Quantitative and Qualitative Distinctions between HIV-1 and SIV Reservoirs: Implications for HIV-1 Cure-Related Studies.

The persistence of the latent viral reservoir is the main hurdle to curing HIV-1 infection. SIV infection of non-human primates (NHPs), namely Indian-origin rhesus macaques, is the most relevant and widely used animal model to evaluate therapies that seek to eradicate HIV-1. The utility of a model ultimately rests on how accurately it can recapitulate human disease, and while reservoirs in the NHP model behave quantitatively very similar to those of long-term suppressed persons with HIV-1 (PWH) in the most salient aspects, recent studies have uncovered key nuances at the clonotypic level that differentiate the two in qualitative terms. In this review, we will highlight differences relating to proviral intactness, clonotypic structure, and decay rate during ART between HIV-1 and SIV reservoirs and discuss the relevance of these distinctions in the interpretation of HIV-1 cure strategies. While these, to some degree, may reflect a unique biology of the virus or host, distinctions among the proviral landscape in SIV are likely to be shaped significantly by the condensed timeframe of NHP studies. ART is generally initiated earlier in the disease course, and animals are virologically suppressed for shorter periods before receiving interventions. Because these are experimental variables dictated by the investigator, we offer guidance on study design for cure-related studies performed in the NHP model. Finally, we highlight the case of GS-9620 (Vesatolimod), an antiviral TLR7 agonist tested in multiple independent pre-clinical studies in which virological outcomes may have been influenced by study-related variables.

Potent latency reversal by Tat RNA-containing nanoparticle enables multi-omic analysis of the HIV-1 reservoir.

The development of latency reversing agents that potently reactivate HIV without inducing global T cell activation would benefit the field of HIV reservoir research and could pave the way to a functional cure. Here, we explore the reactivation capacity of a lipid nanoparticle containing Tat mRNA (Tat-LNP) in CD4 T cells from people living with HIV undergoing antiretroviral therapy (ART). When combined with panobinostat, Tat-LNP induces latency reversal in a significantly higher proportion of latently infected cells compared to PMA/ionomycin (≈ 4-fold higher). We demonstrate that Tat-LNP does not alter the transcriptome of CD4 T cells, enabling the characterization of latently infected cells in their near-native state. Upon latency reversal, we identify transcriptomic differences between infected cells carrying an inducible provirus and non-infected cells (e.g. LINC02964, GZMA, CCL5). We confirm the transcriptomic differences at the protein level and provide evidence that the long non-coding RNA LINC02964 plays a role in active HIV infection. Furthermore, p24+ cells exhibit heightened PI3K/Akt signaling, along with downregulation of protein translation, suggesting that HIV-infected cells display distinct signatures facilitating their long-term persistence. Tat-LNP represents a valuable research tool for in vitro reservoir studies as it greatly facilitates the in-depth characterization of HIV reservoir cells' transcriptome and proteome profiles.

Transforming Growth Factor ß Signaling Promotes HIV-1 Infection in Activated and Resting Memory CD4+ T Cells.

