Survival of the fittest: positive selection of CD4+ T cells expressing a membrane-bound fusion inhibitor following HIV-1 infection

Abstract

Although a variety of genetic strategies have been developed to inhibit HIV replication, few direct comparisons of the efficacy of these inhibitors have been carried out. Moreover, most studies have not examined whether genetic inhibitors are able to induce a survival advantage that results in an expansion of genetically-modified cells following HIV infection. We evaluated the efficacy of three leading genetic strategies to inhibit HIV replication: 1) an HIV-1 tat/rev-specific small hairpin (sh) RNA; 2) an RNA antisense gene specific for the HIV-1 envelope; and 3) a viral entry inhibitor, maC46. In stably transduced cell lines selected such that >95% of cells expressed the genetic inhibitor, the RNA antisense envelope and viral entry inhibitor maC46 provided the strongest inhibition of HIV-1 replication. However, when mixed populations of transduced and untransduced cells were challenged with HIV-1, the maC46 fusion inhibitor resulted in highly efficient positive selection of transduced cells, an effect that was evident even in mixed populations containing as few as 1% maC46-expressing cells. The selective advantage of the maC46 fusion inhibitor was also observed in HIV-1-infected cultures of primary T lymphocytes as well as in HIV-1-infected humanized mice. These results demonstrate robust inhibition of HIV replication with the fusion inhibitor maC46 and the antisense Env inhibitor, and importantly, a survival advantage of cells expressing the maC46 fusion inhibitor both in vitro and in vivo. Evaluation of the ability of genetic inhibitors of HIV-1 replication to confer a survival advantage on genetically-modified cells provides unique information not provided by standard techniques that may be important in the in vivo efficacy of these genes.

Figure 1. Schematic diagrams of the viral transfer plasmids.

The lentiviral vectors HIV-shI-GFP, VRX494, M589, HJ57, and M420, are based on HIV-1_NL4-3 or HIV-1_HXB2. The viral inhibitors encoded by each of the experimental vectors are noted in parentheses. Where indicated, vectors use a heterologous CMV promoter to initiate transcription of the genomic RNA with a self-inactivating 3′ LTR. The VRX494 vector uses a functional HIV-1 LTR, which is upregulated after viral infection . All vectors contain cis-acting regulatory domains (the ψ packaging signal, the central polypurine tract [cppt], the Rev response element [RRE], and, in some cases, the Woodchuck post-transcriptional regulatory element [wPRE]), and eGFP. The vector HIV-shI-GFP contains the U6 promoter regulating a shRNA targeting exon 1 of HIV-1 tat and rev (shI) . The lentiviral vector VRX494 contains 937 bp of antisense (AS) HIV-1 envelope, and eGFP transcriptionally regulated by the HIV-1 LTR . The vector M589 contains an internal SFFV promoter regulating expression of the C46 heptad repeat-anchored with a linker and transmembrane domain:GFP fusion protein (maC46:GFP). The control vectors HJ57 and M420 do not contain an inhibitor cassette.

Figure 2. Potent inhibition of HIV-1 replication by the maC46 fusion inhibitor and antisense-Env VRX494 vectors.

Non-transduced CEMx174 cells (NT) and CEMx174 cells transduced with vectors expressing the indicated inhibitor gene or a control vector (HJ57, GFP) were infected with HIV-1_NL4-3 at MOIs of (A) 10⁻⁴, (B) 10⁻³, and (C) 10⁻² TCID₅₀ per cell. Viral replication was assessed by measuring HIV-1 Gag p24 antigen in cell-free culture supernatants. The minimum level of detection for HIV-1 Gag p24 was 15 pg/ml. The data are representative of at least two experiments.

Figure 3. Preferential survival of cells expressing the maC46 fusion inhibitor following HIV-1 infection.

