Glycosyl-Phosphatidylinositol-Anchored Anti-HIV Env Single-Chain Variable Fragments Interfere with HIV-1 Env Processing and Viral Infectivity.

In previous studies, we demonstrated that single-chain variable fragments (scFvs) from anti-human immunodeficiency virus (HIV) Env monoclonal antibodies act as entry inhibitors when tethered to the surface of target cells by a glycosyl-phosphatidylinositol (GPI) anchor. Interestingly, even if a virus escapes inhibition at entry, its replication is ultimately controlled. We hypothesized that in addition to functioning as entry inhibitors, anti-HIV GPI-scFvs may also interact with Env in an infected cell, thereby interfering with the infectivity of newly produced virions. Here, we show that expression of the anti-HIV Env GPI-scFvs in virus-producing cells reduced the release of HIV from cells 5- to 22-fold, and infectivity of the virions that were released was inhibited by 74% to 99%. Additionally, anti-HIV Env GPI-scFv X5 inhibited virion production and infectivity after latency reactivation and blocked transmitter/founder virus production and infectivity in primary CD4+ T cells. In contrast, simian immunodeficiency virus (SIV) production and infectivity were not affected by the anti-HIV Env GPI-scFvs. Loss of infectivity of HIV was associated with a reduction in the amount of virion-associated Env gp120. Interestingly, an analysis of Env expression in cell lysates demonstrated that the anti-Env GPI-scFvs interfered with processing of Env gp160 precursors in cells. These data indicate that GPI-scFvs can inhibit Env processing and function, thereby restricting production and infectivity of newly synthesized HIV. Anti-Env GPI-scFvs therefore appear to be unique anti-HIV molecules as they derive their potent inhibitory activity by interfering with both early (receptor binding/entry) and late (Env processing and incorporation into virions) stages of the HIV life cycle.IMPORTANCE The restoration of immune function and persistence of CD4+ T cells in HIV-1-infected individuals without antiretroviral therapy requires a way to increase resistance of CD4+ T cells to infection by both R5- and X4-tropic HIV-1. Previously, we reported that anchoring anti-HIV-1 single-chain variable fragments (scFvs) via glycosyl-phosphatidylinositol (GPI) to the surface of permissive cells conferred a high level of resistance to HIV-1 variants at the level of entry. Here, we report that anti-HIV GPI-scFvs also derive their potent antiviral activity in part by blocking HIV production and Env processing, which consequently inhibits viral infectivity even in primary infection models. Thus, we conclude that GPI-anchored anti-HIV scFvs derive their potent blocking activity of HIV replication by interfering with successive stages of the viral life cycle. They may be effectively used in genetic intervention of HIV-1 infection.

Glycosylphosphatidylinositol-Anchored Anti-HIV scFv Efficiently Protects CD4 T Cells from HIV-1 Infection and Deletion in hu-PBL Mice.

Despite success in viral inhibition and CD4 T cell recovery by highly active antiretroviral treatment (HAART), HIV-1 is still not curable due to the persistence of the HIV-1 reservoir during treatment. One patient with acute myeloid leukemia who received allogeneic hematopoietic stem cell transplantation from a homozygous CCR5 Δ32 donor has had no detectable viremia for 9 years after HAART cessation. This case has inspired a field of HIV-1 cure research focusing on engineering HIV-1 resistance in permissive cells. Here, we employed a glycosylphosphatidylinositol (GPI)-scFv X5 approach to confer resistance of human primary CD4 T cells to HIV-1. We showed that primary CD4 T cells expressing GPI-scFv X5 were resistant to CCR5 (R5)-, CXCR4 (X4)-, and dual-tropic HIV-1 and had a survival advantage compared to control cells ex vivo In a hu-PBL mouse study, GPI-scFv X5-transduced CD4 T cells were selected in peripheral blood and lymphoid tissues upon HIV-1 infection. Finally, GPI-scFv X5-transduced CD4 T cells, after being cotransfused with HIV-infected cells, showed significantly reduced viral loads and viral RNA copy numbers relative to CD4 cells in hu-PBL mice compared to mice with GPI-scFv AB65-transduced CD4 T cells. We conclude that GPI-scFv X5-modified CD4 T cells could potentially be used as a genetic intervention against both R5- and X4-tropic HIV-1 infections. IMPORTANCE: Blocking of HIV-1 entry is one of most promising approaches for therapy. Genetic disruption of the HIV-1 coreceptor CCR5 by nucleases in T cells is under 2 clinical trials and leads to reduced viremia in patients. However, the emergence of viruses using the CXCR4 coreceptor is a concern for therapies applying single-coreceptor disruption. Here, we report that HIV-1-permissive CD4 T cells engineered with GPI-scFv X5 are resistant to R5-, X4-, or dual-tropic virus infection ex vivo In a preclinical study using hu-PBL mice, we show that CD4 T cells were protected and that GPI-scFv X5-transduced cells were selected in HIV-1-infected animals. Moreover, we show that GPI-scFv X5-transduced CD4 T cells exerted a negative effect on virus replication in vivo We conclude that GPI-scFv X5-modified CD4 T cells could potentially be used as a genetic intervention against both R5- and X4-tropic HIV-1 infections.

