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A significant number of persons with coronavirus disease 2019 (COVID-19) experience persistent, recurrent, or new symptoms several months after the acute stage of severe acute respiratory syndrome coronavirus 2 (SARS‑CoV‑2) infection. This phenomenon, termed Post-Acute Sequelae of SARS-CoV-2 (PASC) or Long COVID, is associated with high viral titers during acute infection, a persistently hyperactivated immune system, tissue injury by NETosis-induced micro-thrombofibrosis (NETinjury), microbial translocation, complement deposition, fibrotic macrophages, the presence of auto-antibodies, and lymphopenic immune environments. Here, we review the current literature on the immunological imbalances that occur during PASC. Specifically, we focus on data supporting common immunopathogenesis and tissue injury mechanisms shared across this highly heterogenous disorder including NETosis, coagulopathy, and fibrosis. Mechanisms include changes in leukocyte subsets/functions, fibroblast activation, cytokine imbalances, lower cortisol, autoantibodies, co-pathogen reactivation, and residual immune activation driven by persistent viral antigens and/or microbial translocation. Taken together, we develop the premise that SARS-CoV-2 infection results in PASC as a consequence of acute and/or persistent single or multiple organ injury mediated by PASC determinants to include degree of host response (inflammation, NETinjury), residual viral antigen (persistent antigen) and exogenous factors (microbial translocation). Determinants of PASC may be amplified by co-morbidities, age, and sex. Keywords: long COVID, PASC, long haulers, NETosis, T cell, NK cell, DC, neutrophil

Understanding the complexity of the long-lived HIV reservoir during antiretroviral therapy (ART) remains a considerable impediment in research towards a cure for HIV. To address this, we developed a single-cell strategy to precisely define the unperturbed peripheral blood HIV-infected memory CD4+ T cell reservoir from ART-treated people living with HIV (ART-PLWH) via the presence of integrated accessible proviral DNA in concert with epigenetic and cell surface protein profiling. We identified profound reservoir heterogeneity within and between ART-PLWH, characterized by new and known surface markers within total and individual memory CD4+ T cell subsets. We further uncovered new epigenetic profiles and transcription factor motifs enriched in HIV-infected cells that suggest infected cells with accessible provirus, irrespective of reservoir distribution, are poised for reactivation during ART treatment. Together, our findings reveal the extensive inter- and intrapersonal cellular heterogeneity of the HIV reservoir, and establish an initial multiomic atlas to develop targeted reservoir elimination strategies.

The human endogenous protein Galectin-9 (Gal-9) reactivates latently HIV-infected cells in vitro and ex vivo, which may allow for immune-mediated clearance of these cells. However, Gal-9 also activates several immune cells, which could negatively affect HIV persistence by promoting chronic activation/exhaustion. This potential "double-edged sword" effect of Gal-9 raises the question of the overall impact of Gal-9 on HIV persistence in vivo.We used the BLT (bone marrow, liver, thymus) humanized mouse model to evaluate the impact of Gal-9 on HIV persistence in vivo during antiretroviral therapy (ART).Two independent cohorts of ART-suppressed HIV-infected BLT mice were treated with either recombinant Gal-9 or PBS control. Plasma viral loads and levels of tissue-associated HIV DNA and RNA were measured by qPCR. Immunohistochemistry and HIV RNAscope were used to quantify CD4+ T, myeloid, and HIV RNA+ cells in tissues. T cell activation and exhaustion were measured by flow cytometry, and plasma markers of inflammation were measured by multiplex cytokine arrays.Gal-9 did not induce plasma markers of inflammation or T cell markers of activation/exhaustion in vivo. However, the treatment significantly increased levels of tissue-associated HIV DNA and RNA compared to controls (P = 0.0007 and P = 0.011, respectively, for cohort I and P = 0.002 and P = 0.005, respectively, for cohort II). RNAscope validated the Gal-9 mediated induction of HIV RNA in tissue-associated myeloid cells, but not T cells.Our study highlights the overall adverse effects of Gal-9 on HIV persistence and the potential need to block Gal-9 interactions during ART-suppressed HIV infection.

