Comparative analysis of human gut- and blood-derived mononuclear cells: contrasts in function and phenotype.

Introduction: Alterations in the gut immune system have been implicated in various diseases.The challenge of obtaining gut tissues from healthy individuals, commonly performed via surgical explants, has limited the number of studies describing the phenotype and function of gut-derived immune cells in health. Methods: Here, by means of recto-sigmoid colon biopsies obtained during routine care (colon cancer screening in healthy adults), the phenotype and function of immune cells present in the gut were described and compared to those found in blood. Results: The proportion of CD4+, CD8+, MAIT, γδ+ T, and NK cells phenotype, expression of integrins, and ability to produce cytokine in response to stimulation with PMA and ionomycin. T cells in the gut were found to predominantly have a memory phenotype as compared to T cells in blood where a naïve phenotype predominates. Recto-sigmoid mononuclear cells also had higher PD-1 and Ki67 expression. Furthermore, integrin expression and cytokine production varied by cell type and location in blood vs. gut. Discussion: These findings demonstrate the differences in functionality of these cells when compared to their blood counterparts and validate previous studies on phenotype within gut-derived immune cells in humans (where cells have been obtained through surgical means). This study suggests that recto-sigmoid biopsies collected during colonoscopy can be a reliable yet more accessible sampling method for follow up of alterations of gut derived immune cells in clinical settings.

Donating One's Body to HIV Cure Research Through Canadian Medical Assistance in Dying: A Case Study.

BACKGROUND: Finding a cure for HIV is challenged by persisting reservoirs, the mapping of which necessitates invasive procedures. Inviting people with HIV (PWHIV) at the end of life to donate body specimens post-mortem through research autopsies is a novel approach, raising ethical concerns. OBJECTIVE: This case study aims to explore the motivations, barriers, and facilitators of a terminally-ill Canadian PWHIV who requested medical assistance in dying (MAID) and expressed interest in donating his body for HIV cure research. CASE PRESENTATION: An in-depth 3-hour and semi-structured interview was conducted with the participant. The interview transcription was thematically coded to identify motivations and perceived barriers and facilitators to participate in end-of-life HIV cure research. Our analysis identified six themes. Two themes expressed motivations: Collaboration in progress in health and science, seeing cure research as collaboration with professionals; and Opportunity to learn more, mostly about science and health. One theme expressed a barrier: Losing interest in or identification with long-term care research matters, especially those related to the management of long-term care. Three themes expressed by facilitators: Receiving information from professionals one trusts and knows, especially clinical and research teams; Perceiving research procedures as simple, useful, and embedded in care, perceiving clinical, educational, and interpersonal benefits that surpass costs of participation; and Perceiving research as one last way to contribute, that is, feeling useful or give back. CONCLUSION: Several circumstances facilitated the patient's participation: being a single man, having time to participate, having no strong religious belief, and valuing clear, direct communication. His motivations to participate in HIV cure research were altruistic, and also an experience of working with clinical and research teams. Finally, this perspective highlights HIV cure research participant candidates' need for education about research procedures.

Impact of in vitro HIV infection on human thymic regulatory T cell differentiation.

Background: The differentiation and function of immunosuppressive regulatory T cells (Tregs) is dictated by the master transcription factor FoxP3. During HIV infection, there is an increase in Treg frequencies in the peripheral blood and lymphoid tissues. This accentuates immune dysfunction and disease progression. Expression of FoxP3 by thymic Tregs (tTregs) is partially controlled by TGF-ß. This cytokine also contributes to Treg development in the peripheral blood and lymphoid tissues. Although TGF-ß mediates lymphoid tissue fibrosis and peripheral Treg differentiation in HIV-infected individuals, its role in the induction and maintenance of Tregs within the thymus during HIV infection remains unclear. Methods: Thymocytes were isolated from fresh human thymic tissues obtained from pediatric patients undergoing cardiac surgery. Infection by both R5- and X4-tropic HIV-1 strains and TGF-ß treatment of human thymocytes was performed in an in vitro co-culture model with OP9-DL1 cells expressing Notch ligand delta-like 1 without T cell receptor (TCR) activation. Results: Despite high expression of CCR5 and CXCR4 by tTregs, FoxP3 + CD3highCD8- thymocytes were much less prone to in vitro infection with R5- and X4-tropic HIV strains compared to FoxP3-CD3highCD8- thymocytes. As expected, CD3highCD4+ thymocytes, when treated with TGF-ß1, upregulated CD127 and this treatment resulted in increased FoxP3 expression and Treg differentiation, but did not affect the rate of HIV infection. FoxP3 expression and Treg frequencies remained unchanged following in vitro HIV infection alone or in combination with TGF-ß1. Conclusion: FoxP3 expression and tTreg differentiation is not affected by in vitro HIV infection alone or the combination of in vitro HIV infection and TGF-ß treatment.

