A Toxin-Conjugated Recombinant Protein Targeting gp120 and gp41 for Inactivating HIV-1 Virions and Killing Latency-Reversing Agent-Reactivated Latent Cells

Abstract

Application of the combination antiretroviral therapy (cART) has reduced AIDS to a manageable chronic infectious disease. However, HIV/AIDS cannot be cured because of the presence of latent reservoirs, thus calling for the development of antiretroviral drugs that can eliminate latency-reversing agent (LRA)-activated HIV-1 virions and latent cells. In this study, we conjugated a small-molecule toxin, DM1, to a gp120-binding protein, mD1.22, a mutated CD4 domain I, and found that mD1.22-DM1 could inactivate HIV-1 virions. However, it could not kill LRA-activated latent cells. We then designed and constructed a dual-targeting protein, DL35D, by linking mD1.22 and the single-chain variable fragment (scFv) of a gp41 NHR-specific antibody, D5, with a 35-mer linker. Subsequently, we conjugated DM1 to DL35D and found that DL35D-DM1 could inhibit HIV-1 infection, inactivate HIV-1 virions, kill HIV-1-infected cells and LRA-reactivated latent cells, suggesting that this toxin-conjugated dual-targeting recombinant protein is a promising candidate for further development as a novel antiviral drug with potential for HIV functional cure. IMPORTANCE Although HIV-1 replication was successfully controlled by antiretroviral drugs, cure strategy for HIV-1/AIDS is still lacking. The long-lived HIV reservoir is considered one of the major obstacles to an HIV/AIDS cure. CD4-PE40 was the first drug that designed to kill HIV-1 infected cells; however, lower efficiency and high immunogenicity have limited its further development. In this study, we designed several dual-targeting recombinant proteins DLDs by linking gp120-binding protein mD1.22 and gp41-binding antibody D5 scFv with different length of linkers. Among them, DL35D with 35-mer linker showed the best anti-HIV-1 activity. We further conjugated the DM1 toxin to DL35D to produce DL35D-DM1, which maintained DL35D's inhibitory and inactivation activity against cell-free HIV-1 strains. Most importantly, DL35D-DM1 could specifically kill HIV-1-infected cells and LRA-reactivated-latent infected cells, suggesting that it is a proper candidate for development as a novel antiviral drug for use in combination with an LRA for HIV functional cure.

Keywords: HIV-1; HIV-1 latent infection; shock and kill; toxin-conjugated recombinant protein; virus inactivator.

FIG 1

Activity of toxin-conjugated protein mD1.22-DM1 in vitro. (A) Cartoon of the membrane fusion process and the mechanisms of mD1.22-DM1 and DLD-DM1. (a) After HIV-1 gp120 binds to the receptor CD4 molecule and then a co-receptor, CXCR4 or/CCR5, on the target cell, serial conformational changes occur in gp120 and gp41, including the exposure of the gp41 NHR and formation of NHR-trimer, which then interacts with 3 CHR molecules to form a 6-HB, resulting in virus-cell membrane fusion. (b) mD1.22-DM1 binds to gp120 through its the mD1.22 portion and then delivers DM1 to the target cell. (c) DLD-DM1 binds to gp120 through its mD1.22 portion and gp41 NHR domain via its D5 scFv portion, resulting in the release of DM1 to the target cell. (B) Analysis of mD1.22 and mD1.22-DM1 by SDS-PAGE (a) and Western blot using a rabbit anti-CD4 polyclonal antibody (b). (C) Detection of the binding activity of mD1.22-DM1 to HIV-1 gp120 by ELISA. The protein mD1.22 and toxin-conjugated protein mD1.22-DM1 were coated onto the wells of a 96-well plate at 5 μg/ml, respectively, followed by addition of gp120 at 3 μg/ml. The binding activity was determined by using mouse anti-gp120 sera. (D) Inhibitory activity of mD1.22-DM1 against infection of HIV-1 laboratory-adapted strains IIIB (a) and Bal (b). The red, green, and blue curves represent the HIV-1 inhibitory activity of mD1.22-DM1, SMCC-DM1, and T20 peptide, respectively. (E) The inactivation activity of mD1.22-DM1 against HIV-1 laboratory-adapted strain IIIB (a) and Bal (b). The red, green, and blue curves represent the HIV-1 inactivation activity of mD1.22-DM1, SMCC-DM1, and T20 peptide, respectively. (F) The proportion of gp120-positive ACH-2 cells 12 h post-stimulation with romidepsin. ACH-2 cells were treated with romidepsin at different concentrations. Then the cells were incubated with the HIV-1 gp120-specific human antibody N6. The proportion of gp120-positive ACH-2 cells was detected with FITC-labeled goat anti-human IgG antibody using flow cytometry. (G) The killing effect of mD1.22-DM1 on LRA-reactivated and nonreactivated ACH-2 cells. One-way ANOVA was used in the statistical analysis. **** and NS mean P < 0.0001 and no statistical significance, respectively.

