Local delivery of CpG oligodeoxynucleotides induces rapid changes in the genital mucosa and inhibits replication, but not entry, of herpes simplex virus type 2

Abstract

Mucosal surfaces are the entry sites for the vast majority of infectious pathogens and provide the first line of defense against infection. In addition to the epithelial barrier, the innate immune system plays a key role in recognizing and rapidly responding to invading pathogens via innate receptors, such as Toll-like receptors (TLR). Bacterial CpG DNA, a potent activator of innate immunity, is recognized by TLR9. Here, we confirm that local mucosal, but not systemic, delivery of CpG oligodeoxynucleotides (ODN) to the genital tract protects mice from a subsequent lethal vaginal herpes simplex virus type 2 (HSV-2) challenge. Since these effects were so local in action, we examined the genital mucosa. Local delivery of CpG ODN induced rapid proliferation and thickening of the genital epithelium and caused significant recruitment of inflammatory cells to the submucosa. Local CpG ODN treatment also resulted in inhibition of HSV-2 replication but had no effect on HSV-2 entry into the genital mucosa. CpG ODN-induced protection against HSV-2 was not associated with early increases in gamma interferon (IFN-gamma) secretion in the genital tract, and CpG ODN-treated IFN-gamma(-/-) mice were protected from subsequent challenge with a lethal dose of HSV-2. Treatment of human HEK-293 cells transfected with murine TLR9 showed that the antiviral activity of CpG ODN was mediated through TLR9. These studies suggest that local induction of mucosal innate immunity can provide protection against sexually transmitted infections, such as HSV-2 or possibly human immunodeficiency virus, at the mucosal surfaces.

FIG. 1.

Local mucosal delivery of CpG ODN protects against vaginal HSV-2 challenge. CpG ODN or control ODN was delivered vaginally or i.m. 24 h prior to lethal IVAG HSV-2 challenge (10⁴ PFU). The challenged mice were monitored daily for genital pathology, survival, and vaginal virus titer. (A) All mice that received CpG ODN IVAG (•) were protected against lethal HSV-2 challenge compared to IVAG control ODN (▴), mice treated i.m. (with CPG [▪] or control [□] ODN), or naive (▵) mice. (B) Mice treated IVAG with CpG ODN had very low or no genital pathology compared to all other groups. Symbols are as for panel A. (C) Vaginal HSV-2 titers were examined on days 1 to 3 post-viral challenge (n = 5/group). Mice treated IVAG with CpG ODN had no detectable virus. Mice that received CpG ODN i.m. had significantly lower viral titers than did control ODN-treated or naive mice on day 1 postchallenge; there were no significant differences in the viral titers on days 2 and 3 postchallenge. Symbols are as for panel A. The error bars indicate standard deviations.

FIG. 2.

CpG ODN induces rapid changes in the murine genital mucosa. Mice were treated with progesterone (Depo-Provera) and 4 days later received 100 μg of CpG ODN or control ODN IVAG. Twenty-four hours later, vaginal tissues were isolated and processed for histology. Photomicrographs representing cross-sections of vaginal tissues from CpG ODN-treated (a, a1, and a2) and control ODN-treated (b, b1, and b2) mice are shown. CpG-treated mice showed rapid proliferation and thickening of the vaginal epithelium (a and a2) compared to control ODN-treated tissues (b and b2). Significant recruitment of inflammatory cells was also observed in the submucosa of CpG-treated mice (a and a1) compared to control sections (b and b1). Magnification, ×25 (a and b) and ×500 (a1, b1, a2, and b2).

FIG. 3.

Local delivery of CpG ODN induces active proliferation of the genital epithelium. Staining for PCNA was performed to confirm the active proliferation of vaginal epithelial cells following local delivery of CpG ODN. Multiple layers of PCNA-positive cells were observed in all samples from CpG-treated mice (a and c) compared to samples from control ODN-treated mice (b and d). Magnification, ×50 (a and b) and ×250 (c and d).

FIG. 4.

