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

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.     

NK and NKT cell-independent contribution of interleukin-15 to innate protection against mucosal viral infection

Interleukin-15 (IL-15) is essential for the development, maturation, and function of NK and NKT cells, which are critical components of the innate immune defense against viral infections. We recently showed that mice lacking IL-15 and/or NK/NKT cells are significantly more susceptible to intravaginal (IVAG) herpes simplex virus type 2 (HSV-2) infection than control mice. For this study, we examined whether IL-15 has any direct antiviral activity, independent of NK/NKT cells, in innate protection against HSV-2 infection. A sensitive enzyme-linked immunosorbent assay for murine IL-15 was developed and used to show that IVAG HSV-2 infection induces IL-15 in vaginal washes. Using immunohistochemistry, we detected IL-15-positive cells in the submucosa and vaginal epithelium following IVAG HSV-2 infection. Local, but not systemic, delivery of murine recombinant IL-15 (mrIL-15) to the genital mucosae of IL-15(-/-) and RAG-2(-/-) gamma(c)(-/-) mice, which both lack NK and NKT cells, resulted in significant reductions in HSV-2 titers in genital washes and 60% survival following IVAG HSV-2 challenge. Furthermore, we showed that IL-15 is important for CpG oligodeoxynucleotide (ODN)-induced innate protection against genital HSV-2 infection. While 100% of CpG ODN-treated RAG2(-/-) gamma(c)(-/-) mice, which are capable of producing IL-15 but lack NK/NKT cells, survived an IVAG HSV-2 challenge, only 60% of CpG ODN-treated IL-15(-/-) mice survived, and all of these mice had similar vaginal viral titers to those in control mice by day 3 postchallenge. Lastly, a treatment of RAW264.7 cells with mrIL-15 induced the production of tumor necrosis factor alpha and beta interferon (IFN-beta), but not IFN-alpha, and significantly protected them against HSV-2 infection in vitro. The results of these studies indicate that IL-15 can act independently of NK/NKT cells in mediating the innate defense against viral infection.     

Intranasal immunization with CpG oligodeoxynucleotides as an adjuvant dramatically increases IgA and protection against herpes simplex virus-2 in the genital tract

Development of vaccines capable of preventing the transmission or limiting the severity of sexually transmitted viruses, such as HSV and HIV, will likely be dependent on the induction of potent long-lasting mucosal immune responses in the genital tract. Recently, synthetic oligodeoxynucleotides (ODN) containing immunostimulatory CpG motifs were shown to serve as potent adjuvants for the induction of mucosal immune responses. Here, we show that intranasal immunization with CpG ODN, plus recombinant glycoprotein B (rgB) of HSV-1, results in significantly elevated levels of specific anti-gB IgA Abs in vaginal washes that remained high throughout the estrous cycle. Additionally, dramatically elevated numbers of specific IgA Ab-secreting cells were present and persisted in the genital tract in response to intravaginal (IVAG) HSV-2 challenge. HSV-2-specific CTL were observed at moderate levels in the spleens of CpG or non-CpG ODN-immunized mice. In contrast, strong CTL responses were observed locally in the genital tissues of both groups following IVAG HSV-2 challenge. Interestingly, mice immunized intranasally with rgB plus CpG ODN, but not non-CpG ODN, were significantly protected following IVAG HSV-2 challenge. Measurement of virus in protected CpG-immunized mice revealed a log lower level of replication within the first few days after infection. In conclusion, these results indicate that intranasal immunization with CpG ODN plus protein mediates immunity in the female genital tract capable of protecting against a sexually transmitted pathogen.     

