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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.

The cure or functional cure of the “Berlin patient” and “London patient” indicates that infusion of HIV-resistant cells could be a viable treatment strategy. Very recently, we genetically linked a short-peptide fusion inhibitor with a glycosylphosphatidylinositol (GPI) attachment signal, rendering modified cells fully resistant to HIV infection. In this study, GPI-anchored m36.4, a single-domain antibody (nanobody) targeting the coreceptor-binding site of gp120, was constructed with a lentiviral vector. We verified that m36.4 was efficiently expressed on the plasma membrane of transduced TZM-bl cells and targeted lipid raft sites without affecting the expression of HIV receptors (CD4, CCR5, and CXCR4). Significantly, TZM-bl cells expressing GPI-m36.4 were highly resistant to infection with divergent HIV-1 subtypes and potently blocked HIV-1 envelope-mediated cell-cell fusion and cell-cell viral transmission. Furthermore, we showed that GPI-m36.4-modified human CEMss-CCR5 cells were nonpermissive to both CCR5- and CXCR4-tropic HIV-1 isolates and displayed a strong survival advantage over unmodified cells. It was found that GPI-m36.4 could also impair HIV-1 Env processing and viral infectivity in transduced cells, underlying a multifaceted mechanism of antiviral action. In conclusion, our studies characterize m36.4 as a powerful nanobody that can generate HIV-resistant cells, offering a novel gene therapy approach that can be used alone or in combination.

Emerging studies demonstrate that the antiviral activity of viral fusion inhibitor peptides can be dramatically improved when being chemically or genetically anchored to the cell membrane, where viral entry occurs. We previously reported that the short-peptide fusion inhibitor 2P23 and its lipid derivative possess highly potent antiviral activities against human immunodeficiency virus type 1 (HIV-1), HIV-2, and simian immunodeficiency virus (SIV). To develop a sterilizing or functional-cure strategy, here we genetically linked 2P23 and two control peptides (HIV-1 fusion inhibitor C34 and hepatitis B virus [HBV] entry inhibitor 4B10) with a glycosylphosphatidylinositol (GPI) attachment signal. As expected, GPI-anchored inhibitors were efficiently expressed on the plasma membrane of transduced TZM-bl cells and primarily directed to the lipid raft site without interfering with the expression of CD4, CCR5, and CXCR4. GPI-anchored 2P23 (GPI-2P23) completely protected TZM-bl cells from infections of divergent HIV-1, HIV-2, and SIV isolates as well as a panel of enfuvirtide (T20)-resistant mutants. GPI-2P23 also rendered the cells resistant to viral envelope-mediated cell-cell fusion and cell-associated virion-mediated cell-cell transmission. Moreover, GPI-2P23-modified human CD4(+) T cells (CEMss-CCR5) fully blocked both R5- and X4-tropic HIV-1 isolates and displayed a robust survival advantage over unmodified cells during HIV-1 infection. In contrast, it was found that GPI-anchored C34 was much less effective in inhibiting HIV-2, SIV, and T20-resistant HIV-1 mutants. Therefore, our studies have demonstrated that genetically anchoring a short-peptide fusion inhibitor to the target cell membrane is a viable strategy for gene therapy of both HIV-1 and HIV-2 infections. IMPORTANCE Antiretroviral therapy with multiple drugs in combination can efficiently suppress HIV replication and dramatically reduce the morbidity and mortality associated with AIDS-related illness; however, antiretroviral therapy cannot eradiate the HIV reservoirs, and lifelong treatment is required, which often results in cumulative toxicities, drug resistance, and a multitude of complications, thus necessitating the development of sterilizing-cure or functional-cure strategies. Here, we report that genetically anchoring the short-peptide fusion inhibitor 2P23 to the cell membrane can fully prevent infections from divergent HIV-1, HIV-2, and SIV isolates as well as a panel of enfuvirtide-resistant mutants. Membrane-bound 2P23 also effectively blocks HIV-1 Env-mediated cell-cell fusion and cell-associated virion-mediated cell-cell transmission, renders CD4(+) T cells nonpermissive to infection, and confers a robust survival advantage over unmodified cells. Thus, our studies verify a powerful strategy to generate resistant cells for gene therapy of both the HIV-1 and HIV-2 infections.