Understanding the facilitator of HIV-1 infection and subsequent latency establishment may aid the discovery of potential therapeutic targets. Here, we report the elevation of plasma transforming growth factor ß (TGF-ß) during acute HIV-1 infection among men who have sex with men (MSM). Using a serum-free in vitro system, we further delineated the role of TGF-ß signaling in mediating HIV-1 infection of activated and resting memory CD4+ T cells. TGF-ß could upregulate both the frequency and expression of the HIV-1 coreceptor CCR5, thereby augmenting CCR5-tropic viral infection of resting and activated memory CD4+ T cells via Smad3 activation. The production of live HIV-1JR-FL upon infection and reactivation was increased in TGF-ß-treated resting memory CD4+ T cells without increasing CD4 expression or inducing T cell activation. The expression of CCR7, a central memory T cell marker that serves as a chemokine receptor to facilitate T cell trafficking into lymphoid organs, was also elevated on TGF-ß-treated resting and activated memory CD4+ T cells. Moreover, the expression of CXCR3, a chemokine receptor recently reported to facilitate CCR5-tropic HIV-1 infection, was increased on resting and activated memory CD4+ T cells upon TGF-ß treatment. These findings were coherent with the observation that ex vivo CCR5 and CXCR3 expression on total resting and resting memory CD4+ T cells in combination antiretroviral therapy (cART)-naive and cART-treated patients were higher than in healthy individuals. Overall, the study demonstrated that TGF-ß upregulation induced by acute HIV-1 infection might promote latency reservoir establishment by increasing infected resting memory CD4+ T cells and lymphoid organ homing of infected central memory CD4+ T cells. Therefore, TGF-ß blockade may serve as a potential supplementary regimen for HIV-1 functional cure by reducing viral latency. IMPORTANCE Incomplete eradication of HIV-1 latency reservoirs remains the major hurdle in achieving a complete HIV/AIDS cure. Dissecting the facilitator of latency reservoir establishment may aid the discovery of druggable targets for HIV-1 cure. This study showed that the T cell immunomodulatory cytokine TGF-ß was upregulated during the acute phase of infection. Using an in vitro serum-free system, we specifically delineated that TGF-ß promoted HIV-1 infection of both resting and activated memory CD4+ T cells via the induction of host CCR5 coreceptor. Moreover, TGF-ß-upregulated CCR7 or CXCR3 might promote HIV-1 latent infection by facilitating lymphoid homing or IP-10-mediated viral entry and DNA integration, respectively. Infected resting and central memory CD4+ T cells are important latency reservoirs. Increased infection of these cells mediated by TGF-ß will promote latency reservoir establishment during early infection. This study, therefore, highlighted the potential use of TGF-ß blockade as a supplementary regimen with cART in acute patients to reduce viral latency.

Phenotypic signatures of immune selection in HIV-1 reservoir cells.

Human immunodeficiency virus 1 (HIV-1) reservoir cells persist lifelong despite antiretroviral treatment1,2 but may be vulnerable to host immune responses that could be exploited in strategies to cure HIV-1. Here we used a single-cell, next-generation sequencing approach for the direct ex vivo phenotypic profiling of individual HIV-1-infected memory CD4+ T cells from peripheral blood and lymph nodes of people living with HIV-1 and receiving antiretroviral treatment for approximately 10 years. We demonstrate that in peripheral blood, cells harbouring genome-intact proviruses and large clones of virally infected cells frequently express ensemble signatures of surface markers conferring increased resistance to immune-mediated killing by cytotoxic T and natural killer cells, paired with elevated levels of expression of immune checkpoint markers likely to limit proviral gene transcription; this phenotypic profile might reduce HIV-1 reservoir cell exposure to and killing by cellular host immune responses. Viral reservoir cells harbouring intact HIV-1 from lymph nodes exhibited a phenotypic signature primarily characterized by upregulation of surface markers promoting cell survival, including CD44, CD28, CD127 and the IL-21 receptor. Together, these results suggest compartmentalized phenotypic signatures of immune selection in HIV-1 reservoir cells, implying that only small subsets of infected cells with optimal adaptation to their anatomical immune microenvironment are able to survive during long-term antiretroviral treatment. The identification of phenotypic markers distinguishing viral reservoir cells may inform future approaches for strategies to cure and eradicate HIV-1.

HIV silencing and cell survival signatures in infected T cell reservoirs.

Rare CD4 T cells that contain HIV under antiretroviral therapy represent an important barrier to HIV cure1-3, but the infeasibility of isolating and characterizing these cells in their natural state has led to uncertainty about whether they possess distinctive attributes that HIV cure-directed therapies might exploit. Here we address this challenge using a microfluidic technology that isolates the transcriptomes of HIV-infected cells based solely on the detection of HIV DNA. HIV-DNA+ memory CD4 T cells in the blood from people receiving antiretroviral therapy showed inhibition of six transcriptomic pathways, including death receptor signalling, necroptosis signalling and antiproliferative Gα12/13 signalling. Moreover, two groups of genes identified by network co-expression analysis were significantly associated with HIV-DNA+ cells. These genes (n = 145) accounted for just 0.81% of the measured transcriptome and included negative regulators of HIV transcription that were higher in HIV-DNA+ cells, positive regulators of HIV transcription that were lower in HIV-DNA+ cells, and other genes involved in RNA processing, negative regulation of mRNA translation, and regulation of cell state and fate. These findings reveal that HIV-infected memory CD4 T cells under antiretroviral therapy are a distinctive population with host gene expression patterns that favour HIV silencing, cell survival and cell proliferation, with important implications for the development of HIV cure strategies.

