Thermolytic CpG-containing DNA oligonucleotides as potential immunotherapeutic prodrugs

A CpG-containing DNA oligonucleotide functionalized with the 2-(N-formyl-N-methyl)aminoethyl thiophosphate protecting group (CpG ODN fma1555) was prepared from phosphoramidites 1a–d using solid-phase techniques. The oligonucleotide behaved as a prodrug by virtue of its conversion to the well-studied immunomodulatory CpG ODN 1555 through thermolytic cleavage of the 2-(N-formyl-N-methyl)aminoethyl thiophosphate protecting group. Such a conversion occurred at 37°C with a half-time of 73 h. The immunostimulatory properties of CpG ODN fma1555 were evaluated in two in vivo assays, one of which consisted of mice challenged in the ear with live Leishmania major metacyclic promastigotes. Local intradermal administration of CpG ODN fma1555 was as effective as that of CpG ODN 1555 in reducing the size of Leishmania lesions over time. In a different infectious model, CpG ODN 1555 prevented the death of Tacaribe-infected mice (43% survival) when administered between day 0 and 3 post infection. Administration of CpG ODN fma1555 three days before infection resulted in improved immunoprotection (60–70% survival). Moreover, co-administration of CpG ODN fma1555 and CpG ODN 1555 in this model increased the window for therapeutic treatment against Tacaribe virus infection, and thus supports the use of thermolytic oligonucleotides as prodrugs in the effective treatment of infectious diseases.     

Pathogen- and Host-Directed Antileishmanial Effects Mediated by Polyhexanide (PHMB)

Background

Cutaneous leishmaniasis (CL) is a neglected tropical disease caused by protozoan parasites of the genus Leishmania. CL causes enormous suffering in many countries worldwide. There is no licensed vaccine against CL, and the chemotherapy options show limited efficacy and high toxicity. Localization of the parasites inside host cells is a barrier to most standard chemo- and immune-based interventions. Hence, novel drugs, which are safe, effective and readily accessible to third-world countries and/or drug delivery technologies for effective CL treatments are desperately needed.

Methodology/Principal Findings

Here we evaluated the antileishmanial properties and delivery potential of polyhexamethylene biguanide (PHMB; polyhexanide), a widely used antimicrobial and wound antiseptic, in the Leishmania model. PHMB showed an inherent antileishmanial activity at submicromolar concentrations. Our data revealed that PHMB kills Leishmania major (L. major) via a dual mechanism involving disruption of membrane integrity and selective chromosome condensation and damage. PHMB’s DNA binding and host cell entry properties were further exploited to improve the delivery and immunomodulatory activities of unmethylated cytosine-phosphate-guanine oligodeoxynucleotides (CpG ODN). PHMB spontaneously bound CpG ODN, forming stable nanopolyplexes that enhanced uptake of CpG ODN, potentiated antimicrobial killing and reduced host cell toxicity of PHMB.

Conclusions

Given its low cost and long history of safe topical use, PHMB holds promise as a drug for CL therapy and delivery vehicle for nucleic acid immunomodulators.     

The combination of stabilized plasmid lipid particles and lipid nanoparticle encapsulated CpG containing oligodeoxynucleotides as a systemic genetic vaccine

Background

DNA vaccines offer unique potential for generating protective and therapeutic immunity against infectious and malignant diseases. Unfortunately, rapid degradation and poor cellular uptake has significantly limited the efficacy of ‘naked’ plasmid DNA vaccines. We have previously described stabilized plasmid lipid particles (SPLP) as effective nonviral gene delivery vehicles for the transfection of tumours at distal sites following intravenous administration. Based on their low toxicity and favourable transfection profile following systemic administration, we investigate SPLP as gene delivery vehicles for the generation of a systemically administered genetic vaccine.

