J. S. May and P. G. Stevenson Fig. 3. Protection against MuHV-4 lytic replication by vaccination with gB. (a) BALB/c mice were immunized i.p. with vaccinia viruses expressing gB, gB-N or an irrelevant insert (VAC-cont), and 1 month later challenged i.n. (104 p.f.u.) with luciferase+ MuHV-4. Viral replication in lungs and noses was monitored by luciferin injection and CCD camera scanning. A representative image is shown of mice 5 days post-challenge. (b) Mice were immunized then infected with luciferase+ MuHV-4 as in (a). Each point shows the luciferase signal for one mouse. The bars show mean values. Dashed lines show the lower limits of signal detection. Priming with either VAC-gB or VAC-gB-N significantly reduced luciferase signals in lungs at all time points (P,0.03 by Student’s two-tailed t-test). Luciferase signals in noses were also significantly reduced in VAC-gB-primed mice at days 2 (P,0.01) and 5 (P,0.04), and in VAC-gB-N-primed mice at day 2 (P,0.01). (c) BALB/c or C57BL/6 mice were immunized i.p. with vaccinia virus recombinants then challenged i.n. with MuHV-4 as in (b), but with luciferase” MuHV-4. Lungs and noses were titrated for infectious virus at 6 days post-infection by plaque assay. Each point shows the titre of one mouse. The bars show geometric means. Both BALB/c (P,0.04) and C57BL/6 mice (P,0.01) lung titres were significantly reduced by priming with either VAC-gB or VAC-gB-N. C57BL/6 mice nose titres were also reduced, although this was only statistically significant for VAC-gB (P,0.01). (d) Latent virus loads in spleens were measured by infectious centre assay at 2 months post-challenge of vaccinated C57BL/6 mice. The bars show mean±SD titres of five mice per group. neutralizing responses in either naive or carrier mice. Immunization with either the full-length gB extracellular domain or its N-terminal half reduced subsequent MuHV- 4 lytic replication. However, neither construct elicited detectable primary neutralizing responses, even though both were recognized by a potent neutralizing mAb. Some boosting of neutralizing antibodies in carrier mice was possible, but the effect was not large, there was consid- erable variation between individual mice, and gB-N was no more effective than gB. We conclude that gB-directed MuHV-4 neutralization is fundamentally limited by the difficulty of targeting the few vulnerable epitopes it presents. How does an essential component of the MuHV-4 entry complex avoid being a good neutralization target? It seems likely that post-endocytic conformation changes allow key gB epitopes to remain hidden on extracellular virions. 2546 Thus, while gB-N binds to cells (Gillet & Stevenson, 2007b), this must occur downstream in entry as blocking heparan sulfate binding by gp70 and gH/gL largely abrogates virion binding (Gillet et al., 2009b). The increased susceptibility of gL2 virions to gB-directed neutralization (Gillet et al., 2009a) implies that gH/gL helps to hide gB; the regulation of virion binding by gp150 (de Lima et al., 2004) suggests that it could also be involved. While the neutralization epitope defined by mAb SC-9E8 is accessible it is evidently difficult to target, much like broadly neutralizing human immunodeficiency virus epitopes (Burton et al., 2005). Such resistance to neutralization may be a common feature of persistent viruses. Herpes simplex virus (HSV) and human cytomegalovirus (HCMV) may be more easily neutralized in vitro by gB- specific antibodies (Cranage et al., 1986; Highlander et al., Journal of General Virology 91