scholarly journals Modified HIV-1 Based Lentiviral Vectors Have an Effect on Viral Transduction Efficiency and Gene Expression in Vitro and in Vivo

2001 ◽  
Vol 4 (3) ◽  
pp. 164-173 ◽  
Author(s):  
Frank Park ◽  
Mark A. Kay
Keyword(s):  
Gene Expression ◽  
Lentiviral Vectors ◽  
Transduction Efficiency ◽  
Viral Transduction ◽  
Hiv 1

TRIM5α Variation Affects Lentiviral Transduction Efficiency for Long-Term Hematopoietic Repopulating Cells in a Rhesus Stem Cell Transplantation Model

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 2056-2056
Author(s):  
Naoya Uchida ◽  
Matthew M. Hsieh ◽  
Aylin C. Bonifacino ◽  
Sandra D. Price ◽  
Allen E. Krouse ◽  
...  
Keyword(s):  
Stem Cell ◽  
Cell Lines ◽  
Transduction Efficiency ◽  
Gfp Expression ◽  
Lentiviral Transduction ◽  
Cd34 Cells ◽  
Hiv 1

Abstract Abstract 2056 The Tripartite Motif-containing protein 5 α (TRIM5α) is thought to play an important role in restricting retroviral infection among species in nature, and restricts HIV-1 based lentiviral transduction of rhesus hematopoietic repopulating cells. We previously developed a chimeric HIV-1 based lentiviral vectors (χHIV vectors) which includes a simian immunodeficiency virus (SIV) capsid in place of the HIV-1 capsid to circumvent this restriction (J Virol. 2009). Transduction efficiency, however, remained highly variable between animals. In this study, we sought to evaluate the effects of rhesus TRIM5α polymorphisms on lentiviral transduction of rhesus hematopoietic repopulating cells. To evaluate whether rhesus TRIM5α polymorphisms influence the efficiency of lentiviral transduction, we transduced cell lines expressing 6 different rhesus TRIM5α types (Mamu-1, -2, -3, -4, -5, and TrimCyp) with eGFP-encoding HIV1, χHIV, and SIV vectors (Figure). Among all TRIM5α cell lines, transgene expression rates (%GFP) from the χHIV vector fell between that of the HIV-1 vector and that of the SIV vector. For the χHIV and SIV vectors, transduction efficiency was reduced in Mamu-1, -2, and -3 (p<0.01) expressing cell lines when compared to that of control cells. For the HIV-1 vector, there was a reduction in %GFP among all TRIM5α types (p<0.01). These results suggest that both the χHIV and SIV vectors escape restriction through rhesus TRIM5α Mamu-4, -5, and TrimCyp. We then analyzed 16 rhesus macaques who were transplanted with CD34+ cells transduced with the χHIV vector. We evaluated %GFP in granulocytes and lymphocytes 6 months after transplantation, as %GFP expression has been found to plateau in the peripheral blood at 6 months. Transduction efficiency of rhesus CD34+ cells was also evaluated in vitro at the time of transplant based on %GFP expression. For statistical analysis, we assessed factors that potentially affect transduction efficiency, including age, sex, weight, total number of mobilized CD34+ cells, cytokine mobilization regimen (G-CSF & stem cell factor (SCF) vs. G-CSF & plerixafor), cell density during transduction, and TRIM5α polymorphisms. Multivariable analysis demonstrated that TRIM5α type Mamu-4 (50.9±19.0% vs. 26.6±16.7%, p=0.04) as well as mobilization regimen (48.5±17.4% vs. 12.7±7.1%, p=0.01) affected CD34+ cell transduction efficiency in vitro. TRIM5α Mamu-4 only showed significant effects on %GFP among lymphocytes in vivo (23.7±17.9% vs. 5.3±3.1%, p=0.046). When analyzing the %GFP among granulocytes in vivo, there was a significant correlation with weight (p=0.01), mobilized CD34+ cell number (p=0.02), TRIM5α type Mamu-4 (29.3±25.4% vs. 8.4±6.0%, p=0.03), and mobilization regimen (25.4±24.1% vs. 7.5±3.0%, p=0.04). If in vitro %GFP is included in the analysis, both %GFP among granulocytes and lymphocytes are strongly affected (<0.001) and TRIM5α type Mamu-4 and mobilization regimen are no longer significant. Univariate analysis showed similar tendencies regarding %GFP among granulocytes and lymphocytes. Taken together, our data suggest that TRIM5α type Mamu-4, mobilization regimen (G-CSF & SCF), and CD34+ cell transduction efficiency in vitro are important factors that can predict higher %GFP in granulocytes and lymphocytes 6 months following transplantation. In summary, rhesus TRIM5α polymorphisms (especially type Mamu-4) play an important role in allowing efficient lentiviral transduction of long-term repopulating cells and contribute to the variable results observed in the rhesus hematopoietic stem cell gene therapy model. Disclosures: No relevant conflicts of interest to declare.