Methods

The uptake of SPLP and their ability to transfect splenic antigen presenting cells (APC) following systemic administration is assessed through fluorescently‐labelled SPLP in combination with phenotype markers and a very sensitive flow cytometry‐based assay for the detection of the transgene, beta‐galactosidase. The priming of antigen‐specific adaptive and humoural immune responses following vaccination with SPLP alone or in combination with liposomal nanoparticle encapsulated CpG‐ODN containing oligodeoxynucleotides (LN CpG‐ODN) is characterized through the use of antigen‐specific cytotoxicity assays, interferon‐γ secretion assays and enzyme‐linked immunosorbant assay.

Results

We demonstrate that SPLP are taken up by and transfect APC in the spleen following intravenous administration and that, in the presence of a strong immunostimulatory signal provided by LN CpG‐ODN, are able to prime transgene‐specific humoural and cellular immune responses.

Conclusions

SPLP represent an effective candidate for the nonviral delivery of a systemic genetic vaccine when combined with additional immune stimulation provided by LN CpG‐ODN. Copyright © 2008 John Wiley & Sons, Ltd.     

STAT3 Decoy Oligodeoxynucleotides-Loaded Solid Lipid Nanoparticles Induce Cell Death and Inhibit Invasion in Ovarian Cancer Cells

Recent advances in the synthesis of multi-functional nanoparticles have opened up tremendous opportunities for the targeted delivery of genes of interest. Cationic solid lipid nanoparticles (SLN) can efficiently bind nucleic acid molecules and transfect genes in vitro. Few reports have combined SLN with therapy using decoy oligodeoxynucleotides (ODN). In the present study, we prepared SLN to encapsulate STAT3 decoy ODN; then, the properties and in vitro behavior of SLN-STAT3 decoy ODN complexes were investigated. SLN-STAT3 decoy ODN complexes were efficiently taken up by human ovarian cancer cells and significantly suppressed cell growth. Blockage of the STAT3 pathway by SLN-STAT3 decoy ODN complexes resulted in an evident induction of cell death, including apoptotic and autophagic death. The mechanism involved the increased expression of cleaved caspase 3, Bax, Beclin-1 and LC3-II and reduced expression of Bcl-2, pro-caspase 3, Survivin, p-Akt and p-mTOR. In addition, SLN-STAT3 decoy ODN complexes inhibited cell invasion by up-regulating E-cadherin expression and down-regulating Snail and MMP-9 expression. These findings confirmed that SLN as STAT3 decoy ODN carriers can induce cell death and inhibit invasion of ovarian cancer cells. We propose that SLN represent a potential approach for targeted gene delivery in cancer therapy.     

Physicochemical and biological characterization of polyethylenimine-graft-poly(ethylene glycol) block copolymers as a delivery system for oligonucleotides and ribozymes

Two different series of polyethylenimine (PEI) block copolymers grafted with linear poly(ethylene glycol) (PEG) were investigated as delivery systems for oligodeoxynucleotides (ODN) and ribozymes. The resulting interpolyelectrolyte complexes were characterized with respect to their physicochemical properties, protection efficiency against enzymatic degradation, complement activation, and biological activity under in vitro conditions. The effect of PEG molecular weight and the graft density of PEG blocks on complex characteristics was studied with two different series of block copolymers. The resulting ODN complexes were characterized by photon correlation spectroscopy (PCS) and laser Doppler anemometry (LDA) to determine complex size and zeta potential. Electrophoresis was performed to study the protective effects of the different block copolymers against enzymatic degradation of ODN. Intact ODN was quantified via densitometric analysis. Ribozymes, a particularly unstable type of oligonucleotides, were used to examine the influence of block copolymer structure on biological activity. The stabilization of ribozymes was also characterized in a cell culture model. Within the first series of block copolymers, the grafted PEG chains (5 kDa) had marginal influence on the complex size. Two grafted PEG chains were sufficient to achieve a neutral zeta potential. Within the second series, size and zeta potential increased with an increasing number of PEG chains. A high number of short PEG chains resulted in a decrease in complex size to values comparable to that of the homopolymer PEI 25 kDa and a neutral zeta potential, indicating a complete shielding of the charges. Complement activation decreased with an increasing number of short PEG 550 Da chains. Ribozyme complexes with PEG-PEI block copolymers achieved a 50% down-regulation of the target mRNA. This effect demonstrated an efficient stabilization and biological activity of the ribozyme, which was comparable to that of PEI 25 kDa. PEGylated PEI block copolymers represent a promising new class of drug delivery systems for ODN and ribozymes with increased biocompatibility and physical stability.     

