Evaluating the Safety and Efficacy of Anti-HIV Transgenic Cells in a Humanized Mouse Model (NOD/SCIDγc−/−) for HIV Stem Cell Gene Therapy.

Blood ◽  
2008 ◽  
Vol 112 (11) ◽  
pp. 2352-2352
Author(s):  
Joseph S. Anderson ◽  
Jon Walker ◽  
Louisa Wirthlin ◽  
John Javien ◽  
Jan A. Nolta ◽  
...  
Keyword(s):  
T Cells ◽  
Immune System ◽  
B Cells ◽  
Mouse Model ◽  
Lentiviral Vector ◽  
Human Cd34 ◽  
Cell Engraftment ◽  
Anti Hiv ◽  
Hiv 1

Abstract In vivo mouse models for safety and efficacy studies of anti-HIV gene therapeutics have been lacking in their ability to evaluate normal systemic hematopoiesis with the development of a functional immune system. These deficiencies in modeling these normal and essential physiologies can now be generated in the recently developed NOD/SCIDγc−/− mouse model. Human CD34+ progenitor cells after intrahepatic injection will undergo multi-organ engraftment generating a functional thymus along with the production of T cells, B cells, and macrophages. Upon injection with various strains of HIV-1, engrafted mice display a disseminated infection due to viral replication in human T cells and macrophages. Infected mice acquire characteristics of increased viremia over time with human CD4+ T cell depletion. Anti-HIV immune responses can also be generated in mice with high viremia. Here we demonstrate the successful engraftment of NOD/SCIDγc−/− mice after injection with human CD34+ progenitor cells isolated from umbilical cord blood. Human cell engraftment was observed in both primary and secondary lymphoid organs including the bone marrow (B cells and macrophages), thymus (T cells), spleen (T cells and B cells), lymph nodes (T cells and B cells), and peripheral blood (T cells and B cells). Human CD34+ cells were found in the liver concluding either organ retention from the initial injection or circulation in the peripheral blood. After displaying a high level of engraftment (>50%), we are now able to proceed with evaluating a number of anti- HIV gene therapeutic constructs for pre-clinical testing. These lentiviral vector constructs include a CCR5 shRNA which has been previously shown to knock down complete expression of CCR5, a human/rhesus macaque chimeric isoform of TRIM5α capable of potently inhibiting HIV-1 infection at the pre-integration step, and combination constructs containing multiple anti-HIV genes. The NOD/SCIDγc−/− mouse model will allow us to evaluate both the safety of these various anti-HIV lentiviral constructs in generating normal functioning transgenic immune system cells and also the efficacy of these anti- HIV genes in inhibiting HIV-1 infection in vivo. With the ability to facilitate multilineage human cell engraftment with the susceptibility for HIV infection, this in vivo model allows for the evaluation of anti-HIV gene therapeutic constructs in a stem cell-based setting. Lentiviral vector transgenic cells can now be tested for their capacity for normal immune system reconstitution.

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.

Lenalidomide Promotes The Expansion Of CD8 T Cells With An Effector Memory Phenotype In a Murine Xenograft Model Of Chronic Lymphocytic Leukemia

Blood ◽  
2013 ◽  
Vol 122 (21) ◽  
pp. 119-119
Author(s):  
Rita Simone ◽  
Sonia Marsilio ◽  
Piers E.M. Patten ◽  
Gerardo Ferrer ◽  
Shih-Shih Chen ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
B Cells ◽  
T Lymphocytes ◽  
B Cell ◽  
Xenograft Model ◽  
Lymphocytic Leukemia ◽  
Effector Memory ◽  
Cell Engraftment

