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. 2013 Jul 11;122(2):179-87.
doi: 10.1182/blood-2013-01-482224. Epub 2013 May 29.

Positive selection of mC46-expressing CD4+ T cells and maintenance of virus specific immunity in a primate AIDS model

Patrick M Younan  1 , Patricia Polacino, John P Kowalski, Christopher W Peterson, Nicholas J Maurice, Nathaniel P Williams, On Ho, Grant D Trobridge, Dorothee Von Laer, Martin Prlic, Brian C Beard, Stephen DeRosa, Shiu-Lok Hu, Hans-Peter Kiem
Affiliations

Positive selection of mC46-expressing CD4+ T cells and maintenance of virus specific immunity in a primate AIDS model

Patrick M Younan et al. Blood. .

Abstract

Despite continued progress in the development of novel antiretroviral therapies, it has become increasingly evident that drug-based treatments will not lead to a functional or sterilizing cure for HIV(+) patients. In 2009, an HIV(+) patient was effectively cured of HIV following allogeneic transplantation of hematopoietic stem cells (HSCs) from a CCR5(-/-) donor. The utility of this approach, however, is severely limited because of the difficulty in finding matched donors. Hence, we studied the potential of HIV-resistant stem cells in the autologous setting in a nonhuman primate AIDS model and incorporated a fusion inhibitor (mC46) as the means for developing infection-resistant cells. Pigtail macaques underwent identical transplants and Simian-Human Immunodeficiency Virus (SHIV) challenge procedures with the only variation between control and mC46 macaques being the inclusion of a fusion-inhibitor expression cassette. Following SHIV challenge, mC46 macaques, but not control macaques, showed a positive selection of gene-modified CD4(+) T cells in peripheral blood, gastrointestinal tract, and lymph nodes, accounting for >90% of the total CD4(+) T-cell population. mC46 macaques also maintained high frequencies of SHIV-specific, gene-modified CD4(+) T cells, an increase in nonmodified CD4(+) T cells, enhanced cytotoxic T lymphocyte function, and antibody responses. These data suggest that HSC protection may be a potential alternative to conventional antiretroviral therapy in patients with HIV/AIDS.

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Figures

Figure 1
Figure 1
Protection of CD4+ T cells and reduced viremia in SHIV-infected macaques previously engrafted with mC46-expressing HSCs. (A) Absolute number of CD4+CD3+ T cells/μl peripheral blood of SHIV-infected macaques engrafted with control (squares and diamonds) or mC46-expressing (circles and triangles) CD34+ HSCs, as determined by FACS analysis. The lower limit of the normal CD4+ T-cell range is indicated by the dotted line. (B) Levels of SHIV RNA detected in peripheral blood of macaques previously engrafted with control (squares) or mC46-expressing (circles) CD34+ HSCs. Macaques were challenged with a viral dose equivalent to 10–50 TCID50. The limit of quantification of the viral load real-time polymerase chain reaction assay was 102 viral RNA copies/mL.
Figure 2
Figure 2
Positive selection of gene-modified CD4+ T cells following SHIV challenge. (A) The percentage of gene-modified CD4+ T cells in peripheral blood was determined by FACS analysis based on GFP expression in the control (squares and diamonds) and mC46 macaques (circles and triangles). (B) The absolute number of CD4+CD3+ (circles and triangles) and total CD4+CD3+ GFP+ T cells (squares and diamonds) was determined by FACS analysis for control and mC46 macaque set 1. The area between the respective lines of each macaque represents nonmodified CD4+ T cells. As indicated, 70% of CD4+ T cells at day 140 post-SHIV challenge are nonmodified. (C-D) FACS plots demonstrating the relative percentage of total CD4+CD3+ T cells and CD4+CD3+GFP+ T cells in mixed cell populations isolated from (C) duodenal biopsies and (D) LN biopsies before and 6 months after SHIV challenge. See supplemental Figure 2 for additional analysis at days 7 and 21 following SHIV challenge and averages of both sets of macaques.
Figure 3
Figure 3
Improved ratio of CD4+ to CD8+ T cells in mC46 macaques. (A) The ratio of CD4+CD3+ T cells over CD8+CD3+ T cells was determined by FACS analysis throughout the course of these studies. Results shown are for both mC46-expressing macaques (circles and triangles) and both control macaques (squares and diamonds). (B-C) The relative percentage of CD4+ T cells and CD8+ T cells in mixed-cell populations isolated from (B) GI and (C) LN samples was determined by FACS analysis. Results for second set are shown before SHIV challenge and after 6 months; averages from both sets are shown in supplemental Figure 2 in addition to time points taken at days 7 and 21 post-SHIV challenge.
Figure 4
Figure 4
SHIV-specific, mC46-expressing CD4+ T cells are maintained in mC46-expressing macaques. (A-B) PBMC isolated from postchallenge macaques were stimulated with SHIV peptide for 6 hours and SHIV-specific cells were identified using intracellular cytokine staining. Boolean gating was used to identify the frequencies of CD4+ (A) and CD8+ (B) cells expressing 1 or more of the following cytokines: IFN-γ, IL-2, and tumor necrosis factor (TNF)-α. To calculate percentages, all negative values after background subtracting were made equal to 0. CD4+ T-cell responders from control (cont.)2 data were omitted because all data failed the count >1000 filter. (C-D) The same Boolean gating data were used to observe the frequencies of SHIV-specific CD4+ and CD8+ cells within the GFP+ and GFP− subsets. Frequencies of GFP+ cells are displayed as green circles; frequencies of GFP− cells are displayed as red squares; data that failed positivity calls are depicted as open symbols. One to 3 samples per macaque was examined per peptide pool depending on viability and total number of gated CD4+ or CD8+ T cells. Cryopreserved samples from 2, 4, and 6 months after SHIV challenge were used for these experiments.
Figure 5
Figure 5
mC46-fusion inhibitor enhances B-lymphocyte responses against HIV/SIV antigens. (A) Western blot analysis was used to visualize the production of anti-SHIV antibodies in the control (left) and mC46-macaque (right) using premade strips from SIVmac239 lysates. Serum samples and control strips are as follows: lanes 1-7, serum samples from weeks 0, 2, 4, 6, 8, 10, and 12; lane 8, SIVmac239-positive control; lane 9, negative control; lane 10, SHIV positive control. (B) ELISA was used to determine the antibody titer against whole SIV and HIV Env (mC46 macaques: circles and triangle; control [cont.] macaques: squares and diamonds). (C-D) Diluted serum samples collected at 1, 3, and 6 months after SHIV challenge were incubated with VSV-G or HIV89.6 env pseudotyped lentiviral vectors encoding GFP for 1.5 hours at 37°C before addition to TZMbl cells. FACS analysis was performed 48 hours following transduction. Antibody titer and neutralization assays were performed in duplicate for each serum sample. (C-D) Results shown are averages observed for controls and mC46 macaques (C: P < .0137; D: P < .0035). Two-tailed t test was used for statistical analysis between each subset (P < .05 is considered significant). Western blot analysis for the second set of macaques is shown in supplemental Figure 7.
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