The Glycosylphosphatidylinositol-Anchored Variable Region of Llama Heavy Chain-Only Antibody JM4 Efficiently Blocks both Cell-Free and T Cell-T Cell Transmission of Human Immunodeficiency Virus Type 1.

The variable regions (VHHs) of two heavy chain-only antibodies, JM2 and JM4, from llamas that have been immunized with a trimeric gp140 bound to a CD4 mimic have been recently isolated (here referred to as VHH JM2 and VHH JM4, respectively). JM2 binds the CD4-binding site of gp120 and neutralizes HIV-1 strains from subtypes B, C, and G. JM4 binds gp120 and neutralizes HIV-1 strains from subtypes A, B, C, A/E, and G in a CD4-dependent manner. In the present study, we constructed glycosylphosphatidylinositol (GPI)-anchored VHH JM2 and JM4 along with an E4 control and transduced them into human CD4+ cell lines and primary CD4 T cells. We report that by genetically linking the VHHs with a GPI attachment signal, VHHs are targeted to the lipid rafts of the plasma membranes. Expression of GPI-VHH JM4, but not GPI-VHH E4 and JM2, on the surface of transduced TZM.bl cells potently neutralizes multiple subtypes of HIV-1 isolates, including tier 2 or 3 strains, transmitted founders, quasispecies, and soluble single domain antibody (sdAb) JM4-resistant viruses. Moreover, transduction of CEMss-CCR5 cells with GPI-VHH JM4, but not with GPI-VHH E4, confers resistance to both cell-free and T cell-T cell transmission of HIV-1 and HIV-1 envelope-mediated fusion. Finally, GPI-VHH JM4-transduced human primary CD4 T cells efficiently resist both cell-free and T cell-T cell transmission of HIV-1. Thus, we conclude that VHH JM4, when targeted to the lipid rafts of the plasma membrane, efficiently neutralizes HIV-1 infection via both cell-free and T cell-T cell transmission. Our findings should have important implications for GPI-anchored antibody-based therapy against HIV-1. IMPORTANCE: Lipid rafts are specialized dynamic microdomains of the plasma membrane and have been shown to be gateways for HIV-1 budding as well as entry into T cells and macrophages. In nature, many glycosylphosphatidylinositol (GPI)-anchored proteins localize in the lipid rafts. In the present study, we developed GPI-anchored variable regions (VHHs) of two heavy chain-only antibodies, JM2 and JM4, from immunized llamas. We show that by genetically linking the VHHs with a GPI attachment signal, VHHs are targeted to the lipid rafts of the plasma membranes. GPI-VHH JM4, but not GPI-VHH JM2, in transduced CD4+ cell lines and human primary CD4 T cells not only efficiently blocks diverse HIV-1 strains, including tier 2 or 3 strains, transmitted founders, quasispecies, and soluble sdAb JM4-resistant strains, but also efficiently interferes T cell-T cell transmissions of HIV-1 and HIV-1 envelope-mediated fusion. Our findings should have important implications in GPI-anchored antibody-based therapy against HIV-1.

Engineered CD4 T cells expressing a membrane anchored viral inhibitor restrict HIV-1 through cis and trans mechanisms.

