Search

Developing an intervention that results in virologic control following discontinuation of antiretroviral therapy (ART) is a major objective of HIV-1 cure research. In this study, we investigated the therapeutic efficacy of a vaccine consisting of adenovirus serotype 26 (Ad26) and modified vaccinia Ankara (MVA) with or without an SIV Envelope (Env) gp140 protein with alum adjuvant in combination with the TLR7 agonist vesatolimod (GS-9620) in 36 ART-suppressed, SIVmac251-infected rhesus macaques. Ad26/MVA therapeutic vaccination led to robust humoral and cellular immune responses, and the Env protein boost increased antibody responses. Following discontinuation of ART, virologic control was observed in 5/12 animals in each vaccine group, compared with 0/12 animals in the sham control group. These data demonstrate therapeutic efficacy of Ad26/MVA vaccination with vesatolimod but no clear additional benefit of adding an Env protein boost. SIV-specific cellular immune responses correlated with virologic control. Our findings show partial efficacy of therapeutic vaccination following ART discontinuation in SIV-infected rhesus macaques.

Antiretroviral therapy (ART) effectively inhibits HIV-1 replication but is not curative due to the persistence of a latent viral reservoir in resting CD4+ T cells. This reservoir is a major barrier to cure. Sequencing studies have revealed that the population of proviruses persisting in ART-treated individuals is dominated by defective proviruses that cannot give rise to viral rebound due to fatal defects including large deletions and APOBEC3-mediated hypermutation. Near full genome sequencing (nFGS) of individual proviruses is used in reservoir assays to provide an estimate of the fraction of proviruses that are intact. nFGS methods rely on a long-distance outer PCR capturing most (~9 kb) of the genome, followed by nested inner PCRs. The outer PCR is carried out at limit dilution, and interpretation of the results is based on the assumption that all proviruses are quantitatively captured. Here, we evaluate nFGS methods using the intact proviral DNA assay (IPDA), a multiplex digital droplet PCR assay that quantitates intact and defective proviruses with single molecule sensitivity using only short, highly efficient amplicons. We analyzed proviral templates of known sequence to avoid the additional complication of sequence polymorphism. With the IPDA, we quantitated molecular yields at each step of nFGS methods. We demonstrate that nFGS methods are inefficient and miss ~70% of full-length proviruses due to amplification failure at the initial outer PCR step. In contrast, proviruses with large internal deletions encompassing 70% of the genome can be quantitatively amplified under the same conditions. Accurate measurement of the latent reservoir of HIV-1 is essential for evaluating the efficacy of cure strategies, and the bias against full length proviruses in nFGS methods must be considered.

Clonal expansion of infected CD4(+) T cells is a major mechanism of HIV-1 persistence and a barrier to achieving a cure. Potential causes are homeostatic proliferation, effects of HIV-1 integration, and interaction with antigens. Here, we show that it is possible to link antigen responsiveness, the full proviral sequence, the integration site, and the T cell receptor β-chain (TCRβ) sequence to examine the role of recurrent antigenic exposure in maintaining the HIV-1 reservoir. We isolated CMV- and Gag-responding CD4(+) T cells from 10 treated individuals. Proviral populations in CMV-responding cells were dominated by large clones, including clones harboring replication-competent proviruses. TCRβ repertoires showed high clonality driven by converging adaptive responses. Although some proviruses were in genes linked to HIV-1 persistence (BACH2, STAT5B, MKL1), the proliferation of infected cells under antigenic stimulation occurred regardless of the site of integration. Paired TCRβ and integration site analysis showed that infection could occur early or late in the course of a clone’s response to antigen and could generate infected cell populations too large to be explained solely by homeostatic proliferation. Together, these findings implicate antigen-driven clonal selection as a major factor in HIV-1 persistence, a finding that will be a difficult challenge to eradication efforts.

Latent HIV infection is the main barrier to cure, and most HIV-infected cells reside in the gut, where distinct but unknown mechanisms may promote viral latency. Transforming growth factor β (TGF-β), which induces the expression of CD103 on tissue-resident memory T cells, has been implicated in HIV latency. Using CD103 as a surrogate marker to identify cells that have undergone TGF-β signaling, we compared the HIV RNA/DNA contents and cellular transcriptomes of CD103+ and CD103- CD4 T cells from the blood and rectum of HIV-negative (HIV-) and antiretroviral therapy (ART)-suppressed HIV-positive (HIV+) individuals. Like gut CD4+ T cells, circulating CD103+ cells harbored more HIV DNA than did CD103- cells but transcribed less HIV RNA per provirus. Circulating CD103+ cells also shared a gene expression profile that is closer to that of gut CD4 T cells than to that of circulating CD103- cells, with significantly lower expression levels of ribosomal proteins and transcriptional and translational pathways associated with HIV expression but higher expression levels of a subset of genes implicated in suppressing HIV transcription. These findings suggest that blood CD103+ CD4 T cells can serve as a model to study the molecular mechanisms of HIV latency in the gut and reveal new cellular factors that may contribute to HIV latency.IMPORTANCE The ability of HIV to establish a reversibly silent, "latent" infection is widely regarded as the main barrier to curing HIV. Most HIV-infected cells reside in tissues such as the gut, but it is unclear what mechanisms maintain HIV latency in the blood or gut. We found that circulating CD103+ CD4+ T cells are enriched for HIV-infected cells in a latent-like state. Using RNA sequencing (RNA-seq), we found that CD103+ T cells share a cellular transcriptome that more closely resembles that of CD4+ T cells from the gut, suggesting that they are homing to or from the gut. We also identified the cellular genes whose expression distinguishes gut CD4+ or circulating CD103+ T cells from circulating CD103- T cells, including some genes that have been implicated in HIV expression. These genes may contribute to latent HIV infection in the gut and may serve as new targets for therapies aimed at curing HIV.

