Histone deacetylase inhibitor romidepsin induces HIV expression in CD4 T cells from patients on suppressive antiretroviral therapy at concentrations achieved by clinical dosing.

Persistent latent reservoir of replication-competent proviruses in memory CD4 T cells is a major obstacle to curing HIV infection. Pharmacological activation of HIV expression in latently infected cells is being explored as one of the strategies to deplete the latent HIV reservoir. In this study, we characterized the ability of romidepsin (RMD), a histone deacetylase inhibitor approved for the treatment of T-cell lymphomas, to activate the expression of latent HIV. In an in vitro T-cell model of HIV latency, RMD was the most potent inducer of HIV (EC50 = 4.5 nM) compared with vorinostat (VOR; EC50 = 3,950 nM) and other histone deacetylase (HDAC) inhibitors in clinical development including panobinostat (PNB; EC50 = 10 nM). The HIV induction potencies of RMD, VOR, and PNB paralleled their inhibitory activities against multiple human HDAC isoenzymes. In both resting and memory CD4 T cells isolated from HIV-infected patients on suppressive combination antiretroviral therapy (cART), a 4-hour exposure to 40 nM RMD induced a mean 6-fold increase in intracellular HIV RNA levels, whereas a 24-hour treatment with 1 µM VOR resulted in 2- to 3-fold increases. RMD-induced intracellular HIV RNA expression persisted for 48 hours and correlated with sustained inhibition of cell-associated HDAC activity. By comparison, the induction of HIV RNA by VOR and PNB was transient and diminished after 24 hours. RMD also increased levels of extracellular HIV RNA and virions from both memory and resting CD4 T-cell cultures. The activation of HIV expression was observed at RMD concentrations below the drug plasma levels achieved by doses used in patients treated for T-cell lymphomas. In conclusion, RMD induces HIV expression ex vivo at concentrations that can be achieved clinically, indicating that the drug may reactivate latent HIV in patients on suppressive cART.

Week 144 resistance analysis of elvitegravir/cobicistat/emtricitabine/tenofovir DF versus atazanavir+ritonavir+emtricitabine/tenofovir DF in antiretroviral-naïve patients.

OBJECTIVE: To describe baseline and emergent HIV-1 resistance to elvitegravir/ cobicistat/emtricitabine/tenofovir DF (EVG/COBI/FTC/TDF) and ritonavir-boosted atazanavir/emtricitabine/tenofovir DF (ATV+RTV+FTC/TDF) in HIV-1-infected, treatment-naïve subjects through 144 weeks. METHOD: This was a randomized, double-blind, phase 3 study. HIV-1 protease (PR) and reverse transcriptase (RT) were sequenced at screening. Genotypic and phenotypic analyses were performed at virologic failure confirmation and retrospectively at baseline for PR, RT, and integrase (IN) for patients with virologic failure through week 144. RESULTS: In the EVG/ COBI/FTC/TDF group through week 144, HIV-1 from 8 patients (2.3%; 8/353 treated patients) developed primary IN strand transfer inhibitor (INSTI) (n = 6) and/or nucleoside RT inhibitor (NRTI) resistance substitutions (n = 7). The emergence of resistance decreased after the first year, with 5 patients developing HIV-1 resistance through week 48, 1 from weeks 48-96, and 2 from weeks 96-144. Emergent substitutions were E92Q, N155H, or Q148R (n = 2 each) and T66I or T97A (n = 1 each) in IN and M184V/I (n = 7) and K65R (n = 1) in RT. All 8 isolates had reduced susceptibility to EVG, FTC, or TDF. Virus with EVG phenotypic resistance showed cross-resistance to raltegravir. In the ATV+RTV+FTC/TDF group, HIV-1 from 2 patients (0.6%; 2/355 treated patients; both at week 144) developed the resistance substitution M184V/I in RT. CONCLUSIONS: Resistance development to EVG/COBI/FTC/TDF was infrequent (2.3%) through 144 weeks of therapy and decreased over time, consistent with durable efficacy.

Study of marine natural products including resorcyclic acid lactones from Humicola fuscoatra that reactivate latent HIV-1 expression in an in vitro model of central memory CD4+ T cells.

An extract of Humicola fuscoatra (UCSC strain no. 108111A) was shown to reactivate latent HIV-1 expression in an in vitro model of central memory CD4+ T cells. We report the bioassay-guided isolation and structure determination of several resorcyclic acid lactones, including four known compounds, radicicol (1, aka. monorden) and pochonins B (2), C (3), and N (4), and three new analogues, radicicols B-D (5-7). Compounds 1-3 and 5 showed moderate activities in the memory T cell model of HIV-1 latency. Radicicol (1) displayed lower potency in reactivating latent HIV-1 (EC50 = 9.1 µM) relative to the HDAC inhibitors apicidin (EC50 = 0.3 µM), romidepsin (EC50 = 0.003 µM), and SAHA (EC50 = 0.6 µM); however, it achieved equivalent maximum efficacy relative to the positive control compounds (98% of SAHA and romidepsin).

