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Recent cases of successful control of human immunodeficiency virus (HIV) by bone marrow transplant in combination with suppressive antiretroviral therapy (ART) and very early initiation of ART have provided proof of concept that HIV infection might now be cured. Current efforts focusing on gene therapy, boosting HIV-specific immunity, reducing infl...
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The conditioning regimen used as part of the Berlin patient's hematopoietic cell transplant likely contributed to his eradication of HIV infection. We studied the impact of conditioning in simian-human immunodeficiency virus-infected (SHIV-infected) macaques suppressed by combination antiretroviral therapy (cART). The conditioning regimen resulted...
Citations
... However, some HIV-infected cells may also escape death through various mechanisms. HIV preferentially infects memory CD4+ T cells, which normally upregulate anti-apoptotic proteins such as Bcl-2, IAP, and cFLIP to prevent activation-induced cell death and promote long term survival [16,17] . During early infection, some HIV proteins (such as Vpr, Nef, and Tat) can also exert anti-apoptotic effects [18] . ...
... Many different small molecules have been reported to induce selective killing of HIV-infected cells and/or to reduce some measure of HIV-or SIV-infected cells, such as proviral DNA or inducible reservoirs [9,17] . These molecules include p53 modulators [37] , interferons [38][39][40][41][42] , RIG-I inducers [43] , TLR7 agonists [44,45] , PD-1 blockers [46] , Bcl-2 inhibitors [47][48][49][50] , IAP/XIAP inhibitors [27,[51][52][53] , BIRC5 inhibitors [26] , DDX3 inhibitors [54] , PI3/Akt inhibitors [55,56] , proteasome inhibitors [57] , mTOR inhibitors [58,59] , TREM1 inhibitors [60,61] , an inhibitor of HIV Rev and inflammation [14,62] , and autosisinducing peptides [63] (see Table, Supplemental Digital Content 1, http://links.lww.com/QAD/D183; ...
Objectives
Some drugs that augment cell-intrinsic defenses or modulate cell death/survival pathways have been reported to selectively kill cells infected with HIV or SIV, but comparative studies are lacking. We hypothesized that these drugs may differ in their ability to kill cells infected with intact and defective proviruses.
Design
To investigate this hypothesis, drugs were tested ex vivo on Peripheral Blood Mononuclear Cells (PBMC) from nine ART-suppressed individuals.
Methods
We tested drugs currently in clinical use or human trials, including auranofin (p53 modulator), interferon alpha2A, interferon gamma, acitretin (RIG-I inducer), GS-9620/vesatolimod (TLR7 agonist), nivolumab (PD-1 blocker), obatoclax (Bcl-2 inhibitor), birinapant (IAP inhibitor), bortezomib (proteasome inhibitor), and INK128/sapanisertib (mTOR[c]1/2 inhibitor). After six days of treatment, we measured cell counts/viabilities and quantified levels of total, intact, and defective HIV DNA by droplet digital PCR (Intact Proviral DNA Assay).
Results
Obatoclax reduced intact HIV DNA (median = 27–30% of DMSO) but not defective or total HIV DNA. Other drugs showed no statistically significant effects.
Conclusions
Obatoclax and other Bcl-2 inhibitors deserve further study in combination therapies aimed at reducing the intact HIV reservoir in order to achieve a functional cure and/or reduce HIV-associated immune activation.
... Elimination of infected cells in which HIV-1 latency was reversed would occur by viral cytopathic effects and immune responses such as HIV-1 specific cytolytic T cells (CTLs), but it has been shown that these mechanisms do not suffice [13,14]. Additional kill interventions are crucial and could be provided in the form of therapeutic vaccines, pharmacological agents that revert immune exhaustion, stimulation of CTL responses or enhancement of apoptosis as well as broadly neutralizing antibodies (bnAbs) (reviewed in [8,15]). Furthermore, suicide gene therapies with different suicide genes have been explored to target productive and latent HIV-1 infection (reviewed in [16]). ...