A Camptothetin Analog, Topotecan, Promotes HIV Latency via Interference with HIV Transcription and RNA Splicing.

Low level HIV transcription during modern antiretroviral therapy (ART) in persons with HIV is linked to residual inflammation and associated diseases, like cardiovascular disease and cancer. The "block and lock" approach to hold HIV in a state of deep latency may help decrease residual inflammation in a person with HIV on ART and thus improve health. A camptothecin analog topotecan (TPT) was previously implicated as an inhibitor of active HIV replication. Using an in vitro primary T cell model of HIV latency, we demonstrated that (i) TPT reduces HIV transcriptional activity in latently infected cells; (ii) downregulation of HIV RNA by TPT cannot be reversed by latency reversing agents; (iii) several primary and secondary mechanism of action of TPT may be involved in control of HIV replication; (iv) regulation of HIV RNA by TPT is dependent on splicing complexity; (v) increase in proportion of unspliced HIV transcripts was facilitated by intron retention and upregulation of splicing factors, specifically SRSF6, by TPT. Although high TPT dosing (10 µM) was needed to achieve the observed effects, viability of primary CD4+ T cells was not greatly affected. Because toxicity can be observed with TPT in persons with cancer, TPT is unlikely to be used as an anti-HIV agent in clinic, but our study provides proof that camptothetin has "block and lock" activity. Other camptothetin analogs, which are less toxic than TPT, should be designed and tested as HIV "block and lock" agents. IMPORTANCE HIV survives in a state of very low activity, called latency, for long periods in persons with HIV on antiretroviral therapy. This low activity of HIV is linked to residual inflammation and associated diseases, such as heart disease and cancer. New strategies are being explored to further silence the HIV provirus and suppress residual inflammation. This study provides strong evidence that the camptothetin analog, Topotecan, can reduce residual activity of HIV in an experimental model of HIV latency. While Topotecan itself is likely not suitable for use in the clinic due to its toxicity, other camptothetin analogs should be designed and investigated as "block and lock" agents.

Potential HIV latency-reversing agents with STAT1-activating activity from the leaves of Wikstroemia chamaedaphne.

Developing highly effective HIV latency-reversing agent is an inportmant approach for the treatment of AIDS via the "shock and kill" of latent HIV. In this study, two unreported modified daphnane-type diterpenes (chamaedaphnelide A and epi-chamaedaphnelide A) and one unreported tigliane-type diterpene (chamaedaphnelide B), along with four known daphnane-type diterpenes and one known tigliane-type diterpene were obtained from the leaves of Wikstroemia chamaedaphne. Chamaedaphnelide A and epi-chamaedaphnelide A represents the first A ring cleavage daphnane-type backbone. Chamaedaphnelide A, epi-chamaedaphnelide A, chamaedaphnelide B, and 6α,7α-epoxy-5ß-hydroxy-12-deoxyphorbol-13-decanoate showed HIV latency-reversing activity, especially chamaedaphnelide B and 6α,7α-epoxy-5ß-hydroxy-12-deoxyphorbol-13-decanoate displayed equally potential to positive drugs prostratin with reversing latent HIV on more than 100-fold compared to unstimulated cells. Furthermore, the activation of STAT1 was involved in the HIV latency-reversing activity of these diterpenes, firstly demonstrating that daphnane- and tigliane-type diterpenes can rapidly activate STAT1 activity. Indeed, these results also supported that activating STAT1 activity is a pathway for reversing latent HIV.

The HIV Latency Reversal Agent HODHBt Enhances NK Cell Effector and Memory-Like Functions by Increasing Interleukin-15-Mediated STAT Activation.

