Viral and latent reservoir persistence in HIV-1-infected patients on therapy

Despite many years of potent antiretroviral therapy, latently infected cells and low levels of plasma virus have been found to persist in HIV-infected patients. The factors influencing this persistence and their relative contributions have not been fully elucidated and remain controversial. Here, we address these issues by developing and employing a simple, but mechanistic viral dynamics model. The model has two novel features. First, it assumes that latently infected T cells can undergo bystander proliferation without transitioning into active viral production. Second, it assumes that the rate of latent cell activation decreases with time on antiretroviral therapy due to the activation and subsequent loss of latently infected cells specific for common antigens, leaving behind cells that are successively less frequently activated. Using the model, we examined the quantitative contributions of T cell bystander proliferation, latent cell activation, and ongoing viral replication to the stability of the latent reservoir and persisting low-level viremia. Not surprisingly, proliferation of latently infected cells helped maintain the latent reservoir in spite of loss of latent infected cells through activation and death, and affected viral dynamics to an extent that depended on the magnitude of latent cell activation. In the limit of zero latent cell activation, the latent cell pool and viral load became uncoupled. However, as the activation rate increased, the plasma viral load could be maintained without depleting the latent reservoir, even in the absence of viral replication. The influence of ongoing viral replication on the latent reservoir remained insignificant for drug efficacies above the "critical efficacy" irrespective of the activation rate. However, for lower drug efficacies viral replication enabled the stable maintenance of both the latent reservoir and the virus. Our model and analysis methods provide a quantitative and qualitative framework for probing how different viral and host factors contribute to the dynamics of the latent reservoir and the virus, offering new insights into the principal determinants of their persistence.     

Viral and Latent Reservoir Persistence in HIV-1–Infected Patients on Therapy

Despite many years of potent antiretroviral therapy, latently infected cells and low levels of plasma virus have been found to persist in HIV-infected patients. The factors influencing this persistence and their relative contributions have not been fully elucidated and remain controversial. Here, we address these issues by developing and employing a simple, but mechanistic viral dynamics model. The model has two novel features. First, it assumes that latently infected T cells can undergo bystander proliferation without transitioning into active viral production. Second, it assumes that the rate of latent cell activation decreases with time on antiretroviral therapy due to the activation and subsequent loss of latently infected cells specific for common antigens, leaving behind cells that are successively less frequently activated. Using the model, we examined the quantitative contributions of T cell bystander proliferation, latent cell activation, and ongoing viral replication to the stability of the latent reservoir and persisting low-level viremia. Not surprisingly, proliferation of latently infected cells helped maintain the latent reservoir in spite of loss of latent infected cells through activation and death, and affected viral dynamics to an extent that depended on the magnitude of latent cell activation. In the limit of zero latent cell activation, the latent cell pool and viral load became uncoupled. However, as the activation rate increased, the plasma viral load could be maintained without depleting the latent reservoir, even in the absence of viral replication. The influence of ongoing viral replication on the latent reservoir remained insignificant for drug efficacies above the “critical efficacy” irrespective of the activation rate. However, for lower drug efficacies viral replication enabled the stable maintenance of both the latent reservoir and the virus. Our model and analysis methods provide a quantitative and qualitative framework for probing how different viral and host factors contribute to the dynamics of the latent reservoir and the virus, offering new insights into the principal determinants of their persistence.     

Evolution of Envelope Sequences of Human Immunodeficiency Virus Type 1 in Cellular Reservoirs in the Setting of Potent Antiviral Therapy

In human immunodeficiency virus (HIV)-infected patients treated with potent antiretroviral therapy, the persistence of latently infected cells may reflect the long decay half-life of this cellular reservoir or ongoing viral replication at low levels with continuous replenishment of the population or both. To address these possibilities, sequences encompassing the C2 and V3 domains of HIV-1 env were analyzed from virus present in baseline plasma and from viral isolates obtained after 2 years of suppressive therapy in six patients. The presence of sequence changes consistent with evolution was demonstrated for three subjects and correlated with less complete suppression of viral replication, as indicated by the rapidity of the initial virus load decline or the intermittent reappearance of even low levels of detectable viremia. Together, these results provide evidence for ongoing replication. In the remaining three patients, virus recovered after 2 years of therapy was either genotypically contemporary with or ancestral to virus present in plasma 2 years before, indicating that virus recovery had indeed resulted from activation of latently infected cells.     

