A novel real‐time CTL assay to measure designer T‐cell function against HIV Env⁺ cells

Abstract

Background To increase the immunosurveillance in HIV infection, we used retroviral vectors expressing CD4-chimeric antigen receptors (CARs) to genetically modify autologous T cells and redirect CTL towards HIV. The CD4 extracellular domain targets Envelope and the intracellular signaling domains activate T cells. The maC46 fusion inhibitor binds HIV and blocks viral replication. Methods We stimulated rhesus PBMCs with αCD3αCD28 and co-transduced T cells with CD4-CAR and maC46 vectors. CD4-CAR transduced T cells were added to Env+ 293T cells at E:T of 1:1. Killing of target cells was measured as reduced impedance. Results We observed gene expression in 60-70% of rhesus CD3+CD8+ T cells with the individual vectors and in 35% of the cells with both vectors. CD4-CAR transduced populations specifically killed Env+ cells. Conclusions In these studies, we showed that designer T cells were redirected to kill Env+ cells. Control of viremia without HAART would revolutionize treatment for HIV patients.

Chimeric Antigen Receptor T Cells Guided by the Single-Chain Fv of a Broadly Neutralizing Antibody Specifically and Effectively Eradicate Virus Reactivated from Latency in CD4 + T Lymphocytes Isolated from HIV-1-Infected Individuals Receiving Suppressive Combined Antiretroviral Therapy

“…We found that the newly designed CAR-T cells, here referred to as VC-CAR-T cells, were able to induce T cell-mediated cytolysis after coculture with gp120-expressing cells and wild-type HIV-1-infected CD4 ϩ T cells. We also found that VC-CAR-T cells display superior potency compared to the CD4-CAR described previously (26,27,(31)(32)(33)(34)(35)(36). Importantly, we have also confirmed that they can effectively kill the reactivated HIV-1-infected CD4 ϩ T lymphocytes isolated from HIV-1-infected patients.…”

“…Therefore, we chose to use VRC01-28BBZ-3-expressing T cells, here referred to as VC-CAR-T cells, for subsequent experiments. Previous studies have reported that CD4-based CAR-T cells, referred to here as CD4-CAR-T cells, display direct cytotoxic activity against HIV-1 Env-expressing target cells (26,27,(31)(32)(33)(34)(35)(36). To compare the efficiency of CD4-CAR and VC-CAR, we generated the former by replacing the VC-CAR scFv with the human CD4 extracellular domain and transduced this construct into CD8 ϩ T lymphocytes (Fig.…”

“…Previous reports have described the generation of HIV-1-specific CAR-T cells by connection of an extracellular antigenbinding domain to intracellular T cell activation domains (e.g., CD3 and CD28). The former can be a single-chain variable fragment (scFv) (26)(27)(28)(29)(30) or a natural molecule, such as CD4 (26,27,(31)(32)(33)(34)(35)(36), capable of directly inducing the death of cells expressing the viral envelope glycoprotein (Env). However, it remains to be determined whether these CAR-T cells can be used to kill the reactivated HIV-1 latently infected cells from the infected individuals receiving suppressive cART.…”

Novel CD4-Based Bispecific Chimeric Antigen Receptor Designed for Enhanced Anti-HIV Potency and Absence of HIV Entry Receptor Activity

“…Early clinical successes with CARs against leukemia and lymphoma (28,29) have garnered particular recognition (30), and the strategy has been proposed for use against viruses, including HIV (reviewed in references 31, 32, 33, and 34). Indeed, previous reports demonstrated favorable in vitro results with CARs targeted by single-chain variable-region antibody (Ab) constructs directed against the gp120 or gp41 subunits of HIV-1 Env (35)(36)(37)(38) as well as with CARs employing extracellular CD4 domains (35,36,(39)(40)(41)(42). Clinical studies were conducted more than a decade ago with CD4 CARs (43)(44)(45); see also https://clinicaltrials.gov/ct2/show/NCT01013415), and subsequent analyses of peripheral blood samples verified long-term persistence as well as retention of CAR expression and the proliferative potential of adoptively transferred CAR-transduced T cells (CAR-T) (46).…”

Combining Cell and Gene Therapy in an Effort to Eradicate HIV

“…[71][72][73][74][75][76][77][78][79][80] used measure of the viable HIV reservoir) and rectal HIV DNA (-0.5 log), and a trend toward less viral rebound, although no decrease in peripheral blood HIV DNA or rectal HIV RNA. 81 Long-term follow-up suggests that this approach was safe and results in long-lived cells with CAR DNA that persisted for more than a decade.…”