SARS-CoV-2 Vaccine-Induced T-Cell Response after Three Doses in People Living with HIV on Antiretroviral Therapy Compared to Seronegative Controls (CTN 328 COVAXHIV Study).

People living with HIV (PLWH) may be at risk for poor immunogenicity to certain vaccines, including the ability to develop immunological memory. Here, we assessed T-cell immunogenicity following three SARS-CoV-2 vaccine doses in PLWH versus uninfected controls. Blood was collected from 38 PLWH on antiretroviral therapy and 24 age-matched HIV-negative controls, pre-vaccination and after 1st/2nd/3rd dose of SARS-CoV-2 vaccines, without prior SARS-CoV-2 infection. Flow cytometry was used to assess ex vivo T-cell immunophenotypes and intracellular Tumor necrosis factor (TNF)-α/interferon(IFN)-γ/interleukin(IL)-2 following SARS-CoV-2-Spike-peptide stimulation. Comparisons were made using Wilcoxon signed-rank test for paired variables and Mann-Whitney for unpaired. In PLWH, Spike-specific CD4 T-cell frequencies plateaued post-2nd dose, with no significant differences in polyfunctional SARS-CoV-2-specific T-cell proportions between PLWH and uninfected controls post-3rd dose. PLWH had higher frequencies of TNFα+CD4 T-cells and lower frequencies of IFNγ+CD8 T-cells than seronegative participants post-3rd dose. Regardless of HIV status, an increase in naive, regulatory, and PD1+ T-cell frequencies was observed post-3rd dose. In summary, two doses of SARS-CoV-2 vaccine induced a robust T-cell immune response in PLWH, which was maintained after the 3rd dose, with no significant differences in polyfunctional SARS-CoV-2-specific T-cell proportions between PLWH and uninfected controls post-3rd dose.

Lentiviral Infections Persist in Brain despite Effective Antiretroviral Therapy and Neuroimmune Activation.

HIV infection persists in different tissue reservoirs among people with HIV (PWH) despite effective antiretroviral therapy (ART). In the brain, lentiviruses replicate principally in microglia and trafficking macrophages. The impact of ART on this viral reservoir is unknown. We investigated the activity of contemporary ART in various models of lentivirus brain infection. HIV-1 RNA and total and integrated DNA were detected in cerebral cortex from all PWH (n = 15), regardless of ART duration or concurrent plasma viral quantity and, interestingly, integrated proviral DNA levels in brain were significantly higher in the aviremic ART-treated group (P < 0.005). Most ART drugs tested (dolutegravir, ritonavir, raltegravir, and emtricitabine) displayed significantly lower 50% effective concentration (EC50) values in lymphocytes than in microglia, except tenofovir, which showed 1.5-fold greater activity in microglia (P < 0.05). In SIV-infected Chinese rhesus macaques, despite receiving suppressive (n = 7) or interrupted (n = 8) ART, brain tissues had similar SIV-encoded RNA and total and integrated DNA levels compared to brains from infected animals without ART (n = 3). SIV and HIV-1 capsid antigens were immunodetected in brain, principally in microglia/macrophages, regardless of ART duration and outcome. Antiviral immune responses were comparable in the brains of ART-treated and untreated HIV- and SIV-infected hosts. Both HIV-1 and SIV persist in brain tissues despite contemporary ART, with undetectable virus in blood. ART interruption exerted minimal effect on the SIV brain reservoir and did not alter the neuroimmune response profile. These studies underscore the importance of augmenting ART potency in different tissue compartments. IMPORTANCE Antiretroviral therapy (ART) suppresses HIV-1 in plasma and CSF to undetectable levels. However, the impact of contemporary ART on HIV-1 brain reservoirs remains uncertain. An active viral reservoir in the brain during ART could lead to rebound systemic infection after cessation of therapy, development of drug resistance mutations, and neurological disease. ART's impact, including its interruption, on brain proviral DNA remains unclear. The present studies show that in different experimental platforms, contemporary ART did not suppress viral burden in the brain, regardless of ART component regimen, the duration of therapy, and its interruption. Thus, new strategies for effective HIV-1 suppression in the brain are imperative to achieve sustained HIV suppression.

