Infection of ectocervical tissue and universal targeting of T-cells mediated by primary non-macrophage-tropic and highly macrophage-tropic HIV-1 R5 envelopes

Abstract

Background: HIV-1 variants carrying non-macrophage-tropic HIV-1 R5 envelopes (Envs) are predominantly transmitted and persist in immune tissue even in AIDS patients who have highly macrophage-tropic variants in the brain. Non-macrophage-tropic R5 Envs require high levels of CD4 for infection contrasting with macrophage-tropic Envs, which can efficiently mediate infection of cells via low CD4. Here, we investigated whether non-macrophage-tropic R5 Envs from the acute stage of infection (including transmitted/founder Env) mediated more efficient infection of ectocervical explant cultures compared to non-macrophage-tropic and highly macrophage-tropic R5 Envs from late disease.

Results: We used Env+ pseudovirions that carried a GFP reporter gene to measure infection of the first cells targeted in ectocervical explant cultures. In straight titrations of Env+ pseudovirus supernatants, mac-tropic R5 Envs from late disease mediated slightly higher infectivities for ectocervical explants although this was not significant. Surprisingly, explant infection by several T/F/acute Envs was lower than for Envs from late disease. However, when infectivity for explants was corrected to account for differences in the overall infectivity of each Env+ pseudovirus (measured on highly permissive HeLa TZM-bl cells), non-mac-tropic early and late disease Env+ pseudoviruses mediated significantly higher infection. This observation suggests that cervical tissue preferentially supports non-mac-tropic Env+ viruses compared to mac-tropic viruses. Finally, we show that T-cells were the main targets for infection regardless of whether explants were stimulated with T-cell or monocyte/macrophage cytokines. There was no evidence of macrophage infection even for pseudovirions carrying highly mac-tropic Envs from brain tissue or for the highly mac-tropic, laboratory strain, BaL, which targeted T-cells in the explant tissue.

Conclusions: Our data support ectocervical tissue as a favorable environment for non-mac-tropic HIV-1 R5 variants and emphasize the role of T-cells as initial targets for infection even for highly mac-tropic variants.

Figure 1

Macrophage infectivity for Envs selected for cervical explant studies. a Infectivity for macrophages (FFU/mL) for Env+ pseudoviruses (left panel). Mac-tropic Envs mediated significantly higher levels of macrophage infectivity compared to T/F/acute and late stage non-mac-tropic Envs (p values = <0.0001) right panel. b Macrophage infection plotted as a percent of TZM-bl infectivity (left panel). Correction of Env+ pseudovirus infectivity for different levels of infectivity on TZM-bl cells confirms that mac-tropic Envs mediated significantly higher levels of macrophage infectivity compared to T/F/acute and late stage non-mac-tropic Envs (p values = <0.0001) right panel. Left panel in a and b show means and standard errors from infectivities measured on macrophages derived from at least three donors, right panels show geometric means with 95% confidence intervals. p values were calculated using unpaired, two-tailed t tests in Prism 6.0f.

Figure 2

GFP reporter virus infection of ectocervical tissue explants. a Infected GFP+ cells in explant cultures. Infected, GFP+ cells are readily observed and easily distinguished from low-level auto-fluorescence of the tissue. Note some are GFP+ infected emigrant cells. b BaL and NA420 LN40 infection of explants without stimulation or in the presence of PHA/IL-2, GM-CSF, or GM-CSF and IL-4. Data are averaged from 2 or 3 replicate explants. c BaL and NA420 LN40 infection was efficiently inhibited following maraviroc blockade of CCR5 and by the post-entry, NNRTI inhibitor, nevirapine. Data are averaged from at least two donors with five replicate explants per donor.

Figure 3

Ectocervical explant infectivity. a Ectocervical explant infection (FFU/mL) is shown for all Env+ pseudoviruses (left panel). No significant differences were detected between late disease mac-tropic, late-disease non-mac-tropic or T/F/acute Env+ pseudoviruses using unpaired, two-tailed t tests (right panel). b Infectivity for ectocervical explants plotted as a percent of HeLa TZM-bl infectivity. Using this correction, many T/F/acute and late disease non-mac-tropic Envs mediated higher infectivity (per TZM-bl IUs) compared to mac-tropic Envs (left panel). Ectocervical explant infectivity for late-disease non-mac-tropic and T/F/acute Env+ pseudoviruses was significantly higher than late-disease mac-tropic Envs (right panel). Each Env+ pseudovirus was tested on explants prepared from at least five donors with five replicate explants per donor.

Figure 4

T-cells are the major cell type infected in ectocervical explants. Cells extracted from PHA/IL-2 stimulated, infected explant cultures were subjected to positive selection of T-cells (a) or negative selection for monocytes and macrophages (b) using StemCell Technologies EasySep immunomagnetic kits. Infected GFP+ cells consistently segregated with T-cells and not monocyte/macrophages. In other experiments, BaL infected explants were stimulated with GM-CSF and cultured in human AB+ plasma to support monocyte/macrophages. Extracted cells then underwent T-cell positive selection (c) or monocyte/macrophage negative selection (d). Again, GFP+ cells segregated with T-cells and not monocyte/macrophages.