Figure 6

Neutralization of Raji cell infection upon pre-incubation with antibodies generated following EBV-VLP immunization. (A) Infection assay. Because EBV does not plaque, infectivity of EGFP-EBV from a frozen stock was directly quantitated by cytometry. Ten microliters stock virus in the absence of serum yielded ~80% infection (green fluorescence) 73 h after infection of Raji and was selected for neutralization experiments performed 111 days after the initial boost. Five microliters stock virus without serum yielded ~50% infection (green fluorescence) 73 h after infection of Raji and was used for terminal neutralization experiments. (B) Day 111 (long-term) Neutralization Assay. Pooled sera from groups of five BALB/c mice immunized with EBVgp350/220-F VLP, UV-EBV, soluble recombinant gp350/220 ED or TNE (vehicle control) were serially diluted as indicated and then pre-incubated with EGFP-EBV to assess neutralization (Methods). Infected cells were incubated at 37°C for 72 h at which time EGFP+ Raji cells were enumerated by cytometry. X-axis, neutralizing antibody source and dilution; Y- axis, percent EGFP+ Raji cells post-infection. (C) Terminal Neutralization assay. Pooled terminal bleed sera from groups of five BALB/c mice immunized with EBVgp350/220-F VLP, UV-EBV or soluble recombinant gp350/220 ED were pre-incubated with EGFP-EBV to assess neutralization (Methods). Infected cells were incubated at 37°C for 72 h at which time EGFP+ Raji cells were enumerated by cytometry. X-axis, neutralizing antibody source; Y-axis, percent EGFP+ Raji cells post-infection. Terminal sera or mAb controls were pre-incubated 1:1 with EGFP-EBV causing further virus dilution such that ~25% of Raji cells were maximally infected (fluoresced green) when pre-incubated with non-blocking mAb-2 L10 or TNE. Pre-incubation with mAb-72A1 (neutralizing) served as the positive control. Results are expressed as mean ± standard deviations (SD). Horizontal black lines terminating in short vertical lines compare sets of neutralization experiments with 2-sided p-values indicated above the line.