MuHV-4 neutralization Fig. 6. Boosting gB-specific antibody responses by post-exposure vaccination of MuHV-4 carrier mice. (a) C57BL/6 mice were infected i.n. with MuHV-4 and 3 months later boosted i.p. with vaccinia virus expressing either the full-length gB extracellular domain (VAC-gB), just its N-terminal half (VAC-gB-N) or a control insert (VAC-cont). Sera were taken before vaccinia infection (pre-boost), 10 and 30 days later, and tested for gB reactivity by flow cytometric staining of CHO cells expressing gB or gB-N. CHO cells either untransfected or transfected with the MuHV-4 gp70 provided staining controls. Bars show mean±SD fluorescence intensities for sera pooled from six mice each. VAC-gB-N and VAC-gB both significantly increased CHO-gB-N staining at days 10 and 30 compared with VAC-cont (P,0.0001 by Student’s two-tailed t-test). The increase with VAC-gB-N was significantly greater than with VAC-gB (P,0.0001). VAC-gB but not VAC-gB-N significantly increased CHO-gB staining at days 10 and 30 compared with VAC-cont (P,0.0001). (b) Sera from the individual day 30 boosted mice in (a) were assayed for CHO-gB and CHO-gB-N reactivity. Although boosting increased staining, this did not reach statistical significance (P.0.05) because of wide individual variation. Each point shows an individual mouse and the bars show geometric means. (c) C57BL/6 MuHV-4 carrier mice (3 months post-infection) were analysed by flow cytometry for serum binding to CHO-gB-N cells. Each point shows the result for one mouse. (d) A separate set of MuHV-4-infected C57BL/ 6 mice were compared for serum reactivity to CHO-gB, CHO-gB-N and CHO-gp70 cells. Each point shows the result for one mouse. There was no obvious correlation for individual mice between the responses to each target. RAW-264 monocytes. After 2 h, phosphonoacetic acid was added (100 mg ml21) to prevent lytic spread. After 16 h, the cells were harvested and assayed for eGFP expression by flow cytometry. In preliminary experiments, virus titres by EF1a-driven eGFP expression in BHK-21 cells equalled or exceeded plaque assay titres, and in RAW-264 cells equalled or exceeded BAC cassette-associated eGFP http://vir.sgmjournals.org 2549