Sara van den Berg

31 2 CMV on influenza vaccination analysis by approximately 10-year intervals. Significant differences were also observed between age groups in the H1N1pdm titers (e.g. T2, p=0.016), with lower responses in the oldest age group compared to the youngest two age groups (e.g. T2, 19-30 year versus 40-52 year p=0.007) ( Figure 2B ) (see Supplementary Table 8 ). Similar results were observed for the different age groups when analyzing the data by protection level, defined by reaching a titer of ≥40 HAU (data not shown). These data indicate that there are already signs of immunosenescence- driven impaired vaccine responses to a novel antigen challenge in middle-aged individuals. No effect of CMV-seropositivity on antibody titers after pandemic influenza vaccination Next, the effect of latent CMV infection on the influenza virus-specific antibody response to the vaccine with the newly introduced H1N1pdm influenza vaccine strain was investigated. CMV-seropositive individuals were compared to CMV-seronegative individuals for influenza titers before and after vaccination. No differences between CMV-seropositive and CMV-seronegative individuals in influenza titer at any time point in both seasons were found ( Supplementary Figure 1A ). Some individuals did already show a detectable pandemic titer before vaccination, although on average the pre-titer was very low (GMT 9.4 HAU). To correct for this and other potential confounders, influenza titers of CMV-seropositive and CMV-seronegative individuals were analyzed adjusted for pre-titer, sex, age and previous influenza vaccinations with a Generalized Estimation Equation (GEE) model ( Supplementary Table 2 ). No significant differences were found between CMV- seropositive and CMV-seronegative individuals in antibody titers at each individual time point ( Figure 3A) . So although age shows a negative effect on the novel pandemic H1N1pdm antibody response indicative of immunosenescence to de novo response ( Figure 2 ), no effect of CMV-serostatus on the influenza virus titer is observed after pandemic vaccination in adults ( Figure 3A ). Figure 2. Effect of age on influenza virus-specific an- tibody titers after influenza vaccination. A representa- tive figure of influenza H1N1pdm antibody titers after pan- demic vaccination plotted against age (T2) ( A ). Dotted horizontal line represents a protective influenza titer of 40 HAU. The geometric mean of the influenza antibody titer ( B ) after vaccination are given for different age groups after vaccination with H1N1pdm for T2 (p=0.016 ANOVA). Correlations are tested with Pearson correlation. Differ- ences between 2 age groups are tested with Student’s t-test for log-transformed influenza antibody titers. ** p < 0.010. GMT = geometric mean titers.