CD8+ T-cell-mediated cross-clade protection in the genital tract following intranasal immunization with inactivated human immunodeficiency virus antigen plus CpG oligodeoxynucleotides

Human immunodeficiency virus (HIV) is a mucosally transmitted infection that rapidly targets and depletes CD4+ T cells in mucosal tissues and establishes a major reservoir for viral persistence in gut-associated lymphoid tissues. Therefore, vaccines designed to prevent HIV infections must induce potent and durable mucosal immune responses, especially in the genital tract. Here we investigated whether intranasal (i.n.) immunization with inactivated gp120-depleted HIV-1 antigen (Ag) plus CpG oligodeoxynucleotide (ODN) as an adjuvant induced local immune responses in the genital tract and cross-clade protection against intravaginal (IVAG) challenge. Lymphocytes isolated from the iliac lymph nodes (ILNs) and genital tracts of female mice i.n. immunized with HIV-1 Ag plus CpG showed significant HIV-specific proliferation and produced significantly higher levels of gamma interferon (IFN-gamma) and beta-chemokines than mice immunized with HIV-1 Ag alone or mixed with non-CpG ODN. CD8+ lymphocytes were dramatically increased in the genital tracts of mice immunized with HIV-1 Ag plus CpG, and protection following IVAG challenge with recombinant vaccinia viruses (rVVs) expressing HIV-1 gag was shown to be CD8 dependent. Finally, cross-clade protection was observed between clades A, C, and G but not B following IVAG challenge with rVVs expressing HIV-1 gag from different clades. These studies provide evidence that mucosal (i.n.) immunization induced strong local T-cell-mediated immune responses in the genital tract and cross-clade protection against IVAG challenge.     

Induction of innate immunity against herpes simplex virus type 2 infection via local delivery of Toll-like receptor ligands correlates with beta interferon production

Toll-like receptors (TLRs) constitute a family of innate receptors that recognize and respond to a wide spectrum of microorganisms, including fungi, bacteria, viruses, and protozoa. Previous studies have demonstrated that ligands for TLR3 and TLR9 induce potent innate antiviral responses against herpes simplex virus type 2 (HSV-2). However, the factor(s) involved in this innate protection is not well-defined. Here we report that production of beta interferon (IFN-beta) but not production of IFN-alpha, IFN-gamma, or tumor necrosis factor alpha (TNF-alpha) strongly correlates with innate protection against HSV-2. Local delivery of poly(I:C) and CpG oligodeoxynucleotides induced significant production of IFN-beta in the genital tract and provided complete protection against intravaginal (IVAG) HSV-2 challenge. There was no detectable IFN-beta in mice treated with ligands for TLR4 or TLR2, and these mice were not protected against subsequent IVAG HSV-2 challenge. There was no correlation between levels of TNF-alpha or IFN-gamma in the genital tract and protection against IVAG HSV-2 challenge following TLR ligand delivery. Both TNF-alpha(-/-) and IFN-gamma(-/-) mice were protected against IVAG HSV-2 challenge following local delivery of poly(I:C). To confirm that type I interferon, particularly IFN-beta, mediates innate protection, mice unresponsive to type I interferons (IFN-alpha/betaR(-/-) mice) and mice lacking IFN regulatory factor-3 (IRF-3(-/-) mice) were treated with poly(I:C) and then challenged with IVAG HSV-2. There was no protection against HSV-2 infection following poly(I:C) treatment of IFN-alpha/betaR(-/-) or IRF-3(-/-) mice. Local delivery of murine recombinant IFN-beta protected C57BL/6 and IRF-3(-/-) mice against IVAG HSV-2 challenge. Results from these in vivo studies clearly suggest a strong correlation between IFN-beta production and innate antiviral immunity against HSV-2.     

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

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.     