Asymmetric division of activated latently infected cells may explain the decay kinetics of the HIV-1 latent reservoir and intermittent viral blips

Most HIV-infected patients when treated with combination antiretroviral therapy achieve viral loads that are below the current limit of detection of standard assays after a few months. Despite this, virus eradication from the host has not been achieved. Latent, replication-competent HIV-1 can generally be identified in resting memory CD4⁺ T cells in patients with “undetectable” viral loads. Turnover of these cells is extremely slow but virus can be released from the latent reservoir quickly upon cessation of therapy. In addition, a number of patients experience transient episodes of viremia, or HIV-1 blips, even with suppression of the viral load to below the limit of detection for many years. The mechanisms underlying the slow decay of the latent reservoir and the occurrence of intermittent viral blips have not been fully elucidated. In this study, we address these two issues by developing a mathematical model that explores a hypothesis about latently infected cell activation. We propose that asymmetric division of latently infected cells upon sporadic antigen encounter may both replenish the latent reservoir and generate intermittent viral blips. Interestingly, we show that occasional replenishment of the latent reservoir induced by reactivation of latently infected cells may reconcile the differences between the divergent estimates of the half-life of the latent reservoir in the literature.     

Immune control of the number and reactivation phenotype of cells latently infected with a gammaherpesvirus

Despite active immune responses, gammaherpesviruses establish latency. In a related process, these viruses also persistently replicate by using a mechanism that requires different viral genes than acute-phase replication. Many questions remain about the role of immunity in chronic gammaherpesvirus infection, including whether the immune system controls latency by regulating latent cell numbers and/or other properties and what specific immune mediators control latency and persistent replication. We show here that CD8(+) T cells regulate both latency and persistent replication and demonstrate for the first time that CD8(+) T cells regulate both the number of latently infected cells and the efficiency with which infected cells reactivate from latency. Furthermore, we show that gamma interferon (IFN-gamma) and perforin, which play no significant role during acute infection, are essential for immune control of latency and persistent replication. Surprisingly, the effects of perforin and IFN-gamma are site specific, with IFN-gamma being important in peritoneal cells while perforin is important in the spleen. Studies of the mechanisms of action of IFN-gamma and perforin revealed that perforin acts primarily by controlling the number of latently infected cells while IFN-gamma acts primarily by controlling reactivation efficiency. The immune system therefore controls chronic gammaherpesvirus infection by site-specific mechanisms that regulate both the number and reactivation phenotype of latently infected cells.     

Epstein-Barr virus microRNAs are evolutionarily conserved and differentially expressed

The pathogenic lymphocryptovirus Epstein-Barr virus (EBV) is shown to express at least 17 distinct microRNAs (miRNAs) in latently infected cells. These are arranged in two clusters: 14 miRNAs are located in the introns of the viral BART gene while three are located adjacent to BHRF1. The BART miRNAs are expressed at high levels in latently infected epithelial cells and at lower, albeit detectable, levels in B cells. In contrast to the tissue-specific expression pattern of the BART miRNAs, the BHRF1 miRNAs are found at high levels in B cells undergoing stage III latency but are essentially undetectable in B cells or epithelial cells undergoing stage I or II latency. Induction of lytic EBV replication was found to enhance the expression of many, but not all, of these viral miRNAs. Rhesus lymphocryptovirus, which is separated from EBV by > or =13 million years of evolution, expresses at least 16 distinct miRNAs, seven of which are closely related to EBV miRNAs. Thus, lymphocryptovirus miRNAs are under positive selection and are likely to play important roles in the viral life cycle. Moreover, the differential regulation of EBV miRNA expression implies distinct roles during infection of different human tissues.     