Role of RIPK1 in SMAC mimetics-induced apoptosis in primary human HIV-infected macrophages.

Macrophages serve as viral reservoirs due to their resistance to apoptosis and HIV-cytopathic effects. We have previously shown that inhibitor of apoptosis proteins (IAPs) confer resistance to HIV-Vpr-induced apoptosis in normal macrophages. Herein, we show that second mitochondrial activator of caspases (SMAC) mimetics (SM) induce apoptosis of monocyte-derived macrophages (MDMs) infected in vitro with a R5-tropic laboratory strain expressing heat stable antigen, chronically infected U1 cells, and ex-vivo derived MDMs from HIV-infected individuals. To understand the mechanism governing SM-induced cell death, we show that SM-induced cell death of primary HIV-infected macrophages was independent of the acquisition of M1 phenotype following HIV infection of macrophages. Instead, SM-induced cell death was found to be mediated by IAPs as downregulation of IAPs by siRNAs induced cell death of HIV-infected macrophages. Moreover, HIV infection caused receptor interacting protein kinase-1 (RIPK1) degradation which in concert with IAP1/2 downregulation following SM treatment may result in apoptosis of macrophages. Altogether, our results show that SM selectively induce apoptosis in primary human macrophages infected in vitro with HIV possibly through RIPK1. Moreover, modulation of the IAP pathways may be a potential strategy for selective killing of HIV-infected macrophages in vivo.

TLR-4 Agonist Induces IFN-γ Production Selectively in Proinflammatory Human M1 Macrophages through the PI3K-mTOR- and JNK-MAPK-Activated p70S6K Pathway.

IFN-γ, a proinflammatory cytokine produced primarily by T cells and NK cells, activates macrophages and engages mechanisms to control pathogens. Although there is evidence of IFN-γ production by murine macrophages, IFN-γ production by normal human macrophages and their subsets remains unknown. Herein, we show that human M1 macrophages generated by IFN-γ and IL-12- and IL-18-stimulated monocyte-derived macrophages (M0) produce significant levels of IFN-γ. Further stimulation of IL-12/IL-18-primed macrophages or M1 macrophages with agonists for TLR-2, TLR-3, or TLR-4 significantly enhanced IFN-γ production in contrast to the similarly stimulated M0, M2a, M2b, and M2c macrophages. Similarly, M1 macrophages generated from COVID-19-infected patients' macrophages produced IFN-γ that was enhanced following LPS stimulation. The inhibition of M1 differentiation by Jak inhibitors reversed LPS-induced IFN-γ production, suggesting that differentiation with IFN-γ plays a key role in IFN-γ induction. We subsequently investigated the signaling pathway(s) responsible for TLR-4-induced IFN-γ production in M1 macrophages. Our results show that TLR-4-induced IFN-γ production is regulated by the ribosomal protein S6 kinase (p70S6K) through the activation of PI3K, the mammalian target of rapamycin complex 1/2 (mTORC1/2), and the JNK MAPK pathways. These results suggest that M1-derived IFN-γ may play a key role in inflammation that may be augmented following bacterial/viral infections. Moreover, blocking the mTORC1/2, PI3K, and JNK MAPKs in macrophages may be of potential translational significance in preventing macrophage-mediated inflammatory diseases.

Selective Induction of Cell Death in Human M1 Macrophages by Smac Mimetics Is Mediated by cIAP-2 and RIPK-1/3 through the Activation of mTORC.

