LuSIV Cells: A Reporter Cell Line for the Detection and Quantitation of a Single Cycle of HIV and SIV Replication

AbstractA single cycle of viral replication is the time required for a virus to enter the host cell, replicate its genome, and produce infectious progeny virions. The primate lentiviruses, human immunodeficiency virus (HIV) and simian immunodeficiency virus (SIV), require on average 24 h to complete one cycle of replication. We have now developed and characterized a reporter assay system in CEMx174 cells for the quantitative measurement of HIV/SIV infection within a single replication cycle. The SIVmac239 LTR (−225 → +149) was cloned upstream of the firefly luciferase reporter gene and this reporter plasmid is maintained in CEMx174 cells under stable selection. This cell line, designated LuSIV, is highly sensitive to infection by primary and laboratory strains of HIV/SIV, resulting in Tat-mediated expression of luciferase, which correlates with viral infectivity. Furthermore, manipulation of LuSIV cells for the detection of luciferase activity is easy to perform and requires a minimal amount of time as compared to current HIV/SIV detection systems. The LuSIV system is a powerful tool for the analysis of HIV/SIV infection that provides a unique assay system that can detect virus replication prior to 24 h and does not require virus to spread from cell to cell. Thus these cells can be used for the study of replication-deficient viruses and the high throughput screening of antivirals, or other inhibitors of infection

Characterization of extracellular vesicles and synthetic nanoparticles with four orthogonal single-particle analysis platforms

Ingreso parcial de los coautores.We compared four orthogonal technologies for sizing, counting, and phenotyping of extracellular vesicles (EVs) and synthetic particles. The platforms were: singleparticle interferometric reflectance imaging sensing (SP-IRIS) with fluorescence, nanoparticle tracking analysis (NTA) with fluorescence, microfluidic resistive pulse sensing (MRPS), and nanoflow cytometry measurement (NFCM). EVs from the human T lymphocyte line H9 (high CD81, low CD63) and the promonocytic line U937 (low CD81, high CD63) were separated from culture conditioned medium (CCM) by differential ultracentrifugation (dUC) or a combination of ultrafiltration (UF) and size exclusion chromatography (SEC) and characterized by transmission electron microscopy (TEM) and Western blot (WB). Mixtures of synthetic particles.(silica and polystyrene spheres) with known sizes and/or concentrations were also tested.MRPS andNFCMreturned similar particle counts,whileNTAdetected counts approximately one order of magnitude lower for EVs, but not for synthetic particles. SP-IRIS events could not be used to estimate particle concentrations. For sizing, SPIRIS, MRPS, and NFCM returned similar size profiles, with smaller sizes predominating (per power law distribution), but with sensitivity typically dropping off below diameters of 60 nm. NTA detected a population of particles with a mode diameter greater than 100 nm. Additionally, SP-IRIS, MRPS, and NFCM were able to identify at least three of four distinct size populations in a mixture of silica or polystyrene nanoparticles. Finally, for tetraspanin phenotyping, the SP-IRIS platform in fluorescencemode was able to detect at least twomarkers on the same particle, while NFCM detected either CD81 or CD63. Based on the results of this study, we can draw conclusions about existing single-particle analysis capabilities that may be useful for EV biomarker development and mechanistic studies

