CAR-T Cells Guided By Broadly Neutralizing Antibody VRC01 Specifically And Effectively Eradicate The Reactivated Virus-latently-infected CD4~+ T-lymphocytes

ObjectHIV-1 replication can be efficiently suppressed with combined antiretroviral therapy(c ART) in infected individuals. However, the virus can integrate into the genome of some long-lived CD4+ T lymphocyte and form a stable viral reservoir. The viremia will rebound soon once the c ART was disrupted, which become a major barrier for eradicating HIV-1 infection. Recent efforts have focused on the reactivation of the latently-infected viruses with special latency-reversing agents(LRAs) without global T cell activation, followed by drug treatment and immune surveillance by the immune system. This strategy is also known as the ‘shock and kill’.However, with examely high mutation rate in the genome during replication, HIV-1 can quickly evade the immune surveillance. Several studies have indicated that CD8+ T lymphocytes in infected patients on c ART lack HIV-1-specific or effective immune responses, and cannot completely eliminate latently infected cells, even after successful reactivation. Therefore, the re-establishment of a potent antiviral immunity is required for an ultimate ‘kill’ strategy to eradicate the viral reservoir.In recent years, chimeric antigen receptor(CAR)-modified immune cells have emerged as a novel approach to kill cancer cells in a high-affinity, T cell receptor-independent, and major histocompatibility complex(MHC)-unrestricted manner. Such specific targeting of cancer cells can be achieved through the adoptive transfer of autologous immune cells genetically engineered to express a CAR moiety recognizing the natural antigen on malignant cells. The clinical use of CARs against leukemia and lymphoma has been shown to be highly effective and successful. This strategy has also been proposed for the treatment of viral infection, including HIV-1, hepatitis B virus, hepatitis C virus. Previous reports have described the generation of HIV-1-specific CAR-T cells by connection of an extracellular antigen-binding domain to intracellular T cell activation domains. The antigen-binding domain can be a single-chain variable fragment(sc Fv) or a natural molecule, such as CD4, which bridges the CAT-T cells to and induces the death of the cells expressing HIV-1 envelope glycoprotein(Env). However, it remains to be determined whether these CAR-T cells can eradicate the latently-infected viral reservoir. ProceduresIn this report, we connected the single-chain variable fragment of the HIV-1-specific broadly neutralizing antibody VRC01 to three CAR moieties, as the extracellular and intracellular domains respectively. We then transduced the three HIV-1-specific CAR into primary CD8+ T lymphocytes by lentiviral vector. By functional experiment, we identified the most efficient CAR construct which was named as VC-CAR and compared the potency of VC-CAR-T lymphocytes and CD4-CAR-T lymphocytes. After co-culture of VC-CAR-T lymphocytes with two HIV-1 Env-expressing target cell lines or wild-type HIV-1 infected CD4+ T lymphocytes, we examined its cytokines secretion by ELISPOT assay and specific killing activity against target cells by detecting LDH release assay. We also tested the inhibition of HIV-1 rebound by VC-CAR-T lymphocytes through withdrawal antiviral treatment in an in vitro viral infection system, which mimiced the in vivo viral rebound process in infected individuals when the c ART therapy was disrupted. Finally, we performed a viral outgrowth assay in the presence of VC-CAR-T cells, and determined the potency of VC-CAR-T lymphocytes in specifically recognizing and eradicating the reactivated latently infected CD4+ T lymphocytes isolated from HIV-1-infected indivuals receiving suppressive c ART. ResultsWe successfully constructed HIV-1 specific VC-CAR-T lymphocytes, which displayed specific and potent activities in eradication of HIV-1 Env-expressing target cells and antiviral cytokines secretion including interferon-γ, granzyme-B and IL-2. VC- CAR-T lymphocytes also exerted robust cytolysis activity against primary CD4+ T lymphocytes which were infected by HIV-1 in vitro, and significantly inhibited the viral rebound after the removal of antiviral drugs in an in vitro HIV-1 infection model which mimiced the disruption of the c ART in clinical cases. More importantly, the VC-CAR-T lymphocytes also potently and persistently induced the cytolysis of LRA-reactivated HIV-1latently infected CD4+ T lymphocytes isolated from infected individuals receiving suppressive c ART. ConclusionsOur studies have demonstrated the potency of the genetically engineered VC-CAR-T lymphocytes in eradicating HIV-1 infected cells and latent viral reservoir, which made them a particularly excellent candidate for therapeutic application in efforts to reach a functional cure of HIV-1 infection.