Defective T Memory Cell Differentiation after Varicella Zoster Vaccination in Older Individuals

<div><p>Vaccination with attenuated live varicella zoster virus (VZV) can prevent zoster reactivation, but protection is incomplete especially in an older population. To decipher the molecular mechanisms underlying variable vaccine responses, T- and B-cell responses to VZV vaccination were examined in individuals of different ages including identical twin pairs. Contrary to the induction of VZV-specific antibodies, antigen-specific T cell responses were significantly influenced by inherited factors. Diminished generation of long-lived memory T cells in older individuals was mainly caused by increased T cell loss after the peak response while the expansion of antigen-specific T cells was not affected by age. Gene expression in activated CD4 T cells at the time of the peak response identified gene modules related to cell cycle regulation and DNA repair that correlated with the contraction phase of the T cell response and consequently the generation of long-lived memory cells. These data identify cell cycle regulatory mechanisms as targets to reduce T cell attrition in a vaccine response and to improve the generation of antigen-specific T cell memory, in particular in an older population.</p></div

T cell analysis in the Toronto seroprevalence and case/control cohorts.

<p>(A) Bin separation of IFNγ responses in CD8 and CD4 T cells specific to pH1N1 stimulation. Frequencies have been corrected for background IFNγ production in LCMV and unstimulated control cultures. (B) Spearman correlation between pH1N1-responding CD8 T cells and donor age. (C) Combinations of effector molecule expression of IFNγ⁺ CD8 T cells from the responder subset. P values above the bars indicate the level of statistical significance compared to all other bars as determined by ANOVA and Tukey test. (D) Spearman correlation between the CD8 T cell response to pH1N1 and the frequency of responding cells with multiple effector functions. (E) CD8 T cell response in case and control subjects. Groups were compared using a nonparametic Mann-Whitney test. (F) Spearman correlation for pH1N1 response and frequency of CD8 T cells with multiple effector functions in cases and controls.</p

Acute and persisting antibody and memory T cell responses to pandemic H1N1 infection in one PCR case-confirmed donor.

<p>Longitudinal samples of unfractionated PBMC were challenged with influenza virus or controls for 18h. (A) Frequency of pandemic H1N1-responsive CD8 T cells out of total CD8 T cells as measured by IFNγ staining. IFNγ responsive CD8 T cells were also sub-divided by expression of other effector markers, granzyme B and CD107a. (B) Memory phenotypes of influenza-responsive CD8 T cells at various times post-onset of influenza symptoms. (C) Frequency and phenotypes of IFNγ⁺ CD4 T cells after pandemic H1N1 challenge. (D) Antibody titers in serum as detected by microneutralization (MN), hemagglutination inhibition (HAI), and a pandemic H1-specific ELISA assay. BLD  =  below the limits of detection.</p

Infection followed by vaccination boosts antibody but not T cell responses to pandemic H1N1.

<p>(A) Antibody titers against pH1N1 for vaccinated and unvaccinated donors in the entire cohort 8-10 months post-pandemic. Vaccinations were self-reported from October 2009 to January 2010. A non-parametric Mann-Whitney test was used for statistical significance. (B) CD8 and CD4 responses to pH1N1 for vaccinated and unvaccinated donors in the total Toronto cohort, measured 8-10 months post-pandemic. Groups were compared using a Mann-Whitney test. (C) IFNγ⁺ CD8 T cell responses in donors with both antibody and CD8 T cell responses, T cell responses only, antibodies only, or no antibody or T cell response to pH1N1. Data has been normalized using log transformation to represent Gaussian distribution; groups were compared using ANOVA and Tukey test. (D) Normalized CD8 T cell response in cases and controls with differing vaccination history for pH1N1. Groups were compared by ANOVA and Tukey test. PCR-confirmed infections were reported from April-November 2009; vaccination was self-reported from October 2009-January 2010. (E) Pandemic-specific antibody responses as measured by microneutralization in the case/control cohort, separated by self-reported vaccination history for the monovalent pH1N1 vaccine. PCR-confirmed infections were reported from April-November 2009; vaccination was self-reported from October 2009-January 2010. Nonparametric Kruskal-Wallis and Mann-Whitney tests were performed to determine statistical significance.</p

Detection of influenza-responsive CD8 T cells by multicolour flow cytometry.

<p>Total PBMC were stimulated for 18 hours with pH1N1 influenza, or as a control, with LCMV Armstrong, or left unstimulated and then assessed for IFNγ production by intracellular cytokine staining and flow cytometry. Gates are based on fluorescence minus one controls. (A) Representative gating used to identify IFNγ⁺ CD8 T cells from total PBMC. (B) Sample non-responder, weak responder, and strong responder to pH1N1 identified in the Toronto cohort 8-10 months post-pandemic; positive versus non-responder is defined in the results. A representative “weak” responder was arbitrarily chosen from the bottom third of positive responses whereas the “strong” responder was from the top third of responders.</p

Influence of pre-existing VZV immunity on vaccine responses.

