Chimeric Antigen Receptor T Cells Guided by the Single-Chain Fv of a Broadly Neutralizing Antibody Specifically and Effectively Eradicate Virus Reactivated from Latency in CD4+ T Lymphocytes Isolated from HIV-1-Infected Individuals Receiving Suppressive Combined Antiretroviral Therapy

Abstract

Despite the advent of combined antiretroviral therapy (cART), the persistence of viral reservoirs remains a major barrier to curing human immunodeficiency virus type 1 (HIV-1) infection. Recently, the shock and kill strategy, by which such reservoirs are eradicated following reactivation of latent HIV-1 by latency-reversing agents (LRAs), has been extensively practiced. It is important to reestablish virus-specific and reliable immune surveillance to eradicate the reactivated virus-harboring cells. In this report, we attempted to reach this goal by using newly developed chimeric antigen receptor (CAR)-T cell technology. To generate anti-HIV-1 CAR-T cells, we connected the single-chain variable fragment of the broadly neutralizing HIV-1-specific antibody VRC01 to a third-generation CAR moiety as the extracellular and intracellular domains and subsequently transduced this into primary CD8+ T lymphocytes. We demonstrated that the resulting VC-CAR-T cells induced T cell-mediated cytolysis of cells expressing HIV-1 Env proteins and significantly inhibited HIV-1 rebound after removal of antiviral inhibitors in a viral infectivity model in cell culture that mimics the termination of the cART in the clinic. Importantly, the VC-CAR-T cells also effectively induced the cytolysis of LRA-reactivated HIV-1-infected CD4+ T lymphocytes isolated from infected individuals receiving suppressive cART. Our data demonstrate that the special features of genetically engineered CAR-T cells make them a particularly suitable candidate for therapeutic application in efforts to reach a functional HIV cure.

Importance: The presence of latently infected cells remains a key obstacle to the development of a functional HIV-1 cure. Reactivation of dormant viruses is possible with latency-reversing agents, but the effectiveness of these compounds and the subsequent immune response require optimization if the eradication of HIV-1-infected cells is to be achieved. Here, we describe the use of a chimeric antigen receptor, comprised of T cell activation domains and a broadly neutralizing antibody, VRC01, targeting HIV-1 to treat the infected cells. T cells expressing this construct exerted specific cytotoxic activity against wild-type HIV-1-infected cells, resulting in a dramatic reduction in viral rebound in vitro, and showed persistent effectiveness against reactivated latently infected T lymphocytes from HIV-1 patients receiving combined antiretroviral therapy. The methods used in this study constitute an improvement over existing CD4-based CAR-T technology and offer a promising approach to HIV-1 immunotherapy.

Copyright © 2016, American Society for Microbiology. All Rights Reserved.

Figures

FIG 1

Characterization of VRC01-28BBZ CAR-T cells. (A) Schematic representation of the VRC01-28BBZ CAR (not to scale). (B) Western blotting was performed to detect the expression of VRC01-28BBZ with anti-Flag tag antibody in HEK293T cells by transfecting three pCPPT-IRES-VRC01-28BBZ-mStrawberry plasmids. The HEK293T cells transfected with pCPPT-IRES-mStrawberry empty vector served as a negative control. (C) Immunofluorescence staining was performed to detect VRC01-28BBZ-3 CAR (VC-CAR) expression in HeLa cells with anti-Flag tag antibody. DAPI staining represents the nuclei. (D) Flow cytometry was performed to detect the transduction efficiencies of VC-CAR and empty vector in human CD8+ T cells by detection of mStrawberry fluorescence. Transduced CD8+ T cells were measured by detecting mStrawberry fluorescence and human Fab stained with goat antibody through flow cytometry. Untransduced CD8+ T cells served as a negative control. The CD4-CAR-transduced CD8+ T cells were detected with anti-CD4 antibody through flow cytometry. CD4-negative gating was set on untransduced control cells (data not shown).

FIG 2

Selection of the most efficient CAR moiety. (A) qRT-PCR was performed to characterize the expression of HIV-1 Env and eGFP in Jurkat-based target cells. Jurkat cells served as a negative control. (B) Cytotoxicity assays were performed using the CytoTox nonradioactive cytotoxicity kit, using as targets (T) Jurkat-gp160NL4-3 cells constitutively expressing Env from HIV-1NL4-3. Cocultures were performed for 24 h with T cells expressing the indicated CARs (E, effector cells); the RFP-CD8+ T cells (Mock) served as a negative control. Shown are the cytotoxic effects of VRC01-28BBZ-transduced T cells on target cells at 2:1 and 1:1 (E:T) ratios for 24 h. (C) Characterization of VRC01-28BBZ-transduced effector CD8+ T cells after coculture with Jurkat-gp160NL4-3 or Jurkat-GFP cells at a 4:1 (E:T) ratio for 20 h by IFN-γ ELISpot assay. Effector cells alone served as the negative control (NC). The PHA-stimulated effector cells served as the positive control (PC). These data represent three independent experiments. (D) Cytotoxic effect of VC-CAR-T cells and CD4-CAR-T cells on Jurkat-gp160NL4-3 at the indicated range of E:T ratios (24 h). (E) The CD8+ T cells expressing VC-CAR were challenged with cell-free HIV-1NL4-3 (p24 titer of 200 ng ml−1). Infection was analyzed at the indicated time points by staining for intracellular p24 after gating the CD3+ CD8+ subpopulation. Untransduced CD8+ T cells and CD8+ T cells transduced with empty vector (mock) served as a negative control, and CD4+ T cells (gate CD3+ CD8−) served as a positive control. The percent value in each histogram (left three panels) indicates the p24-positive population. (A, B, and D) Data reflect means ± standard errors of the means (SEM), and P values were calculated using the two-tailed unpaired Student's t test with equal variances (n = 3). *, P < 0.05; **, P < 0.01; ***, P < 0.001. Data shown in panel E represent two independent experiments.