Abstract Lenalidomide (Revlimid®), a thalidomide analogue, is an orally administered second generation immunomodulator with anti-angiogenic and anti-neoplastic properties. Initial studies treating patients with chronic lymphocytic leukemia (CLL) suggest that lenalidomide can have considerable efficacy and that its mode of action is mainly indirect, affecting non-malignant cells in the microenvironment, in particular T lymphocytes. Because a recently described xenograft model for CLL has highlighted the importance of CLL-derived, autologous T cells in promoting leukemic B-cell engraftment and growth in vivo, we have studied the influence of lenalidomide on the expansion of CLL B- and T-lymphocytes in this model. After an initial 12 day culture of FACS-isolated CLL-derived T cells with or without anti-CD3/CD28 beads plus IL-2 (30 IU/ml), T lymphocytes were transferred into alymphoid NSG mice via the retro-orbital plexus (day 0). On day 7, CLL cells were delivered retro-orbitally. These recipient animals are referred to as “T + PBMC mice”. Mice that did not receive T cells on day 0 but were given CLL PBMCs at day 7, with or without lenalidomide, served as controls (“PBMC only mice”). Recipient mice received lenalidomide (10mg/kg/day) or vehicle control daily by gavage starting at day 0. All mice were sacrificed at day 28 (28 days after T-cell and 21 days after B-cell transfer), and blood, spleen, and bone marrow were collected. On this material, four analyses were performed: [1] level of human CD45+ cell engraftment; [2] numbers and types of CLL-derived T cells; [3] numbers of CLL B cells; and [4] levels of cytokines reflective of Th1 and Th2 immune responses. There was a clear enhancement in human hematopoietic (CD45+) cell engraftment in those mice exposed to lenalidomide. This was most marked for the PBMC only mice (vehicle: 10.64%; lenalidomide: 38.53%), although it was also evident for T + PBMC mice (vehicle: 55.96%; lenalidomide: 69.65%). T-cell phenotyping was carried out, before and after cell culture and also at sacrifice. Prior to culture, CLL samples contained on average ∼96% CD5+CD19+ cells and ∼3% CD5+CD19- cells; for the latter, ∼67% were CD4+ and ∼33% CD8+. After 12-day culture, these percentages remained largely unchanged. However, the numbers and types of T cells recovered from the spleens at sacrifice were quite different after in vivo exposure to lenalidomide. For the PBMC only, the percentages of CD4+ and CD8+ cells in the spleens differed somewhat based on lenalidomide exposure (CD4: Vehicle 86% vs. Lenalidomide 61%; CD8: Vehicle 10% vs. Lenalidomide 28%). However, this change was dramatic for the T + PBMC mice (CD4: Vehicle 64.1% vs. Lenalidomide 28.9%; CD8: Vehicle 34% vs. Lenalidomide 62%). Furthermore, when the CD8+ cells from these animals were subsetted based on antigen-experience and function, it appeared that lenalidomide exposure had led to the outgrowth of a greater number of effector memory (CD45RO+ CD62L-) than central memory (CD45RO+ CD62L+) T-cells. For CLL-derived B cells, the numbers differed, based not only on lenalidomide exposure but also on prior in vitro activation. Specifically, in PBMC only mice, the addition of lenalidomide led to increased numbers of CLL B cells in the spleen (Vehicle: 7.81% vs. Lenalidomide: 14%). Conversely, in the T + PBMC mice, the numbers of B cells decreased (Vehicle: 2.36% vs. Lenalidomide: 0.34%). An analysis of Th1 and Th2-related cytokines in the plasmas of the mice at sacrifice revealed a fall in IL-4, IL-5, and IL-10 and a marked increase in IFNg, consistent with a Th2 to Th1 transition. The above data suggest that administration of lenalidomide permits greater engraftment of human hematopoietic cells in alymphoid mice. Although this enhancement involves all members of the hematopoietic lineage, T cells, in particular CD8+ effector memory T cells, emerge in excess over time. This CD8 expansion is associated with diminished levels of CLL B cells suggesting that the decrease is due to T-cell mediated cytolysis. In contrast, in the absence of prior T-cell activation, CLL T cells appear to support better CLL B-cell growth. These findings suggest that lenalidomide alters B-cell expansion in vivo depending on the activation and differentiation state of the autologous T-cell compartment. They also implicate the generation of cytolytic T cells as one mechanism whereby lenalidomide leads to clinical improvement in CLL. Disclosures: Allen: Celgene Corporation: Honoraria.

scholarly journals Generation of an HIV-1-Resistant Immune System with CD34+ Hematopoietic Stem Cells Transduced with a Triple-Combination Anti-HIV Lentiviral Vector

2012 ◽  
Vol 86 (10) ◽  
pp. 5719-5729 ◽  
Author(s):  
J. E. Walker ◽  
R. X. Chen ◽  
J. McGee ◽  
C. Nacey ◽  
R. B. Pollard ◽  
...  
Keyword(s):  
Stem Cells ◽  
Immune System ◽  
Hematopoietic Stem Cells ◽  
Lentiviral Vector ◽  
Triple Combination ◽  
Hematopoietic Stem ◽  
Anti Hiv ◽  
Hiv 1

scholarly journals Inhibition of In Vivo HIV Infection in Humanized Mice by Gene Therapy of Human Hematopoietic Stem Cells with a Lentiviral Vector Encoding a Broadly Neutralizing Anti-HIV Antibody

