PMC

Effect of CpG ODN treatment on survival in mice already exposed to HSV-2. C57BL/6 mice were injected s.c. with 3.0 mg of Depo-Provera and challenged 6 days later with 9 × 10⁴ PFU of a virulent HSV-2 strain. Four hours later, the mice were treated i.vag. with either vehicle or 60 μg of CpG ODN 1826 and monitored daily for disease progression and death (12 mice/group). ○, CpG ODN treatment; •, control.

Time course of vaginal IL-18 response to i.vag. CpG ODN administration. C57BL/6 mice were injected s.c. with 3.0 mg of Depo-Provera. Six days later (day 0), CpG ODN 1826 was inoculated i.vag. (60 μg/mouse), and the vaginas were removed and saponin extracted at the indicated time points (two to four mice per time point). The vaginal extracts were analyzed for IL-18 content by ELISA, and the data are presented as the fold increase over values for day 0 control mice.

Induction of vaginal IFN-γ response after i.vag. CpG ODN administration. C57BL/6 mice were injected s.c. with 3.0 mg of Depo-Provera. Six days later (day 0), CpG ODN 1826 was inoculated i.vag. (60 μg/mouse), and the vaginas were removed and saponin extracted at the indicated time points (two mice per time point). The vaginal extracts were analyzed for IFN-γ content by ELISA. The data are expressed as fold increase over the values for day 0 control mice. The data are representative of those from two independent experiments with comparable results.

Kinetics of IL-12 response to i.vag. CpG ODN administration. C57BL/6 mice were injected s.c. with 3.0 mg of Depo-Provera. Six days later (day 0), CpG ODN 1826 was inoculated i.vag. (60 μg/mouse). (A and B) The vaginas (A) and gLN (B) were excised and saponin extracted at the indicated time points (two to four mice per time point). (C) The tissue extracts and serum samples (C) (taken at indicated time points) were analyzed for IL-12 content by ELISA. The data are expressed as fold increase over values for day 0 control mice.

Induction of acquired specific immunity by CpG ODN. Groups of C57BL/6 mice were injected s.c. with 3.0 mg of Depo-Provera. Six days later, the mice were inoculated i.vag. with 60 μg of CpG ODN and challenged 2 days later with 9 × 10⁴ PFU of a virulent HSV-2 strain. Four weeks after primary challenge, the mice were either rechallenged (n = 10 to 15), followed by a daily basis monitoring for disease progression and death (A), or the mice were sacrificed and the titers of HSV-specific IgG in the sera and genital tract extracts were determined (B). Error bars indicate standard errors of the means.

Effects of i.vag. CpG ODN administration on disease progression in mice deficient in T and/or B cells after a genital HSV-2 challenge. Groups of SCID (A), nude (B), and μMT (C) mice were injected s.c. with 3.0 mg of Depo-Provera. Six days later, the mice were inoculated i.vag. with a single dose of 60 μg of CpG ODN 1826. The animals were challenged 2 days later with 9 × 10⁴ PFU of a virulent HSV-2 strain and monitored daily for disease progression and mortality (5 to 10 mice per group). ⋄, ○, and ▵, CpG ODN treatment; ▾, •, and □, control.

Impact of CpG ODN pretreatment on disease progression after a genital HSV-2 challenge. Groups of female C57BL/6 mice (6 to 12 mice/group) were injected s.c. with 3.0 mg of Depo-Provera. Six days later, the mice received a single dose of 60 μg of CpG ODN 1826 by either the s.c., i.p., combined s.c. and i.p., i.vag., or combined i.vag. and i.m. route, and they were challenged 2 days later by i.vag. inoculation of 9 ×10⁴ PFU of a virulent HSV-2 strain. Animals were scored daily for macroscopic signs of the disease (A) and mortality (B). Disease progression was scored as healthy (0), genital erythema (1), moderate genital inflammation (2), severe and purulent genital lesion (3), hind-limb paralysis (4), and death or sacrifice due to paralysis (5).

RANTES production in the vaginas of C57BL/6 WT, IFN-γ^−/−, IL-12^−/−, and IL-18^−/− mice after i.vag. CpG ODN administration. C57BL/6 WT, IFN-γ^−/− (GKO), IL-12^−/−, and IL-18^−/− mice were injected s.c. with 3.0 mg of Depo-Provera. Six days later, CpG ODN 1826 was inoculated i.vag. (60 μg/mouse), and the vaginas were removed and saponin extracted. The vaginal extracts were analyzed for RANTES content by ELISA. (A) Kinetics of vaginal RANTES production in WT mice after i.vag. CpG ODN administration. Data are presented as the fold increase over values for day 0 control mice (two to four mice per time point). (B) Vaginal production of RANTES on day 2 after i.vag. CpG ODN administration. Data are expressed as the mean concentration of RANTES per 10 mg of vagina, with the standard error of the mean. Differences were statistically significant at P values of <0.05 (*) and <0.01 (**) by Student's t test compared with CpG ODN-treated WT mice (five to seven mice per group).

The protective effect of CpG ODN is abolished in the absence of IFN-γ, IL-12, or IL-18. Groups of C57BL/6 WT, IFN-γ^−/− (GKO), IL-12^−/−, and IL-18^−/− mice were injected s.c. with 3.0 mg of Depo-Provera. Six days later, the mice were inoculated i.vag. with a single dose of 60 μg of CpG ODN 1826. The animals were challenged 2 days later with 9 × 10⁴ PFU of a virulent HSV-2 strain. (A) The vaginal HSV-2 titers were examined on day 3 after viral challenge (n = 5 to 7). Data are expressed as the mean virus load (PFU per sample) and standard error of the mean. Differences were statistically significant at P values of <0.05 (*) and <0.01 (**) by Student's t test compared with WT mice. (B) The mice were monitored daily for mortality (12 to 17 mice/group).

CpG ODN-induced protection against genital herpes infection is dependent on the CpG motif. Groups of C57BL/6 mice were injected s.c. with 3.0 mg of Depo-Provera. Six days later, the mice were inoculated i.vag. with a single dose of either 60 μg of CpG ODN 1826 or a non-CpG control ODN. The animals were challenged 2 days later with 9 × 10⁴ PFU of a virulent HSV-2 strain. (A) Vaginal HSV-2 titers on day 3 after viral challenge (n = 5 to 7). Data are expressed as the mean virus load (PFU per sample) and standard error of the mean. The difference was statistically significant at P value of <0.05 (*) by Student's t test. (B and C) The mice were monitored daily for macroscopic signs of the disease (B) and mortality (C) (n ≥ 12) as described in Fig. . ○, CpG ODN treatment; •, non-CpG ODN treatment.