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

EBV has long been used to immortalize human B-lymphocytes in vitro and to isolate human mAb [9] [10] [11] , but the method was not routinely embraced due to its poor efficiency and because the resulting transformed cells are frequently unstable and fail to grow or secrete IgG. Agonists of Toll receptor 9 (TLR9), e.g., CpG ODN 2006 (synthetic oligodeoxynucleotides that contain immunostimulatory deoxycytidyl-deoxyguanosine motifs), have been reported to improve EBV infectivity and cloning efficiency [12] , however, there is no clear consensus as to the best conditions for their use and which subset of B-lymphocytes should be used. We applied the EBV immortalization procedure in the presence of different amounts of CpG with or without IL-2 to 3 healthy donors selected on the basis of the high titer of CMV-specific IgG in the blood and we obtained an immortalization efficiency not comparable to that recently reported [12] . Therefore we re-evaluated all the steps crucial to cell immortalization, and established an optimized procedure of isolating human mAbs from the repertoire of immune donors. The resulting method is highly reproducible, effective and rapid. Further, it was validated by the successful isolation of clones of human B-lymphocytes that secrete high levels of IgG that bind and neutralize the human cytomegalovirus (HCMV). HCMV is a ubiquitous opportunistic virus that infects 50-90% of adult human populations and persists for the life of the human host after primary infection. HCMV infection of an immunocompetent individual generally results in subclinical disease; by contrast, HCMV infection of immunocompromised individuals may cause lethal infections [13] . Indeed, HCMV remains the single most impor-tant pathogen in hematopoietic stem cell transplantation [14] as well as, in organ transplantation [15] so that effective neutralizing mAbs would make a major contribution to eliminate HCMV in these patients.