cell binding is the main mechanism by which immune sera stop MuHV-4 infecting fibroblasts (Gill et al., 2006). However, the apparently neutralized virions can still infect myeloid cells via IgG Fc receptors (Rosa et al., 2007), and the limited capacity of immune sera to block host entry (Gillet et al., 2007b) suggests that an equivalent rescue operates in vivo. In contrast to this redundancy in cell binding, infection always requires membrane fusion. Therefore, the virion fusion machinery – gB and gH – is potentially a more universal neutralization target, and mAbs specific for gB or gH/gL (the predominant form of gH on extracellular virions) can block the infection of both fibroblasts and macrophages (Gillet et al., 2007b). However, several factors limit the vulnerability of gB and gH to neutralization. First, gB and gH/gL are poorly immunogenic in the context of whole virus (Gillet et al., 2007c). Neither is conforma- tionally stable without the other (Gillet et al., 2008b), so native virion epitopes tend to be lost unless the gH/gL/gB complex remains intact. In contrast, native gp150 epitopes are preserved even in small protein fragments (Gillet et al., 2007c). Thus gp150, which is not a neutralization target, dominates the antibody response of MuHV-4 carrier mice. gp150-specific antibodies both drive IgG Fc receptor- dependent infection and tend to suppress neutralizing responses (Gillet et al., 2007c), presumably by competition for antigen. Boosting carrier mice with recombinant gH/gL can overcome the immunodominance of gp150 and improve neutralization titres; boosting with gB works less well (Gillet et al., 2007b). Second, a complicated entry mechanism allows key fusion complex epitopes to remain hidden on extracellular virions. MuHV-4 infects by endocytosis (Gill et al., 2006). Fusion follows gH/gL dissociation in late endosomes (Gillet et al., 2008b), when gH changes to an antigenically very different form (gH-only). Since gH-only is inaccess- ible pre-endocytosis, gH-specific antibodies must neutral- ize by blocking the gH/gL to gH-only transition. gB, which forms a complex with gH/gL (Gillet & Stevenson, 2007a), may also not be fully revealed until after endocytosis (Gillet et al., 2009a). The gB N terminus (gB-NT, residues 25–65) is accessible and is a neutralization target for several herpesviruses (Ohlin et al. 1993; Okazaki et al., 2006) including MuHV-4 (Gillet et al., 2006), but is readily masked by O-linked glycans, making this neutralization strongly cell type-dependent (Gillet & Stevenson, 2007b). Most MuHV-4 gB-specific mAbs recognize it either before or after virion endocytosis, consistent with an extensive fusion-associated conformation switch (Gillet et al., 2008c). The recombinant gB extracellular domain (residues 1–724) displays mainly post-fusion epitopes. All known pre-fusion epitopes, including all known neutralization epitopes, lie within residues 1–423 (gB-N) (Gillet et al., 2008c), of which residues 81–423 form gB domains I+II (Heldwein et al., 2006). Most gB-specific neutralizing mAbs have been IgMs and therefore difficult to reproduce by vaccination (Gillet et al., 2006, 2008c). However, we http://vir.sgmjournals.org MuHV-4 neutralization have recently identified a potent neutralizing IgG mAb, SC- 9E8, that recognizes gB residues 81–423 (Gillet et al., 2009a). Delivering its epitope could therefore improve MuHV-4 neutralization. We tested here whether recom- binant forms of gB could elicit MuHV-4-neutralizing antibodies and protect against MuHV-4 challenge. RESULTS Protection against primary MuHV-4 lytic replication by vaccinia virus-expressed gB (VAC- gB) The MuHV-4 gB can be expressed at cell surfaces independent of other virion components by replacing its transmembrane and cytoplasmic domains with a glycosyl-phosphatidyl- inositol (GPI) anchor (Lopes et al., 2004). VAC-gB-GPI displayed both pre-fusion and post-fusion epitopes, but like transfected gB-GPI (Gillet et al., 2008b) showed a much greater representation of post-fusion epitopes than MuHV-4- infected cells (Fig. 1a). Immunization intraperitoneally (i.p.) with VAC-gB (Fig. 1b) induced a stronger gB-specific serum antibody response than i.p. infection with MuHV-4, as measured by flow cytometric staining of gB-GPI-transfected CHO cells. Immunization also afforded significant protection against intranasally (i.n.) MuHV-4 challenge (Fig. 1c). However, sera from the immunized mice showed no MuHV-4 neutralization (Fig. 1d). Similar results were obtained whether sera were used fresh or after heating (56 uC, 30 min) to inactivate complement, and whether or not reconstituted mouse complement (Sigma Chemical Co.) was added to the antibody-coated virions. Expression of gB-N from vaccinia virus We reasoned that gB-N (domains I+II) might more effectively present pre-fusion epitopes than the full-length extracellular domain, as it cannot switch to a post-fusion form. The neutralization epitope recognized by mAb SC- 9E8 was presented at least as well by VAC-gB-N as by VAC- gB, as was the distinct neutralization epitope recognized by IgM mAb BN-6E1 (Fig. 2a). Both pooled (Fig. 2b) and individual sera (Fig. 2c) from mice immunized with VAC- gB or VAC-gB-N showed gB/gB-N cross-reactivity, but substantially stronger recognition of the cognate form. Protection against primary MuHV-4 lytic replication by VAC-gB-N Priming with either VAC-gB or VAC-gB-N reduced MuHV-4 lytic replication after i.n. challenge (Fig. 3). Viral luciferase expression (Fig. 3a, b) and plaque assays (Fig. 3c) yielded similar results. VAC-gB protected marginally better than VAC-gB-N, particularly in noses, but both significantly reduced infection compared with controls. As in previous studies (Stevenson et al., 1999b), reducing lytic replication did not affect long-term latency 2543