Single cell spatial profiling of FFPE splenic tissue from a humanized mouse model of HIV infection.

BACKGROUND: Latency remains a major obstacle to finding a cure for HIV despite the availability of antiretroviral therapy. Due to virus dormancy, limited biomarkers are available to identify latent HIV-infected cells. Profiling of individual HIV-infected cells is needed to explore potential latency biomarkers and to study the mechanisms of persistence that maintain the HIV reservoir. METHODS: Single cell spatial transcriptomic characterization using the CosMx Spatial Molecular Imager platform was conducted to analyze HIV-infected cells in formalin-fixed paraffin-embedded sections of splenic tissue surgically obtained from an HIV-infected humanized mouse model. Regulation of over a thousand human genes was quantified in both viremic and aviremic specimens. In addition, in situ hybridization and immunohistochemistry were performed in parallel to identify HIV viral RNA- and p24-containing cells, respectively. Finally, initial findings from CosMx gene profiling were confirmed by isolating RNA from CD4 + T cells obtained from a person living with HIV on antiretroviral therapy following either PMA/Ionomycin or DMSO treatment. RNA was quantified using qPCR for a panel of targeted human host genes. RESULTS: Supervised cell typing revealed that most of the HIV-infected cells in the mouse spleen sections were differentiated CD4 + T cells. A significantly higher number of infected cells, 2781 (1.61%) in comparison to 112 (0.06%), and total HIV transcripts per infected cell were observed in viremic samples compared to aviremic samples, respectively, which was consistent with the data obtained from ISH and IHC. Notably, the expression of 55 genes was different in infected cells within tissue from aviremic animals compared to viremic. In particular, both spleen tyrosine kinase (SYK) and CXCL17, were expressed approximately 100-fold higher. This data was further evaluated against bulk RNA isolated from HIV-infected human primary CD4 + T cells. A nearly 6-fold higher expression of SYK mRNA was observed in DMSO-treated CD4 + T cells compared to those stimulated with PMA/Ionomycin. CONCLUSION: This study found that the CosMx SMI platform is valuable for assessing HIV infection and providing insights into host biomarkers associated with HIV reservoirs. Higher relative expression of the SYK gene in aviremic-infected cells from the humanized mouse HIV model was consistent with levels found in CD4 + T cells of aviremic donors.

Dual blockade of IL-10 and PD-1 leads to control of SIV viral rebound following analytical treatment interruption.

Human immunodeficiency virus (HIV) persistence during antiretroviral therapy (ART) is associated with heightened plasma interleukin-10 (IL-10) levels and PD-1 expression. We hypothesized that IL-10 and PD-1 blockade would lead to control of viral rebound following analytical treatment interruption (ATI). Twenty-eight ART-treated, simian immunodeficiency virus (SIV)mac239-infected rhesus macaques (RMs) were treated with anti-IL-10, anti-IL-10 plus anti-PD-1 (combo) or vehicle. ART was interrupted 12 weeks after introduction of immunotherapy. Durable control of viral rebound was observed in nine out of ten combo-treated RMs for >24 weeks post-ATI. Induction of inflammatory cytokines, proliferation of effector CD8+ T cells in lymph nodes and reduced expression of BCL-2 in CD4+ T cells pre-ATI predicted control of viral rebound. Twenty-four weeks post-ATI, lower viral load was associated with higher frequencies of memory T cells expressing TCF-1 and of SIV-specific CD4+ and CD8+ T cells in blood and lymph nodes of combo-treated RMs. These results map a path to achieve long-lasting control of HIV and/or SIV following discontinuation of ART.

Functional fibers/textiles for smart sensing devices and applications in personal healthcare systems.

