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

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
• gene therapy
• broadly neutralizing antibodies (bNAbs)
• fusion inhibitory peptide
• glycosylphosphatidylinositol (GPI)

Acknowledgement

We thank Zene Matsuda at the Institute of Medical Science, University of Tokyo, for providing plasmids and cells for DSP-based cell–cell fusion assay. This work was supported by grants from the CAMS Innovation Fund for Medical Sciences (2017-I2M-1-014, 2021-I2M-1-037), National Natural Science Foundation of China (81630061), and National Science and Technology Major Project of China (2018ZX10301103, 2017ZX10202102-001-003).

Disclosure statement

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

Author contributions

YH conceived the project. YC, HJ, XT, LL, XG, YZ, and HC designed and performed the experiments. YC, HJ, and YH analyzed the data. YC, HJ, and YH drafted and edited the manuscript.

Data availability statement

All data are fully available without restriction.

Additional information

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].