Recombinant vaccinia virus-induced T-cell immunity: quantitation of the response to the virus vector and the foreign epitope

Abstract

Recombinant vaccinia viruses (rVV) have been extensively used as vaccines, but there is little information about the total magnitude of the VV-specific T-cell response and how this compares to the immune response to the foreign gene(s) expressed by the rVV. To address this issue, we quantitated the T-cell responses to both the viral vector and the insert following the infection of mice with VV expressing a cytotoxic T lymphocyte (CTL) epitope (NP118-126) from lymphocytic choriomeningitis virus (LCMV). The LCMV epitope-specific response was quantitated by intracellular cytokine staining after stimulation with the specific peptide. To analyze the total VV-specific response, we developed a simple intracellular cytokine staining assay using VV-infected major histocompatibility complex class I and II matched cells as stimulators. Using this approach, we made the following determinations. (i) VV-NP118 induced potent and long-lasting CD8 and CD4 T-cell responses to the vector; at the peak of the response (approximately 1 week), there were approximately 10(7) VV-specific CD8 T cells (25% of the CD8 T cells) and approximately 10(6) VV-specific CD4 T cells (approximately 5% of the CD4 T cells) in the spleen. These numbers decreased to approximately 5 x 10(5) CD8 T cells (approximately 5% frequency) and approximately 10(5) CD4 T cells (approximately 0.5% frequency), respectively, by day 30 and were then stably maintained at these levels for >300 days. The size of this VV-specific T-cell response was comparable to that of the T-cell response induced following an acute LCMV infection. (ii) VV-specific CD8 and CD4 T cells were capable of producing gamma interferon (IFN-gamma), tumor necrosis factor alpha (TNF-alpha), and interleukin-2; all cells were able to make IFN-gamma, a subset produced both IFN-gamma and TNF-alpha, and another subset produced all three cytokines. (iii) The CD8 T-cell response to the foreign gene (LCMV NP118-126 epitope) was coordinately regulated with the response to the vector during all three phases (expansion, contraction, and memory) of the T-cell response. The total number of CD8 T cells responding to NP118-126 were approximately 20- to 30-fold lower than the number responding to the VV vector (approximately 1% at the peak and 0.2% in memory). This study provides a better understanding of T-cell immunity induced by VV-based vaccines, and in addition, the technique described in the study can be readily extended to other viral vectors to determine the ratio of the T-cell response to the insert versus the vector. This information will be useful in optimizing prime-boost regimens for vaccination.

FIG. 1.

Activation and expansion of T cells following VV infection. (A) BALB/c mice were infected with VV and analyzed on the indicated days after infection by staining splenocytes with CD8α, CD4, and CD11a MAbs. The percentages of the CD8 or CD4 T-cell populations that were LFA-1^hi are indicated in the upper right corners of the corresponding panels. (B) Total numbers of CD8 and CD4 T cells in the spleen that were LFA-1^hi (closed symbols) or LFA-1^lo (open symbols) were determined. (C) Direct ex vivo CTL activity was measured on day 7 postinfection. Killing was assayed on ⁵¹Cr-labeled uninfected (○) or VV-infected (□) target cells. Error bars indicate standard deviations.

FIG. 2.

Intracellular cytokine staining of LCMV- and VV-specific CD8 T cells using virus-infected cells as stimulators. BALB/c mice were infected with either LCMV or VV-WT, and the CD8 T-cell response in the spleen was analyzed on day 8 or day 7 after infection, respectively. Splenocytes from infected mice were stimulated in vitro for 5 h, and then intracellular IFN-γ staining was performed. (A and B) Splenocytes from day 8 LCMV-infected BALB/c mice either were not stimulated or were stimulated with the LCMV NP118-126 peptide (A) or uninfected, LCMV-infected, or VV-WT-infected BALB Cl7 cells (B). (C) Splenocytes collected from BALB/c mice day 7 after VV infection were stimulated with uninfected, LCMV-infected, or VV-WT-infected BALB Cl7 cells. The frequencies of the CD8 T cells that stained IFN-γ⁺ are indicated in the upper right corners of the corresponding panels.

FIG. 3.

LCMV- and VV-specific CD4 and CD8 T-cell responses. LCMV-specific T-cell responses were analyzed on day 8 after infection (A), and VV-specific T-cell responses were analyzed on day 7 after infection (B). Splenocytes from infected mice were stimulated with uninfected A-20 cells (B-cell line expressing both MHC class I and II molecules), LCMV-infected A-20 cells, or VV-infected A-20 cells, and intracellular IFN-γ staining was performed to determine the frequencies of virus-specific CD8 and CD4 T cells. The percentages of the CD8 or CD4 T cells that were IFN-γ⁺ are indicated in the upper right corners of the corresponding panels.

FIG. 4.

IFN-γ, TNF-α, and IL-2 production by virus-specific CD8 and CD4 T cells. Splenocytes collected from BALB/c mice on day 7 after VV infection (A) or on day 8 after LCMV infection (B) were stimulated with the corresponding virus-infected A-20 cells for 5 h in vitro. Cell surfaces were stained with anti-CD8α or anti-CD4, and then cells were intracellularly stained with anti-IFN-γ, anti-TNF-α, or anti-IL-2. The percentages of CD8 or CD4 T cells producing each cytokine are indicated in the upper right corners of the corresponding panels.

FIG. 5.

Three populations of virus-specific effector T cells. Splenocytes collected from BALB/c mice on day 7 after VV infection were stimulated in vitro with VV-infected A-20 cells and costained intracellularly for either IFN-γ and TNF-α (top panels) or IFN-γ and IL-2 (bottom panels). The data shown are gated on either CD8 or CD4 T cells, and the numbers within the panels represent the frequencies of the CD8 or CD4 T cells producing each cytokine.

FIG. 6.

Virus-specific memory CD8 and CD4 T-cell responses. Memory CD8 and CD4 T-cell responses were determined more than 200 days following LCMV (A) or VV (B) infection. Responses were analyzed by intracellular IFN-γ staining after stimulation with virus-infected A-20 cells. The percentages of CD8 and CD4 T cells producing IFN-γ are indicated in the upper right corners of the corresponding panels.

FIG. 7.

Kinetics of virus-specific CD4 and CD8 T-cell responses. The total numbers of VV (○)- and LCMV (⋄)-specific CD8 and CD4 T cells were quantitated by intracellular IFN-γ staining on the indicated days after infection. The error bars indicate standard deviations.

FIG. 8.

Comparison of CD8 T-cell responses to the vector with those to the foreign epitope. BALB/c mice were immunized with VV-NP118, and the frequencies (A) and numbers (B) of VV-specific and NP118-specific CD8 T cells were determined by intracellular IFN-γ staining (A and B) or by IFN-γ ELISPOT analysis (C). In panel A, the results for one representative mouse are shown at each time point (n ≥ 8 mice per time point), and the percentages of CD8 T cells producing IFN-γ are indicated in the upper left corners of the corresponding panels. (C) The VV- and NP118-specific memory T-cell responses in mice infected with VV-NP118 >200 days earlier were quantitated by IFN-γ ELISPOT analysis. The error bars represent standard deviations.