ABSTRACT
Itch is an evolutionarily conserved sensation that facilitates expulsion of pathogens and noxious stimuli from the skin. However, in organ failure, cancer, and chronic inflammatory disorders such as atopic dermatitis (AD), itch becomes chronic, intractable, and debilitating. In addition to chronic itch, patients often experience intense acute itch exacerbations. Recent discoveries have unearthed the neuroimmune circuitry of itch, leading to the development of anti-itch treatments. However, mechanisms underlying acute itch exacerbations remain overlooked. Herein, we identify that a large proportion of patients with AD harbor allergen-specific immunoglobulin E (IgE) and exhibit a propensity for acute itch flares. In mice, while allergen-provoked acute itch is mediated by the mast cell-histamine axis in steady state, AD-associated inflammation renders this pathway dispensable. Instead, a previously unrecognized basophil-leukotriene (LT) axis emerges as critical for acute itch flares. By probing fundamental itch mechanisms, our study highlights a basophil-neuronal circuit that may underlie a variety of neuroimmune processes.
Subject(s)
Basophils/pathology , Neurons/pathology , Pruritus/pathology , Acute Disease , Allergens/immunology , Animals , Chronic Disease , Dermatitis, Atopic/immunology , Dermatitis, Atopic/pathology , Disease Models, Animal , Histamine/metabolism , Humans , Immunoglobulin E/immunology , Inflammation/pathology , Leukotrienes/metabolism , Mast Cells/immunology , Mice, Inbred C57BL , Phenotype , Pruritus/immunology , TRPA1 Cation Channel/metabolism , TRPV Cation Channels/metabolismABSTRACT
Human immunodeficiency virus type 1 (HIV-1) infection persists despite years of antiretroviral therapy (ART). To remove the stigma and burden of chronic infection, approaches to eradicate or cure HIV infection are desired. Attempts to augment ART with therapies that reverse viral latency, paired with immunotherapies to clear infection, have advanced into the clinic, but the field is still in its infancy. We review foundational studies and highlight new insights in HIV cure research. Together with advances in ART delivery and HIV prevention strategies, future therapies that clear HIV infection may relieve society of the affliction of the HIV pandemic.
Subject(s)
Anti-HIV Agents/therapeutic use , Chronic Disease/therapy , HIV Infections/therapy , HIV-1/drug effects , Immunotherapy/methods , Virus Latency/drug effects , Animals , Haplorhini , HumansABSTRACT
The mode of acquisition and causes for the variable clinical spectrum of coronavirus disease 2019 (COVID-19) remain unknown. We utilized a reverse genetics system to generate a GFP reporter virus to explore severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) pathogenesis and a luciferase reporter virus to demonstrate sera collected from SARS and COVID-19 patients exhibited limited cross-CoV neutralization. High-sensitivity RNA in situ mapping revealed the highest angiotensin-converting enzyme 2 (ACE2) expression in the nose with decreasing expression throughout the lower respiratory tract, paralleled by a striking gradient of SARS-CoV-2 infection in proximal (high) versus distal (low) pulmonary epithelial cultures. COVID-19 autopsied lung studies identified focal disease and, congruent with culture data, SARS-CoV-2-infected ciliated and type 2 pneumocyte cells in airway and alveolar regions, respectively. These findings highlight the nasal susceptibility to SARS-CoV-2 with likely subsequent aspiration-mediated virus seeding to the lung in SARS-CoV-2 pathogenesis. These reagents provide a foundation for investigations into virus-host interactions in protective immunity, host susceptibility, and virus pathogenesis.
