Jul 2026· Journal of Immunology· Vol 215· 0 citations
TL;DR
Evidence is provided that targeting HIV-1 C-CT through ADCC and CAR NK/T cell therapies can overcome the limitations of bnAbs, and the first evidence to test these approaches to effectively target HIV-1 C-CT is offered.
Abstract
Despite the success of antiretroviral therapy in controlling HIV-1 replication, achieving a cure remains a significant challenge. Blocking viral replication has proven particularly challenging due to the highly efficient nature of cell-to-cell transmission (C-CT), as antibodies often fail to access the virological synapse. Here, we studied the effects of immune-based strategies, including ADCC and CAR NK/T cells, on HIV-1 transmission between cells.
We developed a highly sensitive assay for precise quantification of cell lysis using luciferase activity and built CAR NK/T based on the VRC01 and PGT121 bnAbs. We then utilized the assay to assess cellular cytotoxicity mediated by antibodies and CAR NK/T against target cells expressing HIV-1 envelope glycoproteins (Envs). We have previously developed an ultrasensitive HIV-1 C-CT assay, using a replication-competent HIV-1 vector and an activated nanoluciferase reporter system to quantify viral transmission in primary T cells. Using our nanoluciferase reporter assay, we studied the effects of a) PBMCs and soluble IgGs, and b) CAR NK/T on HIV-1 C-CT.
We detected cytotoxic activity of T and NK cells engineered to express VRC01 and PGT121 CARs against CEM.NKR cells that express luciferase and HIV-1 Envs as targets. HIV-1 C-CT of the NL43(AD8) molecular clone, which contains all HIV-1 genes, exhibits significant resistance to bnAbs but could be efficiently blocked by ADCC. Moreover, CAR PBMCs exhibited increased specific killing across a range of envelope-expressing target cells at various E: T cell ratios. Among the CARs tested, the PGT121 bnAb-based CAR consistently outperformed others in mediating cellular cytotoxicity.
These results provide proof of concept that targeting HIV-1 C-CT through ADCC and CAR NK/T cell therapies can overcome the limitations of bnAbs. This study offers the first evidence to test these approaches to effectively target HIV-1 C-CT, highlighting their potential in studying HIV-1 treatment strategies.
NIH U01 Award, 2022 - 2027
Vaccines and Immunotherapy (VAC)
ABSTRACT Human immunodeficiency virus 1 (HIV-1) remains a global health burden affecting over 39 million people worldwide, with approximately one million new infections occurring each year. Sexual transmission remains the predominant route of HIV-1 acquisition, during which the virus crosses mucosal barriers and interacts with host immune cells to facilitate infection. Dendritic cells (DCs) contribute to systemic viral spread and seeding of reservoirs by efficiently capturing HIV-1, leading to infection and subsequent transmission of the virus to CD4+ T cells. Siglec-1 (CD169) is a key receptor involved in HIV-1 capture, and its blockade may help prevent viral transmission. Here, we used newly developed single-domain antibodies, also known as nanobodies, against Siglec-1 as a candidate inhibitor to limit HIV-1 transmission. Using a Siglec-1-overexpressing cell line, we demonstrated that these nanobodies specifically blocked Siglec-1-mediated HIV-1 binding and transmission. Extending these findings to a more physiologically relevant context, the anti-Siglec-1 nanobodies neither induced immune nor cellular activation in DCs, indicating a favorable safety profile for functional applications. Most notably, the anti-Siglec-1 nanobody 2C2 effectively blocked HIV-1 binding as well as infection of DCs. Moreover, both replication-dependent and replication-independent HIV-1 transmission by DCs was also abrogated by the nanobody. Our findings not only underscore the relevance of Siglec-1 in HIV-1 capture but also highlight the therapeutic potential of utilizing host-targeting strategies against infectious diseases. IMPORTANCE Sexual transmission is the main route of human immunodeficiency virus 1 (HIV-1) infection, and novel interventions are needed to prevent this crucial first step. Mucosal dendritic cells play a key role by capturing HIV-1 via attachment receptors, leading to dendritic cell infection and subsequent transmission to T cells, thereby facilitating viral spread and establishment of infection. Here, we show that small, highly specific nanobodies targeting the attachment receptor Siglec-1 strongly interfere with this early stage of HIV-1 transmission. Siglec-1 nanobodies prevented HIV-1 binding to and infection of dendritic cells, thereby blocking transmission to T cells without inducing unwanted immune activation. Together, these findings identify Siglec-1 nanobodies as promising interventions and support the development of host-directed nanobody-based strategies to reduce HIV-1 spread. Sexual transmission is the main route of human immunodeficiency virus 1 (HIV-1) infection, and novel interventions are needed to prevent this crucial first step. Mucosal dendritic cells play a key role by capturing HIV-1 via attachment receptors, leading to dendritic cell infection and subsequent transmission to T cells, thereby facilitating viral spread and establishment of infection. Here, we show that small, highly specific nanobodies targeting the attachment receptor Siglec-1 strongly interfere with this early stage of HIV-1 transmission. Siglec-1 nanobodies prevented HIV-1 binding to and infection of dendritic cells, thereby blocking transmission to T cells without inducing unwanted immune activation. Together, these findings identify Siglec-1 nanobodies as promising interventions and support the development of host-directed nanobody-based strategies to reduce HIV-1 spread.