Acetylcholinesterase is not a generic marker of extracellular vesicles

Acetylcholinesterase (AChE) activity is found in abundance in reticulocytes and neurons and was developed as a marker of reticulocyte EVs in the 1970s. Easily, quickly, and cheaply assayed, AChE activity has more recently been proposed as a generic marker for small extracellular vesicles (sEV) or exosomes, and as a negative marker of HIV-1 virions. To evaluate these proposed uses of AChE activity, we examined data from different EV and virus isolation methods using T-lymphocytic (H9, PM1 and Jurkat) and promonocytic (U937) cell lines grown in culture conditions that differed by serum content. When EVs were isolated by differential ultracentrifugation, no correlation between AChE activity and particle count was observed. AChE activity was detected in non-conditioned medium when serum was added, and most of this activity resided in soluble fractions and could not be pelleted by centrifugation. The serum-derived pelletable AChE protein was not completely eliminated from culture medium by overnight ultracentrifugation; however, a serum “extra-depletion” protocol, in which a portion of the supernatant was left undisturbed during harvesting, achieved near-complete depletion. In conditioned medium also, only small percentages of AChE activity could be pelleted together with particles. Furthermore, no consistent enrichment of AChE activity in sEV fractions was observed. Little if any AChE activity is produced by the cells we examined, and this activity was mainly present in non-vesicular structures, as shown by electron microscopy. Size-exclusion chromatography and iodixanol gradient separation showed that AChE activity overlaps only minimally with EV-enriched fractions. AChE activity likely betrays exposure to blood products and not EV abundance, echoing the MISEV 2014 and 2018 guidelines and other publications. Additional experiments may be merited to validate these results for other cell types and biological fluids other than blood.Fil: Liao, Zhaohao. University Johns Hopkins; Estados UnidosFil: Martin Jaular, Lorena. Inserm; Francia. PSL Research University; FranciaFil: Soueidi, Estelle. Inserm; Francia. PSL Research University; FranciaFil: Jouve, Mabel. PSL Research University; FranciaFil: Muth, Dillon C.. University Johns Hopkins; Estados UnidosFil: Schøyen, Tine H.. University Johns Hopkins; Estados UnidosFil: Seale, Tessa. University Johns Hopkins; Estados UnidosFil: Haughey, Norman J.. University Johns Hopkins; Estados UnidosFil: Ostrowski, Matias. Universidad de Buenos Aires; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Investigaciones Biomédicas en Retrovirus y Sida. Universidad de Buenos Aires. Facultad de Medicina. Instituto de Investigaciones Biomédicas en Retrovirus y Sida; ArgentinaFil: Théry, Clotilde. PSL Research University; FranciaFil: Witwer, Kenneth W.. University Johns Hopkins; Estados Unido

ELISA‐based detection of immunoglobulins against extracellular vesicles in blood plasma

Abstract Extracellular vesicles (EVs) are intensively investigated for their therapeutic potential and application as drug delivery vehicle. A broad perception of favourable safety profiles and low immunogenicity make EVs an attractive alternative to synthetic nanoparticles. We recently showed that repeated intravenous administration of human cell‐derived EVs into pig‐tailed macaques unexpectedly elicited antibody responses after three or more injections. This coincided with decreasing EV circulation time, and may thus hamper successful EV‐mediated cargo delivery into tissues. Here, we share the custom ELISA protocol that we used to measure such antibody responses. This protocol may help other researchers evaluate immune responses to EV‐based therapies in preclinical studies

Vaginal transmission of cell-associated HIV-1 in the mouse is blocked by a topical, membrane-modifying agent

Because both HIV-1 virions and HIV-infected cells are present in the semen and cervical mucus of infected individuals, HIV-1 prevention strategies must consider both cell-free and cell-associated virus. Antibodies that target HIV-1 virions have been shown to prevent vaginal transmission of cell-free virus in macaques, but since cell-associated transmission has not been reliably demonstrated in this model system, no strategies to prevent such transmission have been tested. We have employed a mouse model in which SCID mice carry human peripheral blood leukocytes (HuPBLs). In these mice, vaginal transmission of cell-associated, but not cell-free, HIV-1 transmission occurs, mediated by transepithelial migration of HIV-infected cells. Topical application of β-cyclodextrin (β-CD), a cholesterol-sequestering agent that interferes with cell migration and budding of virus from lipid rafts, blocks transmission of cell-associated HIV-1. The HuPBL-SCID model of vaginal HIV-1 transmission should prove useful for investigating cell-associated HIV-1 transmucosal HIV-1 transmission, as well as for screening reagents for their potential efficacy in preventing sexual HIV-1 transmission

Minocycline prevents TRAIL upregulation in pDCs and CD4+ T cells by attenuating anti-viral IFN and activation responses.

