Cytotoxic T-lymphocyte responses to a polymorphic Epstein-Barr virus epitope identify healthy carriers with coresident viral strains

Cytotoxic T-lymphocyte (CTL) responses to Epstein-Barr virus (EBV) tend to focus on a few immunodominant viral epitopes; where these epitope sequences are polymorphic between EBV strains, host CTL specificities should reflect the identity of the resident strain. In studying responses in HLA-B27-posi tive virus carriers, we identified 2 of 15 individuals who had strong CTL m emory to the pan-B27 epitope RRIYDLIEL (RRIY) from nuclear antigen EBNA3C b ut whose endogenous EBV strain, isolated in vitro, encoded a variant sequen ce RKIYDLIEL (RKIY) which did not form stable complexes with B27 molecules and which was poorly recognized by RRIY-specific CTLs. To check if such ind ividuals were also carrying an epitope-positive strain (either related to o r distinct from the in vitro isolate), we screened DNA from freshly isolate d peripheral blood mononuclear cells for amplifiable virus sequences across the EBNA3C epitope, across a different region of EBNA3C with type 1-type 2 sequence divergence, and across a polymorphic region of EBNA1. This showed that one of the unexplained RRIY responders carried two distinct type 1 st rains, one with an RKIY and one with an RRIY epitope sequence. The other re sponder carried an RKIY-positive type 1 strain and a type 2 virus whose epi tope sequence of RRIFDLIEL was antigenically cross-reactive with RRIY. Of 1 5 EBV-seropositive donors analyzed by such assays, 12 appeared to be carryi ng a single virus strain, one was coinfected with distinct type 1 strains, and two were carrying both type 1 and type 2 viruses. This implies that a s mall but significant percentage of healthy virus carriers harbor multiple, perhaps sequentially acquired, EBV strains.