Mannose-specific plant lectins from the Amaryllidaceae family qualify as efficient microbicides for prevention of human immunodeficiency virus infection

Abstract

The plant lectins derived from Galanthus nivalis (Snowdrop) (GNA) and Hippeastrum hybrid (Amaryllis) (HHA) selectively inhibited a wide variety of human immunodeficiency virus type 1 (HIV-1) and HIV-2 strains and clinical (CXCR4- and CCR5-using) isolates in different cell types. They also efficiently inhibited infection of T lymphocytes by a variety of mutant virus strains. GNA and HHA markedly prevented syncytium formation between persistently infected HUT-78/HIV cells and uninfected T lymphocytes. The plant lectins did not measurably affect the antiviral activity of other clinically approved anti-HIV drugs used in the clinic when combined with these drugs. Short exposure of the lectins to cell-free virus particles or persistently HIV-infected HUT-78 cells markedly decreased HIV infectivity and increased the protective (microbicidal) activity of the plant lectins. Flow cytometric analysis and monoclonal antibody binding studies and a PCR-based assay revealed that GNA and HHA do not interfere with CD4, CXCR4, CCR5, and DC-SIGN and do not specifically bind with the membrane of uninfected cells. Instead, GNA and HHA likely interrupt the virus entry process by interfering with the virus envelope glycoprotein. HHA and GNA are odorless, colorless, and tasteless, and they are not cytotoxic, antimetabolically active, or mitogenic to human primary T lymphocytes at concentrations that exceed their antivirally active concentrations by 2 to 3 orders of magnitude. GNA and HHA proved stable at high temperature (50 degrees C) and low pH (5.0) for prolonged time periods and can be easily formulated in gel preparations for microbicidal use; they did not agglutinate human erythrocytes and were not toxic to mice when administered intravenously.

FIG. 1.

Effect of preexposure of GNA and HHA to cell-free HIV-1(III_B) particles, uninfected MT-4 cells, Sup-T1 cells, or persistently HIV-1-infected HUT-78/HIV-1(III_B) cells on the antiviral activity of the plant lectins. The data are the mean of two independent experiments. (A and B) Preexposure of GNA (A) or HHA (B) to cell-free virus (○) or noninfected MT-4 cells (▴) prior to virus infection of MT-4 cell cultures. As a control, the antiviral activities of the plant lectins that were not preexposed to HIV-1(III_B) or MT-4 cells are also shown (▪). (C and D) Preexposure of GNA (C) or HHA (D) to HUT-78/HIV-1(III_B) (○) or uninfected Sup-T1 (▴) cells prior to cocultivation of these cells with the non-drug-preexposed counterpart cells. As a control, the inhibitory effect of the plant lectins on giant cell formation between non-drug-preexposed HUT-78/HIV-1(III_B) and Sup-T1 cells (▪) is also shown.

FIG. 2.

PCR analysis of the presence of HIV-1(IIIB) DNA (RT gene) in HIV-1(IIIB)-infected CEM cell cultures that were exposed to fixed concentrations of HHA. Lanes: A, molecular weight markers; B, HHA at 25 μg/ml (subcultivation with cell suspensions); C, HHA at 25 μg/ml (subcultivation with cell culture supernatants); D, HHA at 6.25 μg/ml (subcultivation with cell culture supernatants); E, HHA at 1.25 μg/ml(subcultivation with cell culture supernatants); F, HHA at 0.50 μg/ml (subcultivation with cell culture supernatants).

FIG. 3.

PCR analysis of the appearance of HIV-1 LTR R/U5 DNA in HIV-1/NL4.3-infected U87.CD4.CXCR4 and HIV-1/Ba-L-infected U87.CD4.CCR5 cell cultures. β-Actin DNA was coamplified as an internal control. Lanes: 1, virus control; 2, HHA at 20 μg/ml; 3, GNA at 20 μg/ml; 4, AMD3100 at 1 μg/ml; 5, pentosan polysulfate at 20 μg/ml; 6, TAK-779 at 1 μg/ml.

FIG. 4.

Inhibitory activity of GNA and HHA on cell proliferation.

FIG. 5.

Inhibitory activity of GNA and HHA on the incorporation of [³H]deoxythymidine, [³H]uridine, or [³H]leucine into DNA, RNA, or proteins, respectively.

FIG. 6.

Hemagglutination of mammalian red blood cells by different concentrations of GNA and HHA. The data reflect a typical experiment.