J. S. May and P. G. Stevenson Fig. 1. Expression of gB epitopes by vaccinia virus-expressed gB and gB-N. (a) BHK-21 cells were left uninfected (UI) or infected (1 p.f.u. per cell, 14 h) with either MuHV-4 or a vaccinia virus expressing a GPI-linked gB extracellular domain (VAC- gB). Cell surface gB expression was analysed by flow cytometric staining of pre-fusion gB with mAb BN-1A7, and post-fusion gB with MG-1A12. (b) C57BL/6 mice were infected i.p. with either MuHV-4 or VAC-gB. Sera collected 2 weeks later were used to stain CHO cells either untransfected or transfected with gp70 or GPI-linked gB. Each point shows the median fluorescence intensity for a 1/500 serum dilution from one mouse. The bars show mean values. CHO-gB staining was significantly greater after VAC-gB infection than after MuHV-4 infection (P,0.01 by Student’s two-tailed t-test). In this and subsequent experiments, consistent results were obtained with least two further serum dilutions. (c) Mice were immunized i.p. with either VAC-gB or a control, and 1 month later infected i.n. with MuHV-4. Infectious MuHV-4 in lungs was plaque assayed at 5 days post-challenge. Each point shows the titre of one mouse. The bars show geometric means. (d) Sera from mice infected with either VAC-gB or MuHV-4 were used to neutralize eGFP+ MuHV-4 for BHK-21 cell and NMuMG cell infections. Infection was assayed by flow cytometry for eGFP expression 18 h after exposure to virus/antibody. Each point shows mean±SD of triplicate infections. The dashed lines show infection with virus alone. titres: by 2 months post-infection, splenic infectious centre titres were equivalent between vaccinated and unvaccinated mice (Fig. 3d). We were more concerned whether the protection reflected virus neutralization. Despite good gB- specific antibody responses, sera from gB-N-primed mice failed to neutralize MuHV-4 either as pools (Fig. 4a, b) or as individual sera (Fig. 4c). gB-primed sera even showed a modest enhancement of RAW-264 cell infection compared with naive controls (Fig. 4b). Post-challenge serum neutralization titres were also similar between vaccinated and unvaccinated mice (Fig. 4d). Therefore, although both gB and gB-N presented neutralizing epitopes, protection was independent of detectable neutralization. Protection by vaccination with gB-N depends on IgG Fc receptors Both neutralizing and non-neutralizing passive antibodies reduce MuHV-4 lytic replication by IgG Fc receptor- dependent mechanisms (Wright et al., 2009). The protection 2544 by VAC-gB-N appeared to be similar, as it was lost in IgG Fc receptor-deficient mice (Fig. 5a). Immune sera transferred from VAC-gB-N-immunized to naive mice also gave IgG Fc receptor-dependent protection (Fig. 5b). Boosting neutralizing antibody responses with VAC-gB and VAC-gB-N We next tested whether VAC-gB-N could boost neutral- izing antibodies in MuHV-4 carrier mice. Previously (Gillet et al., 2007b), it was found that VAC-gB was effective in only a minority of mice. C57BL/6 mice infected i.n. with MuHV-4 were 3 months later boosted i.p. with VAC-gB, VAC-gB-N or an irrelevant VAC control. The mice were bled before boosting, 10 days after (acute response) and 30 days after (longer-term response) (Fig. 6). VAC-gB-N boosted gB-N-specific antibodies significantly better than VAC-gB without improving the recognition of CHO-gB cells, again emphasizing that gB-N and gB are antigenically quite different (Fig. 6a). The magnitude of gB- and gB-N- Journal of General Virology 91