Scopoletin Reactivates Latent HIV-1 by Inducing NF-κB Expression without Global T Cell Activation.

Reversing HIV-1 latency promotes the killing of infected cells and is essential for cure strategies. However, current latency-reversing agents (LRAs) are not entirely effective and safe in activating latent viruses in patients. In this study, we investigated whether Scopoletin (6-Methoxy-7-hydroxycoumarin), an important coumarin phytoalexin found in plants with multiple pharmacological activities, can reactivate HIV-1 latency and elucidated its underlying mechanism. Using the Jurkat T cell model of HIV-1 latency, we found that Scopoletin can reactivate latent HIV-1 replication with a similar potency to Prostratin and did so in a dose- and time-dependent manner. Moreover, we provide evidence indicating that Scopoletin-induced HIV-1 reactivation involves the nuclear factor kappa B (NF-κB) signaling pathway. Importantly, Scopoletin did not have a stimulatory effect on T lymphocyte receptors or HIV-1 receptors. In conclusion, our study suggests that Scopoletin has the potential to reactivate latent HIV-1 without causing global T-cell activation, making it a promising treatment option for anti-HIV-1 latency strategies.

Allogeneic gene-edited HIV-specific CAR-T cells secreting PD-1 blocking scFv enhance specific cytotoxic activity against HIV Env+ cells invivo.

HIV-specific chimeric antigen receptor (CAR) T-cells have been developed to target HIV-1 infected CD4+ T-cells that express HIV Env proteins. However, T cell exhaustion and the patient-specific autologous paradigm of CAR-T cell hurdled clinical applications. Here, we created HIV-specific CAR-T cells using human peripheral blood mononuclear cells and a 3BNC117-E27 (3BE) CAR construct that enabled the expression of programmed cell death protein (PD-1) -blocking scFv E27 and the single-chain variable fragment of the HIV-1-specific broadly neutralizing antibody 3BNC117 to target native HIV Env. Compared with T cells expressing 3BNC117-CAR alone, 3BE CAR-T cells showed greater cytotoxic activity against HIV Env+ cells with stronger proliferation capability, higher killing efficiency, and enhanced cytokine secretion in the presence of HIV Env-expressing cells. Furthermore, we manufactured TCR-deficient 3BE CAR-T cells through gene editing and demonstrated that these CAR-T cells could effectively kill HIV Env â€‹+ â€‹cells in vivo without the occurrence of severe graft-versus-host disease (GvHD) in NSG mice. These data suggest that we have provided a feasible approach to the generation of "off-the-shelf" anti-HIV CAR-T cells in combination with PD-1 checkpoint blockade immunotherapy, which can be a powerful therapeutic candidate for the functional cure of HIV.

HIV-1-Specific CAR-T Cells With Cell-Intrinsic PD-1 Checkpoint Blockade Enhance Anti-HIV Efficacy in vivo.

Adoptive cellular immunotherapy therapy using broadly neutralizing antibody-based chimeric antigen receptor-T cells (bNAb-based CAR-T) has shown great potency and safety for the functional cure of HIV. The efficacy of bNAb-based CAR-T cells could be compromised by adaptive resistance during HIV chronic infection according to the phenomenon that cellular exhaustion was observed in endogenous cytotoxic T-lymphocytes (CTLs) along with upregulated expression of PD-1. Here, we created HIV-specific CAR-T cells using human peripheral blood mononuclear cells (PBMCs) and a 3BNC117-DNR CAR (3BD CAR) construct that enables the expression of PD-1 dominant negative receptor (DNR) and the single-chain variable fragment of the HIV-1-specific broadly neutralizing antibody 3BNC117 to target native HIV envelope glycoprotein (Env). Compared with HIV CAR expression alone, 3BD CAR-T cells displayed potent lytic and functional responses to Env-expressing cell lines and HIV-infected CD4+ T cells. Moreover, 3BD CAR-T cells can kill HIV-latently-infected cell lines, which are reactivated by the secretory cytokines of effector cells followed by contact with initial HIV-expressing fraction. Furthermore, bioluminescence imaging indicated that 3BD CAR-T cells displayed superior anti-HIV function in an HIV NCG mouse model of transplanting Env+/PD-L1+ cells (LEL6). These studies suggested that our proposed combinational strategy of HIV CAR-T therapy with PD-1 blockade therapy is feasible and potent, making it a promising therapeutic candidate for HIV functional cure.

FKBP3 Induces Human Immunodeficiency Virus Type 1 Latency by Recruiting Histone Deacetylase 1/2 to the Viral Long Terminal Repeat.

