Mucosal administration of CpG oligodeoxynucleotide elicits strong CC and CXC chemokine responses in the vagina and serves as a potent Th1-tilting adjuvant for recombinant gD2 protein vaccination against genital herpes

Abstract

Although sexually transmitted pathogens are capable of inducing pathogen-specific immune responses, vaginal administration of nonreplicating antigens elicits only weak, nondisseminating immune responses. The present study was undertaken to examine the potential of CpG-containing oligodeoxynucleotide (CpG ODN) for induction of chemokine responses in the genital tract mucosa and also as a vaginal adjuvant in combination with glycoprotein D of herpes simplex virus type 2 (HSV-2) for induction of antigen-specific immune responses. We found that a single intravaginal administration of CpG ODN in mice stimulates a rapid and potent response of CC chemokines macrophage inflammatory protein 1alpha (MIP-1alpha), MIP-1beta, and RANTES as well as of CXC chemokines MIP-2 and IP-10 in the vagina and/or the genital lymph nodes. Importantly, intravaginal vaccination with recombinant gD2 in combination with CpG ODN gave rise to a strong antigen-specific Th1-like immune response in the genital lymph nodes as well as the spleens of the vaccinated mice. Further, such an immunization scheme conferred both systemic and mucosal immunoglobulin G antibody responses as well as protection against an otherwise lethal vaginal challenge with HSV-2. These results illustrate the potential of CpG ODN for induction of potent chemokine responses in the genital tract and also as a vaginal adjuvant for generation of Th1-type mucosal and systemic immune responses towards a nonreplicating antigen derived from a sexually transmitted pathogen. These data have implications for the development of a mucosal vaccine against genital herpes and possibly other sexually transmitted diseases.

FIG. 1.

Time course of CC and CXC chemokine production in response to intravaginal CpG ODN administration. C57BL/6 mice were injected subcutaneously with 3.0 mg of Depo-Provera. Six days later (day 0), the mice were inoculated vaginally with CpG ODN 1826 (60 μg per mouse). The vaginas (A) and the gLNs (B) were excised, and saponin was extracted at the indicated time points (two to four mice per time point). The tissue extracts were analyzed for their MIP-1α, MIP-1β, IP-10, and MIP-2 contents by ELISA. The data are expressed as increases (n-fold) over levels for naïve mice. The peak levels of chemokines were significantly higher than those of the respective control groups at P of <0.01 for vaginal MIP-1α and MIP-1β as well as gLN MIP-1α and at P of <0.05 for the others by the Student t test.

FIG. 2.

Kinetics of vaginal CC and CXC chemokine production after vaginal CpG ODN administration in outbred NMRI mice. The data are expressed as increases (n-fold) in concentrations of RANTES, MIP-1α, MIP-1β, IP-10, and MIP-2 over those for naïve mice. The experiment was performed as described in the legend for Fig. 1. The peak levels of chemokines were significantly higher than those of the respective control groups at P of <0.01 for RANTES and MIP-1β and at P of <0.05 for MIP-1α by the Student t test.

FIG. 3.

gD-specific IgG antibody response in vaginally vaccinated mice. Groups of female C57BL/6 mice (n = 5) were intravaginally vaccinated two times, 10 days apart, with CpG ODN, gD, gD plus CpG ODN, or gD plus control ODN. Four weeks after the last immunization, the mice were sacrificed and the gD-specific IgG levels in their sera and their vaginal extracts (A) as well as IgG1 and IgG2c antibody levels in their sera (B) were determined using an HSV-2 gD-specific ELISA. The data are expressed as the log₁₀ means + standard errors of the means of the optical density (OD) values of 1 of 10 prediluted samples measured at 450 nm. ND, not done.

FIG. 4.

Mucosal vaccination of C57BL/6 mice with gD plus CpG ODN confers strong protection against genital herpes. Groups of female C57BL/6 mice were intravaginally vaccinated twice, 10 days apart, with CpG ODN, gD, gD plus CpG ODN, or gD plus control ODN. Four weeks after the last vaccination, the mice were challenged vaginally with a lethal dose of HSV-2. The animals were examined for vaginal viral titer on day 3 (A). The data are expressed as means + standard errors of the means of virus loads (PFU/sample) from groups of 7 to 10 mice. The virus titers for mice immunized with gD plus CpG ODN were significantly lower, as determined by Student’s t test, than those of the gD group (**, P < 0.01). Animals were monitored daily for survival (B) and signs of disease (C). The results are pooled from two independent experiments.

FIG. 5.

Vaginal vaccination with gD plus CpG ODN elicits protection against genital herpes in outbred NMRI mice. Groups of female NMRI mice (n = 5) were intravaginally immunized twice, 10 days apart, with CpG ODN, gD, or gD plus CpG ODN. Four weeks after the last vaccination, the mice were challenged vaginally with a lethal dose of HSV-2. The animals were examined for vaginal viral titer on day 3 (A). The results are expressed as means + standard errors of the means of virus loads (PFU/sample). *, P < 0.5 by Student’s t test, compared with results for the gD group. Animals were monitored daily for survival (B) and signs of disease (C).

FIG. 6.

Single vaginal immunization with gD plus CpG ODN failed to generate protection against genital herpes in mice. Groups of female C57BL/6 mice (n = 5) were intravaginally immunized with a single dose of gD plus CpG ODN [gD + CpG (×1)] and were challenged 4 weeks later with a lethal dose of HSV-2. The results are expressed as means + standard errors of the means of virus loads (PFU/sample). The mice were examined for vaginal viral titer on day 3 (A) and were also monitored daily for survival (B) and signs of disease (C).