Personalized medical diagnostics and monitoring have become increasingly important due to inefficient and delayed medical services of traditional centralized healthcare systems. To enhance the comfort and portability, flexible health monitoring systems have been developed in recent years. In particular, smart fiber/textile-based sensing devices show superiority for continuously monitoring personal health and vital physiological parameters owing to their light weight, good flexibility and inherent miniaturization. This review focuses on the recent advances in smart fiber/textile-based sensing devices for wearable electronic applications. First, fabrication strategies of smart sensing fibers/textiles are introduced in detail. In addition, sensing performances, working principles and applications of smart sensing fibers/textiles such as pressure sensing fibers/textiles, stretchable strain sensing fibers/textiles, temperature sensing fibers/textiles, and biofluid, gas and humidity sensing fibers/textiles in health monitoring are also reviewed systematically. Finally, we propose current challenges and future prospects in the area of fiber/textile-based sensors for wearable healthcare monitoring and diagnosis systems. In general, this review aims to give an overall perspective of the promising field by reviewing various fiber/textile-based sensing devices and highlighting the importance for researchers to keep up with the sequential exploration of soft sensing fibers/textiles for applications in wearable smart systems.

The Role of the MYL4 Gene in Porcine Muscle Development and Its Molecular Regulatory Mechanisms.

Muscle growth stands as a pivotal economic trait within pig production, governed by a complex interplay of multiple genes, each playing a role in its quantitative manifestation. Understanding the intricate regulatory mechanisms of porcine muscle development is crucial for enhancing both pork yield and quality. This study used the GSE99749 dataset downloaded from the GEO database, conducting a detailed analysis of the RNA-seq results from the longissimus dorsi muscle (LD) of Tibetan pigs (TP), Wujin pigs (WJ) and large white pigs (LW) at 60 days of gestation, representing diverse body sizes and growth rates. Comparative analyses between TPvsWJ and TPvsLW, along with differential gene expression (DEG) analysis, functional enrichment analysis, and protein-protein interaction (PPI) network analysis, revealed 1048 and 1157 significantly differentially expressed genes (p < 0.001) in TPvsWJ and TPvsLW, respectively. With stricter screening criteria, 37 DEGs were found to overlap between the 2 groups. PPI analysis identified MYL5, MYL4, and ACTC1 as the three core genes. This article focuses on exploring the MYL4 gene. Molecular-level experimental validation, through overexpression and interference of the MYL4 gene combined with EDU staining experiments, demonstrated that overexpression of MYL4 significantly promoted the proliferation of porcine skeletal muscle satellite cells (PSMSC), while interference with MYL4 inhibited their proliferation. Furthermore, by examining the effects of overexpressing and interfering with the MYL4 gene on the muscle hypertrophy marker Fst gene and the muscle degradation marker FOXO3 gene, the pivotal role of the MYL4 gene in promoting muscle growth and preventing muscle degradation was further confirmed. These findings offer a new perspective on the molecular mechanisms behind porcine muscle growth and development, furnishing valuable data and insights for muscle biology research.

A Comprehensive Review of Vision-Based 3D Reconstruction Methods.

With the rapid development of 3D reconstruction, especially the emergence of algorithms such as NeRF and 3DGS, 3D reconstruction has become a popular research topic in recent years. 3D reconstruction technology provides crucial support for training extensive computer vision models and advancing the development of general artificial intelligence. With the development of deep learning and GPU technology, the demand for high-precision and high-efficiency 3D reconstruction information is increasing, especially in the fields of unmanned systems, human-computer interaction, virtual reality, and medicine. The rapid development of 3D reconstruction is becoming inevitable. This survey categorizes the various methods and technologies used in 3D reconstruction. It explores and classifies them based on three aspects: traditional static, dynamic, and machine learning. Furthermore, it compares and discusses these methods. At the end of the survey, which includes a detailed analysis of the trends and challenges in 3D reconstruction development, we aim to provide a comprehensive introduction for individuals who are currently engaged in or planning to conduct research on 3D reconstruction. Our goal is to help them gain a comprehensive understanding of the relevant knowledge related to 3D reconstruction.

Initial productive and latent HIV infections originate in vivo by infection of resting T cells.

Productively infected cells are generally thought to arise from HIV infection of activated CD4+ T cells, and these infected activated cells are thought to be a recurring source of latently infected cells when a portion of the population transitions to a resting state. We discovered and report here that productively and latently infected cells can instead originate from direct infection of resting CD4+ T cell populations in lymphoid tissues in Fiebig I, the earliest stage of detectable HIV infection. We found that direct infection of resting CD4+ T cells was correlated with the availability of susceptible target cells in lymphoid tissues largely restricted to resting CD4+ T cells in which expression of pTEFb enabled productive infection, and we documented persistence of HIV-producing resting T cells during antiretroviral therapy (ART). Thus, we provide evidence of a mechanism by which direct infection of resting T cells in lymphoid tissues to generate productively and latently infected cells creates a mechanism by which the productively infected cells can replenish both populations and maintain two sources of virus from which HIV infection can rebound, even if ART is instituted at the earliest stage of detectable infection.