Use of thermolytic protective groups to prevent G-tetrad formation in CpG ODN type D: structural studies and immunomodulatory activity in primates

CpG oligodeoxynucleotides (ODN) show promise as immunoprotective agents and vaccine adjuvants. CpG ODN type D were shown to improve clinical outcome in rhesus macaques challenged with Leishmania major. These ODN have a self-complementary core sequence and a 3′ end poly(G) track that favors G-tetrad formation leading to multimerization. Although multimerization appears necessary for localization to early endosomes and signaling via Toll-like receptor 9 (TLR-9), it can result in product polymorphisms, aggregation and precipitation, thereby hampering their clinical applications. This study shows that functionalizing the poly(G) track of D ODN with thermolytic 2-(N-formyl-N-methyl)aminoethyl (fma) phosphate/thiophosphate protecting groups (pro-D ODN) reduces G-tetrad formation in solution, while allowing tetrad formation inside the cell where the potassium concentration is higher. Temperature-dependent cleavage of the fma groups over time further promoted formation of stable G-tetrads. Peripheral blood cells internalized pro-D ODN efficiently, inducing high levels of IFNα, IL-6, IFNγ and IP-10 and triggering dendritic cell maturation. Administration of pro-D35 to macaques challenged with L.major significantly increased the number of antigen-specific IFNγ-secreting PBMC and reduced the severity of the skin lesions demonstrating immunoprotective activity of pro-D ODN in vivo. This technology fosters the development of more efficient immunotherapeutic oligonucleotide formulations for the treatment of allergies, cancer and infectious diseases.     

Production of Fibronectin Binding Protein A at the Surface of Lactococcus lactis Increases Plasmid Transfer In Vitro and In Vivo

Lactococci are noninvasive lactic acid bacteria frequently used as protein delivery vectors and, more recently, as DNA delivery vehicles. We previously showed that Lactococcus lactis (LL) expressing the Fibronectin-Binding Protein A of Staphylococcus aureus (LL-FnBPA+) showed higher internalization rates in vitro in Caco-2 cells than the native (wt) lactococci and were able to deliver a eukaryotic Green Fluorescent Protein (GFP) expression plasmid in 1% of human Caco-2 cells. Here, using the bovine beta-lactoglobulin (BLG), one of the major cow''s milk allergen, and GFP we characterized the potential of LL-FnBPA+ as an in vivo DNA vaccine delivery vehicle. We first showed that the invasive strain LL-FnBPA+ carrying the plasmid pValac:BLG (LL-FnBPA+ BLG) was more invasive than LL-BLG and showed the same invasivity as LL-FnBPA+. Then we demonstrated that the Caco-2 cells, co-incubated with LL-FnBPA+ BLG produced up to 30 times more BLG than the Caco-2 cells co-incubated with the non invasive LL-BLG. Using two different gene reporters, BLG and GFP, and two different methods of detection, EIA and fluorescence microscopy, we showed in vivo that: i) in order to be effective, LL-FnBPA+ required a pre-coating with Fetal Calf Serum before oral administration; ii) plasmid transfer occurred in enterocytes without regard to the strains used (invasive or not); iii) the use of LL-FnBPA+ increased the number of mice producing BLG, but not the level of BLG produced. We thus confirmed the good potential of invasive recombinant lactic acid bacteria as DNA delivery vector in vivo.     