The intact proviral DNA assay (IPDA) and quadruplex PCR (Q4PCR) represent major advances in accurately quantifying and characterizing the replication-competent HIV reservoir. This study compares the two novel approaches for measuring intact HIV proviral DNA in samples from 39 antiretroviral therapy (ART)-suppressed people living with HIV, thereby informing ongoing efforts to deplete the HIV reservoir in cure-related trials., The HIV proviral reservoir is the major barrier to cure. The predominantly replication-defective proviral landscape makes the measurement of virus that is likely to cause rebound upon antiretroviral therapy (ART)-cessation challenging. To address this issue, novel assays to measure intact HIV proviruses have been developed. The intact proviral DNA assay (IPDA) is a high-throughput assay that uses two probes to exclude the majority of defective proviruses and determine the frequency of intact proviruses, albeit without sequence confirmation. Quadruplex PCR with four probes (Q4PCR) is a lower-throughput assay that uses limiting dilution long-distance PCR amplification followed by quantitative PCR (qPCR) and near-full-length genome sequencing (nFGS) to estimate the frequency of sequence-confirmed intact proviruses and provide insight into their clonal composition. To explore the advantages and limitations of these assays, we compared IPDA and Q4PCR measurements from 39 ART-suppressed people living with HIV. We found that IPDA and Q4PCR measurements correlated with one another, but frequencies of intact proviral DNA differed by approximately 19-fold. This difference may be in part due to inefficiencies in long-distance PCR amplification of proviruses in Q4PCR, leading to underestimates of intact proviral frequencies. In addition, nFGS analysis within Q4PCR explained that some of this difference is explained by proviruses that are classified as intact by IPDA but carry defects elsewhere in the genome. Taken together, this head-to-head comparison of novel intact proviral DNA assays provides important context for their interpretation in studies to deplete the HIV reservoir and shows that together the assays bracket true reservoir size. IMPORTANCE The intact proviral DNA assay (IPDA) and quadruplex PCR (Q4PCR) represent major advances in accurately quantifying and characterizing the replication-competent HIV reservoir. This study compares the two novel approaches for measuring intact HIV proviral DNA in samples from 39 antiretroviral therapy (ART)-suppressed people living with HIV, thereby informing ongoing efforts to deplete the HIV reservoir in cure-related trials.

Type 1 interferons (IFN-I) are potent innate antiviral effectors that constrain HIV-1 transmission. However, harnessing these cytokines for HIV-1 cure strategies has been hampered by an incomplete understanding of their anti-viral activities at later stages of infection. Here, we characterized the IFN-I sensitivity of 500 clonally-derived HIV-1 isolates from plasma and CD4+ T cells of 26 individuals sampled longitudinally following transmission and/or after antiretroviral therapy (ART) and analytical treatment interruption (ATI). Determining the concentration of IFNα2 and IFNβ that reduced HIV-1 replication by 50% (IC50), we found remarkably consistent changes in the sensitivity of viruses to IFN-I inhibition, both across individuals and over time. IFN-I resistance was uniformly high during acute infection, decreased in all subjects in the first year post-infection, was reacquired concomitant with CD4+ T cell loss, and remained elevated in subjects with accelerated disease. Isolates obtained by viral outgrowth during suppressive ART were relatively IFN-I sensitive, resembling viruses circulating just prior to ART initiation. However, viruses that rebounded following treatment interruption displayed the highest levels of IFNα2 and IFNβ resistance observed at any time during the infection course. These findings indicate a dynamic interplay between host innate immune responses and the evolving HIV-1 quasispecies, with the relative contribution of IFN-I to HIV-1 control impacted by both ART and ATI. Although elevated at transmission, IFN-mediated pressures are the highest during viral rebound, limiting the viruses that successfully reactivate from latency.One Sentence SummaryHIV-1 resistance to IFN-I is highest during acute infection and following analytic treatment interruption, indicating a dynamic interplay between host innate immunity and virus biology.

Uncovering the sanctuaries of the long-lived HIV-1 reservoir is crucial to develop cure strategies. Pediatric immunity is distinct from that of adults, which may alter where the reservoir is established in infancy. Thus, it is important to utilize pediatric models to inform cure-directed approaches for HIV-1-infected children. We used an infant rhesus macaque model of HIV-1 infection via breastfeeding to identify key sites of viral persistence under antiretroviral therapy (ART). The gastrointestinal tract was found to be a site for low-level viral transcription during ART. We also show that naive CD4+ T cells harbored intact provirus and were a major contributor to blood and lymphoid reservoir size. This is particularly striking, as memory CD4+ T cells are generally regarded as the main source of latent HIV/simian immunodeficiency virus (SIV) infection of adult humans and rhesus macaques. Our findings highlight unique features of reservoir composition in pediatric infection that should be considered for eradication efforts., Mother-to-child transmission of human immunodeficiency virus type 1 (HIV-1) continues to cause new pediatric cases of infection through breastfeeding, a setting where it is not always possible to initiate early antiretroviral therapy (ART). Without novel interventions that do not rely on daily ART, HIV-1-infected children face lifelong medications to control infection. A detailed analysis of virus persistence following breast milk transmission of HIV-1 and ART has not been performed. Here, we used infant rhesus macaques orally infected with simian/human immunodeficiency virus (SHIV) (SHIV.C.CH505) to identify cellular and anatomical sites of virus persistence under ART. Viral DNA was detected at similar levels in blood and tissue CD4+ T cells after a year on ART, with virus in blood and lymphoid organs confirmed to be replication competent. Viral RNA/DNA ratios were elevated in rectal CD4+ T cells compared to those of other sites (P ≤ 0.0001), suggesting that the gastrointestinal tract is an active site of virus transcription during ART-mediated suppression of viremia. SHIV.C.CH505 DNA was detected in multiple CD4+ T cell subsets, including cells with a naive phenotype (CD45RA+ CCR7+ CD95–). While the frequency of naive cells harboring intact provirus was lower than in memory cells, the high abundance of naive cells in the infant CD4+ T cell pool made them a substantial source of persistent viral DNA (approximately 50% of the total CD4+ T cell reservoir), with an estimated 1:2 ratio of intact provirus to total viral DNA. This viral reservoir profile broadens our understanding of virus persistence in a relevant infant macaque model and provides insight into targets for cure-directed approaches in the pediatric population. IMPORTANCE Uncovering the sanctuaries of the long-lived HIV-1 reservoir is crucial to develop cure strategies. Pediatric immunity is distinct from that of adults, which may alter where the reservoir is established in infancy. Thus, it is important to utilize pediatric models to inform cure-directed approaches for HIV-1-infected children. We used an infant rhesus macaque model of HIV-1 infection via breastfeeding to identify key sites of viral persistence under antiretroviral therapy (ART). The gastrointestinal tract was found to be a site for low-level viral transcription during ART. We also show that naive CD4+ T cells harbored intact provirus and were a major contributor to blood and lymphoid reservoir size. This is particularly striking, as memory CD4+ T cells are generally regarded as the main source of latent HIV/simian immunodeficiency virus (SIV) infection of adult humans and rhesus macaques. Our findings highlight unique features of reservoir composition in pediatric infection that should be considered for eradication efforts.