Distinct modulatory effects of LPS and CpG on IL-18-dependent IFN-gamma synthesis

Innate cellular production of IFN-gamma is suppressed after repeated exposure to LPS, whereas CpG-containing DNA potentiates IFN-gamma production. We compared the modulatory effects of LPS and CpG on specific cellular and cytokine responses necessary for NK-cell dependent IFN-gamma synthesis. C3H/HeN mice pretreated with LPS for 2 days generated 5-fold less circulating IL-12 p70 and IFN-gamma in response to subsequent LPS challenge than did challenged control mice. In contrast, CpG-pretreated mice produced 10-fold more circulating IFN-gamma without similar changes in IL-12 p70 levels, but with 10-fold increases in serum IL-18 relative to LPS-challenged control or endotoxin-tolerant mice. The role of IL-18 in CpG-induced immune potentiation was studied in splenocyte cultures from control, LPS-conditioned, or CpG-conditioned mice. These cultures produced similar amounts of IFN-gamma in response to rIL-12 and rIL-18. However, only CpG-conditioned cells produced IFN-gamma when cultured with LPS or CpG, and production was ablated in the presence of anti-IL-18R Ab. Anti-IL-18R Ab also reduced in vivo IFN-gamma production by >2-fold in CpG-pretreated mice. Finally, combined pretreatment of mice with LPS and CpG suppressed the production of circulating IFN-gamma, IL-12 p70, and IL-18 after subsequent LPS challenge. We conclude that CpG potentiates innate IFN-gamma production from NK cells by increasing IL-18 availability, but that the suppressive effects of LPS on innate cellular immunity dominate during combined LPS and CpG pretreatment. Multiple Toll-like receptor engagement in vivo during infection can result in functional polarization of innate immunity dominated by a specific Toll-like receptor response.     

Effect of dendritic cells treated with CpG ODN on atopic dermatitis of Nc/Nga mice

Atopic dermatitis (AD) is a chronic inflammatory skin disease and the pathogenesis of AD is associated with the release of various cytokines/chemokines due to activated Th(2) immune responses. Synthetic oligodeoxynucleotides (ODNs) containing unmethylated CpG dinucleotide in the context of particular base sequence (CpG motifs) are known to have the immunostimulatory activities in mice and to convert from Th(2) to Th(1) immune responses in AD. We aimed to investigate that CpG ODN, especially phosphodiester form, can stimulate the protective immunity in NC/Nga mice with AD. We isolated BMDCs from NC/Nga mice and then, cultured with GM-CSF and IL-4 for 6 days, and treated for 2 days by either phosphorothioate ODN or phosphodiester ODN. CpG ODN-treated DCs resulted in more production of IL-12. When CpG ODN-treated DCs were intravenously injected into the NC/Nga mice, the NC/Nga mice with CpG ODN-treated DCs showed significant improvement of AD symptoms and decrease of IgE level. Histopathologically, the NC/Nga mice skin with CpG ODN-treated DCs showed the decreased IL-4 and TARC expression comparing with non-injected mice. These results may suggest that phosphodiester CpG ODN-treated DCs might function as a potent adjuvant for AD in a mouse model.     

CpG oligodeoxynucleotides stimulate protective innate immunity against pulmonary Klebsiella infection

Bacterial pneumonia is a leading cause of mortality in the United States. Innate immune responses, including type-1 cytokine production, are critical to the effective clearance of bacterial pathogens from the lung. Synthetic oligodeoxynucleotides (ODN) containing unmethylated CpG dinucleotide motifs (CpG ODN), which mimic the effects of bacterial DNA, have been shown to enhance type-1 cytokine responses during infection due to intracellular pathogens, resulting in enhanced microbial clearance. The role of CpG ODN in modulating protective innate immunity against extracellular pathogens is unknown. Using a murine model of Gram-negative pneumonia, we found that CpG ODN administration stimulated protective immunity against Klebsiella pneumoniae. Specifically, intratracheal (i.t.) administration of CpG ODN (30 microg) 48 h before i.t. K. pneumoniae challenge resulted in increased survival, compared with animals pretreated with control ODN or saline. Pretreatment with CpG ODN resulted in enhanced bacterial clearance in lung and blood, and higher numbers of pulmonary neutrophils, NKT cells, gammadelta-T cells, and activated NK1.1+ cells and gammadelta-T lymphocytes during infection. Furthermore, pretreatment with CpG ODN enhanced the production of TNF-alpha, and type-1 cytokines, including IL-12, IFN-gamma, and the IFN-gamma-dependent ELR- CXC chemokines IFN-gamma-inducible protein-10 and monokine induced by IFN-gamma in response to Klebsiella challenge, compared with control mice. These findings indicate that i.t. administration of CpG ODN can stimulate multiple components of innate immunity in the lung, and may form the basis for novel therapies directed at enhancing protective immune responses to severe bacterial infections of the lung.     