Opioid use has negative effects on immune responses and may impair immune reconstitution in persons living with HIV (PLWH) infection undergoing antiretroviral treatment (ART). The effects of treatment with μ opioid receptor (MOR) agonists (e.g., methadone, MET) and antagonists (e.g., naltrexone, NTX) on immune reconstitution and immune activation in ART-suppressed PLWH have not been assessed in-depth. We studied the effects of methadone or naltrexone on measures of immune reconstitution and immune activation in a cross-sectional community cohort of 30 HIV-infected individuals receiving suppressive ART and medications for opioid use disorder (MOUD) (12 MET, 8 NTX and 10 controls). Plasma markers of inflammation and immune activation were measured using ELISA, Luminex, or Simoa. Plasma IgG glycosylation was assessed using capillary electrophoresis. Cell subsets and activation were studied using whole blood flow cytometry. Individuals in the MET group, but no in the NTX group, had higher plasma levels of inflammation and immune activation markers than controls. These markers include soluble CD14 (an independent predictor of morbidity and mortality during HIV infection), proinflammatory cytokines, and proinflammatory IgG glycans. This effect was independent of time on treatment. Our results indicate that methadone-based MOUD regimens may sustain immune activation and inflammation in ART-treated HIV-infected individuals. Our pilot study provides the foundation and rationale for future longitudinal functional studies of the impact of MOUD regimens on immune reconstitution and residual activation after ART-mediated suppression.

Despite effective combination antiretroviral therapy (cART), people living with HIV (PLWH) continue to harbor replication-competent and transcriptionally active virus in infected cells, which in turn can lead to ongoing viral antigen production, chronic inflammation, and increased risk of age-related comorbidities. To identify new agents that may inhibit postintegration HIV beyond cART, we screened a library of 512 pure compounds derived from natural products and identified (–)-hopeaphenol as an inhibitor of HIV postintegration transcription at low to submicromolar concentrations without cytotoxicity. Using a combination of global RNA sequencing, plasmid-based reporter assays, and enzyme activity studies, we document that hopeaphenol inhibits protein kinase C (PKC)- and downstream NF-κB-dependent HIV transcription as well as a subset of PKC-dependent T-cell activation markers, including interleukin-2 (IL-2) cytokine and CD25 and HLA-DRB1 RNA production.

SARS-CoV-2, the causative agent of COVID-19, continues to cause global morbidity and mortality despite the increasing availability of vaccines. Alongside vaccines, antivirals are urgently needed to combat SARS-CoV-2 infection and spread, particularly in resource-limited regions which lack access to existing therapeutics. Small molecules isolated from medicinal plants may be able to block cellular entry by SARS-CoV-2 by antagonising the interaction of the viral spike glycoprotein receptor-binding domain (RBD) with the host angiotensin-converting enzyme II (ACE2) receptor. As the medicinal plant Gunnera perpensa L. is being used by some South African traditional healers for SARS-CoV-2/COVID-19 management, we hypothesised that it may contain chemical constituents that inhibit the RBD-ACE2 interaction. Using a previously described AlphaScreen-based protein interaction assay, we show here that the DCM:MeOH extract of G. perpensa readily disrupts RBD (USA-WA1/2020)-ACE2 interactions with a half-maximal inhibition concentration (IC

In this study, we evaluated the ability of negatively charged bio-degradable nanoparticles, ONP- 302, to inhibit tumor growth. Therapeutic treatment with ONP-302 in vivo resulted in a marked delay in tumor growth in three different syngeneic tumor models in immunocompetent mice. ONP- 302 efficacy persisted with depletion of CD8+ T cells in immunocompetent mice and also was effective in immune deficient mice. Examination of ONP-302 effects on components of the tumor microenvironment (TME) were explored. ONP-302 treatment caused a gene expression shift in TAMs toward the pro-inflammatory M1 type and substantially inhibited the expression of genes associated with the pro-tumorigenic function of CAFs. ONP-302 also induced apoptosis in CAFs in the TME. Together, these data support further development of ONP-302 as a novel first-in- class anti-cancer therapeutic that can be used as a single-agent as well as in combination therapies for the treatment of solid tumors due to its ability to modulate the TME.

