Peptides were synthesized by A and A Labs and purified to >95%
homogeneity by reverse-phase high-performance liquid chromatography
(HPLC). Purity was determined using analytical reverse-phase HPLC and
amino acid analysis, sequencing, and/or mass spectrometry. Peptides
were radio-labeled with the chloramine T method as described .
Characteristics of study population
Healthy males and females between 18 and 59 years of age were used
in this study. Exclusion criteria were body weight of < 45.4 kg and
established pregnancy. Recruited donors were vaccinated by arm
scarification with a VACV (Dryvax) vaccination as a prophylactic
measure either because of their potential exposure to VACV in a
laboratory or hospital setting or because of their enrollment into
military and health worker vaccination program. The blood draw was
performed within 1 year of the Dryvax vaccination. All experiments
carried out with human subjects were in compliance with the Helsinki
Declaration. Institutional Review Board approval (FWA# 00000032) and
appropriate informed consent were obtained for this study.
PBMC isolation and HLA typing
PBMC were isolated from heparinized blood by gradient centrifugation with a Histopaque-1077 (Sigma-Aldrich, St. Louis, MO) ,
suspended in fetal bovine serum containing 10% dimethyl sulfoxide, and
cryo-preserved in liquid nitrogen. Donor's PBMC were typed for HLA-A by
high-resolution polymerase chain reaction (Forensic Analytical
Molecular Genetics, San Francisco, CA).
A2.1 Tg mice used in this study were the F1 generation derived from crossing homozygous Tg mice (H-2b haplotype) expressing a chimeric gene (A2.1/Kb) consisting of the α1 and α2 domains of HLA and the α3 domain of H-2Kb with BALB/c mice (The Jackson Laboratory, Bar Harbor, ME) [4,33,34]. The HHD A2.1 strain 
was kindly provided by Dr. Jack Bennink (NIAID, NIH, Bethesda, MD). All
mice were bred and maintained following NIH guidelines and
Institutional Animal Care and Use Committee-approved animal protocols
(AAALAC# 000840 and OLAW# A3779-01).
Viruses and infection
The Western Reserve strain of VACV was obtained from Dr. Bernard Moss (NIAID). Mice were infected i.p. with 2 × 106 PFU of VACV. On day 7 post-infection, the mice were sacrificed and splenic CD8+ T cells were used in mouse IFN-γ ELISPOT assays.
Ex vivo IFN-γ ELISPOT
IFN-γ ELISPOT assays were performed as described [5,9]. In brief, for murine assays, 2 × 105 splenic CD8+ T cells were cultured with 105 human Jurkat cells (expressing the same A2.1/Kb construct as in the Tg mice) pulsed with 10 μg/ml of peptide. For human assays, 2 × 105 PBMC were incubated with 5 μg/ml of peptide. After a 20 h incubation at 37°C, plates were developed, and responses calculated as described [5,9,19]. Criteria for positivity were net spot-forming cells (SFC)/106 cells ≥ 20, stimulation index ≥ 2.0, and p-value ≤ 0.05 using a Student's t test in at least 2 out of 3 experiments.