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Background: Marijuana's putative anti-inflammatory properties may benefit HIV-associated comorbidities. How recreational marijuana use affects gene expression in peripheral blood cells (PBC) among youth with HIV-1 (YWH) is unknown., Approach: YWH with defined substance use (n = 54) receiving similar antiretroviral therapy (ART) were assigned to one of four analysis groups: YWH with detectable plasma HIV-1 (> 50 RNA copies/ml) who did not use substances (H+V+S-), and YWH with undetectable plasma HIV-1 who did not use substances (H+V-S-), or used marijuana alone (H+V-S+[M]), or marijuana in combination with tobacco (H+V-S+[M/T]). Non-substance using youth without HIV infection (H-S-, n = 25) provided a reference group. PBC mRNA was profiled by Affymetrix GeneChip Human Genome U133 Plus 2.0 Array. Differentially expressed genes (DEG) within outcome groups were identified by Significance Analysis of Microarrays and used for Hierarchical Clustering, Principal Component Analysis, and Ingenuity Pathways Analysis., Results: HIV-1 replication resulted in > 3000 DEG involving 27 perturbed pathways. Viral suppression reduced DEG to 313, normalized all 27 pathways, and down-regulated two additional pathways, while marijuana use among virally suppressed YWH resulted in 434 DEG and no perturbed pathways. Relative to H+V-S-, multiple DEG normalized in H+V-S+[M]. In contrast, H+V-S+[M/T] had 1140 DEG and 10 dysregulated pathways, including multiple proinflammatory genes and six pathways shared by H+V+S-., Conclusions: YWH receiving ART display unique transcriptome bioprofiles based on viral replication and substance use. In the context of HIV suppression, marijuana use, alone or combined with tobacco, has opposing effects on inflammatory gene expression., (© 2022. The Author(s).)

In people with HIV (PWH) on antiretroviral therapy (ART), immune dysfunction persists, including elevated expression of immune checkpoint (IC) proteins on total and HIV-specific T cells. Reversing immune exhaustion is one strategy to enhance the elimination of HIV-infected cells that persist in PWH on ART. We aimed to evaluate whether blocking CTL-associated protein 4 (CTLA-4), programmed cell death protein 1 (PD-1), T cell Ig domain and mucin domain 3 (TIM-3), T cell Ig and ITIM domain (TIGIT) and lymphocyte activation gene-3 (LAG-3) alone or in combination would enhance HIV-specific CD4 + and CD8 + T cell function ex vivo. Intracellular cytokine staining was performed using human PBMCs from PWH on ART ( n = 11) and expression of CD107a, IFN-γ, TNF-α, and IL-2 was quantified with HIV peptides and Abs to IC. We found the following: 1) IC blockade enhanced the induction of CD107a and IL-2 but not IFN-γ and TNF-α in response to Gag and Nef peptides; 2) the induction of CD107a and IL-2 was greatest with multiple combinations of two Abs; and 3) Abs to LAG-3, CTLA-4, and TIGIT in combinations showed synergistic induction of IL-2 in HIV-specific CD8 + and CD107a and IL-2 production in HIV-specific CD4 + and CD8 + T cells. These results demonstrate that the combination of Abs to LAG-3, CTLA-4, or TIGIT can increase the frequency of cells expressing CD107a and IL-2 that associated with cytotoxicity and survival of HIV-specific CD4 + and CD8 + T cells in PWH on ART. These combinations should be further explored for an HIV cure., (Copyright © 2021 by The American Association of Immunologists, Inc.)