Therapeutic strategies are being clinically tested either to eradicate latent HIV reservoirs or to achieve virologic control in the absence of antiretroviral therapy. Attaining this goal will require a consensus on how best to measure the numbers of persistently infected cells with the potential to cause viral rebound after antiretroviral-therapy cessation in assessing the results of cure-directed strategies in vivo. Current measurements assess various aspects of the HIV provirus and its functionality and produce divergent results. Here, we provide recommendations from the BEAT-HIV Martin Delaney Collaboratory on which viral measurements should be prioritized in HIV-cure-directed clinical trials. This work was supported by the NIH-funded BEAT-HIV Martin Delaney Collaboratory to cure HIV-1 infection (1UM1Al126620). LJM is also supported by NIH R01 AI065279, U01 AI065279, R01 DA048728, R01 DA049666, Kean Family Professorship, and the Philadelphia Foundation (Roberts I. Jacobs Fund). M-AM is supported by NIH grants (DK123733, AG062383, NS117458, AI143385, AI129636, and NS106970), The Foundation for AIDS Research (amfAR) impact grant # 109840–65-RGRL, and W.W. Smith Charitable Trust grant # A1901, Wistar Cancer Center Support Grant P30 CA010815–49S2, and the Penn Center for AIDS Research (AI 045008). MJB is supported by The Miguel Servet program funded by the Spanish Health Institute Carlos III (CP17/00179). M. L. Is supported by NIH grants AI117841, AI120008, AI124776, AI130005, AI122377, and AI135940. XGY is supported by NIH grants AI116228, AI078799, HL134539, AI125109, and DA047034. RS supported by AI126603, AI126620 and AI12661, AI094189, 43222 Howard Hughes Medical Institute, and the Bill and Melinda Gates Foundation (OPP1115715). VP supported by AI143567, AI124843. Y-C Ho supported by Yale Top Scholar, Rudolf J. Anderson Fellowship, AI141009, DA047037, AI118402, W.W. Smith AIDS Research Grant, Gilead AIDS Research Grant, Gilead Research Scholar Grant, AI150464, AI094189, AI14868. J.D.E is supported by NIH and the Bill and Melinda Gates Foundation grants 75N93019C00070, AI133706, AI110164, AI141258, AI143411, AI149672, CA206466, DK119945, INV-002704, and OD011092–60, and OPPO1108533. Sí

Antiretroviral therapy can effectively block HIV-1 replication and prevent or reverse immunodeficiency in HIV-1-infected individuals. However, viral replication resumes within weeks of treatment interruption. The major barrier to a cure is a small pool of resting memory CD4+ T cells that harbor latent HIV-1 proviruses. This latent reservoir is now the focus of an intense international research effort. We describe how the reservoir is established, challenges involved in eliminating it, and pharmacologic and immunologic strategies for targeting this reservoir. The development of a successful cure strategy will most likely require understanding the mechanisms that maintain HIV-1 proviruses in a latent state and pathways that drive the proliferation of infected cells, which slows reservoir decay. In addition, a cure will require the development of effective immunologic approaches to eliminating infected cells. There is renewed optimism about the prospect of a cure, and the interventions discussed here could pave the way.

Objective Resting CD4 T cells have been recognized as the major cell reservoir of latent HIV-1 during antiretroviral therapy (ART). Using an simian immunodeficiency virus (SIV)/macaque model for AIDS and HIV-related neurocognitive disorders we assessed the contribution of the brain to viral latency and reactivation. Design Pigtailed macaques were dual inoculated with SIVDeltaB670 and SIV17E-Fr and treated with an efficacious central nervous system-penetrant ART. After 500 days of viral suppression animals were treated with two cycles of latency reversing agents and increases in viral transcripts were examined. Methods Longitudinal plasma and cerebrospinal fluid (CSF) viral loads were analyzed by quantitative and digital droplet PCR. After necropsy, viral transcripts in organs were analyzed by PCR, in-situ hybridization, and phylogenetic genotyping based on env V1 loop sequences. Markers for neuronal damage and CSF activation were measured by ELISA. Results Increases in activation markers and plasma and CSF viral loads were observed in one animal treated with latency reversing agents, despite ongoing ART. SIV transcripts were identified in occipital cortex macrophages by in-situ hybridization and CD68 staining. The most abundant SIV genotype in CSF was unique and expanded independent from viruses found in the periphery. Conclusion The central nervous system harbors latent SIV genomes after long-term viral suppression by ART, indicating that the brain represents a potential viral reservoir and should be seriously considered during AIDS cure strategies.

The latent reservoir for HIV-1 in resting CD4+ T cells prevents cure with antiretroviral therapy. Hosmane et al. provide evidence supporting the hypothesis that a larger fraction of cells in the reservoir is generated by cell proliferation than by direct infection., A latent reservoir for HIV-1 in resting CD4+ T lymphocytes precludes cure. Mechanisms underlying reservoir stability are unclear. Recent studies suggest an unexpected degree of infected cell proliferation in vivo. T cell activation drives proliferation but also reverses latency, resulting in productive infection that generally leads to cell death. In this study, we show that latently infected cells can proliferate in response to mitogens without producing virus, generating progeny cells that can release infectious virus. Thus, assays relying on one round of activation underestimate reservoir size. Sequencing of independent clonal isolates of replication-competent virus revealed that 57% had env sequences identical to other isolates from the same patient. Identity was confirmed by full-genome sequencing and was not attributable to limited viral diversity. Phylogenetic and statistical analysis suggested that identical sequences arose from in vivo proliferation of infected cells, rather than infection of multiple cells by a dominant viral species. The possibility that much of the reservoir arises by cell proliferation presents challenges to cure.