Infections with the human immunodeficiency virus type 1 (HIV-1) are incurable due the long-lasting, latent viral reservoir. The shock-and-kill cure approach aims to activate latent proviruses in HIV-1 infected cells and subsequently kill these cells with strategies such as therapeutic vaccines or immune enhancement. Here, we combined the dCas9-VPR CRISPR activation (CRISPRa) system with gRNA-V, the truncated Bid (tBid)-based suicide gene strategy and CD3-retargeted adenovirus (Ad) delivery vectors, in an all-in-one targeted shock-and-kill gene therapy approach to achieve specific elimination of latently HIV-1 infected cells. Simultaneous transduction of latently HIV-1 infected J-Lat 10.6 cells with a CD3-retargeted Ad-CRISPRa-V and Ad-tBid led to a 57.7 ± 17.0% reduction of productively HIV-1 infected cells and 2.4-fold ± 0.25 increase in cell death. The effective activation of latent HIV-1 provirus by Ad-CRISPRa-V was similar to the activation control TNF-α. The strictly HIV-1 dependent and non-leaky killing by tBid could be demonstrated. Furthermore, the high transduction efficiencies of up to 70.8 ± 0.4% by the CD3-retargeting technology in HIV-1 latently infected cell lines was the basis of successful shock-and-kill. This novel targeted shock-and-kill all-in-one gene therapy approach has the potential to safely and effectively eliminate HIV-1 infected cells in a highly HIV-1 and T cell specific manner.
... The different mechanisms of cell death in HIV infection and the consequences of these on pathogenesis, for example viral transmission and reservoir formation, provide additional drug targets. Modulation of cell death pathways alongside effective HAART may provide a cure for HIV infection, by preventing loss of CD4 + T cells in early stages, and removing the viral reservoir in late stages (27). ...
The human immunodeficiency virus (HIV) is still a global pandemic and despite the successful use of anti-retroviral therapy, a well-established cure remains to be identified. Viral modulation of cell death has a significant role in HIV pathogenesis. Here we sought to understand the major mechanisms of HIV-induced death of lymphocytes and the effects on viral transmission. Flow cytometry analysis of lymphocytes from five latent HIV-infected patients, and HIV IIIB-infected MT2 cells demonstrated both necrosis and apoptosis to be the major mechanisms of cell death in CD4⁺ and CD4⁻/CD8⁻ lymphocytes. Significantly, pro-apoptotic tumor necrosis factor (TNF) peptide (P13) was found to inhibit HIV-related cell death and reduced viral transmission. Whereas pro-necrotic TNF peptide (P16) had little effect on HIV-related cell death and viral transmission. Understanding mechanisms by which cell death can be manipulated may provide additional drug targets to reduce the loss of CD4⁺ cells and the formation of a viral reservoir in HIV infection.
... Clonal expansion requires cellular activation and NF-κB expression, which also has the potential to reactivate latent proviruses 45 . Although high-level HIV-1 expression results in cell death 46 , in vitro experiments have shown that only a fraction of dividing latent cells die after reactivation 37,47,48 . Some of those that survive do so after transiently expressing the HIV-1 envelope protein or peptide-major histocompatibility complex complexes on the cell surface making them potential targets for bNAb 9,49,50 or CD8 + T cell-mediated clearance 9,50 , as documented in pre-clinical studies in bNAb-treated SHIV-infected macaques 8,9 . ...
HIV-1 infection remains a public health problem with no cure. Anti-retroviral therapy (ART) is effective but requires lifelong drug administration owing to a stable reservoir of latent proviruses integrated into the genome of CD4⁺ T cells¹. Immunotherapy with anti-HIV-1 antibodies has the potential to suppress infection and increase the rate of clearance of infected cells2,3. Here we report on a clinical study in which people living with HIV received seven doses of a combination of two broadly neutralizing antibodies over 20 weeks in the presence or absence of ART. Without pre-screening for antibody sensitivity, 76% (13 out of 17) of the volunteers maintained virologic suppression for at least 20 weeks off ART. Post hoc sensitivity analyses were not predictive of the time to viral rebound. Individuals in whom virus remained suppressed for more than 20 weeks showed rebound viraemia after one of the antibodies reached serum concentrations below 10 µg ml⁻¹. Two of the individuals who received all seven antibody doses maintained suppression after one year. Reservoir analysis performed after six months of antibody therapy revealed changes in the size and composition of the intact proviral reservoir. By contrast, there was no measurable decrease in the defective reservoir in the same individuals. These data suggest that antibody administration affects the HIV-1 reservoir, but additional larger and longer studies will be required to define the precise effect of antibody immunotherapy on the reservoir.