CpG-C ODN M362 as an immunoadjuvant for HBV therapeutic vaccine reverses the systemic tolerance against HBV

Chronic Hepatitis B virus (CHB) infection is a global public health problem. Oligodeoxynucleotides (ODNs) containing class C unmethylated cytosine-guanine dinucleotide (CpG-C) motifs may provide potential adjuvants for the immunotherapeutic strategy against CHB, since CpG-C ODNs stimulate both B cell and dendritic cell (DC) activation. However, the efficacy of CpG-C ODN as an anti-HBV vaccine adjuvant remains unclear. In this study, we demonstrated that CpG M362 (CpG-C ODN) as an adjuvant in anti-HBV vaccine (cHBV-vaccine) successfully and safely eliminated the virus in HBV-carrier mice. The cHBV-vaccine enhanced DC maturation both in vivo and in vitro, overcame immune tolerance, and recovered exhausted T cells in HBV-carrier mice. Furthermore, the cHBV-vaccine elicited robust hepatic HBV-specific CD8⁺ and CD4⁺ T cell responses, with increased cellular proliferation and IFN-γ secretion. Additionally, the cHBV-vaccine invoked a long-lasting follicular CXCR5⁺ CD8⁺ T cell response following HBV re-challenge. Taken together, CpG M362 in combination with rHBVvac cleared persistent HBV and achieved long-term virological control, making it a promising candidate for treating CHB.     

Assessment of Glial Scar,Tissue Sparing,Behavioral Recovery and Axonal Regeneration following Acute Transplantation of Genetically Modified Human Umbilical Cord Blood Cells in a Rat Model of Spinal Cord Contusion

Objective and Methods

This study investigated the potential for protective effects of human umbilical cord blood mononuclear cells (UCB-MCs) genetically modified with the VEGF and GNDF genes on contusion spinal cord injury (SCI) in rats. An adenoviral vector was constructed for targeted delivery of VEGF and GDNF to UCB-MCs. Using a rat contusion SCI model we examined the efficacy of the construct on tissue sparing, glial scar severity, the extent of axonal regeneration, recovery of motor function, and analyzed the expression of the recombinant genes VEGF and GNDF in vitro and in vivo.

Results

Transplantation of UCB-MCs transduced with adenoviral vectors expressing VEGF and GDNF at the site of SCI induced tissue sparing, behavioral recovery and axonal regeneration comparing to the other constructs tested. The adenovirus encoding VEGF and GDNF for transduction of UCB-MCs was shown to be an effective and stable vehicle for these cells in vivo following the transplantation into the contused spinal cord.

Conclusion

Our results show that a gene delivery using UCB-MCs-expressing VEGF and GNDF genes improved both structural and functional parameters after SCI. Further histological and behavioral studies, especially at later time points, in animals with SCI after transplantation of genetically modified UCB-MCs (overexpressing VEGF and GDNF genes) will provide additional insight into therapeutic potential of such cells.     

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.     

Intratumoral injection of CpG oligonucleotides induces the differentiation and reduces the immunosuppressive activity of myeloid-derived suppressor cells

Immunostimulatory CpG oligonucleotides (ODN) activate cells that express TLR9 and have been shown to improve the host's response to tumor Ags. Unfortunately, the immunosuppressive microenvironment that surrounds many cancers inhibits Ag-specific cellular responses and thus interferes with CpG-mediated immunotherapy. Myeloid-derived suppressor cells (MDSC) represent an important constituent of this immunosuppressive milieu. Large numbers of MDSC are present in and near tumor sites where they inhibit the activity of Ag-specific T and NK cells. Current studies indicate that the delivery of CpG ODN directly into the tumor bed reduces the immunosuppressive activity of monocytic (CD11b(+), Ly6G(-), Ly6C(high)) MDSC. Monocytic MDSC express TLR9 and respond to CpG stimulation by 1) losing their ability to suppress T cell function, 2) producing Th1 cytokines, and 3) differentiating into macrophages with tumoricidal capability. These findings provide insight into a novel mechanism by which CpG ODN contribute to tumor regression, and they support intratumoral injection as the optimal route for their delivery.     

