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

The B-lymphocytes representing the whole repertoire of a selected donor immortalized with the SEQUENTIAL method show a significantly higher viability than those obtained with the COMBINED method, and in consequence are easily clonable by limiting dilution, either in the presence or in the absence of polyclonal activators, such as CpG2006 and IL-2. TLR9 expression is increased after EBV infection. Thus bona fide, the addition of CpG2006 during the cloning phase enhances cell growth. However, we observed that cloning efficiency may be independent of CpG2006 and IL-2 in some donors, suggesting that it is also dependent on the genetic background or physiological state of the donor. This issue warrants further investigation. The presence of CpG2006 and IL-2 during the cloning phase may also have practical relevance for screening procedures. For instance, trace amounts of CpG2006 completely block HCMV infection in vitro, hampering screening by functional assays [20] . Moreover, CpG2006 is a potent inducer of cytokines, such as IL-12 and IFN-γ, potentially interfering with a number of bioassays [35] .