Background: HIV-1 infects a wide range of CD4 + T cells with different phenotypic properties and differing expression levels of entry coreceptors. We sought to determine the viral tropism of subtype C (C-HIV) Envelope (Env) clones for different CD4 + T cell subsets and whether tropism changes during acute to chronic disease progression. HIV-1 envs were amplified from the plasma of five C-HIV infected women from three untreated time points; less than 2 months, 1-year and 3-years post-infection. Pseudoviruses were generated from Env clones, phenotyped for coreceptor usage and CD4 + T cell subset tropism was measured by flow cytometry., Results: A total of 50 C-HIV envs were cloned and screened for functionality in pseudovirus infection assays. Phylogenetic and variable region characteristic analysis demonstrated evolution in envs between time points. We found 45 pseudoviruses were functional and all used CCR5 to mediate entry into NP2/CD4/CCR5 cells. In vitro infection assays showed transitional memory (TM) and effector memory (EM) CD4 + T cells were more frequently infected (median: 46% and 25% of total infected CD4 + T cells respectively) than naïve, stem cell memory, central memory and terminally differentiated cells. This was not due to these subsets contributing a higher proportion of the CD4 + T cell pool, rather these subsets were more susceptible to infection (median: 5.38% EM and 2.15% TM cells infected), consistent with heightened CCR5 expression on EM and TM cells. No inter- or intra-participant changes in CD4 + T cell subset tropism were observed across the three-time points., Conclusions: CD4 + T cell subsets that express more CCR5 were more susceptible to infection with C-HIV Envs, suggesting that these may be the major cellular targets during the first 3 years of infection. Moreover, we found that viral tropism for different CD4 + T cell subsets in vitro did not change between Envs cloned from acute to chronic disease stages. Finally, central memory, naïve and stem cell memory CD4 + T cell subsets were susceptible to infection, albeit inefficiently by Envs from all time-points, suggesting that direct infection of these cells may help establish the latent reservoir early in infection.

HIV latency is the major barrier to a cure for people living with HIV (PLWH) on antiretroviral therapy (ART) because the virus persists in long-lived non-proliferating and proliferating latently infected CD4+ T cells. Latently infected CD4+ T cells do not express viral proteins and are therefore not visible to immune mediated clearance. Therefore, identifying interventions that can reverse latency and also enhance immune mediated clearance is of high interest. Interferons (IFNs) have multiple immune enhancing effects and can inhibit HIV replication in activated CD4+ T cells. However, the effects of IFNs on the establishment and reversal of HIV latency is not understood. Using an in vitro model of latency, we demonstrated that plasmacytoid dendritic cells (pDC) inhibit the establishment of HIV latency through secretion of type I IFNα, IFNβ and IFNω but not IFNε or type III IFNλ1 and IFNλ3. However, once latency was established, IFNα but no other IFNs were able to efficiently reverse latency in both an in vitro model of latency and CD4+ T cells collected from PLWH on suppressive ART. Binding of IFNα to its receptor expressed on primary CD4+ T cells did not induce activation of the canonical or non-canonical NFκB pathway but did induce phosphorylation of STAT1, 3 and 5 proteins. STAT5 has been previously demonstrated to bind to the HIV long terminal repeat and activate HIV transcription. We demonstrate diverse effects of interferons on HIV latency with type I IFNα; inhibiting the establishment of latency but also reversing HIV latency once latency is established., Competing Interests: I have read the journal's policy and the authors of this manuscript have the following competing interests: Sharon R Lewin has received grant funding for investigator initiated studies from Gilead Sciences, Merck and Viiv Healthcare. Her institution has received payment for educational and advisory work from Merck, Viiv Healthcare, Abivax and Tetralogic.