HMGB1 activates replication of latent HIV-1 in a monocytic cell-line, but inhibits HIV-1 replication in primary macrophages

High mobility group box protein 1 (HMGB1) is an abundant component of mammalian cells that can be released into extracellular milieu actively or by cells that undergo necrosis. Exposure of inflammatory and endothelial cells to HMGB1 leads to the release of cytokines, including TNF-alpha and IL-6. To evaluate the impact of exogenous HMGB1 on viral replication in HIV-1 infected cells, we studied models of latent and acute infection. Extracellular HMGB1 dose dependently increased HIV-1 replication in the monocytic cells, U1, which is an established model for studying latent HIV-1 infection. Dexamethasone, a known inhibitor of NF-kappaB signaling in U1 cells, inhibited HMGB1-induced stimulation of the viral production. Addition of HMGB1 to primary monocytic cells with active HIV-1 infection elicited the opposite effect, due to suppression of the viral replication. The mechanism of this unexpected finding was explained by an HMGB1-mediated increased release of chemokines (RANTES, MIP-1alpha, and MIP-1beta) that are known to inhibit HIV-1 replication. The stimulatory effect of the HMGB1 was not present when latently infected T-cells (ACH-2) were used as target cells. Our data suggest that extracellular HMGB1 has a dichotomic effect on the HIV-1 infection in monocytes but not in lymphocytes. Both activation of latent HIV-1 infection and inhibition of active replication can thus be seen in vitro.     

Stochastic interplay between mutation and recombination during the acquisition of drug resistance mutations in human immunodeficiency virus type 1

The emergence of drug resistance mutations in human immunodeficiency virus (HIV) has been a major setback in the treatment of infected patients. Besides the high mutation rate, recombination has been conjectured to have an important impact on the emergence of drug resistance. Population genetic theory suggests that in populations limited in size recombination may facilitate the acquisition of beneficial mutations. The viral population in an infected patient may indeed represent such a population limited in size, since current estimates of the effective population size range from 500 to 10(5). To address the effects of limited population size, we therefore expand a previously described deterministic population genetic model of HIV replication by incorporating the stochastic processes that occur in finite populations of infected cells. Using parameter estimates from the literature, we simulate the evolution of drug-resistant viral strains. The simulations show that recombination has only a minor effect on the rate of acquisition of drug resistance mutations in populations with effective population sizes as small as 1,000, since in these populations, viral strains typically fix beneficial mutations sequentially. However, for intermediate effective population sizes (10(4) to 10(5)), recombination can accelerate the evolution of drug resistance by up to 25%. Furthermore, a reduction in population size caused by drug therapy can be overcome by a higher viral mutation rate, leading to a faster evolution of drug resistance.     

Latent infection of KB cells with adeno-associated virus type 2.

Adeno-associated virus (AAV) is a prevalent human virus whose replication requires factors provided by a coinfecting helper virus. AAV can establish latent infections in vitro by integration of the AAV genome into cellular DNA. To study the process of integration as well as the rescue of AAV replication in latently infected cells after superinfection with a helper virus, we established a panel of independently derived latently infected cell clones. KB cells were infected with a high multiplicity of AAV in the absence of helper virus, cloned, and passaged to dilute out input AAV genomes. AAV DNA replication and protein synthesis were rescued from more than 10% of the KB cell clones after superinfection with adenovirus type 5 (Ad5) or herpes simplex virus types 1 or 2. In the absence of helper virus, there was no detectable expression of AAV-specific RNA or proteins in the latently infected cell clones. Ad5 superinfection also resulted in the production of infectious AAV in most cases. All mutant adenoviruses tested that were able to help AAV DNA replication in a coinfection were also able to rescue AAV from the latently infected cells, although one mutant, Ad5hr6, was less efficient at AAV rescue. Analysis of high-molecular-weight cellular DNA indicated that AAV sequences were integrated into the cell genome. The restriction enzyme digestion patterns of the cellular DNA were consistent with colinear integration of the AAV genome, with the viral termini present at the cell-virus junction. In addition, many of the cell lines appeared to contain head-to-tail concatemers of the AAV genome. The understanding of the integration of AAV DNA is increasingly important since AAV-based vectors have many advantages for gene transduction in vitro and in vivo.     

