Latent HIV in primary T lymphocytes is unresponsive to histone deacetylase inhibitors

Recently, there is considerable interest in the field of anti-HIV therapy to identify and develop chromatin-modifying histone deacetylase (HDAC) inhibitors that can effectively reactivate latent HIV in patients. The hope is that this would help eliminate cells harboring latent HIV and achieve an eventual cure of the virus. However, how effectively these drugs can stimulate latent HIVs in quiescent primary CD4 T cells, despite their relevant potencies demonstrated in cell line models of HIV latency, is not clear. Here, we show that the HDAC inhibitors valproic acid (VPA) and trichostatin A (TSA) are unable to reactivate HIV in latently infected primary CD4 T cells generated in the H80 co-culture system. This raises a concern that the drugs inhibiting HDAC function alone might not be sufficient for stimulating latent HIV in resting CD4 T cells in patients and not achieve any anticipated reduction in the pool of latent reservoirs

Rapid Turnover of 2-LTR HIV-1 DNA during Early Stage of Highly Active Antiretroviral Therapy

BACKGROUND: Despite prolonged treatment with highly active antiretroviral therapy (HAART), the infectious HIV-1 continues to replicate and resides latently in the resting memory CD4+ T lymphocytes, which blocks the eradication of HIV-1. The viral persistence of HIV-1 is mainly caused by its proviral DNA being either linear nonintegrated, circular nonintegrated, or integrated. Previous reports have largely focused on the dynamics of HIV-1 DNA from the samples collected with relatively long time intervals during the process of disease and HAART treatment, which may have missed the intricate changes during the intervals in early treatment. METHODOLOGY/PRINCIPAL FINDINGS: In this study, we investigated the dynamics of HIV-1 DNA in patients during the early phase of HARRT treatment. Using optimized real time PCR, we observed significant changes in 2-LTR during the first 12-week of treatment, while total and integrated HIV-1 DNA remained stable. The doubling time and half-life of 2-LTR were not correlated with the baseline and the rate of changes in plasma viral load and various CD4+ T-cell populations. Longitudinal analyses on 2-LTR sequences and plasma lipopolysaccharide (LPS) levels did not reveal any significant changes in the same treatment period. CONCLUSIONS/SIGNIFICANCE: Our study revealed the rapid changes in 2-LTR concentration in a relatively large number of patients during the early HAART treatment. The rapid changes indicate the rapid infusion and clearance of cells bearing 2-LTR in the peripheral blood. Those changes are not expected to be caused by the blocking of viral integration, as our study did not include the integrase inhibitor raltegravir. Our study helps better understand the dynamics of HIV-DNA and its potential role as a biomarker for the diseases and for the treatment efficacy of HAART

Viral Decay Kinetics in the Highly Active Antiretroviral Therapy-Treated Rhesus Macaque Model of AIDS

To prevent progression to AIDS, persons infected with human immunodeficiency virus type 1 (HIV-1) must remain on highly active antiretroviral therapy (HAART) indefinitely since this modality does not eradicate the virus. The mechanisms involved in viral persistence during HAART are poorly understood, but an animal model of HAART could help elucidate these mechanisms and enable studies of HIV-1 eradication strategies. Due to the specificity of non-nucleoside reverse transcriptase (RT) inhibitors (NNRTIs) for HIV-1, we have used RT-SHIV, a chimeric virus of simian immunodeficiency virus with RT from HIV-1. This virus is susceptible to NNRTIs and causes an AIDS-like disease in rhesus macaques. In this study, two groups of HAART-treated, RT-SHIV-infected macaques were analyzed to determine viral decay kinetics. In the first group, viral loads were monitored with a standard TaqMan RT-PCR assay with a limit of detection of 50 viral RNA copies per mL. Upon initiation of HAART, viremia decayed in a bi-phasic manner with half-lives of 1.7 and 8.5 days, respectively. A third phase was observed with little further decay. In the second group, the macaques were followed longitudinally with a more sensitive assay utilizing ultracentrifugation to concentrate virus from plasma. Bi-phasic decay of viral RNA was also observed in these animals with half-lives of 1.8 and 5.8 days. Viral loads in these animals during a third phase ranged from 2–58 RNA copies/mL, with little decay over time. The viral decay kinetics observed in these macaques are similar to those reported for HIV-1 infected humans. These results demonstrate that low-level viremia persists in RT-SHIV-infected macaques despite a HAART regimen commonly used in humans