<p>(A) pDCs become activated in response to TLR7/9 stimulation by HIV and secrete type I IFN, which in turn upregulates ISGs on leukocytes, including the TNF family members TRAIL and FasL and the B7 family member PDL1. These ligands induce apoptosis and/or exhaustion on target cells expressing the cognate death receptors (TRAIL/DR5; FasL/Fas; PDL1/PD1). (B–G) pDCs were isolated from blood of healthy human donors and exposed to 300 ng p24 eq./mL of AT-2 HIV with or without 20 μM minocycline for 18 hours (<i>n</i> = 6 different donors). (B, C) IFNα and IFNβ protein from pDC supernatants were measured by ELISA. (D) IFNβ mRNA was measured by qRT-PCR. (E) Mx mRNA and (F) TRAIL were measured as examples of ISGs by qRT-PCR and flow cytometry, respectively. (G) Viability of pDCs was determined by Annexin V/7AAD staining. (H) CD4+ T cells were isolated from blood of healthy human donors and activated with anti-CD3 with or without 20 μM minocycline (<i>n</i> = 6 different donors). After 24 hours, minocycline was replenished and some wells were additionally stimulated with IFNα and IFNβ. TRAIL was measured by flow cytometry after an additional 24 hours. (I) Representative flow cytometry gating of pDC purity by BDCA2+/CD123+ double staining immediately following isolation. (J) Representative gating of CD4+ T cell purity by CD4+/CD3+ double staining immediately following isolation. (K) Representative gating of pDC viability (Annexin V-/7AAD-) and TRAIL expression after 18 hours of stimulation in culture with virus and/or minocycline. (L) Representative gating of TRAIL expression in CD4+ T cells following 48 hours in culture. Parametric data were analyzed by paired <i>t</i>-test (TRAIL flow cytometry), and nonparametric data were analyzed by Wilcoxon signed-rank test (IFN ELISAs and IFNβ, Mx qRT-PCR).</p

Minocycline attenuates type I IFN production and TRAIL expression in lymphocytes.

<p>PBMCs were isolated from the blood of healthy human donors, pretreated for two hours <i>in vitro</i> with 0, 20, or 40 μM minocycline, and exposed to increasing amounts of either AT-2 inactivated HIV (<i>n</i> = 4 different donors) or infectious influenza virus (<i>n</i> = 3 different donors). After overnight culture, supernatants were analyzed for secreted IFNα (<b>A, D</b>) and IFNβ protein (<b>B, E</b>) by ELISA. (<b>C, F</b>) Lymphocytes were analyzed by flow cytometry for TRAIL expression. (<b>G</b>) Representative flow cytometry gating of lymphocyte TRAIL expression in PBMC mixed cultures following AT-2 HIV stimulation. (<b>H</b>) Representative gating of lymphocyte TRAIL expression in PBMC mixed cultures following influenza stimulation. A two-way repeated measures ANOVA was used to compare the effect of different doses of minocycline (<i>p</i>-value shown on graph) and varying levels of AT-2 HIV or influenza on levels of TRAIL, IFNα, and IFNβ.</p

Minocycline prevents CTLA-4 and IDO expression in CD4+ T cells and pDCs.

<p>(<b>A</b>) IDO can be induced in pDCs by engagement of B7 receptors with CTLA-4 on CD4+ T cells or by stimulation with IFNα, β, γ, TNFα, TGFβ, or HIV. IDO converts the amino acid tryptophan (TRP) into L-formylkynurenine, initiating the production of a cascade of TRP metabolites that block T cell proliferation, induce T cell apoptosis, and convert CD4+ cells into Tregs. KYN: kynurenine; PIC: picolinic acid; TRP: tryptophan; 3HAA: 3-hydroxy anthranilic acid. (<b>B</b>) CD4+ T cells were isolated from blood of healthy human donors and activated with anti-CD3 with or without 20 μM minocycline (<i>n</i> = 6 different donors). After 24 hours minocycline was replenished and some wells were stimulated with IFNα and IFNβ. After 48 hours total cells were analyzed by flow cytometry for CTLA-4. (<b>C</b>) pDCs were isolated from blood of healthy human donors and exposed to AT-2 HIV with or without 20 μM minocycline (<i>n</i> = 6 different donors). After 18 hours RNA was harvested for IDO1 qRT-PCR. CTLA-4 data were analyzed by paired <i>t</i>-test. IDO mRNA was analyzed by Wilcoxon signed-rank test. (<b>D</b>) Representative flow cytometry gating of CTLA-4 expression on isolated CD4+ T cells.</p

