The sensitivity of the assay was 15.6 mIU/ml and the minimum detection level 31.2 mIU/ml. Results were expressed as log2 units or as reciprocal titres. We defined the protective level of HAI measles antibody as a titre of log2 ≥ 3 which equates to 125 mIU [12]. Ex vivo measles effector cell assays: After separation of blood on Lymphoprep PBMC were used in the ex vivo interferon-gamma (IFN-γ) ELIspot assay as previously described [14]. The cells were infected for 2 h with Edmonston (E-D) wild type measles virus or E-Z measles vaccine virus which had been grown for 3 days on a culture of Vero cells in RPMI/10% Foetal Calf Serum (R10F).

The multiplicity of infection was 0.1

and 1.0 for the two strains respectively. The infected cells were then washed BMN 673 price and plated in duplicate at 105 cells/well in R10 with 10% AB serum (R10AB, Sigma). Control PBMC were mock infected with R10F harvested after culture of uninfected Vero cells for 3 days. In addition duplicate wells containing 105 PBMCs were also stimulated with a pool of overlapping 20-mer measles fusion peptides (NMI Peptides) dissolved in normal saline and 0.4% DMSO and used at a final concentration of 2 μg/ml Pfizer Licensed Compound Library in R10AB. Control cells were incubated in medium containing 0.02% DMSO which was the same concentration as that in the test wells. Phytohemagglutinin (5 μg/ml) was used as a positive control. Spots were counted using the AID ELIspot plate reader (Autoimmune Diagnostika). The mean number of spots in the duplicate wells of the negative control was subtracted from the mean spot count in the positive wells; an assay with a control value of ≥50 spots per well was regarded as invalid. Measles

memory cell assays: As described previously 106 PBMC were cultured for 10 days in R10AB with 105 UV irradiated PBMC infected with measles virus [15] or with pooled measles nucleoprotein or fusion peptides as described above. Controls consisted of PBMC mock infected with Vero cell medium and treated in the same way as above. Intracellular cytokine staining (ICS): Following stimulation, cells were permeabilised and stained for flow cytometry analysis as previously described [13]. The staining panel used at 9 and 9.5 months was anti-CD8 17-DMAG (Alvespimycin) HCl FITC, anti-CD4 PE, anti-CD69 PerCP and anti-IFN-γ APC. At 18 months, the panel was anti-IFN-γ FITC, anti-CD4 PE, anti-CD8 PerCP and anti-IL-2 APC. All antibodies were supplied by BD Biosciences. Cytokines in plasma or supernatants: Plasma was frozen at −40° C until assayed using the Bio-Plex 200 Suspension Array system (Bio-Rad) according to the manufacturer’s instructions. FOXP3 mRNA expression: RNA was extracted from whole blood collected in Paxgene tubes (PreAnalytix, QIAGEN) and frozen at −40° C until RNA extracted.