Overexpression of Interleukin-15 Compromises CD4-Dependent Adaptive Immune Responses against Herpes Simplex Virus 2

Interleukin-15 (IL-15) is necessary for the development and function of NK/NKT cells and the maintenance of naive and memory CD8⁺ T cells. In the absence of IL-15, protective innate immunity is not available; however, a functional adaptive immune response against vaginal herpes simplex virus 2 (HSV-2) is generated. Mice overexpressing IL-15 (IL-15tg mice) have higher numbers of NK cells, greater NK-derived gamma interferon, and more CD8⁺ T cells. Here we examined the consequences of IL-15 overexpression for innate and adaptive immunity against genital HSV-2. Surprisingly, IL-15tg mice immunized against HSV-2 were not protected against genital HSV-2 challenge compared to control immunized mice. IL-15tg mice had a higher frequency of NK cells in the genital mucosa than control mice. However, immunized IL-15tg mice had significantly lower numbers of HSV-2-specific CD4⁺ T cells than B6 mice. We then confirmed that CD4⁺ T cells, but not CD8⁺ T cells, are essential for protection against intravaginal HSV-2 challenge. Since we observed less protection in immunized IL-15tg mice, we then examined if the adaptive immune responses generated in an environment with overexpression of IL-15 could provide protection against HSV-2 in an environment with normal levels of IL-15 expression. We adoptively transferred immunized cells from IL-15tg and B6 mice into naive RAG-1^−/− mice and found that the cells from immunized IL-15tg mice were able to provide protection in this IL-15-normal environment. Our data suggest that overexpression of IL-15 results in a reduced CD4⁺ T cell-mediated adaptive immune response against genital HSV-2.     

A critical role for IL-15 in TLR-mediated innate antiviral immunity against genital HSV-2 infection

Innate antiviral immunity, particularly at mucosal surfaces, has a critical role in early control of viral infections. Both type I interferons (IFNs) and interleukin-15 (IL-15) are essential components of innate antiviral immunity. It has been shown that toll-like receptor (TLR) ligand-induced innate antiviral immunity requires IFN-α/β and -λ receptor signaling. However, it is not known if IL-15 has a role in TLR ligand-mediated antiviral responses. Here, we report that ligands for TLR-3 and TLR-9 cannot confer protection against genital herpes simplex virus-2 (HSV-2) in the absence of IL-15 in vivo. Interestingly, wild-type mice depleted of natural killer (NK) cells and treated with TLR ligands are protected upon HSV-2 challenge, suggesting that the critical role of IL-15 is independent of NK cell-mediated activity. To examine the cytokine response in the absence of IL-15, we investigated TLR ligand-induced IFN-β and -λ production in the vaginal washes, but found no impairment in IL-15(-/-) mice. Finally, we report no impairment in the expression of the IFN-stimulated genes in IL-15(-/-) mice. Collectively, the data suggest that TLR ligands induce an IFN-mediated response in the vaginal tract of both wild-type and IL-15(-/-) mice, but its induction is insufficient for providing protection against HSV-2 in the absence of IL-15.     