Human immunodeficiency virus type 1 (HIV-1) cannot be completely eliminated because of existence of the latent HIV-1 reservoir. However, the facts of HIV-1 latency, including its establishment and maintenance, are incomplete. FKBP3, encoded by the FKBP3 gene, belongs to the immunophilin family of proteins and is involved in immunoregulation and such cellular processes as protein folding. In a previous study, we found that FKBP3 may be related to HIV-1 latency using CRISPR screening. In this study, we knocked out the FKBP3 gene in multiple latently infected cell lines to promote latent HIV-1 activation. We found that FKBP3 could indirectly bind to the HIV-1 long terminal repeat through interaction with YY1, thereby recruiting histone deacetylase 1/2 to it. This promotes histone deacetylation and induces HIV-1 latency. Finally, in a primary latent cell model, we confirmed the effect of FKBP3 knockout on the latent activation of HIV-1. Our results suggest a new mechanism for the epigenetic regulation of HIV-1 latency and a new potential target for activating latent HIV-1. IMPORTANCE The primary reason why AIDS cannot be completely cured is the existence of a latent HIV-1 reservoir. Currently, the facts of HIV-1 latency, including its establishment and maintenance, are incomplete. Using a CRISPR library in our earlier screening of genes related to HIV-1 latency, we identified FBKP3 as a candidate gene related to HIV-1 latency. Therefore, in this mechanistic study, we first confirmed the HIV-1 latency-promoting effect of FKBP3 and determined that FKBP3 promotes histone deacetylation by recruiting histone deacetylase 1/2 to the HIV-1 long terminal repeat. We also confirmed, for the first time, that FKBP3 can act as a transcription factor (TF) recruitment scaffold and participate in epigenetic regulation of HIV-1 latency. These findings suggest a new mechanism for the epigenetic regulation of HIV-1 latency and a new potential target for activating latent HIV-1.

Chemokine Receptor CCR2b Enhanced Anti-tumor Function of Chimeric Antigen Receptor T Cells Targeting Mesothelin in a Non-small-cell Lung Carcinoma Model.

Chimeric antigen receptor (CAR) T cell therapy faces a number of challenges for the treatment of non-small-cell lung carcinoma (NSCLC), and efficient migration of circulating CAR T cells plays an important role in anti-tumor activity. In this study, a CAR specific for tumor antigen mesothelin (Msln-CAR) was co-expressed with cell chemokine receptors CCR2b or CCR4. Findings showed that CCR2b and CCR4 enhanced the migration of Msln-CAR T cell in vitro by transwell assay. When incubated with mesothelin-positive tumor cells, Msln-CCR2b-CAR and Msln-CCR4-CAR T cell specifically exerted potent cytotoxicity and produced high levels of proinflammatory cytokines, including IL-2, IFN-γ, and TNF-α. Furthermore, NSCLC cell line-derived xenograft (CDX) model was constructed by implanting subcutaneously modified A549 into NSG mice. Compared to conventional Msln-CAR T cells, living imaging indicated that Msln-CCR2b-CAR T cells displayed superior anti-tumor function due to enhanced migration and infiltration into tumor tissue shown by immunohistochemistry (IHC) analysis. In addition, histopathological examinations of mice organs showed that no obvious organic damages were observed. This is the first time that CAR T cell therapy combined with chemokine receptor is applied to NSCLC treatment.

PEBP1 suppresses HIV transcription and induces latency by inactivating MAPK/NF-κB signaling.

The latent HIV-1 reservoir is a major barrier to viral eradication. However, our understanding of how HIV-1 establishes latency is incomplete. Here, by performing a genome-wide CRISPR-Cas9 knockout library screen, we identify phosphatidylethanolamine-binding protein 1 (PEBP1), also known as Raf kinase inhibitor protein (RKIP), as a novel gene inducing HIV latency. Depletion of PEBP1 leads to the reactivation of HIV-1 in multiple models of latency. Mechanistically, PEBP1 de-phosphorylates Raf1/ERK/IκB and IKK/IκB signaling pathways to sequestrate NF-κB in the cytoplasm, which transcriptionally inactivates HIV-1 to induce latency. Importantly, the induction of PEBP1 expression by the green tea compound epigallocatechin-3-gallate (EGCG) prevents latency reversal by inhibiting nuclear translocation of NF-κB, thereby suppressing HIV-1 transcription in primary CD4+ T cells isolated from patients receiving antiretroviral therapy (ART). These results suggest a critical role for PEBP1 in the regulation of upstream NF-κB signaling pathways governing HIV transcription. Targeting of this pathway could be an option to control HIV reservoirs in patients in the future.