Magnetic tubular nickel@silica-graphene nanocomposites with high preconcentration capacity for organothiophosphate pesticide removal in environmental water: Fabrication, magnetic solid-phase extraction, and trace detection.

Organothiophosphate pesticides (OPPs) are the most common water contaminants, significantly endangering human health and bringing serious public safety issues. Thus, developing effective technologies for the removal or trace detection of OPPs from water is urgent. Herein, a novel graphene-based silica-coated core-shell tubular magnetic nanocomposite (Ni@SiO2-G) was fabricated for the first time and used for the efficient magnetic solid-phase extraction (MSPE) of the OPPs chlorpyrifos, diazinon, and fenitrothion from environmental water. The experimental factors affecting extraction efficiency such as adsorbent dosage, extraction time, desorption solvent, desorption mode, desorption time, and adsorbent type were evaluated. The synthesized Ni@SiO2-G nanocomposites showed a higher preconcentration capacity than the Ni nanotubes, Ni@SiO2 nanotubes, and graphene. Under the optimized conditions, 5 mg of tubular nano-adsorbent displayed good linearity within the range of 0.1-1 µg·mL-1, low limits of detection (0.04-0.25 pg·mL-1), low limits of quantification (0.132-0.834 pg·mL-1), good reusability (n = 5; relative standard deviations between 1.46% and 9.65%), low dosage (5 mg), and low real detection concentration (< 3.0 ng·mL-1). Moreover, the possible interaction mechanism was investigated by density functional theory calculation. Results showed that Ni@SiO2-G was a potential magnetic material for the preconcentration and extraction of formed OPPs at ultra-trace levels from environmental water.

TyG-BMI and hypertension in Normoglycemia subjects in Japan: A cross-sectional study.

BACKGROUND: Insulin Resistance (IR) are associated with Hypertension (HTN). Triglyceride glucose-body mass index (TyG-BMI) is a readily available and clinically significant indicator of IR. This study aimed to investigate whether TyG-BMI is independently associated with HTN. METHODS: A total of 15,464 patients with normal blood glucose from 2004 to 2016 participated in this study. Participants were divided into four groups using the quartile method: TyG-BMI below 153.1, between 153.1 and 174.2, between 174.2 and 199.3, and over 199.3. The covariates included age, sex, BMI, WC, HDL-C, TC, TG, HbA1c, FPG, ALT, AST, GGT, SBP, DBP, smoking status, alcohol consumption, and exercise habits. RESULTS: The average age was 43.7 ± 8.9 years, and 45.4% were men. The prevalence of HTN was 6.2% (964/15464) of the population. TyG-BMI remained significantly associated with HTN after multivariate adjustment for TyG-BMI as a continuous variable (adjusted OR = 2.87, 95% CI: 1.90-4.34). Each 10-unit rise in TyG-BMI (continuous variable) was linked to a 31% increase in the prevalence of HTN (adjusted OR = 1.31, 95% CI: 1.25-1.37). In the subgroup analysis stratified by age, sex, waist circumference, and smoking status, the association between TyG-BMI and HTN were stable. CONCLUSION: In this study, TyG-BMI was highly correlated with HTN, but more experiments and different populations are needed to verify this.

Potent targeted activator of cell kill molecules eliminate cells expressing HIV-1.

