Pseudotyped single-cycle simian immunodeficiency viruses expressing gamma interferon augment T-cell priming responses in vitro

Abstract

To increase the safety and efficacy of human immunodeficiency virus vaccines, several groups have conducted studies using the macaque model with single-cycle replicating simian immunodeficiency viruses (SIVs). However, these constructs had poor or diminished efficacy compared to live attenuated vaccines. We previously showed that immunization of macaques with live attenuated SIV with a deletion in the nef gene and expressing gamma interferon (IFN-gamma) results in significantly enhanced safety and efficacy. To further enhance safety, we constructed and characterized single-cycle SIVs, pseudotyped with the glycoprotein of vesicular stomatitis virus, expressing different levels of macaque IFN-gamma. Expression of IFN-gamma did not alter the infectivity or antigenicity of pseudotyped SIV. The transduction of dendritic cells (DCs) by IFN-gamma-expressing particles resulted in the up-regulation of costimulatory and major histocompatibility complex molecules. Furthermore, T cells primed with DCs transduced by SIV particles expressing high levels of IFN-gamma and then stimulated with SIV induced significantly higher numbers of spot-forming cells in an enzyme-linked immunospot assay than did T cells primed with DCs transduced with SIV particles lacking the cytokine. In conclusion, we demonstrated that the transduction of DCs in vitro with pseudotyped single-cycle SIVs expressing IFN-gamma increased DC activation and augmented T-cell priming activity.

FIG. 1.

Strategy for generating pseudotyped single-cycle SIVs expressing IFN-γ. pSIV239 encoding the full-length SIV_mac239 proviral DNA is shown at the top. Deletions are indicated by shaded regions, and the inserted macaque IFN-γ and GFP genes are shown as open boxes. IFN-γ was also inserted in the antisense orientation (designated aMγ). Pseudotyped SIVs were generated by transient cotransfection of pVSV-G and a plasmid encoding the defective proviral DNA into 293T cells.

FIG. 2.

IFN-γ was expressed at lower and higher levels by different constructs. (A) Untransduced HeLa cells and HeLa cells transduced with dSIV, dSIV_Lγ, or dSIV_Hγ were fixed, permeabilized, and stained with a rabbit anti-rhesus macaque IFN-γ polyclonal antibody and an Alexa Fluor 594-conjugated goat anti-rabbit secondary antibody (red) to demonstrate IFN-γ expression (lower row). Note that IFN-γ accumulated in the Golgi apparatus due to monensin treatment. Cells in the upper row were photographed to demonstrate GFP (green) expression. (B) Supernatants of HeLa cells transduced with dSIV, dSIV_Lγ, or dSIV_Hγ at an MOI of 0.1 were collected at different time points. The antiviral activity of macaque IFN-γ was measured by the prevention of the cytopathic effect of EMCV in human A549 cells. IFN-γ titers were expressed as the reciprocal of the dilution of sample giving 50% protection against EMCV. The limit of detection is 2 U/ml and is designated by the dotted line. (C) Supernatants of DCs transduced by different viral particles were collected and assayed by IFN-γ ELISA at 72 h posttransduction. The optical density (O.D.) of each well was read on a standard microplate reader. Results represent samples assayed in duplicate. The background value was subtracted.

FIG. 3.

Equivalent amounts of Gag were produced by all single-cycle SIVs in vitro. HeLa, Rat-2, or monocyte-derived dendritic cells were transduced with different constructs at an MOI of 0.1. Two days posttransduction, supernatants were collected, and the p27 Gag concentration was measured by ELISA. The data shown represent the mean values from triplicate samples assayed in duplicate; error bars indicate the SEM.

FIG. 4.

IFN-γ expression did not affect the antigenicities of pseudotyped SIVs. PBMCs (2 × 10⁵ cells/well) from two SIV_Δnef-infected rhesus macaques and five naïve macaques were cocultured with different antigens for 18 h in tissue culture plates and then transferred onto ELISPOT assay plates for another 5 h of incubation. IFN-γ-secreting cells were measured by an ELISPOT assay. Results from two infected macaques and one representative naïve macaque are shown as SFC/10⁶ PBMCs. Columns represent the mean values of cells stimulated by different antigens or medium alone from triplicate experiments assayed in duplicate. Error bars indicate the SEM.

FIG. 5.

DC activation markers were up-regulated by IFN-γ expression. Monocyte-derived DCs from four macaques were transduced by various constructs at an MOI of 0.1 or the same volume of PBS for 2 days. DCs were collected and stained with PE-conjugated anti-CD80, anti-CD86, anti-CD83, or allophycocyanin-Cy7-conjugated anti-HLA-DR monoclonal antibodies. Cells were analyzed by flow cytometry, and representative data from one macaque are shown in A. Blue lines indicate histograms of individual surface markers of DCs transduced by different particles; red lines indicate histograms of surface markers of DCs treated with PBS. The relative MFI of each fluorochrome compared to that of PBS-treated cells from four macaques is summarized in B. Columns represent the mean value of the relative MFI of each fluorochrome from four macaque samples. Error bars represent the SEM. * indicates a P value of <0.01 and ** indicates a P value of <0.001 compared to the dSIV group.

FIG. 6.

Transduction of DCs with IFN-γ-expressing dSIVs enhanced the ability of DCs to prime T cells in vitro. CD4⁺ or CD8⁺ T cells from four naïve rhesus macaques were stimulated twice with mock-, dSIV-, dSIV_Lγ-, or dSIV_Hγ-transduced DCs. These cells were then restimulated with dSIV-transduced DCs (black columns) at a ratio of 10:1 for 24 h, and CD4⁺ (A) or CD8⁺ (B) IFN-γ-secreting cells were enumerated by ELISPOT assay. CD4⁺ or CD8⁺ T cells cocultured with untransduced DCs in the same ratio (patterned columns) and CD4⁺ or CD8⁺ T cells alone (blank columns) were included as controls. Data are shown as the mean values of SFC produced by cells from four macaques and assayed in triplicate. Error bars indicate the SEM. Statistical analyses on CD4⁺ or CD8⁺ T-cell responses were performed by parametric analysis of variance, followed by Tukey's multiple comparisons test. Statistically significant differences (P < 0.05) between dSIV_Hγ- and mock-primed cells are indicated by an *; differences between dSIV_Hγ- and dSIV-primed cells are indicated by **.