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 microneutralization assay was adapted from a previously reported technique [48] . The effect of B cell supernatants (or purified IgG) on HCMV infectivity was measured by staining the HCMV Immediate Early Antigens (IEA, IE1+IE2) by indirect immunoperoxidase. IEA-positive nuclei were counted under the microscope. B cell supernatants containing irrelevant IgG antibodies were used as a negative control and a commercial preparation of human IgG, purified from the serum of HCMV seropositive patients (Cytotect; Biotest, Dreieich, Germany), as a positive control, respectively. Positivity was defined as ≥ 40% inhibition of IEA + cells, compared to negative control wells. HCMV microneutralization assays were also performed with the endotheliotropic HCMV VR1814, a derivative of a clinical isolate recovered from a cervical swab of a pregnant woman [49] , and HUVEC, as described above. Experiments were performed with cells at passage 2-6. Plaque reduction assay was performed as reported [50] .