Deep Molecular Characterization of HIV-1 Dynamics under Suppressive HAART

In order to design strategies for eradication of HIV-1 from infected individuals, detailed insight into the HIV-1 reservoirs that persist in patients on suppressive antiretroviral therapy (ART) is required. In this regard, most studies have focused on integrated (proviral) HIV-1 DNA forms in cells circulating in blood. However, the majority of proviral DNA is replication-defective and archival, and as such, has limited ability to reveal the dynamics of the viral population that persists in patients on suppressive ART. In contrast, extrachromosomal (episomal) viral DNA is labile and as a consequence is a better surrogate for recent infection events and is able to inform on the extent to which residual replication contributes to viral reservoir maintenance. To gain insight into the diversity and compartmentalization of HIV-1 under suppressive ART, we extensively analyzed longitudinal peripheral blood mononuclear cells (PBMC) samples by deep sequencing of episomal and integrated HIV-1 DNA from patients undergoing raltegravir intensification. Reverse-transcriptase genes selectively amplified from episomal and proviral HIV-1 DNA were analyzed by deep sequencing 0, 2, 4, 12, 24 and 48 weeks after raltegravir intensification. We used maximum likelihood phylogenies and statistical tests (AMOVA and Slatkin-Maddison (SM)) in order to determine molecular compartmentalization. We observed low molecular variance (mean variability ≤0.042). Although phylogenies showed that both DNA forms were intermingled within the phylogenetic tree, we found a statistically significant compartmentalization between episomal and proviral DNA samples (P<10−6 AMOVA test; P = 0.001 SM test), suggesting that they belong to different viral populations. In addition, longitudinal analysis of episomal and proviral DNA by phylogeny and AMOVA showed signs of non-chronological temporal compartmentalization (all comparisons P<10−6) suggesting that episomal and proviral DNA forms originated from different anatomical compartments. Collectively, this suggests the presence of a chronic viral reservoir in which there is stochastic release of infectious virus and in which there are limited rounds of de novo infection. This could be explained by the existence of different reservoirs with unique pharmacological accessibility properties, which will require strategies that improve drug penetration/retention within these reservoirs in order to minimise maintenance of the viral reservoir by de novo infection

An Investigation of Nurses' Knowledge, Attitudes, and Practices Regarding Disinfection Procedures in Italy

<p>Abstract</p> <p>Background</p> <p>This study assessed the level of knowledge, attitudes, and practice regarding disinfection procedures among nurses in Italian hospitals.</p> <p>Methods</p> <p>A face-to-face interview gathered the following information: demographic and practice characteristics; knowledge about the healthcare-associated infections (HAIs) and the disinfection practices; attitudes towards the utility of guidelines/protocols and perception of the risks of acquiring/transmitting HAIs; compliance with antisepsis/disinfection procedures; and sources of information.</p> <p>Results</p> <p>Only 29% acknowledged that urinary and respiratory tract infections were the two most common HAIs and this knowledge was significantly higher in those with a higher level of education. Attitudes towards the utility of guidelines/protocols for disinfection procedures showed a mean score of 9.1. The results of the linear regression model indicated a more positive attitude in female nurses, in those with a lower number of years of activity, and in those needing additional information about disinfection procedures. Nurses with higher educational level and with a higher perception of risk of transmitting an infectious disease while working were more likely to perform appropriate antisepsis of the surgical wound and handwashing before and after medication.</p> <p>Conclusions</p> <p>Plan of successful prevention activities about HAIs and provide pointers to help optimize disinfection procedures and infection prophylaxis and management are needed.</p

Switching Virally Suppressed, Treatment-Experienced Patients to a Raltegravir-Containing Regimen Does Not Alter Levels of HIV-1 DNA

Background: Current HIV-1 antiretroviral therapy (ART) greatly reduces virus replication but does not significantly affect the viral reservoir. Raltegravir, a recently introduced integrase inhibitor, could, at least theoretically, reduce residual viremia in patients on ART and affect the viral reservoir size. The aim of this study was to assess whether switching therapy in treatment-experienced patients that were virally suppressed to a raltegravir-containing regimen reduces the size of the viral reservoir, and if such treatment leads to a change in levels of HIV 2-LTR circles in this patient group. Methods: 14 ART experienced individuals with a suppressed viral load (,50 HIV-1 RNA copies/mL plasma) at baseline (for at least 2 months) were switched to a raltegravir-containing regimen. Blood samples were taken at baseline and at $2 timepoints up to 4866 weeks. Levels of total HIV-1 DNA and 2-LTR circles in peripheral blood mononuclear cells (PBMCs) were measured using real-time PCR assays. Results: There was no significant change in HIV-1 total DNA levels over the study duration (p = 0.808), median slope 0.24 (conservative nonparametric 95 % CI: 211.78, 26.23). Low levels of 2-LTR circles were detected in 2 patients. One had 16 copies/10 6 PBMCs at baseline and the other had 34 copies/10 6 PBMCs at week 51. Conclusions: The switch to a raltegravir containing regimen was not associated with a significant change in HIV-1 total DNA levels in this cohort. There were no observed changes in the levels of HIV-1 2-LTR circles associated with raltegravi

Resting Regulatory CD4 T Cells: A Site of HIV Persistence in Patients on Long-Term Effective Antiretroviral Therapy

