HIV-1 persists in infected individuals in a stable pool of resting CD4(+) T cells as a latent but replication-competent provirus. This latent reservoir is the major barrier to the eradication of ...HIV-1. Clinical trials are currently underway investigating the effects of latency-disrupting compounds on the persistence of the latent reservoir in infected individuals. To accurately assess the effects of such compounds, accurate assays to measure the frequency of latently infected cells are essential. The development of a simpler assay for the latent reservoir has been identified as a major AIDS research priority. We report here the development and validation of a rapid viral outgrowth assay that quantifies the frequency of cells that can release replication-competent virus following cellular activation. This new assay utilizes bead and column-based purification of resting CD4(+) T cells from the peripheral blood of HIV-1 infected patients rather than cell sorting to obtain comparable resting CD4(+) T cell purity. This new assay also utilizes the MOLT-4/CCR5 cell line for viral expansion, producing statistically comparable measurements of the frequency of latent HIV-1 infection. Finally, this new assay employs a novel quantitative RT-PCR specific for polyadenylated HIV-1 RNA for virus detection, which we demonstrate is a more sensitive and cost-effective method to detect HIV-1 replication than expensive commercial ELISA detection methods. The reductions in both labor and cost make this assay suitable for quantifying the frequency of latently infected cells in clinical trials of HIV-1 eradication strategies.
Celotno besedilo
Dostopno za:
DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
A stable latent reservoir for HIV-1 in resting CD4
T cells is the principal barrier to a cure
. Curative strategies that target the reservoir are being tested
and require accurate, scalable reservoir ...assays. The reservoir was defined with quantitative viral outgrowth assays for cells that release infectious virus after one round of T cell activation
. However, these quantitative outgrowth assays and newer assays for cells that produce viral RNA after activation
may underestimate the reservoir size because one round of activation does not induce all proviruses
. Many studies rely on simple assays based on polymerase chain reaction to detect proviral DNA regardless of transcriptional status, but the clinical relevance of these assays is unclear, as the vast majority of proviruses are defective
. Here we describe a more accurate method of measuring the HIV-1 reservoir that separately quantifies intact and defective proviruses. We show that the dynamics of cells that carry intact and defective proviruses are different in vitro and in vivo. These findings have implications for targeting the intact proviruses that are a barrier to curing HIV infection.
HIV-1 persists in a latent reservoir despite antiretroviral therapy (ART). This reservoir is the major barrier to HIV-1 eradication. Current approaches to purging the latent reservoir involve ...pharmacologic induction of HIV-1 transcription and subsequent killing of infected cells by cytolytic T lymphocytes (CTLs) or viral cytopathic effects. Agents that reverse latency without activating T cells have been identified using in vitro models of latency. However, their effects on latently infected cells from infected individuals remain largely unknown. Using a new ex vivo assay, we demonstrate that none of the latency-reversing agents (LRAs) tested induced outgrowth of HIV-1 from the latent reservoir of patients on ART. Using a quantitative reverse transcription PCR assay specific for all HIV-1 mRNAs, we demonstrate that LRAs that do not cause T cell activation do not induce substantial increases in intracellular HIV-1 mRNA in patient cells; only the protein kinase C agonist bryostatin-1 caused significant increases. These findings demonstrate that current in vitro models do not fully recapitulate mechanisms governing HIV-1 latency in vivo. Further, our data indicate that non-activating LRAs are unlikely to drive the elimination of the latent reservoir in vivo when administered individually.
Celotno besedilo
Dostopno za:
DOBA, IJS, IZUM, KILJ, NUK, PILJ, PNG, SAZU, UILJ, UKNU, UL, UM, UPUK
HIV-1 protease inhibitors (PIs) are among the most effective antiretroviral drugs. They are characterized by highly cooperative dose-response curves that are not explained by current pharmacodynamic ...theory. An unresolved problem affecting the clinical use of PIs is that patients who fail PI-containing regimens often have virus that lacks protease mutations, in apparent violation of fundamental evolutionary theory. Here, we show that these unresolved issues can be explained through analysis of the effects of PIs on distinct steps in the viral life cycle. We found that PIs do not affect virion release from infected cells but block entry, reverse transcription, and post-reverse transcription steps. The overall dose-response curves could be reconstructed by combining the curves for each step using the Bliss independence principle, showing that independent inhibition of multiple distinct steps in the life cycle generates the highly cooperative dose-response curves that make these drugs uniquely effective. Approximately half of the inhibitory potential of PIs is manifest at the entry step, likely reflecting interactions between the uncleaved Gag and the cytoplasmic tail (CT) of the Env protein. Sequence changes in the CT alone, which are ignored in current clinical tests for PI resistance, conferred PI resistance, providing an explanation for PI failure without resistance.
Sustained, drug-free control of HIV-1 replication is naturally achieved in less than 0.5% of infected individuals (here termed 'elite controllers'), despite the presence of a replication-competent ...viral reservoir
. Inducing such an ability to spontaneously maintain undetectable plasma viraemia is a major objective of HIV-1 cure research, but the characteristics of proviral reservoirs in elite controllers remain to be determined. Here, using next-generation sequencing of near-full-length single HIV-1 genomes and corresponding chromosomal integration sites, we show that the proviral reservoirs of elite controllers frequently consist of oligoclonal to near-monoclonal clusters of intact proviral sequences. In contrast to individuals treated with long-term antiretroviral therapy, intact proviral sequences from elite controllers were integrated at highly distinct sites in the human genome and were preferentially located in centromeric satellite DNA or in Krüppel-associated box domain-containing zinc finger genes on chromosome 19, both of which are associated with heterochromatin features. Moreover, the integration sites of intact proviral sequences from elite controllers showed an increased distance to transcriptional start sites and accessible chromatin of the host genome and were enriched in repressive chromatin marks. These data suggest that a distinct configuration of the proviral reservoir represents a structural correlate of natural viral control, and that the quality, rather than the quantity, of viral reservoirs can be an important distinguishing feature for a functional cure of HIV-1 infection. Moreover, in one elite controller, we were unable to detect intact proviral sequences despite analysing more than 1.5 billion peripheral blood mononuclear cells, which raises the possibility that a sterilizing cure of HIV-1 infection, which has previously been observed only following allogeneic haematopoietic stem cell transplantation
, may be feasible in rare instances.
