Integrase Defective Lentiviral Vector Promoter Impacts Transgene Expression in Target Cells and Magnitude of Vector-Induced Immune Responses

Abstract

Integrase defective lentiviral vectors (IDLVs) are a promising vaccine delivery platform given their ability to induce high magnitude and durable antigen-specific immune responses. IDLVs based on the simian immunodeficiency virus (SIV) are significantly more efficient at transducing human and simian dendritic cells (DCs) compared to HIV-based vectors, resulting in a higher expansion of antigen-specific CD8+ T cells. Additionally, IDLV persistence and continuous antigen expression in muscle cells at the injection site contributes to the durability of the vaccine-induced immune responses. Here, to further optimize transgene expression levels in both DCs and muscle cells, we generated ten novel lentiviral vectors (LVs) expressing green fluorescent protein (GFP) under different hybrid promoters. Our data show that three of the tested hybrid promoters resulted in the highest transgene expression levels in mouse DCs, monkey DCs and monkey muscle cells. We then used the three LVs with the highest in vitro transgene expression levels to immunize BALB/c mice and observed high magnitude T cell responses at 3 months post-prime. Our study demonstrates that the choice of the vector promoter influences antigen expression levels in target cells and the ensuing magnitude of T cell responses in vivo.

Keywords: antigen expression; dendritic cells; immune responses; lentiviral vectors; muscle cells; vector promoter.

Figure 1

Impact of vector promoter on transgene expression in 293T Lenti-X cells over time. 293T Lenti-X cells were transduced with a multiplicity of infection (MOI) of 1 using the LVs expressing GFP under the indicated promoters. (a) Results of time-course flow cytometric analyses performed at the indicated time points to compare the mean fluorescence intensity (MFI) among the different LVs. (b) MFI normalized by the percentage of transduced cells expressing GFP, to account for differences in transduction efficiency. Histograms show the means of three replicates. Error bars indicate standard error mean (S.E.M).

Figure 2

Impact of vector promoter on transgene expression in mouse-bone-marrow-derived dendritic cells (BMDCs). Mouse-BMDCs were transduced with a MOI of 2 using the LVs expressing GFP under the indicated promoters. (a) Results of time-course flow cytometric analyses performed at Days 3 and 7 post-transduction, to compare MFIs among the different LVs. (b) MFI normalized by the percentage of transduced cells expressing GFP. Histograms show the means of two replicates. Error bars indicate S.E.M.

Figure 3

Impact of vector promoter on transgene expression in monkey-monocyte-derived dendritic cells. Monkey-monocyte-derived DCs were transduced with an MOI of 4 using the LVs expressing GFP under the indicated promoters. (a) Results of time-course flow cytometric analyses performed at the indicated time points to compare MFIs among the different LVs. (b) MFI normalized for the percentage of transduced cells expressing GFP. Histograms represent means of two replicates. Error bars indicate S.E.M.

Figure 4

Impact of vector promoter on transgene expression in human-monocyte-derived dendritic cells (MDDCs). Human MDDCs were transduced with an MOI of 4 using the LVs expressing GFP under the indicated promoters. (a) Results of time-course flow cytometric analyses performed at the indicated time points to compare MFIs among the different LVs. (b) MFI normalized for the percentage of transduced cells expressing GFP. Histograms represent the means of two replicates. Error bars indicate S.E.M.

Figure 5

Impact of vector promoter on transgene expression in cynomolgus macaque skeletal muscle cells. Monkey skeletal muscle cells were transduced with an MOI of 0.5 using the LVs expressing GFP under the indicated promoters. (a) Results of time-course flow cytometric analyses performed at the indicated time points to compare mean fluorescence intensity (MFI) among the different LVs. (b) MFI normalized by the percentage of transduced cells expressing GFP. Histograms show the means of two replicates. Error bars indicate S.E.M. (c) Fluorescence microscopy images of monkey muscle cells transduced with the indicated LVs at 7 days post-transduction.

Figure 6

Magnitude of T cell responses in mice immunized with IDLVs expressing GFP under different promoters. (a) A total of 25 BALB/c mice were immunized intramuscularly with 50 ng RT/mouse corresponding to 5 × 10⁶ transducing units (TU) of the indicated IDLVs or saline. Spleens were harvested 12 weeks post-immunization to measure T cell responses. (b) Magnitudes of GFP-specific T cell responses induced by the indicated IDLVs at 12 weeks post-immunization, as measured by IFN-γ ELISpot. Data are expressed (left panel) as numbers of specific spot-forming cells (SFCs) per million cells and (right panel) as % of GFP-specific SFCs normalized per ConA-induced SFCs, to account for potential differences in T cell responsiveness to stimuli among samples. Background responses in unstimulated wells (medium only) were subtracted. GFP = green fluorescent protein; ConA = concavalin A.