Subject(s)
Betacoronavirus/genetics , Coronavirus Infections/pathology , Coronavirus Infections/virology , Pneumonia, Viral/pathology , Pneumonia, Viral/virology , Respiratory System/virology , Reverse Genetics/methods , Aged , Angiotensin-Converting Enzyme 2 , Animals , Antibodies, Monoclonal/immunology , Antibodies, Neutralizing/immunology , Betacoronavirus/immunology , Betacoronavirus/pathogenicity , COVID-19 , Cell Line , Cells, Cultured , Chlorocebus aethiops , Coronavirus Infections/immunology , Coronavirus Infections/therapy , Cystic Fibrosis/pathology , DNA, Recombinant , Female , Furin/metabolism , Humans , Immunization, Passive , Lung/metabolism , Lung/pathology , Lung/virology , Male , Middle Aged , Nasal Mucosa/metabolism , Nasal Mucosa/pathology , Nasal Mucosa/virology , Pandemics , Peptidyl-Dipeptidase A/metabolism , Pneumonia, Viral/immunology , Respiratory System/pathology , SARS-CoV-2 , Serine Endopeptidases/metabolism , Vero Cells , Virulence , Virus Replication , COVID-19 SerotherapyABSTRACT
Individuals infected with human immunodeficiency virus type-1 (HIV-1) show metabolic alterations of CD4+ T cells through unclear mechanisms with undefined consequences. We analyzed the transcriptome of CD4+ T cells from patients with HIV-1 and revealed that the elevated oxidative phosphorylation (OXPHOS) pathway is associated with poor outcomes. Inhibition of OXPHOS by the US Food and Drug Administration-approved drug metformin, which targets mitochondrial respiratory chain complex-I, suppresses HIV-1 replication in human CD4+ T cells and humanized mice. In patients, HIV-1 peak viremia positively correlates with the expression of NLRX1, a mitochondrial innate immune receptor. Quantitative proteomics and metabolic analyses reveal that NLRX1 enhances OXPHOS and glycolysis during HIV-1-infection of CD4+ T cells to promote viral replication. At the mechanistic level, HIV infection induces the association of NLRX1 with the mitochondrial protein FASTKD5 to promote expression of mitochondrial respiratory complex components. This study uncovers the OXPHOS pathway in CD4+ T cells as a target for HIV-1 therapy.
Subject(s)
CD4-Positive T-Lymphocytes/virology , Genomics , HIV Infections/virology , HIV-1/growth & development , Metabolome , Metabolomics , Oxidative Phosphorylation , Proteome , Transcriptome , Virus Replication , Animals , Antiviral Agents/pharmacology , CD4-Positive T-Lymphocytes/drug effects , CD4-Positive T-Lymphocytes/immunology , CD4-Positive T-Lymphocytes/metabolism , Disease Models, Animal , Female , Gene Expression Profiling , Gene Regulatory Networks , HEK293 Cells , HIV Infections/drug therapy , HIV Infections/immunology , HIV Infections/metabolism , HIV-1/drug effects , HIV-1/immunology , HIV-1/metabolism , Host-Pathogen Interactions , Humans , Jurkat Cells , Male , Metformin/pharmacology , Mice , Mitochondrial Proteins/genetics , Mitochondrial Proteins/metabolism , Oxidative Phosphorylation/drug effects , Proteomics , RNA-Binding Proteins/genetics , RNA-Binding Proteins/metabolism , Viral Load , Virus Replication/drug effectsABSTRACT
7SK is a conserved noncoding RNA that regulates transcription by sequestering the transcription factor P-TEFb. 7SK function entails complex changes in RNA structure, but characterizing RNA dynamics in cells remains an unsolved challenge. We developed a single-molecule chemical probing strategy, DANCE-MaP (deconvolution and annotation of ribonucleic conformational ensembles), that defines per-nucleotide reactivity, direct base pairing interactions, tertiary interactions, and thermodynamic populations for each state in RNA structural ensembles from a single experiment. DANCE-MaP reveals that 7SK RNA encodes a large-scale structural switch that couples dissolution of the P-TEFb binding site to structural remodeling at distal release factor binding sites. The 7SK structural equilibrium shifts in response to cell growth and stress and can be targeted to modulate expression of P-TEFbresponsive genes. Our study reveals that RNA structural dynamics underlie 7SK function as an integrator of diverse cellular signals to control transcription and establishes the power of DANCE-MaP to define RNA dynamics in cells.
