The antigenic spectrum of EBV virions is enlarged through the construction of BNRF1-latent protein gene fusions.

(A) The insertion of latent protein epitopes into the major tegument protein BNRF1 enables them to be incorporated into the tegument of VLPs/LPs. (B) Modification of BNRF1 to include an antigenic fragment from EBNA3C (E3C) does not influence lytic replication in producer cells. The late lytic gene product gp350 was detected at the surface of induced 293/wtEBV and 293/EBV-E3C producer cells by staining with α-gp350 and α-mouse IgG-Cy3 antibodies. (C) EBV virions that encode a BNRF1-EBNA3C fusion protein stimulate BNRF1- and EBNA3C-specific CD4+ T cells. Autologous LCLs were pulsed with various amounts (1 x 104 to 1 x 106 genome equivalents (geq)) of wtEBV or EBV-E3C and then cocultured with CD4+ T cells that were specific for BNRF1 VSD (1006–1017 aa) or EBNA3C 5H11 (325–339 aa) epitopes. In parallel, LCLs were pulsed with control peptides (μg to ng quantities) prior to coculture with CD4+ T cells. T-cell activation was determined by measuring secreted IFN-γ by ELISA. (D) A neutralizing antibody that recognizes gp350 impairs the antigenicity of wtEBV and EBV-E3C. The neutralizing antibody 72A1 was titrated (50, 5 and 0 μg/mL) and incubated with 1 x 106 geq of wtEBV and EBV-E3C. Thereafter, supernatants were used in T-cell activation assays. The data displayed in each chart represents triplicate values and error bars represent standard deviation. Furthermore, each graph is a representative experiment of at least three.