Therapeutic effect of CpG motifs on the development of chronic graft-versus-host disease in mice

Transferring DBA/2 spleen cells into (C57BL/10xDBA/2) F1 (referred to as BDF1) mice induces a chronic graft-versus-host disease (GVHD), characterized by the production of Th(2) cytokines, hypergammaglobulinemia, and immune complex-mediated glomerulonephritis that resembles systemic lupus erythematosus. DNA motif consisting of an unmethylated CpG dinucleotide flanked by two 5' purines and two 3' pyrimidines (CpG ODN) induces Th(1) cytokine production in mice. This study examines the effect of administering CpG ODN to mice undergoing chronic GVHD, based on the premise that altering Th(1)/Th(2) activity might beneficially impact on disease progression.GVHD BDF1 mice injected with DBA/2 spleen cells were treated with weekly intraperitoneal injection of 50 microg CpG ODN. This treatment significantly suppressed the production of IgG anti-DNA autoantibody and reduced the development of glomerulonephritis. Serum IgG2a titers were higher in the CpG ODN than in non-CpG control group, whereas IgG1 titers were unchanged. As predicted, IFN-gamma levels were significantly higher in the CpG ODN-treated group, while IL-4 levels were lower, resulting in a shift in the Th(1)/Th(2) cytokine ratio. Results suggest that CpG ODN administration may be of therapeutic benefit in chronic GVHD.     

Interleukin-15 and natural killer and NKT cells play a critical role in innate protection against genital herpes simplex virus type 2 infection

Interleukin-15 (IL-15), natural killer (NK) cells, and NK T (NKT) cells, components of the innate immune system, are known to contribute to defense against pathogens, including viruses. Here we report that IL-15(-/-) (NK(-) and NKT(-/+)) mice and RAG-2(-/-)/gamma(c)(-/-) (NK(-) and NKT(-)) mice that lack all lymphoid cells were very susceptible to vaginal infection with a low dose of herpes simplex virus type 2 (HSV-2). IL-15(-/-) and RAG-2(-/-)/gamma(c)(-/-) mice were 100-fold more susceptible and RAG-2(-/-), CD-1(-/-) (NKT(-)), and gamma interferon (IFN-gamma)(-/-) mice were 10-fold more susceptible to vaginal HSV-2 infection than control C57BL/6 mice. NK and/or NKT cells were the early source of IFN-gamma in vaginal secretions following genital HSV-2 infection. This study demonstrates that IL-15 and NK-NKT cells are critical for innate protection against genital HSV-2.     

Influence of DNA encoding cytokines on systemic and mucosal immunity following genetic vaccination against herpes simplex virus

The aim of our investigation was to improve the effectiveness of DNA vaccines against herpes simplex virus (HSV) infection. We chose coimmunization with DNA encoding cytokines known to emphasize components of immune defense that best correlate with immune protection. These include interferon-producing T and NK cells and the IgG2a isotype immunoglobulin. Our results show that the coadministration of plasmid DNA encoding IL-12 or IL-18 along with glycoprotein B (gB) DNA improves immune induction. Recipients of the coimmunization procedure had elevated humoral as well as IFN-gamma-producing T cell responses and showed greater resistance to vaginal challenge with a lethal dose of HSV-1. The adjuvant effects were observed when the vaccines were administered either systemically or mucosally. By most assays, the adjuvant effect of IL-18 was superior to IL-12, although gB DNA plus IL-18 failed to induce levels of immunity achieved by UV-inactivated HSV immunization. Mucosal immunization proved as an effective means of inducing systemic immunity, but was less effective than the systemic route for inducing protection from vaginal challenge. Our results also demonstrated that protection from such challenges was mainly a property of IFN-gamma. Thus, immunized IFN-gamma-/- mice remained susceptible to challenges even while generating readily measurable immune responses. The approach of using DNA vaccines combined with DNA encoding cytokines holds promise and represents a potentially useful approach for vaccines.     