Subject(s)
Positive Transcriptional Elongation Factor B , RNA-Binding Proteins , Binding Sites/genetics , HeLa Cells , Humans , Positive Transcriptional Elongation Factor B/genetics , RNA, Small Nuclear/genetics , RNA, Untranslated , RNA-Binding Proteins/geneticsABSTRACT
Understanding the mechanisms that drive HIV expression and latency is a key goal for achieving an HIV cure. Here we investigate the role of the SETD2 histone methyltransferase, which deposits H3K36 trimethylation (H3K36me3), in HIV infection. We show that prevention of H3K36me3 by a potent and selective inhibitor of SETD2 (EPZ-719) leads to reduced post-integration viral gene expression and accelerated emergence of latently infected cells. CRISPR/Cas9-mediated knockout of SETD2 in primary CD4 T cells confirmed the role of SETD2 in HIV expression. Transcriptomic profiling of EPZ-719-exposed HIV-infected cells identified numerous pathways impacted by EPZ-719. Notably, depletion of H3K36me3 prior to infection did not prevent HIV integration but resulted in a shift of integration sites from highly transcribed genes to quiescent chromatin regions and to polycomb repressed regions. We also observed that SETD2 inhibition did not apparently affect HIV RNA levels, indicating a post-transcriptional mechanism affecting HIV expression. Viral RNA splicing was modestly reduced in the presence of EPZ-719. Intriguingly, EPZ-719 exposure enhanced responsiveness of latent HIV to the HDAC inhibitor vorinostat, suggesting that H3K36me3 can contribute to a repressive chromatin state at the HIV locus. These results identify SETD2 and H3K36me3 as novel regulators of HIV integration, expression and latency.
Subject(s)
HIV Infections , HIV-1 , Histone-Lysine N-Methyltransferase , Virus Latency , Histone-Lysine N-Methyltransferase/metabolism , Histone-Lysine N-Methyltransferase/genetics , Humans , Virus Latency/physiology , HIV Infections/virology , HIV Infections/metabolism , HIV Infections/genetics , HIV-1/physiology , HIV-1/genetics , CD4-Positive T-Lymphocytes/virology , CD4-Positive T-Lymphocytes/metabolism , Gene Expression Regulation, ViralABSTRACT
People with HIV-1 (PWH) on antiretroviral therapy (ART) can maintain undetectable virus levels, but a small pool of infected cells persists. This pool is largely comprised of defective proviruses that may produce HIV-1 proteins but are incapable of making infectious virus, with only a fraction (~10%) of these cells harboring intact viral genomes, some of which produce infectious virus following ex vivo stimulation (i.e. inducible intact proviruses). A majority of the inducible proviruses that persist on ART are formed near the time of therapy initiation. Here we compared proviral DNA (assessed here as 3' half genomes amplified from total cellular DNA) and inducible replication competent viruses in the pool of infected cells that persists during ART to determine if the original infection of these cells occurred at comparable times prior to therapy initiation. Overall, the average percent of proviruses that formed late (i.e. around the time of ART initiation, 60%) did not differ from the average percent of replication competent inducible viruses that formed late (69%), and this was also true for proviral DNA that was hypermutated (57%). Further, there was no evidence that entry into the long-lived infected cell pool was impeded by the ability to use the CXCR4 coreceptor, nor was the formation of long-lived infected cells enhanced during primary infection, when viral loads are exceptionally high. We observed that infection of cells that transitioned to be long-lived was enhanced among people with a lower nadir CD4+ T cell count. Together these data suggest that the timing of infection of cells that become long-lived is impacted more by biological processes associated with immunodeficiency before ART than the replication competency and/or cellular tropism of the infecting virus or the intactness of the provirus. Further research is needed to determine the mechanistic link between immunodeficiency and the timing of infected cells transitioning to the long-lived pool, particularly whether this is due to differences in infected cell clearance, turnover rates and/or homeostatic proliferation before and after ART.
Subject(s)
HIV Infections , HIV-1 , Humans , Proviruses/genetics , HIV-1/genetics , Anti-Retroviral Agents/pharmacology , Anti-Retroviral Agents/therapeutic use , HIV Infections/drug therapy , CD4-Positive T-Lymphocytes , DNA, Viral/genetics , DNA, Viral/metabolism , Viral Load , TropismABSTRACT
An Amendment to this paper has been published and can be accessed via a link at the top of the paper.
ABSTRACT
Human immunodeficiency virus (HIV) persists indefinitely in individuals with HIV who receive antiretroviral therapy (ART) owing to a reservoir of latently infected cells that contain replication-competent virus1-4. Here, to better understand the mechanisms responsible for latency persistence and reversal, we used the interleukin-15 superagonist N-803 in conjunction with the depletion of CD8+ lymphocytes in ART-treated macaques infected with simian immunodeficiency virus (SIV). Although N-803 alone did not reactivate virus production, its administration after the depletion of CD8+ lymphocytes in conjunction with ART treatment induced robust and persistent reactivation of the virus in vivo. We found viraemia of more than 60 copies per ml in all macaques (n = 14; 100%) and in 41 out of a total of 56 samples (73.2%) that were collected each week after N-803 administration. Notably, concordant results were obtained in ART-treated HIV-infected humanized mice. In addition, we observed that co-culture with CD8+ T cells blocked the in vitro latency-reversing effect of N-803 on primary human CD4+ T cells that were latently infected with HIV. These results advance our understanding of the mechanisms responsible for latency reversal and lentivirus reactivation during ART-suppressed infection.