Attenuation of Pathogenic Immune Responses during Infection with Human and Simian Immunodeficiency Virus (HIV/SIV) by the Tetracycline Derivative Minocycline

<div><p>HIV immune pathogenesis is postulated to involve two major mechanisms: 1) chronic innate immune responses that drive T cell activation and apoptosis and 2) induction of immune regulators that suppress T cell function and proliferation. Both arms are elevated chronically in lymphoid tissues of non-natural hosts, which ultimately develop AIDS. However, these mechanisms are not elevated chronically in natural hosts of SIV infection that avert immune pathogenesis despite similarly high viral loads. In this study we investigated whether minocycline could modulate these pathogenic antiviral responses in non-natural hosts of HIV and SIV. We found that minocycline attenuated <i>in vitro</i> induction of type I interferon (IFN) and the IFN-stimulated genes indoleamine 2,3-dioxygenase (IDO1) and TNF-related apoptosis inducing ligand (TRAIL) in human plasmacytoid dendritic cells and PBMCs exposed to aldrithiol-2 inactivated HIV or infectious influenza virus. Activation-induced TRAIL and expression of cytotoxic T-lymphocyte antigen 4 (CTLA-4) in isolated CD4+ T cells were also reduced by minocycline. Translation of these <i>in vitro</i> findings to <i>in vivo</i> effects, however, were mixed as minocycline significantly reduced markers of activation and activation-induced cell death (CD25, Fas, caspase-3) but did not affect expression of IFNβ or the IFN-stimulated genes IDO1, FasL, or Mx in the spleens of chronically SIV-infected pigtailed macaques. TRAIL expression, reflecting the mixed effects of minocycline on activation and type I IFN stimuli, was reduced by half, but this change was not significant. These results show that minocycline administered after infection may protect against aspects of activation-induced cell death during HIV/SIV immune disease, but that <i>in vitro</i> effects of minocycline on type I IFN responses are not recapitulated in a rapid progressor model <i>in vivo</i>.</p></div

Induction of HIF-1α by HIV-1 Infection in CD4+ T Cells Promotes Viral Replication and Drives Extracellular Vesicle-Mediated Inflammation

Human immunodeficiency virus type 1 (HIV-1) is a very important global pathogen that preferentially targets CD4+ T cells and causes acquired immunodeficiency syndrome (AIDS) if left untreated. Although antiretroviral treatment efficiently suppresses viremia, markers of immune activation and inflammation remain higher in HIV-1-infected patients than in uninfected individuals. The hypoxia-inducible factor 1α (HIF-1α) is a transcription factor that plays a fundamental role in coordinating cellular metabolism and function. Here we show that HIV-1 infection induces HIF-1α activity and that this transcription factor upholds HIV-1 replication. Moreover, we demonstrate that HIF-1α plays a key role in HIV-1-associated inflammation by promoting the release of extracellular vesicles which, in turn, trigger the secretion of inflammatory mediators by noninfected bystander lymphocytes and macrophages. In summary, we identify that the coordinated actions of HIF-1α and extracellular vesicles promote viral replication and inflammation, thus contributing to HIV-1 pathogenesis.Chronic immune activation and inflammation are hallmarks of HIV-1 infection and a major cause of serious non-AIDS events in HIV-1-infected individuals on antiretroviral treatment (ART). Herein, we show that cytosolic double-stranded DNA (dsDNA) generated in infected CD4+ T cells during the HIV-1 replication cycle promotes the mitochondrial reactive oxygen species (ROS)-dependent stabilization of the transcription factor hypoxia-inducible factor 1α (HIF-1α), which in turn, enhances viral replication. Furthermore, we show that induction of HIF-1α promotes the release of extracellular vesicles (EVs). These EVs foster inflammation by inducing the secretion of gamma interferon by bystander CD4+ T cells and secretion of interleukin 6 (IL-6) and IL-1β by bystander macrophages through an HIF-1α-dependent pathway. Remarkably, EVs obtained from plasma samples from HIV-1-infected individuals also induced HIF-1α activity and inflammation. Overall, this study demonstrates that HIF-1α plays a crucial role in HIV-1 pathogenesis by promoting viral replication and the release of EVs that orchestrate lymphocyte- and macrophage-mediated inflammatory responses