Efficient HIV-1 Trans Infection of CD4+ T Cells Occurs in the Presence of Antiretroviral Therapy

Abstract

Background: Antiretroviral therapy (ART) has dramatically improved the quality of life of people with HIV-1 infection (PWH). However, it is not curative, and interruption of ART results in rapid viral rebound. Cell-to-cell transfer of HIV-1, or trans infection, is a highly efficient mechanism of virus infection of CD4⁺ T cells by professional antigen-presenting cells (APCs), that is, dendritic cells (DCs), macrophages, and B lymphocytes.

Methods: APC from HIV seronegative donors treated with ART in vitro (CCR5 agonist, NRTI, PI and NNRTI, alone or in combination), were loaded with HIV R5-tropic HIV_Bal and mixed with autologous or heterologous CD4⁺ T lymphocytes to assess trans infection. Ex vivo APC from chronic HIV-infected MACS participants before and after initiation of ART, were also loaded with HIV R5-tropic HIV_Bal and tested for trans infection against autologous or heterologous CD4⁺ T lymphocytes. Virus replication was measured by p24 ELISA.

Results: Here we show in vitro that antiretroviral drugs did not block the ability of DCs and B cells to trans-infect CD4⁺ T cells, although they were effective in blocking direct cis infection of CD4⁺ T cells. Moreover, ex vivo DCs and B cells from ART-suppressed PWH mediated efficient HIV-1 trans infection of CD4⁺ T cells, which were resistant to direct cis infection.

Conclusions: Our study supports a role for HIV-1 trans infection in maintenance of the HIV-1 reservoir during ART.

Keywords: ART; B lymphocytes; HIV; antigen-presenting cells; dendritic cells; trans infection.

Figure 1.

Effect of antiretroviral drugs on B cell and DC mediated trans infection in vitro. Panel A and B. B cells or DC were treated with the indicated drugs, loaded with HIV^Bal at 10^-3 m.o.i. and mixed with activated, purified autologous CD4⁺ T cells as described in Methods. Panel C. Cell-free cis infection of CD⁺ T cells with HIV^Bal at 10^-1 m.o.i. was conducted in parallel to determine CD⁺ T cells susceptibility to infection. Cell culture supernatants were collected at the indicated time points and HIV-1 Gag p24 was measured by ELISA. Data are mean values ±SE; n=4 experiments. GraphPad prism 7.0 Software was used for statistical analysis (one-way ANOVA followed by Students t test. *p<0.05) ‡= below limit of detection. Abbreviation: ns, nonsignificant.

Figure 2.

Effect of combination antiretroviral drugs on B cells and DC mediated trans infection in vitro. B cells (panel A) or DC (panel B) were treated with the indicated drugs in combination, loaded with HIV^Bal at 10^-3 m.o.i. and mixed with activated, purified autologous CD4⁺ T cells. Purified CD4⁺ T cells were also infected in cis in the presence of the drug combination (panel C). Drug concentrations are described in Methods. Data are mean values ±SE; n=4 experiments. Panel D and E: B cells and DC were treated with up to 9 times the concentration of maraviroc or of the three drug combination, respectively and used in trans infection experiments as described above. Panel F: purified CD4⁺ T cells were also infected with cell free virus in the presence of maraviroc or of the three drug combination in decreasing concentrations. Cell cultures supernatants were collected at the indicated time points and HIV-1 Gag p24 was measured by ELISA. Data are representative of 2 independent experiments and are mean values ±SE of triplicate wells. GraphPad prism 7.0 Software was used for statistical analysis (one-way ANOVA followed by Students t test. *p<0.05) ‡= below limit of detection. Abbreviations: DC, dendritic cell; DRV/RPV/TVF, darunavir/rilpivirine/tenofovir.

Figure 3.

Ex-vivo APC from ART suppressed patients trans infect CD4⁺ T cells. B cells (panel A) and DC (panel B) derived from participants under suppressive ART (black bars) were loaded with HIV^Bal at 10^-3 m.o.i as described in Methods and co-cultured with autologous CD4⁺ T cells for 12 days. B cells and DC derived from archived PBMC from participants prior to ART initiation were also tested in parallel (red bars). Supernatants were collected at the indicated time points and tested for HIV-1 Gag p24. Data are mean values ±SE; n=10 experiments. Panel C. Purified CD4⁺ T cells from the participants described in panels A and B were infected in cis with HIV^Bal at 10^-1 m.o.i. in parallel with CD4⁺ T cells archived prior to ART initiation. Supernatants were collected at the indicated time points and tested for HIV-1 Gag p24. Data are mean values ±SE; n=10 experiments. GraphPad prism 7.0 Software was used for statistical analysis. *p<0.05. Abbreviations: ART, antiretroviral therapy; DC, dendritic cell.

Figure 4.

APC from NP do not trans infect CD4⁺ T cells. B cells from NP under ART were loaded with HIV^Bal at 10^-3 m.o.i. and mixed with autologous (black bar) or heterologous, CD4⁺ T cells from SN donor (grey bar). CD4⁺ T cells from NP under ART were co-cultured with B cells from a SN donor loaded with HIV^Bal at 10^-3 m.o.i ( thatched bar) (B) Purified CD4⁺ T cells from NP were infected in cis with HIV^Bal at 10^-1 m.o.i. in parallel with CD4⁺ T cells cryopreserved prior to ART initiation. Supernatants were collected at the indicated time points and tested for HIV-1 Gag p24. Data are representative of 2 independent experiments and are mean values ±SE of triplicate wells. GraphPad prism 7.0 Software was used for statistical analysis. *p<0.05. Abbreviations: ART, antiretroviral therapy; NP, nonprogressor; SN, HIV-1-seronegative donor.