Subject(s)
CD8-Positive T-Lymphocytes/immunology , Interleukin-15/agonists , Simian Immunodeficiency Virus/physiology , Virus Replication , Animals , CD4-Positive T-Lymphocytes/virology , HIV Infections/immunology , HIV Infections/virology , Humans , Interleukin-15/immunology , Lymphocyte Depletion , Macaca mulatta , Mice , Simian Acquired Immunodeficiency Syndrome/immunology , Simian Acquired Immunodeficiency Syndrome/virology , Virus Latency , Virus Replication/immunologyABSTRACT
Long-lasting, latently infected resting CD4+ T cells are the greatest obstacle to obtaining a cure for HIV infection, as these cells can persist despite decades of treatment with antiretroviral therapy (ART). Estimates indicate that more than 70 years of continuous, fully suppressive ART are needed to eliminate the HIV reservoir1. Alternatively, induction of HIV from its latent state could accelerate the decrease in the reservoir, thus reducing the time to eradication. Previous attempts to reactivate latent HIV in preclinical animal models and in clinical trials have measured HIV induction in the peripheral blood with minimal focus on tissue reservoirs and have had limited effect2-9. Here we show that activation of the non-canonical NF-κB signalling pathway by AZD5582 results in the induction of HIV and SIV RNA expression in the blood and tissues of ART-suppressed bone-marrow-liver-thymus (BLT) humanized mice and rhesus macaques infected with HIV and SIV, respectively. Analysis of resting CD4+ T cells from tissues after AZD5582 treatment revealed increased SIV RNA expression in the lymph nodes of macaques and robust induction of HIV in almost all tissues analysed in humanized mice, including the lymph nodes, thymus, bone marrow, liver and lung. This promising approach to latency reversal-in combination with appropriate tools for systemic clearance of persistent HIV infection-greatly increases opportunities for HIV eradication.
Subject(s)
HIV Infections/virology , HIV-1/physiology , NF-kappa B/metabolism , Simian Acquired Immunodeficiency Syndrome/virology , Simian Immunodeficiency Virus/physiology , Virus Latency , Alkynes/pharmacology , Animals , Anti-Retroviral Agents/pharmacology , HIV Infections/metabolism , HIV-1/drug effects , Macaca mulatta , Mice , Oligopeptides/pharmacology , Simian Acquired Immunodeficiency Syndrome/metabolism , Simian Immunodeficiency Virus/drug effects , Virus Latency/drug effectsABSTRACT
HIV post-treatment controllers (PTCs) are rare individuals who maintain low levels of viremia after stopping antiretroviral therapy (ART). Understanding the mechanisms of HIV post-treatment control will inform development of strategies aiming at achieving HIV functional cure. In this study, we evaluated 22 PTCs from 8 AIDS Clinical Trials Group (ACTG) analytical treatment interruption (ATI) studies who maintained viral loads ≤400 copies/mL for ≥24 wk. There were no significant differences in demographics or frequency of protective and susceptible human leukocyte antigen (HLA) alleles between PTCs and post-treatment noncontrollers (NCs, n = 37). Unlike NCs, PTCs demonstrated a stable HIV reservoir measured by cell-associated RNA (CA-RNA) and intact proviral DNA assay (IPDA) during analytical treatment interruption (ATI). Immunologically, PTCs demonstrated significantly lower CD4+ and CD8+ T cell activation, lower CD4+ T cell exhaustion, and more robust Gag-specific CD4+ T cell responses and natural killer (NK) cell responses. Sparse partial least squares discriminant analysis (sPLS-DA) identified a set of features enriched in PTCs, including a higher CD4+ T cell% and CD4+/CD8+ ratio, more functional NK cells, and a lower CD4+ T cell exhaustion level. These results provide insights into the key viral reservoir features and immunological profiles for HIV PTCs and have implications for future studies evaluating interventions to achieve an HIV functional cure.