Summary Background Allogeneic blood or marrow transplantation (alloBMT) is a potentially life-saving treatment for individuals with HIV and haematological malignancies; challenges include identifying donors and maintaining antiretroviral therapy (ART). The objectives of our study were to investigate interventions to expand donor options and to prevent ART interruptions for patients with HIV in need of alloBMT. Methods This single-arm, interventional trial took place at the Johns Hopkins Sidney Kimmel Comprehensive Cancer Center (Baltimore, MD, USA). Individuals with HIV who were at least 18 years of age and referred for alloBMT for a standard clinical indication were eligible. The only exclusion criterion was a history of documented resistance to enfuvirtide. We used post-transplant cyclophosphamide as graft-versus-host disease (GVHD) prophylaxis to expand donor options and an optimised ART strategy of avoiding pharmacoenhancers and adding subcutaneous enfuvirtide during post-transplant cyclophosphamide and during oral medication intolerance. Our primary outcome was the proportion of participants who maintained ART through day 60 after alloBMT. We measured the HIV latent reservoir using a quantitative viral outgrowth assay. This study is registered on ClinicalTrials.gov , NCT01836068 . Findings Between June 1, 2013, and August 27, 2015, nine patients who were referred for transplant provided consent. Two patients had relapsed malignancy before donor searches were initiated. Seven patients had suitable donors identified (two matched sibling, two matched unrelated, two haploidentical, and one single-antigen mismatched unrelated) and proceeded to alloBMT. All patients maintained ART through day 60 and required ART changes (median 1, range 1–3) in the first 90 days. One patient stopped ART and developed HIV rebound with grade 4 meningoencephalitis at day 146. Among six patients who underwent alloBMT and had longitudinal measurements available, the HIV latent reservoir was not detected post-alloBMT in four patients with more than 95% donor chimerism, consistent with a 2·06–2·54 log10 reduction in the HIV latent reservoir. In the two patients with less than 95% donor chimerism, the HIV latent reservoir remained stable. Interpretation By using post-transplant cyclophosphamide as GVHD prophylaxis, we successfully expanded alloBMT donor options for patients with HIV. Continuing ART with a regimen that includes enfuvirtide post-alloBMT was safe, but life-threatening viral rebound can occur with ART interruption. Funding amfAR (the Foundation for AIDS Research), Johns Hopkins University Center for AIDS Research, and National Cancer Institute.

An estimated 35 million people worldwide are infected with HIV, yet a widely applicable cure strategy remains elusive. Recent case reports have suggested that curing HIV infection is possible, renewing excitement about research efforts. We describe those cases and discuss their relevance to the global HIV epidemic. We also review ongoing cure strategies that are transitioning from the lab to the clinic, and the assays and clinical assessments that can be used to evaluate cure interventions.

While antiretroviral therapy (ART) can reduce HIV-1 to undetectable levels, the virus generally reappears if treatment is stopped. Resurgence of the virus is due to the reactivation of T cells harboring latent integrated provirus, and recent studies indicate that proliferation of these latently infected cells helps maintain the HIV-1 reservoir. In this issue of the JCI, Lee et al. evaluated CD4+ T cell subsets to determine whether certain populations are more likely to harbor full-length, replication-competent provirus. The authors identified an enrichment of clonally expanded Th1 cells containing intact HIV-1 proviruses, suggesting that this polarized subset contributes to the persistence of the reservoir. Strategies to target these provirus-harboring cells need to be considered for future therapies aimed toward HIV-1 cure.

While memory T-cells represent a hallmark of adaptive immunity, little is known about the genetic mechanisms regulating the longevity of memory CD4 T cells. Here, we studied the dynamics of gene expression in antigen specific CD4 T cells during infection, memory differentiation, and long-term survival up to nearly a year in mice. We observed that differentiation into long lived memory cells is associated with increased expression of genes inhibiting cell proliferation and apoptosis as well as genes promoting DNA repair response, lipid metabolism, and insulin resistance. We identified several transmembrane proteins in long-lived murine memory CD4 T cells, which co-localized exclusively within the responding antigen-specific memory CD4 T cells in human. The unique gene signatures of long-lived memory CD4 T cells, along with the new markers that we have defined, will enable a deeper understanding of memory CD4 T cell biology and allow for designing novel vaccines and therapeutics.

Heterologous immunity is an important aspect of the adaptive immune response. We hypothesized that this process could modulate the HIV-1-specific CD8+ T cell response, which has been shown to play an important role in HIV-1 immunity and control. We found that stimulation of peripheral blood mononuclear cells (PBMCs) from HIV-1-positive subjects with microbial peptides that were cross-reactive with immunodominant HIV-1 epitopes resulted in dramatic expansion of HIV-1-specific CD8+ T cells. Interestingly, the TCR repertoire of HIV-1-specific CD8+ T cells generated by ex vivo stimulation of PBMCs using HIV-1 peptide was different from that of cells stimulated with cross-reactive microbial peptides in some HIV-1-positive subjects. Despite these differences, CD8+ T cells stimulated with either HIV-1 or cross-reactive peptides effectively suppressed HIV-1 replication in autologous CD4+ T cells. These data suggest that exposure to cross-reactive microbial antigens can modulate HIV-1-specific immunity.

One of the main challenges in the fight against HIV infection is to develop strategies that are able to eliminate the persistent viral reservoir that harbours integrated, replication-competent provirus within host cellular DNA. This reservoir is resistant to antiretroviral therapy (ART) and to clearance by the immune system of the host; viruses originating from this reservoir lead to rebound viraemia once treatment is stopped, giving rise to new rounds of infection. Several studies have focused on elucidating the cells and tissues that harbour persistent virus, the true size of the reservoir and how best to target it, but these topics are the subject of ongoing debate. In this Viewpoint article, several experts in the field discuss the constitution of the viral reservoir, how best to measure it and the best ways to target this source of persistent infection.

Several observational studies suggest that medroxyprogesterone acetate (MPA) injectable contraceptives may increase a woman's risk of sexual HIV-1 acquisition. In-vitro studies are conflicting, mainly due to differences in the type of progestin studied or activation status of the primary cells. We sought to determine whether MPA increases infection of unstimulated peripheral blood mononuclear cells (PBMCs).Freshly isolated PBMCs from normal blood donors were treated with physiologic MPA concentrations ranging from 0.003 to 5 ng/ml and infected with GFP-tagged R5-tropic or X4-tropic HIV-1 pseudoviruses by spinoculation. The infection was limited to a single cycle. Cells were stained with CD3, CD8 and CD14. Infection was quantified as the percentage of GFP cells by flow cytometry.Absolute infection was greater among unstimulated MPA-treated CD3⁺CD8⁻ T cells vs. untreated cells across MPA concentrations of 0.003-3 ng/ml using R5 (P 0.003) and 0.03-0.3 ng/ml using X4 (P 0.005) pseudovirus. There was increased relative infection of CD3⁺CD8⁻ T cells in MPA-treated whole PBMC cultures but not after monocytes were depleted (P 0.02). HIV-1 infection of stimulated PBMC showed no differences in R5 or X4 infection across all MPA concentrations (P 0.5).The CD3⁺CD8⁻ T-cell population of MPA-treated unstimulated PBMCs were more susceptible to HIV-1 infection than untreated cells. The increased infection was partly due to monocytes and was lost when PBMC were exogenously stimulated. These data provide confirmation of a biological association between MPA exposure and increased susceptibility to HIV-1 infection, particularly among women who inject drugs.

