Selective increase in rVV-EBNA1 recognition in MS. PBMCs from 24 MS patients and 24 healthy virus carriers were separately infected with rVV constructs expressing the EBV latent antigens EBNA1, 2, 3A, 3B, 3C, LMP1, and LMP2A, as well as the lytic EBV-encoded protein BZLF1. (A) Approximately 10–15% of PMBCs infected with the LMP1-encoding rVV consistently showed intracellular expression of LMP1 by flow cytometry, indicating that rVVs achieve EBV antigen expression in PBMCs. (B) EBNA1 protein expression in rVV-EBNA1ΔGA infected PBMCs compared with a fusion protein consisting of the C terminus of EBNA1 (aa 400–641) coupled to the heavy chains of an antibody (IgG-EBNA1). (C) IFN-γ specific ELISPOT responses in PBMCs to the indicated EBV antigens, to the virus vector backbone (rVV-TK⁻), and to influenza A virus (A/Aichi/68; H3N2) infection. Bars represent means. *, P = 0.005. (D) IFN-γ –specific ELISPOT responses to the indicated antigens, but in CD8-depleted PBMCs. *, P = 0.003.