HIV infection requires lifelong antiretroviral therapy because of the persistence of latently infected CD4 + T cells. The induction of virus expression from latently infected cells occurs following T cell receptor (TCR) activation, but not all latently infected cells respond to TCR stimulation. We compared two models of latently infected cells using an enhanced green fluorescent protein (EGFP) reporter virus to infect CCL19-treated resting CD4 + (rCD4 + ) T cells (preactivation latency) or activated CD4 + T cells that returned to a resting state (postactivation latency). We isolated latently infected cells by sorting for EGFP-negative (EGFP - ) cells after infection. These cells were cultured with antivirals and stimulated with anti-CD3/anti-CD28, mitogens, and latency-reversing agents (LRAs) and cocultured with monocytes and anti-CD3. Spontaneous EGFP expression was more frequent in postactivation than in preactivation latency. Stimulation of latently infected cells with monocytes/anti-CD3 resulted in an increase in EGFP expression compared to that for unstimulated controls using the preactivation latency model but led to a reduction in EGFP expression in the postactivation latency model. The reduced EGFP expression was not associated with reductions in the levels of viral DNA or T cell proliferation but depended on direct contact between monocytes and T cells. Monocytes added to the postactivation latency model during the establishment of latency reduced spontaneous virus expression, suggesting that monocyte-T cell interactions at an early time point postinfection can maintain HIV latency. This direct comparison of pre- and postactivation latency suggests that effective strategies needed to reverse latency will depend on how latency is established. IMPORTANCE One strategy being evaluated to eliminate latently infected cells that persist in HIV-infected individuals on antiretroviral therapy (ART) is to activate HIV expression or production with the goal of inducing virus-mediated cytolysis or immune-mediated clearance of infected cells. The gold standard for the activation of latent virus is T cell receptor stimulation with anti-CD3/anti-CD28. However, this stimulus activates only a small proportion of latently infected cells. We show clear differences in the responses of latently infected cells to activating stimuli based on how latent infection is established, an observation that may potentially explain the persistence of noninduced intact proviruses in HIV-infected individuals on ART., (Copyright © 2018 American Society for Microbiology.)

Background: Whether ongoing virus replication occurs in HIV-infected individuals on antiretroviral therapy (ART) is unclear; therefore, whether residual virus replication is a barrier to achieving a cure for HIV is also unknown. We aimed to establish whether ART intensification with dolutegravir would reveal or affect residual virus replication in HIV-infected individuals on suppressive treatment., Methods: In this randomised, placebo-controlled, double-blind trial, we enrolled HIV-infected adults (aged 18 years and older) receiving combination ART (at least three agents) for at least 3 years from the Alfred Hospital and Melbourne Sexual Health Centre, Melbourne, VIC, Australia. Eligible participants had fewer than 50 copies per mL HIV-1 plasma RNA for more than 3 years and fewer than 20 copies per mL at screening and two CD4 counts higher than 350 cells per μL in the previous 24 months including screening. Participants were randomly assigned (1:1) to receive 50 mg oral dolutegravir or placebo once a day for 56 days in addition to background ART. Follow-up was done at days 1, 3, 7, 14, 28, 56, and 84. The primary outcome was the change from baseline in frequency of 2-long terminal repeat (2-LTR) circles in peripheral blood CD4 cells at day 7. This trial is registered with ClinicalTrials.gov, number NCT02500446., Findings: Between Sept 21, 2015, and Sept 19, 2016, 46 individuals were screened for inclusion. 40 were eligible for inclusion and were randomly assigned to the dolutegravir (n=21) or placebo group (n=19). All enrolled participants completed the study procedures and no individuals were lost to follow up. All participants were on suppressive ART with 12% receiving protease inhibitors and the others non-nucleoside reverse transcriptase inhibitors. Median 2-LTR circles fold-change from baseline to day 7 was -0·17 (IQR -0·90 to 0·90) in the dolutegravir group and -0·26 (-1·00 to 1·17) in the placebo group (p=0·17). The addition of dolutegravir to pre-existing ART regimens was safe and there were no treatment discontinuations or treatment-related serious adverse events., Interpretation: Our findings show that in HIV-infected individuals on modern suppressive ART regimens, residual replication is rare, if at all present, and was not recorded in blood after dolutegravir intensification. Because tissue biopsies were not done we cannot exclude the possibility of residual virus replication in tissue. Strategies other than ART alone are needed to eliminate HIV persistence on treatment., Funding: ViiV Healthcare., (Copyright © 2018 Elsevier Ltd. All rights reserved.)

