Skip to main page content
U.S. flag

An official website of the United States government

Dot gov

The .gov means it’s official.
Federal government websites often end in .gov or .mil. Before sharing sensitive information, make sure you’re on a federal government site.

Https

The site is secure.
The https:// ensures that you are connecting to the official website and that any information you provide is encrypted and transmitted securely.

Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
. 2015 May;23(5):943-951.
doi: 10.1038/mt.2015.19. Epub 2015 Feb 4.

Lentivirus-mediated Gene Transfer in Hematopoietic Stem Cells Is Impaired in SHIV-infected, ART-treated Nonhuman Primates

Patrick M Younan  1 , Christopher W Peterson  1 , Patricia Polacino  2 , John P Kowalski  1 , Willimark Obenza  1 , Hannah W Miller  3 , Brian P Milless  1 , Phil Gafken  1 , Stephen C DeRosa  3 , Shiu-Lok Hu  4 , Hans-Peter Kiem  5
Affiliations

Lentivirus-mediated Gene Transfer in Hematopoietic Stem Cells Is Impaired in SHIV-infected, ART-treated Nonhuman Primates

Patrick M Younan et al. Mol Ther. 2015 May.

Abstract

Recent studies have demonstrated that genetically modified hematopoietic stem cells (HSCs) can reduce HIV viremia. We have developed an HIV/AIDS-patient model in Simian/human immunodeficiency virus (SHIV)-infected pigtailed macaques that are stably suppressed on antiretroviral therapy (ART: raltegravir, emtricitabine and tenofovir). Following SHIV infection and ART, animals undergo autologous HSC transplantation (HSCT) with lentivirally transduced cluster of differentiation (CD)34(+) cells expressing the mC46 anti-HIV fusion protein. We show that SHIV(+), ART-treated animals had very low gene marking levels after HSCT. Pretransduction CD34(+) cells contained detectable levels of all three ART drugs, likely contributing to the low gene transfer efficiency. Following HSCT recovery and the cessation of ART, plasma viremia rebounded, indicating that myeloablative total body irradiation cannot completely eliminate viral reservoirs after autologous HSCT. The kinetics of recovery following autologous HSCT in SHIV(+), ART-treated macaques paralleled those observed following transplantation of control animals. However, T-cell subset analyses demonstrated a high percentage of C-C chemokine receptor 5 (CCR5)-expressing CD4(+) T-cells after HSCT. These data suggest that an extended ART interruption time may be required for more efficient lentiviral transduction. To avoid complications associated with ART interruption in the context of high percentages of CD4(+)CCR5(+)T-cells after HSCT, the use of vector systems not impaired by the presence of residual ART may also be beneficial.

