Use of immunostimulatory sequence-containing oligonucleotides as topical therapy for genital herpes simplex virus type 2 infection

Abstract

Synthetic oligonucleotides containing CpG motifs in specific sequence contexts have been shown to induce potent immune responses. We have evaluated mucosal administration of two immunostimulatory sequence (ISS)-containing phosphorothioate-stabilized oligonucleotides for antiherpetic efficacy in animal models. The ISS oligonucleotides, suspended in phosphate-buffered saline, were tested in mouse and guinea pig vaginal models of herpes simplex virus type 2 (HSV-2) infection. For comparison, groups of untreated, non-ISS oligonucleotide-treated, and acyclovir-treated animals also were monitored. The results indicated that vaginal epithelial application of ISS (up to 6 h after viral inoculation) with mice lethally challenged with HSV-2 delayed disease onset and reduced the number of animals that developed signs of disease (P = 0.003). ISS application significantly increased survival rates over those of controls (P = 0.0014). The ISS also impacted an established infection in the guinea pig model of HSV-2 disease. A single administration of ISS (21 days after viral inoculation) significantly reduced the frequency and severity of HSV-2 lesions compared to results with non-ISS oligonucleotide-treated and untreated guinea pigs (P < 0.01). HSV-2 is shed from the vaginal cavity of the guinea pig in the absence of lesions, similar to the case with humans. As an additional indication of ISS efficacy, the magnitude of viral shedding also was significantly reduced in ISS-treated animals (P < 0.001). These effects appeared to be immunologically mediated, since ISS had no direct effect on HSV-2 replication in vitro using standard plaque assays. These data suggest that ISS may be useful in the treatment and control of genital herpes in humans.

FIG. 1.

Intravaginal and topical perigenital application of 1018 ISS in PBS reduced and delayed the incidence (A) and progression (B) of herpetic disease and increased survival (C) following lethal vaginal HSV-2 challenge. Groups of mice (n = 15 each) were inoculated with HSV-2 and then treated with 1018 ISS 2 h (•) or 6 h (▾) later. As controls, animals were treated similarly with PBS (⋄) or were not treated (□). Daily observations of each animal for initial disease signs of hair loss and erythema (incidence of disease) and for progression (neurologic injury indicated by chronic release of urine) were made for 14 days p.i. Survival was monitored 21 days p.i.

FIG. 2.

Application of either of two ISS-containing oligonucleotides but not non-ISS control oligonucleotides 2 h after lethal HSV-2 challenge significantly reduced and delayed the incidence (A) and progression (B) of herpetic disease and increased survival (C). Mice, treated intravaginally and topically with either 1018 ISS (•) or C106 ISS (▴), were protected against hair loss and erythema (A) and chronic urine release (B) compared to mice treated with the 1019 non-ISS (○) or 1040 non-ISS (▵) control oligonucleotides. As controls, mice were treated with PBS (⋄) or were not treated (□). As a control for intervention, a 21-dose course of acyclovir (♦) was provided to a final group of mice.

FIG. 3.

Vaginal shedding of HSV-2, as detected by quantitative PCR, was significantly reduced by a single application of 1018 ISS but not 1019 non-ISS (P < 0.001). Daily vaginal swabs (22 to 43 days p.i.) were analyzed for HSV-2 DNA content by amplification of a region of the DNA polymerase gene. Resulting amplimers were blotted and then quantified by densitometry and compared to a series of known concentrations of HSV-2 genomic DNA. Genomic equivalents for each positive vaginal swab were extrapolated from the linear relationship between the viral standards and amplimer densities. The average number of genomic equivalents is presented for each group.