Eradication of human immunodeficiency virus type-1 (HIV-1)-infected cells

Predictions made soon after the introduction of human immunodeficiency virus type-1 (HIV-1) protease inhibitors about potentially eradicating the cellular reservoirs of HIV-1 in infected individuals were too optimistic. The ability of the HIV-1 genome to remain in the chromosomes of resting CD4+ T cells and macrophages without being expressed (HIV-1 latency) has prompted studies to activate the cells in the hopes that the immune system can recognize and clear these cells. The absence of natural clearance of latently infected cells has led to the recognition that additional interventions are necessary. Here, we review the potential of utilizing suicide gene therapy to kill infected cells, excising the chromosome-integrated HIV-1 DNA, and targeting cytotoxic liposomes to latency-reversed HIV-1-infected cells

A monoclonal antibody to the gp120-CD4 complex has differential effects on HIV-induced syncytium formation and viral infectivity

A murine monoclonal antibody (MAb F-91-55) raised against the complex of soluble CD4 and human immunodeficiency virus type 1 (HIV-1) gp120 had previously been found to inhibit syncytium formation without inhibiting the interaction of CD4 with gp120, and its binding site was localized within the first two domains (D1/D2) of CD4. We investigated whether this antibody inhibited the infectivity of HIV-1 in the CD4+ T cell lines A3.01, Sup-T1 and H9. We also examined the effect of the antibody on syncytium formation between these cells and chronically infected H9 cells. Syncytium formation was found to depend critically on the incubation medium used. The effect of the MAb on HIV-1 infectivity was very limited with A3.01 and Sup-T1 cells, although it inhibited syncytium formation between A3.01 or Sup-T1 and chronically infected H9 cells. In contrast, the MAb inhibited significantly the infectivity of HIV-1 in H9 cells, but it also inhibited syncytium formation between H9 and chronically infected H9 cells to a greater extent than in the case of the other cell lines. Our results indicate that cellular systems used for syncytium assays differ in their susceptibility to inhibitory antibodies. In the A3.01 and Sup-T1 cell systems, the differences in the ability of the MAb to block viral entry or syncytium formation raise the possibility that the mechanisms of interaction of gp120/gp41 with cell membrane CD4 may be different in cell-cell and virus-cell membrane fusion

Inhibition of HIV-1 Env-Mediated Cell-Cell Fusion by Lectins, Peptide T-20, and Neutralizing Antibodies

Broadly cross-reactive, neutralizing human monoclonal antibodies, including 2F5, 2G12, 4E10 and IgG1 b12, can inhibit HIV-1 infection in vitro at very low concentrations. We examined the ability of these antibodies to inhibit cell-cell fusion between Clone69TRevEnv cells induced to express the viral envelope proteins, gp120/gp41 (Env), and highly CD4-positive SupT1 cells. The cells were loaded with green and red-orange cytoplasmic fluorophores, and fusion was monitored by fluorescence microscopy.status: publishe

Human immunodeficiency virus type-1 (HIV-1) infection increases the sensitivity of macrophages and THP-1 cells to cytotoxicity by cationic liposomes