Immune activation plays a critical role in HIV-1 pathogenesis, but the pathways that are responsible for HIV-1-associated immune activation are not well understood. Recent advances in the development of a humanized mouse model for HIV-1 infection might provide new approaches to study the mechanisms of HIV-1 associated immune activation, and to test interventions aimed at reducing HIV-1 pathogenesis.

Objective: To study the cytokine/chemokine profiles in response to HIV-1 viremia, and elucidate the pathways leading to HIV-1-induced inflammation., Design/methods: Plasma levels of 19 cytokines in individuals with early HIV-1 infection and individuals undergoing treatment interruptions were evaluated via multiplex assay. To investigate the cellular sources of relevant cytokines, sorted cells from HIV-1 infected individuals were assessed for mRNA expression. Relevant signaling pathways were assessed by comparing cytokine production patterns of peripheral blood mononuclear cells stimulated with intact HIV-1 or specific Toll-like receptor (TLR) stimulants with and without a TLR7/9 antagonist., Results: IP-10 plasma concentration was most significantly associated with HIV-1 viral load and was the most significant contributor in a multivariate model. IP-10 mRNA was highly expressed in monocytes and mDCs and these cells were the dominant producers after in-vitro stimulation with TLR7/8 ligands (CL097 and ssRNAGag1166), AT-2 HIV-1, and HIV-1NL43 virus. Partial least square discriminant analysis of culture supernatants revealed distinct cytokine/chemokine secretion profiles associated with intact viruses compared with TLR7/8 ligands alone, with IP-10 production linked to the former. A TLR7/9 antagonist blocked IP-10 production following whole virus stimulation, suggesting the involvement of TLR7/9 in the recognition of HIV-1 by these cells., Conclusion: Monocytes and mDCs produce significant amounts of IP-10 in response to HIV-1 viremia and after in-vitro stimulation with HIV-1. Stimulation with HIV-1-derived TLR7/8-ligands versus HIV-1 resulted in distinct cytokine/chemokine profiles, indicating additional pathways other than TLR7/8 that lead to the activation of innate immune cells by HIV-1.

Background: Clinical studies have shown faster disease progression and stronger immune activation in human immunodeficiency virus (HIV)-1-infected females when compared with males for the same level of HIV-1 replication. Here we determine whether the elevated levels of HIV-1-induced interferon-alpha (IFN-α) production observed in females are associated with higher interferon-stimulated gene (ISG) expression levels in T cells, hence suggesting type-I IFN as a mechanism for the higher HIV-1-associated immune activation observed., Methods: T-cell and dendritic cell populations were isolated from treatment-naive chronically HIV-1-infected individuals enrolled in the Adult Clinical Trials Group 384 by fluorescence-activated cell sorting. The expression of 98 genes involved in Toll-like receptor and type I IFN signaling pathways were quantified using Nanostring technology., Results: Several ISGs were significantly correlated with HIV-1 viral load and/or CD4(+) T-cell count. Higher expression levels of a subset of these ISGs were observed in cells derived from females as compared to males after adjusting for viral load and were correlated to higher levels of T-cell activation., Conclusion: These data show that higher IFN-α production is associated with higher ex vivo expression of several ISGs in females. This might contribute to higher levels of immune activation and the observed faster HIV-1 disease progression in females for a given level of viral replication.

