Figure 6. Mitochondrial DNA and OXPHOS protein abundance confirm the elevated OXPHOS signature of responders. (A) Relative mtDNA abundance in young (left panel) and older subjects (right panel) is significantly altered between vaccine responders (open boxes) and non-responders (filled boxes) at day 7 for young responders, with a trend at day 2 for older responders, compared to non-responders. mtDNA values are normalized with respect to the pre-vaccination time point. A subset from the cohort of older responders (n=7), older non-responders (n=8), young responders (n=6) and young non-responders (n=5) was evaluated. Dashed lines indicate the lower and upper range of values, with outliers indicated by open circles, bars indicating the 25th and 75th percentiles and dark horizontal lines the medians ** p<0.05, *p<0.1 (B) Induction of the mitochondrial chaperone protein HSP60 and mitochondrial Complex II component SDHA after influenza vaccination in young (left panel) compared to older adults (right panel), stratified by vaccine status (n=5 for each of the categories of older responder, older non-responder, young responder and young non-responder; relative pixel intensities of HSP60 and SDHA using β-actin as a loading control are normalized to day 0). For HSP60, a statistically significant increase in protein expression was found for young responders compared to non-responders at day 7 (p=0.04), and for older responders compared to non-responders at days 2 (p=0.007), 7 (p=0.0001), and 28 (p=0.0001). For SDHA, a significant increase in expression in young responders compared to non-responders was found at day 2 (p=0.007), and in older responders vs. older non-responders at days 2 (p=0.006), 7 (p=0.002), and 28 (p=0.007; p values were calculated using a t-test). (C) Representative Western blot of HSP60 and SDHA expression for young and older vaccine responders and non-responders.