2010 ◽  
Vol 84 (13) ◽  
pp. 6645-6653 ◽  
Author(s):  
Aviva Joseph ◽  
Jian Hua Zheng ◽  
Ken Chen ◽  
Monica Dutta ◽  
Cindy Chen ◽  
...  
Keyword(s):  
Hiv Infection ◽  
Lentiviral Vector ◽  
High Titer ◽  
Hematopoietic Stem ◽  
Specific Antibodies ◽  
Human B Cells ◽  
Fold Reduction ◽  
Anti Hiv ◽  
Hiv 1

ABSTRACT Due to the inherent immune evasion properties of the HIV envelope, broadly neutralizing HIV-specific antibodies capable of suppressing HIV infection are rarely produced by infected individuals. We examined the feasibility of utilizing genetic engineering to circumvent the restricted capacity of individuals to endogenously produce broadly neutralizing HIV-specific antibodies. We constructed a single lentiviral vector that encoded the heavy and light chains of 2G12, a broadly neutralizing anti-HIV human antibody, and that efficiently transduced and directed primary human B cells to secrete 2G12. To evaluate the capacity of this approach to provide protection from in vivo HIV infection, we used the humanized NOD/SCID/γc null mouse model, which becomes populated with human B cells, T cells, and macrophages after transplantation with human hematopoietic stem cells (hu-HSC) and develops in vivo infection after inoculation with HIV. The plasma of the irradiated NOD/SCID/γc null mice transplanted with hu-HSC transduced with the 2G12-encoding lentivirus contained 2G12 antibody, likely secreted by progeny human lymphoid and/or myeloid cells. After intraperitoneal inoculation with high-titer HIV-1JR-CSF, mice engrafted with 2G12-transduced hu-HSC displayed marked inhibition of in vivo HIV infection as manifested by a profound 70-fold reduction in plasma HIV RNA levels and an almost 200-fold reduction in HIV-infected human cell numbers in mouse spleens, compared to control hu-HSC-transplanted NOD/SCID/γc null mice inoculated with equivalent high-titer HIV-1JR-CSF. These results support the potential efficacy of this new gene therapy approach of using lentiviral vectors encoding a mixture of broadly neutralizing HIV antibodies for the treatment of HIV infection, particularly infection with multiple-drug-resistant isolates.

scholarly journals Lentivector Knockdown of CCR5 in Hematopoietic Stem and Progenitor Cells Confers Functional and Persistent HIV-1 Resistance in Humanized Mice

2015 ◽  
Vol 89 (13) ◽  
pp. 6761-6772 ◽  
Author(s):  
Renier Myburgh ◽  
Sandra Ivic ◽  
Michael S. Pepper ◽  
Gustavo Gers-Huber ◽  
Duo Li ◽  
...  
Keyword(s):  
Gene Therapy ◽  
T Cells ◽  
Immune System ◽  
Humanized Mice ◽  
Target Cells ◽  
Hematopoietic Stem ◽  
Stem And Progenitor Cells ◽  
Hiv 1 ◽  
Selection Of

ABSTRACTGene-engineered CD34+hematopoietic stem and progenitor cells (HSPCs) can be used to generate an HIV-1-resistant immune system. However, a certain threshold of transduced HSPCs might be required for transplantation into mice for creating an HIV-resistant immune system. In this study, we combined CCR5 knockdown by a highly efficient microRNA (miRNA) lentivector with pretransplantation selection of transduced HSPCs to obtain a rather pure population of gene engineered CD34+cells. Low-level transduction of HSPCs and subsequent sorting by flow cytometry yielded >70% transduced cells. Mice transplanted with these cells showed functional and persistent resistance to a CCR5-tropic HIV strain: viral load was significantly decreased over months, and human CD4+T cells were preserved. In one mouse, viral mutations, resulting presumably in a CXCR4-tropic strain, overcame HIV resistance. Our results suggest that HSPC-based CCR5 knockdown may lead to efficient control of HIVin vivo. We overcame a major limitation of previous HIV gene therapy in humanized mice in which only a proportion of the cells in chimeric micein vivoare anti-HIV engineered. Our strategy underlines the promising future of gene engineering HIV-resistant CD34+cells that produce a constant supply of HIV-resistant progeny.IMPORTANCEMajor issues in experimental long-termin vivoHIV gene therapy have been (i) low efficacy of cell transduction at the time of transplantation and (ii) transduction resulting in multiple copies of heterologous DNA in target cells. In this study, we demonstrated the efficacy of a transplantation approach with a selection step for transduced cells that allows transplantation of an enriched population of HSPCs expressing a single (low) copy of a CCR5 miRNA. Efficient maintenance of CD4+T cells and a low viral titer resulted only when at least 70% of the HIV target cells were genetically modified. These findings imply that clinical protocols of HIV gene therapy require a selective enrichment of genetically targeted cells because positive selection of modified cells is likely to be insufficient below this threshold. This selection approach may be beneficial not only for HIV patients but also for other patients requiring transplantation of genetically modified cells.