Elimination of human immunodeficiency virus (HIV) reservoirs is a critical endpoint to eradicate HIV. One therapeutic intervention against latent HIV is "shock and kill." This strategy is based on the transcriptional activation of latent HIV with a latency-reversing agent (LRA) with the consequent killing of the reactivated cell by either the cytopathic effect of HIV or the immune system. We have previously found that the small molecule 3-hydroxy-1,2,3-benzotriazin-4(3H)-one (HODHBt) acts as an LRA by increasing signal transducer and activator of transcription (STAT) factor activation mediated by interleukin-15 (IL-15) in cells isolated from aviremic participants. The IL-15 superagonist N-803 is currently under clinical investigation to eliminate latent reservoirs. IL-15 and N-803 share similar mechanisms of action by promoting the activation of STATs and have shown some promise in preclinical models directed toward HIV eradication. In this work, we evaluated the ability of HODHBt to enhance IL-15 signaling in natural killer (NK) cells and the biological consequences associated with increased STAT activation in NK cell effector and memory-like functions. We showed that HODHBt increased IL-15-mediated STAT phosphorylation in NK cells, resulting in increases in the secretion of CXCL-10 and interferon gamma (IFN-γ) and the expression of cytotoxic proteins, including granzyme B, granzyme A, perforin, granulysin, FASL, and TRAIL. This increased cytotoxic profile results in increased cytotoxicity against HIV-infected cells and different tumor cell lines. HODHBt also improved the generation of cytokine-induced memory-like NK cells. Overall, our data demonstrate that enhancing the magnitude of IL-15 signaling with HODHBt favors NK cell cytotoxicity and memory-like generation, and thus, targeting this pathway could be further explored for HIV cure interventions. IMPORTANCE Several clinical trials targeting the HIV latent reservoir with LRAs have been completed. In spite of a lack of clinical benefit, they have been crucial to elucidate hurdles that "shock and kill" strategies have to overcome to promote an effective reduction of the latent reservoir to lead to a cure. These hurdles include low reactivation potential mediated by LRAs, the negative influence of some LRAs on the activity of natural killer and effector CD8 T cells, an increased resistance to apoptosis of latently infected cells, and an exhausted immune system due to chronic inflammation. To that end, finding therapeutic strategies that can overcome some of these challenges could improve the outcome of shock and kill strategies aimed at HIV eradication. Here, we show that the LRA HODHBt also improves IL-15-mediated NK cell effector and memory-like functions. As such, pharmacological enhancement of IL-15-mediated STAT activation can open new therapeutic avenues toward an HIV cure.

Prolonged viral suppression with anti-HIV-1 antibody therapy.

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.

Altered Response Pattern following AZD5582 Treatment of SIV-Infected, ART-Suppressed Rhesus Macaque Infants.

The "shock and kill" strategy for HIV-1 cure incorporates latency-reversing agents (LRA) in combination with interventions that aid the host immune system in clearing virally reactivated cells. LRAs have not yet been investigated in pediatric clinical or preclinical studies. Here, we evaluated an inhibitor of apoptosis protein (IAP) inhibitor (IAPi), AZD5582, that activates the noncanonical NF-κB (ncNF-κB) signaling pathway to reverse latency. Ten weekly doses of AZD5582 were intravenously administered at 0.1 mg/kg to rhesus macaque (RM) infants orally infected with SIVmac251 at 4 weeks of age and treated with a triple ART regimen for over 1 year. During AZD5582 treatment, on-ART viremia above the limit of detection (LOD, 60 copies/mL) was observed in 5/8 infant RMs starting at 3 days post-dose 4 and peaking at 771 copies/mL. Of the 135 measurements during AZD5582 treatment in these 5 RM infants, only 8 were above the LOD (6%), lower than the 46% we have previously reported in adult RMs. Pharmacokinetic analysis of plasma AZD5582 levels revealed a lower Cmax in treated infants compared to adults (294 ng/mL versus 802 ng/mL). RNA-Sequencing of CD4+ T cells comparing pre- and post-AZD5582 dosing showed many genes that were similarly upregulated in infants and adults, but the expression of key ncNF-κB genes, including NFKB2 and RELB, was significantly higher in adult RMs. Our results suggest that dosing modifications for this latency reversal approach may be necessary to maximize virus reactivation in the pediatric setting for successful "shock and kill" strategies. IMPORTANCE While antiretroviral therapy (ART) has improved HIV-1 disease outcome and reduced transmission, interruption of ART results in rapid viral rebound due to the persistent latent reservoir. Interventions to reduce the viral reservoir are of critical importance, especially for children who must adhere to lifelong ART to prevent disease progression. Here, we used our previously established pediatric nonhuman primate model of oral SIV infection to evaluate AZD5582, identified as a potent latency-reversing agent in adult macaques, in the controlled setting of daily ART. We demonstrated the safety of the IAPi AZD5582 and evaluate the pharmacokinetics and pharmacodynamics of repeated dosing. The response to AZD5582 in macaque infants differed from what we previously showed in adult macaques with weaker latency reversal in infants, likely due to altered pharmacokinetics and less inducibility of infant CD4+ T cells. These data supported the contention that HIV-1 cure strategies for children are best evaluated using pediatric model systems.