Inflammatory macrophages have been implicated in many diseases, including rheumatoid arthritis and inflammatory bowel disease. Therefore, targeting macrophage function and activation may represent a potential strategy to treat macrophage-associated diseases. We have previously shown that IFN-γ-induced differentiation of human M0 macrophages toward proinflammatory M1 state rendered them highly susceptible to the cytocidal effects of second mitochondria-derived activator of caspases mimetics (SMs), antagonist of the inhibitors of apoptosis proteins (IAPs), whereas M0 and anti-inflammatory M2c macrophages were resistant. In this study, we investigated the mechanism governing SM-induced cell death during differentiation into M1 macrophages and in polarized M1 macrophages. IFN-γ stimulation conferred on M0 macrophages the sensitivity to SM-induced cell death through the Jak/STAT, IFN regulatory factor-1, and mammalian target of rapamycin complex-1 (mTORC-1)/ribosomal protein S6 kinase pathways. Interestingly, mTORC-1 regulated SM-induced cell death independent of M1 differentiation. In contrast, SM-induced cell death in polarized M1 macrophages is regulated by the mTORC-2 pathway. Moreover, SM-induced cell death is regulated by cellular IAP (cIAP)-2, receptor-interacting protein kinase (RIPK)-1, and RIPK-3 degradation through mTORC activation during differentiation into M1 macrophages and in polarized M1 macrophages. In contrast to cancer cell lines, SM-induced cell death in M1 macrophages is independent of endogenously produced TNF-α, as well as the NF-κB pathway. Collectively, selective induction of cell death in human M1 macrophages by SMs may be mediated by cIAP-2, RIPK-1, and RIPK-3 degradation through mTORC activation. Moreover, blocking cIAP-1/2, mTORC, or IFN regulatory factor-1 may represent a promising therapeutic strategy to control M1-associated diseases.

Identification of novel genes involved in apoptosis of HIV-infected macrophages using unbiased genome-wide screening.

BACKGROUND: Macrophages, besides resting latently infected CD4+ T cells, constitute the predominant stable, major non-T cell HIV reservoirs. Therefore, it is essential to eliminate both latently infected CD4+ T cells and tissue macrophages to completely eradicate HIV in patients. Until now, most of the research focus is directed towards eliminating latently infected CD4+ T cells. However, few approaches have been directed at killing of HIV-infected macrophages either in vitro or in vivo. HIV infection dysregulates the expression of many host genes essential for the survival of infected cells. We postulated that exploiting this alteration may yield novel targets for the selective killing of infected macrophages. METHODS: We applied a pooled shRNA-based genome-wide approach by employing a lentivirus-based library of shRNAs to screen novel gene targets whose inhibition should selectively induce apoptosis in HIV-infected macrophages. Primary human MDMs were infected with HIV-eGFP and HIV-HSA viruses. Infected MDMs were transfected with siRNAs specific for the promising genes followed by analysis of apoptosis by flow cytometry using labelled Annexin-V in HIV-infected, HIV-exposed but uninfected bystander MDMs and uninfected MDMs. The results were analyzed using student's t-test from at least four independent experiments. RESULTS: We validated 28 top hits in two independent HIV infection models. This culminated in the identification of four target genes, Cox7a2, Znf484, Cstf2t, and Cdk2, whose loss-of-function induced apoptosis preferentially in HIV-infected macrophages. Silencing these single genes killed significantly higher number of HIV-HSA-infected MDMs compared to the HIV-HSA-exposed, uninfected bystander macrophages, indicating the specificity in the killing of HIV-infected macrophages. The mechanism governing Cox7a2-mediated apoptosis of HIV-infected macrophages revealed that targeting respiratory chain complex II and IV genes also selectively induced apoptosis of HIV-infected macrophages possibly through enhanced ROS production. CONCLUSIONS: We have identified above-mentioned novel genes and specifically the respiratory chain complex II and IV genes whose silencing may cause selective elimination of HIV-infected macrophages and eventually the HIV-macrophage reservoirs. The results highlight the potential of the identified genes as targets for eliminating HIV-infected macrophages in physiological environment as part of an HIV cure strategy.

Association between radical cystectomy prophylactic antimicrobial regimen and postoperative infection.

INTRODUCTION: Infections are common after radical cystectomy. The objective of this study was to determine the association between antimicrobial prophylactic regimen and infection after radical cystectomy. METHODS: A retrospective cohort study was performed on patients who underwent radical cystectomy at one tertiary Canadian center between January 2016 and April 2020. Patients received antimicrobial prophylaxis based on surgeon preference (cefazolin/metronidazole or ampicillin/ciprofloxacin/metronidazole, or other). A univariable and multivariable logistic regression model was created to determine the association between antimicrobial regimen and postoperative infection within 30 days. The association between patient demographic factors, as well as preoperative and intraoperative variables and infection, was also determined. Infection characteristics, including type, timing, and antimicrobial susceptibilities were reported. RESULTS: One hundred and sixty-five patients were included. Mean age was 69.8 years, 121 (73.3%) were male, and 72 (43.6%) received orthotopic neobladder diversion. Ninety-six patients (58%) received cefazolin/metronidazole prophylaxis, 50 (30%) received ampicillin/ciprofloxacin/metronidazole, and 19 (11.5%) received another regimen. Fifty-four patients (32.7%) developed a postoperative infection (surgical site infection or urinary tract infection). Surgical site infection occurred in 35 patients (21.2%) and urinary tract infection occurred in 34 (21.0%). There was no association between antimicrobial regimen and incidence of postoperative infection (surgical site infection or urinary tract infection, relative risk 0.99, 95% confidence interval 0.50-1.99). CONCLUSIONS: The overall incidence of infection was 32.7% following radical cystectomy. The preoperative prophylactic antibiotic regimen used was not associated with incidence of postoperative infection.