FIG 2

Expression and verification of dual-targeting recombinant proteins, DLDs. (A) Construction strategy for dual-targeting recombinant proteins, DLDs. A DLD is comprised of mD1.22 and D5 scFv linked with a flexible linker (GGGGS)n, in which “n” means 4, 5, 6, 7, or 8. Analysis of DLDs with SDS-PAGE (B). Detection of DLDs with Western blot using a rabbit anti-human CD4 polyclonal antibody (C), mouse anti-mD1.22 serum (D), and mouse anti-D5 scFv serum (E). Detection of binding of DLDs in ELISA with anti-CD4 antibody (F), mouse anti-mD1.22 antibody (G), and mouse anti-D5 antibody (H), respectively.

FIG 3

Detection of the antiviral mechanism of DLDs. (A) Binding activity of DLDs with gp120 was measured with ELISA using mouse anti-His-Tag monoclonal antibody. (B) Inhibitory activity of DLDs on the binding of gp120 and CD4 receptor on the target cell was measured with cell-based ELISA using TZM-bl cells expressing CD4 and rabbit anti-HIV-1 gp120 antibody. (C) The binding between DLDs and N63 peptide derived from gp41 NHR was measured with ELISA using mouse anti-His-Tag monoclonal antibody. (D) The binding between DLDs and N63-trimer was measured with ELISA using mouse anti-His-Tag monoclonal antibody. (E) Measurement of inhibitory activity of DLDs on 6-HB formation between N36 and C34-FAM peptides by FN-PAGE. DLDs were incubated with N36 peptide at 37°C for 30 min before addition of C34-FAM. After incubation for 30 min, the mixtures were analyzed by FN-PAGE using an imaging system through U.V. detection (panels a and b). After imaging, the gels were stained with Coomassie blue (panels c and d).

FIG 4

Anti-HIV-1 activity of DLDs in vitro. (A-B) Inhibitory activity of DLDs against infection by HIV-1 laboratory-adapted strains IIIB (X4) and Bal (R5). MT-2 and M7 cells were used for infection by HIV-1 IIIB and Bal, respectively. (C-D) Inactivation activity of DLDs against cell-free HIV-1 IIIB and Bal virions. (E) Inhibitory activity of DLDs on HIV-1 Env-mediated cell-cell fusion. The HIV-1_IIIB chronically infected H9 (H9/HIV-1_IIIB) cells and CD4⁺ MT-2 cells were used as the effector and target cells in the cell-cell fusion assay. (F) Inhibitory activity of DL35D on HIV-1 cell-to-cell transmission.

FIG 5

Activity of DL35D-DM1 against HIV-1 strains in vitro. (A-B) Inhibitory activity of DL35D-DM1 against infection by HIV-1 laboratory-adapted strains (IIIB and Bal). (C-D) Inactivation activity of DL35D-DM1 against cell-free HIV-1 particles (HIV-1 IIIB and Bal). (E) Inactivating LRA-reactivated HIV-1 virions released from ACH-2 cells by DL35D-DM1. (F) Inhibition of HIV-1 cell-to-cell transmission by DL35D-DM1.

FIG 6

Killing effect of DL35D-DM1 on HIV-1-infected cells. (A) Killing effect of protein DL35D on HIV-1-infected H9/HIV-1_IIIB cells and uninfected H9 cells. (B) Killing effect of DL35D-DM1 on H9/HIV-1_IIIB cells and H9 cells. (C) Killing effect of protein DL35D on LRA-reactivated HIV-1 latent-infected ACH-2 cells. (D) Killing effect of DL35D-DM1 on LRA-reactivated HIV-1 latent-infected ACH-2 cells. Nonreactivated ACH-2 cells were used as control. (E) Killing effect of DL35D-DM1 on cells without expression of HIV-1 Env. MT-2 cells (a), RD cells (b), U87 CD4+CCR5+ cells (c), and Huh-7 cells (d) were used in this experiment. Two-way ANOVA was used in the statistical analysis. ****, ***, and * mean P < 0.0001, P < 0.001, and P < 0.05, respectively.

FIG 7

The putative mechanisms of action of DL35D-DM1 to inhibit HIV-1 infection, inactivate cell-free HIV-1 virions and kill cells expressing HIV-1 Env. (a) The mechanism of DL35D-DM1 to inactivate cell-free HIV-1 virions. DL35D-DM1 binds HIV-1 gp120 to trigger its conformational change and then binds to exposed gp41 NHR trimer, resulting in the inactivation of HIV-1 virion. (b) The mechanism of DL35D-DM1 to inhibit HIV-1 infection. DL35D-DM1 binds to gp120 to block the interaction between gp120 and cell receptors or binds to gp41 to block 6-HB formation, resulting in inhibition of HIV-1 infection. (c) The mechanism of DL35D-DM1 to kill LRA-reactivated HIV-1 latently infected cell or HIV-1-infected cell. DL35D-DM1 binds to HIV-1 Env expressed on the surface of cells (e.g., LRA-reactivated HIV-1 latently infected cell or HIV-1-infected cell) and gets into the endosome in the cell, where the released DM1 to disrupt the microtube system, resulting cell death. The figure was created with BioRender.com.