Semi-solid gels function as physical barriers to human immunodeficiency virus transport in vitro

Abstract

Vaginal gels may act as physical barriers to HIV during sexual transmission. However, the extent and significance of this effect are not well understood. During male-to-female sexual transmission of HIV, semen containing infectious HIV is present within the lower female reproductive tract. In cases where a topical gel has previously been applied to the vaginal epithelium, virions must move through gel layers before reaching vulnerable tissue. This additional barrier could affect the functioning of anti-HIV microbicide gels and placebos. To better understand HIV transport in gels, we: (1) quantified diffusion coefficients of HIV virions within semi-solid delivery vehicles; and (2) tested the barrier functioning of thin gel layers in a Transwell system. Two gels used as placebos in microbicides clinical trials, hydroxyethyl cellulose (HEC) and methylcellulose (MC), were found to hinder HIV transport in vitro. The diffusion coefficients for HIV virions in undiluted HEC and MC were 4±2 x 10⁻¹² and 7±1 x 10⁻¹² cm²/s, respectively. These are almost 10,000 times lower than the diffusion coefficient for HIV in water. Substantial gel dilution (80%:diluent/gel, v/v) was required before diffusion coefficients rose to even two orders of magnitude lower than those in water. In the Transwell system, gel layers of approximately 150-μm thickness reduced HIV transport. There was a log reduction in the amount of HIV that had breached the Transwell membrane after 0-, 4-, and 8-h incubations. The ability of a gel to function as a physical barrier to HIV transport from semen to tissue will also depend on its distribution over the epithelium and effects of dilution by vaginal fluids or semen. Results here can serve as a baseline for future design of products that act as barriers to HIV transmission. The potential barrier function of placebo gels should be considered in the design and interpretation of microbicides clinical trials.

Figure 1

Biological context of Transwell system used to evaluate barrier functioning of thin gel layers. (A) Schematic of microbicide functioning in vivo. Microbicide gels are applied topically prior to challenge by HIV. Gels form layers of approximately 100-500 μm thick on epithelial surfaces. HIV must traverse these gel layers to reach vulnerable tissue. (B) Transwell system simulates HIV transmission in the presence of vaginal gels. A thin gel layer is applied to the Transwell membrane. A suspension of HIV is added to the top compartment. After incubation, levels of HIV in the bottom compartment are quantified using the TZM-bl assay.

Figure 2

Examples of tracks obtained for fluorescently-labeled HIV virions in HEC and MC, and for 100-nm bead dried to glass slide. Particles were tracked for 41s.

Figure 3

Examples of plots for MSD vs. τ for HIV in (A) HEC and (B) MC. Data represent different virions within single experiments.

Figure 4

Diffusion coefficients (mean ± SE, n = 3 independent experiments) obtained using particle tracking of fluorescently-labeled HIV in HEC and MC undiluted, diluted to 50% (v/v) in PBS, and diluted to 20% (v/v) in PBS.

Figure 5

Examples of (A) HEC and (B) MC gel distributions applied to membranes in Transwell system.

Figure 6

Levels of HIV-1 in bottom compartment of Transwell for two semi-solid gels, HEC and MC (n ≥ 4 independent experiments). Experimental results are normalized to controls where no gel was applied to the membrane, such that % control = RLU_{bottom,experiment}/RLU_{bottom,control}. Additional control experiments were performed in which an equivalent amount of gel was mixed in to solutions. For 0-, 4-, and 8- hour incubations, there was a log reduction in levels of HIV-1 in the bottom compartment.

Figure 7

Interpretation of experimentally-measured diffusion coefficients using mathematical model of diffusion of HIV virions from semen, through microbicide layer, to tissue compartment. (A) Schematic of mathematical model. Measured diffusion coefficients were input to the model. Plots show percent of virions in the tissue compartment, as a function of time, for various concentrations of (B) HEC and (C) MC. In (B), curves for 20% HEC, 50% HEC, and undiluted HEC appear superimposed. In (C), curves for 50% MC and undiluted MC appear superimposed.