Long-lived poxvirus immunity, robust CD4 help, and better persistence of CD4 than CD8 T cells

Abstract

The currently used smallpox vaccine is associated with a high incidence of adverse events, and there is a serious need for a safe and effective alternative vaccine. Here, we carried out a longitudinal evaluation of vaccinia virus-specific CD4 and CD8 T cells in smallpox-vaccinated individuals by using a highly sensitive intracellular cytokine staining assay. Our results demonstrate that, in addition to the CD8 response, the smallpox vaccinations raised a robust CD4 response with a Th1-dominant cytokine profile. These CD4 T cells were stable and exhibited only a twofold contraction between peak effector and memory phases compared with an approximate sevenfold contraction for CD8 cells. A significant proportion of vaccinated individuals lost detectable CD8 memory while maintaining CD4 memory. After a booster immunization, these individuals generated a robust CD8 response, which some of them rapidly lost. Thus, the current smallpox vaccine provides long-lasting CD4 help that may be critical for long-lived B-cell memory. We suggest that the provision of adequate CD4 help for CD8 and humoral effector functions will be critical to the success of the next generation of smallpox vaccines.

FIG. 1.

Cellular immune responses after primary vaccination. (A) ICS assay to measure vaccinia virus-specific CD4 and CD8 T cells. PBMCs were stimulated with vaccinia virus as described in Materials and Methods and stained for CD3, CD8, IFN-γ, and IL-2. Cells were gated on lymphocytes based on the scatter pattern, analyzed for CD3 expression, and analyzed for the expression of CD8 and IFN-γ. Cells in the right quadrants represent CD8 cells, and those in the left quadrants represent CD4 cells (CD3 positive, CD8 negative). The frequencies in the upper quadrants are data for IFN-γ-producing cells expressed as the percentage of total CD4 cells (left quadrants) or total CD8 cells (right quadrants). (B) Frequency of vaccinia virus-specific CD4 and CD8 T cells at 2 weeks postvaccination. The numbers inside the graph represent the respective geometric mean values.

FIG. 2.

Cytokine expression profile of vaccinia virus-specific T cells. (A) ICS analysis at 2 weeks (peak effector) and 12 weeks (memory) postvaccination. The numbers in the upper quadrants represent the frequencies of the respective cytokine-producing cells as the percentage of IFN-γ-producing cells, and the cells numbers in the lower quadrants represent the frequencies of the respective cytokine-producing cells expressed as the percentage of total CD4 cells. (B) ELISPOT analysis at 2 weeks postvaccination. Data represent the geometric mean frequency of three vaccinated individuals. SFU, spot-forming units.

FIG. 3.

Longevity of vaccinia virus-specific T-cell memory. (A) Longitudinal analysis of CD4 and CD8 T cells after primary vaccination in two individuals. (B) Cross-sectional analysis of vaccinia virus-specific CD4 and CD8 T cells over time. The solid symbols represent data for the responses in naïve individuals. (C) Comparison of vaccinia virus-specific CD4 and CD8 responses within each vaccinated individual.

FIG. 4.

Cellular immunity after booster immunization. (A) Longitudinal analysis of vaccinia virus-specific CD4 and CD8 T cells after booster vaccination. Each symbol represents data for a boosted individual. (B) Comparison of the frequencies of vaccinia virus-specific T cells before and after the boost within each individual. The vertical and the horizontal dotted lines represent the sensitivity of the assay.