Supplemental Tables 1-6 Supplemental Table 1. Results of some recent trials of chemotherapy in MPM. Supplemental Table 2. Post-Trial Therapies Supplemental Table 3. Characteristics of Patients Selected for Flow Cytometry Analysis Supplemental Table 4. List of Immune Response Genes assayed by Nanostring Supplemental Table 5: Correlation of Antibody Response to MOS or Radiographic Response Supplemental Table 6. Flow Cytometry Results

Purpose: “In situ vaccination” using immunogene therapy has the ability to induce polyclonal antitumor responses directed by the patient's immune system.Experimental Design: Patients with unresectable malignant pleural mesothelioma (MPM) received two intrapleural doses of a replication-defective adenoviral vector containing the human IFNα2b gene (Ad.IFN) concomitant with a 14-day course of celecoxib followed by chemotherapy. Primary outcomes were safety, toxicity, and objective response rate; secondary outcomes included progression-free and overall survival. Biocorrelates on blood and tumor were measured.Results: Forty subjects were treated: 18 received first-line pemetrexed-based chemotherapy, 22 received second-line chemotherapy with pemetrexed (n = 7) or gemcitabine (n = 15). Treatment was generally well tolerated. The overall response rate was 25%, and the disease control rate was 88%. Median overall survival (MOS) for all patients with epithelial histology was 21 months versus 7 months for patients with nonepithelial histology. MOS in the first-line cohort was 12.5 months, whereas MOS for the second-line cohort was 21.5 months, with 32% of patients alive at 2 years. No biologic parameters were found to correlate with response, including numbers of activated blood T cells or NK cells, regulatory T cells in blood, peak levels of IFNα in blood or pleural fluid, induction of antitumor antibodies, nor an immune-gene signature in pretreatment biopsies.Conclusions: The combination of intrapleural Ad.IFN, celecoxib, and chemotherapy proved safe in patients with MPM. OS rate was significantly higher than historical controls in the second-line group. Results of this study support proceeding with a multicenter randomized clinical trial of chemo-immunogene therapy versus standard chemotherapy alone. Clin Cancer Res; 22(15); 3791–800. ©2016 AACR.

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Additional Trial related information and methods

Supplemental Figure 1. Immunoblots of Patient Serum before and after Therapy Supplemental Figure 2. Distribution of 1/Nab Titers for the 40 Patients Supplemental Figure 3. Distribution of the Day 2 serum levels of Interferon-alpha Supplemental Figure 4. Lack of Correlation of lymphocyte or macrophage infiltration with survival. Supplemental Figure 5. Lack of Correlation of PDL1 staining with survival. Supplemental Figure 6. Correlation of the mRNA immunoscore with survival.

Current antiviral drug discovery efforts face many challenges, including development of new drugs during an outbreak and coping with drug resistance due to rapidly accumulating viral mutations. Emerging artificial intelligence and machine learning (AI/ML) methods can accelerate anti-infective drug discovery and have the potential to reduce overall development costs in Low and Middle-Income Countries (LMIC), which in turn may help to develop new and/or accessible therapies against communicable diseases within these countries. While the marketplace currently offers a plethora of data-driven AI/ML tools, most to date have been developed within the context of non-communicable diseases like cancer, and several barriers have limited the translation of existing tools to the discovery of drugs against infectious diseases. Here, we provide a perspective on the benefits, limitations, and pitfalls of AI/ML tools in the discovery of novel therapeutics with a focus on antivirals. We also discuss available and emerging data sharing models including intellectual property-preserving AI/ML. In addition, we review available data sources and platforms and provide examples for low-cost and accessible screening methods and other virus-based bioassays suitable for implementation of AI/ML-based programs in LMICs. Finally, we introduce an emerging AI/ML-based Center in Cameroon (Central Africa) which is currently developing methods and tools to promote local, independent drug discovery and represents a model that could be replicated among LMIC globally.

Cancer immunotherapy often depends on recognition of peptide epitopes by cytotoxic T lymphocytes (CTLs). The tumor microenvironment (TME) is enriched for peroxynitrite (PNT), a potent oxidant produced by infiltrating myeloid cells and some tumor cells. We demonstrate that PNT alters the profile of MHC class I bound peptides presented on tumor cells. Only CTLs specific for PNT-resistant peptides have a strong antitumor effect in vivo, whereas CTLs specific for PNT-sensitive peptides are not effective. Therapeutic targeting of PNT in mice reduces resistance of tumor cells to CTLs. Melanoma patients with low PNT activity in their tumors demonstrate a better clinical response to immunotherapy than patients with high PNT activity. Our data suggest that intratumoral PNT activity should be considered for the design of neoantigen-based therapy and also may be an important immunotherapeutic target.

