Synthesis and in vitro characterization of antigen-conjugated polysaccharide as a CpG DNA carrier

Oligodeoxynucleotides containing unmethylated CpG sequences (CpG DNAs) are known as an immune adjuvant. CpG DNAs coupled with a particular antigen enabling both CpG DNA and antigen delivery to the same antigen-presenting cell have been shown to be more effective. Based on our previous finding that beta-(1-->3)-D-glucan schizophyllan (SPG) can be used as a CpG DNA carrier, here we present the synthesis of an antigen-conjugated SPG and the characterization of the conjugate. Ovalbumin (OVA, 43 kDa) was used as a model antigen, and two OVA were conjugated to one SPG molecule (M(w) = 150,000), denoted by OVA-SPG. Circular dichroism and gel electrophoresis showed that OVA-SPG could form a complex with a (dA)(40)-tailed CpG DNA at the 3' end (1,668-(dA)(40)). When OVA-SPG was added to macrophages (J774.A1), the amount of the ingested OVA-SPG was increased compared with that of OVA itself, suggesting that Dectin-1 (proinflammatory nonopsonic receptor for beta-glucans) is involved to ingest OVA-SPG. Furthermore, the complex of the conjugate and DNA was co-localized in the same vesicles, implying that OVA (antigen) and CpG DNA (adjuvant) were ingested into the cell at the same time. This paper shows that OVA-SPG can be used as a CpG DNA carrier to induce antigen-specific immune responses.     

Enhanced cytokine secretion from primary macrophages due to Dectin-1 mediated uptake of CpG DNA/β-1,3-glucan complex

Unmethylated CpG sequences (CpG DNA) can induce Th1 response and thus become a potential immunotherapeutic agent. A key step in the treatment is to transport CpG DNA to its receptor TLR9 located in the endocytosis pathway of target immune cells. For the effective transport, we prepared a novel complex from a β-1,3-glucan schizophyllan (SPG) and CpG DNA, and administered the complex to murine peritoneal macrophages that had been previously activated by thioglycollate and expressed a major β-1,3-glucan receptor Dectin-1 on the cellular surface. Flow cytometric analysis and microscopic observation showed that the complex was taken up by the macrophage through Dectin-1 mediated pathway. Indeed, ELISA demonstrated that IL-12 production was increased sigmoidally with increasing SPG/CpG DNA ratio in the complexation, and reached the maximum at the SPG-rich composition. In the present work, we describe a new approach to deliver CpG DNA to immune cells by use of a β-1,3-glucan/DNA complex.     

Potent CpG oligonucleotides containing phosphodiester linkages: in vitro and in vivo immunostimulatory properties

Bacterial and synthetic DNAs, containing CpG dinucleotides in specific sequence contexts, activate the vertebrate immune system. Unlike phosphorothioate (PS) CpG DNAs, phosphodiester (PO) CpG DNAs require either palindromic sequences and/or poly(dG) sequences at the 3(')-end for activity. Here, we report 'PO-immunomers' having two PO-CpG DNA molecules joined through their 3(')-ends. These PO-imunomers permitted us, for the first time, to assess immunostimulatory properties of PO-CpG DNAs in vitro and in vivo without the need for palindromic and/or poly(dG) sequences. In medium containing 10% fetal bovine serum, PO-immunomers were more resistant than PO-CpG DNAs to nucleases. Compared to PS-CpG DNA in BALB/c and C3H/HeJ mice spleen cell culture assays, PO-immunomers showed increased IL-12 secretion and minimal amounts of IL-6 secretion. PO-immunomers activated NF-kappa B and induced cytokine secretion in J774 cell cultures. In addition, PO-immunomers showed antitumor activity in nude mice bearing human breast (MCF-7) and prostate (DU145) cancer xenografts.     

The roles of Toll-like receptor 9, MyD88, and DNA-dependent protein kinase catalytic subunit in the effects of two distinct CpG DNAs on dendritic cell subsets