Identification and expression analysis of cobia (Rachycentron canadum) Toll-like receptor 9 gene

Cobia culture is hindered by bacterial infection (Photobacterium damselae subsp. piscicida) and in order to study the effect of P. damselae subsp. piscicida challenge and CpG ODN stimulation on cobia Toll like receptor 9 (RCTLR9), we used PCR to clone RCTLR9 gene and qRT-PCR to quantify gene expression. The results indicated that RCTLR9 cDNA contains 3141 bp. It encodes 1047 amino acids containing 16 typical structures of leucine-rich repeats (LRRs) including an LRRTYP, LRRCT and a motif involved in PAMP binding was identified at position 240–253 amino acid. Broad expression of RCTLR9 was found in larval, juvenile and adult stages irrespective of the tissues. In larval stage, RCTLR9 mRNA expression decreased at 5 d and then increased at 10 dph. At juvenile stage cobia, the expression was significantly high (p < 0.05) in spleen and intestine compared to gill, kidney, liver and skin. However, at adult stage, the significant high expression was found in gill and intestine. Cobia challenged with P. damselae subsp. piscicida showed significant increase in RCTLR9 expression at 24 h post challenge in intestine, spleen and liver, while in kidney the expression was peak at 12 h and later it decreased at 24 h. The highest expression was 40 fold increase in spleen and the lowest expression was ∼3.6 fold increase in liver. Cobia stimulated with CpG oligonucleotides showed that the induction of these genes was CpG ODN type and time dependent. In spleen and liver, CpG ODNs 1668 and 2006 injected group showed high expression of RCTLR9, IL-1β, chemokine CC compared to other groups. Meanwhile, CpG ODN 2006 has induced high expression of IgM. The CpG ODNs 2395 have induced significant high expression of Mx in spleen and liver. These results demonstrates the potential of using CpG ODN to enhance cobia resistance to P. damselae subsp. piscicida infection and use as an adjuvant in vaccine development.     

A C-type CpG ODN accelerates wound healing via regulating fibroblasts and immune response

Abolished or delayed wound healing is a serious problem in clinical surgery, therefore, the new therapy for wound healing is needed. Synthetic oligodeoxynucleotides containing one or more CpG motifs (CpG ODN) has been reported to activate the immune system and improves skin wound healing. The aim of the present study was to evaluate the role of a new C-type CpG ODN in wound healing. We found that the CpG ODN promoted cell proliferation and collagen I production in human skin fibroblasts cells. Besides, we also investigated the effect of CpG ODN on the activation of immune cells. The macrophages and plasmacytoid dendritic cells (pDCs) were incubated with CpG ODN. CpG ODN activated macrophage and pDCs via regulating TLR9/MyD88/NF-κB pathway and TLR9/MyD88/IRF7 pathway, respectively. To further evaluate the effect of CpG ODN on wound healing in vivo a wound healing model was established in mice. The results showed that CpG ODN treatment accelerated wound healing in mice. CpG ODN increased cytokines secretion in wound skin and elevated the ratio of CD4 ⁺ and CD8 ⁺ T cells in the spleen. Our results showed that CpG ODN accelerated wound healing, which was partly due to the regulation of fibroblasts and immune response. The findings suggested that the CpG ODN might be a proper medicament for the treatment of wound healing.     