scholarly journals Lentiviral Vectors Encoding Human Immunodeficiency Virus Type 1 (HIV-1)-Specific T-Cell Receptor Genes Efficiently Convert Peripheral Blood CD8 T Lymphocytes into Cytotoxic T Lymphocytes with Potent In Vitro and In Vivo HIV-1-Specific Inhibitory Activity

2008 ◽  
Vol 82 (6) ◽  
pp. 3078-3089 ◽  
Author(s):  
Aviva Joseph ◽  
Jian Hua Zheng ◽  
Antonia Follenzi ◽  
Teresa DiLorenzo ◽  
Kaori Sango ◽  
...  
Keyword(s):  
T Lymphocytes ◽  
Ex Vivo ◽  
Lentiviral Vectors ◽  
Cell Receptor ◽  
Specific Ctls ◽  
Immunodeficiency Virus ◽  
Hiv 1

ABSTRACT The human immunodeficiency virus type 1 (HIV-1)-specific CD8 cytotoxic T-lymphocyte (CTL) response plays a critical role in controlling HIV-1 replication. Augmenting this response should enhance control of HIV-1 replication and stabilize or improve the clinical course of the disease. Although cytomegalovirus (CMV) or Epstein-Barr virus (EBV) infection in immunocompromised patients can be treated by adoptive transfer of ex vivo-expanded CMV- or EBV-specific CTLs, adoptive transfer of ex vivo-expanded, autologous HIV-1-specific CTLs had minimal effects on HIV-1 replication, likely a consequence of the inherently compromised qualitative function of HIV-1-specific CTLs derived from HIV-1-infected individuals. We hypothesized that this limitation could be circumvented by using as an alternative source of HIV-1-specific CTLs, autologous peripheral CD8+ T lymphocytes whose antigen specificity is redirected by transduction with lentiviral vectors encoding HIV-1-specific T-cell receptor (TCR) α and β chains, an approach used successfully in cancer therapy. To efficiently convert peripheral CD8 lymphocytes into HIV-1-specific CTLs that potently suppress in vivo HIV-1 replication, we constructed lentiviral vectors encoding the HIV-1-specific TCR α and TCR β chains cloned from a CTL clone specific for an HIV Gag epitope, SL9, as a single transcript linked with a self-cleaving peptide. We demonstrated that transduction with this lentiviral vector efficiently converted primary human CD8 lymphocytes into HIV-1-specific CTLs with potent in vitro and in vivo HIV-1-specific activity. Using lentiviral vectors encoding an HIV-1-specific TCR to transform peripheral CD8 lymphocytes into HIV-1-specific CTLs with defined specificities represents a new immunotherapeutic approach to augment the HIV-1-specific immunity of infected patients.

scholarly journals Specific activation of HIV-1 from monocytic reservoir cells by bromodomain inhibitor in humanized mice in vivo

2018 ◽  
Author(s):  
Guangming Li ◽  
Zheng Zhang ◽  
Natalia Reszka-Blanco ◽  
Feng Li ◽  
Liqun Chi ◽  
...  
Keyword(s):  
Gene Expression ◽  
T Cells ◽  
Cd4 T Cells ◽  
Humanized Mice ◽  
Viral Transcription ◽  
Dependent Manner ◽  
Monocytic Cells ◽  
Hiv 1