S. Man, A. Affandi, H. Brink et al.· Journal of Virology· 0 citations
Human Immunodeficiency Virus 1 (HIV-1) became a global pandemic in the early 1980s, continuing to affect millions of people every year. Though progress has been made in the last four decades, there are no FDA approved therapeutic classes for the assembly stage in the HIV-1 replication cycle. Previous studies have found the HIV-1 can become drug resistant by increasing its prevalence to infect naïve cells by cell-cell contact. Therefore, it is more important now than ever to study the spread of HIV-1 from cell-cell contacts.
First, we establish a robust cell-cell contact imaging assay with T-cells and monocyte-like macrophage (MLM). We, then, combine this pulse-chase assays to investigate the mobility of internalized Env to sites of cell-cell contact and determine that exocytosis to these sites was not seen on a large or readily rapid rate. Next, we combined our established cell-cell contact system with lithography masks and hydrophobic compound Lipidure to only allow cells to lay down on areas of the imaging coverslip that were ablated. This was used for adherent immune cells as well as improved upon to establish an imaging method for suspension T-cells. We found an increase in the number of cell-cell contacts with our method.
Following this, we use single channel superresolution microscopy to quantify the relative monomeric and dimeric CD4 population. Here, we provided an orthogonal approach to the biochemical verification for the chimera’s monomeric nature. Lastly, we use simultaneous, dual-color superresolution imaging to investigate the nanometer organization of viral proteins and receptors at sites of cell-cell contact.
ABSTRACT Chimeric antigen receptor T cell (CAR-T cell) therapy targeting and eliminating HIV-infected cells offers a promising approach to provide people living with HIV (PLWH) with a functional cure by preventing the recurrence of viremia caused by reactivation of latent HIV-1-infected cells. We previously described a bispecific CAR-T cell targeting two highly conserved gp120 epitopes (duoCAR-T cell) with potent anti-HIV-1 activity that is currently in clinical trials. However, elevated levels of transforming growth factor β (TGF-β) present in many PLWH may hinder the activity of both infused HIV-1-specific CAR-T cells, such as duoCAR-T cells and endogenous HIV-1-specific CD8+ T cells, thereby limiting their effectiveness to achieve a functional HIV-1 cure. We hypothesized that HCW9218, a novel bifunctional immunomodulatory protein composed of TGF-βRII and IL-15/IL-15Rα, would enhance anti-HIV-1 immunity by TGF-βRII binding and neutralizing TGF-β, while IL-15/IL-15Rα would stimulate effector cells and reactivate latent HIV-1-infected cells. We used duoCAR-T cells generated from CD4+ and CD8+ T cells from people without HIV (PWoH) and PLWH donors to demonstrate the in vitro capacity of HCW9218 to block TGF-β activity, and enhance duoCAR-T cell proliferation, cytotoxicity, and anti-HIV-1 activity. HCW9218 also functioned as a latency-reversing agent, stimulating HIV-1 production by CD4+ T cells from ART-suppressed PLWH. Production of HIV-1 by HCW9218-treated CD4+ T cells from ART-suppressed PLWH donors was suppressed by co-culture with autologous duoCAR-T cells. Together, these findings highlight the potential of HCW9218 to augment T cell and CAR-T-based therapies and contribute to strategies aimed at achieving a functional cure for HIV-1. IMPORTANCE The persistence of HIV-1 reservoirs remains the primary barrier to an HIV-1 cure because antiretroviral therapy (ART) suppresses viral replication but does not eliminate latent HIV-1-infected cells. Treatment with anti-HIV-1 duoCAR-T cells is a potential strategy to target and eliminate HIV-1-infected cells, but their activity may be impaired by the immunosuppressive environment in lymphoid tissues of people living with HIV (PLWH). Transforming growth factor β (TGF-β), a pleiotropic cytokine elevated in PLWH, is a key mediator of this immunosuppression. Here, we show that HCW9218, a bifunctional fusion protein with TGF-β-neutralizing activity and IL-15 superagonist activity, preserves duoCAR-T cell function in the presence of TGF-β and reactivates HIV-1 production by latent HIV-1-infected cells in ART-suppressed CD4+ T cells from PLWH. These findings highlight HCW9218 as a unique dual-function immunotherapy that may enhance the efficacy of duoCAR-T cells while facilitating clearance of the HIV reservoir. The persistence of HIV-1 reservoirs remains the primary barrier to an HIV-1 cure because antiretroviral therapy (ART) suppresses viral replication but does not eliminate latent HIV-1-infected cells. Treatment with anti-HIV-1 duoCAR-T cells is a potential strategy to target and eliminate HIV-1-infected cells, but their activity may be impaired by the immunosuppressive environment in lymphoid tissues of people living with HIV (PLWH). Transforming growth factor β (TGF-β), a pleiotropic cytokine elevated in PLWH, is a key mediator of this immunosuppression. Here, we show that HCW9218, a bifunctional fusion protein with TGF-β-neutralizing activity and IL-15 superagonist activity, preserves duoCAR-T cell function in the presence of TGF-β and reactivates HIV-1 production by latent HIV-1-infected cells in ART-suppressed CD4+ T cells from PLWH. These findings highlight HCW9218 as a unique dual-function immunotherapy that may enhance the efficacy of duoCAR-T cells while facilitating clearance of the HIV reservoir.
