Virucidal activity of the dendrimer microbicide SPL7013 against HIV-1

Abstract

Topical microbicides for use by women to prevent the transmission of human immunodeficiency virus (HIV) and other sexually transmitted infections are urgently required. Dendrimers are highly branched nanoparticles being developed as microbicides. SPL7013 is a dendrimer with broad-spectrum activity against HIV type I (HIV-1) and -2 (HIV-2), herpes simplex viruses type-1 (HSV-1) and -2 (HSV-2) and human papillomavirus. SPL7013 [3% (w/w)] has been formulated in a mucoadhesive carbopol gel (VivaGel®) for use as a topical microbicide. Previous studies showed that SPL7013 has similar potency against CXCR4-(X4) and CCR5-using (R5) strains of HIV-1 and that it blocks viral entry. However, the ability of SPL7013 to directly inactivate HIV-1 is unknown. We examined whether SPL7013 demonstrates virucidal activity against X4 (NL4.3, MBC200, CMU02 clade EA and 92UG046 clade D), R5 (Ba-L, NB25 and 92RW016 clade A) and dual-tropic (R5X4; MACS1-spln) HIV-1 using a modified HLA-DR viral capture method and by polyethylene glycol precipitation. Evaluation of virion integrity was determined by ultracentrifugation through a sucrose cushion and detection of viral proteins by Western blot analysis. SPL7013 demonstrated potent virucidal activity against X4 and R5X4 strains, although virucidal activity was less potent for the 92UG046 X4 clade D isolate. Where potent virucidal activity was observed, the 50% virucidal concentrations were similar to the 50% effective concentrations previously reported in drug susceptibility assays, indicating that the main mode of action of SPL7013 is by direct viral inactivation for these strains. In contrast, SPL7013 lacked potent virucidal activity against R5 HIV-1 strains. Evaluation of the virucidal mechanism showed that SPL7013-treated NL4.3, 92UG046 and MACS1-spln virions were intact with no significant decrease in gp120 surface protein with respect to p24 capsid content compared to the corresponding untreated virus. These studies demonstrate that SPL7013 is virucidal against HIV-1 strains that utilize the CXCR4 coreceptor but not viruses tested in this study that solely use CCR5 by a mechanism that is distinct from virion disruption or loss of gp120. In addition, the mode of action by which SPL7013 prevents infection of cells with X4 and R5X4 strains is likely to differ from R5 strains of HIV-1.

Figure 1. SPL7013 virucidal activity against NL4.3 by HLA-DR capture and PEG precipitation

(A). Comparison of the SPL7013 virucidal activity against NL4.3 as measured by HLA-DR capture and PEG precipitation. The HLA-DR capture method was performed as published previously (Fletcher et al., 2006) with the following modifications. After incubation with SPL7013 or PRO 2000, plates were washed 5 – 6 times with Dulbecco modified Eagle medium (DMEM) supplemented with 10% (v/v) fetal calf serum, 100 U/ml penicillin, 100 µg/ml streptomycin and 2 mM glutamine (DMEM-10) to remove microbicide. TZM-bl cells were then added (2.5 × 10⁵/well) to quantify HIV-1 infectivity by measuring luciferase activity in cell lysates as published (Tyssen et al., 2010). Sufficient virus, grown in PHA-stimulated PBMC, was used to yield ≥100,000 relative light units (RLU; virus titers 2 × 10⁵ – 1 × 10⁶ infectious units/ml, depending on strain). PEG precipitation assays, initially performed with SPL7013 in the absence of HIV-1, established that 3% (w/v) PEG did not precipitate the dendrimer up to 500 µg/ml (data not shown). For assays performed with virus, an equal volume of NL4.3 (sufficient to yield ~100,000 RLU) was mixed with an equal volume of medium containing microbicide. The mixture was incubated for 1 h at 37°C followed by the addition of a half volume of PEG/NaCl [9% PEG 6000 (w/v) and 0.5 M NaCl] and overnight incubation at 4°C. HIV-1 was pelleted by centrifugation in a microfuge at 13,000 × g for 10 min at room temperature. Supernatant was removed and the pellets resuspended in DMEM-10 and added to wells seeded with TZM-bl cells. Luciferase activity was measured after 48 h incubation as described (Tyssen et al., 2010). (B). NL4.3 virucidal activity of SPL7013 compared to PRO 2000 in the HLA DR capture assay. (C). NB25 virucidal activity of SPL7013 compared to PRO 2000 in the HLA DR capture assay. Data for graphs A and B were derived from three independent assays and error bars denote standard error of the mean. For graph C, data were derived from two independent assays and error bars denote the standard error.

Figure 2. SPL7013 treated HIV-1 does not lead to viral disruption or loss of gp120 surface protein

(A). Quantitation of the expression of gp120 relative to p24 in SPL7013 treated virus normalized to untreated HIV-1 (U/T Control). Virus was treated with 200 µg/ml of SPL7013 for 1 h at 37°C followed by ultracentrifugation at 120,000 × g for 1 h at 4°C through a 25% (w/v) sucrose cushion. The pellets were resuspended in TNEN buffer (50 mM Tris pH 8.0, 50 mM NaCl, 10 mM EDTA and 0.5% IGEPAL) containing protease inhibitors (1 µg/ml each of aprotinin, leupeptin and pepstatin), subjected to SDS-PAGE, followed by quantitative Western blot analysis using the Odyssey Infrared Imaging System as described previously (Figueiredo et al., 2006). Human anti-p24 from pooled sera was used to detect HIV capsid, mouse anti-gp120 ID6 was used to detect NL4.3 gp120 and sheep anti-gp120 (Shutt et al., 1998) was used to detect gp120 of MACS1-spln and the clade D strain. Secondary antibodies used were IRDye 800CW conjugated affinity purified anti-human IgG (Rockland), Alexa Fluor 680 Goat anti-mouse IgG (Invitrogen) and Alex Fluor 680 donkey anti-sheep IgG (Invitrogen). The gp120 expression was normalized for p24 expression then expressed as a ratio to the gp120:p24 normalised value for the untreated control, which was set to 1.0. Data were derived from four independent assays for NL4.3 and the clade D strain (92UG046) and three independent assays for MACS1 (MACS1-spln). No significant difference was observed for the gp120:p24 ratio for SPL7013 treated compared to untreated NL4.3 (p=0.44, n=4), clade D (p=0.56, n=4) and MACS1-spln (p=0.35, n=3) using the Wilcoxon Rank Sum Test. Error bars denote standard error of the mean. (B). Representative Western blot of untreated (−) and SPL7013 treated (+) virus showing gp120 and p24 viral proteins.