppressive ART was intensified with integrase inhibitor raltegravir, suggesting that low-level replication persisted despite ART.

18. Archin N, Liberty A, Kashuba A, et al. Administration of vorinostat disrupts HIV-1 latency in patients on antiretroviral therapy. Nature 2012; 487:482–485.
19. Rasmussen TA, Tolstrup M, Brinkmann CR, et al. Panobinostat, a histone deacetylase inhibitor, for latent-virus reactivation in HIV-infected patients on suppressive antiretroviral therapy: a phase 1/2, single group, clinical trial. Lancet HIV 2014; 1:e13–e21.
20. Wei DG, Chiang V, Fyne E, et al. Histone deacetylase inhibitor romidepsin induces HIV expression in CD4 T cells from patients on suppressive antiretroviral therapy at concentrations achieved by clinical dosing. PLoS Pathog 2014; 10:e1004071.
21. Piatak M Jr, Luk K, Williams B, Lifson J, et al. Quantitative competitive polymerase chain reaction for accurate quantitation of HIV DNA and RNA species. Biotechniques 1993; 14:70–81.
22. Ibáñez A, Puig T, Elias J, et al. Quantification of integrated and total HIV-1 DNA after long-term highly active antiretroviral therapy in HIV-1-infected patients. AIDS 1999; 13:1045–1049.
23. Butler SL, Hansen MS, Bushman FD. A quantitative assay for HIV DNA integration in vivo. Nat Med 2001; 7:631–634.
24. Strain MC, Lada SM, Luong T, et al. Highly precise measurement of HIV DNA by droplet digital PCR. PLoS One 2013; 8:e55943.
25▪. Hong F, Aga E, Cillo A, et al. Novel assays to measure total cell-associated HIV-1 DNA and RNA. J Clin Microbiol 2016; JCM. 02904-15 [Epub ahead of print].

New assays for the quantification of HIV DNA based on the detection of HIV pol with improved sensitivity of detection of up to three copies of HIV DNA molecules.

26. Siliciano JD, Kajdas J, Finzi D, et al. Long-term follow-up studies confirm the stability of the latent reservoir for HIV-1 in resting CD4+ T cells. Nat Med 2003; 9:727–728.
27. Eriksson S, Graf EH, Dahl V, et al. Comparative analysis of measures of viral reservoirs in HIV-1 eradication studies. PLoS Pathog 2013; 9:e1003174.
28. Liszewski MK, Jianqing JY, O’Doherty U. Detecting HIV-1 integration by repetitive-sampling Alu-gag PCR. Methods 2009; 47:254–260.
29. Graf EH, O’Doherty U. Quantitation of integrated proviral DNA in viral reservoirs. Curr Opin HIV AIDS 2013; 8:100–105.
30. S⊘gaard OS, Graversen ME, Leth S, et al. The depsipeptide romidepsin reverses HIV-1 latency in vivo. PLoS Pathog 2015; 11:e1005142.
31. Sharkey M. Tracking episomal HIV DNA: implications for viral persistence and eradication of HIV. Curr Opin HIV AIDS 2013; 8:93–99.
32. Pace MJ, Graf EH, O’Doherty U. HIV 2-long terminal repeat circular DNA is stable in primary CD4+ T cells. Virology 2013; 441:18–21.
33. Buzón MJ, Massanella M, Llibre JM, et al. HIV-1 replication and immune dynamics are affected by raltegravir intensification of HAART-suppressed subjects. Nat Med 2010; 16:460–465.
34. Ho Y-C, Shan L, Hosmane NN, et al. Replication-competent noninduced proviruses in the latent reservoir increase barrier to HIV-1 cure. Cell 2013; 155:540–551.
35. Siliciano JD, Siliciano RF. Enhanced culture assay for detection and quantitation of latently infected, resting CD4+ T-cells carrying replication-competent virus in HIV-1-infected individuals. Methods Mol Biol 2005; 304:3–15.
36. Ylisastigui L, Archin NM, Lehrman G, et al. Coaxing HIV-1 from resting CD4 T cells: histone deacetylase inhibition allows latent viral expression. AIDS 2004; 18:1101–1108.
37▪. Banga R, Procopio F, Cavassini M, Perreau M. In vitro reactivation of replication competent and infectious HIV-1 by HDAC inhibitors. J Virol 2015; 90:1858–1871.

The study evaluated the efficiency of different LRAs using a modified viral outgrowth assay that used a novel method of P24 detection, that is, electrochemiluminescence, and showed that certain HDAC inhibitors might reverse HIV-1 latency ex vivo in resting CD4+ T cells isolated from long-term-treated aviremic HIV-1-infected study participants, supporting their further development in HIV-1-cure clinical trials.