Oligodeoxynucleotides containing unmethylated CpG motifs (CpG DNAs) can function as powerful immune adjuvants by activating APC. Compared with conventional phosphorothioate-backbone CpG DNAs, another type of CpG DNAs, called an A or D type (A/D-type), possesses higher ability to induce IFN-alpha production. Conventional CpG DNAs can exert their activity through Toll-like receptor 9 (TLR9) signaling, which depends on a cytoplasmic adapter, MyD88. However, it remains unknown how A/D-type CpG DNAs exhibit their immunostimulatory function. In this study we have investigated murine dendritic cell (DC) responses to these two distinct CpG DNAs. Not only splenic, but also in vitro bone marrow-derived, DCs could produce larger amounts of IFN-alpha in response to A/D-type CpG DNAs compared with conventional CpG DNAs. This IFN-alpha production was mainly due to the B220(+) DC subset. On the other hand, the B220(-) DC subset responded similarly to both CpG DNAs in terms of costimulatory molecule up-regulation and IL-12 induction. IFN-alpha, but not IL-12, induction was dependent on type I IFN. However, all activities of both CpG DNAs were abolished in TLR9- and MyD88-, but were retained in DNA-PKcs-deficient DCs. This study demonstrates that the TLR9-MyD88 signaling pathway is essential for all DC responses to both types of CpG DNAs.     

Ternary complex consisting of DNA, polycation, and a natural polysaccharide of schizophyllan to induce cellular uptake by antigen presenting cells

A natural polysaccharide called schizophyllan (SPG) can form a complex with polynucleotides, and the complex has been shown to deliver biofunctional short DNAs such as antisense DNAs and CpG-DNAs. Although it is a novel and efficient method, there is a drawback: attachment of homo-polynucleotide tails [for example, poly(dA) or poly(C)] to the end of DNA is necessary to stabilize the complex, because DNA heterosequences cannot bind to SPG. The aim of this paper is to present an alternative method in which SPG/DNA complexes can be made without using the tails. The basic strategy is as follows: since SPG can form hydrophobic domains in aqueous solutions, hydrophobic objects should be encapsulated by this domain. DNA alone is highly hydrophilic; however, once DNA/polycation complexes are made, they should be included by the SPG hydrophobic domain. The aim of this paper is to prove the formation of the polycation/DNA/SPG ternary complex. Gel electrophoresis showed that presence of SPG influenced the migration pattern of polycation+DNA mixtures. With increasing the SPG ratio, the zeta potential (zeta) of the polycation+DNA+SPG mixture decreased drastically to reach almost zeta = 0 and the particle size distributions were altered due to the ternary complex formation. Confocal laser scanning microscopy revealed that the polycation/DNA/SPG ternary complexes showed high uptake efficiency when the complexes were exposed to macrophage-like cells (J774.A1). IL-12 secretion was enhanced when CpG-DNA was added as the ternary complex. These features can be ascribed to the fact that J774.A1 has a SPG recognition site called Dectin-1 on the cellular surface and the ternary complex can be ingested by this pathway.     

Enhanced resistance to nuclease degradation of nucleic acids complexed to asialoglycoprotein-polylysine carriers.

We have previously shown targeting of DNA to hepatocytes using an asialoorosomucoid-polylysine (AsOR-PL) carrier system. The AsOR-PL conjugate condenses DNA and facilitates entry via specific receptor-ligand interactions. In these studies, our objective was to determine if AsOR-PL conjugates protect bound DNA from nuclease attack. Double-stranded plasmid or single-stranded oligonucleotide DNA, alone or bound to conjugate, was incubated under conditions mimicking those encountered during in vitro and in vivo transfections. The results showed that complexed DNA was effectively protected from degradation by serum nucleases. Degradation of single-stranded oligonucleotides was inhibited 3- to 6-fold in serum during 5 hours of incubation. For complexed plasmids, greater than 90% remained full-length during 1.5 and 3 hour incubations in serum or culture medium containing 10% serum, respectively. Uncomplexed plasmid was completely degraded after 15 minutes in serum or 60 minutes in medium. In cell lysates, the conjugate was not effective in inhibiting endonuclease activity; plasmids were readily converted from supercoiled to open circular and linear forms. However, the resultant nicked forms were substantially protected from further degradation during one hour of incubation compared to plasmid alone. Under all conditions complexed DNA did not readily dissociate from the conjugate. Overall, for both single and double-stranded DNA, AsOR-PL conjugates conferred substantial protection from nuclease degradation.     

