In vitro activities of candidate microbicides against cell-associated HIV

Abstract

Most research on HIV transmission and microbicides focuses on the inhibition of cell-free virus (CFV) present in genital secretions. However, an effective microbicide should also block the transmission of cell-associated virus (CAV) originating from seminal T cells and macrophages. Because inhibition of CAV remains controversial, especially for viral entry inhibitors, we developed a novel in vitro assay to evaluate the activities of different classes of candidate microbicides against cell-free HIV and HIV-infected leukocytes (i.e., resting peripheral blood mononuclear cells [PBMC], activated PBMC, and monocyte-derived macrophages). The assay is based on two CD4(+) CXCR4(+) T-cell lines (R5MaRBLE and X4MaRBLE) that both contain a firefly luciferase reporter gene but differ in the expression of the CCR5 coreceptor. Consequently, the quantification of the luciferase activities and the Gag p24 concentrations in cocultures of R5-tropic HIV-infected leukocytes with each cell line separately allowed us to discriminate between the infection of the cell lines (i.e., target cells), the ongoing infection in the HIV-infected leukocytes (i.e., effector cells), and the total infection of the coculture (i.e., effector plus target cells). All 14 antiretrovirals tested were able to block target cell infection by all three sources of CAV, although a small decrease in activity (2- to 18-fold) was observed for all entry inhibitors. On the other hand, the production of Gag p24 by the infected effector cells could be blocked only by protease inhibitors. Overall, these results show that entry and protease inhibitors are eligible drug classes for inclusion in future combination microbicides.

Fig 1

Experimental setup. (A) In vitro generation of HIV-infected effector cells. Human PBMC were either activated with PHA and IL-2 or left untreated, while monocytes from the same donor were differentiated to macrophages. During the last 3 days of culture, all three cell types were infected with the R5-tropic subtype B strain BaL, resulting in three sources of HIV-infected effector cells: resting PBMC, activated PBMC, and macrophages. (B) Measuring infection of target cells. In a coculture of BaL-infected effector cells (green cells) with a firefly-luciferase-containing target T-cell line, R5M (red cells), luminescence originates only from infected R5M target cells (yellow cells). (C) Measuring infection of the coculture. In a coculture of BaL-infected effector cells with the HIV-permissive T-cell line R5M, the Gag p24 concentration in the supernatant quantifies the infection of both effector and target cells. (D) Measuring infection of effector cells. In a coculture of BaL-infected effector cells with the X4M T-cell line (blue cells), which lacks the coreceptor CCR5, the Gag p24 concentration in the supernatant quantifies the infection of the effector cells only, as the X4M cells do not become productively infected.

Fig 2

Infectious titer and proviral DNA load of HIV-infected effector cells. The TCID₅₀ of BaL-infected effector cells (i.e., resting PBMC/CD4⁺ T cells, activated PBMC/CD4⁺ T cells, and macrophages) was determined in a 7-day coculture with R5M target cells. Firefly luciferase was used to assess target cell infection, and the TCID₅₀ was calculated using the method of Reed and Muench, expressed as the number of cells needed for 50% infection and plotted on the left y axis (black symbols). The integrated proviral DNA load was determined using a two-step Alu-Gag PCR. Each sample was tested in duplicate. Viral loads were calculated using an external curve for HIV proviral DNA by serially diluting 8E5 T cells and were plotted on the right y axis (red symbols). The threshold of detection was 100 copies/10⁶ cells. The triangles and squares represent two PBMC donors from which the effector cells were generated. Horizontal lines represent the means of the depicted points.

Fig 3

EC₅₀ values of 14 antiretrovirals for cell-free HIV and HIV-infected effector cells using firefly luciferase measurement on R5M cells. One hundred TCID₅₀ of either cell-free virus, resting PBMC, activated PBMC, or macrophages was added to R5M target cells and incubated for 7 days in the presence of a serial dilution of the respective compound. Firefly luciferase was used to assess target cell infection, and the EC₅₀ was calculated using nonlinear regression analysis on data from at least three to six independent experiments (GraphPad Prism). Nonentry inhibitors (A) and entry inhibitors (B) are shown (Table 1). The error bars depict the 95% confidence intervals, while the numbers and brackets represent the fold changes between cell-free virus and cell-associated virus (black) or between two types of cell-associated virus (gray). Significant differences were calculated using a Z test with Bonferroni correction; ∗, P < 0.05; ∗∗, P < 0.01; ∗∗∗, P < 0.001.

Fig 4

Dose-response curves of four antiretrovirals for cell-free HIV and HIV-infected effector cells using Gag p24 measurement on R5M and X4M cells. One hundred TCID₅₀ of resting PBMC (blue) or activated PBMC (red) was added to either R5M or X4M target cells and incubated for 7 days in the presence of a serial dilution of the respective compound. Cumulative Gag p24 was subsequently measured in the supernatant of R5M cocultures to assess effector plus target cell infection (full regression lines) or in the supernatants of X4M cocultures to assess ongoing infection in the effector cells (dashed regression lines) and plotted against the compound concentrations. Nonlinear regression was performed on data from one representative experiment (GraphPad Prism). As similar results were obtained for the other eight compounds, only one representative inhibitor of each antiretroviral class is depicted. OD, optical density.

Fig 5

Relative importance of cell-to-cell spread in a coculture of R5M target cells with HIV-infected effector cells. (A) Cell-free HIV or HIV-infected effector cells (x axis) were physically separated from the R5M target cells using a virion-permeable membrane with 3-μm pores in a 24-well Transwell system with the cell-free virus or effector cells placed in the apical compartment and the target cells in the basal compartment. In parallel, cocultures of cell-free virus or effector cells with R5M target cells were set up in the basal compartment. After 5 days of incubation, R5M infection was assessed using firefly luciferase. Values for target cell infection by cell-free HIV or HIV-infected effector cells when separated from the target cells are expressed as percentages of those obtained in the respective cocultures (y axis). Mean percentages ± standard errors of the mean (SEM) of three independent experiments are depicted. (B) Additionally, the TCID₅₀ of the infected effector cells was determined as described in the legend to Fig. 2 using a 96-well Transwell system with the effector cells placed in the apical compartment and the target cells in the basal compartment. Viral titers were calculated using the method of Reed and Muench and expressed as percentages of the titer obtained in cocultures of effector and target cells. One experiment is represented.