Determination of equid herpesvirus 1-specific, CD8(+), cytotoxic T lymphocyte precursor frequencies in ponies

The frequency of antigen-specific, genetically restricted cytotoxic T lymph ocyte precursors (CTLp) was measured in peripheral blood mononuclear cells (PBMC) of ponies before and after infection with equid herpesvirus 1 (EHV1) . Split-well limiting dilution analysis (LDA) was developed to measure CTLp frequency using EHV1-infected Cr-51-labelled lymphoblasts as targets. Exte nsive characterisation showed that recombinant human interleukin-2, autolog ous antigen presenting cells and equine serum containing virus neutralising antibody were necessary for maturation of CTLp into effector CTL in vitro. CTLs were not induced when the equine serum (containing VN antibody) was r eplaced with either foetal calf serum or foetal equine serum (without VN an tibody), or seronegative equine serum. CTLp frequency decreased significant ly when CD8(+) lymphocytes were depleted from the induction cultures. There was good inter- and intra-assay reproducibility using both fresh and recov ered cryopreserved PBMC. Both EHV1 and EHV4 could be used to induce effecto r CTL which lysed EHV1-infected target cells. CTLp frequencies were measured in 2 groups of ponies: Group 1 consisted of two ponies (approx. 9 years old), which had multiple previous experimental infections with EHV1; Group 2 comprised five young (1-2 years) and two olde r (7 years) ponies which had presumed natural exposure to EHV1/EHV4 but no previous experimental infections. The results showed that CTLp frequencies were higher in the ponies of Group 1 compared with the others. Moreover, po nies with the higher CTLp frequencies were better protected against re-chal lenge infection with EHV1, showing reduced or absent clinical and virologic al signs. Consequently, measurement of EHV1-specific CTLp frequency is a po tential in vitro correlate of immunity which may be useful for screening ne w vaccines in horses before embarking upon challenge protection studies to confirm efficacy. (C) 1999 Elsevier Science B.V. All rights reserved.