Reversal of HIV-1 latency by small molecules is a potential cure strategy. This approach will likely require effective drug combinations to achieve high levels of latency reversal. Using resting CD4+ T cells (rCD4s) from infected individuals, we developed an experimental and theoretical framework to identify effective latency-reversing agent (LRA) combinations. Utilizing ex vivo assays for intracellular HIV-1 mRNA and virion production, we compared 2-drug combinations of leading candidate LRAs and identified multiple combinations that effectively reverse latency. We showed that protein kinase C agonists in combination with bromodomain inhibitor JQ1 or histone deacetylase inhibitors robustly induce HIV-1 transcription and virus production when directly compared with maximum reactivation by T cell activation. Using the Bliss independence model to quantitate combined drug effects, we demonstrated that these combinations synergize to induce HIV-1 transcription. This robust latency reversal occurred without release of proinflammatory cytokines by rCD4s. To extend the clinical utility of our findings, we applied a mathematical model that estimates in vivo changes in plasma HIV-1 RNA from ex vivo measurements of virus production. Our study reconciles diverse findings from previous studies, establishes a quantitative experimental approach to evaluate combinatorial LRA efficacy, and presents a model to predict in vivo responses to LRAs.

Despite antiretroviral therapy (ART), human immunodeficiency virus (HIV)-1 persists in a stable latent reservoir, primarily in resting memory CD4(+) T cells. This reservoir presents a major barrier to the cure of HIV-1 infection. To purge the reservoir, pharmacological reactivation of latent HIV-1 has been proposed and tested both in vitro and in vivo. A key remaining question is whether virus-specific immune mechanisms, including cytotoxic T lymphocytes (CTLs), can clear infected cells in ART-treated patients after latency is reversed. Here we show that there is a striking all or none pattern for CTL escape mutations in HIV-1 Gag epitopes. Unless ART is started early, the vast majority (>98%) of latent viruses carry CTL escape mutations that render infected cells insensitive to CTLs directed at common epitopes. To solve this problem, we identified CTLs that could recognize epitopes from latent HIV-1 that were unmutated in every chronically infected patient tested. Upon stimulation, these CTLs eliminated target cells infected with autologous virus derived from the latent reservoir, both in vitro and in patient-derived humanized mice. The predominance of CTL-resistant viruses in the latent reservoir poses a major challenge to viral eradication. Our results demonstrate that chronically infected patients retain a broad-spectrum viral-specific CTL response and that appropriate boosting of this response may be required for the elimination of the latent reservoir.

Remarkable advances have been made in the treatment of human immunodeficiency virus (HIV)-1 infection, but in the entire history of the epidemic, only 1 patient has been cured. Herein we review the fundamental mechanisms that render HIV-1 infection difficult to cure and then discuss recent clinical and experimental situations in which some form of cure has been achieved. Finally, we consider approaches that are currently being taken to develop a general cure for HIV-1 infection.

Background It is unknown if extremely early initiation of antiretroviral therapy (ART) may lead to long-term ART-free HIV remission or cure. As a result, we studied 2 individuals recruited from a pre-exposure prophylaxis (PrEP) program who started prophylactic ART an estimated 10 days (Participant A; 54-year-old male) and 12 days (Participant B; 31-year-old male) after infection with peak plasma HIV RNA of 220 copies/mL and 3,343 copies/mL, respectively. Extensive testing of blood and tissue for HIV persistence was performed, and PrEP Participant A underwent analytical treatment interruption (ATI) following 32 weeks of continuous ART. Methods and findings Colorectal and lymph node tissues, bone marrow, cerebral spinal fluid (CSF), plasma, and very large numbers of peripheral blood mononuclear cells (PBMCs) were obtained longitudinally from both participants and were studied for HIV persistence in several laboratories using molecular and culture-based detection methods, including a murine viral outgrowth assay (mVOA). Both participants initiated PrEP with tenofovir/emtricitabine during very early Fiebig stage I (detectable plasma HIV-1 RNA, antibody negative) followed by 4-drug ART intensification. Following peak viral loads, both participants experienced full suppression of HIV-1 plasma viremia. Over the following 2 years, no further HIV could be detected in blood or tissue from PrEP Participant A despite extensive sampling from ileum, rectum, lymph nodes, bone marrow, CSF, circulating CD4+ T cell subsets, and plasma. No HIV was detected from tissues obtained from PrEP Participant B, but low-level HIV RNA or DNA was intermittently detected from various CD4+ T cell subsets. Over 500 million CD4+ T cells were assayed from both participants in a humanized mouse outgrowth assay. Three of 8 mice infused with CD4+ T cells from PrEP Participant B developed viremia (50 million input cells/surviving mouse), but only 1 of 10 mice infused with CD4+ T cells from PrEP Participant A (53 million input cells/mouse) experienced very low level viremia (201 copies/mL); sequence confirmation was unsuccessful. PrEP Participant A stopped ART and remained aviremic for 7.4 months, rebounding with HIV RNA of 36 copies/mL that rose to 59,805 copies/mL 6 days later. ART was restarted promptly. Rebound plasma HIV sequences were identical to those obtained during acute infection by single-genome sequencing. Mathematical modeling predicted that the latent reservoir size was approximately 200 cells prior to ATI and that only around 1% of individuals with a similar HIV burden may achieve lifelong ART-free remission. Furthermore, we observed that lymphocytes expressing the tumor marker CD30 increased in frequency weeks to months prior to detectable HIV-1 RNA in plasma. This study was limited by the small sample size, which was a result of the rarity of individuals presenting during hyperacute infection. Conclusions We report HIV relapse despite initiation of ART at one of the earliest stages of acute HIV infection possible. Near complete or complete loss of detectable HIV in blood and tissues did not lead to indefinite ART-free HIV remission. However, the small numbers of latently infected cells in individuals treated during hyperacute infection may be associated with prolonged ART-free remission., Timothy Henrich and colleagues study the effect of very early antiretroviral treatment on the reservoir of HIV-infected cells in two patients., Author summary Why was this study done? Early initiation of ART following infection may limit the total body burden of HIV. It is not known if starting ART extremely early after HIV infection will lead to ART-free remission or cure. We studied 2 individuals who started ART an estimated 10 and 12 days after HIV infection with very low peak viral load measurement; extensive testing of blood and tissue for HIV persistence was performed. One participant stopped ART in order to test if and when HIV would rebound. What did the researchers find? No HIV could be definitively detected for up to 2 years in the participant who initiated ART approximately 10 days after HIV infection. Intermittent, very low levels of HIV were detected in blood but not tissue in the participant who initiated ART an estimated 12 days following infection. The participant with no detectable HIV following ART experienced viral rebound 225 days after stopping ART. What do these findings mean? HIV relapsed despite initiation of ART at one of the earliest stages of acute HIV infection possible. Near complete loss of detectable HIV in blood and tissues did not lead to indefinite ART-free HIV remission.