Soluble IL-7Rα/sCD127 in Health, Disease, and Its Potential Role as a Therapeutic Agent.

Soluble cytokine receptors can influence immune responses by modulating the biological functions of their respective ligands. These effects can be either agonistic or antagonistic and a number of soluble cytokine receptors have been shown to play critical roles in both maintenance of health and disease pathogenesis. Soluble IL-7Ra (sCD127) is one such example. With its impact on the IL-7/CD127 pathway, which is fundamental for the development and homeostasis of T cells, the role of sCD127 in health and disease has been extensively studied in recent years. Within this review, the role of sCD127 in maintaining host immune function is presented. Next, by addressing genetic factors affecting sCD127 expression and the associated levels of sCD127 production, the roles of sCD127 in autoimmune disease, infections and cancer are described. Finally, advances in the field of soluble cytokine therapy and the potential for sCD127 as a biomarker and therapeutic agent are discussed.

HIV-Infected Macrophages Are Infected and Killed by the Interferon-Sensitive Rhabdovirus MG1.

The use of unique cell surface markers to target and eradicate HIV-infected cells has been a longstanding objective of HIV-1 cure research. This approach, however, overlooks the possibility that intracellular changes present within HIV-infected cells may serve as valuable therapeutic targets. For example, the identification of dysregulated antiviral signaling in cancer has led to the characterization of oncolytic viruses capable of preferentially killing cancer cells. Since impairment of cellular antiviral machinery has been proposed as a mechanism by which HIV-1 evades immune clearance, we hypothesized that HIV-infected macrophages (an important viral reservoir in vivo) would be preferentially killed by the interferon-sensitive oncolytic Maraba virus MG1. We first showed that HIV-infected monocyte-derived macrophages (MDM) were more susceptible to MG1 infection and killing than HIV-uninfected cells. As MG1 is highly sensitive to type I interferons (IFN-I), we then investigated whether we could identify IFN-I signaling differences between HIV-infected and uninfected MDM and found evidence of impaired IFN-α responsiveness within HIV-infected cells. Finally, to assess whether MG1 could target a relevant, primary cell reservoir of HIV-1, we investigated its effects in alveolar macrophages (AM) obtained from effectively treated individuals living with HIV-1. As observed with in vitro-infected MDM, we found that HIV-infected AM were preferentially eliminated by MG1. In summary, the oncolytic rhabdovirus MG1 appears to preferentially target and kill HIV-infected cells via impairment of antiviral signaling pathways and may therefore provide a novel approach to an HIV-1 cure.IMPORTANCE Human immunodeficiency virus type 1 (HIV-1) remains a treatable, but incurable, viral infection. The establishment of viral reservoirs containing latently infected cells remains the main obstacle in the search for a cure. Cure research has also focused on only one cellular target of HIV-1 (the CD4+ T cell) while largely overlooking others (such as macrophages) that contribute to HIV-1 persistence. In this study, we address these challenges by describing a potential strategy for the eradication of HIV-infected macrophages. Specifically, we show that an engineered rhabdovirus-initially developed as a cancer therapy-is capable of preferential infection and killing of HIV-infected macrophages, possibly via the same altered antiviral signaling seen in cancer cells. As this rhabdovirus is currently being explored in phase I/II clinical trials, there is potential for this approach to be readily adapted for use within the HIV-1 cure field.

Selective killing of human M1 macrophages by Smac mimetics alone and M2 macrophages by Smac mimetics and caspase inhibition.