Modulation of interleukin-12 synthesis by DNA lacking the CpG motif and present in a mycobacterial cell wall complex

 A mycobacterial cell wall complex prepared from the non-pathogenic microorganism Mycobacterium phlei, where mycobacterial DNA is preserved and complexed to cell wall fragments, possesses anticancer and immunomodulatory activity. DNA from a number of prokaryotes has been found to modulate the immune system and to induce cytokine synthesis. We have therefore determined whether the DNA associated with this complex has the ability to induce the synthesis of interleukin-12 (IL-12), a potent anticancer cytokine. Mycobacterial DNA complexed with cell wall fragments or DNA purified from M. phlei induced IL-12 synthesis by murine and human monocytes and macrophages in vitro, and was capable of inducing IL-12 synthesis in vivo in mice following i.p. administration. Neutralization of DNA with cationic liposomes or digestion with DNase I significantly decreased the ability of the cell wall complex to induce IL-12. CpG methylation of DNA extracted from these cell walls or from M. phlei did not affect the induction of IL-12 synthesis by monocytes and macrophages. In contrast, CpG methylation of DNA from Escherichia coli abolished its ability to induce IL-12 synthesis. These results demonstrate that unmethylated CpG motifs present in M. phlei DNA are not a prerequisite for the induction of IL-12 synthesis. The size of the mycobacterial DNA, in the range of 5 bp to genomic DNA, did not influence its capacity to induce IL-12. Our results emphasize that M. phlei DNA associated with the cell wall complex makes a significant contribution to the overall immunomodulatory and anticancer activity of this mycobacterial cell wall preparation and that these activities are not correlated with the presence of CpG motifs. Received: 23 November 1999 / Accepted: 28 March 2000     

Herpes simplex virus type 1 DNA is immunostimulatory in vitro and in vivo

Recently, prokaryotic DNAs containing unmethylated CpG motifs have been shown to be intrinsically immunostimulatory both in vitro and in vivo, tending to promote Th1-like responses. In contrast, CpG dinucleotides in mammalian DNAs are extensively methylated on cytosines and hence immunologically inert. Since the herpes simplex virus (HSV) genome is unmethylated and G+C rich, we predicted that CpG motifs would be highly prevalent in the HSV genome; hence, we examined the immunostimulatory potential of purified HSV DNA in vitro and in vivo. Mouse splenocyte cultures treated with HSV DNA or HSV-derived oligodeoxyribonucleotides (ODNs) showed strong proliferative responses and production of inflammatory cytokines (gamma interferon [IFN-γ], tumor necrosis factor [TNF], and interleukin-6 [IL-6]) in vitro, whereas splenocytes treated with mammalian CV-1 DNA or non-CpG ODN did not. After immunization with ovalbumin (OVA), only splenocytes from mice immunized with HSV DNA or HSV-ODN as the adjuvants proliferated strongly and produced typical Th1 responses, including CD8⁺ cytotoxic T-lymphocyte responses, upon restimulation with OVA. Furthermore, HSV-ODN synergized with IFN-γ to induce nitric oxide (NO), IL-6, and TNF production from macrophages. These results demonstrate that HSV DNA and HSV-ODN are immunostimulatory, driving potent Th1 responses both in vitro and in vivo. Considering that HSV DNA has been found to persist in nonneuronal cells, these results fuel speculation that HSV DNA might play a role in pathogenesis, in particular, in diseases like herpes stromal keratitis (HSK) that involve chronic inflammatory responses in the absence of virus or viral antigens.     

Cytokine induction by a bacterial DNA-specific modified base

Unmethylated CpG dinucleotides in DNA contribute to a rapid inflammatory response in mammals. Here we show that N(6)-methyladenine (N(6)-MeA), a bacterium-specific modified base, also causes cytokine production. An oligodeoxyribonucleotide (ODN) containing N(6)-MeA induced cytokines when injected into mice. Co-injection of N(6)-MeA and CpG ODNs enhanced cytokines 2- to 3-fold, as compared with the injection of a CpG ODN alone. Plasmid DNA containing N(6)-MeA, complexed with cationic lipids, induced IL-12. These results indicate that the bacterium-specific base, in addition to the unmethylated CpG motif, triggers the mammalian immune response, and suggest that N(6)-MeA-containing DNA could be useful for cellular immunotherapy and DNA vaccine.     

A method to select chemically modified aptamers directly

In vitro selection is a strategy to identify high-affinity ligands of a predetermined target among a large pool of randomized oligonucleotides. Most in vitro selections are performed with unmodified RNA or DNA sequences, leading to ligands of high affinity and specificity (aptamers) but of very short lifetime in the ex vivo and in vivo context. Only a very limited number of modified triphosphate nucleotides conferring nuclease resistance to the oligomer can be incorporated by polymerases. This encourages the development of alternative methods for the identification of nuclease-resistant aptamers. In this paper, we describe such a method. After selection of 2'O-methyl oligonucleotides against the TAR RNA structure of HIV-1, the complementary DNA sequences are fished out of a pool of randomized oligodeoxynucleotides by Watson-Crick hybridization. The DNA-fished sequences are amplified by PCR as double and single strands, the latter being used to fish back the chemically modified candidates from the initial library. This procedure allows an indirect amplification of the selected candidates. This enriched pool of modified sequences is then used for the next selection round against the target.     