人单核细胞系中HIV-1前病毒转录调节新型研究模型的建立

【目的】抗反转录病毒疗法(ART)能够有效控制人免疫缺陷病毒(Human immunodeficiency virus-1,HIV-1)的复制,但是不能将其完全清除。至2012年底,全球仍有3 500万HIV-1感染者,同年约160万人死于艾滋病(Acquired immune deficiency syndrome,AIDS)及其相关疾病。HIV-1感染难以根治的主要原因之一是机体内HIV-1潜伏储存库(Reservoir)的存在。HIV-1潜伏储存库主要由CD4+T细胞和单核巨噬细胞构成,与CD4+T细胞相比,目前研究者对单核巨噬系细胞中HIV-1病毒复制机制尚不明了,且缺乏适宜的研究体系。因此,为探讨单核细胞活化或分化信号对HIV-1复制的影响,我们建立了旨在研究HIV-1前病毒转录调控机制的人单核巨噬细胞系模型。【方法】构建env区域移码突变和nef区域携带EGFP或Nano Luc报告基因的HIV-1 NLn GFP-Kp或NLn Nano Luc-Kp重组病毒,分别感染2种人单核细胞系THP-1或U937细胞。通过有限稀释法制备单克隆细胞系,利用流式细胞术或Nano Luc荧光素酶活性分析检测报告基因的表达。筛选EGFP或Nano Luc阴性表达的细胞克隆,经激活剂佛波酯(Phorbol-12-myristate-13-acetate,PMA)刺激后鉴定潜伏感染的细胞克隆。【结果】研究中鉴定了4个HIV-1潜伏感染的细胞克隆。其中2个是表达EGFP的THP-1克隆,2个是以Nano Luc为报告基因的U937克隆。这些克隆在PMA刺激处理后皆有报告基因的表达,而在恒态条件下未检测到报告基因的表达。【结论】成功建立了4个HIV-1潜伏感染的人单核细胞系克隆,该模型有助于理解单核巨噬系细胞的HIV-1病毒复制机制,可能成为进一步研究HIV-1前病毒转录调控机制的有力工具。     

Signaling through Toll-like receptors triggers HIV-1 replication in latently infected mast cells

Evidence that human progenitor mast cells are susceptible to infection with CCR5-tropic strains of HIV-1 and that circulating HIV-1-infected FcepsilonRIalpha(+) cells with a similar progenitor phenotype have been isolated from AIDS patients has led to speculation that mast cells may serve as a potential reservoir for infectious HIV-1. In this study, progenitor mast cells, developed in vitro from CD34(+) cord blood stem cells, were experimentally infected with the CCR5-tropic strain HIV-1Bal after 28 days in culture as they reached their HIV-1-susceptible progenitor stage. HIV-1 p24 Ag levels were readily detectable by day 7 postinfection (PI), peaked at 2-3 wk PI as mature (tryptase/chymase-positive) HIV-1 infection-resistant mast cells emerged, and then steadily declined to below detectable limits by 10 wk PI, at which point integrated HIV-1 proviral DNA was confirmed by PCR quantitation in ( approximately 34% of) latently infected mast cells. Stimulation by ligands for Toll-like receptor (TLR) 2, TLR4, or TLR9 significantly enhanced viral replication in a dose- and time-dependent manner in both HIV-1-infected progenitor and latently infected mature mast cells, without promoting degranulation, apoptosis, cellular proliferation, or dysregulation of TLR agonist-induced cytokine production in infected mast cells. Limiting dilution analysis of TLR activated, latently infected mature mast cells indicated that one in four was capable of establishing productive infections in A301 sentinel cells. Taken together, these results indicate that mast cells may serve both as a viral reservoir and as a model for studying mechanisms of postintegration latency in HIV infection.     

