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

Using the SEQUENTIAL method, we obtained natural antibodies neutralizing HCMV infectivity by immortalizing B-lymphocytes from a healthy donor (CMV5) and from a patient recovering from acute HCMV infection (CMV7). High titers of anti-HCMV-specific IgG coupled with the virus-neutralizing activity of the sera were the criteria for selecting the two donors from a larger panel ( Table 1 ). The immortalized B-lymphocytes were cloned by limiting dilution. After 3 weeks, clusters of cells growing in wells were appreciable and the IgG concentration in the clone supernatants was 10-40 μg/ml/10 6 cells, as determined by means of an immunoenzymatic method, measuring IgG concentrations in the media normalized for cell densities. Clones producing HCMV-specific IgG were identified by different primary screening assays. In a first experiment a total of 1,664 clones were tested either by their binding to specific HCMV envelope glycoprotein domains (such as those from gB or gH, which have been shown to induce neutralizing antibodies) [44] , or by their ability to neutralize HCMV infectivity in vitro [45, 46] . The effect on HCMV infectivity was determined by an HCMV microneutralization assay based on the evaluation of the ability of clone supernatants to interfere on the infectivity of HCMV AD169 strain (a laboratory strain from ATCC, cod. VR-538) in human Embryo Lung Fibroblasts (HELF). This strategy yielded 44 clones producing mAbs specific for the gB, and 15 for gH. Nine of these displayed a neutralizing activity of > 40%. Those supernatants that were negative in the gB and gH ELISAs, or in the neutralization assay were also screened in an ELISA that detects human IgG that bind to HCMV virion proteins extract (BEIA-CMV), and 8 samples were further selected by this screening assay. In a second experiment, the immortalized Blymphocytes were cloned by limiting dilution and after 3 weeks, 324 clones were screened directly for their neutralizing activity. Of these, 20 produced IgG with a neutralizing activity of > 40%, among these, 2 wells were found to bind gB, while the remaining 18 did not bind in either the gB or gH domains.