Complex decay dynamics of HIV virions, intact and defective proviruses, and 2LTR circles following initiation of antiretroviral therapy.

In persons living with HIV-1 (PLWH) who start antiretroviral therapy (ART), plasma virus decays in a biphasic fashion to below the detection limit. The first phase reflects the short half-life (<1 d) of cells that produce most of the plasma virus. The second phase represents the slower turnover (t1/2 = 14 d) of another infected cell population, whose identity is unclear. Using the intact proviral DNA assay (IPDA) to distinguish intact and defective proviruses, we analyzed viral decay in 17 PLWH initiating ART. Circulating CD4+ T cells with intact proviruses include few of the rapidly decaying first-phase cells. Instead, this population initially decays more slowly (t1/2 = 12.9 d) in a process that largely represents death or exit from the circulation rather than transition to latency. This more protracted decay potentially allows for immune selection. After ∼3 mo, the decay slope changes, and CD4+ T cells with intact proviruses decay with a half-life of 19 mo, which is still shorter than that of the latently infected cells that persist on long-term ART. Two-long-terminal repeat (2LTR) circles decay with fast and slow phases paralleling intact proviruses, a finding that precludes their use as a simple marker of ongoing viral replication. Proviruses with defects at the 5' or 3' end of the genome show equivalent monophasic decay at rates that vary among individuals. Understanding these complex early decay processes is important for correct use of reservoir assays and may provide insights into properties of surviving cells that can constitute the stable latent reservoir.

Isolation, Synthesis, and Structure-Activity Relationship Study on Daphnane and Tigliane Diterpenes as HIV Latency-Reversing Agents.

Three new diterpenes, stellejasmins A (1) and B (2) and 12-O-benzoylphorbol-13-heptanoate (3), were isolated from the roots of Stellera chamaejasme L. The structures of 1-3 were elucidated by extensive NMR and mass spectroscopic analyses. Compounds 1 and 2 are the first derivatives containing a hydroxy group at C-2 in the family of daphnane and tigliane diterpenes. The presence of a chlorine atom in 1 is unique in the plant metabolite. Compound 3 has an odd-number acyl group, which is biosynthetically notable. Human immunodeficiency virus (HIV) LTR-driven transcription activity was tested with 1-3 and 17 known diterpenes isolated from S. chamaejasme L. and Wikstroemia retusa A.Gray. Among these, gnidimacrin (4), stelleralide A (5), and wikstroelide A (20) were highly potent, with EC50 values of 0.14, 0.33, and 0.39 nM, respectively. The structure-activity relationship (SAR) was investigated using 20 natural and eight synthetic diterpenes. This is the first SAR study on natural daphnane and tigliane diterpenes.

Voacanga globosa Spirobisindole Alkaloids Exert Antiviral Activity in HIV Latently Infected Cell Lines by Targeting the NF-kB Cascade: In Vitro and In Silico Investigations.

Since the efficiency in the transcription of the HIV genome contributes to the success of viral replication and infectivity, we investigated the downregulating effects of the spirobisindole alkaloids globospiramine (1), deoxyvobtusine (2), and vobtusine lactone (3) from the endemic Philippine medicinal plant, Voacanga globosa, during HIV gene transcription. Alkaloids 1-3 were explored for their inhibitory activity on TNF-α-induced viral replication in two latently HIV-infected cell lines, OM10.1 and J-Lat. The induction of HIV replication from OM10.1 and J-Lat cells elicited by TNF-α was blocked by globospiramine (1) within noncytotoxic concentrations. Furthermore, globospiramine (1) was found to target the NF-ĸB activation cascade in a dose-dependent manner when the transcriptional step at which inhibitory activity is exerted was examined in TNF-α-induced 293 human cells using transient reporter (luciferase) gene expression systems (HIV LTR-luc, ĸB-luc, and mutant ĸB-luc). Interrogation through molecular docking against the NF-ĸB p50/p65 heterodimer and target sites of the subunits comprising the IKK complex revealed high binding affinities of globospiramine (1) against the S281 pocket of the p65 subunit (BE = -9.2 kcal/mol) and the IKKα activation loop (BE = -9.1 kcal/mol). These findings suggest globospiramine (1) as a molecular inspiration to discover new alkaloid-based anti-HIV derivatives.