Cationic liposomes may be valuable for the delivery of anti-sense oligonucleotides, ribozymes, and therapeutic genes into human immunodeficiency virus type 1 (HIV-1)-infected and uninfected cells. We evaluated the toxicity of three cationic liposomal preparations, Lipofectamine, Lipofectin, and 1,2-dimyristyloxypropyl-3-dimethyl-hydroxyethyl ammonium bromide (DMRIE) reagent, to HIV-infected and uninfected cells. Monocyte/macrophages were infected with HTV-1(BaL) and treated with liposomes in medium containing 20% fetal bovine serum (FBS) for 4 h or 24 h at 37°C. Uninfected monocytic THP-1 cells and chronically infected THP-1/HIV-1(IIIB) cells were treated with phorbol 12-myristate 13-acetate (PMA) and exposed to liposomes in the presence of 10% FBS. Toxicity was evaluated by the Alamar Blue assay and viral p24 production. The toxic effect of cationic liposomes was very limited with uninfected cells, although concentrations of liposomes that were not toxic within a few days of treatment could cause toxicity at later times. In HIV-1(BaL)-infected macrophages, Lipofectamine (up to 8 μM) and Lipofectin (up to 40 μM) were not toxic after a 4-h treatment, while DMRIE reagent at 40 μM was toxic. While a 4-h treatment of THP-1/HIV-1(IIIB) cells with the cationic liposomes was not toxic, even up to 14 days post-treatment, all three cationic liposomes were toxic to cells at the highest concentration tested after a 24-h treatment. Similar results were obtained with the Alamar Blue assay, Trypan Blue exclusion and a method that enumerates nuclei. Infected cells with relatively high overall viability could be impaired in their ability to produce virions, indicating that virus production appears to be more sensitive to treatment with the cationic liposomes than cell viability. Our results indicate that HIV-infected cells are more susceptible than uninfected cells to killing by cationic liposomes. The molecular basis of this differential effect is unknown; it is proposed that alterations in cellular membranes during virus budding cause enhanced interactions between cationic liposomes and cellular membranes

Human immunodeficiency virus type-1 (HIV-1) infection increases the sensitivity of macrophages and THP-1 cells to cytotoxicity by cationic liposomes

AbstractCationic liposomes may be valuable for the delivery of anti-sense oligonucleotides, ribozymes, and therapeutic genes into human immunodeficiency virus type 1 (HIV-1)-infected and uninfected cells. We evaluated the toxicity of three cationic liposomal preparations, Lipofectamine, Lipofectin, and 1,2-dimyristyloxypropyl-3-dimethyl-hydroxyethyl ammonium bromide (DMRIE) reagent, to HIV-infected and uninfected cells. Monocyte/macrophages were infected with HIV-1BaL and treated with liposomes in medium containing 20% fetal bovine serum (FBS) for 4 h or 24 h at 37°C. Uninfected monocytic THP-1 cells and chronically infected THP-1/HIV-1IIIB cells were treated with phorbol 12-myristate 13-acetate (PMA) and exposed to liposomes in the presence of 10% FBS. Toxicity was evaluated by the Alamar Blue assay and viral p24 production. The toxic effect of cationic liposomes was very limited with uninfected cells, although concentrations of liposomes that were not toxic within a few days of treatment could cause toxicity at later times. In HIV-1Bal-infected macrophages, Lipofectamine (up to 8 μM) and Lipofectin (up to 40 μM) were not toxic after a 4-h treatment, while DMRIE reagent at 40 μM was toxic. While a 4-h treatment of THP-1 /HIV-1 IIIB cells with the cationic liposomes was not toxic, even up to 14 days post-treatment, all three cationic liposomes were toxic to cells at the highest concentration tested after a 24-h treatment. Similar results were obtained with the Alamar Blue assay, Trypan Blue exclusion and a method that enumerates nuclei. Infected cells with relatively high overall viability could be impaired in their ability to produce virions, indicating that virus production appears to be more sensitive to treatment with the cationic liposomes than cell viability. Our results indicate that HIV-infected cells are more susceptible than uninfected cells to killing by cationic liposomes. The molecular basis of this differential effect is unknown; it is proposed that alterations in cellular membranes during virus budding cause enhanced interactions between cationic liposomes and cellular membranes

Anionic liposomes inhibit human immunodeficiency virus type 1 (HIV-1) infectivity in CD4\u3csup\u3e+\u3c/sup\u3e A3.01 and H9 cells

