Protective CD4+ and CD8+ T cells against influenza virus induced by vaccination with nucleoprotein DNA - PubMed (original) (raw)

Protective CD4+ and CD8+ T cells against influenza virus induced by vaccination with nucleoprotein DNA

J B Ulmer et al. J Virol. 1998 Jul.

Abstract

DNA vaccination is an effective means of eliciting both humoral and cellular immunity, including cytotoxic T lymphocytes (CTL). Using an influenza virus model, we previously demonstrated that injection of DNA encoding influenza virus nucleoprotein (NP) induced major histocompatibility complex class I-restricted CTL and cross-strain protection from lethal virus challenge in mice (J. B. Ulmer et al., Science 259:1745-1749, 1993). In the present study, we have characterized in more detail the cellular immune responses induced by NP DNA, which included robust lymphoproliferation and Th1-type cytokine secretion (high levels of gamma interferon and interleukin-2 [IL-2], with little IL-4 or IL-10) in response to antigen-specific restimulation of splenocytes in vitro. These responses were mediated by CD4+ T cells, as shown by in vitro depletion of T-cell subsets. Taken together, these results indicate that immunization with NP DNA primes both cytolytic CD8+ T cells and cytokine-secreting CD4+ T cells. Further, we demonstrate by adoptive transfer and in vivo depletion of T-cell subsets that both of these types of T cells act as effectors in protective immunity against influenza virus challenge conferred by NP DNA.

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Figures

FIG. 1

FIG. 1

Lymphoproliferative responses after NP DNA vaccination. Female BALB/c mice were uninjected or injected with NP DNA (50 μg), control DNA (50 μg), or inactivated influenza virus (A/PR/8/34) (flu; 15 μg) on weeks 0 and 3 or were infected awake with 1,000 TCID50 of influenza virus (A/PR/8/34) on week 0. Spleens were collected and pooled from three mice per group on week 7 and restimulated in vitro with NP. Lymphoproliferation data are presented as a stimulation index.

FIG. 2

FIG. 2

Cytokine secretion from restimulated spleen cells. Female BALB/c mice were injected with NP DNA (50 μg) or control DNA (50 μg) on weeks 0 and 3, and spleens were collected and pooled from three mice per group on week 7. Cells from DNA-injected mice were restimulated in vitro specifically with recombinant NP protein, and cells from NP DNA-injected mice were nonspecifically activated with the mitogen concanavalin A (Con A). Cytokines secreted into the culture supernatant were detected by ELISA and are presented as picograms/milliliter of culture supernatant.

FIG. 3

FIG. 3

Anti-NP immunoglobulin subtype profile. Female BALB/c mice were injected with NP DNA (50 μg) or NP protein (10 μg) on weeks 0 and 3, and sera were collected on week 5. Anti-NP antibody subtypes were measured by ELISA as described in Materials and Methods. Data are presented as geometric mean ELISA titers ± standard errors of the means for groups of five mice.

FIG. 4

FIG. 4

In vitro depletion of T-cell subsets. Female BALB/c mice were injected with NP DNA (200 μg) on weeks 0, 3, and 6, and spleens were collected and pooled from groups of three mice on week 23. T-cell subsets were prepared and restimulated as described in Materials and Methods. Cells from NP DNA-injected and uninjected mice were restimulated with NP protein and analyzed for proliferation plotted as a stimulation index (A) and secretion of IFN-γ (B) or IL-2 (C), as measured by ELISA and plotted as picograms/milliliter of culture supernatant.

FIG. 5

FIG. 5

In vivo depletion of T-cell subsets. Groups of 10 female BALB/c mice were injected with NP DNA (200 μg) on weeks 0, 3, and 6 and then were untreated or treated with anti-CD4, anti-CD8, or control (rat IgG) antibody on week 9. As a negative control, mice were injected three times with control DNA (200 μg). All groups were challenged under anesthesia with 1,000 TCID50 of influenza virus A/HK/68 and monitored for survival (A) and weight loss (B). The results of two experiments were similar, and the data were combined in Fig. 5 to achieve an n of 20 per data point.

FIG. 6

FIG. 6

Adoptive transfer of T-cell subsets. Spleen cells from uninjected mice (solid triangles) or mice primed with influenza virus A/PR/8/34 (flu-infected; solid squares), immunized with NP DNA, or injected with HIV Gag DNA (open squares) were harvested. The NP DNA-primed spleen cells were enriched for CD4+ (open circles) or CD8+ T lymphocytes (open triangles). Spleen cells from mice immunized with HIV Gag DNA were restimulated with syngeneic cells pulsed with Gag peptide 193-212, while cells from the remaining groups were restimulated in vitro for 7 days with syngeneic cells infected with A/PR/8/34. These lymphocytes were adoptively transferred into age-matched naive mice (2.5 × 107 cells/mouse for the groups denoted by open and solid squares; 107 for groups denoted by open circles and triangles) that had been intranasally challenged with A/HK/68 (H3N2) 4 h previously. Data are plotted as percent survival (A) and weight loss (B) versus days after challenge for groups of 10 mice.

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