Objective and Design: The objective of this study was to determine changes in toll-like receptor (TLR) responses of monocytes, myeloid dendritic cells and plasmacytoid dendritic cells during primary and chronic HIV-1 infection. TLRs serve as important innate receptors to sense pathogens, and have been implicated in mediating immune activation in HIV-1 infection. Studies assessing the consequences of HIV-1 infection on the ability of innate immune cells to respond to TLR stimulation have come to varying conclusions., Methods: Using intracellular flow cytometry, cytokine production by cryopreserved peripheral blood mononuclear cells from healthy controls and HIV-1-infected individuals were examined after TLR stimulation., Results: We observed that the effect of HIV-1 infection on TLR responses not only depended on the stage of HIV-1 infection, but was also dependent on the individual receptor and cell type examined. Monocyte and myeloid dendritic cell responses to TLR8 stimulation were associated with HIV-1 viral load and CD4 T-cell count, whereas plasmacytoid dendritic cell responses to TLR7 stimulation were not. Responses to TLR2 stimulation were not affected by HIV-1 infection, whereas responses to TLR9 stimulation were universally decreased in all HIV-1-infected individuals examined regardless of treatment or clinical parameters., Conclusion: Responsiveness to TLR7/8 stimulation, which have been shown to recognize HIV-1 ssRNA, did not decrease in chronic infection, and may represent a contributing factor to ongoing T-cell immune activation in the setting of chronic viremic HIV-1 infection.

There is growing evidence that highlights the role of the immune response during acute human immunodeficiency virus type 1 (HIV-1) infection in the control or development of disease. The adaptive immune responses do not appear until after HIV-1 infection is already well established, so the role of earlier and faster-responding innate immunity needs to be more closely scrutinized. In particular, 2 aspects of innate immunity for which there are growing research developments will be examined in this review: the actions of type I interferons and natural killer cells. These two components of the innate immune response contribute to viral control both by killing infected cells and by modulating other immune cells that develop. However, the role of interferon α in immune activation is a double-edged sword, causing recruitment of adaptive immune cells that can assist in viral control but concurrently contributing to immune activation-dependent disease progression. Understanding the complexity of how innate responses affect the outcome of HIV-1 infection will help in the development of vaccines that can use innate immunity to enhance viral control with minimal pathogenesis.

Background: Chronic HIV-1 infection is characterized by high levels of persistent immune activation. Both HIV-1-encoded Toll-like receptor 7/8 (TLR7/8) ligands and TLR ligands encoded by products of microbial translocation have been implicated in inducing and sustaining immune activation in infected individuals, but the consequences of simultaneous exposure to different TLR ligands are not well understood., Objective: To examine the impact of preexposure of monocytes to HIV-1-encoded TLR8 ligands on their ability to respond to subsequent stimulation with microbial TLR2/4 ligands., Method: Stable monocytic cell lines (THP-1-Blue-CD14 cells) or primary monocytes were stimulated with ligands for TLR2, TLR4, and TLR8, including chemically inactivated HIV-1, alone, or in sequential combinations. Responses by THP-1 cells to TLR stimulation were quantified using Quanti-Blue colometric assay, and TLR-induced tumor necrosis factor-alpha production of primary monocytes was quantified by intracellular cytokine staining using flow cytometry., Results: The exposure of monocytes to HIV-1 or HIV-1-derived TLR8 ligands sensitized these cells for TLR4 stimulation, resulting in a significantly higher response to lipopolysaccharide compared to cells that were not prestimulated with TLR8 ligands or HIV-1., Conclusion: TLR crosstalk can enhance the pro-inflammatory monocytes response to products of microbial translocation and might play an important role in the modulation of immune function in HIV-1 infection.