Antiretroviral therapy inhibits HIV-1 replication but is not curative due to establishment of a persistent reservoir after virus integration into the host genome. Reservoir reduction is therefore an important HIV-1 cure strategy. Some HIV-1 nonnucleoside reverse transcriptase inhibitors induce HIV-1 selective cytotoxicity in vitro but require concentrations far exceeding approved dosages. Focusing on this secondary activity, we found bifunctional compounds with HIV-1-infected cell kill potency at clinically achievable concentrations. These targeted activator of cell kill (TACK) molecules bind the reverse transcriptase-p66 domain of monomeric Gag-Pol and act as allosteric modulators to accelerate dimerization, resulting in HIV-1+ cell death through premature intracellular viral protease activation. TACK molecules retain potent antiviral activity and selectively eliminate infected CD4+ T cells isolated from people living with HIV-1, supporting an immune-independent clearance strategy.

Genetic polymorphism of clopidogrel metabolism related gene CYP2C19 gene in Chinese from Foshan area of Guangdong Province.

BACKGROUND: The CYP2C19 gene is highly polymorphic, and CYP2C19 is involved in the broad interindividual variability of the clinical efficacy of certain clinical medications, such as clopidogrel. However, data on the CYP2C19 genotype in the Chinese population of the Foshan area of Guangdong Province are scarce. The purpose of this study was to determine CYP2C19 genetic polymorphisms in patients in the Foshan area and to compare the CYP2C19 genotype frequencies in different populations to determine the allele distribution pattern to identify the most appropriate prescription. METHODS: The CYP2C19 gene was detected in 1231 patients on a gene chip platform, and the genotype frequencies of CYP2C19 in Foshan populations from different populations were compared. RESULTS: The frequencies of CYP2C19*1, *2 and *3 in the Foshan population were 63.89%, 30.46% and 5.65%, respectively. For the three metabolic types, the frequency associated with the rapid metabolism type (*1/*1) was 41.51 [95% confidence interval (CI) 40.11 to 42.91%]; that for the intermediate metabolism type (*1/*2, *1/*3) was 44.76% (95% CI 43.34 to 46.18) and that for the slow metabolism type (*2/*2, *2/*3, *3/*3) was 13.73% (95% CI 12.75 to 14.71%). In the Foshan population, the frequencies of the CYP2C19 *2 and *3 alleles were similar to those previously reported for Chinese and other Asian populations. CONCLUSION: Our study is a report on the genetic basis of CYP2C19 polymorphism in the Foshan population. Our results will potentially contribute to the improvement of pharmacotherapy effectiveness by providing personalized medicine for the Foshan population.

Preclinical immunogenicity and efficacy of a candidate COVID-19 vaccine based on a vesicular stomatitis virus-SARS-CoV-2 chimera.

BACKGROUND: To investigate a vaccine technology with potential to protect against coronavirus disease 2019 (COVID-19) and reduce transmission of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) with a single vaccine dose, we developed a SARS-CoV-2 candidate vaccine using the live vesicular stomatitis virus (VSV) chimeric virus approach previously used to develop a licensed Ebola virus vaccine. METHODS: We generated a replication-competent chimeric VSV-SARS-CoV-2 vaccine candidate by replacing the VSV glycoprotein (G) gene with coding sequence for the SARS-CoV-2 Spike glycoprotein (S). Immunogenicity of the lead vaccine candidate (VSV∆G-SARS-CoV-2) was evaluated in cotton rats and golden Syrian hamsters, and protection from SARS-CoV-2 infection also was assessed in hamsters. FINDINGS: VSV∆G-SARS-CoV-2 delivered with a single intramuscular (IM) injection was immunogenic in cotton rats and hamsters and protected hamsters from weight loss following SARS-CoV-2 challenge. When mucosal vaccination was evaluated, cotton rats did not respond to the vaccine, whereas mucosal administration of VSV∆G-SARS-CoV-2 was found to be more immunogenic than IM injection in hamsters and induced immunity that significantly reduced SARS-CoV-2 challenge virus loads in both lung and nasal tissues. INTERPRETATION: VSV∆G-SARS-CoV-2 delivered by IM injection or mucosal administration was immunogenic in golden Syrian hamsters, and both vaccination methods effectively protected the lung from SARS-CoV-2 infection. Hamsters vaccinated by mucosal application of VSV∆G-SARS-CoV-2 also developed immunity that controlled SARS-CoV-2 replication in nasal tissue. FUNDING: The study was funded by Merck Sharp & Dohme, Corp., a subsidiary of Merck & Co., Inc., Rahway, NJ, USA, and The International AIDS Vaccine Initiative, Inc. (IAVI), New York, USA. Parts of this research was supported by the Biomedical Advanced Research and Development Authority (BARDA) and the Defense Threat Reduction Agency (DTRA) of the US Department of Defense.

