Sara van den Berg

177 7 Duration of CMV infection Here we used a unique long-term longitudinal design to study the effect of duration of CMV infection on the immune system and on general health. We assessed how CMV-specific antibody levels developed within a cohort of older individuals (60-89 years at endpoint) over a follow-up time of 25-30 years. We relate duration of CMV infection to CMV-specific antibody levels, numbers of various T-cell subsets, function of CMV-specific T-cells, frailty and prevalence of cardiovascular disease. EXPERIMENTAL PROCEDURES Study design This study was performed with a selected group of participants from the longitudinal DoetinchemCohort Study (DCS) [32, 33], which we refer to as the DCS subcohort (n=289). Individuals have been followed as part of the DCS since 1987, with assessments every five years, resulting in six measurement rounds (1987-1992, 1993-1997, 1998-2002, 2003-2007, 2008-2012, 2013-2017). Every DCS measurement round, blood plasma samples were taken and stored for later use. The selection of the DCS subcohort from the DCS was described in more detail elsewhere [34]. Briefly, all active DCS participants 60-87 years of age ( n=289 ) were randomly stratified by sex, age, and a frailty index score (see below), leading to a selection of equal numbers of men and women, distributed evenly over the included age range and over three frailty groups (healthy, intermediate, frail). The DCS subcohort participants were invited for an additional blood sample between October 2016 and March 2017, which was not only used to retrieve plasma, but also to perform immune cell phenotyping on fresh whole blood and to store PBMCs for later use ( Figure 1A ). Furthermore, at round 5 and round 6, the participants’ general health was determined by a frailty index, which has been validated based on 36 deficits [34]. For most individuals the additional blood sample (round 7) taken for the DCS subcohort was the end point of study, for some (n=55) end point sampling was some months later (round 6).This study was approved by the Medical Ethics Committee of the University Medical Center Utrecht. All participants gave written informed consent for every DCS round and for this DCS subcohort study separately. Exclusion criteria From the original 289 participants, individuals were excluded from the longitudinal analyses if their follow-up time was less than 25 years (n=17) ( Supplementary Figure 1 ), or because of an inconclusive CMV-serostatus (n=4). Thus, the results are based on 268 individuals with an average follow up time of 27.7 years (minimum of 25, maximum of 30 years). Frailty index A frailty index as previously explained (Samson et al 2019) is based on 36 “health deficits”. The concept of this index was based on previous studies [30, 35-37] and was adapted for and validated in the DCS. The index is a variable with values between zero and one, zero representing the ‘best’ and one representing the ‘worst’ health status. The frailty index was calculated for each individual based on the data collected during the DCS measurements of