Differential in vitro inhibitory activity against HIV-1 of alpha-(1-3)- and alpha-(1-6)-D-mannose specific plant lectins: implication for microbicide development

Abstract

Background: Plant lectins such as Galanthus nivalis agglutinin (GNA) and Hippeastrum hybrid agglutinin (HHA) are natural proteins able to link mannose residues, and therefore inhibit HIV-target cell interactions. Plant lectins are candidate for microbicide development.

Objective: To evaluate the activity against HIV of the mannose-specific plant lectins HHA and GNA at the cellular membrane level of epithelial cells and monocyte-derived dendritic cells (MDDC), two potential target cells of HIV at the genital mucosal level.

Methods: The inhibitory effects of HHA and GNA were evaluated on HIV adsorption to genital epithelial HEC-1A cell line, on HIV transcytosis throughout a monolayer of polarized epithelial HEC-1A cells, on HIV adsorption to MDDC and on transfer of HIV from MDDC to autologous T lymphocytes.

Results: HHA faintly inhibited attachment to HEC-1A cells of the R5-tropic HIV-1Ba-L strain, in a dose-dependent manner, whereas GNA moderately inhibited HIV adsorption in the same context, but only at high drug doses. Only HHA, but not GNA, inhibited HIV-1JR-CSF transcytosis in a dose-dependent manner. By confocal microscopy, HHA, but not GNA, was adsorbed at the epithelial cell surface, suggesting that HHA interacts specifically with receptors mediating HIV-1 transcytosis. Both plant lectins partially inhibited HIV attachment to MDDC. HHA inhibited more efficiently the transfer of HIV from MDDC to T cell, than GNA. Both HHA and GNA lacked toxicity below 200 microg/ml irrespective the cellular system used and do not disturb the monolayer integrity of epithelial cells.

Conclusion: These observations demonstrate higher inhibitory activities of the lectin plant HHA by comparison to GNA, on HIV adsorption to HEC-1A cell line, HIV transcytosis through HEC-1A cell line monolayer, HIV adsorption to MDDC and HIV transfer from MDDC to T cells, highlighting the potential interest of HHA as effective microbicide against HIV.

Figure 1

(A). Non-toxic concentration of the study plant lectins in epithelial cell line (HEC-1A) and primary immune cells (MDDC and MDM). HEC-1A and primary immune cells were cultured with concentrations of products for 24 h. After washing, culture viability was determined by using the MTT-cytotoxicity assay. The values given are the percentage of viability. (B) and (C). Effect of HHA and GNA on HEC-1A epithelial monolayer integrity. HEC-1A cells were grown on transwell supports and allowed to form intact monolayer (5 to 7 days). Non-toxic concentrations of each lectins were added to the apical side of the transwell, and epithelial integrity was measured at 30 min and 2, 4, 9, and 24 h by using the Minicell-ERS instrument. The data shown are the averages from duplicate wells ± 1 standard error of the mean after product addition.

Figure 2

Ability of lectins to inhibit attachment of HIV-1 cell-free particles on epithelial cells. HEC-1A cells were co-incubated with non-toxic concentrations of each lectins or mannan (100 μg/ml) and 5 ng of HIV-1_Ba-L (A) or HIV-1_NDK (B) virus were added for 1 h. Incubation with mannan was used as control. The quantity of attached virus was evaluated by measurement of p24 antigen by ELISA. Means of three independent experiments are presented ; error bars represent standard deviations. (C) HEC-1A cells were incubated with or without each labelled-lectin at 4°C. Cells were then analyzed by confocal microscopy. * < 0.05.

Figure 3

Ability of lectins to inhibit transcytosis of HIV-1_J-RCSF free particles through a tight monolayer of endometrial epithelial cell (HEC-1A). HIV-1_J-RCSF (5 ng p24 antigen) was co-incubated with a non-toxic concentrations of each lectins or mannan (100 μg/ml) on the HEC-1A apical pole cultured in dual-chamber by the transwell system for 1 h. Results of an experiment performed in duplicate are expressed as the quantity of virus recovered in the basal chamber in the presence of HHA (A) and GNA (B), or in the absence of lectins. Means of three independent experiments are presented ; error bars represent standard deviations. *< 0.05, **< 0.01.

Figure 4

(A). Lack of toxicity of HHA and GNA towards dendritic cells (MDDC). MDDC were cultured with concentrations of products for 24 h. After washing, culture viability was determined by using the MTT cytotoxicity assay. The values given are the percentage of viability. (B). Interactions of plant lectins with surface receptors of dendritic cells. Dendritic cells (MDDC) were incubated with or without each labelled-lectin at 4°C. Cells were then analyzed by confocal microscopy. (C and D). Ability of lectins to inhibit uptake of HIV-1_Ba-L cell-free particles on dendritic cells. MDDC were co-incubated with non-toxic concentrations of HHA (C), or GNA (D) or mannan (100 μg/ml) and 1 ng p24 antigen of HIV-1_Ba-L were added for 1 h. The quantity of attached-virus was evaluated by measurement of p24 antigen by ELISA. Means of three independent experiments are presented ; error bars represent standard deviations. *< 0.05

Figure 5

Ability of plant lectins to inhibit transfer of HIV-1_Ba-L free particles by dendritic cells to autologous T lymphocytes. Dendritic cells (MDDC) were incubated with non-toxic concentrations of each plant lectin and 0.5 ng p24 antigen of virus were added for 3 h. The HIV-production by T cells was evaluated on the 6^th day of co-culture by measurement of p24 antigen by ELISA. Means of three independent experiments are presented; error bars represent standard deviations. *< 0.05, **< 0.01.