Impact of primary elvitegravir resistance-associated mutations in HIV-1 integrase on drug susceptibility and viral replication fitness.

Elvitegravir (EVG) is an effective HIV-1 integrase (IN) strand transfer inhibitor (INSTI) in advanced clinical development. Primary INSTI resistance-associated mutations (RAMs) at six IN positions have been identified in HIV-1-infected patients failing EVG-containing regimens in clinical studies: T66I/A/K, E92Q/G, T97A, S147G, Q148R/H/K, and N155H. In this study, the effect of these primary IN mutations, alone and in combination, on susceptibility to the INSTIs EVG, raltegravir (RAL), and dolutegravir (DTG); IN enzyme activities; and viral replication fitness was characterized. Recombinant viruses containing the six most common mutations exhibited a range of reduced EVG susceptibility: 92-fold for Q148R, 30-fold for N155H, 26-fold for E92Q, 10-fold for T66I, 4-fold for S147G, and 2-fold for T97A. Less commonly observed primary IN mutations also showed a range of reduced EVG susceptibilities: 40- to 94-fold for T66K and Q148K and 5- to 10-fold for T66A, E92G, and Q148H. Some primary IN mutations exhibited broad cross-resistance between EVG and RAL (T66K, E92Q, Q148R/H/K, and N155H), while others retained susceptibility to RAL (T66I/A, E92G, T97A, and S147G). Dual combinations of primary IN mutations further reduced INSTI susceptibility, replication capacity, and viral fitness relative to either mutation alone. Susceptibility to DTG was retained by single primary IN mutations but reduced by dual mutation combinations with Q148R. Primary EVG RAMs also diminished IN enzymatic activities, concordant with their structural proximity to the active site. Greater reductions in viral fitness of dual mutation combinations may explain why some primary INSTI RAMs do not readily coexist on the same HIV-1 genome but rather establish independent pathways of resistance to EVG.

Enhancing a student productivity model for adaptive problem-solving assistance.

Research on intelligent tutoring systems has been exploring data-driven methods to deliver effective adaptive assistance. While much work has been done to provide adaptive assistance when students seek help, they may not seek help optimally. This had led to the growing interest in proactive adaptive assistance, where the tutor provides unsolicited assistance upon predictions of struggle or unproductivity. Determining when and whether to provide personalized support is a well-known challenge called the assistance dilemma. Addressing this dilemma is particularly challenging in open-ended domains, where there can be several ways to solve problems. Researchers have explored methods to determine when to proactively help students, but few of these methods have taken prior hint usage into account. In this paper, we present a novel data-driven approach to incorporate students' hint usage in predicting their need for help. We explore its impact in an intelligent tutor that deals with the open-ended and well-structured domain of logic proofs. We present a controlled study to investigate the impact of an adaptive hint policy based on predictions of HelpNeed that incorporate students' hint usage. We show empirical evidence to support that such a policy can save students a significant amount of time in training and lead to improved posttest results, when compared to a control without proactive interventions. We also show that incorporating students' hint usage significantly improves the adaptive hint policy's efficacy in predicting students' HelpNeed, thereby reducing training unproductivity, reducing possible help avoidance, and increasing possible help appropriateness (a higher chance of receiving help when it was likely to be needed). We conclude with suggestions on the domains that can benefit from this approach as well as the requirements for adoption.

Building a Virtual Community of Practice: Teacher Learning for Computational Thinking Infusion.

The COVID-19 pandemic led to an urgent need for professional development (PD) experiences to support teacher learning across hybrid and digital contexts. This study investigates teachers' experiences in a Virtual Pivot, a PD workshop designed to support computational thinking integration into disciplinary teaching. Participants were 151 middle and high school content area teachers, including 49 teachers who participated in previous face-to-face workshops. Virtual Pivot employed research-based design principles for virtual teacher PD, including asynchronous and synchronous engagement, explicit instruction in technological tools and scaffolds for teacher collaboration. Data sources included pre-PD surveys (n = 151), post-PD surveys (n = 119), interviews (n = 57) and six-month follow-up surveys (n = 105). Findings describe elements of Virtual Pivot which supported teacher learning and engagement (virtual community of practice, PD structure, during-PD support, pre-PD support and badges). We conclude by discussing this study's theoretical, methodological and practical contributions for designing and investigating virtual computational thinking PD experiences.

TLR7 agonist administration to SIV-infected macaques receiving early initiated cART does not induce plasma viremia.

