Figure 11

Compendium of study population and methods. An overview of sampling, pre-analytical steps, experimental procedures, data mining and statistical analysis employed in the present investigation. The study population was composed of 15 subjects, both sexes, age ranging from 18 to 57 years old. Whole blood samples were collected from each volunteer before [NV_(D0)] and 30–45 days after [PV_(D30–45)] 17DD-YF 1/5 fractional dose primary vaccination in vacuum system containing sodium heparin as anticoagulant. PBMC and plasma samples were isolated by Histopaque-1077 density gradient. Plasma samples were pre-treated with ecteola-cellulose for heparin removal. Heparin-free plasma were used for detection of YF-specific neutralizing antibodies by micro Focus Reduction Neutralization—Horseradish Peroxidase (μFRN-HRP) test. Plasma aliquot was employed to characterize the profile of systemic soluble mediators by Luminex xMAP technology. PBMC samples were submitted to long-term in vitro culture to evaluate YF-specific phenotypic/functional features of cellular immunity by Mass Cytometry (CyTOF) using a cocktail of metal-isotope-tagged antibodies (MitAbs). Cell culture supernatant was collected to quantify soluble mediators by high-throughput microbeads xMAP technology. Distinct approaches were used for data mining and statistical analysis, including: quantification of neutralizing antibodies (µFRN-HRP status), analysis of phenotypic/functional features of cellular immunity, use of systems immunology tools and kinetics timeline of soluble mediators.