ABSTRACTThe combination antiretroviral therapy (cART) effectively suppresses HIV-1 infection and enables HIV-infected individuals to live long productive lives. However, the persistence of HIV-1 reservoir cells with latent or low-replicating HIV-1 in patients under cART make HIV-1 infection an incurable disease. Recent studies have focused on the development of strategies such as epigenetic modulators to activate and purge these reservoirs. Bromodomain inhibitors (BETi) are epigenetic modulating compounds able to activate viral transcription in HIV-1 latency cell lines in a positive transcription elongation factor b (P-TEFb)-dependent manner. Little is known about the efficacy of activating HIV-1 reservoir cells under cART by BETi in vivo. In this study, we seek to test the potential of a BETi (I-BET151) in activating HIV-1 reservoir cells under effective cART in humanized mice in vivo. We discover that I-BET151 efficiently activates HIV-1 transcription in monocytic cells, but not in CD4+T cells, during suppressive cART in vivo. We further reveal that HIV-1 proviruses in monocytic cells are more sensitive to I-BET151 treatment than in T cells in vitro. Finally, we demonstrate that I-BET151-activated viral transcription in monocytic cells is dependent on both CDK2 and CDK9, whereas only CDK9 is involved in activation of HIV-1 by I-BET151 in T cells. Our findings indicate a role of myeloid cells in HIV-1 persistence, and highlights the limitation of measuring or targeting T cell reservoirs alone in terms of HIV-1 cure, as well as provides a potential strategy to reactivate monocytic reservoirs during cART.IMPORTANCEIt has been reported the low level of active P-TEFb critically contributes to the maintenance of HIV-1 latency or low-replication in HIV-1 reservoir cells under cART. Bromodomain inhibitors are used to activate HIV-1 replication in vitro but their effect on activation of the HIV-1 resevoirs with cART in vivo is not clear. We found that BETi (I-BET151) treatment reactivated HIV-1 gene expression in humanized mice during suppressive cART. Interestingly, I-BET151 preferentially reactivated HIV-1 gene expression in monocytic cells, but not in CD4 T cells. Furthermore, I-BET151 significantly increased HIV-1 transcription in monocytic cells, but not in latently infected CD4 T cells, via CDK2-dependent mechanisms. Our findings suggest that BETi can preferentially activate monocytic HIV-1 reservoir cells, and a combination of latency reversal agents targeting different cell types and pathways is needed to achieve reactivation of different HIV-1 reservoir cells during cART.

scholarly journals Specific ActivationIn Vivoof HIV-1 by a Bromodomain Inhibitor from Monocytic Cells in Humanized Mice under Antiretroviral Therapy

2019 ◽  
Vol 93 (12) ◽  
Author(s):  
Guangming Li ◽  
Zheng Zhang ◽  
Natalia Reszka-Blanco ◽  
Feng Li ◽  
Liqun Chi ◽  
...  
Keyword(s):  
Gene Expression ◽  
T Cells ◽  
Antiretroviral Therapy ◽  
Cd4 T Cells ◽  
Myeloid Cells ◽  
Humanized Mice ◽  
Monocytic Cells ◽  
Hiv 1

ABSTRACTCombination antiretroviral therapy (cART) effectively suppresses HIV-1 replication and enables HIV‑infected individuals to live long, productive lives. However, the persistence of HIV-1 reservoirs of both T and myeloid cells with latent or low-replicating HIV-1 in patients under cART makes HIV-1 infection an incurable disease. Recent studies have focused on the development of strategies to activate and purge these reservoirs. Bromodomain and extraterminal domain proteins (BETs) are epigenetic readers involved in modulating gene expression. Several bromodomain inhibitors (BETi) are reported to activate viral transcriptionin vitroin HIV-1 latency cell lines in a P-TEFb (CDK9/cyclin T1)-dependent manner. Little is known about BETi efficacy in activating HIV-1 reservoir cells under cARTin vivo. Here we report that a BETi (I-BET151) efficiently activated HIV-1 reservoirs under effective cART in humanized micein vivo. Interestingly, I-BET151 during suppressive cARTin vivoactivated HIV-1 gene expression only in monocytic cells and not in CD4+T cells. We further demonstrate that BETi preferentially enhanced HIV-1 gene expression in monocytic cells rather than in T cells and that whereas CDK9 was involved in activating HIV-1 by I-BET151 in both monocytic and T cells, CDK2 enhanced HIV-1 transcription in monocytic cells but inhibited it in T cells. Our findings reveal a role for CDK2 in differential modulation of HIV-1 gene expression in myeloid cells and in T cells and provide a novel strategy to reactivate monocytic reservoirs with BETi during cART.IMPORTANCEBromodomain inhibitors have been reported to activate HIV-1 transcriptionin vitro, but their effect on activation of HIV-1 reservoirs during cARTin vivois unclear. We found that BETi (I-BET151) treatment reactivated HIV-1 gene expression in humanized mice during suppressive cART. Interestingly, I-BET151 preferentially reactivated HIV-1 gene expression in monocytic cells, but not in CD4 T cells, in cART-treated mice. Furthermore, I-BET151 significantly increased HIV-1 transcription in monocytic cells, but not in HIV-1-infected CD4 T cells, via CDK2-dependent mechanisms. Our findings suggest that BETi can preferentially activate monocytic HIV-1 reservoir cells and that a combination of reservoir activation agents targeting different cell types and pathways is needed to achieve reactivation of different HIV-1 reservoir cells during cART.