Despite antiretroviral therapy (ART), a latent reservoir of replication-competent HIV-1 persists in resting memory CD4+ T-cells and precludes cure1-6. Lorenzo-Redondo et al.7 analyzed HIV-1 sequences collected from three individuals during the first six months of ART, discovered specific patterns of sequence evolution, and concluded that viral replication persists during therapy. We believe these evolutionary patterns are artifacts of rapidly decaying viral subpopulations present during the first months of therapy and are not characteristic of the long-lived reservoir. The study therefore provides no evidence that ongoing replication is an additional barrier to cure for treated individuals who consistently maintain low viral loads.

A mechanistic understanding of HIV-1 latency depends on a model system that recapitulates the in vivo condition of latently infected, resting CD4(+) T lymphocytes. Latency seems to be established after activated CD4(+) T cells, the principal targets of HIV-1 infection, become productively infected and survive long enough to return to a resting memory state in which viral expression is inhibited by changes in the cellular environment. This protocol describes an ex vivo primary cell system that is generated under conditions that reflect the in vivo establishment of latency. Creation of these latency model cells takes 12 weeks and, once established, the cells can be maintained and used for several months. The resulting cell population contains both uninfected and latently infected cells. This primary cell model can be used to perform drug screens, to study cytolytic T lymphocyte (CTL) responses to HIV-1, to compare viral alleles or to expand the ex vivo life span of cells from HIV-1-infected individuals for extended study.

In the early years of the AIDS epidemic, a diagnosis of HIV-1 infection was equivalent to a death sentence. The development of combination antiretroviral therapy (cART) in the 1990s to combat HIV-1 infection was one of the most impressive achievements of medical science. Today, patients who are treated early with cART can expect a near-normal lifespan. In this Opinion article, we propose a fundamental theory to explain the mechanistic basis of cART and why it works so well, including a model to assess and predict the efficacy of antiretroviral drugs alone or in combination.

HIV-1 infection can now be readily controlled with combination antiretroviral therapy. However, the virus persists indefinitely in a stable latent reservoir in resting CD4 + T cells. This reservoir generally prevents cure of the infection with combination antiretroviral therapy alone. However, several recent cases of potential HIV-1 cure have generated renewed optimism. Here we review these cases and consider new developments in our understanding of the latent reservoir. In addition, we consider clinical aspects of curative strategies to provide a more realistic picture of what a generally applicable cure for HIV-1 infection is likely to entail.

A population of CD4T lymphocytes harboring latent HIV genomes can persist in patients on antiretroviral therapy, posing a barrier to HIV eradication. To examine cellular complexes controlling HIV latency, we conducted a genome-wide screen with a pooled ultracomplex shRNA library and invitro system modeling HIV latency and identified the mTOR complex as a modulator of HIV latency. Knockdown of mTOR complex subunits or pharmacological inhibition of mTOR activity suppresses reversal of latency in various HIV-1 latency models and HIV-infected patient cells. mTOR inhibitors suppress HIV transcription both through the viral transactivator Tat and via Tat-independent mechanisms. This inhibition occurs at least in part via blocking the phosphorylation of CDK9, a p-TEFb complex member that serves as a cofactor for Tat-mediated transcription. The control of HIV latency by mTOR signaling identifies a pathway that may have significant therapeutic opportunities.

Once considered an inevitable consequence of HIV treatment, drug resistance is declining. This decline supports the hypothesis that antiretroviral therapy can arrest replication and prevent the evolution of resistance. Further support comes from excellent clinical outcomes, the failure of treatment intensification to reduce residual viremia, the lack of viral evolution in patients on optimal therapy, pharmacodynamics studies explaining the extraordinarily high antiviral activity of modern regimens, and recent reports of potential cures. Evidence supporting ongoing replication includes higher rates of certain complications in treated patients and an increase in circular forms of the viral genome after intensification with integrase inhibitors. Recent studies also provide an explanation for the observation that some patients fail protease-inhibitor based regimens without evidence for resistance.

Highly active antiretroviral therapy (HAART) is able to suppress human immunodeficiency virus type 1 (HIV-1) to undetectable levels in the majority of patients, but eradication has not been achieved because latent viral reservoirs persist, particularly in resting CD4 + T lymphocytes. It is generally understood that HIV-1 does not efficiently infect resting CD4 + T cells, and latent infection in those cells may arise when infected CD4 + T lymphoblasts return to resting state. In this study, we found that stimulation by endothelial cells can render resting CD4 + T cells permissible for direct HIV infection, including both productive and latent infection. These stimulated T cells remain largely phenotypically unactivated and show a lower death rate than activated T cells, which promotes the survival of infected cells. The stimulation by endothelial cells does not involve interleukin 7 (IL-7), IL-15, CCL19, or CCL21. Endothelial cells line the lymphatic vessels in the lymphoid tissues and have frequent interactions with T cells in vivo . Our study proposes a new mechanism for infection of resting CD4 + T cells in vivo and a new mechanism for latent infection in resting CD4 + T cells.

Current assays for quantification of HIV-1 virions rely on real-time reverse transcriptase (RT)-PCR detection of conserved regions of HIV-1 RNA and can be limited by detection of contaminating viral or plasmid DNA. We developed a novel RT-PCR assay using a reverse primer that hybridizes with the poly(A) tail of HIV-1 mRNAs, anchored by conserved viral nucleotides at the most distal region of the transcript. This assay can detect and quantify HIV-1 RNA with high specificity and sensitivity.

The latent reservoir for HIV-1 in resting CD4(+) T cells remains a major barrier to HIV-1 eradication, even though highly active antiretroviral therapy (HAART) can successfully reduce plasma HIV-1 levels to below the detection limit of clinical assays and reverse disease progression. Proposed eradication strategies involve reactivation of this latent reservoir. Multiple mechanisms are believed to be involved in maintaining HIV-1 latency, mostly through suppression of transcription. These include cytoplasmic sequestration of host transcription factors and epigenetic modifications such as histone deacetylation, histone methylation and DNA methylation. Therefore, strategies targeting these mechanisms have been explored for reactivation of the latent reservoir. In this review, we discuss current pharmacological approaches toward eradication, focusing on small molecule latency-reversing agents, their mechanisms, advantages and limitations.

