3554 Nucleic Acids Research, 2005, Vol. 33, No. 11 Figure 2. PAGE analysis of fully deprotected and RP-HPLC-purified oligonucleoside phosphorothioates under denaturing conditions (20% polyacrylamide–7 M urea, 1· TBE buffer, pH 8.3). Left lane: CpG ODN 1555 synthesized from commercial 2-cyanoethyl deoxyribonucleoside phos- phoramidites and deprotected by treatment with pressurized ammonia gas ( 10 bar) for 12 h at 25 C. Middle lane: CpG ODN fma1555 synthesized from phosphoramidites 1a–d and deprotected by treatment with pressurized ammonia gas ( 10 bar) for 12 h at 25 C followed by heating in 1· PBS (pH 7.2) for 626 h at 37 C. Right lane: ODN fma1556 synthesized and deprotected under conditions identical to those used for CpG ODN fma1555. Oligonucleo- tides are visualized as purple bands upon staining the gel with Stains-all. Bromophenol blue is used as a marker and shows as a large band, in each lane, at the bottom of the gel. Puriﬁed CpG ODN fma1555, CpG ODN 1555 and ODN fma1556 were assayed for endotoxins using the Limulus amebocyte lysate assay and were found to contain <0.1 endo- toxin unit per mg of oligonucleotide. Virus and growth conditions Tacaribe virus (TCRV strain TRVL 11573) was obtained from American Type Culture Collection (ATCC VR-114, Manassas, VA) as a suckling mouse brain desiccate and expanded in Vero cell monolayers in P75 cell culture ﬂasks (Corning) for eight days at 37 C in R5 or R10 medium (RPMI 1640, 5 or 10% heat-inactivated fetal calf serum, 1.5 mM L-glutamine, 100 U penicillin/ml and 100 mg of streptomycin/ml) (Invitrogen) as described (37). Cell-free supernatant was harvested, aliquoted and frozen at 70 C for use in mouse infections. TCRV titers were determined using the Tissue culture ID50 method (38). Brieﬂy, the virus-containing suspension was diluted (10 1 to 10 11) in R5 medium in a 96-well round-bottom plate (Costar). Trip- licate to quadruplicate dilutions were subsequently overlaid on Vero cell monolayers cultured in a 96-well ﬂat-bottom plate (Costar). The virus was allowed to adhere 1–2 h at 37 C whereupon the virus suspension was removed from the plate and fresh R5 medium added. Cells were monitored daily for the development of cytopathic effects and were scored after 6–8 days of incubation at 37 C. The virus titer was deﬁned as the last dilution showing mild cytopathic effects in culture in two out of three replicate wells. A similar method was used to titer viruses in tissue homogenates made from infected neonatal brains (38). Animals and infection protocols Balb/C mice were obtained from the National Cancer Institute (Frederick, MD), housed in sterile microisolator cages in the CBER-speciﬁc pathogen-free animal facility, and bred at 6–12 weeks of age. All experiments were approved by the FDA Animal Care and Use Committee. Protocol A. To assess immune activation in vivo, healthy 1–3 month old female Balb/C mice were administered CpG ODN 1555 or fma1555 intraperitoneally (IP). Control mice received saline. For experiments assessing the kinetics of immune activation, spleen cells were collected 24, 72 and 120 h after treatment and the number of cytokine-secreting cells enumerated by ELIspot as previously described (39). For studies addressing dose response, spleens were harvested 72 h after treatment with CpG ODN 1555 or fma1555 (30, 100 or 300 mg/mouse, IP). Control mice received saline or ODN fma1556 (100 mg/mouse). Brieﬂy, four dilutions of sin- gle cell suspensions were overlaid on 96-well Immunolon H2B plates coated with antibodies to IL-12, IL-6 (BD Pharmingen, San Diego, CA), TNFa (R&D systems, Minneapolis, MN) or anti-IgM (Southern Biotechnology Associates, Birmingham AL). After 5 h at 37 C, the cells were washed away and overlaid with biotin-conjugated antibodies to IL-12, IL-6 (BD Pharmingen), TNF-a (Genzyme, Cambridge, MA) or IgM (Southern Biotechnology Associates) followed by phosphatase-conjugated avidin (BD Pharmingen). After a ﬁnal wash, spots were visualized by the addition of 5- bromo-4-chloro-3-indolyl phosphate (Kirkegaard and Perry Laboratories, Gaithersburg, MD) in low melt agarose (Sigma, St Louis, MO). Spots generated by individual cytokine secreting cells were counted manually using a 40· magniﬁcation and are expressed as per 106 spleen cells. Protocol B. Infections with Leishmania major were performed as described (40). L.major clone VI (MHOM/IL/80/Friedlin) Promastigotes (kindly provided by Sylvie Bertholet) were grown at 26 C in medium 199 supplemented with 20% Hi- FCE (HyClone), 100 U penicillin/ml, 100 mg of streptomycin/ ml, 2 mM L-glutamine, 40 mM HEPES, 0.1 mM adenine in 50 mM HEPES, 5 mg of hemin/ml of 50% triethanolamine and 1 mg of 6-biotin (M199/S)/ml. Infective-stage (metacyclic) promastigotes of L.major were isolated from stationary cul- tures (4- to 5-days old) by negative selection of infective forms using peanut agglutinin (Vector Laboratories). Three groups of Balb/c mice (6–10 weeks of age, four mice per group) were infected in the ear dermis with 1000 metacyclic promastigotes using a 27.5-gauge needle in a total volume of 5 ml. Each of two groups of animals was treated intradermally at the site with 25 mg of CpG ODN fma1555 or CpG ODN 1555. The third group was left untreated. The number of parasites and total volume was maintained constant for all animals. Cuta- neous inoculation of live metacyclic L.major parasites causes a cutaneous lesion that models those observed in human cuta- neous leishmaniasis. The size of the lesion was monitored weekly. Statistical analysis was performed by one-way ANOVA (analysis of variance) using the SigmaStat software. The Student–Newman–Keuls test was selected to determine the hypothesis probability values. Protocol C. Neonatal BALB/c mice were bred in-house under speciﬁc pathogen-free conditions. Newborn mice were