We next determined whether the mixtures of ASYMP epitopes from HSV-1 gB, VP11/12, and VP13/14 will induce functional CD8
+ T cells in terms of IFN-γ, TNF-α-production, and CD107 expression (i.e., cytotoxic activity). As shown in
Figures 4A through
4C and in
Table 2, 9 days after the challenge, at the time of acute infection, three of the nine peptides (one from each Ag) elicited significant IFN-γ–producing CD8
+ T cells in PBMC from HLA-Tg rabbits. Of the three peptides selected from VP11/12, VP11/12
220–228 elicited significantly higher IFN-γ–producing CD8
+ T cells (
Tables 2,
3). Lower, but significant levels of IFN-γ–producing CD8
+ T cells were generated against VP11/12
702–710, but no cytotoxic activity was detected against the remaining VP11/12
66–74 peptide (
Tables 2,
3). Similar results were obtained when CD8
+ T cells were tested for production of TNF-α (
Figs. 4D–F;
Table 2). In addition, CD8
+ T cells derived from immunized HLA-Tg rabbits showed cytolytic activity against gB
561–569 peptide and low but consistent cytotoxic T-cell responses against the remaining gB peptides (gB
17–25 and gB
342–350;
Figs. 4G–I;
Tables 2,
3). As expected, none of the peptides elicited significant CD8
+ T-cell responses in mock-vaccinated HLA-Tg rabbits (
Fig. 4). Most animals (6 of 10) developed CD8
+ T-cell responses against a single gB peptide (gB
561–569;
Tables 2,
3). In contrast, 6 of 10 animals showed potent IFN-γ–producing CD107
+CD8
+ cytotoxic T cells to two VP11/12 peptides (VP11/12
66–74 and VP11/12
220–228;
Tables 2,
3). Interestingly, most animals developed CD8
+ T-cell responses against all three VP13/14 peptides (
Tables 2,
3). Six of 10 animals showed potent IFN-γ–producing CD107
+CD8
+ cytotoxic T cells to VP13/14
504–512 while five showed significant response to VP13/14
286–294 and VP13/14
544–552. Collectively, the results point to polyfunctionality of CD8
+ T cells induced by mixtures of ASYMP epitopes from HSV-1 gB, VP11/12, and VP13/14.