The ingenol-based protein kinase C agonist GSK445A is a potent inducer of HIV and SIV RNA transcription.

Activation of the NF-κB signaling pathway by Protein Kinase C (PKC) agonists is a potent mechanism for human immunodeficiency virus (HIV) latency disruption in vitro. However, significant toxicity risks and the lack of evidence supporting their activity in vivo have limited further evaluation of PKC agonists as HIV latency-reversing agents (LRA) in cure strategies. Here we evaluated whether GSK445A, a stabilized ingenol-B derivative, can induce HIV/simian immunodeficiency virus (SIV) transcription and virus production in vitro and demonstrate pharmacological activity in nonhuman primates (NHP). CD4+ T cells from people living with HIV and from SIV+ rhesus macaques (RM) on antiretroviral therapy (ART) exposed in vitro to 25 nM of GSK445A produced cell-associated viral transcripts as well as viral particles at levels similar to those induced by PMA/Ionomycin, indicating that GSK445A can potently reverse HIV/SIV latency. Importantly, these concentrations of GSK445A did not impair the proliferation or survival of HIV-specific CD8+ T cells, but instead, increased their numbers and enhanced IFN-γ production in response to HIV peptides. In vivo, GSK445A tolerability was established in SIV-naïve RM at 15 µg/kg although tolerability was reduced in SIV-infected RM on ART. Increases in plasma viremia following GSK445A administration were suggestive of increased SIV transcription in vivo. Collectively, these results indicate that GSK445A is a potent HIV/SIV LRA in vitro and has a tolerable safety profile amenable for further evaluation in vivo in NHP models of HIV cure/remission.

Parallel analysis of transcription, integration, and sequence of single HIV-1 proviruses.

HIV-1-infected cells that persist despite antiretroviral therapy (ART) are frequently considered "transcriptionally silent," but active viral gene expression may occur in some cells, challenging the concept of viral latency. Applying an assay for profiling the transcriptional activity and the chromosomal locations of individual proviruses, we describe a global genomic and epigenetic map of transcriptionally active and silent proviral species and evaluate their longitudinal evolution in persons receiving suppressive ART. Using genome-wide epigenetic reference data, we show that proviral transcriptional activity is associated with activating epigenetic chromatin features in linear proximity of integration sites and in their inter- and intrachromosomal contact regions. Transcriptionally active proviruses were actively selected against during prolonged ART; however, this pattern was violated by large clones of virally infected cells that may outcompete negative selection forces through elevated intrinsic proliferative activity. Our results suggest that transcriptionally active proviruses are dynamically evolving under selection pressure by host factors.

Progesterone Sporadically Induces Reactivation from Latency in Female Calves but Proficiently Stimulates Bovine Herpesvirus 1 Productive Infection.