BACKGROUND: In HIV-infected patients on long-term HAART, virus persistence in resting long-lived CD4 T cells is a major barrier to curing the infection. Cell quiescence, by favouring HIV latency, reduces the risk of recognition and cell destruction by cytotoxic lymphocytes. Several cell-activation-based approaches have been proposed to disrupt cell quiescence and then virus latency, but these approaches have not eradicated the virus. CD4+CD25+ regulatory T cells (Tregs) are a CD4+ T-cell subset with particular activation properties. We investigated the role of these cells in virus persistence in patients on long-term HAART. METHODOLOGY/PRINCIPAL FINDINGS: We found evidence of infection of resting Tregs (HLADR(-)CD69(-)CD25(hi)FoxP3+CD4+ T cells) purified from patients on prolonged HAART. HIV DNA harbouring cells appear more abundant in the Treg subset than in non-Tregs. The half-life of the Treg reservoir was estimated at 20 months. Since Tregs from patients on prolonged HAART showed hyporesponsiveness to cell activation and inhibition of HIV-specific cytotoxic T lymphocyte-related functions upon activation, therapeutics targeting cell quiescence to induce virus expression may not be appropriate for purging the Treg reservoir. CONCLUSIONS: Our results identify Tregs as a particular compartment within the latent reservoir that may require a specific approach for its purging

Histone Deacetylase Inhibitors Impair the Elimination of HIV-Infected Cells by Cytotoxic T-Lymphocytes

Resting memory CD4+ T-cells harboring latent HIV proviruses represent a critical barrier to viral eradication. Histone deacetylase inhibitors (HDACis), such as suberanilohydroxamic acid (SAHA), romidepsin, and panobinostat have been shown to induce HIV expression in these resting cells. Recently, it has been demonstrated that the low levels of viral gene expression induced by a candidate HDACi may be insufficient to cause the death of infected cells by viral cytopathic effects, necessitating their elimination by immune effectors, such as cytotoxic T-lymphocytes (CTL). Here, we study the impact of three HDACis in clinical development on T-cell effector functions. We report two modes of HDACi-induced functional impairment: i) the rapid suppression of cytokine production from viable T-cells induced by all three HDACis ii) the selective death of activated T-cells occurring at later time-points following transient exposures to romidepsin or, to a lesser extent, panobinostat. As a net result of these factors, HDACis impaired CTL-mediated IFN-γ production, as well as the elimination of HIV-infected or peptide-pulsed target cells, both in liquid culture and in collagen matrices. Romidepsin exerted greater inhibition of antiviral function than SAHA or panobinostat over the dose ranges tested. These data suggest that treatment with HDACis to mobilize the latent reservoir could have unintended negative impacts on the effector functions of CTL. This could influence the effectiveness of HDACi-based eradication strategies, by impairing elimination of infected cells, and is a critical consideration for trials where therapeutic interruptions are being contemplated, given the importance of CTL in containing rebound viremia

Murine Leukemia Virus Spreading in Mice Impaired in the Biogenesis of Secretory Lysosomes and Ca2+-Regulated Exocytosis

Retroviruses have been observed to bud intracellularly into multivesicular bodies (MVB), in addition to the plasma membrane. Release from MVB is thought to occur by Ca(2+)-regulated fusion with the plasma membrane.To address the role of the MVB pathway in replication of the murine leukemia virus (MLV) we took advantage of mouse models for the Hermansky-Pudlak syndrome (HPS) and Griscelli syndrome. In humans, these disorders are characterized by hypopigmentation and immunological alterations that are caused by defects in the biogenesis and trafficking of MVBs and other lysosome related organelles. Neonatal mice for these disease models lacking functional AP-3, Rab27A and BLOC factors were infected with Moloney MLV and the spread of virus into bone marrow, spleen and thymus was monitored. We found a moderate reduction in MLV infection levels in most mutant mice, which differed by less than two-fold compared to wild-type mice. In vitro, MLV release form bone-marrow derived macrophages was slightly enhanced. Finally, we found no evidence for a Ca(2+)-regulated release pathway in vitro. Furthermore, MLV replication was only moderately affected in mice lacking Synaptotagmin VII, a Ca(2+)-sensor regulating lysosome fusion with the plasma membrane.Given that MLV spreading in mice depends on multiple rounds of replication even moderate reduction of virus release at the cellular level would accumulate and lead to a significant effect over time. Thus our in vivo and in vitro data collectively argue against an essential role for a MVB- and secretory lysosome-mediated pathway in the egress of MLV

The co-receptor signaling model of HIV-1 pathogenesis in peripheral CD4 T cells

HIV-mediated CD4 depletion is the hallmark of AIDS and is the most reliable predictor of disease progression. While HIV replication is associated with CD4 depletion in general, plasma viremia by itself predicts the rate of CD4 loss only minimally in untreated patients. To resolve this paradox, I hypothesize the existence of a subpopulation of R5X4-signaling viruses. I also suggest that the gradual evolution and emergence of this subpopulation are largely responsible for the slow depletion of peripheral CD4 T cells