Matthew J. Pace

Directly Infected Resting CD4+T Cells Can Produce HIV Gag without Spreading Infection in a Model of HIV Latency

Despite the effectiveness of highly active antiretroviral therapy (HAART) in treating individuals infected with HIV, HAART is not a cure. A latent reservoir, composed mainly of resting CD4+T cells, drives viral rebound once therapy is stopped. Understanding the formation and maintenance of latently infected cells could provide clues to eradicating this reservoir. However, there have been discrepancies regarding the susceptibility of resting cells to HIV infection in vitro and in vivo. As we have previously shown that resting CD4+T cells are susceptible to HIV integration, we asked whether these cells were capable of producing viral proteins and if so, why resting cells were incapable of supporting productive infection. To answer this question, we spinoculated resting CD4+T cells with or without prior stimulation, and measured integration, transcription, and translation of viral proteins. We found that resting cells were capable of producing HIV Gag without supporting spreading infection. This block corresponded with low HIV envelope levels both at the level of protein and RNA and was not an artifact of spinoculation. The defect was reversed upon stimulation with IL-7 or CD3/28 beads. Thus, a population of latent cells can produce viral proteins without resulting in spreading infection. These results have implications for therapies targeting the latent reservoir and suggest that some latent cells could be cleared by a robust immune response.

HIV reservoirs and latency models.

The main impediment to a cure for HIV is the existence of long-lasting treatment resistant viral reservoirs. In this review, we discuss what is currently known about reservoirs, including their formation and maintenance, while focusing on latently infected CD4+ T cells. In addition, we compare several different in vivo and in vitro models of latency. We comment on how each model may reflect the properties of reservoirs in vivo, especially with regard to cell phenotype, since recent studies demonstrate that multiple CD4+ T cell subsets contribute to HIV reservoirs and that with HAART and disease progression the relative contribution of different subsets may change. Finally, we focus on the direct infection of resting CD4+ T cells as a source of reservoir formation and as a model of latency, since recent results help explain the misconception that resting CD4+ T cells appeared to be resistant to HIV in vitro.

Human immunodeficiency virus integrates directly into naive resting CD4+ T cells but enters naive cells less efficiently than memory cells.

ABSTRACT Resting CD4+ T cells restrict human immunodeficiency virus (HIV) infection at or before reverse transcription, resulting in slower kinetics of reverse transcription. In a previous study, we showed that, despite this restriction at reverse transcription, HIV integration occurs in resting CD4+ T cells, albeit with slower kinetics. In that study, the resting T cells were a mixture of memory and naïve cells. Here we asked whether the more quiescent naïve cell subset could be directly infected by HIV and, if so, whether the level of integration in naïve cells was comparable to that in memory cells. We found that HIV integrates in the naïve subset of resting CD4+ T cells without prior activation of the cells. The level of integration (proviruses/cell) in naïve cells was lower than that in memory cells. This difference between naïve and memory cells was observed whether we inoculated the cells with R5 or X4 HIV and could not be explained solely by differences in coreceptor expression. The presence of endogenous dendritic cells did not change the number of proviruses/cell in memory or naïve cells, and deoxynucleoside pools were equally limiting. Our results instead indicate the existence of a novel restriction point in naïve T cells at viral fusion that results in reduced levels of fusion to naïve CD4+ T cells. We conclude that HIV can integrate into both naïve and memory cells directly. Our data further support our hypothesis that integrated proviral infection of resting T cells can be established without T-cell activation.

HIV latency and integration site placement in five cell-based models

BackgroundHIV infection can be treated effectively with antiretroviral agents, but the persistence of a latent reservoir of integrated proviruses prevents eradication of HIV from infected individuals. The chromosomal environment of integrated proviruses has been proposed to influence HIV latency, but the determinants of transcriptional repression have not been fully clarified, and it is unclear whether the same molecular mechanisms drive latency in different cell culture models.ResultsHere we compare data from five different in vitro models of latency based on primary human T cells or a T cell line. Cells were infected in vitro and separated into fractions containing proviruses that were either expressed or silent/inducible, and integration site populations sequenced from each. We compared the locations of 6,252 expressed proviruses to those of 6,184 silent/inducible proviruses with respect to 140 forms of genomic annotation, many analyzed over chromosomal intervals of multiple lengths. A regularized logistic regression model linking proviral expression status to genomic features revealed no predictors of latency that performed better than chance, though several genomic features were significantly associated with proviral expression in individual models. Proviruses in the same chromosomal region did tend to share the same expressed or silent/inducible status if they were from the same cell culture model, but not if they were from different models.ConclusionsThe silent/inducible phenotype appears to be associated with chromosomal position, but the molecular basis is not fully clarified and may differ among in vitro models of latency.

