Development of an efficient fluorescence-based microneutralization assay using recombinant human cytomegalovirus strains expressing green fluorescent protein.

MedImmune Vaccines has created four, live, attenuated human cytomegalovirus (HCMV) vaccine candidates, each derived from defined portions of the parental strains, Towne and Toledo. To determine each candidate's ability to induce HCMV specific immunity, a fluorescence-based microneutralization assay was developed using recombinants of Toledo and Towne which express enhanced green fluorescent protein (EGFP). Replication of the EGFP recombinants in cell culture was the same as the respective parental strains. Using the EGFP recombinants, this fluorescence-based microneutralization assay was compared with the traditional plaque reduction assay. Serum samples were analyzed by both the fluorescence microneutralization and plaque reduction assays and regression analysis showed a correlation of R2 > or = 0.90 between the two assays. As an alternative to measuring fluorescence, infected cells were examined microscopically and the number of green fluorescent cells was counted automatically. Regression lines between fluorescent cell counting and fluorescence in the well also showed a high correlation (R2 > or = 0.92). An excellent linear concordance in titers was observed between the two assays. Using the plaque reduction assay, serum samples were identified that preferentially neutralized the Toledo strain compared to the Towne strain. The same preferences were observed with the fluorescence-based microneutralization assay. This new assay is adaptable to rapid, automated collection of neutralization data and would therefore be suitable for the examination of large numbers of clinical serum samples.