Efficacy evaluation of live Escherichia coli expression Brucella P39 protein combined with CpG oligodeoxynucleotides vaccine against Brucella melitensis 16M, in BALB/c mice

Brucella is gram-negative bacteria responsible for brucellosis in a wide variety of animals and humans. BALB/c mice were immunized with live Escherichia coli expression the p39 gene of Brucella melitensis, a gene coding for the periplasmic binding protein. Mice were injected with either E. coli BL21 (DE3) pEt15b or E. coli BL21 (DE3) pEt15b-p39 alone or adjuvanted with either CpG oligodeoxynucleotides (CpG ODN) or non-CpG ODN. E. coli BL21 (DE3) pEt15b-p39 with CpG ODN or with non-CpG ODN mice groups showed a significant IFN-γ production and T-cell proliferation as a reaction to P39 antigen. In addition, antibody responses (IgG, IgG1 and IgG2a), were only found in these two mice groups. A higher level of protection against B. melitensis 16M were observed in mice immunized with E. coli BL21 (DE3) pEt15b-p39 and CpG ODN comparing with those immunized with E. coli BL21 (DE3) pEt15b-p39 alone or with non-CpG ODN. No protection against B. melitensis 16M was observed in mice immunized with E. coli BL21 (DE3) pEt15b alone or with the adjuvant. Rev.1 protection at 4 and 8 weeks post-challenge was more effective than that observed with E. coli BL21 (DE3) pEt15b-p39 and CpG ODN.     

Enhanced tumor-specific long-term immunity of hemagglutinating [correction of hemaggluttinating] virus of Japan-mediated dendritic cell-tumor fused cell vaccination by coadministration with CpG oligodeoxynucleotides

Immunization with dendritic cells (DCs) using various Ag-loading approaches has shown promising results in tumor-specific immunotherapy and immunoprevention. Fused cells (FCs) that are generated from DCs and tumor cells are one of effective cancer vaccines because both known and unknown tumor Ags are presented on the FCs and recognized by T cells. In this study, we attempted to augment antitumor immunity by the combination of DC-tumor FC vaccination with immunostimulatory oligodeoxynucleotides containing CpG motif (CpG ODN). Murine DCs were fused with syngeneic tumor cells ex vivo using inactivated hemagglutinating virus of Japan (Sendai virus). Mice were intradermally (i.d.) immunized with FCs and/or CpG ODN. Coadministration of CpG ODN enhanced the phenotypical maturation of FCs and unfused DCs, and the production of Th1 cytokines, such as IFN-gamma and IL-12, leading to the induction of tumor-specific CTLs without falling into T cell anergy. In addition, immunization with FCs + CpG ODN provided significant protection against lethal s.c. tumor challenge and spontaneous lung metastasis compared with that with either FCs or CpG ODN alone. Furthermore, among mice that rejected tumor challenge, the mice immunized with FCs + CpG ODN, but not the mice immunized with FCs or CpG ODN alone, completely rejected tumor rechallenge, indicating that CpG ODN provided long-term maintenance of tumor-specific immunity induced by FCs. Thus, the combination of DC-tumor FCs and CpG ODN is an effective and feasible cancer vaccine to prevent the generation and recurrence of cancers.     

APC stimulated by CpG oligodeoxynucleotide enhance activation of MHC class I-restricted T cells

Oligonucleotides containing unmethylated CpG motifs (cytosine-phosphorothioate-guanine oligodeoxynucleotide (CpG ODN)) are potent immunostimulatory agents capable of enhancing the Ag-specific Th1 response when used as immune adjuvants. We evaluated the cellular mechanisms responsible for this effect. Development of a CTL response was enhanced when mice were immunized with peptide-pulsed dendritic cells (DCs) treated with CpG ODN. However, in vitro, CpG ODN had no direct effect on highly purified T cells. In vitro, CpG ODN treatment of peptide- or protein-pulsed DCs enhanced the ability of the DCs to activate class I-restricted T cells. The presence of helper T cells enhanced this effect, indicating that treatment with CpG ODN does not obviate the role of T cell help. The enhanced ability of CpG ODN-treated DCs to activate T cells was present but blunted when DCs derived from IL-12 knockout mice were used. Fixation of Ag-pulsed, CpG ODN-treated DCs limited their ability to activate T cells. In contrast, fixation had little effect on DC activation of T cells when DCs were not exposed to CpG ODN. This indicates that production of soluble factors by DCs stimulated with CpG ODN plays a particularly important role in their ability to activate class I-restricted T cells. We conclude that CpG ODN enhances the development of a cellular immune response by stimulating APCs such as DCs, to produce IL-12 and other soluble factors.     