HIV-1 infection of target cells can occur through either cell-free virions or cell-cell transmission in a virological synapse, with the latter mechanism of infection reported to be 100- to 1,000-fold more efficient. Neutralizing antibodies and entry inhibitors effectively block cell-free HIV-1, but with few exceptions, they display much less inhibitory activity against cell-mediated HIV-1 transmission. Previously, we showed that engineering HIV-1 target cells by genetically linking single-chain variable fragments (scFvs) of antibodies to glycosyl phosphatidylinositol (GPI) potently blocks infection by cell-free virions and cell-mediated infection by immature dendritic cell (iDC)-captured HIV-1. Expression of scFvs on CD4+ cell lines by transduction with X5 derived anti-HIV-1 Env antibody linked to a GPI attachment signal directs GPI-anchored scFvs into lipid rafts of the plasma membrane. In this study, we further characterize the effect of GPI-scFv X5 on cell-cell HIV-1 transmission from DCs to target cells. We report that expression of GPI-scFv X5 in transduced CD4+ cell lines and human primary CD4+ T cells potently restricts viral replication in iDC- or mDC-captured HIV-1 in trans. Using live-cell imaging, we observed that when GPI-GFP or GPI-scFv X5 transduced T cells are co-cultured with iDCs, GPI-anchored proteins enrich in contact zones and subsequently migrate from T cells into DCs, suggesting that transferred GPI-scFv X5 interferes with HIV-1 infection of iDCs. We conclude that GPI-scFv X5 on the surface of transduced CD4+ T cells not only potently blocks cell-mediated infection by DCs, but it transfers from transduced cells to the surface of iDCs and neutralizes HIV-1 replication in iDCs. Our findings have important implications for HIV-1 antibody-based immunotherapies as they demonstrate a viral inhibitory effect that extends beyond the transduced CD4+ T cells to iDCs which can enhance HIV-1 replication.

Chimeric Antigen Receptor T Cell Bearing Herpes Virus Entry Mediator Co-stimulatory Signal Domain Exhibits High Functional Potency.

Chimeric antigen receptor (CAR) is a hybrid molecule consisting of an antigen-binding domain and a signal transduction domain. The artificial T cells expressing CAR (CAR-T cells) are expected to be a useful tool for treatment of various diseases, such as cancer. The addition of a co-stimulatory signal domain (CSSD) to CAR is shown to be critical for modulating CAR-T cell activities. However, the interplay among types of CSSDs, effector functions, and characteristics of CAR-T cells is largely unknown. To elucidate the interplay, we analyzed effector functions, differentiation to memory T cell subsets, exhaustion, and energy metabolism of the CAR-T cells with different CSSDs. Comparing to the CAR-T cells bearing a CD28- or 4-1BB-derived CSSD, which are currently used for CAR-T cell development, we found that the CAR-T cells with a herpes virus entry mediator (HVEM)-derived CSSD exhibited enhanced effector functions and efficient and balanced differentiation to both central and effector memory subsets, associated with an elevated energy metabolism and a reduced level of exhaustion. Thus, we developed the CAR-T cells bearing the CSSD derived from HVEM with high functional potency. The HVEM-derived CSSD may be useful for developing effective CAR-T cells.

Efficient Transduction of Human and Rhesus Macaque Primary T Cells by a Modified Human Immunodeficiency Virus Type 1-Based Lentiviral Vector.

