PBMC were screened by IFN-γ ELISPOT using 34 pools of overlapping peptides representing 16 HSV-2 ORFs. Results are from 3 HSV-2 seropositive subjects (A–C), an HSV-1+/2− subject (D) and an HSVneg subject (E). The dashed line represents the median SFC/106 PBMC from the DMSO control wells; the solid line represents the threshold for positivity (4 times the mean of the DMSO control wells and ≥55 SFC/106 PBMC). IFN-γ ELISPOT responses to PHA control wells were 1,258 SFC/106 PBMC (HSV-2+ Subject in A), 2,553 SFC/106 PBMC (HSV-2+ Subject in B), 2,329 SFC/106 PBMC (HSV-2+ Subject in C), 1,145 SFC/106 PBMC (HSV-1+/2−Subject in D) and 2,811 SFC/106 PBMC (HSVneg Subject in E).