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. 2022 Dec;11(1):30-49.
doi: 10.1080/22221751.2021.2011616.

Cell membrane-anchored anti-HIV single-chain antibodies and bifunctional inhibitors targeting the gp41 fusion protein: new strategies for HIV gene therapy

Yue Chen  1   2 , Hongliang Jin  1   2 , Xiaoran Tang  1   2 , Li Li  1 , Xiuzhu Geng  1   2 , Yuanmei Zhu  1   2 , Huihui Chong  1   2 , Yuxian He  1   2
Affiliations

Cell membrane-anchored anti-HIV single-chain antibodies and bifunctional inhibitors targeting the gp41 fusion protein: new strategies for HIV gene therapy

Yue Chen et al. Emerg Microbes Infect. 2022 Dec.

Abstract

Emerging studies indicate that infusion of HIV-resistant cells could be an effective strategy to achieve a sterilizing or functional cure. We recently reported that glycosylphosphatidylinositol (GPI)-anchored nanobody or a fusion inhibitory peptide can render modified cells resistant to HIV-1 infection. In this study, we comprehensively characterized a panel of newly isolated HIV-1-neutralizing antibodies as GPI-anchored inhibitors. Fusion genes encoding the single-chain variable fragment (scFv) of 3BNC117, N6, PGT126, PGT128, 10E8, or 35O22 were constructed with a self-inactivating lentiviral vector, and they were efficiently expressed in the lipid raft sites of target cell membrane without affecting the expression of HIV-1 receptors (CD4, CCR5 and CXCR4). Significantly, transduced cells exhibited various degrees of resistance to cell-free HIV-1 infection and cell-associated HIV-1 transmission, as well as viral Env-mediated cell-cell fusion, with the cells modified by GPI-10E8 showing the most potent and broad anti-HIV activity. In mechanism, GPI-10E8 also interfered with the processing of viral Env in transduced cells and attenuated the infectivity of progeny viruses. By genetically linking 10E8 with a fusion inhibitor peptide, we subsequently designed a group of eight bifunctional constructs as cell membrane-based inhibitors, designated CMI01∼CMI08, which rendered cells completely resistant to HIV-1, HIV-2, and simian immunodeficiency virus (SIV). In human CD4+ T cells, GPI-10E8 and its bifunctional derivatives blocked both CCR5- and CXCR4-tropic HIV-1 isolates efficiently, and the modified cells displayed robust survival selection under HIV-1 infection. Therefore, our studies provide new strategies for generating HIV-resistant cells, which can be used alone or with other gene therapy approaches.

Keywords: HIV; broadly neutralizing antibodies (bNAbs); fusion inhibitory peptide; gene therapy; glycosylphosphatidylinositol (GPI).

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Conflict of interest statement

No potential conflict of interest was reported by the author(s).

