G. complex) and (which encodes ICP8, a single-strand DNA-binding protein) deleted, was described previously (9, 10). A stock of 0.96) over 5 logs of DNA content. For statistical purposes, reactions yielding fewer than 6 copies of DNA were assumed to contain 3 copies. The geometric mean results for three independent experiments were determined. Anti-gD2 antibody responses. Blood was collected at 3 weeks after the last vaccination. Sera were separated and stored at ?20C. Titers of antibody to gD2 were measured by kinetic enzyme-linked immunosorbent assay. Briefly, Immulon-1 96-well plates (Dynex Technologies, Chantilly, Va.) were coated with 1 g of gD2 per ml overnight at 4C and then washed and blocked with buffer containing 2% bovine serum albumin. Sera diluted 1:1,000 (as determined in preliminary experiments) were added to each well in duplicate and incubated for 1 h at room temperature. Plates were washed with PBS containing 0.05% Tween 20 and incubated for 1 h with a 1:2,500 dilution of anti-guinea pig immunoglobulin G antibody conjugated with horseradish peroxidase (Sigma, St. Louis, Mo.). After the plates were washed, the increases in optical density per minute were measured and calculated for six time points at 30-s intervals, starting immediately after the addition of substrate (1 Step ABST; Pierce, Rockford, Ill.). Neutralizing antibody responses. Titers of antibodies that neutralize the infectivity of HSV-2 strain 333 were determined by using 0.1 ml of serum recovered from each animal. HSV-2 (100 PFU) was incubated with serial twofold dilutions of sera for 1 h and added in triplicate to Vero cell monolayers in six-well plates. The plates were incubated for 1 h at 37C, RQ-00203078 and the inocula were replaced with fresh medium containing 0.5% human immunoglobulin G (Abbott Labs, Chicago, Ill.) to prevent diffusion of cell-free RQ-00203078 virus. Two days later, the plates were stained and the plaques were counted. Regression lines representing the best fit for the data were calculated with Microsoft Excel software, and dilutions that reduced the numbers of plaques by 50% were calculated from the fitted lines. Quantification of HSV-specific IFN-+ CD8+ T cells. HSV-specific gamma-interferon-positive (IFN-+) CD8+ T cells infiltrating trigeminal ganglia (TG) were detected and quantified as previously described (18), with slight modification. Briefly, P815 cells were infected with HSV-2 strain 333 at a multiplicity of infection of 1 1 for 5 h. Four or six TG were pooled and dispersed in a 2-mg/ml concentration of collagenase type I (Sigma) for 1 to 1 1.5 h and passed through a 100-m-pore-size RQ-00203078 filter. TG cells were resuspended in Iscove’s modified Dulbecco’s medium supplemented with 10% fetal bovine serum, 10 U of interleukin-2 per ml, glutamine, and antibiotics. About 104 neurons and variable numbers of lymphocytes were recovered from each ganglion, depending on whether and when the animals were vaccinated and/or infected. TG lymphocytes were stimulated by coincubation in a 5-ml fluorescence-activated cell sorter (FACS) tube with 5 105 HSV-infected P815 cells FLJ32792 per TG for another 5 h in the presence of 10 mg of brefeldin A per ml (Sigma). The cells were washed and stained with fluorescein isothiocyanate (FITC)-conjugated anti-CD8 and perinidin chlorophyll protein (PerCP)-conjugated anti-CD45 monoclonal antibody (BD Pharmingen, San Diego, Calif.). After being washed again, the cells were fixed and permeabilized with Cytofix/Cytoperm buffer (BD Pharmingen) and washed with PermWash buffer (BD Pharmingen) according to the manufacturer’s protocol. Cells were stained with phycoerythrin (PE)-conjugated anti-IFN- monoclonal antibody and analyzed promptly with a FACSCaliber flow cytometry system RQ-00203078 (Becton Dickinson, Franklin Lakes, N.J.) by using CellQuest software (Becton Dickinson). Forward- and side-scatter gates were set by back gating from the CD45+ CD8+ population. Data continued to be acquired until the events recorded decreased to fewer than 500/s after PBS was added. Aliquots of cells equivalent to 0.5 TG were stained with FITC-conjugated anti-CD8 PE-conjugated CD3 and PerCP-conjugated anti-CD45 monoclonal antibodies (BD Pharmingen), and the number of CD8+ CD3+ CD45high cells were counted with a FACSCaliber (Becton Dickinson). To obtain spleen cells, freshly harvested spleens were crushed gently, and cells were passed through a 100-m-pore-size filter. The spleen cells were frozen and stored in liquid nitrogen until used. To quantify HSV-specific IFN-+ CD8+ T cells in the spleen, 2 106 cells were stimulated by cocultivation with P815 cells that had been infected.