Concurrent Measures Of Total And Integrated HIV DNA Monitor Reservoirs And Ongoing Replication In Eradication Trials

Objectives:Interest in targeting HIV reservoirs is fueling trials that may decrease reservoir size and/or induce viral replication. Therefore, we aimed to develop strategies to sensitively measure changes in these parameters in patients on and off antiretroviral therapy (ART). Achieving these goals may help evaluate the effects of future clinical trials. Design:To determine the relationship between measurements of total and integrated HIV DNA and their role as markers of reservoir size and ongoing replication, these parameters were measured during the first year of ART, during long-term effective ART, and during a clinical trial aimed at targeting reservoirs. Methods:Total and integrated HIV DNA were measured in patient samples using quantitative PCR techniques. CD4+T cell counts and plasma viremia were also monitored. Results:Unintegrated HIV DNA became undetectable during the first year of ART. Total and integrated HIV DNA levels were generally equal in well controlled patients on ART, and low-level plasma viremia correlated best with integration measures. Finally, patients who controlled plasma viremia (<400 copies/ml) during interferon-&agr; monotherapy exhibited a decrease in the level of integrated but not total HIV DNA and a rise in the ratio of total to integrated HIV DNA over time. Conclusion:Our findings suggest that appearance of unintegrated HIV DNA reflects residual HIV expression and de-novo reverse transcription, providing insight into the mechanism by which interferon-&agr; reduces the HIV reservoir. We conclude that concurrent measurements of total and integrated HIV DNA provide information regarding reservoir size and ongoing replication in trials targeting HIV.

Gag-Positive Reservoir Cells Are Susceptible to HIV-Specific Cytotoxic T Lymphocyte Mediated Clearance In Vitro and Can Be Detected In Vivo

Resting CD4+ T cells infected with HIV persist in the presence of suppressive anti-viral therapy (ART) and are barriers to a cure. One potential curative approach, therapeutic vaccination, is fueled by recognition of the ability of a subset of elite controllers (EC) to control virus without therapy due to robust anti-HIV immune responses. Controllers have low levels of integrated HIV DNA and low levels of replication competent virus, suggesting a small reservoir. As our recent data indicates some reservoir cells can produce HIV proteins (termed GPR cells for Gag-positive reservoir cells), we hypothesized that a fraction of HIV-expressing resting CD4+ T cells could be efficiently targeted and cleared in individuals who control HIV via anti-HIV cytotoxic T lymphocytes (CTL). To test this we examined if superinfected resting CD4+ T cells from EC express HIV Gag without producing infectious virus and the susceptibility of these cells to CTL. We found that resting CD4+ T cells expressed HIV Gag and were cleared by autologous CD8+ T cells from EC. Importantly, we found the extent of CTL clearance in our in vitro assay correlates with in vivo reservoir size and that a population of Gag expressing resting CD4+ T cells exists in vivo in patients well controlled on therapy.

Patients on HAART often have an excess of unintegrated HIV DNA: Implications for monitoring reservoirs

HIV establishes a latent reservoir early in infection that is resistant to anti-retroviral therapy and has a slow rate of decay. It is thought that the majority of HIV DNA in treated patients is integrated since unintegrated HIV DNA appears to be unstable. Thus, to monitor the HIV latent reservoir, total HIV DNA is commonly measured in PBMC from infected individuals. We investigated how often total approaches integrated HIV DNA in treated patients. To do this, we first assessed how accurate our integration assay is and determined the error in our measurements of total and integrated HIV DNA. We demonstrated an excess of total over integrated HIV DNA was present in a subset of patients, suggesting that measurements of total HIV DNA do not always correlate to the level of integration. Determining the cause of this excess and its frequency may have important implications for understanding HIV latent reservoir maintenance.

