Sara van den Berg

92 Chapter 4 MATERIALS & METHODS Study design Healthy young and older adults Samples of healthy individuals covering a broad age range were combined from two cohorts. Samples of young adults (n=34), between 18 and 52 years of age, from unvaccinated controls or pre-vaccination participants were used from a study carried out in 2009-2011 (the Pandemic influenza vaccination trial, Netherlands Trial Register NTR2070) [42]. The study was approved by the Central Committee on Research Involving Human Subjects of the Netherlands. Samples of older adults (N=62), ≥60 years of age, were control samples from a study carried out in 2014-2015 (Influenza-like-illness-3, Netherlands Trial Register 4818) (Van Kaaijk et al., submitted). This study was approved by the acknowledged ethical committee METC Noord Holland. Both studies were carried out in accordance with the recommendations of Good Clinical Practice with written informed consent from all subjects, in accordance with the Declaration of Helsinki. Influenza virus A infected older adults during influenza virus infection Laboratory-confirmed Influenza virus A infected older adults were selected from the same study as the healthy older adults. In this prospective observational study participants were monitored for influenza-like-illness (ILI) in the flu season of 2014-2015 (Influenza-like-illness-3, Netherlands Trial Register 4818). Participants were instructed about influenza-like-illness (ILI) symptoms according to the Dutch Pel criteria, defined by fever (≥37.8°C) with at least 1 other symptom of headache, muscle pain, sore throat, coughing, runny nose, or chest pain [43] and to report ILI as soon as possible after onset. Nasopharyngeal and oropharyngeal samples were obtained within 72 hours of reporting ILI by standard procedures [44]. Influenza virus A infection was laboratory confirmed, and subtyped by PCR and sequencing in n=72 individuals by methods described previously [44]. These 72 influenza virus A confirmed patients were included in the current study. The H3N2 strain was detected in the majority of patients (n=64, of which n=20 clade 3C.3b, n=37 clade 3C.2a, n=7 inconclusive clade), and the H1N1 strain in the remaining individuals (n=8). Blood samples were collected within the first 72 hours of fever onset, and followed up after 2 weeks and 8 weeks. PBMC and serum isolation Peripheral blood mononuclear cells were obtained by Lymphoprep (Progen) density gradient centrifugation from heparinized blood, according to the manufacturer’s instructions. PBMCs were frozen in 90% fetal calf serum and 10% dimethyl sulfoxide at -135°C until further use. Serum was isolated out of tubes with clot-activation factor and stored at -80°C until further use. Cytomegalovirus (CMV)-specific antibodies Anti-CMV IgG antibody concentrations were measured either using a commercial ELISA or by a multiplex immunoassay developed in-house [45]. For healthy young adults, CMV- specific antibody levels were measured using a commercial ELISA (IBL international