Figures

Figure 1.
Figure 1.
Expression of GPI-scFvs in transduced TZM-bl cells and their effects on CD4, CCR5, and CXCR4. (A) Schematic diagram of lentiviral transfer vector expressing a GPI-scFv or bifunctional inhibitor. The encoding sequence of scFv or a bifunctional inhibitor was genetically linked with the sequences encoding a His tag and the GPI attachment signal of DAF. LTR, long terminal repeat; RRE, Rev response element; cPPT, central polypurine track; PGK, human PGK promoter; DAF, delay-accelerating factor; WPRE, woodchuck hepatitits virus posttranscription regulatory element. (B) The expression of GPI-scFvs on the surface of transduced TZM-bl cells with or without PI-PLC treatment was determined by FACS analysis using an anti-His tag antibody. (C) Effects of GPI-scFvs on the expression of HIV receptors. The expression levels of CD4, CCR5, and CXCR4 on the surface of GPI-scFv-transduced TZM-bl cells were detected by FACS analysis using a PE-conjugated anti-human CD4, CCR5, or CXCR4 antibody and judged by the fluorescence intensity.
Figure 2.
Figure 2.
Confocal analyses of GPI-scFvs in transduced TZM-bl cells. Alexa555-CtxB stands for the lipid raft marker GM1 stained with Alexa 555-conjugated cholera toxin B subunit; Alexa488-Anti-His stands for the transduced cells stained with mouse anti-His tag antibody followed by Alexa 488-conjugated goat anti-mouse IgG antibody. Scale bar: 20 μm.
Figure 3.
Figure 3.
Anti-HIV activities of GPI-scFvs in transduced cells. The inhibitory activities of GPI-scFvs against a panel of replication-competent HIV-1 isolates (A), the “global panel” of HIV-1 pseudoviruses (B), and viral Env-mediated cell-cell fusion (C) in Table 1 were summarized for convenient overall view. Error bars indicate the means ± standard deviations (SD). (D-F) The inhibitory activities of GPI-scFvs on cell-cell HIV-1 transmission. TZM-bl cells expressing GPI-scFvs were used as a target and cocultured with CEMss-CCR5 donor cells that were preinfected with the HIV-1 isolates RHPA.c/2635 (D), THRO.c/2626 (E) and MJ4 (F). % cell-cell transmission was monitored by quantifying the production of the reporter luciferase in TZM-bl cells. Error bars indicate the means ± standard deviations (SD) from three independent experiments with triplicate samples, and statistical comparisons relative to the GPI-FluIgG03 control were conducted by ANOVA (ns, not significant; *, P< 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001).
Figure 4.
Figure 4.
Inhibitory activity of GPI-10E8 in transduced human CD4+ T cells against CXCR4-tropic and CCR5-tropic HIV-1 isolates. CEMss-CCR5 cells transduced with GPI-10E8/GFP or GPI-FluIgG03/GFP were infected with 1,000 TCID50 of NL4-3 (A), SG3.1 (B), MJ4 (C), or RHPA.c/2635 (D) and intracellular HIV-1 p24 antigen and GFP expression were monitored over time by flow cytometry. (E) Infection curves of the transduced CEMss-CCR5 cells. Data from a representative experiment of at least two independent experiments are shown.
Figure 5.
Figure 5.
Selective survival of human CD4+ T cells expressing GPI-10E8 during CXCR4-tropic or CCR5-tropic HIV-1 infection. CEMss-CCR5 cells were transduced with GPI-10E8/GFP and mixed with untransduced cells at a ratio of approximately 20% GFP-positive cells. The mixed population was challenged with 1,000 TCID50 of CXCR4-tropic NL4-3 (A) or CCR5-tropic RHPA.c/2635 (B), and the proportion of transgene-expressing cells was monitored over time by flow cytometry. (C) Survival curve of the transduced CEMss-CCR5 cells. Data from a representative experiment of at least two independent experiments are shown.
Figure 6.
Figure 6.
Design and characterization of GPI-anchored bifunctional inhibitors targeting gp41. (A) Design strategy. The sequence of a fusion inhibitor peptide P41 was genetically connected to the N- or C-termini of 10E8 scFv through a flexible (GGGGS)n linker, generating eight GPI-anchored bifunctional constructs named CMI01 ∼ CMI08. (B) The expression of CMI constructs was detected in the cell lysates and culture supernatants of transduced TZM-bl cells by western blotting with an anti-His tag antibody. (C) Surface expression of CMI constructs in transduced TZM-bl cells with or without PI-PLC treatment was determined by FACS analysis using an anti-His tag antibody. (D) Effects of CMI constructs on the surface expression of CD4, CCR5, and CXCR4 in transduced TZM-bl cells were detected by FACS analysis using a PE-conjugated anti-human CD4, CCR5, or CXCR4 antibody and judged by fluorescence intensity.
Figure 7.
Figure 7.
Antiviral activity of GPI-anchored bifunctional inhibitors in transduced cells. The inhibitory activities of CMI constructs against two HIV-1 pseudoviruses (A), two replicative HIV-2 isolates (B), and two SIV pseudoviruses (C), as well as the inhibitory activities of the CMI02 and CMI06 mutants with the disruptive P41 mutations on the HIV-1SF162 and SIVmac239 pseudoviruses (D) and their Env-mediated cell-cell fusion (E), and the cell-cell HIV-1 transmission of three replicative HIV-1 isolates (F) were respectively determined in transduced TZM-bl or 293FTtarget cells. (G) The inhibitory activities of CMI02 and CMI06 constructs against the cell-cell fusion mediated by a panel of HIV-1 Envs that were partially resistant to GPI-10E8. (H) The inhibitory activities of GPI-10E8 and CMI constructs on VSV-G infection. (I-K) The anti-HIV activities of CMI02 and CMI06 in human primary CD4+ T cells. Transduced human primary CD4+ T cells were infected with 1,000 TCID50 of NL4-3, and intracellular p24 and GFP expression were monitored over time by flow cytometry. Infection curves of the transduced CD4+ T cells (I) represent the means ± SD from three independent experiments with triplicate samples, and statistical comparison was only performed between GPI-FluIgG03-ransduced cells and CMI02 or CMI06-transduced cells at day 6 after NL4-3 infection (****, P < 0.0001) as the most GPI-FluIgG03-transduced cells were dead at day 9. The survival curves of human CD4+ T cells transduced with CMI02 (J) and CMI06 (K) were statistically analyzed. The results of three independent experiments are shown with different colours of lines.
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Grants and funding

This work was supported by National Natural Science Foundation of China: [Grant Number 81630061]; National Science and Technology Major Project of China: [Grant Number 2017ZX10202102-001-003,2018ZX10301103]; CAMS Innovation Fund for Medical Sciences: [Grant Number 2017-I2M-1-014,2021-I2M-1-037].