HIV 2-Long Terminal Repeat Circular DNA is Stable in Primary CD4+ T Cells

Treatment resistant latent reservoirs remain a barrier to cure HIV, but the maintenance and properties of these reservoirs are not completely understood. 2-LTR circular HIV DNA has been used to assess ongoing viral replication in HAART treated patients. However, the half-life of this DNA form is still debated with conflicting in vivo and in vitro data. Prior in vitro studies have focused on cell lines or short lived activated cells in cultures of brief duration, while in vivo studies have the added complications of cell migration, division, and death. Therefore, we monitored the stability of 2-LTR circles in primary CD4+T cells in a month long culture and compared it to the stability of integrated HIV DNA and T cell receptor excision circles (TRECs), another circular DNA form that is thought to be stable. We found that 2-LTRs, along with TRECs, were stable, suggesting 2-LTRs do not necessarily indicate ongoing replication.

R5 HIV env and Vesicular Stomatitis Virus G Protein Cooperate To Mediate Fusion to Naïve CD4+ T Cells

ABSTRACT Naïve CD44 T cells are resistant to both HIV R5 env and vesicular stomatitis virus G protein (VSV-G)-mediated fusion. However, viral particles carrying both HIV R5 env and VSV-G infect naïve cells by an unexplained mechanism. We show that VSV-G-pseudotyped virus cannot fuse to unstimulated cells because the viral particles cannot be endocytosed. However, virions carrying both HIV R5 env and VSV-G can fuse because CD4 binding allows viral uptake. Our findings reveal a unique mechanism by which R5 HIV env and VSV-G cooperate to allow entry to naïve CD4+ T cells, providing a tool to target naïve CD4+ T cells with R5 HIV to study HIV coreceptor signaling and latency.