Inhibition of the BCL6 BTB domain results in killing Diffuse Large B-cell Lymphoma (DLBL) cells, reducing the T-cell dependent germinal center (GC) reaction in mice, and reversing GC hyperplasia in nonhuman primates. The available BCL6 BTB-specific inhibitors are poorly water soluble thus limiting their absorption in vivo and our understanding of therapeutic strategy targeting GC. We synthesized a prodrug (AP-4-287) from a potent BCL6 BTB inhibitor (FX1) with improved aqueous solubility and pharmacokinetics (PK) in mice. We also evaluated its in vivo biological activity on humoral immune responses using the sheep red blood cells (SRBC)-vaccination mouse model. AP-4-287 had a significant higher aqueous solubility and was readily converted to FX1 in vivo after intraperitoneally (i.p.) administration, but a shorter half-life in vivo. Importantly, AP-4-287 treatment led to a reversible effect on (1) the reduction in the frequency of splenic Ki67+ CD4+ T cells, Tfh cells, and GC B cells; (2) lower GC formation following vaccination; and (3) a decrease in the titers of antigen-specific IgG and IgM antibodies. Our study advances the pre-clinical development of drug targeting BCL6 BTB domain for the treatment of diseases that are associated with abnormal BCL6 elevation. This article is protected by copyright. All rights reserved.

ObjectiveThe human endogenous β-galactoside-binding protein Galectin-9 (Gal-9) reactivates latently HIV-infected cells, which may allow for immune-mediated clearance of these cells. However, Gal-9 also activates T cell Receptor (TCR) signaling pathways, which could negatively affect HIV persistence by promoting T cell expansion and chronic activation/exhaustion. This potential “double-edged sword” effect of Gal-9 during HIV infection raises the question of the overall beneficial versus detrimental impact of Gal-9 on HIV persistence in vivo.DesignWe used the BLT (bone marrow, liver, thymus) humanized mouse model to evaluate the overall impact of Gal-9 on HIV persistence in vivo during antiretroviral therapy (ART).MethodsTwo independent cohorts of BLT mice with high human immune reconstitution were infected with HIV, placed on ART, and then treated with either recombinant human Gal-9 or PBS during ART suppression. Plasma viral loads and levels of tissue-associated HIV DNA and RNA were measured by qPCR. Markers of T cell activation/exhaustion were measured by flow cytometry, and plasma markers of inflammation were measured by multiplex cytokine arrays.ResultsGal-9 treatment was tolerable in ART-suppressed humanized mice and did not significantly induce plasma markers of inflammation or T cell markers of activation/exhaustion. However, Gal-9 treatment during ART significantly increased levels of tissue-associated HIV DNA and RNA compared to controls (P=0.0007 and P=0.011, respectively, for cohort I and P=0.002 and P=0.005, respectively, for cohort II).ConclusionsOur study highlights the overall adverse effects of Gal-9 on HIV persistence and the potential need to block Gal-9 interactions during ART-suppressed HIV infection.

The potential of immunotherapy strategies utilizing BNAbs such as 3BNC117 and 10-1074 to limit viral replication while also facilitating clearance of HIV infected cells has heightened interest in identifying the predominant NK effector subset(s) capable of mediating Antibody Dependent Cellular Cytotoxicity (ADCC). Utilizing Advanced Poly-chromatic Flow Cytometry, we identified that CD57 positive NK cells from ART-suppressed PLWH expressed significantly higher levels of the CD16 FcγR Receptor, 2B4 ADCC co-receptor and HLA-DR activation marker while NKG2C positive NK cells expressed significantly higher levels of the CD2 ADCC co-receptor (p