Detecting Sources of Immune Activation and Viral Rebound in HIV Infection.

Anti-retroviral therapy (ART) generally suppresses HIV replication to undetectable levels in peripheral blood, but immune activation associated with increased morbidity and mortality is sustained during ART, and infection rebounds when treatment is interrupted. To identify drivers of immune activation and potential sources of viral rebound, we modified RNAscope in situ hybridization to visualize HIV-producing cells as a standard against which to compare the following assays of potential sources of immune activation and virus rebound following treatment interruption: (i) envelope detection by induced transcription-based sequencing (EDITS) assay; (ii) HIV-Flow; (iii) Flow-FISH assays that can scan tissues and cell suspensions to detect rare cells expressing env mRNA, gag mRNA/Gag protein and p24; and (iv) an ultrasensitive immunoassay that detects p24 in cell/tissue lysates at subfemtomolar levels. We show that the sensitivities of these assays are sufficient to detect one rare HIV-producing/env mRNA+/p24+ cell in one million uninfected cells. These high-throughput technologies provide contemporary tools to detect and characterize rare cells producing virus and viral antigens as potential sources of immune activation and viral rebound. IMPORTANCE Anti-retroviral therapy (ART) has greatly improved the quality and length of life for people living with HIV, but immune activation does not normalize during ART, and persistent immune activation has been linked to increased morbidity and mortality. We report a comparison of assays of two potential sources of immune activation during ART: rare cells producing HIV and the virus' major viral protein, p24, benchmarked on a cell model of active and latent infections and a method to visualize HIV-producing cells. We show that assays of HIV envelope mRNA (EDITS assay), gag mRNA, and p24 (Flow-FISH, HIV-Flow. and ultrasensitive p24 immunoassay) detect HIV-producing cells and p24 at sensitivities of one infected cell in a million uninfected cells, thereby providing validated tools to explore sources of immune activation during ART in the lymphoid and other tissue reservoirs.

Combined noncanonical NF-κB agonism and targeted BET bromodomain inhibition reverse HIV latency ex vivo.

Latency reversal strategies for HIV cure using inhibitor of apoptosis protein (IAP) antagonists (IAPi) induce unprecedented levels of latent reservoir expression without immunotoxicity during suppressive antiretroviral therapy (ART). However, full targeting of the reservoir may require combinatorial approaches. A Jurkat latency model screen for IAPi combination partners demonstrated synergistic latency reversal with bromodomain (BD) and extraterminal domain protein inhibitors (BETi). Mechanistic investigations using CRISPR-CAS9 and single-cell RNA-Seq informed comprehensive ex vivo evaluations of IAPi plus pan-BET, bD-selective BET, or selective BET isoform targeting in CD4+ T cells from ART-suppressed donors. IAPi+BETi treatment resulted in striking induction of cell-associated HIV gag RNA, but lesser induction of fully elongated and tat-rev RNA compared with T cell activation-positive controls. IAPi+BETi resulted in HIV protein induction in bulk cultures of CD4+ T cells using an ultrasensitive p24 assay, but did not result in enhanced viral outgrowth frequency using a standard quantitative viral outgrowth assay. This study defines HIV transcriptional elongation and splicing as important barriers to latent HIV protein expression following latency reversal, delineates the roles of BET proteins and their BDs in HIV latency, and provides a rationale for exploration of IAPi+BETi in animal models of HIV latency.

Interleukin-10 contributes to reservoir establishment and persistence in SIV-infected macaques treated with antiretroviral therapy.

