Peptide-derivatized SB105-A10 dendrimer inhibits the infectivity of R5 and X4 HIV-1 strains in primary PBMCs and cervicovaginal histocultures

Abstract

Peptide dendrimers are a class of molecules that exhibit a large array of biological effects including antiviral activity. In this report, we analyzed the antiviral activity of the peptide-derivatized SB105-A10 dendrimer, which is a tetra-branched dendrimer synthetized on a lysine core, in activated peripheral blood mononuclear cells (PBMCs) that were challenged with reference and wild-type human immunodeficiency virus type 1 (HIV-1) strains. SB105-A10 inhibited infections by HIV-1 X4 and R5 strains, interfering with the early phases of the viral replication cycle. SB105-A10 targets heparan sulfate proteoglycans (HSPGs) and, importantly, the surface plasmon resonance (SPR) assay revealed that SB105-A10 strongly binds gp41 and gp120, most likely preventing HIV-1 attachment/entry through multiple mechanisms. Interestingly, the antiviral activity of SB105-A10 was also detectable in an organ-like structure of human cervicovaginal tissue, in which SB105-A10 inhibited the HIV-1ada R5 strain infection without altering the tissue viability. These results demonstrated the strong antiviral activity of SB105-A10 and suggest a potential microbicide use of this dendrimer to prevent the heterosexual transmission of HIV-1.

Figure 1. Structure of SB105-A10 and SB104 dendrimers.

The general structure of peptide is indicated. These dendrimers were synthesized by the addition of four identical short peptide chains to a tetrameric lysine central core. The aminoacid sequence of four peptide chains in SB105-A10 and SB104 is also shown.

Figure 2. SB105-A10 inhibits HIV-1_IIIb and HIV-1_ada replication in activated PBMCs.

HIV-1_IIIb and HIV-1_ada were pre-incubated for 1 hours at 37°C with different concentrations (0, 0.1, 1, 5, 10 and 20 µg/ml) of SB105-A10 (A) or SB104 (B) and challenged with activated PBMCs for 2 hours at 37°C. HIV replication was monitored by HIV-1 p24 ELISA and values were determined from cell culture supernatants at day 7 pi. Data were expressed as the means ± standard deviations (±SD) of HIV-1 gag p24 amount relative to untreated controls (set to 100%). Three experiments in duplicate were performed. The last two panels (C and D) show that the two dendrimers are not toxic for PBMCs at concentrations tested as documented by trypan blue technique. Data were expressed as the means (±SD) of viable cells relative to untreated controls (set to 100%) obtained from three independent experiments in duplicate. * p<0.05.

Figure 3. SB105-A10 treatment inhibits the HIV replication of HIV-1 reference strains and isolates from HIV-1 positive pateints.

Different concentrations (0, 0.1, 1, 5, 10 and 20 µg/ml) of SB105-A10 were added to HIV-1 reference (A) and isolated (B) strains (100 pg/ml p24) for 1 hours at 37°C and then added to activated PBMCs. After two hours, several PBS washings were carried out and p24 determination was performed at day 7. Significant decreases of HIV-1 p24 amount were detected in infected samples treated with 5, 10 and 20 µg/ml of SB105-A10 with respect to the untreated controls. Data were expressed as the means (±SD) of HIV-1 gag p24 amount relative to untreated controls (set to 100%) obtained from three independent experiments in duplicate. * p<0.05.

Figure 4. Investigation of SB105-A10-related antiviral mechanisms in activated PBMCs infected by HIV-1_IIIb or HIV-1_ada strains.

Attachment, pre-attachment and post-attachment assays were carried out as described in panel A, B and C respectively. Significant decreases of HIV-1 gag p24 in the cellular supernatants were detected at day 4 pi, in SB105-A10-treated samples with respect to untreated controls in pre-attachment and attachment assays (panel D). Data were expressed as the means (±SD) of HIV-1 gag p24 amount relative to untreated controls (set to 100%) obtained from three independent experiments in duplicate. *p<0.05.

Figure 5. Pre-incubation of SB105-A10 either with HIV-1_IIIb or HIV-1_ada strains inhibits the viral infectivity in dilution assay.

HIV-1 strains were incubated for 1 hour at 37°C with SB105-A10 (20 µg/ml; 4.2 µM). Following this incubation the samples were diluted 50-fold to reduce dendrimer concentrations to a level below that at which SB105-A10 significantly inhibits HIV replication and challenged with activated PBMCs. Significant decrease of HIV-1 p24 amount was detectable at day 4 pi. Data were expressed as the means (±SD) of HIV-1 p24 amount relative to untreated controls (set to 100%) for each HIV-1 strain obtained from three independent experiments in duplicate. * p<0.05.

Figure 6. SPR analysis of the interaction of SB105-A10 with HIV-1 gp41 and gp120 proteins.

Left panels: blank-subtracted sensorgrams showing the binding of gp41 ectodomain (aminoacids 546–682; 10 nM) and full-lenght recombinant gp120 glycoprotein (100 nM) to the sensorchips containing streptavidin and biotinylated SB105-A10 (straight line) or streptavidin alone (dotted line). Right panels: saturation curves of the binding of gp41 and gp120 to immobilized SB105-A10 peptide. The saturation curves were obtained using the values of RU bound at equilibrium after the injection of different concentrations (0.61, 1.25, 2.5, 5.0, 10 nM for gp41 and 12.5, 25, 50, 100 nM, for gp120). The χ² values of the fitting of the saturation curves were equal to 2.6 and 4.3 for gp41 and gp120 respectively.

Figure 7. Flow cytometry analysis of binding between SB105-A10 and heparan sulphates.

Typical experiments were shown. (A), Different concentrations of FITC-conjugated SB105-A10 (from 0 to 250 µg/ml) were assayed for 1 hour at 4°C on activated PBMCs (5×10⁶/ml). The saturation was achieved at 25 µg/ml. (B), Activated PBMCs (5×10⁶/ml) were treated with 25 µg/ml of FITC-conjugated SB105-A10 for 1 hour at 4°C and then washed with PBS containing 2 M NaCl, a treatment known to remove cationic polypeptides from cell surface HSPGs. (C), Activated PBMCs (5×10⁶/ml) were incubated with heparinase III for 2 h at 37°C or left untreated before the binding assay with 25 µg/ml of FITC-conjugated SB105-A10. Both heparinase III and 2 M NaCl treatments reduce but not abolish the specific fluorescence signal suggesting an interaction between SB105-A10 and cell membrane that is not related to HSPGs.

Figure 8. Determination of HIV-1 proviral DNA content in Epivaginal tissue infected by HIV-1_ada.

EpiVaginal™ tissues were challenged with HIV-1_ada (25 ng/ml) plus the SB105-A10 dendrimer (2 or 10 µg/tissue) for 24 hours, and total DNA was extracted and purified from vaginal tissues at day 4 pi. HIV-1 DNA burden was determined by SYBR Green-based quantitative real time PCR using either HIV-1 gag or pol gene-specific primer pairs , . Data are expressed as the fold decrease (±SD) of the DNA proviral content in dendrimer-treated cell culture with respect to DNA proviral amount determined in untreated cell cultures, obtained from two independent experiments in quadruplicate.