Sara Lamcaj, Erin B Cole, Christopher R Hiner et al.· Journal of Virology· 0 citations
ABSTRACT Human immunodeficiency virus type 1 (HIV-1) persists in latent reservoirs, mainly within CD4+ T cells, which are refractory to antiretroviral therapy, and can lead to rapid viral rebound upon treatment interruption. The “shock and kill” strategy aims to eliminate latent reservoirs by inducing viral transcription through latency-reversing agents (LRAs), thereby exposing infected cells to immune-mediated clearance. We developed and validated a simple, low-cost, and highly reproducible in vitro screening protocol to evaluate the efficacy and safety of LRAs, using a two-color flow cytometry assay on ACH2 cells, a well-characterized model of HIV-1 latency. Using this method, we identified PEP005 and CUDC-907 as the most potent LRAs across multiple experimental settings. Their reactivation capacity was further confirmed through transcriptomic analysis, which revealed a significant upregulation of viral RNA copies following stimulation. In addition, we collaborated with Dompé for using the Exscalate platform, an innovative computer-aided drug discovery approach, together with the experimental validation, which led to the identification of Tandutinib, a tyrosine kinase and mTOR inhibitor, as a novel LRA candidate with appreciable latency-reversing activity in the ACH2 model. While the ACH2 cell line does not fully recapitulate the complexity of HIV latency in vivo, it offers a robust and scalable system for early-stage screening and prioritization of candidate LRAs. Importantly, the future application of these LRAs in ex vivo samples derived from people living with HIV, with a particular focus on pediatric samples, will be crucial to deepen our understanding of latency reactivation in clinically relevant settings and age-specific immune environments. IMPORTANCE This study addresses a major obstacle to curing human immunodeficiency virus type 1 (HIV-1) infection: the persistence of latent viral reservoirs that are not eliminated by current therapies. We developed a simple and reproducible assay to identify compounds capable of reactivating latent virus, a key step in cure strategies. Using this approach, we identified effective latency-reversing compounds and, through collaboration with Dompé using the EXSCALATE platform, we also identified Tandutinib as a promising new candidate for further investigation. This study addresses a major obstacle to curing human immunodeficiency virus type 1 (HIV-1) infection: the persistence of latent viral reservoirs that are not eliminated by current therapies. We developed a simple and reproducible assay to identify compounds capable of reactivating latent virus, a key step in cure strategies. Using this approach, we identified effective latency-reversing compounds and, through collaboration with Dompé using the EXSCALATE platform, we also identified Tandutinib as a promising new candidate for further investigation.
A. Neri, Arianna Rotili, Elena Morrocchi et al.· Microbiology spectrum· 0 citations
Antiretroviral therapy (ART) has proven effective in suppressing HIV-1 replication, but further development of HIV-1 inhibitors is continually driven by the challenge of drug resistance and viral adaptation. The HIV-1 capsid is a promising target for treatment due to its high sequence conservation as well as its crucial role in the viral life cycle. Recently, we have developed a novel capsid-targeting biologic that prevents HIV-1 replication by efficient degradation of newly synthesized capsid. Here, we have investigated the sensitivity to viral escape as well as the breadth of this biologic against HIV-1 subtypes. The capsid-targeting biologic efficiently blocked replication of different primary HIV-1 isolates, and continuous exposure of these viruses to the biologic resulted in viral breakthrough of two out of ten primary HIV-1 isolates tested. Notably, the breakthrough variants did not have amino acid changes in the nanobody epitope but primarily in the matrix region. The breakthrough variants remained sensitive to the biologic albeit to a lesser extent. In the absence of the biologic, breakthrough variants showed increased replication kinetics when compared to their parental virus, suggesting that adaption to the biologic is likely due to the increased viral production and that the target area of the biologic is too conserved for actual escape. This is further underscored by the broad specificity of the biologic as importantly the biologic blocked infection of different HIV-1 subtypes that occur worldwide (A, B, C, D, CRF01_AE, CRF02_AG). These results demonstrate the broad neutralization potential of anti-capsid biologics with a high barrier to resistance, making capsid-targeting inhibitors important for novel antiretroviral drug strategies worldwide.
F. M. Stel, E. Zijlstra-Willems, A. V. Nuenen et al.· International Journal of Mol...· 0 citations
D10 is the first Tat activator available and the first LRA that targets an HIV protein, and induces strong HIV production by latent cell lines and latent cells from people living with HIV-1.
P. Tong, Laetitia Marty, Nawel Chekrit et al.· bioRxiv· 0 citations