Dual effects of the novel ingenol derivatives on the acute and latent HIV-1 infections.

The latent reservoir of HIV-1 in resting memory CD4+ T cells serves as a major barrier to curing HIV-1 infection. Reactivation of latent HIV-1 is proposed as a promising strategy for the clearance of the viral reservoirs. Because of the limitations of current latency reversal agents (LRAs), identification of new LRAs is urgently required. Here, we analyzed Euphorbia kansui extracts and obtained three ingenol derivative compounds named EK-1A, EK-5A and EK-15A. We found that ingenol derivatives can effectively reactivate latent HIV-1 at very low (nanomolar) concentrations in HIV latency model in vitro. Furthermore, ingenol derivatives exhibited synergy with other LRAs in reactivating latent HIV-1. We verified that EK-15A can promote latent HIV-1 reactivation in the ex vivo resting CD4+ T cells isolated from the peripheral blood of HIV-infected individuals on suppressive antiretroviral therapy. In addition, ingenol derivatives down-regulated the expression of cell surface HIV co-receptors CCR5 and CXCR4, therefore potentially preventing new infection of HIV-1. Our results indicated that the ingenol derivatives extracted from Euphorbia kansui have dual functions: reactivation of latent HIV-1 and inhibition of HIV-1 infection.

Zinc-Finger Nucleases Induced by HIV-1 Tat Excise HIV-1 from the Host Genome in Infected and Latently Infected Cells.

Highly active anti-retroviral therapy (HAART) cannot clear infected cells harboring HIV-1 proviral DNA from HIV-1-infected patients. We previously demonstrated that zinc-finger nucleases (ZFNs) can specifically and efficiently excise HIV-1 proviral DNA from latently infected human T cells by targeting long terminal repeats (LTRs), a novel and alternative antiretroviral strategy for eradicating HIV-1 infection. To prevent unwanted off-target effects from constantly expressed ZFNs, in this study, we engineered the expression of ZFNs under the control of HIV-1 LTR, by which ZFN expression can be activated by the HIV-1 (Trans-Activator of Transcription) Tat protein. Our results show that functional expression of ZFNs induced by Tat excise the integrated proviral DNA of HIV-NL4-3-eGFP in approximately 30% of the population of HIV-1-infected cells. The results from HIV-1-infected human primary T cells and latently infected T cells treated with the inducible ZFNs further validated that proviral DNA can be excised. Taken together, positively regulated expression of ZFNs in the presence of HIV-1 Tat may provide a safer and novel implementation of genome-editing technology for eradicating HIV-1 proviral DNA from infected host cells.

Hydrogen sulfide attenuates myocardial fibrosis in diabetic rats through the JAK/STAT signaling pathway.

The aim of the present study was to determine the role of hydrogen sulfide (H2S) in improving myocardial fibrosis and its effects on oxidative stress, endoplasmic reticulum (ER) stress and cell apoptosis in diabetic rats, by regulating the Janus kinase̸signal transducer and activator of transcription (JAK̸STAT) signaling pathway. A total of 40 male Sprague­Dawley rats were randomly divided into four groups (n=10) as follows: normal (control group), diabetes mellitus [streptozotocin (STZ) group], diabetes mellitus treated with H2S (STZ + H2S group), and normal rats treated with H2S (H2S group). Diabetes in rats was induced by intraperitoneal (i.p.) injection of STZ at a dose of 40 mg̸kg. NaHS (100 µmol̸kg, i.p.), which was used as an exogenous donor of H2S, was administered to rats in the STZ + H2S and H2S groups. After 8 weeks, the pathological morphological changes in myocardial fibers were observed following hematoxylin and eosin and Masson's trichrome staining. Apoptosis of myocardial tissue was analyzed by the terminal deoxynucleotidyl transferase dUTP nick end labeling assay. Oxidative stress was evaluated through detecting the content of malondialdehyde (MDA), 4-hydroxynonenal (4-HNE), glutathione (GSH) and superoxide dismutase (SOD) in the myocardial cells by ELISA. The expression of collagen III, matrix metalloproteinase (MMP)8, MMP14, tissue inhibitor of metalloproteinase (TIMP)2, transforming growth factor (TGF)-ß, cystathionine­Î³­lyase (CSE), eukaryotic initiation factor 2α (eIF2α), GRP94, Bcl-2, caspase-3, tumor necrosis factor (TNF)-α, nuclear factor­κB (NF­κB) and proteins related to the JAK̸STAT pathway, was detected by western blot analysis. The results indicated that the array of myocardial cells was markedly disordered in STZ group rats; compared with the control group, both myocardial interstitial fibrosis and the deposition of collagen III were increased. Furthermore, the expression ratio of MMPs̸TIMPs was dysregulated, while the expression levels of TGF-ß, eIF2α, GRP94, caspase-3, TNF-α, NF-κB, MDA and 4-HNE were significantly increased. Furthermore, the expressions of JAK-1̸2 and STAT1̸3̸5̸6 were also markedly upregulated, while those of CSE, SOD, GSH and Bcl-2 were downregulated. Compared with the STZ group, these changes were reversed in the STZ + H2S group. The results of the present study demonstrated that H2S can improve myocardial fibrosis in diabetic rats, and the underlying mechanism may be associated with the downregulation of the JAK̸STAT signaling pathway, thereby suppressing oxidative stress and ER stress, inflammatory reaction and cell apoptosis.