Subject(s)
CD8-Positive T-Lymphocytes , HIV Infections , Humans , Killer Cells, Natural , Lymphocyte Activation , RNA , HIV Infections/drug therapy , HIV Infections/immunology , ViremiaABSTRACT
Although antiretroviral therapy (ART) is effective at suppressing HIV replication, a viral reservoir persists that can reseed infection if ART is interrupted. Curing HIV will require elimination or containment of this reservoir, but the size of the HIV reservoir is highly variable between individuals. To evaluate the size of the HIV reservoir, several assays have been developed, including PCR-based assays for viral DNA, the intact proviral DNA assay, and the quantitative viral outgrowth assay (QVOA). QVOA is the gold standard assay for measuring inducible replication-competent proviruses, but this assay is technically challenging and time-consuming. To begin progress toward a more rapid and less laborious tool for quantifying cells infected with replication-competent HIV, we developed the Microwell Outgrowth Assay, in which infected CD4 T cells are co-cultured with an HIV-detecting reporter cell line in a polydimethylsiloxane (PDMS)/polystyrene array of nanoliter-sized wells. Transmission of HIV from infected cells to the reporter cell line induces fluorescent reporter protein expression that is detected by automated scanning across the array. Using this approach, we were able to detect HIV-infected cells from ART-naïve people with HIV (PWH) and from PWH on ART with large reservoirs. Furthermore, we demonstrate that infected cells can be recovered from individual rafts and used to analyze the diversity of viral sequences. Although additional development and optimization will be required for quantifying the reservoir in PWH with small latent reservoirs, this assay may be a useful prototype for microwell assays of infected cells.IMPORTANCEMeasuring the size of the HIV reservoir in people with HIV (PWH) will be important for determining the impact of HIV cure strategies. However, measuring this reservoir is challenging. We report a new method for quantifying HIV-infected cells that involves culturing cells from PWH in an array of microwells with a cell line that detects HIV infection. We show that this approach can detect rare HIV-infected cells and derive detailed virus sequence information for each infected cell.
Subject(s)
HIV Infections , Virology , Humans , CD4-Positive T-Lymphocytes , Cell Line , DNA, Viral , HIV Infections/virology , Proviruses/genetics , Viral Load , Virus Latency , Virology/methodsABSTRACT
Lifelong treatment is required for people living with HIV as current antiretroviral therapy (ART) does not eradicate HIV infection. Latently infected cells are essentially indistinguishable from uninfected cells and cannot be depleted by currently available approaches. This study evaluated antibody mediated transient CD4+ T cell depletion as a strategy to reduce the latent HIV reservoir. Anti-CD4 antibodies effectively depleted CD4+ T cells in the peripheral blood and tissues of humanized mice. We then demonstrate that antibody-mediated CD4+ T cell depletion of HIV infected ART-suppressed animals results in substantial reductions in cell-associated viral RNA and DNA levels in peripheral blood cells over the course of anti-CD4 antibody treatment. Recovery of CD4+ T cells was observed in all tissues analyzed except for the lung 26 days after cessation of antibody treatment. After CD4+ T cell recovery, significantly lower levels of cell-associated viral RNA and DNA were detected in the tissues of anti-CD4 antibody-treated animals. Further, an 8.5-fold reduction in the levels of intact HIV proviral DNA and a 3.1-fold reduction in the number of latently infected cells were observed in anti-CD4-antibody-treated animals compared with controls. However, there was no delay in viral rebound when ART was discontinued in anti-CD4 antibody-treated animals following CD4+ T cell recovery compared with controls. Our results suggest that transient CD4+ T cell depletion, a long-standing clinical intervention that might have an acceptable safety profile, during suppressive ART can reduce the size of the HIV reservoir in humanized mice.