Ophthalmic Delivery of Brinzolamide by Liquid Crystalline Nanoparticles: In Vitro and In Vivo Evaluation

Brinzolamide (BLZ) is a drug used to treat glaucoma; however, its use is restricted due to some unwanted adverse events. The goal of this study was to develop BLZ-loaded liquid crystalline nanoparticles (BLZ LCNPs) and to figure out the possibility of LCNPs as a new therapeutic system for glaucoma. BLZ LCNPs were produced by a modified emulsification method and their physicochemical aspects were estimated. In vitro release study revealed BLZ LCNPs displayed to some extent prolonged drug release behavior in contrast to that of BLZ commercial product (Azopt®). The ex vivo apparent permeability coefficient of BLZ LCNP systems demonstrated a 3.47-fold increase compared with that of Azopt®. The pharmacodynamics was checked over by calculating the percentage fall in intraocular pressure and the pharmacodynamic test showed that BLZ LCNPs had better therapeutic potential than Azopt®. Furthermore, the in vivo ophthalmic irritation was evaluated by Draize test. In conclusion, BLZ LCNPs would be a promising delivery system used for the treatment of glaucoma, with advantages such as lower doses but maintaining the effectiveness, better ocular bioavailability, and patient compliance compared with Azopt®.     

Encapsulation in liposomal nanoparticles enhances the immunostimulatory,adjuvant and anti-tumor activity of subcutaneously administered CpG ODN

Immunostimulatory oligodeoxynucleotides (ODN) containing cytosine-guanine (CpG) motifs are powerful stimulators of innate as well as adaptive immune responses, exerting their activity through triggering of the Toll-like receptor 9. We have previously shown that encapsulation in liposomal nanoparticles (LN) enhances the immunostimulatory activity of CpG ODN (LN-CpG ODN) (Mui et al. in J Pharmacol Exp Ther 298:1185, 2001). In this work we investigate the effect of encapsulation on the immunopotency of subcutaneously (s.c.) administered CpG ODN with regard to activation of innate immune cells as well as its ability to act as a vaccine adjuvant with tumor-associated antigens (TAAs) to induce antigen (Ag)-specific, adaptive responses and anti-tumor activity in murine models. It is shown that encapsulation specifically targets CpG ODN for uptake by immune cells. This may provide the basis, at least in part, for the significantly enhanced immunostimulatory activity of LN-CpG ODN, inducing potent innate (as judged by immune cell activation and plasma cytokine/chemokine levels) and adaptive, Ag-specific (as judged by MHC tetramer positive T lymphocytes, IFN-γ secretion and cytotoxicity) immune responses. Finally, in efficacy studies, it is shown that liposomal encapsulation enhances the ability of CpG ODN to adjuvanate adaptive immune responses against co-administered TAAs after s.c. immunization, inducing effective anti-tumor activity against both model and syngeneic tumor Ags in murine tumor models of thymoma and melanoma.     

CpG oligodeoxynucleotides induce strong up-regulation of interleukin 33 via Toll-like receptor 9

We previously reported the strong immunostimulatory effects of a CpG oligodeoxynucleotide (ODN), designated MsST, from the lacZ gene of Streptococcus (S.) thermophilus ATCC19258. Here we show that 24 h of stimulation with MsST in mouse splenocytes and peritoneal macrophages strongly induces expression of interleukin (IL)-33, a cytokine in the IL-1 superfamily. Other IL-1 superfamily members, including IL-1α, IL-1β and IL-18, are down-regulated after 24 h of stimulation of MsST. We also found that MsST-induced IL-33 mRNA expression is inhibited by the suppressive ODN A151, which can inhibit Toll-like receptor 9 (TLR9)-mediated responses. This is the first report to show that IL-33 can be induced by CpG ODNs. The strong induction of IL-33 by MsST suggests that it may be a potential therapeutic ODN for the treatment of inflammatory disease. The presence of a strong CpG ODN in S. thermophilus also suggests that the bacterium may be a good candidate as a starter culture for the development of new physiologically functional foods.     