... Although productive HIV-1 replication can be cytotoxic for the cells, HIV-1 production does not always kill these cells. However, under certain conditions, such as p53 activation, Bcl2 inhibition and PKC activation can modulate the condition of the latent-infected cells to turn into apoptosis-sensitive cells (Badley et al., 2013). Notably, we recently reported that some LRAs, such as PEP005, strongly induce the upregulation of active caspase-3, resulting in enhanced apoptosis (Hattori et al., 2018), which may explain why the addition of an LRA successfully accelerated the elimination of latently HIV-1-infected cells in the WIPE assay even in the absence of the host immune system. ...
Persistence of HIV-1 latent reservoir cells during antiretroviral therapy (ART) is a major obstacle for curing HIV-1. Even though latency-reversing agents (LRAs) are under development to reactivate and eradicate latently infected cells, there are few useful models for evaluating LRA activity in vitro. Here, we establish a long-term cell culture system called the “widely distributed intact provirus elimination” (WIPE) assay. It harbors thousands of different HIV-1-infected cell clones with a wide distribution of HIV-1 provirus similar to that observed in vivo. Mathematical modeling and experimental results from this in vitro infection model demonstrates that the addition of an LRA to ART shows a latency-reversing effect and contributes to the eradication of replication-competent HIV-1. The WIPE assay can be used to optimize therapeutics against HIV-1 latency and investigate mechanistic insights into the clonal selection of heterogeneous HIV-1-infected cells.
... Interestingly, a new therapy, based on the oxidative stress response, has been proposed to target the HIV-1 reservoir. Auranofin (AF) is a gold compound with a pro-oxidant function [57] capable of inducing apoptosis of memory T cells based on their oxidative status [58]. AF combined with ART completely suppresses simian immunodeficiency virus/SIV viremia in macaques. ...
The immunodeficiency observed in HIV-1-infected patients is mainly due to uninfected bystander CD4⁺ T lymphocyte cell death. The viral envelope glycoproteins (Env), expressed at the surface of infected cells, play a key role in this process. Env triggers macroautophagy/autophagy, a process necessary for subsequent apoptosis, and the production of reactive oxygen species (ROS) in bystander CD4⁺ T cells. Here, we demonstrate that Env-induced oxidative stress is responsible for their death by apoptosis. Moreover, we report that peroxisomes, organelles involved in the control of oxidative stress, are targeted by Env-mediated autophagy. Indeed, we observe a selective autophagy-dependent decrease in the expression of peroxisomal proteins, CAT and PEX14, upon Env exposure; the downregulation of either BECN1 or SQSTM1/p62 restores their expression levels. Fluorescence studies allowed us to conclude that Env-mediated autophagy degrades these entire organelles and specifically the mature ones. Together, our results on Env-induced pexophagy provide new clues on HIV-1-induced immunodeficiency.
Abbreviations: Ab: antibodies; AF: auranofin; AP: anti-proteases; ART: antiretroviral therapy; BafA1: bafilomycin A1; BECN1: beclin 1; CAT: catalase; CD4: CD4 molecule; CXCR4: C-X-C motif chemokine receptor 4; DHR123: dihydrorhodamine 123; Env: HIV-1 envelope glycoproteins; GAPDH: glyceraldehyde-3-phosphate dehydrogenase; GFP: green fluorescent protein; GFP-SKL: GFP-serine-lysine-leucine; HEK: human embryonic kidney; HIV-1: type 1 human immunodeficiency virus; HTRF: homogeneous time resolved fluorescence; MAP1LC3/LC3: microtubule associated protein 1 light chain 3; NAC: N-acetyl-cysteine; PARP: poly(ADP-ribose) polymerase; PEX: peroxin; ROS: reactive oxygen species; siRNA: small interfering ribonucleic acid; SQSTM1/p62: sequestosome 1.
... Therefore, we assume that bryostatin may lead to cell cycle arrest and apoptosis in the process of reactivation of latent HIV infected CD4 + cell. Previous studies indicate that latent HIV-1 can sense the apoptosis of its host cell and responds by completing its replication cycle 36,37 . Another study revealed that G2 arrest of cell cycle induced HIV-1 transcriptional activation 38 . ...
