Fig. 4. gB-primed sera fail to neutralize MuHV-4. (a) BALB/c mice were primed with vaccinia virus expressing gB, gB-N or a control insert. After 1 month, sera pooled from six mice per group were tested for their capacity to reduce BHK-21 or RAW-264 cell infections by eGFP+ MuHV-4, as assayed by flow cytometry 16 h after exposure to virus. Neutralization by sera pooled from MuHV- 4-infected mice (Imm ser) is also shown. Dashed lines show the level of infection by virus alone. (b) In a similar protocol to (a), VAC- gB-primed and VAC-gB-N-primed sera, each pooled from six C57BL/6 mice, showed no significant MuHV-4 neutralization. (c) Sera from the mice in (b) were tested individually for neutralization of BHK-21 cell infection. Again none was observed. The dashed line shows neutralization by pooled, MuHV-4-immune sera. (d) Sera pooled from unprimed or VAC-primed mice (n57) were collected either before or 16 days after MuHV-4 challenge and used to neutralize MuHV-4 virions for BHK-21 cell infection. Each line shows mean±SEM results. No significant difference was observed between VAC-gB-N-primed and control mice. http://vir.sgmjournals.org MuHV-4 neutralization 1988; Utz et al., 1989) because they fuse at the plasma membrane rather than in late endosomes: if gB conforma- tion switches are reversible as for vesicular stomatitis virus glycoprotein G (Roche et al., 2007) and some switching precedes actual fusion, then mAbs specific for the post- fusion HSV or HCMV gB could lock it non-productively in that form. Such a mechanism would be consistent with HSV and HCMV neutralization epitopes mapping to prominent features of post-fusion gB (Heldwein et al., 2006). In contrast, endocytic entry hides the MuHV-4 gB conformation changes from antibody. HCMV may be protected partly by competition between neutralizing and non-neutralizing antibodies (Speckner et al., 1999), but viral transmission would also segregate plasma membrane fusion from the antibody in immune donors, thereby protecting gB epitopes revealed after cell binding. Reducing viral lytic replication may reduce transmission events associated with primary infection, but optimally protecting naive subjects against infection by virus carriers is likely to require mucosal vaccination, either boosting IgA responses in carriers or generating primary IgA responses in naive subjects. First it is necessary to induce good neutralization. Attention so far has focussed on IgG, since IgG responses are more readily elicited by vaccination. Alpha- and betaherpesvirus gB vaccine studies (Willey et al., 1988; Nazerian et al., 1992; Pass et al., 2009) have generally not distinguished neutralization from IgG Fc receptor- dependent effector functions. This first description of gB immunization against a gammaherpesvirus gave protection consistent with the alpha- and betaherpesvirus studies, but showed that neutralization was not involved. Therefore, it seems unlikely that vaccination with gB alone will reduce infection prevalence. METHODS Mice. C57BL/6, BALB/c, Sv129 (Harlan UK Ltd) and C57BL/ 66Sv129 FcRc2/2FccRII2/2 mice (Taconic Europe) were housed at the Cambridge University Department of Pathology and infected when 6–8 weeks old under the Home Office Project Licence 80/1992. All experiments conformed to local and national ethical regulations. MuHV-4 (104 p.f.u.) was given i.n. under general anaesthesia. Vaccinia viruses (106 p.f.u.) were given i.p. For luciferase imaging, mice were injected i.p. with luciferin (2 mg per mouse), anaesthetized with isoflurane and scanned with an IVIS Lumina (Caliper Life Sciences). Quantitative comparisons were made by using Living Image software. Sera were collected from tail veins, or by cardiac puncture under terminal anaesthesia. Cells. BHK-21 fibroblasts (ATCC CCL-10), TK2143 cells (ATCC CRL-8303), NMuMG epithelial cells (ATCC CRL-1636), NIH-3T3- CRE cells (Stevenson et al., 2002), RAW-264 monocytes (ATCC TIB- 71), CHO-K1 cells (ATCC CCL-61) and CHO-K1 derivatives expressing either gp70, the main product of ORF4 (Gillet et al., 2007a), a fusion protein of the gH and gL extracellular domains with a GPI anchor (Gillet et al., 2007c), or a GPI-linked gB extracellular domain (CHO-gB) (Lopes et al., 2004), were grown in Dulbecco’s modified Eagle’s medium (DMEM; Invitrogen Corporation) sup- plemented with 10 % FCS (PAA Laboratories), 2 mM glutamine, 100 U penicillin ml21 and 100 mg streptomycin ml21. To make 2547