MuHV-4 neutralization Fig. 2. Primary antibody responses to gB vaccines in C57BL/6 and BALB/c mice. (a) BHK-21 cells were left uninfected (UI) or infected (1 p.f.u. per cell, 14 h) with vaccinia virus expressing either the full-length gB extracellular domain (VAC-gB), just its N- terminal domains (VAC-gB-N), or a control insert (VAC-cont). gB expression was analysed by flow cytometry. mAbs BN-6E1 and SC-9E8 recognize neutralizing epitopes on pre-fusion gB. MG-1A12 recognizes a post-fusion epitope that is not contained within gB-N. (b) Sera from mice infected i.p. 1 month previously with VAC-gB, VAC-gB-N or a control virus were pooled from six mice per group and assayed for reactivity to gB, gB-N and gp70 (as a negative control) by flow cytometry of transfected CHO cells. Bars show mean±SD values. For both C57BL/6 and BALB/c mice, VAC-gB-N primed CHO-gB-N- specific responses significantly better than did VAC-gB, and VAC-gB primed CHO-gB-N-specific responses significantly better than did VAC-gB-N (P,0.0001 by Student’s two-tailed t-test). (c) Individual sera from the C57BL/6 mice in (b) were assayed for reactivity to gB and gB-N. Each point shows staining of the relevant CHO cells by serum from one mouse. The bars show mean values. VAC-gB-primed mice showed significantly stronger staining of both CHO-gB (P,0.0001) and CHO-gB-N (P,0.01), as did VAC-gB-N-primed mice (P,0.0001 by Student’s two-tailed t-test). Each group also showed significantly stronger staining of the cognate form of gB (P,0.0001). specific antibody responses varied considerably between individual mice (Fig. 6b). The gB-N-specific antibody responses of non-boosted carriers (Fig. 6c) were also highly variable, and showed no correlation with responses to full- length gB or the MuHV-4 gp70 (Fig. 6d). Thus, even homozygous sibling mice varied considerably in antibody response. Pooled C57BL/6 mouse sera (Fig. 7a) neutralized MuHV-4 for BHK-21 cell infection better after boosting with either VAC-gB or VAC-gB-N compared with the control. Boosting improved the neutralization of RAW-264 cell infection only at the 10 day time point. The effects were not large, and individual neutralization titres (Fig. 7b, c) overlapped between groups, consistent with the varied boosting efficacy observed by flow cytometry (Fig. 6). Thus, VAC-gB and VAC-gB-N were both capable of boosting neutralizing antibodies, but worked well in few mice, presumably because http://vir.sgmjournals.org few mice made primary gB-specific neutralizing responses. VAC-gB and VAC-gB-N similarly increased significantly gB- N- and gB-specific antibody responses in BALB/c carrier mice (Supplementary Fig. S1, available in JGV Online) with only a small improvement in neutralization (Supplementary Fig. S2, available in JGV Online). As the boosting of neutralization was similar between VAC-gB and VAC-gB-N, the limiting factor appeared to be the composition of the primary antibody response to MuHV-4 rather than the level of subsequent gB-N delivery. DISCUSSION We sought to understand why MuHV-4-infected mice make poor neutralizing antibody responses to gB. Specifically, we tested whether recombinant forms of gB containing known neutralization epitopes could stimulate 2545