Immunodeficiency viruses undergo fusion with liposomes containing anionic phospholipids (Larsen et al., 1990). We have investigated the effect of liposomes composed of cardiolipin, phosphatidylserine or phosphatidylinositol, on the infectivity of three strains of HIV-1 in A3.01 and H9 cells, measured by p24 (gag) production in the medium. The infectivity of HIV-1 in A3.01 or H9 cells was inhibited by the presence of cardiolipin liposomes during a 2 h infection period, with IC50\u27s of 23.0, 4.8, and 5.0 μM phospholipid, respectively, for the different strains. Liposomes composed of phosphatidylserine or phosphatidylinositol were ineffective under similar conditions. However, prolonged pre-incubation of the virus with these liposomes also inhibited infectivity. Inhibition of virus binding to cells could not account for the inhibition of infectivity. We propose that the fusion products of HIV-1 and anionic liposomes are impaired in their ability to fuse with the plasma membrane

Cationic liposome-mediated expression of HIV-regulated luciferase and diphtheria toxin a genes in HeLa cells infected with or expressing HIV

HIV-regulated expression of the diphtheria toxin A fragment gene (HIV-DT-A) is a potential gene therapy approach to AIDS. Since cationic liposomes are safe and non-immunogenic for in vivo gene delivery, we examined whether LipofectAMINE or DMRIE reagent could mediate the transfection of HIV-DT-A (pTHA43) or the HIV-regulated luciferase gene (pLUCA43) into HIV-infected or uninfected HeLa cells. pLUCA43 was expressed at a 103-fold higher level in HeLa/LAV cells than in uninfected HeLa cells, while the extent of expression of RSV-regulated luciferase was the same in both cell lines. Co-transfection of HeLa cells with pTHA43 and the proviral HIV clone, HXBΔBgl, resulted in complete inhibition of virus production. In contrast, the delivery of HIV-DT-A to chronically infected HeLa/LAV or HeLa/IIIB cells, or to HeLa CD4+ cells before infection, did not have a specific effect on virus production, since treatment of cells with control plasmids also reduced virus production. This reduction could be ascribed to cytotoxicity of the reagents. The efficiency of transfection, as measured by the percentage of cells expressing β-gal, was ~5%. Thus, cationic liposome mediated transfection was too inefficient to inhibit virus production when the DT-A was delivered by cationic liposomes to chronically- or de novo-infected cells. However, when both the virus and DT-A genes were delivered into the same cells by cationic liposomes, DT-A was very effective at inhibiting virus production. Our results indicate that the successful use of cationic liposomes for gene therapy will require the improvement of their transfection efficiency

Delivery of an anti-HIV-1 ribozyme into HIV-infected cells via cationic liposomes

Cationic liposome-mediated intracellular delivery of a fluorescein-labeled chimeric DNA-RNA ribozyme targeted to the HIV-1 5\u27 LTR was investigated, using THP-1, THP-1/HIV-1(IIIB) or HeLa/LAV cells. Different fluorescence patterns were observed when the cells were exposed to Lipofectamine, Lipofectin or DMRIE:DOPE (1:1) complexed to the ribozyme. With Lipofectamine intense cell-associated fluorescence was found. Incubation with Lipofectin resulted in less intense diffuse fluorescence, while with DMRIE an intense but sporadic fluorescence was observed. Differentiated THP-1/HIV-1(IIIB) cells were more susceptible to killing by liposome-ribozyme complexes than THP-1 cells. Under non-cytotoxic conditions (a 4-h treatment) complexes of 5, 10 or 15 μM Lipofectin or DOTAP:DOPE (1:1) and ribozyme, at lipid:ribozyme ratios of 8:1 or 4:1, did not affect p24 production in THP-1/HIV-1(IIIB) cells in spite of the intracellular accumulation of the ribozyme. A 24-h exposure of THP-1/HIV-1(IIIB) cells to 5 μM Lipofectin or DOTAP:DOPE (1:1) complexed with either the functional or a modified control ribozyme reduced virus production by approximately 30%. Thus, the antiviral effect of the liposome-complexed ribozyme was not sequence-specific. In contrast, the free ribozyme at a relatively high concentration inhibited virus production by 30%, while the control ribozyme was ineffective, indicating a sequence-specific effect. Both Lipofectin and DOTAP complexed with ribozyme were toxic at 10 and 15 μM after a 24-h treatment. A 4-h treatment of HeLa/LAV cells with Lipofectin at 5, 10 or 15 μM was not toxic to the cells, but also did not inhibit p24 production. In contrast, treatment of HeLa CD4+ cells immediately after infection with HIV-1(IIIB) at the same lipid concentrations and lipid:ribozyme ratios was cytotoxic. Our results indicate that the delivery of functional ribozyme into cells by cationic liposomes is an inefficient process and needs extensive improvement before it can be used in ex vivo and in vivo applications. Copyright (C) 1998 Elsevier Science B.V