Background Immune markers have been correlated with prognosis in a variety of solid tumors, including cervical cancer. Objective To review the literature on hematologic and immune markers and their association with recurrence and survival among patients with cervical cancer treated with chemoradiation. Evidence review This systematic review was conducted in accordance with PRISMA guidelines via searches of Ovid MEDLINE, Ovid Embase, and the Cochrane Library using keywords regarding cervical cancer, immune markers, and HIV. Studies involving patients treated with cisplatin‐based chemoradiotherapy were selected and reviewed by at least two independent reviewers, with disagreements resolved by a third reviewer. Findings A total of 737 studies were identified, of which 314 assessed immune biomarkers in immunocompetent patients (30 included in the final analysis) and 327 studies in immunosuppressed patients (5 included in the final analysis). The strongest prognostic indicators were lymphopenia and elevated neutrophil‐to‐lymphocyte ratio. Other potential markers included HPV‐specific lymphocyte response, cytokine profile, expression of immune‐blocking antigens on cell surfaces, and tumor‐associated lymphocyte, macrophage, and neutrophil infiltration. Studies of immunosuppressed patients described more severe cytopenic changes overall and concluded that viral suppression led to improved outcomes. Conclusions The immunologic interplay at work in cervical cancer development, progression, and treatment is complex. Strong evidence was found in favor of lymphopenia and elevated neutrophil‐to‐lymphocyte ratio being prognostic for worse outcomes with other markers showing potential associations as well. Although the interpretation of immune status with regard to treatment approach remains unclear, future studies should aim to tailor treatment that minimizes possible detrimental immune effects., Immune markers have been correlated with prognosis in a variety of solid tumors, including cervical cancer. In this systematic review of immune markers for cervical cancer patients being treated with chemoradiation, we surveyed the literature for immunologic and hematologic prognostic markers and found the strongest negative prognostic indicators were lymphopenia and elevated neutrophil‐to‐lymphocyte ratio.

In the pilot NCT01935089 trial, we tested whether pegylated interferon alpha2b (Peg-IFN-α2b) with antiretroviral therapy (ART) was safe and could impact HIV and immune measures in blood and in gut-associated lymphoid tissue (GALT). Twenty HIV-1(+) ART-suppressed individuals received 1 μg/kg/week Peg-IFN-α2b with ART for 20 weeks, with intermediate 4-week analytical ART interruption (ATI). Safety, immune activation, HIV viral load and integrated HIV DNA in blood, and HIV RNA and DNA in gut biopsies were measured. A total of 7/20 participants experienced grade 3–4 adverse events, while 17/20 participants completed the study. Of the 17 participants who completed the study, 8 remained suppressed during ATI, while all 17 were suppressed at end of treatment (EoT). As expected, treatment increased activation of T and natural killer (NK) cells and IFN-stimulated molecule expression on monocytes in periphery. While circulating CD4(+) T cells showed a trend for a decrease in integrated HIV DNA, GALT showed a significant decrease in HIV-1 RNA(+) cells as measured by in situ hybridization along with a reduction in total HIV DNA and cell-associated RNA by EoT. The observed decrease in HIV-1 RNA(+) cells in GALT was positively associated with the decrease in activated NK cells and macrophages. This study documents for the first time that 20 weeks of immunotherapy with Peg-IFN-α2b+ART (inclusive of a 4-week ATI) is safe and results in an increase in blood and GALT immune activation and in a significant decrease in HIV-1 RNA(+) cells in GALT in association with changes in innate cell activation.

