To investigate Dom I and Dom II for antibody binding, expression plasmids were constructed which allowed for the synthesis of either domain in eukaryotic cells. In both cases the cloning strategy involved the attachment of a HA epitope tag at the amino terminus of the respective peptide in order to facilitate detection. Dom I comprised aa 133-343 of gB. The Dom IIspecific peptide consisted of the gB-specific residues 112-132 and 344-438 joined by a 5 aa (Ile-Ala-Gly-Ser-Gly) synthetic linker sequence. Following transient expression of the peptides in Cos7 cells, antibody binding was analyzed by indirect immunofluorescence. All four antibodies from Group A were reactive with the Dom I-specific peptide, whereas all Group B mabs recognized Dom II ( Fig. 4 and Table 2 ). The crystal structure of HSV-1 gB as well as the model of HCMV gB predict that for Dom I and Dom II discontinuous sequence stretches are essential for formation of either domain. Shorter protein fragments would be expected not to fold correctly. We tested this assumption by expressing a Dom II variant with five amino acids deleted at the carboxyl-terminus and observed complete loss of binding of all Group A-specific antibodies. Likewise, the continuous part of Dom I (residues 140-255) showed no antibody binding capacity (data not shown). Thus, further resolution of antibody epitopes will be possible only by generation of point mutants in Dom I and Dom II, respectively. Having identified Dom I and Dom II as new targets for neutralizing antibodies, we re-tested clonal antibody supernatants from 4 individuals to obtain information about the overall frequency of Dom I-and Dom II-specific antibodies in HCMV infected individuals. As shown in Table 1 , the frequency of Dom I and Dom II specific memory B cells was variable among different donors and in most cases considerably lower as compared to AD-1-specific B cells.