Reduced repeat length of nascent nucleosomal DNA is generated by replicating chromatin in vivo

Micrococcal nuclease digestion of nuclei from sea urchin embryos revealed transient changes in chromatin structure which resulted in a reduction in the repeat length of nascent chromatin DNA as compared with bulk DNA. This was considered to be entirely the consequence of in vivo events at the replication fork (Cell 14, 259, 1978). However, a micrococcal nuclease-generated sliding of nucleosome cores relative to nascent DNA, which might account for the smaller DNA fragments, was not excluded. In vivo [3H]thymidine pulse-labeled nuclei were fixed with a formaldehyde prior to micrococcal nuclease digestion. This linked chromatin proteins to DNA and thus prevented any in vitro sliding of histone cores. All the nascent DNAs exhibiting shorter repeat lengths after micrococcal nuclease digestion, were resolved at identical mobilities in polyacrylamide gels of DNA from fixed and unfixed nuclei. We conclude that these differences in repeat lengths between nascent and bulk DNA was generated in vivo by changes in chromatin structure during replication, rather than by micrococcal nuclease-induced sliding of histone cores in vitro.     

Nucleosomes protect DNA from DNA methylation in vivo and in vitro

Positioned nucleosomes limit the access of proteins to DNA. However, the impact of nucleosomes on DNA methylation in vitro and in vivo is poorly understood. Here, we performed a detailed analysis of nucleosome binding and nucleosomal DNA methylation by the de novo methyltransferases. We show that compared to linker DNA, nucleosomal DNA is largely devoid of CpG methylation. ATP-dependent chromatin remodelling frees nucleosomal CpG dinucleotides and renders the remodelled nucleosome a 2-fold better substrate for Dnmt3a methyltransferase compared to free DNA. These results reflect the situation in vivo, as quantification of nucleosomal DNA methylation levels in HeLa cells shows a 2-fold decrease of nucleosomal DNA methylation levels compared to linker DNA. Our findings suggest that nucleosomal positions are stably maintained in vivo and nucleosomal occupancy is a major determinant of global DNA methylation patterns in vivo.     

CpG-induced IFNgamma expands TLR4-specific IL-18 responses in vivo

Serum IL-18 responses to LPS increase after pretreatment with CpG-containing DNA. Compared to saline-pretreated controls, mice pretreated with CpG for two days produced 20-fold more serum IL-18 when challenged with lipopolysaccharide (LPS). In contrast, IFNgamma-deficiency or anti-IFNgamma pretreatment reduced CpG-expanded IL-18 responses to LPS by 67 and 83%, respectively. Mice pretreated with either IFNgamma or CpG comparably increased LPS-inducible serum IL-18 responses. LPS, compared to challenge with other TLR agonists, was best able to trigger high serum IL-18 levels in CpG-pretreated mice and this response was TLR4-dependent. CpG, compared to pretreatment with other TLR agonists, optimally expanded LPS-induced IL-18 responses that correlated with higher levels of circulating IFNgamma levels prior to LPS challenge. High-level serum IL-18 responses were caspase-1-dependent and P2X7 receptor-independent. We conclude that CpG promotes high-level IL-18 synthesis by an IFNgamma-dependent and IFNgamma-sufficient mechanism in vivo that is optimally triggered by LPS.     

A type IV modification dependent restriction nuclease that targets glucosylated hydroxymethyl cytosine modified DNAs

The Escherichia coli CT596 prophage exclusion genes gmrS and gmrD were found to encode a novel type IV modification-dependent restriction nuclease that targets and digests glucosylated (glc)-hydroxymethylcytosine (HMC) DNAs. The protein products GmrS (36 kDa) and GmrD (27 kDa) were purified and found to be inactive separately, but together degraded several different glc-HMC modified DNAs (T4, T2 and T6). The GMR enzyme is able to degrade both alpha-glucosy-HMC T4 DNA and beta-glucosyl-HMC T4 DNA, whereas no activity was observed against non-modified DNAs including unmodified T4 cytosine (C) DNA or non-glucosylated T4 HMC DNA. Enzyme activity requires NTP, favors UTP, is stimulated by calcium, and initially produces 4 kb DNA fragments that are further degraded to low molecular mass products. The enzyme is inhibited by the T4 phage internal protein I* (IPI*) to which it was found to bind. Overall activities of the purified GmrSD enzyme are in good agreement with the properties of the cloned gmr genes in vivo and suggest a restriction enzyme specific for sugar modified HMC DNAs. IPI* thus represents a third generation bacteriophage defense against restriction nucleases of the Gmr type.     

