Enhanced presentation of major histocompatibility complex class I-restricted human immunodeficiency virus type 1 (HIV-1) Gag-specific epitopes after DNA immunization with vectors coding for vesicular stomatitis virus glycoprotein-pseudotyped HIV-1 Gag particles - PubMed (original) (raw)

Enhanced presentation of major histocompatibility complex class I-restricted human immunodeficiency virus type 1 (HIV-1) Gag-specific epitopes after DNA immunization with vectors coding for vesicular stomatitis virus glycoprotein-pseudotyped HIV-1 Gag particles

D Marsac et al. J Virol. 2002 Aug.

Abstract

In vivo priming of cytotoxic T lymphocytes (CTL) by DNA injection predominantly occurs by antigen transfer from DNA-transfected cells to antigen-presenting cells. A rational strategy for increasing DNA vaccine potency would be to use a delivery system that facilitates antigen uptake by antigen-presenting cells. Exogenous antigen presentation through the major histocompatibility complex (MHC) class I-restricted pathway of some viral antigens is increased after adequate virus-receptor interaction and the fusion of viral and cellular membranes. We used DNA-based immunization with plasmids coding for human immunodeficiency virus type 1 (HIV-1) Gag particles pseudotyped with vesicular stomatitis virus glycoprotein (VSV-G) to generate Gag-specific CTL responses. The presence of the VSV-G-encoding plasmid not only increased the number of mice displaying anti-Gag-specific cytotoxic response but also increased the efficiency of specific lysis. In vitro analysis of processing confirmed that exogenous presentation of Gag epitopes occurred much more efficiently when Gag particles were pseudotyped with the VSV-G envelope. We show that the VSV-G-pseudotyped Gag particles not only entered the MHC class II processing pathway but also entered the MHC class I processing pathway. In contrast, naked Gag particles entered the MHC class II processing pathway only. Thus, the combined use of DNA-based immunization and nonreplicating pseudotyped virus to deliver HIV-1 antigen to the immune system in vivo could be considered in HIV-1 vaccine design.

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Figures

FIG. 1.

FIG. 1.

Efficiency of the Gag-specific cytotoxic T-cell response after DNA coinjection. Mice were immunized with 10 (A) or 100 (B) μg of pCMV.ΔR8-2 + pCMV.AS (open diamond) or pCMV.ΔR8-2 + pCMV.VSV (solid squares) plasmid DNA encoding naked or VSV-G-pseudotyped Gag particles, respectively. (C) Mice were immunized with 100 μg of pCMV.ΔR8-2 + pCMV.VSV. DNA was injected into normal muscle. Cytotoxic activity of in vitro-stimulated spleen T cells was measured 2 weeks after immunization. The specific lysis was calculated by subtracting the nonspecific lysis on P815 target cells from the specific lysis obtained on P815 cells pulsed with HIV-1 p24_gag_ peptide (A and B). (C) Target cells were infected with either wild-type vaccinia virus (open triangles) or recombinant vaccinia virus expressing the HIV-1 Gag protein (solid circles). Specific lysis values represent mean values ± standard errors from three to five individual mice in each immunization group. E:T ratio, effector/target ratio.

FIG. 2.

FIG. 2.

Dose-dependent cytotoxic T-cell responses after coinjection of DNAs coding for naked or VSV-G-pseudotyped Gag particles. Mice were immunized with 1, 10, or 100 μg of either pCMV.ΔR8-2 + pCMV.AS (open columns) or pCMV.ΔR8-2 + pCMV.VSV (solid columns) plasmid DNA. DNA was injected into either normal muscle (left panel) or cardiotoxin-pretreated muscle (regenerating muscle, right panel). Cytotoxic activity of spleen cells was measured by using peptide-loaded or unloaded P815 cells as targets. Cytolytic responses were considered positive after subtraction of the background when the specific lysis was 10% or more at an effector/target ratio of 100/1. The number of responding mice/tested mice is indicated at the top of each column and represents cumulative results obtained from three to five independent experiments. ∗, P < 0.05; ∗∗, P < 0.001 by χ2 Pearson test.

FIG. 3.

FIG. 3.

Analysis of in vitro processing of Gag particles. An IFN-γ ELISPOT assay was performed with Gag-specific effector T cells obtained from mice immunized with pCMV.ΔR8-2 DNA encoding naked Gag particles. The number of IFN-γ SFC per 106 splenocytes was measured in response to a short-term stimulation of splenocytes (40 h) with either naked or VSV-G-pseudotyped Gag particles. The number of specific SFC was calculated after subtracting the background obtained in wells containing splenocytes in culture medium. Different concentrations of viral particles were tested for their ability to present Gag epitopes (100, 20, and 4 ng of HIV-1 p24 per ml). Results are mean values ± standard errors from three individual mice. Please note that the number of IFN-γ SFC is expressed per 106 splenocytes.

FIG. 4.

FIG. 4.

Analysis of T-cell subpopulations activated after in vitro processing of Gag particles. The IFN-γ ELISPOT assay was performed as described in the legend to Fig. 3. Effector T cells were pooled splenocytes from five mice immunized with pCMV.ΔR8-2 DNA encoding naked Gag particles. The number of Gag-specific IFN-γ SFC was measured in response to a short-term stimulation of the splenocytes with HIV-1 Gag peptide (1 μg/ml), VSV-G-pseudotyped HIV-1 Gag particles (p24; 100 ng/ml), or naked HIV-1 Gag particles (p24; 100 ng/ml). The ELISPOT assay was performed on undepleted (A), CD4+ T-cell-depleted (B), and CD8+ T-cell-depleted (C) splenocytes. Please note that IFN-γ SFC are expressed for, respectively, 106 T lymphocytes (A), 106 CD8+ T cells (B), and 106 CD4+ T cells (C) after staining and quantification of each cell population by FACS analysis.

FIG. 5.

FIG. 5.

CD4+ T-cell responses induced in vivo by injection of DNAs encoding naked or VSV-G-pseudotyped particles. Groups of 5 or 11 mice were injected with 100 μg of DNA vectors encoding either naked (pCMV.ΔR8-2 + pCMV.AS) or VSV-G-pseudotyped Gag particles (pCMV.ΔR8-2 + pCMV.VSV) into normal muscle (left panel) or in regenerating muscle (right panel). Two weeks after DNA immunization, ex vivo ELISPOT assay was performed on splenocytes to measure Gag-specific IFN-γ-secreting CD4+ T cells. Splenocytes were incubated for 40 h with naked Gag particles (100 ng/ml) or in culture medium. Results are given as the mean number of specific IFN-γ-secreting CD4+ T cells per spleen ± standard error.

FIG. 6.

FIG. 6.

Analysis of VSV-specific T-cell responses induced in vivo by injection of DNAs encoding either naked or VSV-G-pseudotyped Gag particles. Groups of five mice were injected with 100 μg of either pCMV.ΔR8-2 + pCMV.AS DNA (left panel) or pCMV.ΔR8-2 + pCMV.VSV (right panel) DNA into normal muscle. Two weeks after DNA immunization, ex vivo ELISPOT assay was performed on splenocytes to measure VSV-specific IFN-γ-secreting T cells. Splenocytes were incubated for 40 h with wild-type vaccinia virus (rvv WT) or with recombinant vaccinia virus expressing VSV-G (rvv VSV) at an MOI of 1/1. The ELISPOT assay was performed on undepleted, CD4+ T-cell-depleted, and CD8+ T-cell-depleted splenocytes. The number of IFN-γ SFC is expressed per 106 splenocytes.

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