Serum alters the uptake and biologic activity of CpG oligodeoxynucleotides in B cell chronic lymphocytic leukemia

Immunostimulatory CpG-containing oligodeoxynucleotides (CpG ODN) have a number of effects on B cells, including upregulation of immunogenic molecules, and, therefore, appear attractive as potential components of immunotherapy for B cell chronic lymphocytic leukemia (B-CLL). Previous in vitro studies investigating the effect of CpG ODN on B-CLL cells used serum-low conditions and did not account for the longer-half life of CpG ODN in vitro. The present study was designed to explore how the presence of serum and exposure time affect CpG ODN-mediated changes on B-CLL cells. The optimal concentration for CpG ODN-mediated effects in the presence of 100% serum or plasma was higher (10-20 microg/ml) than for serum-low conditions. Maximal CpG ODN-mediated effects required the presence of ODN for no longer than 3 hours. The inhibition of CpG ODN-mediated effects by serum correlated with lower uptake of ODN into B-CLL cells in the presence of serum. A threshold effect on biologic response was observed, with a given amount of ODN internalized, resulting in phenotypic changes. In conclusion, systemic short-term application of CpG ODN appears to be sufficient to induce phenotypic changes, but higher doses of CpG ODN than previously thought may be necessary because of inhibition of their uptake by serum.     

Enhancement of CD8+ T cell immunity in the lung by CpG oligodeoxynucleotides increases protective efficacy of a modified vaccinia Ankara vaccine against lethal poxvirus infection even in a CD4-deficient host

Immunostimulatory CpG oligodeoxynucleotides (ODN) have proven effective as adjuvants for protein-based vaccines, but their impact on immune responses induced by live viral vectors is not known. We found that addition of CpG ODN to modified vaccinia Ankara (MVA) markedly improved the induction of longer-lasting adaptive protective immunity in BALB/c mice against intranasal pathogenic vaccinia virus (Western Reserve; WR). Protection was mediated primarily by CD8(+) T cells in the lung, as determined by CD8-depletion studies, protection in B cell-deficient mice, and greater protection correlating with CD8(+) IFN-gamma-producing cells in the lung but not with those in the spleen. Intranasal immunization was more effective at inducing CD8(+) T cell immunity in the lung, and protection, than i.m. immunization. Addition of CpG ODN increased the CD8(+) response but not the Ab response. Depletion of CD4 T cells before vaccination with MVA significantly diminished protection against pathogenic WR virus. However, CpG ODN delivered with MVA was able to substitute for CD4 help and protected CD4-depleted mice against WR vaccinia challenge. This study demonstrates for the first time a protective adjuvant effect of CpG ODN for a live viral vector vaccine that may overcome CD4 deficiency in the induction of protective CD8(+) T cell-mediated immunity.     

Peritumoral CpG DNA elicits a coordinated response of CD8 T cells and innate effectors to cure established tumors in a murine colon carcinoma model

The immune system of vertebrates is able to detect bacterial DNA based on the presence of unmethylated CpG motifs. We examined the therapeutic potential of oligodeoxynucleotides with CpG motifs (CpG ODN) in a colon carcinoma model in BALB/c mice. Tumors were induced by s.c. injection of syngeneic C26 cells or Renca kidney cancer cells as a control. Injection of CpG ODN alone or in combination with irradiated tumor cells did not protect mice against subsequent tumor challenge. In contrast, weekly injections of CpG ODN into the margin of already established tumors resulted in regression of tumors and complete cure of mice. The injection site was critical, since injection of CpG ODN at distant sites was not effective. Mice with two bilateral C26 tumors rejected both tumors upon peritumoral injection of one tumor, indicating the development of a systemic immune response. The tumor specificity of the immune response was demonstrated in mice bearing a C26 tumor and a Renca tumor at the same time. Mice that rejected a tumor upon peritumoral CpG treatment remained tumor free and were protected against rechallenge with the same tumor cells, but not with the other tumor, demonstrating long term memory. Tumor-specific CD8 T cells as well as innate effector cells contributed to the antitumor activity of treatment. In conclusion, peritumoral CpG ODN monotherapy elicits a strong CD8 T cell response and innate effector mechanisms that seem to act in concert to overcome unresponsiveness of the immune system toward a growing tumor.