Destabilized pp65Vac enables recognition of APC by splenic effectors derived from fusion peptide immunizations.

(A) A bulk line was derived after repetitive IVSs (5 × ), as described in “Materials and methods,” from the 100-nmol immunization shown in Figure 1 that was a homogeneous CD8 T-cell population by flow cytometry (data not shown). A CRA was performed in which targets (JA2.1 T cells) were either infected with VV or pulsed with peptides (data not shown). JA2.1 cells were infected with Ub-R-pp65Vac (●) or pp65Vac (▪) for 16 hours at an MOI of 3. Nonspecific lysis is shown (Ub-R-IEVac, ▴) for VV-infected targets and was less than 5% for peptide-loaded T2 cells (data not shown). Error bars represent averages of 4 separate experiments carried out on different days. Details of the construction of Ub-R-pp65Vac are presented in “Materials and methods.” (B) HLA A2.1/K^bmice were immunized subcutaneously once with 50 nmol Tet₆₃₉V alone (50) or with 25 μg CpG ss-ODN (50D) as described in the legend to Figure 3. Targets are either JA2.1 T cells infected with Ub-R-pp65Vac (● or ▵) or Ub-R-IEVac (▪ and ⋄) as described in the legend to Figure 1.