The inflammatory and anti-inflammatory Mϕs have been implicated in many diseases including rheumatoid arthritis, multiple sclerosis, and leprosy. Recent studies suggest targeting Mϕ function and activation may represent a potential target to treat these diseases. Herein, we investigated the effect of second mitochondria-derived activator of caspases (SMAC) mimetics (SMs), the inhibitors of apoptosis (IAPs) proteins, on the killing of human pro- and anti-inflammatory Mϕ subsets. We have shown previously that human monocytes are highly susceptible whereas differentiated Mϕs (M0) are highly resistant to the cytocidal abilities of SMs. To determine whether human Mϕ subsets are resistant to the cytotoxic effects of SMs, we show that M1 Mϕs are highly susceptible to SM-induced cell death whereas M2a, M2b, and M2c differentiated subsets are resistant, with M2c being the most resistant. SM-induced cell death in M1 Mϕs was mediated by apoptosis as well as necroptosis, activated both extrinsic and intrinsic pathways of apoptosis, and was attributed to the IFN-γ-mediated differentiation. In contrast, M2c and M0 Mϕs experienced cell death through necroptosis following simultaneous blockage of the IAPs and the caspase pathways. Overall, the results suggest that survival of human Mϕs is critically linked to the activation of the IAPs pathways. Moreover, agents blocking the cellular IAP1/2 and/or caspases can be exploited therapeutically to address inflammation-related diseases.

Intravenous administration of Penicillin results in therapeutic intravitreal levels in chronic postoperative endophthalmitis.

IMPORTANCE: The role of systemic antibiotics in the treatment of bacterial endophthalmitis remains controversial. While penicillin is a highly effective antibiotic against bacteria that frequently cause endophthalmitis, the ability of systemically administered Penicillin G to penetrate into the vitreous at adequate therapeutic concentrations has not been studied. Its role in the treatment of endophthalmitis, particularly for bacteria for which it is the antibiotic of choice, therefore remains unknown. OBJECTIVE: We sought to determine whether intravenous administration of Penicillin G leads to adequate therapeutic concentrations in the vitreous for the treatment of bacterial endophthalmitis. DESIGN AND SETTING: This study was conducted in an ambulatory setting, at the Ottawa Hospital Eye Institute, a university-affiliated tertiary care center, where a 77-year old gentleman with chronic post-cataract surgery Actinomyces neuii endophathalmitis was treated with intravenous Penicillin G (4 × 106 units every 4 h) and intravitreal ampicillin (5000µg/0.1 m1). MAIN OUTCOMES AND MEASURES: Intravitreal concentration of Penicillin G and ampicillin were obtained at the time of intraocular lens removal, measured by high-performance liquid chromatography. RESULTS: The intravitreal concentration of penicillin and ampicillin was 3.5µg/ml and 0.3µg/ml, respectively. Both the concentration of penicillin and ampicillin were within the level of detection of their respective assays (penicillin 0.06-5µg/ml, ampicillin 0.12-2.5µg/ml). CONCLUSION AND RELEVANCE: This study shows that intravenous Penicillin G administered every four-hours allows for adequate intravitreal concentrations of penicillin. Future studies are required to determine if the results of this study translate into improved clinical outcomes.

Reverse Docking for the Identification of Molecular Targets of Anticancer Compounds.

Molecular docking is a useful and powerful computational method for the identification of potential interactions between small molecules and pharmacological targets. In reverse docking, the ability of one or a few compounds to bind a large dataset of proteins is evaluated in silico. This strategy is useful for identifying molecular targets of orphan bioactive compounds, proposing new molecular mechanisms, finding alternative indications of drugs, or predicting drug toxicity. Herein, we describe a detailed reverse docking protocol for the identification of potential targets for 4-hydroxycoumarin (4-HC). Our results showed that RAC1 is a target of 4-HC, which partially explains the biological activities of 4-HC on cancer cells. The strategy reported here can be easily applied to other compounds and protein datasets.

Repurposing the HCV NS3-4A protease drug boceprevir as COVID-19 therapeutics.