38. Bullen CK, Laird GM, Durand CM, Siliciano JD, Siliciano RF. New ex vivo approaches distinguish effective and ineffective single agents for reversing HIV-1 latency in vivo. Nat Med 2014; 20:425–429.
39. Soriano-Sarabia N, Bateson RE, Dahl NP, et al. Quantitation of replication-competent HIV-1 in populations of resting CD4+ T cells. J Virol 2014; 88:14070–14077.
40. Chun T-W, Carruth L, Finzi D, et al. Quantification of latent tissue reservoirs and total body viral load in HIV-1 infection. Nature 1997; 387:183–188.
41. Finzi D, Blankson J, Siliciano JD, et al. Latent infection of CD4+ T cells provides a mechanism for lifelong persistence of HIV-1, even in patients on effective combination therapy. Nat Med 1999; 5:512–517.
42. Finzi D, Hermankova M, Pierson T, et al. Identification of a reservoir for HIV-1 in patients on highly active antiretroviral therapy. Science 1997; 278:1295–1300.
43. Cillo AR, Sobolewski MD, Bosch RJ, et al. Quantification of HIV-1 latency reversal in resting CD4+ T cells from patients on suppressive antiretroviral therapy. Proc Natl Acad Sci U S A 2014; 111:7078–7083.
44. Laird GM, Eisele EE, Rabi SA, et al. Rapid quantification of the latent reservoir for HIV-1 using a viral outgrowth assay. PLoS Pathog 2013; 9:e1003398.
45. Laird GM, Bullen CK, Rosenbloom DI, et al. Ex vivo analysis identifies effective HIV-1 latency: reversing drug combinations. J Clin Invest 2015; 125:1901–1912.
46▪. Howell BJ, Wu G, Swanson M, et al. Developing and applying ultrasensitive P24 protein immunoassay for HIV latency. Abstracts of the Seventh International Workshop on HIV Persistence during Therapy, 8–11 December 2015, Miami, Florida, USA. J Virus Eradication 2015; 1 (Suppl 1):5.

Novel assay for ultrasensitive P24 detection in culture supernatant of culture-based assays.

47. Pasternak AO, Lukashov VV, Berkhout B. Cell-associated HIV RNA: a dynamic biomarker of viral persistence. Retrovirology 2013; 10:41.
48. Fletcher CV, Staskus K, Wietgrefe SW, et al. Persistent HIV-1 replication is associated with lower antiretroviral drug concentrations in lymphatic tissues. Proc Natl Acad Sci U S A 2014; 111:2307–2312.
49. Kieffer TL, Finucane MM, Nettles RE, et al. Genotypic analysis of HIV-1 drug resistance at the limit of detection: virus production without evolution in treated adults with undetectable HIV loads. J Infect Dis 2004; 189:1452–1465.
50. Dinoso JB, Kim SY, Wiegand AM, et al. Treatment intensification does not reduce residual HIV-1 viremia in patients on highly active antiretroviral therapy. Proc Natl Acad Sci U S A 2009; 106:9403–9408.
51. Archin NM, Bateson R, Tripathy M, et al. HIV-1 expression within resting CD4 T-cells following multiple doses of vorinostat. J Infect Dis 2014; 210:728–735.
52▪▪. Procopio FA, Fromentin R, Kulpa DA, et al. A novel assay to measure the magnitude of the inducible viral reservoir in HIV-infected individuals. EBioMedicine 2015; 2:872–881.

The study developed a novel assay called the tat/rev-induced limiting dilution analysis for the quantification of HIV reservoir based on the detection of multiply spliced tat/rev HIV RNA.

53. Spivak AM, Planelles V. HIV-1 eradication: early trials (and tribulations). Trends Mol Med 2016; 22:10–27.
54. Kearney MF, Spindler J, Shao W, et al. Lack of detectable HIV-1 molecular evolution during suppressive antiretroviral therapy. PLoS Pathog 2014; 10:e1004010.
55. Anderson JA, Archin NM, Ince W, et al. Clonal sequences recovered from plasma from patients with residual HIV-1 viremia and on intensified antiretroviral therapy are identical to replicating viral RNAs recovered from circulating resting CD4+ T cells. J Virol 2011; 85:5220–5223.
56. Evering TH, Mehandru S, Racz P, et al. Absence of HIV-1 evolution in the gut-associated lymphoid tissue from patients on combination antiviral therapy initiated during primary infection. PLoS Pathog 2012; 8:e1002506.
57. Cohn LB, Silva IT, Oliveira TY, et al. HIV-1 integration landscape during latent and active infection. Cell 2015; 160:420–432.
58. Wagner TA, McLaughlin S, Garg K, et al. Proliferation of cells with HIV integrated into cancer genes contributes to persistent infection. Science 2014; 345:570–573.
59. Lorenzo-Redondo R, Fryer HR, Bedford T, et al. Persistent HIV-1 replication maintains the tissue reservoir during therapy. Nature 2016; 530:51–56.
60. Maldarelli F, Wu X, Su L, et al. Specific HIV integration sites are linked to clonal expansion and persistence of infected cells. Science 2014; 345:179–183.
61. Palmer S, Wiegand AP, Maldarelli F, et al. New real-time reverse transcriptase-initiated PCR assay with single-copy sensitivity for human immunodeficiency virus type 1 RNA in plasma. J Clin Microbiol 2003; 41:4531–4536.
62. Douek DC, Brenchley JM, Betts MR, et al. HIV preferentially infects HIV-specific CD4+ T cells. Nature 2002; 417:95–98.
63. O’Doherty U, Swiggard WJ, Jeyakumar D, et al. A sensitive, quantitative assay for human immunodeficiency virus type 1 integration. J Virol 2002; 76:10942–10950.
Keywords:

integrated HIV DNA; quantitative viral outgrowth assay; tat/rev-induced limiting dilution analysis

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