The discovery of a stable latent reservoir for HIV-1 in resting memory CD4(+) T cells provides a mechanism for lifelong persistence of HIV-1. The long-lived latently infected cells persist in spite of prolonged highly active antiretroviral therapy and present a major barrier to a cure of HIV-1 infection. In this review, we discuss the current understanding of HIV-1 persistence and latent viral infection in the context of effective antiretroviral therapy and the recent progress in purging latent viral reservoirs. Recent studies demonstrate that reactivation of latent HIV-1 is a promising strategy for the depletion of these viral reservoirs. A thorough evaluation of the anti-latency activity of drug candidates should include the measurement of changes in intracellular viral RNA, plasma virus levels, and the size of latent viral reservoirs, as well as potential adverse effects. Currently, there are several technical barriers to the evaluation of anti-latency drugs in vivo. We also discuss these challenging issues that remain unresolved.

We report that a major subpopulation of monocyte-derived macrophages (MDMs) contains high levels of dUTP, which is incorporated into HIV-1 DNA during reverse transcription (U/A pairs), resulting in pre-integration restriction and post-integration mutagenesis. After entering the nucleus, uracilated viral DNA products are degraded by the uracil base excision repair (UBER) machinery with less than 1% of the uracilated DNA successfully integrating. Although uracilated proviral DNA showed few mutations, the viral genomic RNA was highly mutated, suggesting that errors occur during transcription. Viral DNA isolated from blood monocytes and alveolar macrophages (but not T cells) of drug-suppressed HIV-infected individuals also contained abundant uracils. The presence of viral uracils in short-lived monocytes suggests their recent infection through contact with virus producing cells in a tissue reservoir. These findings reveal new elements of a viral defense mechanism involving host UBER that may be relevant to the establishment and persistence of HIV-1 infection. DOI: http://dx.doi.org/10.7554/eLife.18447.001, eLife digest Human immunodeficiency virus type 1 (HIV-1) infects and kills immune cells known as CD4+ T cells, leading to the disease AIDS. Current drug treatments enable HIV-1 infected patients to live relatively long and healthy lives. However, no cure for HIV-1 exists because the virus lives indefinitely in a resting state within the genetic material – or genome – of the infected cell, where it is not susceptible to drug treatments. Most HIV-1 research focuses on T cells, but another type of immune cell – the macrophage – may also harbor resting HIV-1 in its genome. Compared to other cells, macrophages are unusual because they produce large amounts of a molecule called deoxyuridine triphosphate (dUTP). Most cells, including T cells, keep dUTP levels very low because it closely resembles molecules that are used to make DNA and so it can be accidentally incorporated into the cell’s DNA. When this happens, the cell removes the dUTP from the DNA using enzymes in a process called uracil base excision repair (UBER). To hide inside the cell’s genome, HIV-1 needs to produce a DNA copy of its own genome, but it was not known what happens when HIV-1 tries to do this within a macrophage that contains high levels of dUTP and UBER enzymes. Here, Hansen et al. reveal that about 90% of macrophages have exceptionally high levels of dUTP and are poorly infected by HIV-1. The high levels of dUTP result in the virus incorporating dUTP into its DNA, which is then attacked and fragmented by UBER enzymes. However, about one in a hundred viral DNA molecules do manage to successfully integrate into the genome of the macrophage. This viral DNA later gives rise to new virus particles through an error-prone process that, by introducing new mutations into the virus genome, may help HIV-1 to evolve and persist. Further experiments examined cells that give rise to macrophages from infected patients who had been on anti-HIV drug therapy for several years. Hansen et al. found that there was lots of dUTP in the DNA sequences of HIV-1 viruses found in these “precursor” cells. These precursor cells only live for several days before being eliminated, so the presence of viruses containing dUTP suggests these cells were infected recently. A future challenge will be to identify new anti-HIV drugs that specifically target macrophages and to understand the role of error-prone production of new viral genomes. DOI: http://dx.doi.org/10.7554/eLife.18447.002

A priority for HIV cure research is measuring latent infection that can fuel viral recrudescence if patients cease antiretroviral therapy (ART). One important quantity that is difficult to measure is the rate at which latently infected cells activate, giving rise to spreading infection [1]. Pinkevych et al. recently estimated this rate by analyzing several clinical cohorts and concluded that one cell activates every 6 days [2]. This rate is 24-fold lower than our previous estimate of 4 cells/day [1], resulting in far more optimistic predictions for the prospects of reservoir-reducing therapy. We question their estimation approach and suggest that a higher rate is likely for most infected individuals.

In HIV-infected individuals receiving suppressive antiretroviral therapy, the virus persists indefinitely in a reservoir of latently infected cells. The proliferation of these cells may contribute to the stability of the reservoir and thus to the lifelong persistence of HIV-1 in infected individuals. Because the HIV-1 replication process is highly error-prone, the detection of identical viral genomes in distinct host cells provides evidence for the clonal expansion of infected cells. We evaluated alignments of unique, near-full-length HIV-1 sequences to determine the relationship between clonality in a short region and clonality in the full genome. Although it is common to amplify and sequence short, subgenomic regions of the viral genome for phylogenetic analysis, we show that sequence identity of these amplicons does not guarantee clonality across the full viral genome. We show that although longer amplicons capture more diversity, no subgenomic region can recapitulate the diversity of full viral genomes. Consequently, some identical subgenomic amplicons should be expected even from the analysis of completely unique viral genomes, and the presence of identical amplicons alone is not proof of clonally expanded HIV-1. We present a method for evaluating evidence of clonal expansion in the context of these findings., Author Summary Although antiretroviral therapy effectively blocks HIV-1 replication, the virus persists indefinitely in a reservoir of latently infected cells. This reservoir is a major barrier to HIV-1 cure. Recent studies have identified the proliferation of latently infected cells as a mechanism that may contribute to the lifelong persistence of HIV-1. In contrast with cellular proliferation, viral replication is highly error-prone; therefore, the detection of identical viral genomes in distinct host cells provides evidence that those cells are the progeny of an infected cell that underwent clonal expansion. For this reason, the accurate and reliable identification of identical HIV-1 genomes derived from distinct cells is critical for understanding HIV-1 persistence and advancing cure research. Studies of HIV-1 clonal expansion often present clonality of short, subgenomic sequences as evidence for the clonality of full viral genomes. In this study, we quantified the relationship between sequence identity in short regions and sequence identity of near-full-length genomes to demonstrate that no subgenomic region completely captures the diversity of the full viral genome. Consequently, identical subgenomic sequences are not proof of identical full-length HIV-1 genomes. In the context of these findings, we developed a method to evaluate identical subgenomic sequences as possible evidence for clonal expansion.