Reduction/elimination of HIV-1 reservoirs that persist despite combination antiretroviral therapy (cART) will likely require induction of viral expression by residual infected cells and enhanced clearance of these cells. TLR7 agonists have potential to mediate these activities. We evaluated immunologic and virologic effects of repeated doses of the TLR7 agonist GS-9620 in SIV-infected rhesus macaques receiving cART, which was initiated at 13 days after infection and was continued for 75 weeks prior to GS-9620 administration. During cART, GS-9620 induced transient upregulation of IFN-stimulated genes in blood and tissues, increases in plasma cytokines, and changes in immune cell population activation and phenotypes but did not result in measurable increases in plasma viremia or viral RNA-to-viral DNA ratio in PBMCs or tissues nor decreases in viral DNA in PBMC or tissues. SIV-specific CD8+ T cell responses, negligible prior to GS-9620 treatment, were not measurably boosted by treatment; a second course of GS-9620 administration overlapping with later cART discontinuation was associated with increased CD8+ T cell responses during viral recrudescence. These results confirm and extend evidence for GS-9620-mediated enhancement of antiviral immune responses in SIV-infected macaques but suggest that GS-9620-mediated viral induction may depend critically on the timing of initiation and duration of cART and resulting characteristics of viral reservoirs.

TLR7 agonists induce transient viremia and reduce the viral reservoir in SIV-infected rhesus macaques on antiretroviral therapy.

Antiretroviral therapy (ART) can halt HIV-1 replication but fails to target the long-lived latent viral reservoir. Several pharmacological compounds have been evaluated for their ability to reverse HIV-1 latency, but none has demonstrably reduced the latent HIV-1 reservoir or affected viral rebound after the interruption of ART. We evaluated orally administered selective Toll-like receptor 7 (TLR7) agonists GS-986 and GS-9620 for their ability to induce transient viremia in rhesus macaques infected with simian immunodeficiency virus (SIV) and treated with suppressive ART. In an initial dose-escalation study, and a subsequent dose-optimization study, we found that TLR7 agonists activated multiple innate and adaptive immune cell populations in addition to inducing expression of SIV RNA. We also observed TLR7 agonist-induced reductions in SIV DNA and measured inducible virus from treated animals in ex vivo cell cultures. In a second study, after stopping ART, two of nine treated animals remained aviremic for more than 2 years, even after in vivo CD8+ T cell depletion. Moreover, adoptive transfer of cells from aviremic animals could not induce de novo infection in naïve recipient macaques. These findings suggest that TLR7 agonists may facilitate reduction of the viral reservoir in a subset of SIV-infected rhesus macaques.

Lack of impact of pre-existing T97A HIV-1 integrase mutation on integrase strand transfer inhibitor resistance and treatment outcome.

T97A is an HIV-1 integrase polymorphism associated with integrase strand transfer inhibitor (INSTI) resistance. Using pooled data from 16 clinical studies, we investigated the prevalence of T97A (pre-existing and emergent) and its impact on INSTI susceptibility and treatment response in INSTI-naive patients who enrolled on elvitegravir (EVG)- or raltegravir (RAL)-based regimens. Prior to INSTI-based therapy, primary INSTI resistance-associated mutations (RAMs) were absent and T97A pre-existed infrequently (1.4%; 47 of 3367 integrase sequences); most often among non-B (5.3%) than B (0.9%) HIV-1 subtypes. During INSTI-based therapy, few patients experienced virologic failure with emergent INSTI RAMs (3%; 122 of 3881 patients), among whom T97A emerged infrequently in the presence (n = 6) or absence (n = 8) of primary INSTI RAMs. A comparison between pre-existing and emergent T97A patient populations (i.e., in the absence of primary INSTI RAMs) showed no significant differences in EVG or RAL susceptibility in vitro. Furthermore, among all T97A-containing viruses tested, only 38-44% exhibited reduced susceptibility to EVG and/or RAL (all of low magnitude; <11-fold), while all maintained susceptibility to dolutegravir. Of the patients with pre-existing T97A, 17 had available clinical follow-up: 16 achieved virologic suppression and 1 maintained T97A and INSTI sensitivity without further resistance development. Overall, T97A is an infrequent integrase polymorphism that is enriched among non-B HIV-1 subtypes and can confer low-level reduced susceptibility to EVG and/or RAL. However, detection of T97A does not affect response to INSTI-based therapy with EVG or RAL. These results suggest a very low risk of initiating INSTI-based therapy in patients with pre-existing T97A.

The Use of Chick Embryos to Study Wnt Activity Gradients.

The chick spinal cord provides a valuable model for assessing Wnt signaling activity. Loss or gain of function constructs that are transfected by electroporation can be directed to a single side of the spinal cord, thus leaving the contralateral side as an internal control. Here, we describe a method for measuring Wnt signaling via the use of BAT-Gal, a ß-catenin dependent Wnt reporter.