Acute infection of the ocular, oral, or nasal cavity by bovine herpesvirus 1 (BoHV-1) culminates in lifelong latency in sensory neurons within trigeminal ganglia. The BoHV-1 latency reactivation cycle, including calves latently infected with commercially available modified live vaccines, can lead to reproductive complications, including abortions. Recent studies demonstrated progesterone stimulated BoHV-1 productive infection and sporadically induced reactivation from latency in male rabbits. The progesterone receptor (PR) and progesterone transactivate the immediate early transcription unit 1 (IEtu1) promoter and the infected cell protein 0 (bICP0) early promoter. These viral promoters drive expression of two viral transcriptional regulatory proteins (bICP0 and bICP4) that are crucial for productive infection. Based on these observations, we hypothesize that progesterone induces reactivation in a subset of calves latently infected with BoHV-1. These studies demonstrated progesterone was less efficient than dexamethasone at initiating reactivation from latency in female calves. Notably, heat stress correlated with enhancing the ability of progesterone to induce reactivation from latency. Previous studies demonstrated that heat stress activates the glucocorticoid receptor (GR), which suggested GR activation augments progesterone-mediated reactivation from latency. Additional studies revealed GR and PR cooperatively stimulated productive infection and synergistically transactivated the IEtu1 promoter when cultures were treated with dexamethasone. Mutating one or both GR binding sites in the IEtu1 promoter blocked transactivation. Collectively, these studies indicated that progesterone intermittently triggered reactivation from latency, and heat stress augmented reactivation from reactivation. Finally, these studies suggest progesterone enhances virus spread in tissues and cells where PR is abundantly expressed. IMPORTANCE Steroid hormone fluctuations are predicted to enhance or initiate bovine herpesvirus 1 (BoHV-1) replication and virus spread in cattle. For example, stress increases the incidence of BoHV-1 reactivation from latency in cattle, and the synthetic corticosteroid dexamethasone consistently induces reactivation from latency. The glucocorticoid receptor (GR) and dexamethasone stimulate key viral regulatory promoters and productive infection, in part because the viral genome contains numerous consensus GR-responsive elements (GREs). The progesterone receptor (PR) and GR belong to the type I nuclear hormone receptor family. PR and progesterone specifically bind to and transactivate viral promoters that contain GREs and stimulate BoHV-1 productive infection. Although progesterone did not induce reactivation from latency in female calves as efficiently as dexamethasone, heat stress enhanced progesterone-mediated reactivation from latency. Consequently, we predict that low levels of stressful stimuli can cooperate with progesterone to induce reactivation from latency or promote virus spread.

Latency reversal plus natural killer cells diminish HIV reservoir in vivo.

HIV is difficult to eradicate due to the persistence of a long-lived reservoir of latently infected cells. Previous studies have shown that natural killer cells are important to inhibiting HIV infection, but it is unclear whether the administration of natural killer cells can reduce rebound viremia when anti-retroviral therapy is discontinued. Here we show the administration of allogeneic human peripheral blood natural killer cells delays viral rebound following interruption of anti-retroviral therapy in humanized mice infected with HIV-1. Utilizing genetically barcoded virus technology, we show these natural killer cells efficiently reduced viral clones rebounding from latency. Moreover, a kick and kill strategy comprised of the protein kinase C modulator and latency reversing agent SUW133 and allogeneic human peripheral blood natural killer cells during anti-retroviral therapy eliminated the viral reservoir in a subset of mice. Therefore, combinations utilizing latency reversal agents with targeted cellular killing agents may be an effective approach to eradicating the viral reservoir.

Pharmacological suppression of glycogen synthase kinase-3 reactivates HIV-1 from latency via activating Wnt/ß-catenin/TCF1 axis in CD4+ T cells.

ABSTRACTHIV-1 latency posts a major obstacle for HIV-1 eradication. Currently, no desirable latency reversing agents (LRAs) have been implicated in the "Shock and Kill" strategy to mobilize the latently infected cells to be susceptible for clearance by immune responses. Identification of key cellular pathways that modulate HIV-1 latency helps to develop efficient LRAs. In this study, we demonstrate that the Wnt downstream ß-catenin/TCF1 pathway is a crucial modulator for HIV-1 latency. The pharmacological activation of the ß-catenin/TCF1 pathway with glycogen synthase kinase-3 (GSK3) inhibitors promoted transcription of HIV-1 proviral DNA and reactivated latency in CD4+ T cells; the GSK3 kinase inhibitor 6-bromoindirubin-3'-oxime (6-BIO)-induced HIV-1 reactivation was subsequently confirmed in resting CD4+ T cells from cART-suppressed patients and SIV-infected rhesus macaques. These findings advance our understanding of the mechanisms responsible for viral latency, and provide the potent LRA that can be further used in conjunction of immunotherapies to eradicate viral reservoirs.

Exacerbated AIDS Progression by PD-1 Blockade during Therapeutic Vaccination in Chronically Simian Immunodeficiency Virus-Infected Rhesus Macaques after Interruption of Antiretroviral Therapy.

