Low CD8 T-cell proliferative potential and high viral load limit the effectiveness of therapeutic vaccination

Abstract

Therapeutic vaccination has the potential to boost immune responses and enhance viral control during chronic infections. However, many therapeutic vaccination approaches have fallen short of expectations, and effective boosting of antiviral T-cell responses is not always observed. To examine these issues, we studied the impact of therapeutic vaccination, using a murine model of chronic infection with lymphocytic choriomeningitis virus (LCMV). Our results demonstrate that therapeutic vaccination using a recombinant vaccinia virus expressing the LCMV GP33 CD8 T-cell epitope can be effective at accelerating viral control. However, mice with lower viral loads at the time of vaccination responded better to therapeutic vaccination than did those with high viral loads. Also, the proliferative potential of GP33-specific CD8 T cells from chronically infected mice was substantially lower than that of GP33-specific memory CD8 T cells from mice with immunity to LCMV, suggesting that poor T-cell expansion may be an important reason for suboptimal responses to therapeutic vaccination. Thus, our results highlight the potential positive effects of therapeutic vaccination on viral control during chronic infection but also provide evidence that a high viral load at the time of vaccination and the low proliferative potential of responding T cells are likely to limit the effectiveness of therapeutic vaccination.

FIG. 1.

Enhanced viral control in therapeutically vaccinated mice. (A) C57BL/6 mice were infected with LCMV clone 13, resulting in chronic infection. Between days 30 and 35 p.i., when the viral titers in sera were between 2 × 10³ and 2 × 10⁵, mice were immunized with VV expressing the LCMV GP33-41 epitope (VVGP33; filled symbols) or with a control VV expressing an irrelevant epitope (control VV; open symbols). Viral titers in sera were monitored by a plaque assay at the indicated times postinfection. (B) Viral titers in the indicated tissues were determined by a plaque assay on day 4 and days 50 to 75 post-therapeutic vaccination. The data are representative of four independent experiments. Statistically significant differences (P < 0.05) between the control VV- and VVGP33-vaccinated groups are indicated (*).

FIG. 2.

Effective boosting of circulating virus-specific CD8 T cells by therapeutic vaccination correlates with low viral load. (A) Mice with immunity to LCMV (>30 days post-LCMV Armstrong vaccination) and chronically infected mice (∼30 days post-LCMV clone 13 infection) were vaccinated with VVGP33, and changes in the numbers of Db/GP33 tetramer-positive CD8 T cells were monitored in the blood on days 0, 4, 7, and 14 post-therapeutic vaccination. The frequencies of Db/GP33 tetramer-positive CD8 T cells in the blood are shown for individual mice following infection with either VVGP33 (red lines) or a control VV (blue lines). (B) Changes in the GP33-specific CD8 T-cell responses in the blood of chronically infected mice, measured by MHC tetramer staining (left panel) or intracellular IFN-γ staining (right panel) at 4 to 7 days post-therapeutic vaccination, were plotted versus the viral load at the time of VVGP33 vaccination. Linear regression analysis revealed a significant correlation between improved responses, as measured by IFN-γ production, and a lower viral load at the time of vaccination. P values indicate the significance of the correlation.

FIG. 3.

GP33-specific CD8 T-cell numbers in the spleen and liver were only marginally increased following VVGP33 therapeutic vaccination. (A) The total number of Db/GP33 tetramer-positive CD8 T cells in the spleen or liver was determined 4 days after the infection of LCMV-immune or chronically infected mice with a control VV or VVGP33. The data are representative of three independent experiments. (B) The total number of GP33-specific CD8 T cells in the spleen was determined by intracellular IFN-γ staining 4 days after the infection of LCMV-immune (Im) or chronically infected mice with a control VV or VVGP33. (C) The level of functional exhaustion at 4 days post-VVGP33 infection of chronically infected mice was measured by determining the percentage of the MHC tetramer-positive CD8 T-cell population that could produce IFN-γ following 5 h of peptide stimulation. Horizontal bars indicate the averages for the groups.

FIG. 4.

Therapeutic vaccination with VVGP33 does not enhance responses to other epitopes. (A) Responses to five different LCMV epitopes in the spleen were determined on day 4 post-therapeutic vaccination by intracellular IFN-γ staining following 5 h of stimulation. The data are representative of three independent experiments. (B) The percentages of IFN-γ-producing CD8 T cells specific for each of the five LCMV peptides from panel A are summarized for multiple mice (n = 3 to 12 for each response).

FIG. 5.

Therapeutic vaccination results in only a modest increase in virus-specific CD8 T-cell division in vivo. (A) Chronically infected mice were infected with control VV or VVGP33 and also fed BrdU in their drinking water. After 7 days, the mice were sacrificed and the percentage of Db/GP33 tetramer-positive cells that had incorporated BrdU was determined. A group of control mice with immunity to LCMV Armstrong were also infected with VVGP33 and fed BrdU, and the turnover of Db/GP33 tetramer-positive CD8 T cells was determined for these mice on day 7 p.i. (n = 3/group) and is representative of two independent experiments. (B) An example of BrdU staining in chronically infected mice 7 days after control VV or VVGP33 vaccination. The levels of BrdU incorporation were not substantially different between control VV-infected and chronically infected mice that did not receive VV (data not shown).

FIG. 6.

Poor T-cell expansion following therapeutic vaccination of chronically infected mice is due to intrinsic cell defects in proliferative potential. (A) CD8 T cells were purified from mice with immunity to LCMV Arm (on day 180 p.i.) and LCMV clone 13-infected mice (on day 35 p.i.) by the use of magnet-associated cell sorter beads (>95% pure; data not shown). These cells were labeled with CFSE and mixed with CD8-depleted splenocytes from naïve mice. The GP33 peptide was added (0.2 μg/ml), and proliferation was assessed after 84 h by staining with CD8 and Db/GP33 tetramers. Similar division profiles were also observed for other LCMV-specific CD8 T-cell responses (data not shown). (B) CD8 T cells were purified from Thy1.1⁺ LCMV-immune mice (on day 130 p.i.) and from Thy1.2⁺ chronically infected mice (LCMV clone 13; day 40), labeled with CFSE, and mixed to contain equal numbers of Db/GP33 tetramer-positive CD8 T cells (∼10⁵ of each). This mixture was adoptively transferred to naïve recipients, and these recipients were immediately infected with LCMV clone 13. The division of donor GP33-specific CD8 T cells was determined by CFSE dilution after 65 h. The plots were gated on Thy1.1⁺ (top) or Thy1.2⁺ (bottom) Db/GP33 tetramer-positive CD8 T cells from the spleen. Similar division profiles were observed for other LCMV epitopes and other tissues (data not shown).