scholarly journals Oncogene-specific T cells fail to eradicate lymphoma-initiating B cells in mice

Blood ◽  
2018 ◽  
Vol 132 (9) ◽  
pp. 924-934
Author(s):  
Dana Hoser ◽  
Christian Schön ◽  
Christoph Loddenkemper ◽  
Philipp Lohneis ◽  
Anja A. Kühl ◽  
...  
Keyword(s):  
T Cells ◽  
Immune System ◽  
B Cells ◽  
Mouse Model ◽  
B Cell ◽  
Cell Lymphoma ◽  
B Cell Lymphoma ◽  
Key Points

Key PointsAbsence of oncogene-specific T cells leads to increased B-cell lymphoma incidence in a new mouse model. Premalignant lymphoma-initiating B cells are not eradicated by the immune system, retaining the risk of lymphoma development.

Human CLL Intraclonal Fractions Differ in Their Abilities to Respond to, Elicit, and Suppress Pro-Engraftment and Growth Signals From Autologous T Cells in a Murine Adoptive Transfer Model

Blood ◽  
2012 ◽  
Vol 120 (21) ◽  
pp. 316-316
Author(s):  
Shih-Shih Chen ◽  
Piers E.M. Patten ◽  
Rita Simone ◽  
Sonia Marsilio ◽  
Jacqueline C. Barrientos ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
B Cells ◽  
Lymphoid Tissues ◽  
Transfer Model ◽  
Activated T Cells ◽  
Cell Engraftment