Hematopoietic Stem Cells and HIV Infection

(See the major article by McNamara et al on pages 1807–16.) New emphasis has been placed on curing human immunodeficiency virus (HIV) infection. Before we can eradicate HIV, we need to understand which cells are infected in vivo and which contribute to reservoirs. The best-characterized latent reservoir is resting CD4+ T cells. However, there is evidence that other reservoirs exist, as well [1–3]. Recent data suggest that hematopoietic stem cells and/or progenitors (HPCs) may contribute to the reservoir [4], but other investigators could not detect infected HPCs in vivo [5, 6]. It is challenging to determine whether HPCs are infected, because T cells (which are likely infected at higher levels) may contaminate HPC preparations [5, 6]. A related, more-difficult challenge is to determine whether true hematopoietic stem cells (HSCs) are infected by HIV in vivo. Resolving these issues will have important implications for HIV cure efforts, including transplantation approaches. In this issue of the Journal, McNamara et al [7] address whether HPCs are detectably infected in vivo and, if so, whether T-cell contamination explains their results. Their data suggest that HPCs are only infected in a subset of HIV infected individuals—those who have been infected for a relatively short time before initiating antiretroviral therapy (ART). This finding may help resolve conflicting data from other groups who did not detect HPC infection [5, 6]. McNamara et al [7] positively selected for immature HPCs, using CD133. The authors chose this simple approach because it maximized cell yield, allowing them to separate a subset of HPCs away from contaminating T cells. By taking this approach, they successfully limited the number of T cells in the CD133+ fraction to <1%, while T cells were enriched in the CD133– fraction (36%–82%). They then measured, by limiting-dilution polymerase chain reaction analysis, the amounts of HIV DNA in the CD133+ and CD133– fractions. In 4 of 11 patients, they detected signal in the CD133+ but not the CD133– fraction. In 5 of 11 subjects, a Fisher exact test indicated that the signal in the CD133+ fraction was unlikely to be due to contaminating T cells or CD133– cells. Despite these in vivo data, an argument against HSC infection is that lineages such as B cells and neutrophils are not infected with HIV. One potential explanation may be that infected HPCs die as they differentiate into progenitors. In vitro work by Carter et al [8] suggested that HPCs infected with a gutted reporter virus (that did not encode HIV proteins) develop into progenitors more efficiently than cells infected with a Δenv virus. A wild-type virus expressing Env should be still more toxic. Consistent with the idea that HIV infection might kill progenitors, bone marrow mononuclear counts correlated with years of infection in patients with high viral loads [9]. However, even if cytopathic events contribute to HSC death, a portion of the viruses may be mutated such that they cannot express HIV proteins. These proviruses should be detectable in downstream lineages (ie, B cells); more vigorous studies should be pursued to determine whether these cells contain defective proviruses at very low levels. If HPCs are infected, why could other groups not repeat this finding? Perhaps HPC infection is only detectable early in HIV disease. Notably, the patient population with HPC infection is very different from the patient population in the study by Durand et al [10]. The patients (without infected HPCs) studied by Durand et al received a diagnosis of HIV infection on average 18 years (median, 21 years) prior to the study, while the patients with infected HPCs in the study by McNamara et al received a diagnosis more recently, on average 7 years (median, 6 years) prior to the study. Similarly, the patients without infected HPCs in the study by McNamara et al also received a diagnosis on average 18 years (median, 21 years) prior to the study. Other, related interpretations are possible. Perhaps, the time during which a patient is not receiving ART determines whether HPCs can be detectably infected. It is possible that after many years of HIV infection, the majority of HPCs have already been infected and died, such that the level of remaining HIV-susceptible HPCs is too low to detect. It is also possible that the results are donor related; for example, some humans could have more HPCs susceptible to HIV infection, perhaps related to the level of CD4 on their surface. Whether true human stem cells express CD4 has not been fully answered, but it is an important question. A lack of CD4 may explain why no infection was detected by Josefsson et al, who selected for CD4– HPCs [6]. Because HIV gp120 binding and HIV infection of enriched HPCs is inhibited by anti-CD4 in vitro [9, 10] and because CD34+CD4+ sorted cells are enriched for pluripotency in long-term culture assays [10], at least a subset of HPCs may express low levels of CD4. Thus, CD4, if present, is so at low levels, meaning there should be lower levels of infection in HPCs than in CD4+ T cells [11]. Limitations of the report by McNamara et al [7] include the small number of patients studied and, because of the available volume of bone marrow aspirate, the small number of genomes assayed in both the CD133+ and CD133– fractions. Initially, it appears a bit surprising that they did not detect any signal in the CD133– fraction in several subjects, since 36%–72% of these cells expressed CD3. However, this is possible because of the small number of genomes assayed and the short time course of infection in the patients with infected HPCs. Moreover, data are accumulating that the T-cell reservoir increases as the interval during which ART is not received increases [12]. In other words, the level of infection among contaminating T cells rises and becomes more problematic over time, just as the level of infection among HPCs may be decreasing. Thus, the short course of infection likely allowed detection of HPC infection. It is also possible that many more HIV-infected individuals have infected HPCs, but we are only able to detect this in a subset of patients who have a small T-cell reservoir. Enhancements in cell sorting technology may achieve the necessary purities to overcome this issue in the future. The most important outstanding question is whether HPCs are an HIV reservoir. Because many patients do not have detectable infection in these cells [5, 6], it seems likely that HPCs at most may be a reservoir in a subset of infected individuals. Furthermore, as yet, no infectious virus has been cultured from exogenously stimulated HPCs from patients (again, likely because of the small volumes obtained from bone marrow aspirates, which is a substantial hurdle). Additionally, comparison of the data from an earlier report by Carter et al [8] with data from the current study (which includes later measurements of the same patients) suggests that the level of HPC infection may decrease over time during ART. Clearly, more longitudinal studies need to be performed to define which patients have infected HPCs and to determine the half-life of these HPCs in patients receiving ART. If the half-life of the HPC reservoir were short, then autologous HPC transplantation in an ART-treated individual could be a feasible approach to eradication if the T cells could be adequately removed. If only a subset of patients were infected, identification of this subset would also be important in eradication trials. Other important issues include the tropism of virus in infected HPCs in vivo. McNamara et al suggest that they will be CXCR4-tropic viruses, since earlier progenitors are infected more efficiently with CXCR4-tropic viruses [4]. This may explain why patients do less well once a CXCR4-tropic virus predominates. Notably, sequencing proviruses from patient HPCs will provide information on tropism and the frequency of defective forms in HPCs, which may differ from the frequency in T cells. Additionally, while CD133– cells are enriched for HSCs, proving that HIV-infected HPCs in vivo include true HSCs with long-term renewal and engraftment capability will also be important. If only downstream progenitors are infected, their half-lives will be shorter, and the cells may die out. A more conclusive approach will likely require serial transplantation of infected cells (with reporter HIV) in NSG mice [13]. McNamara et al provide insight into the infection of HPCs in vivo and provide evidence suggesting that HPC infection occurs in a subset of patients and is not a result of T-cell contamination [7]. While it will be important to know whether HPCs are a reservoir for HIV, further studies to establish if, when, and at what level HPCs are infected should also be pursued to both understand HIV pathogenesis and help in the design of eradication approaches.