Subject(s)
HIV Infections , HIV-1 , Humans , Mice , Animals , CD4-Positive T-Lymphocytes , Virus Latency , Virus Replication , Anti-Retroviral Agents/pharmacology , Anti-Retroviral Agents/therapeutic use , RNA, Viral , DNA , Viral LoadABSTRACT
BACKGROUND: Monoclonal antibodies (mAbs) represent a crucial antiviral strategy for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection, but it is unclear whether combination mAbs offer a benefit over single-active mAb treatment. Amubarvimab and romlusevimab significantly reduced the risk of hospitalizations or death in the ACTIV-2/A5401 trial. Certain SARS-CoV-2 variants are intrinsically resistant against romlusevimab, leading to only single-active mAb therapy with amubarvimab in these variants. We evaluated virologic outcomes in individuals treated with single- versus dual-active mAbs. METHODS: Participants were nonhospitalized adults at higher risk of clinical progression randomized to amubarvimab plus romlusevimab or placebo. Quantitative SARS-CoV-2 RNA levels and targeted S-gene next-generation sequencing was performed on anterior nasal samples. We compared viral load kinetics and resistance emergence between individuals treated with effective single- versus dual-active mAbs depending on the infecting variant. RESULTS: Study participants receiving single- or dual-active mAbs had similar demographics, baseline nasal viral load, symptom score, and symptom duration. Compared with single-active mAb treatment, treatment with dual-active mAbs led to faster viral load decline at study days 3 (P < .001) and 7 (P < .01). Treatment-emergent resistance mutations were more likely to be detected after amubarvimab plus romlusevimab treatment than with placebo (2.6% vs 0%; P < .001) and were more frequently detected in the setting of single-active compared with dual-active mAb treatment (7.3% vs 1.1%; P < .01). Single-active and dual-active mAb treatment resulted in similar decrease in rates of hospitalizations or death. CONCLUSIONS: Compared with single-active mAb therapy, dual-active mAbs led to similar clinical outcomes but significantly faster viral load decline and a lower risk of emergent resistance.
Subject(s)
Antibodies, Monoclonal, Humanized , COVID-19 Drug Treatment , SARS-CoV-2 , Viral Load , Humans , SARS-CoV-2/immunology , SARS-CoV-2/drug effects , Female , Male , Middle Aged , Viral Load/drug effects , Antibodies, Monoclonal, Humanized/therapeutic use , Antibodies, Monoclonal, Humanized/pharmacology , Drug Resistance, Viral , Antiviral Agents/therapeutic use , Antiviral Agents/pharmacology , COVID-19/immunology , COVID-19/virology , Antibodies, Monoclonal/therapeutic use , Antibodies, Monoclonal/immunology , Aged , Adult , Drug Therapy, CombinationABSTRACT
BACKGROUND: The histone deacetylase inhibitor vorinostat (VOR) can reverse human immunodeficiency virus type 1 (HIV-1) latency in vivo and allow T cells to clear infected cells in vitro. HIV-specific T cells (HXTCs) can be expanded ex vivo and have been safely administered to people with HIV (PWH) on antiretroviral therapy. METHODS: Six PWH received infusions of 2 × 107 HXTCs/m² with VOR 400â mg, and 3 PWH received infusions of 10 × 107 HXTCs/m² with VOR. The frequency of persistent HIV by multiple assays including quantitative viral outgrowth assay (QVOA) of resting CD4+ T cells was measured before and after study therapy. RESULTS: VOR and HXTCs were safe, and biomarkers of serial VOR effect were detected, but enhanced antiviral activity in circulating cells was not evident. After 2 × 107 HXTCs/m² with VOR, 1 of 6 PWH exhibited a decrease in QVOA, and all 3 PWH exhibited such declines after 10 × 107 HXTCs/m² and VOR. However, most declines did not exceed the 6-fold threshold needed to definitively attribute decline to the study intervention. CONCLUSIONS: These modest effects provide support for the strategy of HIV latency reversal and reservoir clearance, but more effective interventions are needed to yield the profound depletion of persistent HIV likely to yield clinical benefit. Clinical Trials Registration. NCT03212989.