Quercetin synergistically reactivates human immunodeficiency virus type 1 latency by activating nuclear factor­κB.

Highly active antiretroviral therapy (HAART) is very effective in suppressing human immunodeficiency virus type 1 (HIV­1) replication. However, the treatment is required to be administered for the remainder of an individual's lifetime due to latent HIV­1 reservoirs. The 'shock­and­kill' strategy, which involves using agents to reactivate latent HIV­1 and subsequently killing latently infected cells in the presence of HAART, was recently proposed. Unfortunately, no agents have currently demonstrated an ability to reactivate latent HIV­1 in vivo in the absence of toxicity. Therefore, the identification of novel latency activators is required. In order to identify a potential novel agent, the present study investigated the effect of quercetin on latent HIV­1 reactivation using an established model of HIV­1 latency. As a marker for reactivation of HIV­1 in C11 Jurkat cells, the expression of green fluorescent protein, controlled by HIV­1 long terminal repeat, was observed by fluorescence microscopy. The results of the present study demonstrated that quercetin effectively reactivated latent HIV­1 gene expression alone, and led to synergistic reactivation when combined with prostratin or valproic acid. In addition, the present study provides evidence that quercetin may reactivate HIV­1 expression by inducing nuclear factor­κB nuclear translocation, and that the toxicity of quercetin is lower when compared with various additional activators of HIV­1. Combined, the results of the present study indicate that quercetin may be an effective agent to disrupt HIV­1 latency and may be useful in future eradication strategies.

The bromodomain and extraterminal domain inhibitor bromosporine synergistically reactivates latent HIV-1 in latently infected cells.

The long-lived latent HIV-1 reservoir is the major barrier for complete cure of Acquired Immune Deficiency Syndrome (AIDS). Here we report that a novel bromodomain and extraterminal domain (BET) inhibitor bromosporine which can broadly target BETs, is able to potently reactivate HIV-1 replication in different latency models alone and more powerful when combined with prostratin or TNF-α. Furthermore, the treatment with bromosporine induced HIV-1 full-length transcripts in resting CD4+ T cells from infected individuals with suppressive antiretroviral therapy (ART) ex vivo, with no obvious cytotoxicity or global activation of T cell. Finally, our data suggest that Tat plays a critical role in the bromosporine-mediated reactivation of latent HIV-1, which involved the increase of CDK9 T-loop phosphorylation. In summary, we found that the BET inhibitor bromosporine, alone or with other activators, might be a candidate for future HIV-1 eradication strategies.

BET inhibitors RVX-208 and PFI-1 reactivate HIV-1 from latency.

Persistent latent reservoir in resting CD4+ T cells is a major obstacle in curing HIV-1 infection. Effective strategies for eradication of the HIV-1 reservoir are urgently needed. We report here for the first time that two BET inhibitors, RVX-208, which has entered phase II clinical trials for diverse cardiovascular disorders, and PFI-1, which has been widely studied in oncology, can reactivate HIV-1 from latency. RVX-208 and PFI-1 treatment alone or in combination with other latency reversing agents efficiently reactivated HIV-1 transcription through an up-regulation of P-TEFb by increasing CDK9 Thr-186 phosphorylation in latently infected Jurkat T cells in vitro. The two BET inhibitors also reactivated HIV-1 transcription in cART treated patient-derived resting CD4+ T cells ex vivo, without influence on global immune cell activation. Our findings, in combination with previous reports, further confirm that BET inhibitors are a group of leading compounds for combating HIV-1 latency for viral eradication.