Longitudinal evaluation of prostaglandin E2 (PGE2) and periodontal status in HIV\u3csup\u3e+\u3c/sup\u3e patients

The study aim was to determine whether prostaglandin E2 (PGE2) in gingival crevicular fluid (GCF) could serve as a risk factor for periodontitis in human immunodeficiency virus-positive (HIV+) patients. Clinical measurements, including gingival index (GI), plaque index, bleeding index, probing depth (PD), attachment loss (AL) and GCF samples were taken from two healthy sites (including sites with gingival recession, GI = 0; PD ≤ 3 mm; AL ≤ 2 mm), three gingivitis sites (GI \u3e 0; PD ≤ 3 mm; AL = 0) and three periodontitis sites (GI \u3e 0; PD ≥ 5 mm; AL ≥ 3 mm) of each of the 30 patients at baseline and 6-month visits. GCF samples were also taken by means of paper strips. GCF PGE2 levels were determined by a sandwich ELISA. The progressing site was defined as a site which had 2 mm or more attachment loss during the 6-month study period. The mean amounts of PGE2 were significantly higher in gingivitis and periodontitis sites than in healthy sites (p \u3c 0.0001). GCF levels of PGE2 were significantly correlated with probing depth, attachment loss, CD4+ cells, viral load, age and smoking pack-years at baseline and 6-month visits (0.0001 \u3c p \u3c 0.05). Repeated measures analysis of 19 active sites versus 221 inactive sites indicated that PGE2 levels were significantly higher in active sites than in inactive sites (p \u3c 0.0001). It is likely that the compromised immune system contributes to the pathogenesis of periodontitis in HIV+ patients. It is well known that the activated inflammatory cells produce inflammatory mediators which stimulate the production of PGE2. Longitudinal evaluation of GCF PGE2 with respect to the progression of untreated periodontitis sites in HIV+ subjects will contribute to the understanding of the pathogenesis of periodontitis in HIV+ patients. These data indicate that sites with high GCF levels of PGE2 in HIV+ patients are at significantly greater risk for progression of periodontitis. © 2007 Elsevier Ltd. All rights reserved

Enhancement of human immunodeficiency virus type 1 infection by cationic liposomes: The role of CD4, serum and liposome-cell interactions

We have reported previously the enhancement of the infectivity of human immunodeficiency virus type 1 (HIV- 1) by liposomes composed of the cationic lipid N[2,3-(dioleyloxy) propyl]-N,N,N-trimethylammonium chloride (DOTMA). To determine the mechanism by which this process occurs, we have investigated the role of CD4, serum concentration and liposome-cell interactions in the DOTMA-mediated stimulation of HIV-1 infection of A3.01 cells. Serum alone significantly inhibited the binding and infectivity of HIV-1, but DOTMA-mediated enhancement of infectivity was more pronounced in the presence of serum than in its absence. HIV-1 binding to cells was increased in the presence of DOTMA liposomes, DEAE-dextran and polybrene, all of which also enhanced infectivity to a similar extent at comparable concentrations. Fluorescence dequenching measurements indicated that DOTMA liposomes fused with HIV-1, but not with cell membranes, in the presence of serum. The enhancing effect of DOTMA liposomes on HIV-1 infectivity was CD4-dependent, and appeared to involve virus-liposome fusion and liposome binding to the cell surface. DOTMA liposomes did not mediate infection of the CD4- K562 and Raji cell lines