Novobiocin and coumermycin A1 inhibit viral replication and the reactivation of herpes simplex virus type 1 from the trigeminal ganglia of latently infected mice.

Herpes simplex virus type 1 was reactivated from the trigeminal ganglia of latently infected mice in a quantitative and time-dependent manner. Novobiocin and coumermycin A1 reversibly inhibited the reactivation of herpes simplex virus type 1. They did not inhibit viral replication in permissive cells (CV-1) but did inhibit replication in cells of neuronal origin (C1300) and acutely infected trigeminal ganglia.     

Meaning of DNA detection during the follow-up of HIV-1 infected patients: a brief review

A growing body of evidence indicates that proviral DNA load quantitation is an important parameter in establishing the dynamics of HIV infection. Proviral DNA load can be determined during the follow-up of infected individuals to evaluate reservoir status in addition to viral replication. Hence, the study of viral reservoirs, represented by HIV-1 latently infected cells, including resting memory CD4+ T cells, monocytes and macrophages, by which HIV-1 can be reactivated, opens new perspectives in the assessment and the comprehension of HIV-1 infection. However, the identification of viral reservoirs, that can store both wild and drug resistance viruses, is one of the most important steps in developing treatment strategies because it is now clear that viral reservoirs not only prevent sterilizing immunity but also represent a major obstacle to curing the infection with the potent antiretroviral drugs currently in use. Even if only careful evaluation of virological and immunological markers is necessary to fully characterize the course of HIV-1 infection and to provide a more complete laboratory-based assessment of disease progression, the availability of a new standardized assay such as DNA proviral load will be important to assess the true extent of virological suppression in treated patients and to verify the efficacy of new immune-based therapies aimed at purging HIV-1 DNA reservoirs. Several studies demonstrate, in fact, that HIV-1 cellular DNA load may be an indicator of spread of infection whereas the plasma RNA load is indicates active infection. This article will review the importance of monitoring HIV-1 proviral load DNA during the follow-up of HIV-1 infected subjects, suggesting that additional information complementing HIV RNA load could provide crucial information to monitor viral replication and the effectiveness of HAART therapy.     

A flexible model of HIV-1 latency permitting evaluation of many primary CD4 T-cell reservoirs

Latently infected cells form the major obstacle to HIV eradication. Studies of HIV latency have been generally hindered by the lack of a robust and rapidly deployable cell model that involves primary human CD4 T lymphocytes. Latently infected cell lines have proven useful, but it is unclear how closely these proliferating cells recapitulate the conditions of viral latency in non-dividing CD4 T lymphocytes in vivo. Current primary lymphocyte models more closely reflect the in vivo state of HIV latency, but they are limited by protracted culture periods and often low cell yields. Additionally, these models are always established in a single latently infected cell type that may not reflect the heterogeneous nature of the latent reservoir. Here we describe a rapid, sensitive, and quantitative primary cell model of HIV-1 latency with replication competent proviruses and multiple reporters to enhance the flexibility of the system. In this model, post-integration HIV-1 latency can be established in all populations of CD4 T cells, and reactivation of latent provirus assessed within 7 days. The kinetics and magnitude of reactivation were evaluated after stimulation with various cytokines, small molecules, and T-cell receptor agonists. Reactivation of latent HIV proviruses was readily detected in the presence of strong activators of NF-κB. Latently infected transitional memory CD4 T cells proved more responsive to these T-cell activators than latently infected central memory cells. These findings reveal potentially important biological differences within the latently infected pool of memory CD4 T cells and describe a flexible primary CD4 T-cell system to evaluate novel antagonists of HIV latency.     