Specific and Stable Suppression of HIV Provirus Expression In Vitro by Chimeric Zinc Finger DNA Methyltransferase 1.

HIV-1 inserts its proviral DNA into the infected host cells, by which HIV proviral DNA can then be duplicated along with each cell division. Thus, provirus cannot be eradicated completely by current antiretroviral therapy. We have developed an innovative strategy to silence the HIV provirus by targeted DNA methylation on the HIV promoter region. We genetically engineered a chimeric DNA methyltransferase 1 composed of designed zinc-finger proteins to become ZF2 DNMT1. After transient transfection of the molecular clone encoding this chimeric protein into HIV-1 infected or latently infected cells, efficient suppression of HIV-1 expression by the methylation of CpG islands in 5'-LTR was observed and quantified. The effective suppression of HIV in latently infected cells by ZF2-DNMT1 is stable and can last through about 40 cell passages. Cytotoxic caused by ZF2-DNMT1 was only observed during cellular proliferation. Taken together, our results demonstrate the potential of this novel approach for anti-HIV-1 therapy.

The BET inhibitor OTX015 reactivates latent HIV-1 through P-TEFb.

None of the currently used anti-HIV-1 agents can effectively eliminate latent HIV-1 reservoirs, which is a major hurdle to a complete cure for AIDS. We report here that a novel oral BET inhibitor OTX015, a thienotriazolodiazepine compound that has entered phase Ib clinical development for advanced hematologic malignancies, can effectively reactivate HIV-1 in different latency models with an EC50 value 1.95-4.34 times lower than JQ1, a known BET inhibitor that can reactivate HIV-1 latency. We also found that OTX015 was more potent when used in combination with prostratin. More importantly, OTX015 treatment induced HIV-1 full-length transcripts and viral outgrowth in resting CD4(+) T cells from infected individuals receiving suppressive antiretroviral therapy (ART), while exerting minimal toxicity and effects on T cell activation. Finally, biochemical analysis showed that OTX015-mediated activation of HIV-1 involved an increase in CDK9 occupancy and RNAP II C-terminal domain (CTD) phosphorylation. Our results suggest that the BET inhibitor OTX015 may be a candidate for anti-HIV-1-latency therapies.

Specific Reactivation of Latent HIV-1 by dCas9-SunTag-VP64-mediated Guide RNA Targeting the HIV-1 Promoter.

HIV-1 escapes antiretroviral agents by integrating into the host DNA and forming a latent transcriptionally silent HIV-1 provirus. Transcriptional activation is prerequisite for reactivation and the eradication of latent HIV-1 proviruses. dCas9-SunTag-VP64 transcriptional system has been reported that it can robustly activate the expression of an endogenous gene using a single guide RNA (sgRNA). Here, we systematically investigated the potential of dCas9-SunTag-VP64 with the designed sgRNAs for reactivating latent HIV-1. We found dCas9-SunTag-VP64 with sgRNA 4 or sgRNA 5 targeted from -164 to -146 or -124 to -106 bp upstream of the transcription start sites of HIV-1 could induce high expression of luciferase reporter gene after screening of sgRNAs targeting different regions of the HIV-1 promoter. Further, we confirmed that dCas9-SunTag-VP64 with sgRNA 4 or sgRNA 5 can effectively reactivate latent HIV-1 transcription in several latently infected human T-cell lines. Moreover, we confirmed that the reactivation of latent HIV-1 by dCas9-SunTag-VP64 with the designed sgRNA occurred through specific binding to the HIV-1 LTR promoter without genotoxicity and global T-cell activation. Taken together, our data demonstrated dCas9-SunTag-VP64 system can effectively and specifically reactivate latent HIV-1 transcription, suggesting that this strategy could offer a novel approach to anti-HIV-1 latency.

A survey of research progress and development tendency of attribute-based encryption.

With the development of cryptography, the attribute-based encryption (ABE) draws widespread attention of the researchers in recent years. The ABE scheme, which belongs to the public key encryption mechanism, takes attributes as public key and associates them with the ciphertext or the user's secret key. It is an efficient way to solve open problems in access control scenarios, for example, how to provide data confidentiality and expressive access control at the same time. In this paper, we survey the basic ABE scheme and its two variants: the key-policy ABE (KP-ABE) scheme and the ciphertext-policy ABE (CP-ABE) scheme. We also pay attention to other researches relating to the ABE schemes, including multiauthority, user/attribute revocation, accountability, and proxy reencryption, with an extensive comparison of their functionality and performance. Finally, possible future works and some conclusions are pointed out.