Subject(s)
HIV Infections , HIV-1 , Humans , Vorinostat/therapeutic use , Vorinostat/pharmacology , Histone Deacetylase Inhibitors/therapeutic use , Histone Deacetylase Inhibitors/pharmacology , CD4-Positive T-Lymphocytes , Cell- and Tissue-Based Therapy , Virus LatencyABSTRACT
Limited cellular levels of the HIV transcriptional activator Tat are one contributor to proviral latency that might be targeted in HIV cure strategies. We recently demonstrated that lipid nanoparticles containing HIV tat mRNA induce HIV expression in primary CD4 T cells. Here, we sought to further characterize tat mRNA in the context of several benchmark latency reversal agents (LRAs), including inhibitor of apoptosis protein antagonists (IAPi), bromodomain and extra-Terminal motif inhibitors (BETi), and histone deacetylase inhibitors (HDACi). tat mRNA reversed latency across several different cell line models of HIV latency, an effect dependent on the TAR hairpin loop. Synergistic enhancement of tat mRNA activity was observed with IAPi, HDACi, and BETi, albeit to variable degrees. In primary CD4 T cells from durably suppressed people with HIV, tat mRNA profoundly increased the frequencies of elongated, multiply-spliced, and polyadenylated HIV transcripts, while having a lesser impact on TAR transcript frequencies. tat mRNAs alone resulted in variable HIV p24 protein induction across donors. However, tat mRNA in combination with IAPi, BETi, or HDACi markedly enhanced HIV RNA and protein expression without overt cytotoxicity or cellular activation. Notably, combination regimens approached or in some cases exceeded the latency reversal activity of maximal mitogenic T cell stimulation. Higher levels of tat mRNA-driven HIV p24 induction were observed in donors with larger mitogen-inducible HIV reservoirs, and expression increased with prolonged exposure time. Combination LRA strategies employing both small molecule inhibitors and Tat delivered to CD4 T cells are a promising approach to effectively target the HIV reservoir.
Subject(s)
HIV Infections , HIV-1 , Histone Deacetylase Inhibitors , Nanoparticles , Virus Latency , tat Gene Products, Human Immunodeficiency Virus , Humans , Anti-HIV Agents/pharmacology , CD4-Positive T-Lymphocytes/drug effects , CD4-Positive T-Lymphocytes/virology , CD4-Positive T-Lymphocytes/metabolism , Histone Deacetylase Inhibitors/pharmacology , HIV Antigens/genetics , HIV Infections/drug therapy , HIV Infections/virology , HIV-1/drug effects , HIV-1/genetics , RNA, Messenger/genetics , RNA, Messenger/metabolism , tat Gene Products, Human Immunodeficiency Virus/genetics , Virus Latency/drug effectsABSTRACT
Posttreatment controllers (PTCs) are rare HIV-infected individuals who can limit viral rebound after antiretroviral therapy interruption (ATI), but the mechanisms of this remain unclear. To investigate these mechanisms, we quantified various HIV RNA transcripts (via reverse transcription droplet digital PCR [RT-ddPCR]) and cellular transcriptomes (via RNA-seq) in blood cells from PTCs and noncontrollers (NCs) before and two time points after ATI. HIV transcription initiation did not significantly increase after ATI in PTCs or in NCs, whereas completed HIV transcripts increased at early ATI in both groups and multiply-spliced HIV transcripts increased only in NCs. Compared to NCs, PTCs showed lower levels of HIV DNA, more cell-associated HIV transcripts per total RNA at all times, no increase in multiply-spliced HIV RNA at early or late ATI, and a reduction in the ratio of completed/elongated HIV RNA after early ATI. NCs expressed higher levels of the IL-7 pathway before ATI and expressed higher levels of multiple cytokine, inflammation, HIV transcription, and cell death pathways after ATI. Compared to the baseline, the NCs upregulated interferon and cytokine (especially TNF) pathways during early and late ATI, whereas PTCs upregulated interferon and p53 pathways only at early ATI and downregulated gene translation during early and late ATI. In NCs, viral rebound after ATI is associated with increases in HIV transcriptional completion and splicing, rather than initiation. Differences in HIV and cellular transcription may contribute to posttreatment control, including an early limitation of spliced HIV RNA, a delayed reduction in completed HIV transcripts, and the differential expression of the IL-7, p53, and TNF pathways. IMPORTANCE The findings presented here provide new insights into how HIV and cellular gene expression change after stopping ART in both noncontrollers and posttreatment controllers. Posttreatment control is associated with an early ability to limit increases in multiply-spliced HIV RNA, a delayed (and presumably immune-mediated) ability to reverse an initial rise in processive/completed HIV transcripts, and multiple differences in cellular gene expression pathways. These differences may represent correlates or mechanisms of posttreatment control and may provide insight into the development and/or monitoring of therapeutic strategies that are aimed at a functional HIV cure.