FIG 3

Cytokine release of VC-CAR-T cells after coculture with Env-expressing Jurkat target cells. (A) Characterization of VC-CAR or control transduced effector CD8+ T cells cocultured with Jurkat-gp160NL4-3 or Jurkat-GFP cells at 2:1 and 4:1 (E:T) ratios for 20 h by IFN-γ ELISpot assay. The PHA-stimulated effector cells served as the positive control (PC). Effector cells alone served as the negative control (NC). (B) Summary of IFN-γ ELISpot assays described for panel A. The count number of the y axis was measured as spot-forming cells (SFCs)/104 CD8+ T cells. (C) Characterization of VC-CAR or control transduced effector CD8+ T cells cocultured with Jurkat-gp160BaL cells. (D) Summary of IFN-γ ELISpot assay described for panel C. The count number of the y axis was measured as SFCs/104 CD8+ T cells. (E) Granzyme B production in cocultures of Jurkat-gp160NL4-3 target cells with VC-CAR-transduced effector CD8+ T cells at the indicated E:T ratios for 20 h. The effector cells expressing red fluorescent protein served as negative controls. (F) IL-2 production from triple VC-CAR or control effector CD8+ T cells cocultured with Jurkat-gp160NL4-3 cells for 18 h. (B, E, and F) Data reflects means ± SEM. P values were calculated using the two-tailed unpaired Student's t test with equal variances (n = 3). *, P < 0.05; **, P < 0.01; ***, P < 0.001.

FIG 4

Specific killing of Env-expressing cells by VC-CAR-transduced effector CD8+ T cells. Direct killing of target cell lines was performed using the CytoTox nonradioactive cytotoxicity kit with Jurkat-gp160NL4-3 (A), Jurkat-gp160BaL (B), and Jurkat-GFP (C) cells as targets. Cocultures were performed for 24 h with CD8+ T cells expressing the VC-CAR (E, effector cells); the red fluorescent protein-transduced CD8+ T cells served as a negative control. Data reflect means ± SEM.

FIG 5

Specific killing of HIV-1NL4-3-infected primary CD4+ T cells by VC-CAR-transduced effector CD8+ T cells. (A) Primary CD4+ T cells were infected with HIV-1NL4-3 (p24 titer of 200 ng ml−1). At day 8 postinfection, CD4+ T cells were mixed with autologous VC-CAR or control transduced effector CD8+ T cells at a 1:2 or 1:4 ratio. Seventy-two hours after coculture, specific cytotoxicity was analyzed by flow cytometry for the intracellular staining of Gag+ T cells gated on CD3+ CD8− or CD3+ CD8+ subpopulations, respectively, as indicated at the top. (B) Summary of residual CD3+ CD8− Gag+ T cells from the flow cytometry analysis. (C) VC-CAR or control transduced effector CD8+ T cells were cocultured with HIV-1NL4-3-infected primary CD4+ T cells at a 2:1 (E:T) ratio for 20 h. IFN-γ secretion was analyzed by ELISpot assay. The PHA-stimulated effector cells served as the positive control (PC). Effector cells alone served as the negative control (NC). (D) Specific killing of HIV-1NL4-3-infected primary CD4+ T cells by VC-CAR-transduced effector CD8+ T cells. Cocultures were performed for 24 h. (B and D) Data reflect means ± SEM from triplicates. P values were calculated using the two-tailed unpaired Student's t test with equal variances (n = 3). *, P < 0.05; **, P < 0.01; ***, P < 0.001.

FIG 6

Effective suppression of HIV-1 rebound after the withdrawal of antiviral treatment in vitro. The primary CD4+ T cells were infected with HIV-1NL4-3 (1 ng ml−1 p24). (A) Experimental design. (B) The expression of the activation markers CD25 and HLA-DR on unstimulated and activated CD4+ T cells and CD4+ T cells at day 14 postinfection. The percentage of cells in each quadrant is indicated. (C) After the withdrawal of antiviral drugs, 0.5 × 106 CD4+ T cells were mixed with autologous VC-CAR engineered or control CD8+ T cells at a 1:2 or 1:4 ratio. On day 24, cells were collected and viral RNAs were isolated and amplified by real-time RT-qPCR with primer SK38 and SK39. The cutoff for cell-associated viral RNA is 800 copies ml−1. (D) Every 2 days the cultures were tested for the presence of p24 in the supernatant by ELISA. The dotted line represents the addition of antiviral drugs. (C and D) Data reflect means ± SEM. P values were calculated using the two-tailed unpaired Student's t test with equal variances (n = 3). *, P < 0.05; **, P < 0.01; ***, P < 0.001.

FIG 7

Elimination of reactivated HIV-1 latently infected CD4+ T lymphocytes from HIV-1-infected individuals receiving suppressive cART. (A) Experimental design. (B) On day 0, CD4+ T cells from HIV-1-infected patients were stimulated by PHA-M or by a combination of specific LRAs (SAHA plus bryostatin-1). After 1 day, 1 × 106 CD4+ T cells were mixed with autologous VC-CAR-engineered or control CD8+ T cells at a 1:1 ratio. On day 3, cells were collected and viral RNAs were isolated and amplified by real-time qRT-PCR with primers SK38 and SK39. The cutoff for cell-associated viral RNA is 800 copies ml−1. (C) The cultures were further tested for the presence of p24 with viral outgrowth by ELISA at the indicated time points. (B and C) Data reflect means ± SEM. P values were calculated using the two-tailed unpaired Student's t test with equal variances (n = 3). *, P < 0.05; **, P < 0.01; ***, P < 0.001.