Although antiretroviral therapy (ART) suppresses viral replication to clinically undetectable levels, human immunodeficiency virus type 1 (HIV-1) persists in CD4(+) T cells in a latent form that is not targeted by the immune system or by ART. This latent reservoir is a major barrier to curing individuals of HIV-1 infection. Many individuals initiate ART during chronic infection, and in this setting, most proviruses are defective. However, the dynamics of the accumulation and the persistence of defective proviruses during acute HIV-1 infection are largely unknown. Here we show that defective proviruses accumulate rapidly within the first few weeks of infection to make up over 93% of all proviruses, regardless of how early ART is initiated. By using an unbiased method to amplify near-full-length proviral genomes from HIV-1-infected adults treated at different stages of infection, we demonstrate that early initiation of ART limits the size of the reservoir but does not profoundly affect the proviral landscape. This analysis allows us to revise our understanding of the composition of proviral populations and estimate the true reservoir size in individuals who were treated early versus late in infection. Additionally, we demonstrate that common assays for measuring the reservoir do not correlate with reservoir size, as determined by the number of genetically intact proviruses. These findings reveal hurdles that must be overcome to successfully analyze future HIV-1 cure strategies.

Antiretroviral therapy (ART) is successful in the suppression of HIV but cannot target and eradicate the latent proviral reservoir. The location of retroviral integration into the human genome is thought to play a role in the clonal expansion of infected cells and HIV persistence. We developed a high-throughput targeted sequence capture assay that uses a pool of HIV-specific probes to enrich Illumina libraries prior to deep sequencing. Using an expanded clonal population of ACH-2 cells, we demonstrate that this sequence capture assay has an extremely low false-positive rate. This assay assessed four cellular models commonly used to study HIV latency and latency-reversing agents: ACH-2 cells, J-Lat cells, the Bcl-2-transduced primary CD4 + model, and the cultured T CM (central memory) CD4 + model. HIV integration site characteristics and genes were compared between these cellular models and to previously reported patient data sets. Across these cellular models, there were significant differences in integration site characteristics, including orientation relative to that of the host gene, the proportion of clonally expanded sites, and the proportion located within genic regions and exons. Despite a greater diversity of minority integration sites than expected in ACH-2 cells, their integration site characteristics consistently differed from those of the other models and from the patient samples. Gene ontology analysis of highly represented genes from the patient samples found little overlap with HIV-containing genes from the cell lines. These findings show that integration site differences exist among the commonly used cellular models of HIV latency and in comparison to integration sites found in patient samples. IMPORTANCE Despite the success of ART, currently there is no successful therapy to eradicate integrated proviruses. Cellular models of HIV latency are used to test the efficacy of latency-reversing agents, but it is unclear how well these models reflect HIV integration into the human genome in vivo . We have developed a novel probe-based sequence enrichment assay to sequence and analyze integrated HIV. We compared HIV integration site characteristics between four cellular models and to previously described patient data sets. Significant differences were detected in the distribution of HIV integration sites between cellular models of HIV latency and compared to data sets from patient samples. The results from this study have implications for how well these cellular models of HIV infection truly reflect HIV integration in vivo and their applicability in drug discovery for novel latency-reversing agents.

Monitoring the efficacy of novel reservoir-reducing treatments for HIV is challenging. The limited ability to sample and quantify latent infection means that supervised antiretroviral therapy (ART) interruption studies are generally required. Here we introduce a set of mathematical and statistical modeling tools to aid in the design and interpretation of ART-interruption trials. We show how the likely size of the remaining reservoir can be updated in real-time as patients continue off treatment, by combining the output of laboratory assays with insights from models of reservoir dynamics and rebound. We design an optimal schedule for viral load sampling during interruption, whereby the frequency of follow-up can be decreased as patients continue off ART without rebound. While this scheme can minimize costs when the chance of rebound between visits is low, we find that the reservoir will be almost completely reseeded before rebound is detected unless sampling occurs at least every two weeks and the most sensitive viral load assays are used. We use simulated data to predict the clinical trial size needed to estimate treatment effects in the face of highly variable patient outcomes and imperfect reservoir assays. Our findings suggest that large numbers of patients—between 40 and 150—will be necessary to reliably estimate the reservoir-reducing potential of a new therapy and to compare this across interventions. As an example, we apply these methods to the two “Boston patients”, recipients of allogeneic hematopoietic stem cell transplants who experienced large reductions in latent infection and underwent ART-interruption. We argue that the timing of viral rebound was not particularly surprising given the information available before treatment cessation. Additionally, we show how other clinical data can be used to estimate the relative contribution that remaining HIV+ cells in the recipient versus newly infected cells from the donor made to the residual reservoir that eventually caused rebound. Together, these tools will aid HIV researchers in the evaluating new potentially-curative strategies that target the latent reservoir., Author Summary New therapies are being developed to permanently cure HIV infection. Many aim to reduce the pool of latent virus that persists despite years of treatment with antiretroviral drugs. Because latent virus is so difficult to sample and measure, often the only way to know if these new therapies have worked is to interrupt all treatment, and wait indefinitely to see if the infection rebounds. In this study we use a set of mathematical and statistical models to suggest optimal ways to design and interpret these treatment interruption trials. For various scenarios, we predict how long patients should be followed to be confident that they are cured, how frequent viral load sampling should occur, and how large clinical trials will need to be to estimate and compare drug efficacy. We demonstrate how to infer a range for number of remaining latent cells based on the timing of rebound after a long remission. As a case study, we apply these results to data from two HIV-positive patients who underwent bone marrow transplants and remained off treatment for months before suddenly rebounding. These findings can help inform the testing of new potentially-curative HIV therapies.

Combination antiretroviral therapy (ART) for HIV-1 infection reduces plasma virus levels to below the limit of detection of clinical assays. However, even with prolonged suppression of viral replication with ART, viremia rebounds rapidly after treatment interruption. Thus, ART is not curative. The principal barrier to cure is a remarkably stable reservoir of latent HIV-1 in resting memory CD4+ T cells. In this review, we consider explanations for the remarkable stability of the latent reservoir. Stability does not appear to reflect replenishment from new infection events but rather normal physiologic processes that provide for immunologic memory. Of particular importance are proliferative processes that drive clonal expansion of infected cells. Recent evidence suggests that in some infected cells, proliferation is a consequence of proviral integration into host genes associated with cell growth. Efforts to cure HIV-1 infection by targeting the latent reservoir may need to consider the potential of latently infected cells to proliferate.