Generation of potent neutralizing human monoclonal antibodies against cytomegalovirus infection from immune B cells

Funaro, Ada; Gribaudo, Giorgio; Luganini, Anna; Ortolan, Erika; Lo Buono, Nicola; Vicenzi, Elisa; Cassetta, Luca; Landolfo, Santo; Buick, Richard; Falciola, Luca; Murphy, Marianne; Garotta, Gianni; Malavasi, Fabio

CD22 + lymphocytes were purified by magnetic selection (Miltenyi Biotec, Bologna, Italy) from PBMCs pooled from 5 healthy donors, then CD22 + cells were divided into three aliquots that were exposed to different EBV-based methods for immortalization. For the BASIC process, CD22 + /IgG + lymphocytes were isolated by depletion of IgM + cells with a MoFlo ® High-Performance Cell Sorter (Dakocytomation, Glostrup, Denmark) and cultured (10 6 /ml) in 24 well plates in Iscove's Modified Dulbecco's medium (IMDM) supplemented with L-glutamine, 100 μg/ml streptomycin and 100 U/ml penicillin, non-essential amino acids and 10% FBS (complete medium) for 15 h, in the presence of EBV-containing supernatant (from B95-8 marmoset lymphoma cells, ATCC, Washington, DC, cat. no. CRL-1612) 1:1 (v/v) at 37°C and 5% CO 2 . The cells were then washed c. Sera were tested in the HCMV microneutralization assay using the AD169 strain and HELF [48] . Numbers indicate dilution of serum required for 50% inhibition of HCMV infectivity. The number of viable lymphoblasts was measured microscopically by trypan blue dye exclusion. Where indicated, the DNA intercalating, fluorescent dye propidium iodide (PI, Sigma Aldritch) was used with a FACSCalibur flow cytometer and CellQuest Software (Becton Dickinson, Franklin Lakes, NJ) to assess cell viability. Viable cells were defined as those with a high forward and orthogonal scatter, characteristic of lymphoblasts, and excluding PI.