The reactivation of HIV latency cell will be necessary to curing HIV infection. Although many latency-reversal agents (LRAs) have proven effective to reactivate the latency cell, there is a lack of any systematic analysis of the molecular targets of these LRAs and related pathways in the context of transcriptome. In this study, we performed an integrated analysis of the target profile of bryostatin and transcriptome of the reactivated CD4⁺ T cells after exposing to bryostatin. The result showed a distinct gene expression profile between latency cells and bryostatin reactivated cells. We found bryostatin can target multiple types of protein other than only protein kinase C. Functional network analysis of the target profile and differential expressed genes suggested that bryostatin may activate a few novel pathways such as pyrimidine metabolism, purine metabolism and p53 signaling pathway, besides commonly known pathways DNA replication, cell cycle and so on. The results suggest that bryostatin may reactivate the HIV-latent cells through up-regulation of pyrimidine and purine metabolism or through starting the cell-cycle arrest and apoptosis induced by up-regulation of p53 signaling pathway. Our study provides some novel insights into the role of bryostatin and its affected pathways in controlling HIV latency and reactivation.
... Brenner (2009) identified the key genes driving organ development and programmed cell death. These results contributed to understand the mode of action of bacteria and viruses as the HIV and inspired new cancer therapies based on controlled cell death (Badley et al. 2013;Kroemer and Jäättelä 2005;Kroemer et al. 2013;Delbridge et al. 2016). Early in the twenty-first century, studies on model organisms like D. melanogaster highly contributed to unveil the genetic mechanisms related to cancer pathogenesis and many other gene-related disorders. ...
Well-established and newly developed genome technologies are revolutionising the field of biomedicine, by providing genomic data and genetic engineered structures that support investigating individual propensity for developing certain diseases, on one hand, and by predicting individual responses to the environmental stimulus due to gene common variants. Indeed, the former has provided innovative ways of combining genotype-phenotype-based therapies for a wide range of diseases, including malaria and its side effects. Ultimately, computationally guided gene modifications via in silico design of plasmids have contributed with the optimal production of recombinant DNA, benefiting from useful species variant traits. On the other hand, natural or semisynthetic plant secondary metabolites-derived compounds have been used in diseases’ therapies, particularly treating infectious diseases as malaria. In recent years, major efforts have been made to reduce the burden of infectious diseases worldwide, especially in the developing world. In this context, malaria prevention and treatment have stimulated collective measures, which are widely reported by the World Health Organization (WHO). Therefore, aiming at addressing the latest advances in the field, in this chapter, the relevance of pharmacogenomics and computational design in drug discovery, including information on the benefits of using plants secondary metabolites for the production of anti-malarial compounds, are presented. Moreover, given the plethora of prospective side effects resulting from this burden of disease, including neurocognitive impairment in patients affected by cerebral Plasmodium falciparum infection, a set of key elements in patient-response-based drug screening is discussed, in the context of stem cells technology. All together, we anticipate the above mentioned new technologies to be the precursors of short-term novelty in computationally designed gene-personalised healthcare, bringing about significant improvement in the current malarial therapies.
... The latent viral reservoir is not recognized by the immune system and therefore these cells are not eliminated. The "shock and kill" strategy aims to induce viral protein production by reactivation of the latent HIV-1 provirus to prompt elimination of the infected cells through recognition by the immune system or cell death as a result of viral replication (Matalon et al., 2011;Badley et al., 2013). The "shock and kill" strategy has been investigated primarily using CD4+ T cells and T cell lines. ...
... The "shock and kill" strategy has been investigated primarily using CD4+ T cells and T cell lines. Histone deacetylases (HDACs), for instance, are important for suppression of HIV-1 proviral DNA expression, and several HDAC inhibitors including SAHA, vorinostat, oxamflatin, metacept-1, and metacept-3 (Shehu-Xhilaga et al., 2009;Badley et al., 2013) have been studied for their ability to reactivate and eliminate latently infected cells. Although HDAC inhibitors have shown activity to reactivate HIV-1 from latent reservoirs in patients on ART, no decline in HIV-1 proviral DNA in CD4+ T cells was observed (Archin et al., 2009a(Archin et al., ,b, 2012(Archin et al., , 2017. ...
... Moreover, LRAs alone are mostly not sufficient to induce cell death and therefore an effective immune response is essential for the elimination of the reactivated cells and this is lacking in the majority of HIV-1 infected patients. Furthermore, HIV-1 infected macrophages can reside in immune privileged compartments like the CNS and can therefore not be reached by cytotoxic T cells (Shan et al., 2012;Badley et al., 2013). ...
