J. S. May and P. G. Stevenson Fig. 7. Boosting gB-specific neutralizing antibody responses by post-exposure vaccination of MuHV-4 carrier mice. (a) Sera taken pre-boosting, 10 or 30 days post-boosting with vaccinia viruses expressing the full-length gB extracellular domain (VAC- gB), its N-terminal half (VAC-gB-N) or an irrelevant protein (VAC-cont), were pooled from groups of six C57BL/6 mice each. eGFP+ MuHV-4 virions were incubated with dilutions of the pooled sera and then used to infect either BHK-21 cells (0.3 p.f.u. per cell) or RAW-264 cells (3 p.f.u. per cell). Infected cells were enumerated 16 h later by flow cytometry. Dashed lines show infection with untreated virus. Each point gives the result for 10 000 cells. Thus by x2 test, the visible reductions in infection with VAC-gB or VAC-gB boosting were all highly significant (P,0.0001). The day 30 RAW-264 cell infection differences were not considered significant as they were not maintained over more than 1 serum dilution. (b) Individual sera taken at day 30 post- boosting were analysed for neutralization of BHK-21 cell infection as in (a). With 4 ml serum, both the VAC-gB (P,0.05) and VAC-gB-N boosted groups showed a significant reduction in infection (P,0.03 by Student’s two-tailed t-test) compared with the control group. With 1.3 ml serum, only the VAC-gB-N boosted group showed a reduction (P,0.03). (c) Sera taken at day 30 post-boosting were analysed for neutralization of RAW-264 cell infection (3 p.f.u. per cell). The boosted groups showed a wider distribution than the unboosted, but no significant reduction. expression maximized by lipopolysaccharide treatment (Rosa et al., 2007). Flow cytometry. Cells exposed to eGFP+ viruses were washed twice in PBS and analysed directly for green channel fluorescence. For specific staining, cells were incubated (1 h, 4 uC) with MuHV-4 gB- specific mAbs or with immune sera, followed by fluorescein- conjugated rabbit anti-mouse IgG pAb (Dako Cytomation). mAbs BN-1A7 (IgG2a), BN-6E1 (IgM) and SC-9E8 (IgG2a) all recognize epitopes in gB-N and are specific for pre-fusion gB, whereas MG- 1A12 is specific for post-fusion gB (Gillet et al., 2008c). All cells were washed twice in PBS after each antibody incubation and analysed on a FACS Scan that runs the CellQuest software (BD Biosciences). ACKNOWLEDGEMENTS This work was supported by the Wellcome Trust (GR076956MA and WT089111MA) and by the Medical Research Council (G0701185). 2550 REFERENCES Adler, H., Messerle, M., Wagner, M. & Koszinowski, U. H. (2000). Cloning and mutagenesis of the murine gammaherpesvirus 68 genome as an infectious bacterial artificial chromosome. J Virol 74, 6964–6974. Akula, S. M., Pramod, N. P., Wang, F. Z. & Chandran, B. (2001). Human herpesvirus 8 envelope-associated glycoprotein B interacts with heparan sulfate-like moieties. Virology 284, 235–249. Bennett, N. J., May, J. S. & Stevenson, P. G. (2005). Gamma- herpesvirus latency requires T cell evasion during episome mainten- ance. PLoS Biol 3, e120. Burton, D. R., Stanfield, R. L. & Wilson, I. A. (2005). Antibody vs. HIV in a clash of evolutionary titans. Proc Natl Acad Sci U S A 102, 14943– 14948. Cranage, M. P., Kouzarides, T., Bankier, A. T., Satchwell, S., Weston, K., Tomlinson, P., Barrell, B., Hart, H., Bell, S. E. & other authors (1986). Identification of the human cytomegalovirus glycoprotein B Journal of General Virology 91