<p>(A) VZV-specific T cell frequencies were determined by IFN-γ–specific ELISpot before (day 0) and at days 8±1, 14±1 and 28±3 after vaccination. **p<0.0001 by paired Wilcoxon-Mann-Whitney test. (B) Antibody indices determined by VZV-IgG-specific ELISA increased between day 0 and 28 (p<0.01). Data from individual vaccinees are joined with a line. (C and D) Fold change in VZV-specific antibody concentrations from day 0 to day 28 were negatively correlated with initial antibody concentrations (C, r² = 0.73, p <0.001), but not with initial VZV-specific T cell frequencies (D, p = 0.16). (E and F) Fold change in VZV-specific T cell frequencies from day 0 to day 28 showed no correlation with initial VZV-specific antibodies (E, p = 0.47) or initial T cell frequencies (F, p = 0.12). (G) Fold change in VZV-specific antibodies from day 0 to day 28 did not correlate with fold change in VZV-specific T cell frequencies from day 0 to day 28 (p = 0.38).</p

Contribution of initial expansion and subsequent contraction of VZV-specific T cells after vaccination to final T cell memory frequencies.

<p>VZV-specific T cell frequencies were measured by IFN-γ–specific ELISpot. Peak ELISpot counts were defined as the highest observed count at either day 8 (25/29 people) or day 14 (4/29 people) after vaccination. Absolute increase to the peak value (expansion) was calculated compared to day 0 counts. Absolute decline from peak (contraction) was calculated compared to day 28 counts. (A) Unsupervised hierarchical clustering analysis based on frequencies of VZV-specific T cells at all three time points. Vaccinees separated into three main clusters [C1 (black), C2 (red), C3 (blue)]. The color code for the different clusters is maintained in Fig 2B–2F. (B) Mean±SD of VZV-specific T cell frequencies at day 0, peak, and day 28 are shown for each cluster. (C-F) Boxplots showing overall increases in VZV-specific T cell frequencies from day 0 to day 28 (C), expansion in VZV-specific T cell frequencies from day 0 to peak value (D), contraction in VZV-specific T cell frequencies from peak to day 28 (E), and age range of individuals in each cluster (F). p-values calculated by one-tailed Wilcoxon-Mann-Whitney test are shown. (G) Vaccinees were grouped according to their age < 59 (orange) or >59 (green) years and T cell frequency trajectories after vaccinations are shown as described in Fig 2B. (H) When corrected for age, VZV-specific T cell expansion showed a weak correlation with overall increase in VZV-specific T cell frequency from day 0 to day 28 that did not reach significance (r = 0.31, p = 0.10). (I) Contraction after peak responses corrected for age inversely correlated with overall increase in VZV-specific T cell frequency from day 0 to day 28 (r = -0.53, p = 0.003).</p

Correlation of whole blood-derived gene signatures with VZV-specific T cell responses.

<p>(A) Deconvolution of whole blood gene expression for leukocyte subsets was performed. Volcano plots show fold change of gene expression in monocytes (left), lymphocytes (middle) and neutrophils (right) between day 0 and day 1. The genes with significant changes in expression (fold change>1.5, p<0.05) after vaccination are colored in red. (B) Fold changes in monocyte-derived genes (day 0 to day 1, based on deconvolution analysis) were correlated with log-transformed changes in frequencies of antigen-specific T cells after VZV vaccination. Results are shown as volcano plots of correlation coefficients for VZV-specific T cell expansion (VZV-specific T cell frequencies day 0 to peak, left panel), contraction (peak to day 28, middle panel) and overall responses (day 28 to day 0, right panel). The genes with significant correlations (p<0.05) are colored in red. (C) The Venn diagram shows the overlap in genes that significantly correlate with expansion, contraction or global responses.</p

Demographics of study participants.

<p>Demographics of study participants.</p

Preferential association of cell cycle and DNA repair pathways with VZV-specific T cell responses.

<p>(A) Correlation coefficients of modules that were significantly correlated with the decline in frequencies after peak responses as well as the overall increase from day 0 to day 28 as described in <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1005892#ppat.1005892.g006" target="_blank">Fig 6</a>, are shown as heat maps. Module nomenclature is from [<a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1005892#ppat.1005892.ref013" target="_blank">13</a>]. (B) Networks of genes in modules M22.0 (top left panel), M103 (top right panel) and M4.1 (bottom panel). The sizes of nodes represent the absolute value of correlation coefficients between gene expression level at day 14 after vaccination and peak to day 28 T cell responses. Orange and blue colors indicate positive and negative correlation, respectively. The grey line represents the co-expression relationship as described previously [<a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1005892#ppat.1005892.ref013" target="_blank">13</a>]. Gene names for the top genes with the highest correlation coefficient are listed.</p