Abyssomicin 2 reactivates latent HIV-1 by a PKC- and HDAC-independent mechanism.

Screening of a marine natural products library afforded three new analogues of the tetronic acid containing polyketide abyssomicin family and identified abyssomicin 2 as a selective reactivator of latent HIV virus. Examination of the mode of action of this new latent HIV reactivating agent demonstrated that it functions via a distinct mechanism compared to that of existing reactivating agents and is effective at reactivating latent virus in a subset of primary patient cell lines.

RNase H active site inhibitors of human immunodeficiency virus type 1 reverse transcriptase: design, biochemical activity, and structural information.

Pyrimidinol carboxylic acids were designed as inhibitors of HIV-1 RNase H function. These molecules can coordinate to two divalent metal ions in the RNase H active site. Inhibition of enzymatic activity was measured in a biochemical assay, but no antiviral effect was observed. Binding was demonstrated via a solid state structure of the isolated p15-Ec domain of HIV-1 RT showing inhibitor and two Mn(II) ions bound to the RNase H active site.

Identification and characterization of subpopulations of Pax3 and Pax7 expressing cells in developing chick somites and limb buds.

Pax3 and Pax7 are closely related paired-boxed family transcription factors that are known to play important roles in embryonic and adult myogenesis. Previous reports describing the expression of Pax3 and Pax7 transcripts reveal expression in many overlapping domains. In this manuscript, we extend these studies by examining the protein expression profiles for Pax3 and Pax7 in developing chick somites and limbs with cellular resolution. Our studies show the existence of distinct subpopulations of cells in the somite and developing limb that are defined by the relative expression levels of Pax3 and Pax7. We also show that Pax3 and Pax7 negatively regulate each other's expression in the dermomyotome, thus providing a possible mechanism for the maintenance of observed expression patterns in the dermomyotome. Further characterization of Pax3- and/or Pax7-positive cells in the dermomyotome and myotome with respect to proliferation and differentiation reveals subpopulations of cells with distinct properties.

Porcupine-mediated lipid-modification regulates the activity and distribution of Wnt proteins in the chick neural tube.

A long-term goal of developmental biology is to understand how morphogens establish gradients that promote proper tissue patterning. A number of reports describe the formation of the Wg (Wnt1) gradient in Drosophila and have shown that Porcupine, a predicted membrane-bound O-acyl transferase, is required for the correct distribution of Wg protein. The discovery that Wnts are palmitoylated on a conserved cysteine residue suggests that porcupine activity and Wnt palmitoylation are important for the generation of Wnt gradients. To establish the role of porcupine in Wnt gradient formation in vertebrates, we tested the role of porcupine/Wnt palmitoylation in human embryonic kidney 293T cells and in the chick neural tube. Our results lead us to conclude that: (1) vertebrate Wnt1 and Wnt3a possess at least one additional site for porcupine-mediated lipid-modification; (2) porcupine-mediated lipid-modification of Wnt proteins promotes their activity in 293T cells and in the chick neural tube; and (3) porcupine-mediated lipid-modification reduces the range of activity of Wnt1 and Wnt3a in the chick neural tube. These findings highlight the importance of porcupine-mediated lipid modifications in the formation of vertebrate Wnt activity gradients.

Differential inhibition of Wnt-3a by Sfrp-1, Sfrp-2, and Sfrp-3.

Secreted frizzled related proteins (Sfrps) are extracellular attenuators of Wnt signaling that play important roles in both embryogenesis and oncogenesis. Although Sfrps are generally thought to bind and sequester Wnts away from active receptor complexes, very little is known about the specificity of Sfrp family members for various Wnts. In the developing chick neural tube, sfrp-1, 2, and 3 transcripts are expressed in and adjacent to the dorsal neural tube, where Wnt-1 and Wnt-3a are expressed. To better define the possible roles of Sfrp-1, 2, and 3 in the neural tube, we first tested the ability of purified Sfrps to inhibit Wnt-3a-induced accumulation of beta-catenin in L cells. We find that both Sfrp-1 and Sfrp-2 can inhibit Wnt-3a activity while Sfrp-3 cannot. To determine where Sfrp-1 and Sfrp-2 impinge on the Wnt signaling pathway, we tested the ability of these Sfrps to inhibit Wnt signaling induced by the addition of LiCl, an inhibitor of GSK-3. Sfrp-1 and Sfrp-2 are unable to inhibit the accumulation of beta-catenin in LiCl-treated cells, suggesting that the ability of Sfrps to inhibit the accumulation of beta-catenin is GSK-3 dependent. We have further shown that Sfrp-2 inhibits the ability of ectopic Wnt-3a to stimulate proliferation in the developing chick neural tube. These results provide the framework for understanding how Sfrps function to regulate Wnt-3a activity in developing embryos and in cancer.