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.     

Endosomal translocation of vertebrate DNA activates dendritic cells via TLR9-dependent and -independent pathways

TLRs discriminate foreign from self via their specificity for pathogen-derived invariant ligands, an example being TLR9 recognizing bacterial unmethylated CpG motifs. In this study we report that endosomal translocation of CpG DNA via the natural endocytotic pathway is inefficient and highly saturable, whereas endosomal translocation of DNA complexed to the cationic lipid N-[1-(2,3-dioleoyloxy)propyl]-N,N,N-trimethylammonium methylsulfate (DOTAP) is not. Interestingly, DOTAP-mediated enhanced endosomal translocation of otherwise nonstimulatory vertebrate DNA or of certain noncanonical CpG motifs triggers robust dendritic cell activation in terms of both up-regulation of CD40/CD69 and cytokine production, such as type I IFN and IL-6. We report that the stimulatory activity of phosphorothioated noncanonical CpG oligodeoxynucleotides is TLR9 dependent, whereas phosphodiester DNA, such as vertebrate DNA, in addition trigger TLR9-independent pathways. We propose that the inefficiency of the natural route for DNA internalization hinders low affinity TLR9 ligands in endosomes to reach threshold concentrations required for TLR9 activation. Endosomal compartmentalization of TLR9 may thus reflect an evolutionary strategy to avoid TLR9 activation by self-DNA.     

Identification of immunostimulatory oligodeoxynucleotide from Escherichia coli genomic DNA

Bacterial DNA containing immunostimulatory CpG motifs can stimulate antigen-presenting cells to express costimulatory molecules and to produce various cytokines in vivo and in vitro. In this study, we fragmented macromolecular E.coli genomic DNA with DNase I, and analyzed the ability of the resulting DNA fragments to induce the NF-kappaB activation and humoral immune response. Furthermore, using computational analysis and luciferase assay for synthetic ODNs based on the sequence of the immunostimulatory DNA fragments (DF-ODNs), an active component of DF-ODNs sequences was investigated. Experimental results demonstrated that DF-ODN is optimal for the NF-kappaB-responsive promoter activation in the mouse macrophage cell line and the humoral immune response in vivo. In agreement with the activity of the DFODNs processed by DNase I, a synthetic ODN based on the DF-ODN sequences is potent at inducing IL-12 mRNA expression in primary dendritic cells. These results suggest that the discovery and characterization of a highly active natural CpG-ODN may be achieved by the analyses of bacterial DNA fragments generated by a nuclease activity.     

The acceleration of the inhibition of platelet prothrombinase complex by heparin.

The influence of heparin on the inhibition of factor Xa has been studied under conditions where factor Xa is bound to collagen-thrombin-stimulated platelets to form the prothrombinase complex. Unfractionated heparin was found to cause a concentration-dependent acceleration of the inhibition of the platelet prothrombinase complex up to a maximum rate constant of 4.1 X 10(7) M-1 X min-1 at heparin concentrations of 0.2 microM and above. This is equivalent to a 4800-fold acceleration over the rate constant for the inhibition in the absence of heparin, and is 6.8-fold lower than the rate constant for the inhibition of uncomplexed factor Xa in the presence of saturating concentrations of heparin which was determined as 2.8 X 10(8) M-1 X min-1. The effects of three Mr fractions of heparin were also studied. These were a gel-filtered heparin of Mr 15000, a gel-filtered heparin of Mr 6000 and a heparin oligosaccharide (primarily 8-10 monosaccharide units) prepared by nitrous acid depolymerization, each with high affinity for antithrombin III. These fractions all accelerated the rate of the antithrombin III inhibition of the platelet prothrombinase complex, with maximum rate constants of 6.8 X 10(7), 1.4 X 10(7) and 9.8 X 10(6) M-1 X min-1, respectively. On comparison with the effect of these heparin fractions on the rate of inhibition of uncomplexed factor Xa a progressively increasing disparity between the rate of inhibition of uncomplexed and complexed factor Xa was observed, rising from 1.7-fold with the oligosaccharide to 6.8-fold with the unfractionated heparin. A possible mechanism for this differential activity between uncomplexed and complexed factor Xa with the various heparin fractions is discussed in terms of an involvement of heparin binding to factor Xa.