Local delivery of CpG ODN inhibits HSV-2 replication in the genital mucosa. Depo-Provera-treated mice received 100 μg of CpG ODN or control ODN. After 24 h, the mice were vaginally infected with HSV-2 expressing GFP. Vaginal tissues were dissected 24 or 48 h postinfection and processed for histology. Virtually no GFP-positive cells were detected in the genital mucosa from CpG-treated mice (b) compared to control ODN-treated controls, which had numerous GFP-positive cells (a). The photomicrographs are representative of vaginal sections 48 h post-HSV-2 challenge. Magnification, ×250.

FIG. 5.

CpG ODN treatment of vaginal mucosa does not inhibit HSV-2 entry into vaginal epithelial cells. Transgenic mice containing the immediate-early HSV-2 ICP4 gene promoter fused to the bacterial β-galactosidase gene were treated IVAG with CpG or control ODN (n = 4). After 24 h, the mice were infected IVAG with HSV-2 (10⁴ PFU). Vaginal tissues were removed 24 h postchallenge. From each vaginal tissue, 11 slides with five cross sections on each slide were prepared and processed for X-Gal staining. HSV-2 was able to enter the vaginal epithelial cells in both CpG-treated (a and d) and control ODN-treated (b and e) mice to comparable levels (with no significant differences in the mean numbers of blue cells from CpG ODN-treated and control ODN-treated mice). UV-inactivated HSV-2, proven to be replication deficient, was also able to enter the epithelial cells and activate the ICP4-driven β-galactosidase gene (c and f). The arrows indicate positive cells (ICP4 activated). Magnification, ×100 (a to c) and ×250 (d to e).

FIG. 6.

CpG ODN-induced innate protection against HSV-2 infection occurs in the absence of IFN-γ. B6 and IFN-γ^−/− mice were treated IVAG with 100 μg of CpG ODN 24 h prior to HSV-2 infection. (A) No induction of early (days 1 to 3 postchallenge) IFN-γ was detected in the vaginal washes of CpG ODN-treated mice compared to control ODN-treated mice. The error bars indicate standard deviations. *P < 0.01; **, P < 0.001. (B) Local delivery of CpG ODN resulted in complete protection against subsequent challenge with a lethal dose of vaginal HSV-2 in IFN-γ^−/− mice. (C) IFN-γ^−/− mice treated with CpG ODN had no detectable virus in the vaginal washes compared to control ODN-treated mice.

FIG. 7.

Antiviral effects of CpG are mediated through TLR9. Monolayers of HEK-293 cells transformed with murine TLR9 (A) or normal HEK-293 cells (B) were treated with CpG ODN or control ODN (25 μg/ml) or left untreated. After 24 h, the antiviral effect of CpG ODN was detected using a standard viral plaque assay with a known stock of HSV-2. (A) CpG-treated cells were significantly (>1 log unit) more resistant to HSV-2 infection in the presence of mTLR-9 than control ODN-treated or untreated cells. (B) Untransformed HEK-293 cells treated with CpG ODN showed no protection against HSV-2 infection compared to control ODN-treated or untreated cells. (C) Histogram showing significant reduction (P < 0.01) in HSV-2 titer in mTLR9-transformed HEK-293 cells following CpG ODN, but not control ODN, treatment. The error bars indicate standard deviations.

FIG. 7.

Antiviral effects of CpG are mediated through TLR9. Monolayers of HEK-293 cells transformed with murine TLR9 (A) or normal HEK-293 cells (B) were treated with CpG ODN or control ODN (25 μg/ml) or left untreated. After 24 h, the antiviral effect of CpG ODN was detected using a standard viral plaque assay with a known stock of HSV-2. (A) CpG-treated cells were significantly (>1 log unit) more resistant to HSV-2 infection in the presence of mTLR-9 than control ODN-treated or untreated cells. (B) Untransformed HEK-293 cells treated with CpG ODN showed no protection against HSV-2 infection compared to control ODN-treated or untreated cells. (C) Histogram showing significant reduction (P < 0.01) in HSV-2 titer in mTLR9-transformed HEK-293 cells following CpG ODN, but not control ODN, treatment. The error bars indicate standard deviations.