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

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

Transfection of human macrophages by lipoplexes via the combined use of transferrin and pH-sensitive peptides

The crucial function of macrophages in a variety of biological processes and pathologies render these cells important targets for gene therapeutic interventions. Commonly used synthetic gene delivery vectors have not been successful in transfecting these non-dividing cells. A combination strategy involving cationic liposomes to condense and carry DNA, transferrin to facilitate cellular uptake, and the pH-sensitive peptide GALA to promote endosome destabilization, resulted in significant expression of a luciferase gene. Transfection of macrophages was dependent on the degree of differentiation of the cells. The quaternary complexes of cationic liposomes, DNA, transferrin, and GALA exhibited a net negative charge, which may obviate a limitation of cationic synthetic vectors in vivo. The lack of cytotoxicity and the expected back of immunogenicity of these complexes may render them useful for gene delivery to macrophages in vivo

Differential effects of a hydrophobic tripeptide on human immunodeficiency virus type 1 (HIV-1)-induced syncytium formation and viral infectivity

The synthetic hydrophobic peptide, Z-D-Phe-L-Phe-Gly, was shown previously to inhibit the infectivity of paramyxoviruses and the fusion of Sendai virus with liposomes. We examined the ability of this peptide to inhibit HIV-1 infectivity in A3.01, Sup-T1, and H9 cells and syncytium formation between these cells and chronically infected H9 cells. Although the peptide inhibited syncytium formation in a dose-dependent manner, its effect on virus infectivity was very limited. Our results suggest that the mechanisms of interaction of the HIV-1 envelope glycoprotein gp120/gp41 with the target cell membrane leading to membrane fusion may be different in cell-cell and virus-cell fusion. © 1995 by Academic Press, Inc

Inhibition of human immunodeficiency virus type-1 replication in macrophages and H9 cells by free or liposome-encapsulated L-689,502, an inhibitor of the viral protease

Macrophages are recognized as a major reservoir of HIV-1 in infected individuals. We examined the effect of an inhibitor of the viral protease, L-689,502, on virus production by monocyte-derived macrophages infected with HIV-1(BaL). Continuous treatment with L-689,502 drastically inhibited virus production in a dose-dependent manner in the range of 10-200 nM, in some cases by more than 1000-fold, compared to untreated cells. Since liposomes can be targeted to macrophages in vivo, we examined whether the inhibitor was effective following delivery in liposomes. The inhibitor encapsulated in multilamellar liposomes was more effective than the free drug in inhibiting virus production in macrophages, throughout the concentration range studied. The EC90 of the liposomal inhibitor was 2.9- to 4.5-fold lower than that of the free compound. L-689,502 encapsulated in sterically stabilized liposomes with prolonged circulation time inhibited virus production at a level comparable to the free inhibitor. When macrophages were infected and treated for only a limited time, L-689,502 in multilamellar liposomes was the most effective of the three treatments. In chronically infected H9 cells treated continuously, the free inhibitor was more effective than the liposome-encapsulated drug, but virus production was reduced only to 40-60% of controls. In contrast, treatment of acutely infected H9 cells with either free or encapsulated L-689,502 inhibited virus production by up to three orders of magnitude. Our results indicate that liposomes may be useful for the delivery of HIV protease inhibitors with low aqueous solubility and low oral bio-availability, and for the targeting of these drugs to lymph nodes

Secretory leukocyte protease inhibitor (SLPI): oxidation of SLPI does not explain its variable anti-HIV activity

Secretory leukocyte protease inhibitor (SLPI) has been proposed as a potential inhibitor of HIV-1 infection in human saliva. Although the ability of recombinant (r) SLPI to inhibit HIV-1 infection of macrophages and primary Tcells has been demonstrated by two independent laboratories, evidence to the contrary has also been reported. This study re-examines the anti-HIV effect of rSLPI and investigates the effects of repeated freeze-thawing and oxidation on the anti-HIV activity of rSLPI. rSLPI inhibited HIV-1BaL infection of human macrophages in a highly variable manner. HPLC and electrospray ionization mass spectrometry (ESI) analyses indicated that variability in our inhibition data could not be attributed to the degradation or oxidation of rSLPI. These results suggest that the variable anti-HIV effect of rSLPI may be due to differential expression of the cell-surface molecule(s) to which SLPI binds rather than to changes in the rSLPI molecule