Abstract Abstract 316 Chronic lymphocytic leukemia (CLL) clones contain activated/proliferative leukemic cells in lymphoid tissues and resting cells in the periphery. Different subsets of CLL cells have distinct proliferation rates. Recently divided “proliferative” cells have a surface membrane phenotype of CXCR4DIMCD5BRIGHT (CXCR4DIM) and contain higher numbers of CD38+ and Ki-67+ cells. Circulating “resting” CLL cells express CXCR4BRIGHTCD5DIM (CXCR4BR) and genetic signatures of older, quiescent cells that need to home to lymphoid tissues or die. CXCR4DIM and CXR4BR subsets are relatively minor (1–10% of total) components of CLL clones, with the major fraction (≥90%) of CLL cells having intermediate levels of CXCR4 and CD5 (CXCR4INT). Based on these differences, we proposed a model of transitioning CXCR4DIM → CXCR4INT → CXCR4BR CLL cells in the blood. Because higher birth rates correlate with more aggressive disease, and transiting back to solid tissues permits clonal survival and re-activation, this model suggests CXCR4DIM and CXCR4BR subsets as therapeutic targets. Aiming to further understand functional differences in CLL subsets in vitro and in vivo, we found that CLL subsets differ in cell size (CXCR4DIM>CXCR4INT>CXCR4BR), in vivo apoptosis and transmigration in vitro (both CXCR4DIM< CXCR4INT< CXCR4BR). Thus, while more CXCR4BR cells undergo apoptosis, CXCR4BR cells can migrate better to tissues to receive survival signals. In vivo functional differences were then studied in a NOD/SCID/γcnull (NSG) mouse model using pre-activated CLL-derived autologous T cells. Primary CLL blood cells from 1 M-CLL and 2 U-CLL patients were sorted for CXCR4BR, CXCR4INT or CXCR4DIM fractions. Each fraction (5×106 cells) was injected into NSG mice with 5×105 CD3/28-activated autologous T cells. At weeks 2–6 post transfer, blood analyses showed more extensive expansion of CLL B and T cells in mice received CXCR4DIM than in those injected with CXCR4BR or CXCR4INT. At weeks 9–12, mice were sacrificed. Although T cells dominated in blood, spleen and bone marrow of all recipients, a larger fraction of CLL B cells existed in CXCR4BR injected mice, suggesting better long-term CLL cell engraftment capacity of this fraction. Because regulation of T cells plays key roles in CLL cell survival/growth in patients and in the NSG adoptive transfer model, we next analyzed the same fractions for their abilities to activate T cells and elicit help for engraftment and growth. Unactivated CD5+ T cells (1–1.5×105) and B-CLL fractions (3–5×106 cells) were sorted from 6 patient samples (3 U-CLL and 3 M-CLL), injected into mice and followed bi-weekly until week 6. In 5 cases, except one with few CXCR4BR and CXCR4DIM cells, CXCR4DIM injected mice had more extensive T cell growth starting from week 2. Mice injected with CXCR4BR from 2 U-CLL cases also showed T cell expansions, but at comparatively lesser levels and at later time points (from week 4–5). At week 6, CLL B cells were found in spleen and bone marrow in mice with activated T cells; the numbers of CLL B cells correlated with T cell numbers. Also, identical CXCR4 levels were found in CLL cells regardless of origination from CXCR4BR or CXCR4DIM. Notably, no human B or T cells were detected in CXCR4INT injected mice. In fact, adding CXCR4INT cells to CXCR4DIM mice suppressed CXCR4DIM induced T cell expansion and cytokine production. Specifically, mice receiving both CXCR4DIM and CXCR4INT cells had diminished T cell expansion and at least 3 fold reduced serum levels of IFNγ and IL5. Overall, our data confirm the need for activated T cells for CLL B cell growth in mice; suggest superior long term CLL B cell engraftment by CXCR4BR cells with activated T cell support, and identify a greater ability of CXCR4DIM cells to activate autologous T cells, although some U-CLL CXCR4BR cells could do so to a lesser degree. Superior activation of T cells by CXCR4DIM B cells may be due to higher numbers of CD23+, CD25+, CD27+, CD29+ and CD44+ cells in CXCR4DIM fraction that facilitate cellular interactions. Finally, unlike CXCR4BR and CXCR4DIM cells, the major fraction in patient blood, CXCR4INT, inhibited T cell activation. These results indicate previously unappreciated levels of intraclonal CLL cell heterogeneity that may have important clinical relevance, allow more precise biologic analyses, and provide a rationale for preferential therapeutic targeting of these fractions. Disclosures: No relevant conflicts of interest to declare.

scholarly journals Anti-idiotypic antibodies elicit anti-HIV-1–specific B cell responses

2019 ◽  
Vol 216 (10) ◽  
pp. 2316-2330 ◽  
Author(s):  
Pia Dosenovic ◽  
Anna-Klara Pettersson ◽  
Abigail Wall ◽  
Eddy S. Thientosapol ◽  
Junli Feng ◽  
...  
Keyword(s):  
B Cells ◽  
B Cell ◽  
Neutralizing Antibodies ◽  
Target Cells ◽  
Human B Cells ◽  
Cd4 Binding Site ◽  
Idiotypic Antibody ◽  
Anti Hiv ◽  
Hiv 1

Human anti-HIV-1 broadly neutralizing antibodies (bNAbs) protect against infection in animal models. However, bNAbs have not been elicited by vaccination in diverse wild-type animals or humans, in part because B cells expressing the precursors of these antibodies do not recognize most HIV-1 envelopes (Envs). Immunogens have been designed that activate these B cell precursors in vivo, but they also activate competing off-target responses. Here we report on a complementary approach to expand specific B cells using an anti-idiotypic antibody, iv8, that selects for naive human B cells expressing immunoglobulin light chains with 5–amino acid complementarity determining region 3s, a key feature of anti-CD4 binding site (CD4bs)–specific VRC01-class antibodies. In mice, iv8 induced target cells to expand and mature in the context of a polyclonal immune system and produced serologic responses targeting the CD4bs on Env. In summary, the results demonstrate that an anti-idiotypic antibody can specifically recognize and expand rare B cells that express VRC01-class antibodies against HIV-1.

Sign in / Sign up

Export Citation Format

Share Document