Subject(s)
HIV Infections , RNA, Viral , Transcriptome , Humans , HIV Infections/drug therapy , HIV Infections/genetics , HIV Infections/immunology , HIV-1/genetics , Interferons/genetics , Interleukin-7/genetics , RNA, Viral/genetics , Transcriptome/immunology , Tumor Suppressor Protein p53/geneticsABSTRACT
IMPORTANCE: The lack of a reliable method to accurately detect when replication-competent HIV has been cleared is a major challenge in developing a cure. This study introduces a new approach called the HIVepsilon-seq (HIVε-seq) assay, which uses long-read sequencing technology and bioinformatics to scrutinize the HIV genome at the nucleotide level, distinguishing between defective and intact HIV. This study included 30 participants on antiretroviral therapy, including 17 women, and was able to discriminate between defective and genetically intact viruses at the single DNA strand level. The HIVε-seq assay is an improvement over previous methods, as it requires minimal sample, less specialized lab equipment, and offers a shorter turnaround time. The HIVε-seq assay offers a promising new tool for researchers to measure the intact HIV reservoir, advancing efforts towards finding a cure for this devastating disease.
Subject(s)
HIV Infections , HIV , Proviruses , Female , Humans , CD4-Positive T-Lymphocytes , DNA, Viral/genetics , HIV Infections/drug therapy , HIV Infections/epidemiology , HIV Infections/virology , Nucleotides , Proviruses/genetics , Viral Load , Sequence Analysis, DNA , Male , Sex Factors , HIV/geneticsABSTRACT
BACKGROUND: Atopic dermatitis (AD) is thought to precede the onset of other allergic illness (OAI) in a temporal progression (ie, atopic march), yet the timing and progression has been questioned. It is also unclear how parental allergic illness impacts the development of these illnesses in offspring. OBJECTIVE: (1) Explore risk of incident AD and (2) timing of allergic disease onset in children of mothers with AD compared with mothers without AD from the United Kingdom. METHODS: We created a birth cohort of mother-child pairs using IQVIA Medical Research Data database and developed Cox proportional models to examine the above associations (hazard ratio, HR [95% confidence interval, CI]). RESULTS: Among 1,224,243 child-mother pairs, mean child (standard deviation) follow-up time was 10.8 (8.3) years and 50.1% were males (N = 600,905). Children were 59% (HR = 1.59 [1.57, 1.60]) more likely to have AD if their mothers had AD compared with no AD with mean age of first AD diagnosis at 3.3 (4.8) years. Most children with any diagnosis of AD present with AD first (91.0%); however, in those with asthma, only 67.8% developed AD first. CONCLUSION: Children born to mothers with AD are more prone to develop AD and some develop OAI first, suggesting that not all follow the same sequential pathway.
Subject(s)
Asthma , Dermatitis, Atopic , Hypersensitivity , Male , Humans , Child, Preschool , Female , Dermatitis, Atopic/epidemiology , Dermatitis, Atopic/diagnosis , Cohort Studies , Asthma/epidemiology , United Kingdom/epidemiology , Risk FactorsABSTRACT
BACKGROUND: Crisaborole ointment, 2%, is a nonsteroidal topical phosphodiesterase 4 inhibitor approved for the treatment of mild-to-moderate atopic dermatitis. OBJECTIVE: To evaluate the efficacy and safety of crisaborole in stasis dermatitis (SD). METHODS: In this randomized, double-blind, vehicle-controlled, decentralized phase 2a study (NCT04091087), 65 participants aged ≥45 years with SD without active ulceration received crisaborole or vehicle (1:1) twice-daily for 6 weeks. The primary end point was percentage change from baseline in total sign score at week 6 based on in-person assessment. RESULTS: Crisaborole-treated participants had significantly reduced total sign score from baseline versus vehicle based on in-person (nondermatologist) assessment (-32.4% vs -18.1%, P = .0299) and central reader (dermatologists) assessment of photographs (-52.5% vs -10.3%, P = .0004). Efficacy according to success and improvement per Investigator's Global Assessment score and lesional percentage body surface area reached statistical significance based on central reader but not in-person assessments. Skin and subcutaneous tissue disorders were common all-causality treatment-emergent adverse events with crisaborole. LIMITATIONS: Small sample size and short treatment duration were key limitations. In-person assessment was not conducted by dermatologists. CONCLUSION: Crisaborole improved signs and symptoms of SD and was well tolerated. Central reader assessment represents a promising approach for siteless clinical research.