Current antiretroviral therapy (ART) effectively suppresses Human Immunodeficiency Virus type 1 (HIV-1) in infected individuals. However, even long term ART does not eradicate HIV-1 infected cells and the virus persists in cellular reservoirs. Beside memory CD4+ T cells, cells of the myeloid lineage, especially macrophages, are believed to be an important sanctuary for HIV-1. Monocytes and macrophages are key players in the innate immune response to pathogens and are recruited to sites of infection and inflammation. Due to their long life span and ability to reside in virtually every tissue, macrophages have been proposed to play a critical role in the establishment and persistence of the HIV-1 reservoir. Current HIV-1 cure strategies mainly focus on the concept of “shock and kill” to purge the viral reservoir. This approach aims to reactivate viral protein production in latently infected cells, which subsequently are eliminated as a consequence of viral replication, or recognized and killed by the immune system. Macrophage susceptibility to HIV-1 infection is dependent on the local microenvironment, suggesting that molecular pathways directing differentiation and polarization are involved. Current latency reversing agents (LRA) are mainly designed to reactivate the HIV-1 provirus in CD4+ T cells, while their ability to abolish viral latency in macrophages is largely unknown. Moreover, the resistance of macrophages to HIV-1 mediated kill and the presence of infected macrophages in immune privileged regions including the central nervous system (CNS), may pose a barrier to elimination of infected cells by current “shock and kill” strategies. This review focusses on the role of monocytes/macrophages in HIV-1 persistence. We will discuss mechanisms of viral latency and persistence in monocytes/macrophages. Furthermore, the role of these cells in HIV-1 tissue distribution and pathogenesis will be discussed.
... Cellular and immune defenses can trigger apoptosis in response to viral products, while anti-apoptotic pathways may contribute to the survival of immortalized cell lines, especially in the face of productive HIV infection. Emerging evidence suggests that anti-apoptotic pathways may also contribute to the survival of HIV-infected cells and persistence of proviral clones in vivo [68][69][70], and these pathways may be targets for new therapies aimed at killing infected cells and curing HIV [71]. ...
Background:
HIV-infected cell lines are widely used to study latent HIV infection, which is considered the main barrier to HIV cure. We hypothesized that these cell lines differ from each other and from cells from HIV-infected individuals in the mechanisms underlying latency.
Results:
To quantify the degree to which HIV expression is inhibited by blocks at different stages of HIV transcription, we employed a recently-described panel of RT-ddPCR assays to measure levels of 7 HIV transcripts ("read-through," initiated, 5' elongated, mid-transcribed/unspliced [Pol], distal-transcribed [Nef], polyadenylated, and multiply-sliced [Tat-Rev]) in bulk populations of latently-infected (U1, ACH-2, J-Lat) and productively-infected (8E5, activated J-Lat) cell lines. To assess single-cell variation and investigate cellular genes associated with HIV transcriptional blocks, we developed a novel multiplex qPCR panel and quantified single cell levels of 7 HIV targets and 89 cellular transcripts in latently- and productively-infected cell lines. The bulk cell HIV transcription profile differed dramatically between cell lines and cells from ART-suppressed individuals. Compared to cells from ART-suppressed individuals, latent cell lines showed lower levels of HIV transcriptional initiation and higher levels of polyadenylation and splicing. ACH-2 and J-Lat cells showed different forms of transcriptional interference, while U1 cells showed a block to elongation. Single-cell studies revealed marked variation between/within cell lines in expression of HIV transcripts, T cell phenotypic markers, antiviral factors, and genes implicated in latency. Expression of multiply-spliced HIV Tat-Rev was associated with expression of cellular genes involved in activation, tissue retention, T cell transcription, and apoptosis/survival.
Conclusions:
HIV-infected cell lines differ from each other and from cells from ART-treated individuals in the mechanisms governing latent HIV infection. These differences in viral and cellular gene expression must be considered when gauging the suitability of a given cell line for future research on HIV. At the same time, some features were shared across cell lines, such as low expression of antiviral defense genes and a relationship between productive infection and genes involved in survival. These features may contribute to HIV latency or persistence in vivo, and deserve further study using novel single cell assays such as those described in this manuscript.