The persistence of cells latently infected with HIV-1, named the latent reservoir, is the major barrier to HIV-1 eradication, and the formation and maintenance of the latent reservoir might be exacerbated by activation of the immunoinhibitory pathway and dysfunction of CD8+ T cells during HIV-1 infection. Our previous findings demonstrated that prophylactic vaccination combined with PD-1 blockade generated distinct immune response profiles and conferred effective control of highly pathogenic SIVmac239 infection in rhesus macaques. However, to our surprise, herein we found that a therapeutic vaccination in combination with PD-1 blockade resulted in activation of the viral reservoir, faster viral rebound after treatment interruption, accelerated AIDS progression, and, ultimately, death in chronically SIV-infected macaques after antiretroviral therapy (ART) interruption. Our study further demonstrated that the SIV provirus was preferentially enriched in PD-1+CD4+ T cells due to their susceptibility to viral entry, potent proliferative ability, and inability to perform viral transcription. In addition, the viral latency was effectively reactivated upon PD-1 blockade. Together, these results suggest that PD-1 blockade may be a double-edged sword for HIV-1 immunotherapy and provide important insight toward the rational design of immunotherapy strategies for an HIV-1 cure. IMPORTANCE As it is one of the most challenging public health problems, there are no clinically effective cure strategies against HIV-1 infection. We demonstrated that prophylactic vaccination combined with PD-1 blockade generated distinct immune response profiles and conferred better control of highly pathogenic SIVmac239 infection in rhesus macaques. In the present study, to our surprise, PD-1 blockade during therapeutic vaccination accelerated the reactivation of latent reservoir and AIDS progression in chronically SIV-infected macaques after ART interruption. Our study further demonstrated that the latent SIV provirus was preferentially enriched in PD-1+CD4+ T cells because of its susceptibility to viral entry, inhibition of SIV transcription, and potent ability of proliferation, and the viral latency was effectively reactivated by PD-1 blockade. Therefore, PD-1 blockade might be a double-edged sword for AIDS therapy. These findings provoke interest in further exploring novel treatments against HIV-1 infection and other emerging infectious diseases.

Bryostatin-1 Decreases HIV-1 Infection and Viral Production in Human Primary Macrophages.

While combination antiretroviral therapy maintains undetectable viremia in people living with HIV (PLWH), a lifelong treatment is necessary to prevent viremic rebound after therapy cessation. This rebound seemed mainly caused by long-lived HIV-1 latently infected cells reverting to a viral productive status. Reversing latency and elimination of these cells by the so-called shock-and-kill strategy is one of the main investigated leads to achieve an HIV-1 cure. Small molecules referred to as latency reversal agents (LRAs) proved to efficiently reactivate latent CD4+ T cells. However, the LRA impact on de novo infection or HIV-1 production in productively infected macrophages remains elusive. Nontoxic doses of bryostatin-1, JQ1, and romidepsin were investigated in human monocyte-derived macrophages (MDMs). Treatment with bryostatin-1 or romidepsin resulted in a downregulation of CD4 and CCR5 receptors, respectively, accompanied by a reduction of R5 tropic virus infection. HIV-1 replication was mainly regulated by receptor modulation for bryostatin-1, while romidepsin effects rely on upregulation of SAMHD1 activity. LRA stimulation of chronically infected cells did not enhance HIV-1 production or gene expression. Surprisingly, bryostatin-1 caused a major decrease in viral production. This effect was not viral strain specific but appears to occur only in myeloid cells. Bryostatin-1 treatment of infected MDMs led to decreased amounts of capsid and matrix mature proteins with little to no modulation of precursors. Our observations revealed that bryostatin-1-treated myeloid and CD4+ T cells respond differently upon HIV-1 infection. Therefore, additional studies are warranted to more fully assess the efficiency of HIV-1 eradicating strategies. IMPORTANCE HIV-1 persists in a cellular latent form despite therapy that quickly propagates infection upon treatment interruption. Reversing latency would contribute to eradicate these cells, closing the gap to a cure. Macrophages are an acknowledged HIV-1 reservoir during therapy and are suspected to harbor latency establishment in vivo. However, the impact of latency reversal agents (LRAs) on HIV-1 infection and viral production in human macrophages is poorly known but nonetheless crucial to probe the safety of this strategy. In this in vitro study, we discovered encouraging antireplicative features of distinct LRAs in human macrophages. We also described a new viral production inhibition mechanism by protein kinase C agonists that is specific to myeloid cells. This study provides new insights into HIV-1 propagation restriction potentials by LRAs in human macrophages and underline the importance of assessing latency reversal strategy on all HIV-1-targeted cells.