HIV-1 persists in a latent reservoir in resting CD4+ T cells despite antiretroviral therapy (ART). The reservoir decays slowly over the first 7 years of ART (t1/2 = 44 months). However, whether decay ...continues with long-term ART is unclear. Recent integration site studies indicate gradual selection against inducible, intact proviruses, raising speculation that decades of ART might allow treatment interruption without viral rebound. Therefore, we measured the reservoir in 42 people on long-term ART (mean 22 years) using a quantitative viral outgrowth assay. After 7 years of ART, there was no long-term decrease in the frequency of inducible, replication-competent proviruses but rather an increase with an estimated doubling time of 23 years. Another reservoir assay, the intact proviral DNA assay, confirmed that reservoir decay with t1/2 of 44 months did not continue with long-term ART. The lack of decay reflected proliferation of infected cells. Most inducible, replication-competent viruses (79.8%) had env sequences identical to those of other isolates from the same sample. Thus, although integration site analysis indicates changes in reservoir composition, the proliferation of CD4+ T cells counteracts decay, maintaining the frequency of inducible, replication-competent proviruses at roughly constant levels over the long term. These results reinforce the need for lifelong ART.
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.
Antiretroviral therapy cannot cure HIV-1 infection due to the persistence of a small number of latently infected cells harboring replication-competent proviruses. Measuring persistent HIV-1 is ...challenging, as it consists of a mosaic population of defective and intact proviruses that can shift from a state of latency to active HIV-1 transcription. Due to this complexity, most of the current assays detect multiple categories of persistent HIV-1, leading to an overestimate of the true size of the latent reservoir. Here, we review the development of the viral outgrowth assay, the gold-standard quantification of replication-competent proviruses, and discuss the insights provided by full-length HIV-1 genome sequencing methods, which allowed us to unravel the composition of the proviral landscape. In this review, we provide a dissection of what defines HIV-1 persistence and we examine the unmet needs to measure the efficacy of interventions aimed at eliminating the HIV-1 reservoir.
Celotno besedilo
Dostopno za:
DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
Background. Candidemia is a severe invasive fungal infection with high mortality. Recognition of Candida species is mediated through pattern recognition receptors such as Toll-like receptors (TLRs). ...This study assessed whether genetic variation in TLR signaling influences susceptibility to candidemia. Methods. Thirteen mostly nonsynonymous single nucleotide polymorphisms (SNPs) in genes encoding TLRs and signaling adaptors MyD88 and Mal/TIRAP were genotyped in 338 patients (237 white, 93 African American, 8 other race) with candidemia and 351 noninfected controls (263 white, 88 African American). The SNPs significant in univariate analysis were further analyzed with multivariable logistic regression to determine association with clinical outcomes. Functional consequences of these polymorphisms were assessed via in vitro stimulation assays. Results. Analyses of TLR SNPs revealed that 3 TLR1 SNPs (R80T, S248N, I602S) were significantly associated with candidemia susceptibility in whites. This association was not found in African Americans, likely due to lower power in this smaller study population. Furthermore, these TLR1 polymorphisms displayed impaired cytokine release by primary monocytes. No associations with susceptibility to candidemia were observed for SNPs in TLR2, TLR4, TLR6, TLR9, MyD88, or TIRAP. Conclusions. Nonsynonymous SNPs in TLR1 are associated with impaired TLR1 function, decreased cytokine responses, and predisposition to candidemia in whites.
The latent reservoir for HIV-1 in resting memory CD4+ T cells is the major barrier to curing HIV-1 infection. Studies of HIV-1 latency have focused on regulation of viral gene expression in cells in ...which latent infection is established. However, it remains unclear how infection initially becomes latent. Here we described a unique set of properties of CD4+ T cells undergoing effector-to-memory transition including temporary upregulation of CCR5 expression and rapid downregulation of cellular gene transcription. These cells allowed completion of steps in the HIV-1 life cycle through integration but suppressed HIV-1 gene transcription, thus allowing the establishment of latency. CD4+ T cells in this stage were substantially more permissive for HIV-1 latent infection than other CD4+ T cells. Establishment of latent HIV-1 infection in CD4+ T could be inhibited by viral-specific CD8+ T cells, a result with implications for elimination of latent HIV-1 infection by T cell-based vaccines.
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•HIV replicates continuously, and the evolutionary basis of latency has been unclear•CD4+ T lymphoblasts transitioning to a memory state can be latently infected•Infection in a narrow time window after T cell activation leads to HIV latency•HIV-specific CTL can inhibit the establishment of latency
The latent reservoir for HIV is a barrier to cure, but it is unclear why HIV establishes latency given its ability to evade immune responses through evolution. Shan et al. show that latency is an unfortunate consequence of infection of CD4+ T cells within a narrow time window after activation.
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