scholarly journals TRIM5α Variations Influence Transduction Efficiency With Lentiviral Vectors in Both Human and Rhesus CD34 + Cells In Vitro and In Vivo

2014 ◽  
Vol 22 (2) ◽  
pp. 348-358 ◽  
Author(s):  
Molly E Evans ◽  
Chutima Kumkhaek ◽  
Matthew M Hsieh ◽  
Robert E Donahue ◽  
John F Tisdale ◽  
...  
Keyword(s):  
Lentiviral Vectors ◽  
Transduction Efficiency ◽  
Cd34 Cells

scholarly journals RelA-Associated Inhibitor Blocks Transcription of Human Immunodeficiency Virus Type 1 by Inhibiting NF-κB and Sp1 Actions

2002 ◽  
Vol 76 (16) ◽  
pp. 8019-8030 ◽  
Author(s):  
Norio Takada ◽  
Takaomi Sanda ◽  
Hiroshi Okamoto ◽  
Jian-Ping Yang ◽  
Kaori Asamitsu ◽  
...  
Keyword(s):  
Gene Expression ◽  
Human Immunodeficiency Virus ◽  
Dna Binding ◽  
Human Immunodeficiency Virus Type ◽  
Virus Type ◽  
Immunodeficiency Virus ◽  
Hiv 1

ABSTRACT RelA-associated inhibitor (RAI) is an inhibitor of nuclear factor κB (NF-κB) newly identified by yeast two-hybrid screen as an interacting protein of the p65 (RelA) subunit. In this study, we attempted to examine the effect of RAI on transcription and replication of human immunodeficiency virus type 1 (HIV-1). We found that RAI inhibited gene expression from the HIV-1 long terminal repeat (LTR) even at the basal level. Upon in vitro DNA-binding reactions, RAI could directly block the DNA-binding of p65 subunit of NF-κB but not that of the p50 subunit or AP1. We found that RAI could also inhibit the DNA-binding of Sp1 and thus inhibit the basal HIV-1 promoter activity. We further examined the effects of RAI on Sp1 and found that RAI colocalizes with Sp1 in the nucleus and interacts with Sp1 in vitro and in vivo. Moreover, we found that RAI efficiently blocked the HIV-1 replication when cotransfected with a full-length HIV-1 clone. These findings indicate that RAI acts as an efficient inhibitor of HIV-1 gene expression in which both NF-κB and Sp1 play major roles.

scholarly journals Multispecific anti-HIV duoCAR-T cells display broad in vitro antiviral activity and potent in vivo elimination of HIV-infected cells in a humanized mouse model

2019 ◽  
Vol 11 (504) ◽  
pp. eaav5685 ◽  
Author(s):  
Kim Anthony-Gonda ◽  
Ariola Bardhi ◽  
Alex Ray ◽  
Nina Flerin ◽  
Mengyan Li ◽  
...  
Keyword(s):  
T Cells ◽  
Mouse Model ◽  
Hiv Infection ◽  
Lentiviral Vectors ◽  
Car T ◽  
Anti Hiv ◽  
Hiv 1

Adoptive immunotherapy using chimeric antigen receptor–modified T cells (CAR-T) has made substantial contributions to the treatment of certain B cell malignancies. Such treatment modalities could potentially obviate the need for long-term antiretroviral drug therapy in HIV/AIDS. Here, we report the development of HIV-1–based lentiviral vectors that encode CARs targeting multiple highly conserved sites on the HIV-1 envelope glycoprotein using a two-molecule CAR architecture, termed duoCAR. We show that transduction with lentiviral vectors encoding multispecific anti-HIV duoCARs confer primary T cells with the capacity to potently reduce cellular HIV infection by up to 99% in vitro and >97% in vivo. T cells are the targets of HIV infection, but the transduced T cells are protected from genetically diverse HIV-1 strains. The CAR-T cells also potently eliminated PBMCs infected with broadly neutralizing antibody-resistant HIV strains, including VRC01/3BNC117-resistant HIV-1. Furthermore, multispecific anti-HIV duoCAR-T cells demonstrated long-term control of HIV infection in vivo and prevented the loss of CD4+T cells during HIV infection using a humanized NSG mouse model of intrasplenic HIV infection. These data suggest that multispecific anti-HIV duoCAR-T cells could be an effective approach for the treatment of patients with HIV-1 infection.

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