[Construction of a chimeric cell model expressing human immunodeficiency virus-1 envelope protein based on transposon system and evaluation of chimeric antigen receptor t-cell cytotoxicity in vitro].

Authors

Jiarui Liu, Yang Gao, Changjun Wang, Lei Yu, Yan Ma, Mengying Zhou, Ying Zhou, Siqi Yang, Jiezhen Li, Chaojiang Gu, Shasha Zhao

Published in

Sheng wu gong cheng xue bao = Chinese journal of biotechnology. Volume 42. Issue 7. Pages 3203-3216. Jul 25, 2026.

Abstract

The gp120/gp41 complex on the surface of human immunodeficiency virus type I (HIV-1) mediates viral entry into host cells and it is a primary target for the development of vaccines and antiviral interventions including broadly neutralizing antibodies (bNAbs), gene therapy, membrane fusion inhibitors, and CAR-T cell-based therapies. Therefore, stable cell models displaying the HIV envelope protein are needed to enable CAR-T cells to recognize and trigger their cytotoxic responses. We constructed a transposon-based plasmid system for the stable expression of six envelope proteins. A gp160(ΔCT) variant with a substituted furin cleavage site and a deleted gp41 cytoplasmic tail was constructed. Stable cell lines were generated by co-transfecting HEK293T cells with the transposon plasmid and a transposase plasmid, followed by puromycin selection. The membrane surface expression of the proteins and their recognition by the bNAbs were confirmed via indirect flow cytometry, which measured the binding activities of three neutralizing antibodies to gp160(ΔCT) proteins from diverse HIV-1 strains. The cell model was co-incubated with CAR-T cells at varying effector-to-target ratios. Subsequent calcein-AM release assays and ELISA quantification of granzyme B and perforin confirmed the dose-dependent and antigen-specific cytotoxicity mediated by all three CAR-T cell constructs. Furthermore, flow cytometric analysis using CFSE labeling revealed that all three CAR-T cell products exhibited pronounced, dose-dependent proliferation upon stimulation with the gp160(ΔCT) cell model. Collectively, the HEK293T model stably expressing membrane-anchored HIV-1 gp160(ΔCT) functioned as an effective target for CAR-T cells and robustly induced antigen-specific CAR-T cell proliferation, in comparison with the MLV-gp80(ΔCT)-293T negative control. The gp160(ΔCT)-expressing HEK293T model thus provides a robust platform for benchmarking the targeted recognition and cytotoxic activity of antiviral antibodies, agents, and CAR-T therapies in HIV research.

PMID:
42522626
Bibliographic data and abstract were imported from PubMed on 29 Jul 2026.

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