3558 Nucleic Acids Research, 2005, Vol. 33, No. 11 fma1555-treated mice (Figure 5), the difference was not statistically signiﬁcant. This study demonstrated that the thermolytic CpG-ODN fma1555 is biologically active and can elicit a protective immune response in mice infected with an intracellular pathogen. To further assess whether CpG ODN fma1555 behaves as a prodrug, a model of TCRV infection in neonatal mice was used. The TCRV is a New World arena virus that causes a lethal meningoencephalitis in newborn mice. We showed that treatment with CpG-ODN 1555 (50 mg/mouse) protected 43% of the newborn mice from death. As shown in several murine Figure 5. Immunoprotection of mice treated with CpG ODN fma1555 against progressive L.major infection. Balb/c mice (4 mice/group) were challenged in the ear with 103 live L.major metacyclic promastigotes. The mice were either left untreated or received a single treatment at the site (ID) with CpG ODN 1555 or CpG ODN fma1555. The lesion size was monitored weekly. Note the reduction in lesion size in treated versus untreated mice. Statistical analysis was performed by one-way ANOVA (* indicates P < 0.05, ** indicates P < 0.01). The difference between mice treated with CpG ODN 1555 and CpG ODN fma1555 was not statistically significant. models, the therapeutic window for protection is narrow; treatment was effective when the oligonucleotides were administered at the time, or up to three days after TCRV infection. Mice that received CpG ODN 1555 three days prior to infection were not protected and died from lethal menin- goencephalitis at the same rate as untreated mice (Figure 6a). As shown in Figure 6b, mice treated with CpG ODN fma1555 (50 mg/mouse) at the time of TCRV infection were not pro- tected from death, but mice that received the treatment three days before infection had improved survival. The protective effect of ODN fma1555 was CpG-mediated, as mice treated with ODN fma1556 lacking CpG motifs were not protected. These ﬁndings indicate that the 2-(N-formyl-N-methyl) aminoethyl ( fma) thiophosphate protecting group and/or its thermolytically cleaved counterpart [tentatively, 2-(N-formyl- N-methyl)aminoethanol] do not impart signiﬁcant immuno- protective properties to CpG ODNs fma1555. Interestingly, when CpG ODN fma1555 and CpG ODN 1555 were co-administered (25 mg of each ODN/mouse) three days prior to or on the same day of the TCRV challenge, the survival rate averaged better than 50% (Figure 6c), thereby suggesting that the combination of CpG ODN 1555 and CpG ODN fma1555 widened the window of therapeutic treatment against the disease. Future experiments will be necessary to determine the mechanism and kinetics of the local and systemic immunostimulation elicited by CpG ODN fma1555. However, our results are consistent with the notion that CpG ODN fma1555 behaves as a prodrug in vivo through an unprecedented thermolytic process. These ﬁndings thus pave the way to the development of new thermolytic CpG- containing oligonucleotide prodrugs exhibiting various half- time of thiophosphate deprotection to enable the preparation of more efﬁcient immunotherapeutic oligonucleotide formu- lations for the treatment of asthma, cancer, autoimmune and various infectious diseases. (a) Untreated 1555 day -3 1555 day 0 (b) Untreated fma 1555 day -3 fma 1555 day 0 fma 1556 day-3 (c) Untreated fma 1555 + 1555 day -3 fma 1555 + 1555 day 0 100 L 80 A V I VR 60 U S % 40 20 0 0 5 10 15 20 25 30 35 0 5 10 15 20 25 30 35 0 5 10 15 20 25 30 35 DAYS POST INFECTION Figure 6. Survival of TCRV-infected mice treated with CpG ODN fma1555. Newborn Balb/c mice (1–4 days old) were infected with TCRV (2000 TC50/mouse). (a) CpG ODN 1555 (50 mg/mouse IP) protects newborn mice from infection when administered at the time of infection (open squares, n = 9), but cannot protect mice if administered three days prior to infection (open triangles, n = 7). (b) CpG ODN fma1555 (50 mg/mouse) administration protected mice when administered three days prior to infection (open triangles, n = 8), but not when administered at the time of infection (open squares, n = 9). Control mice received ODN fma1556 lacking CpG motifs on day 3 (open diamonds, n = 6). (c) Combination of CpG ODN 1555 (25 mg/mouse) and CpG ODN fma1555 (25 mg/mouse) administered on day 3 (closed triangles, n = 10) or at the time of infection (closed squares, n = 11) extends the period of treatment against the disease.