Glycoprotein G of herpes simplex virus 2 as a novel vaccine antigen for immunity to genital and neurological disease

The envelope glycoproteins of herpes simplex virus 1 (HSV-1) and HSV-2, with the exception of glycoprotein G, elicit cross-reactive B- and T-cell responses. Human vaccine trials, using the cross-reactive glycoproteins B and D, have shown no protection against genital HSV-2 infection or disease. In this study, the mature form of glycoprotein G (mgG-2) of HSV-2 was used for immunization of mice, either alone or in combination with adjuvant CpG, followed by an intravaginal challenge with a lethal dose of a fully virulent HSV-2 strain. Mice immunized with mgG-2 plus CpG showed low disease scores and a significantly higher survival rate (73%) than mice immunized with mgG-2 alone (20%) or controls (0%). Accordingly, limited numbers of infectious HSV-2 particles were detected in the spinal cord of mice immunized with mgG-2 plus CpG. The observed protection was associated with a gamma interferon (IFN-γ) response by splenic CD4(+) T cells upon antigen restimulation in vitro and in vaginal washes 1 day postinfection. The majority of sera collected from mice immunized with mgG-2 plus CpG showed macrophage-mediated antibody-dependent cellular cytotoxicity and antibody-dependent complement-mediated cytolysis, while no neutralization activity was observed. In conclusion, we have shown that immunization with the type-specific mgG-2 protein in combination with CpG could elicit protective immunity against an otherwise lethal vaginal HSV-2 challenge. The mgG-2 protein may therefore constitute a promising HSV-2 vaccine antigen to be considered for future human trials.     

Formulation with CpG oligodeoxynucleotides prevents induction of pulmonary immunopathology following priming with formalin-inactivated or commercial killed bovine respiratory syncytial virus vaccine

Commercial killed bovine respiratory syncytial virus (K-BRSV) and formalin-inactivated BRSV (FI-BRSV) tend to induce Th2-type immune responses, which may not be protective and may even be detrimental during subsequent exposure to the virus. In this study we assessed the ability of CpG oligodeoxynucleotides (ODNs) to aid in the generation of effective and protective BRSV-specific immune responses. Mice were immunized subcutaneously with FI-BRSV formulated with CpG ODN, Emulsigen (Em), CpG ODN and Em, or non-CpG ODN and Em. Two additional groups were immunized with K-BRSV or K-BRSV and CpG ODN. After two vaccinations, the mice were challenged with BRSV. FI-BRSV induced Th2-biased immune responses characterized by production of serum immunoglobulin G1 (IgG1) and IgE, as well as interleukin-4 (IL-4), by in vitro-restimulated splenocytes. Formulation of FI-BRSV with CpG ODN, but not with non-CpG ODN, enhanced serum IgG2a and IFN-gamma production by splenocytes, whereas serum IgE was reduced. Although the immune response induced by K-BRSV was not as strongly Th2 biased, the addition of CpG ODN to this commercial vaccine also resulted in a more Th1-type response. Furthermore, the addition of CpG ODN to the BRSV vaccine formulations resulted in enhanced neutralizing antibody responses. Significant production of IL-5, eotaxin, and eosinophilia was observed in the lungs of FI-BRSV- and K-BRSV-immunized mice. However, IL-5 and eotaxin levels, as well as the number of eosinophils, were decreased in the mice vaccinated with the CpG ODN-formulated vaccines. Finally, when formulated with CpG ODN, both FI-BRSV and K-BRSV significantly reduced virus production after challenge with BRSV.