PubMed Disclaimer

Figures

Figure 1
Figure 1
Effects of irradiation on CD4+ T-cells and plasma viremia. (a) Schematic outline of studies. Following baseline sample collections and 8 weeks following SHIV1157ipd3N4 challenge (“Acute phase”), macaques were placed on daily ART consisting of TFV, FTC and twice daily RAL (see Materials and Methods). Hematopoietic stem cell transplant (“HSC Tx”) was performed after stable suppression of plasma viremia was achieved. Following transplant recovery and cessation of ART, macaques were monitored for a period of ~6 months. A study time line for SHIV-naive control animals that underwent HSCT is also displayed. GI and lymph node biopsies (“B”) were collected at the indicated time points. (b) Total CD4+CD3+ T-cell levels were assessed by flow cytometry at the indicated time points over the course of these studies following SHIV1157ipd3N4 challenge and prior to and following HSCT. (c) Plasma viremia and (d) proviral DNA contents were determined by real-time PCR as indicated in the Methods section. Arrows indicate HSCT and dotted line indicates end of ART.
Figure 2
Figure 2
Phenotypic analysis of peripheral T-lymphocytes following SHIV-challenge. (a) The percentage of CD4+, CD4+CD8+, and CD8+ T-cells expressing CCR5+ was determined by flow cytometry throughout the course of these studies. (b–d) The percentage of central memory (TCM; CCR7+CD45RA–), effector memory (TEM; CCR7–CD45RA–), and naive cells (CCR7+CD45RA+) was determined in (b) CD4+CD3+, (c) CD8+CD3+, and (d) CD4+CD8+CD3+ T-cells.
Figure 3
Figure 3
T-lymphocyte subset analysis in lymph node and GI biopsies. Single-cell suspensions were analyzed by flow cytometry to determine the percentage of CD4+ T-cells, CD8+ T-cells, double positive T-cells, and CD4+CCR5+ T-cells in (a) lymph nodes and (b) GI biopsies.
Figure 4
Figure 4
CD34+ HSCs from cART-treated animals display decreased transgene expression in vivo. (a) CD34+ HSCs isolated from four SHIV- and cART-naive macaques, and from six SHIV+, cART-treated animals (Cohorts 1–3, Figure 1) were transduced with lentiviral vectors expressing mC46 and GFP (circles) or GFP alone (triangles). The percentage of GFP+ HSCs was determined following 10 days in liquid culture. (b) Bulk transduced cells from (a) were infused into the autologous recipient. Following hematopoietic recovery (see Materials and Methods), steady state GFP expression was determined from total leukocytes. Data represent the average of multiple longitudinal blood draws from each animal plus standard deviation. (c–d) Using samples collected from animals in Cohorts 1–3 and prepared immediately prior to lentiviral transduction, the average absolute amounts of antiretroviral drugs in CD34+ HSCs and PBMCs (c) and the ratio of ART in CD34+ HSCs versus PBMCs (d) were determined by MASS-Spec/HPLC. All PBMC samples were collected in conjunction with bone marrow harvests.
Figure 5
Figure 5
ART-mediated inhibition of lenti- and Foamy viral vectors. The effects of (a) Raltegravir, (b) PMPA and (c) FTC on lentiviral and foamy virus-based vectors was assessed. HT1080 cells were cultured in the presence of the indicated concentration of each respective antiretroviral drug. Next, transductions were performed with VSV-G pseudotyped, GFP-expressing lentiviral or foamy viral vectors. Transduction efficiencies were determined by measuring fluorescence 48 hours following transduction. Results are average of duplicate samples. VSV-G, vesicular stomatitis virus glycoprotein.
Figure 6
Figure 6
ART increases lentiviral 2-LTR circle formation in transduced macaque CD34+ cells in vitro. (a–b) CD34+ HSCs were harvested from a SHIV- and ART-naive animal, and were cultured in the absence (“Control”) or presence of ART (“Control + ART”). CD34+ HSCs were also harvested from a SHIV-infected, ART-treated animal (“SHIV+, in vivo ART”) (“SHIV+ART+). Cells were transduced with GFP lentivirus, and assayed by real-time PCR at 2, 4, and 11 days post-transduction for total lentiviral copies and 2-LTR forms of viral DNA (a) using previously described protocols (see Materials and Methods). (b) The ratio of 2-LTR circles to total viral copies was calculated. Results represent average of duplicate samples.
See this image and copyright information in PMC

Similar articles

See all similar articles

Cited by

See all "Cited by" articles

References

    1. Little RF, Dunleavy K. Update on the treatment of HIV-associated hematologic malignancies. Hematology Am Soc Hematol Educ Program. 2013;2013:382–388. - PubMed
    1. Chao C, Xu L, Abrams D, Leyden W, Horberg M, Towner W, et al. Survival of non-Hodgkin lymphoma patients with and without HIV infection in the era of combined antiretroviral therapy. AIDS. 2010;24:1765–1770. - PMC - PubMed
    1. Jacobson CA, Abramson JS. HIV-associated Hodgkin's lymphoma: prognosis and therapy in the era of cART. Adv Hematol. 2012;2012:507257. - PMC - PubMed
    1. Suneja G, Shiels MS, Angulo R, Copeland GE, Gonsalves L, Hakenewerth AM, et al. Cancer treatment disparities in HIV-infected individuals in the United States. J Clin Oncol. 2014;32:2344–2350. - PMC - PubMed
    1. Hütter G, Zaia JA. Allogeneic haematopoietic stem cell transplantation in patients with human immunodeficiency virus: the experiences of more than 25 years. Clin Exp Immunol. 2011;163:284–295. - PMC - PubMed

Publication types

MeSH terms