Human immunodeficiency virus type 1 (HIV-1)-based lentiviral vectors efficiently transduce genes to human, but not rhesus, primary T cells and hematopoietic stem cells (HSCs). The poor transduction of HIV-1 vectors to rhesus cells is mainly due to species-specific restriction factors such as rhesus TRIM5α. Previously, several strategies to modify HIV-1 vectors were developed to overcome rhesus TRIM5α restriction. While the modified HIV-1 vectors efficiently transduce rhesus HSCs, they remain suboptimal for rhesus primary T cells. Recently, HIV-1 variants that encode combinations of LNEIE mutations in capsid (CA) protein and SIVmac239 Vif were found to replicate efficiently in rhesus primary T cells. Thus, the present study tested whether HIV-1 vectors packaged by a packaging construct containing these CA substitutions could efficiently transduce both human and rhesus primary CD4 T cells. To accomplish this, LNEIE mutations were made in the packaging construct CEMΔ8.9, and recombinant HIV-1 vectors packaged by Δ8.9 WT or Δ8.9 LNEIE were generated. Transduction rates, CA stability, and vector integration in CEMss-CCR5 and CEMss-CCR5-rhTRIM5α/green fluorescent protein cells, as well as transduction rates in human and rhesus primary CD4 T cells by Δ8.9 WT or Δ8.9 LNEIE-packaged HIV-1 vectors, were compared. Finally, the influence of rhesus TRIM5α variations in transduction rates to primary CD4 T cells from a cohort of 37 Chinese rhesus macaques was studied. While it maintains efficient transduction for human T-cell line and primary CD4 T cells, Δ8.9 LNEIE-packaged HIV-1 vector overcomes rhesus TRIM5α-mediated CA degradation, resulting in significantly higher transduction efficiency of rhesus primary CD4 T cells than Δ8.9 WT-packaged HIV-1 vector. Rhesus TRIM5α variations strongly influence transduction efficiency of rhesus primary CD4 T cells by both Δ8.9 WT or Δ8.9 LNEIE-packaged HIV-1 vectors. Thus, it is concluded that Δ8.9 LNEIE-packaged HIV-1 vector overcomes rhesus TRIM5α restriction and efficiently transduces both human and rhesus primary T cells.

Blocking type I interferon signaling enhances T cell recovery and reduces HIV-1 reservoirs.

Despite the efficient suppression of HIV-1 replication that can be achieved with combined antiretroviral therapy (cART), low levels of type I interferon (IFN-I) signaling persist in some individuals. This sustained signaling may impede immune recovery and foster viral persistence. Here we report studies using a monoclonal antibody to block IFN-α/ß receptor (IFNAR) signaling in humanized mice (hu-mice) that were persistently infected with HIV-1. We discovered that effective cART restored the number of human immune cells in HIV-1-infected hu-mice but did not rescue their immune hyperactivation and dysfunction. IFNAR blockade fully reversed HIV-1-induced immune hyperactivation and rescued anti-HIV-1 immune responses in T cells from HIV-1-infected hu-mice. Finally, we found that IFNAR blockade in the presence of cART reduced the size of HIV-1 reservoirs in lymphoid tissues and delayed HIV-1 rebound after cART cessation in the HIV-1-infected hu-mice. We conclude that low levels of IFN-I signaling contribute to HIV-1-associated immune dysfunction and foster HIV-1 persistence in cART-treated hosts. Our results suggest that blocking IFNAR may provide a potential strategy to enhance immune recovery and reduce HIV-1 reservoirs in individuals with sustained elevations in IFN-I signaling during suppressive cART.

CCR5 gene disruption via lentiviral vectors expressing Cas9 and single guided RNA renders cells resistant to HIV-1 infection.

CCR5, a coreceptor for HIV-1 entry, is a major target for drug and genetic intervention against HIV-1. Genetic intervention strategies have knocked down CCR5 expression levels by shRNA or disrupted the CCR5 gene using zinc finger nucleases (ZFN) or Transcription activator-like effector nuclease (TALEN). In the present study, we silenced CCR5 via CRISPR associated protein 9 (Cas9) and single guided RNAs (sgRNAs). We constructed lentiviral vectors expressing Cas9 and CCR5 sgRNAs. We show that a single round transduction of lentiviral vectors expressing Cas9 and CCR5 sgRNAs into HIV-1 susceptible human CD4+ cells yields high frequencies of CCR5 gene disruption. CCR5 gene-disrupted cells are not only resistant to R5-tropic HIV-1, including transmitted/founder (T/F) HIV-1 isolates, but also have selective advantage over CCR5 gene-undisrupted cells during R5-tropic HIV-1 infection. Importantly, using T7 endonuclease I assay we did not detect genome mutations at potential off-target sites that are highly homologous to these CCR5 sgRNAs in stably transduced cells even at 84 days post transduction. Thus we conclude that silencing of CCR5 via Cas9 and CCR5-specific sgRNAs could be a viable alternative strategy for engineering resistance against HIV-1.