Interleukin-10 (IL-10) is an immunosuppressive cytokine that signals through STAT3 to regulate T follicular helper (Tfh) cell differentiation and germinal center formation. In SIV-infected macaques, levels of IL-10 in plasma and lymph nodes (LNs) were induced by infection and not normalized with antiretroviral therapy (ART). During chronic infection, plasma IL-10 and transcriptomic signatures of IL-10 signaling were correlated with the cell-associated SIV-DNA content within LN CD4+ memory subsets, including Tfh cells, and predicted the frequency of CD4+ Tfh cells and their cell-associated SIV-DNA content during ART, respectively. In ART-treated rhesus macaques, cells harboring SIV-DNA by DNAscope were preferentially found in the LN B cell follicle in proximity to IL-10. Finally, we demonstrated that the in vivo neutralization of soluble IL-10 in ART-treated, SIV-infected macaques reduced B cell follicle maintenance and, by extension, LN memory CD4+ T cells, including Tfh cells and those expressing PD-1 and CTLA-4. Thus, these data support a role for IL-10 in maintaining a pool of target cells in lymphoid tissue that serve as a niche for viral persistence. Targeting IL-10 signaling to impair CD4+ T cell survival and improve antiviral immune responses may represent a novel approach to limit viral persistence in ART-suppressed people living with HIV.

Impact of Tamoxifen on Vorinostat-Induced Human Immunodeficiency Virus Expression in Women on Antiretroviral Therapy: AIDS Clinical Trials Group A5366, The MOXIE Trial.

BACKGROUND: Biological sex and the estrogen receptor alpha (ESR1) modulate human immunodeficiency virus (HIV) activity. Few women have enrolled in clinical trials of latency reversal agents (LRAs); their effectiveness in women is unknown. We hypothesized that ESR1 antagonism would augment induction of HIV expression by the LRA vorinostat. METHODS: AIDS Clinical Trials Group A5366 enrolled 31 virologically suppressed, postmenopausal women on antiretroviral therapy. Participants were randomized 2:1 to receive tamoxifen (arm A, TAMOX/VOR) or observation (arm B, VOR) for 5 weeks followed by 2 doses of vorinostat. Primary end points were safety and the difference between arms in HIV RNA induction after vorinostat. Secondary analyses included histone 4 acetylation, HIV DNA, and plasma viremia by single copy assay (SCA). RESULTS: No significant adverse events were attributed to study treatments. Tamoxifen did not enhance vorinostat-induced HIV transcription (between-arm ratio, 0.8; 95% confidence interval [CI], .2-2.4). Vorinostat-induced HIV transcription was higher in participants with increases in H4Ac (fold increase, 2.78; 95% CI, 1.34-5.79) vs those 9 who did not (fold increase, 1.04; 95% CI, .25-4.29). HIV DNA and SCA plasma viremia did not substantially change. CONCLUSIONS: Tamoxifen did not augment vorinostat-induced HIV RNA expression in postmenopausal women. The modest latency reversal activity of vorinostat, postmenopausal status, and low level of HIV RNA expression near the limits of quantification limited assessment of the impact of tamoxifen. This study is the first HIV cure trial done exclusively in women and establishes both the feasibility and necessity of investigating novel HIV cure strategies in women living with HIV. CLINICAL TRIALS REGISTRATION: NCT03382834.

Neurotoxicity with high-dose disulfiram and vorinostat used for HIV latency reversal.

OBJECTIVE: The aim of this study was to examine whether administering both vorinostat and disulfiram to people with HIV (PWH) on antiretroviral therapy (ART) is well tolerated and can enhance HIV latency reversal. DESIGN: Vorinostat and disulfiram can increase HIV transcription in PWH on ART. Together, these agents may lead to significant HIV latency reversal. METHODS: Virologically suppressed PWH on ART received disulfiram 2000 mg daily for 28 days and vorinostat 400 mg daily on days 8-10 and 22-24. The primary endpoint was plasma HIV RNA on day 11 relative to baseline using a single copy assay. Assessments included cell-associated unspliced RNA as a marker of latency reversal, HIV DNA in CD4+ T-cells, plasma HIV RNA, and plasma concentrations of ART, vorinostat, and disulfiram. RESULTS: The first two participants (P1 and P2) experienced grade 3 neurotoxicity leading to trial suspension. After 24 days, P1 presented with confusion, lethargy, and ataxia having stopped disulfiram and ART. Symptoms resolved by day 29. After 11 days, P2 presented with paranoia, emotional lability, lethargy, ataxia, and study drugs were ceased. Symptoms resolved by day 23. CA-US RNA increased by 1.4-fold and 1.3-fold for P1 and P2 respectively. Plasma HIV RNA was detectable from day 8 to 37 (peak 81 copies ml-1) for P2 but was not increased in P1 Antiretroviral levels were therapeutic and neuronal injury markers were elevated in P1. CONCLUSION: The combination of prolonged high-dose disulfiram and vorinostat was not safe in PWH on ART and should not be pursued despite evidence of latency reversal.