The rapid growth of COVID-19 cases is causing an increasing death toll and also paralyzing the world economy. De novo drug discovery takes years to move from idea and/or pre-clinic to market, and it is not a short-term solution for the current SARS-CoV-2 pandemic. Drug repurposing is perhaps the only short-term solution, while vaccination is a middle-term solution. Here, we describe the discovery path of the HCV NS3-4A protease inhibitors boceprevir and telaprevir as SARS-CoV-2 main protease (3CLpro) inhibitors. Based on our hypothesis that α-ketoamide drugs can covalently bind to the active site cysteine of the SARS-CoV-2 3CLpro, we performed docking studies, enzyme inhibition and co-crystal structure analyses and finally established that boceprevir, but not telaprevir, inhibits replication of SARS-CoV-2 and mouse hepatitis virus (MHV), another coronavirus, in cell culture. Based on our studies, the HCV drug boceprevir deserves further attention as a repurposed drug for COVID-19 and potentially other coronaviral infections as well.

Sperm can act as vectors for HIV-1 transmission into vaginal and cervical epithelial cells.

PROBLEM: Sperm are the major cells in semen. Human sperm possess a number of HIV-1 gp120 binding ligands including sulfogalactosylglycerolipid (SGG). However, the mechanisms of how sperm capture HIV-1 onto their surface are unclear. Furthermore, the ability of sperm to deliver HIV-1 to vaginal/cervical epithelial cells lining the lower female reproductive tract, as a first step in HIV-1 transmission, needs to be determined. METHOD OF STUDY: Sperm from healthy donors were incubated with dual-tropic HIV-1CS204 (clinical isolate), and virus capture was determined by p24 antigen ELISA. The involvement of SGG in HIV-1 capture was assessed by determining Kd values of HIV-1 gp120-SGG binding as well as computational docking of SGG to the gp120 V3 loop. The ability of sperm-associated HIV-1 to infect peripheral blood mononuclear cells (PBMCs) and TZM-bl indicator cells was determined. Lastly, infection of vaginal (Vk2/E6E7), ectocervical (Ect1/E6E7), and endocervical (End1/E6E7) epithelial cells mediated by HIV-1-associated sperm was evaluated. RESULTS: Sperm were able to capture HIV-1 in a dose-dependent manner, and the capture reached a maximum within 5 minutes. Captured HIV-1, however, could be removed from sperm by Percoll-gradient centrifugation. Affinity of gp120 for SGG was substantial, implicating sperm SGG in HIV-1 capture. Sperm-associated HIV-1 could productively infect PBMCs and TZM-bl cells, and was capable of being transmitted into vaginal/cervical epithelial cells. CONCLUSION: Sperm are able to capture HIV-1, which remains infectious and is able to be transmitted into vaginal/cervical epithelial cells, a result indicating the importance of sperm in HIV transmission.

Effect of metformin on the size of the HIV reservoir in non-diabetic ART-treated individuals: single-arm non-randomised Lilac pilot study protocol.

INTRODUCTION: People living with HIV (PLWH) on antiretroviral therapy (ART) do not progress to AIDS. However, they still suffer from an increased risk of inflammation-associated complications. HIV persists in long-lived CD4+ T cells, which form the major viral reservoir. The persistence of this reservoir despite long-term ART is the major hurdle to curing HIV. Importantly, the size of the HIV reservoir is larger in individuals who start ART late in the course of infection and have a low CD4+/CD8+ ratio. HIV reservoir size is also linked to the levels of persistent inflammation on ART. Thus, novel strategies to reduce immune inflammation and improve the host response to control the HIV reservoir would be a valuable addition to current ART. Among the different strategies under investigation is metformin, a widely used antidiabetic drug that was recently shown to modulate T-cell activation and inflammation. Treatment of non-diabetic individuals with metformin controls inflammation by improving glucose metabolism and by regulating intracellular immunometabolic checkpoints such as the adenosin 5 monophosphate activated protein kinase and mammalian target of rapamycin, in association with microbiota modification. METHODS AND ANALYSIS: 22 PLWH on ART for more than 3 years, at high risk of inflammation or the development of non-AIDS events (low CD4+/CD8+ ratio) will be recruited in a clinical single-arm pilot study. We will test whether supplementing ART with metformin in non-diabetic HIV-infected individuals can reduce the size of the HIV reservoir as determined by various virological assays. The expected outcome of this study is a reduction in both the size of the HIV reservoir and inflammation following the addition of metformin to ART, thus paving the way towards HIV eradication. ETHICS AND DISSEMINATION: Ethical approval: McGill university Health Centre committee number MP-37-2016-2456. Canadian Canadian Institutes of Health Research/Canadian HIV Trials Network (CTN) protocol CTNPT027. Results will be made available through publication in peer-reviewed journals and through the CTN website. TRIAL REGISTRATION NUMBER: NCT02659306.