HIV latency in the humanized BLT mouse

Even after extended treatment with powerful antiretroviral drugs, HIV is not completely eliminated from infected individuals. Latently infected CD4(+) T cells constitute one reservoir of replication-competent HIV that needs to be eliminated to completely purge virus from antiretroviral drug-treated patients. However, a major limitation in the development of therapies to eliminate this latent reservoir is the lack of relevant in vivo models that can be used to test purging strategies. Here, we show that the humanized BLT (bone marrow-liver-thymus) mouse can be used as both an abundant source of primary latently infected cells for ex vivo latency analysis and also as an in vivo system for the study of latency. We demonstrate that over 2% of human cells recovered from the spleens of HIV-infected BLT mice can be latently infected and that this virus is integrated, activation inducible, and replication competent. The non-tumor-inducing phorbol esters prostratin and 12-deoxyphorbol-13-phenylacetate can each induce HIV ex vivo from these latently infected cells, indicating that this model can be used as a source of primary cells for testing latency activators. Finally, we show activation-inducible virus is still present following suppression of plasma viral loads to undetectable levels by using the antiretroviral drugs zidovudine, indinavir sulfate, and didanosine, demonstrating that this model can also be used to assess the in vivo efficacy of latency-purging strategies. Therefore, the HIV-infected BLT mouse should provide a useful model for assessment of HIV latency activators and approaches to eliminate persistent in vivo HIV reservoirs.     

Mechanisms for macrophage-mediated HIV-1 induction

Viral latency is a long-term pathogenic condition in patients infected with HIV-1. Low but sustained virus replication in chronically infected cells can be activated by stimulation with proinflammatory cytokines such as TNF-alpha, IL-1 beta, or other host factors. However, the precise mechanism by which cellular activation induces latently infected cells to produce virions has remained unclear. In the present report, we present evidence that activation of HIV-1 replication in latently infected U1 or ACH2 cells by human macrophages is mediated by a rapid nuclear localization of NF-kappaB p50/p65 dimer with concomitant increased expression of proinflammatory cytokines. Multiplexed RT-PCR amplification of mRNA isolated from cocultures of macrophages and U1 and ACH2 cells showed significant induction of IL-1beta, IL-6, IL-8, TNF-alpha, and TGF-beta expression within 3 h of coincubation. Fixation of macrophages, U-1, or ACH2 cells with paraformaldehyde before coculture completely abrogated the induction of NF-kappaB subunits and HIV-1 replication, suggesting that cooperative interaction between the two cell types is an essential process for cellular activation. Pretreatment of macrophage-U1 or macrophage-ACH2 cocultures with neutralizing anti-TNF-alpha Ab down-regulated the replication of HIV-1. In addition, pretreatment of macrophage-U1 or macrophage-ACH2 cocultures with the NF-kappaB inhibitor (E)3-[(4-methylphenyl)sulfonyl]-2-propenenitrile (BAY 11-7082) prevented the induction of cytokine expression, indicating a pivotal role of NF-kappaB-mediated signaling in the reactivation of HIV-1 in latently infected cells by macrophages. These results provide a mechanism by which macrophages induce HIV-1 replication in latently infected cells.     

Can the viral reservoir of latently infected CD4<Superscript>+</Superscript> T cells be eradicated with antiretroviral HIV drugs?

The majority of cells infected with the human immunodeficiency virus are activated CD4⁺ T cells, which can be treated with antiretoviral drugs. However, an obstacle to eradication is the presence of viral reservoirs, such as latently infected CD4⁺ T cells. Such cells may be less susceptible to antiretroviral drugs and may persist at low levels during treatment. We introduce a model of impulsive differential equations that describe T cell and drug interactions. We make the extreme assumption that latently infected cells are unaffected by drugs, in order to answer the research question: Can the viral reservoir of latently infected cells be eradicated using current antiretroviral therapy? We analyse the model in both the presence and absence of drugs, showing that, if the frequency of drug taking is sufficiently high, then the number of uninfected CD4⁺ T cells approaches the number of T cells in the uninfected immune system. In particular, this implies that the latent reservoir will be eliminated. It follows that, with sufficient application of drugs, latently infected cells cannot sustain a viral reservoir on their own. We illustrate the results with numerical simulations.