Discovery of macrocyclic HDACs 1, 2, and 3 selective inhibitors for HIV latency reactivation.

A series of unique macrocyclic HDACs 1, 2, and 3 selective inhibitors were identified with good enzymatic activity and high selectivity over HDACs 6 and 8. These macrocyclic HDAC inhibitors used an ethyl ketone as the zinc-binding group. Compounds 25 and 26 stood out as leads due to their low double-digit nM EC50s in the 2C4 cell-based HIV latency reactivation assay. The PK profiles of these macrocyclic HDAC inhibitors still needed improvement.

Improved Detection of HIV Gag p24 Protein Using a Combined Immunoprecipitation and Digital ELISA Method.

Greater than 90% of HIV-1 proviruses are thought to be defective and incapable of viral replication. While replication competent proviruses are of primary concern with respect to disease progression or transmission, studies have shown that even defective proviruses are not silent and can produce viral proteins, which may contribute to inflammation and immune responses. Viral protein expression also has implications for immune-based HIV-1 clearance strategies, which rely on antigen recognition. Thus, sensitive assays aimed at quantifying both replication-competent proviruses and defective, yet translationally competent proviruses are needed to understand the contribution of viral protein to HIV-1 pathogenesis and determine the effectiveness of HIV-1 cure interventions. Previously, we reported a modified HIV-1 gag p24 digital enzyme-linked immunosorbent assay with single molecule array (Simoa) detection of cell-associated viral protein. Here we report a novel p24 protein enrichment method coupled with the digital immunoassay to further extend the sensitivity and specificity of viral protein detection. Immunocapture of HIV gag p24 followed by elution in a Simoa-compatible format resulted in higher protein recovery and lower background from various biological matrices and sample volumes. Quantification of as little as 1 fg of p24 protein from cell lysates from cells isolated from peripheral blood or tissues from ART-suppressed HIV participants, as well as simian-human immunodeficiency virus-infected non-human primates (NHPs), with high recovery and reproducibility is demonstrated here. The application of these enhanced methods to patient-derived samples has potential to further the study of the persistent HIV state and examine in vitro response to therapies, as well as ex vivo study of translationally competent cells from a variety of donors.

Discovery of Ethyl Ketone-Based Highly Selective HDACs 1, 2, 3 Inhibitors for HIV Latency Reactivation with Minimum Cellular Potency Serum Shift and Reduced hERG Activity.

We describe the discovery of histone deacetylase (HDACs) 1, 2, and 3 inhibitors with ethyl ketone as the zinc-binding group. These HDACs 1, 2, and 3 inhibitors have good enzymatic and cellular activity. Their serum shift in cellular potency has been minimized, and selectivity against hERG has been improved. They are also highly selective over HDACs 6 and 8. These inhibitors contain a variety of substituted heterocycles on the imidazole or oxazole scaffold. Compounds 31 and 48 stand out due to their good potency, high selectivity over HDACs 6 and 8, reduced hERG activity, optimized serum shift in cellular potency, and good rat and dog PK profiles.

Gag p24 Is a Marker of Human Immunodeficiency Virus Expression in Tissues and Correlates With Immune Response.

We demonstrate that human immunodeficiency virus (HIV) gag p24 protein is more readily detected in gut and lymph node tissues than in blood CD4+ T cells and correlates better with CD4 count during antiretroviral therapy (ART). Gut p24 levels also measurably decline with ART in natural controllers. During ART, gut p24 expression is more strongly associated both with HIV-specific CD8+ T-cell frequency and plasma soluble CD14 levels than gut HIV RNA expression. This study supports using gag p24 as a marker of HIV expression in HIV+ tissues to study effects of viral persistence and to monitor efficacy of treatment in HIV-based clearance studies.