Following treatment of hepatitis B virus (HBV) monoinfection, HBV-specific T-cell responses increase significantly; however, little is known about the recovery of HBV-specific T-cell responses following HBV-active highly active antiretroviral therapy (HAART) in HIV-HBV coinfected patients. HIV-HBV coinfected patients who were treatment naïve and initiating HBV-active HAART were recruited as part of a prospective cohort study in Thailand and followed for 48 weeks (n = 24). Production of gamma interferon (IFN-gamma) and tumor necrosis factor alpha (TNF-alpha) in both HBV- and HIV-specific CD8(+) T cells was quantified using intracellular cytokine staining on whole blood. Following HBV-active HAART, the median (interquartile range) log decline from week 0 to week 48 for HBV DNA was 5.8 log (range, 3.4 to 6.7) IU/ml, and for HIV RNA it was 3.1 (range, 2.9 to 3.5) log copies/ml (P < 0.001 for both). The frequency of HIV Gag-specific CD8(+) T-cell responses significantly decreased (IFN-gamma, P < 0.001; TNF-alpha, P = 0.05). In contrast, there was no significant change in the frequency (IFN-gamma, P = 0.21; TNF-alpha, P = 0.61; and IFN-gamma and TNF-alpha, P = 0.11) or magnitude (IFN-gamma, P = 0.13; TNF-alpha, P = 0.13; and IFN-gamma and TNF-alpha, P = 0.13) of HBV-specific CD8(+) T-cell responses over 48 weeks of HBV-active HAART. Of the 14 individuals who were HBV e antigen (HBeAg) positive, 5/14 (36%) lost HBeAg during the 48 weeks of follow-up. HBV-specific CD8(+) T cells were detected in 4/5 (80%) of patients prior to HBeAg loss. Results from this study show no sustained change in the HBV-specific CD8(+) T-cell response following HBV-active HAART. These findings may have implications for the duration of treatment of HBV in HIV-HBV coinfected patients, particularly in HBeAg-positive disease.

Hepatitis B virus (HBV)-specific T cells play a key role both in the control of HBV replication and in the pathogenesis of liver disease. Human immunodeficiency virus type 1 (HIV-1) coinfection and the presence or absence of HBV e (precore) antigen (HBeAg) significantly alter the natural history of chronic HBV infection. We examined the HBV-specific T-cell responses in treatment-naïve HBeAg-positive and HBeAg-negative HIV-1-HBV-coinfected (n = 24) and HBV-monoinfected (n = 39) Asian patients. Peripheral blood was stimulated with an overlapping peptide library for the whole HBV genome, and tumor necrosis factor alpha and gamma interferon cytokine expression in CD8+ T cells was measured by intracellular cytokine staining and flow cytometry. There was no difference in the overall magnitude of the HBV-specific T-cell responses, but the quality of the response was significantly impaired in HIV-1-HBV-coinfected patients compared with monoinfected patients. In coinfected patients, HBV-specific T cells rarely produced more than one cytokine and responded to fewer HBV proteins than in monoinfected patients. Overall, the frequency and quality of the HBV-specific T-cell responses increased with a higher CD4+ T-cell count (P = 0.018 and 0.032, respectively). There was no relationship between circulating HBV-specific T cells and liver damage as measured by activity and fibrosis scores, and the HBV-specific T-cell responses were not significantly different in patients with either HBeAg-positive or HBeAg-negative disease. The quality of the HBV-specific T-cell response is impaired in the setting of HIV-1-HBV coinfection and is related to the CD4+ T-cell count.

Manifestations of viral infections can differ between women and men, and marked sex differences have been described in the course of HIV-1 disease. HIV-1-infected women tend to have lower viral loads early in HIV-1 infection but progress faster to AIDS for a given viral load than men. Here we show substantial sex differences in the response of plasmacytoid dendritic cells (pDCs) to HIV-1. pDCs derived from women produce markedly more interferon-alpha (IFN-alpha) in response to HIV-1-encoded Toll-like receptor 7 (TLR7) ligands than pDCs derived from men, resulting in stronger secondary activation of CD8(+) T cells. In line with these in vitro studies, treatment-naive women chronically infected with HIV-1 had considerably higher levels of CD8(+) T cell activation than men after adjusting for viral load. These data show that sex differences in TLR-mediated activation of pDCs may account for higher immune activation in women compared to men at a given HIV-1 viral load and provide a mechanism by which the same level of viral replication might result in faster HIV-1 disease progression in women compared to men. Modulation of the TLR7 pathway in pDCs may therefore represent a new approach to reduce HIV-1-associated pathology.