Background: Acute myeloid leukemia (AML) is the most common form of leukemia in adults. In 2017, 120,000 new AML cases of were reported worldwide, with an expectation to reach double the number by 2040. Presently, with an 8.5 months of median survival rate and approximately 60% recurrence rate following chemotherapy treatment, leads to the opportunity to improve overall AML cancer therapy. The Retinoblastoma (Rb1) protein can control canonical cell cycle progression and be a determinant to apoptosis depending on its phosphorylation state, binding partners, and localization (specifically mitochondrial Rb1). We have developed a new small molecule, AP-3-84, which binds the LxCxE domain of Rb1 and can induce cell death in multiple acute myeloid leukemia cell lines by in vitro studies. Methods: AML cell lines (THP-1, AML-193, Kasumi-1) were cultured at different cell concentration (75000/ml, 125,000/ml and 200,000/ml) and exposed to 5, 10, 15, 20 uM concentration of AP-3-84. Cell differentiation was induced by 100 ng/ml PMA over 24 hours. Nonhemopoietic cancer cells (ID8, PDAC) were analyzed after exposure to AP-3-84. Cell death outcomes were measured by Annexin-V using flowcytometry or viability measures by Alamar blue assay. Protein levels were measured by Western blots at intervals between 30 min and 6 hours after exposure to AP-3-84 for total Rb1, phosphorylated Rb1, ATF3, BIM, NOXA, Bak, Bax, cleaved PARP, cleaved Caspase-3. Cytoplasmic and mitochondrial lysates were examined. Confocal immunocytochemistry on Rb1 expression in nucleus versus cytoplasm was performed. Results: Rb1 protein expression was documented by immunocytochemistry in nucleus and cytoplasm of THP1 cells consistent with its detection in nuclear and mitochondrial lysates (Western). AP-3-84 induction of cell death was observed at 10uM or higher concentration, with a 5 minute exposure sufficient to cause cell death in 70-80% of cells 24 hours later. Cell death by AP-3-84 was specific to dividing AML cells as no cell death was induced in PMA differentiated THP1 cells or nonhemopoietic cancer cells. No effect in cell division rate by AP-3-84 was present in nonhemopoitic cancer cells indicating a lack of cell cycle regulation. Mechanistically, AP-3-84 induced higher levels of ER-stress signals (higher ATF4, ATF3) and increased mitochondrial protein levels of NOXA, BIM, cleaved-PARP, and cleaved Caspase-3. Bax and Bak levels were unchanged after AP-3-84 exposure. Conclusion: We identify a new small molecule, AP-3-84, with therapeutic potential in AML by its binding to the LxCxE pocket of Rb1 triggering mitochondria-mediated cell apoptosis. Citation Format: Avishek Bhuniya, Lily Lu, Adi Narayana Reddy Poli, Evgenii N. Tcyganov, Colin Hart, Brijesh Karanam, Paridhima Sharma, Taylor Hill, Joseph Salvino, Luis J. Montaner. Targeting acute myeloid leukemia cell death via small molecule AP-3-84 binding to the LxCxE pocket of Rb1 [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 2537.

Despite effective antiretroviral therapy, HIV-1 persistence in latent reservoirs remains a major obstacle to a cure. We postulate that HIV-1 silencing factors suppress HIV-1 reactivation and that inhibition of these factors will increase HIV-1 reactivation. To identify HIV-1 silencing factors, we conducted a genome-wide CRISPR inhibition (CRISPRi) screen using four CRISPRi-ready, HIV-1-d6-GFP-infected Jurkat T cell clones with distinct integration sites. We sorted cells with increased green fluorescent protein (GFP) expression and captured single guide RNAs (sgRNAs) via targeted deep sequencing. We identified 18 HIV-1 silencing factors that were significantly enriched in HIV-1-d6-GFP(high) cells. Among them, SLTM (scaffold attachment factor B-like transcription modulator) is an epigenetic and transcriptional modulator having both DNA and RNA binding capacities not previously known to affect HIV-1 transcription. Knocking down SLTM by CRISPRi significantly increased HIV-1-d6-GFP expression (by 1.9- to 4.2-fold) in three HIV-1-d6-GFP-Jurkat T cell clones. Furthermore, SLTM knockdown increased the chromatin accessibility of HIV-1 and the gene in which HIV-1 is integrated but not the housekeeping gene POLR2A. To test whether SLTM inhibition can reactivate HIV-1 and further induce cell death of HIV-1-infected cells ex vivo, we established a small interfering RNA (siRNA) knockdown method that reduced SLTM expression in CD4(+) T cells from 10 antiretroviral therapy (ART)-treated, virally suppressed, HIV-1-infected individuals ex vivo. Using limiting dilution culture, we found that SLTM knockdown significantly reduced the frequency of HIV-1-infected cells harboring inducible HIV-1 by 62.2% (0.56/10(6) versus 1.48/10(6) CD4(+) T cells [P = 0.029]). Overall, our study indicates that SLTM inhibition reactivates HIV-1 in vitro and induces cell death of HIV-1-infected cells ex vivo. Our study identified SLTM as a novel therapeutic target. IMPORTANCE HIV-1-infected cells, which can survive drug treatment and immune cell killing, prevent an HIV-1 cure. Immune recognition of infected cells requires HIV-1 protein expression; however, HIV-1 protein expression is limited in infected cells after long-term therapy. The ways in which the HIV-1 provirus is blocked from producing protein are unknown. We identified a new host protein that regulates HIV-1 gene expression. We also provided a new method of studying HIV-1–host factor interactions in cells from infected individuals. These improvements may enable future strategies to reactivate HIV-1 in infected individuals so that infected cells can be killed by immune cells, drug treatment, or the virus itself.