A. Flow cytometric analysis for expression of GFP and HIV-1 p24 Gag at the indicated times post-infection. Non-transduced CEMx174 cells were mixed with transduced cells expressing the maC46 inhibitors such that approximately 25–30% of the cells were GFP⁺. Mixtures of cells were then infected with HIV-1_NL4-3 at a MOI of 10⁻³ TCID₅₀ per cell. Expression of HIV-1 p24 Gag and GFP were determined by flow cytometry on days 5, 9, 14 and 21. B. Survival advantage of cells expressing maC46 following HIV-1 infection. Mixtures of non-transduced CEMx174 cells and transduced cells expressing the shI, maC46, and antisense env inhibitors, or transduced with the HJ57 control vector (GFP) were generated such that approximately 25–30% of the cells were GFP⁺. Cells were then infected with HIV-1_NL4-3 at MOI of 10⁻³ TCID₅₀ per cell or remained uninfected. The percentage of GFP⁺ cells in the absence of HIV-1 infection was stable for each vector examined over the 21 day culture period (data not shown). C. Mixtures of non-transduced CEMx174 cells and cells transduced with vectors expressing either the sh(tat/rev), antisense env (VRX494), or the maC46 fusion inhibitors, were generated at the ratios indicated at time 0 and then infected with HIV-1_NL4-3 at an MOI of 10⁻³ TCID₅₀/cell. Initial mixtures of vector-transduced CEMx174 cells ranged from 1% up to 95% GFP⁺ cells. The percentage of GFP⁺ cells was determined by flow cytometry at the indicated times post-infection.

Figure 4. Adsorption of HIV-1 virions to the surface of transduced cells expressing maC46.

A. Localization of virus in PM-1 cells transduced with M589, which expresses maC46 and GFP, was analyzed by confocal microscopy. Mixtures of maC46-GFP transduced and non-transduced cells (approximately 10% GFP⁺) were infected with HIV-1_D117/II. After 10 days, cells were stained for HIV-1 p24 (red) and analyzed for expression of GFP (green). maC46-GFP⁺ cells show p24 signal only on the cell membrane, whereas non-transduced cells have a cytoplasmic p24 staining. B. A3-CCR5 or A3-CCR5-maC46 cells were incubated with HIV-pseudotyped viral particles. After 5 hours cells were fixed, stained for p24, and analyzed for the presence of bound viral particles. C. A3-CCR5 cells expressing maC46 were analyzed for their potential to transfer replication-incompetent lentiviral particles pseudotyped with an HIV envelope to PM-1 cells. A3-CCR5 or A3-CCR5-maC46 cells were incubated with the viral particles for 5 hours and after washing, transferred to PM-1 cells expressing RFP at a final ratio of 1∶1. GFP expression of PM-1 cells was analyzed by flow cytometry after 3 days.

Figure 5. In vitro selection of maC46-expressing primary human T cells.

Human CD4⁺ T cells were transduced with either a control lentiviral vector expressing GFP alone (M420) or a vector expressing maC46 and GFP. On day 5 after transduction, cells were diluted with non-transduced cells to achieve 10% GFP⁺ cells, and the mixtures were infected with HIV-1_D117/II. Data are presented as the mean ± the standard error of the mean (SEM) of triplicate samples. A. Flow cytometric analysis of the percentage of CD4⁺ T cells as a percentage of all viable cells. Infected cultures (closed symbols) showed a sustained loss of CD4⁺ T cells. B. Preferential survival of maC46-expressing CD4⁺ T cells following HIV-1 infection. C. Increased expression of GFP on PM-1 cells transduced with the maC46-GFP vector (M589) after HIV-1-infection. The geometric mean of GFP expression (Geo mean) on human CD4⁺ T cells was analyzed at the indicated time points.

Figure 6. Survival advantage of maC46-expressing human CD4⁺ T cells in HIV-1-infected humanized mice.

Human T cells were transduced with a control vector (M420) expressing GFP alone (squares) or the M589 vector expressing maC46-GFP (circles) and then infected with the HIV strain D117/II. Infected and uninfected cells were injected intraperitoneally into adult NOG mice. Four infected and 5 uninfected mice were followed for 5 weeks for each vector. A. Relative numbers of human CD4⁺ T cells. The percentage of human CD4⁺ T cells as a fraction of total viable cells was calculated. Means and the SEM for each group are shown. B. HIV infection leads to a selective survival of maC46-GFP expressing human CD4⁺ T cells (red closed circles) in vivo. Means and the SEM for each group are shown. C. Viral load in the serum of individual infected mice is not altered by expression of maC46-GFP. Differences in viral loads between mice receiving CD4⁺ T cells transduced with the maC46-expressing vector and the GFP control vector were not significant (Mann-Whitney test).