In untreated HIV-1 infection, rapid viral evolution allows escape from immune responses. Viral replication can be blocked by antiretroviral therapy. However, HIV-1 persists in a latent reservoir in resting CD4(+) T cells, and rebound viremia occurs following treatment interruption. The reservoir, which is maintained in part by clonal expansion, can be measured using quantitative viral outgrowth assays (QVOAs) in which latency is reversed with T cell activation to allow viral outgrowth. Recent studies have shown that viruses detected in QVOAs prior to treatment interruption often differ from rebound viruses. We hypothesized that autologous neutralizing antibodies directed at the HIV-1 envelope (Env) protein might block outgrowth of some reservoir viruses. We modified the QVOA to reflect pressure from low concentrations of autologous antibodies and showed that outgrowth of a substantial but variable fraction of reservoir viruses is blocked by autologous contemporaneous immunoglobulin G (IgG). A reduction in outgrowth of >80% was seen in 6 of 15 individuals. This effect was due to direct neutralization. We established a phylogenetic relationship between rebound viruses and viruses growing out in vitro in the presence of autologous antibodies. Some large infected cell clones detected by QVOA carried neutralization-sensitive viruses, providing a cogent explanation for differences between rebound virus and viruses detected in standard QVOAs. Measurement of the frequency of reservoir viruses capable of outgrowth in the presence of autologous IgG might allow more accurate prediction of time to viral rebound. Ultimately, therapeutic immunization targeting the subset of variants resistant to autologous IgG might contribute to a functional cure.

A sterilizing or functional cure for HIV is currently precluded by resting CD4(+) T cells that harbor latent but replication-competent provirus. The “shock-and-kill” pharmacological ap-proach aims to reactivate provirus expression in the presence of antiretroviral therapy and target virus-expressing cells for elimination. However, no latency reversal agent (LRA) to date effectively clears viral reservoirs in humans, suggesting a need for new LRAs and LRA combinations. Here, we screened 216 compounds from the pan-African Natural Product Library and identified knipholone anthrone (KA) and its basic building block anthralin (dithranol) as novel LRAs that reverse viral latency at low micromolar concentrations in multiple cell lines. Neither agent's activity depends on protein kinase C; nor do they inhibit class I/II histone deacetylases. However, they are differentially modulated by oxidative stress and metal ions and induce distinct patterns of global gene expression from established LRAs. When applied in combination, both KA and anthralin synergize with LRAs representing multiple functional classes. Finally, KA induces both HIV RNA and protein in primary cells from HIV-infected donors. Taken together, we describe two novel LRAs that enhance the activities of multiple “shock-and-kill” agents, which in turn may inform ongoing LRA combination therapy efforts.

While there is an emerging consensus that engagement of the Mu opioid receptor by opioids may modulate various stages the HIV life cycle (e.g.: increasing cell susceptibility to infection, promoting viral transcription, and depressing immune responses to virally-infected cells), the overall effect on latency and viral reservoirs remains unclear. Importantly, the hypothesis that the increase in immune activation observed in chronic opioid users by direct or indirect mechanisms (i.e., microbial translocation) would lead to a larger HIV reservoir after ART-suppression has not been supported to date. The potential for a subsequent decrease in reservoirs after ART-suppression has been postulated and is supported by early reports of opioid users having lower latent HIV burden. Here, we review experimental data supporting the link between opioid use and HIV modulation, as well as the scientific premise for expecting differential changes in immune activation and HIV reservoir between different medications for opioid use disorder. A better understanding of potential changes in HIV reservoirs relative to the engagement of the Mu opioid receptor and ART-mediated immune reconstitution will help guide future cure-directed studies in persons living with HIV and opioid use disorder. Graphical Abstract Review. HIV replication, immune activation and dysbiosis: opioids may affect immune reconstitution outcomes despite viral suppression.