Phosphodiester CpG oligonucleotides as adjuvants: polyguanosine runs enhance cellular uptake and improve immunostimulative activity of phosphodiester CpG oligonucleotides in vitro and in vivo

Abstract

Bacterial DNA and oligonucleotides (ODN) containing CpG-motifs strongly activate cells of the immune system. Accordingly CpG-DNA is a powerful adjuvant in vaccination protocols for B-cell as well as for cytotoxic T-cell responses. A decisive propensity of CpG-DNA is its capacity to induce preferentially T helper type 1 (Th1)-dominated immune responses. To exert its function CpG-DNA has to be taken up by responsive cells, e.g. antigen-presenting cells (APC). The rate of uptake is influenced by the DNA's backbone modification and critically determines activity of CpG-DNA. CpG ODN with a phosphothioate backbone (PTO) are currently used for most in vivo and in vitro studies, since PTO modification protects ODN from the attack of nucleases. However, after administration of PTO-modified CpG-ODN long-lasting effects including lymphadenopathy as well as sustained local interferon-γ (IFN-γ) and interleukin-12 (IL-12) production have been reported. To circumvent these restrictions we investigated the effects of DNA sequence as well as DNA backbone modification on cellular uptake and resulting immunostimulation. We show here that uptake of phosphodiester (PO)-CpG-ODN can be strongly enhanced by poly guanosine runs added at the 3′ end of the ODN. In addition these ODN showed an improved immunostimulatory activity in vivo and in vitro. This included protection of mice against lethal Th2-dependent leishmaniasis as well as priming of antigen specific Th1 responses. More importantly, guanosine-rich PO-CpG-ODN neither induced lymphadenopathy nor prolonged cytokine production after local administration. Since these improved PO ODN are efficient in vitro and in vivo and lack long lasting undesired effects they could be used preferably as adjuvants in vaccination protocols.

Introduction

Bacterial DNA and synthetic oligonucleotides (ODN) containing a CpG dinucleotide (CpG-ODN) motif stimulate cells of the innate and adaptive immune system. The reported effects include activation, polyclonal proliferation and immunglobulin secretion of B cells,¹ cytokine secretion and up-regulation of costimulatory molecules of macrophages and dendritic cells (DC),^2–4 direct and indirect costimulatory effects for T cells,⁵ as well as enhancing effects on haemopoiesis.⁶ These properties explain the powerful activity of CpG-DNA as adjuvant in adaptive immune responses.^7,8 CpG-ODN support strongly the induction of cytotoxic T-cell responses, which are crucial for defending intracellular pathogens.⁹ A further outstanding feature of CpG-DNA is its capacity to induce T helper type 1 (Th1)-dominated immune responses.^8,10 Moreover CpG-DNA is capable to redirect ongoing Th2 responses.¹⁰ Thus, CpG-ODN have been recognized as a new class of adjuvants supporting vaccination against allergy, infectious diseases and tumours.^7,11 In addition CpG-sequence motifs in plasmids used for DNA vaccination critically determine efficacy as well as the induced Th1/Th2 profiles^12,13 of the immune response.

Although CpG-DNA promises an impressive applicability in vaccines, information on the initial steps of its mode of action are still sparse. At least in murine antigen-presenting cells (APC) cellular uptake of CpG-DNA is obligatory to induce activation^1,14 yet specific receptors for uptake are not defined. Cellular responses induced by CpG-DNA are dependent on Toll-like receptor 9 (TLR9);¹⁵ however, the cellular compartment where CpG-DNA meets its putative receptor is not known. While TLR9 dependent activation critically depends on CpG-sequence motifs, cellular uptake appears to be independent of DNA sequence motifs. On the other side the rate of uptake critically determines the CpG-ODN's efficacy.^16,17 Reports analysing antisense DNA approaches revealed that DNA backbone modifications as well as supramolecular ODN structures influence cellular uptake of ODN.^18–23 Accordingly, phosphothioate (PTO)-modified ODN are taken up more efficiently compared to phosphodiester (PO) ODN or methylphosphonate-phosphodiester ODN. PTO-ODN show an increased affinity for cell membrane binding sites. Although DNA is able to bind to cell membranes, so far no PTO- or PO-specific uptake receptors have been identified. Moreover these studies indicated that cellular uptake seems to be independent of the ODN's sequence. Interestingly, ODN comprising runs of polyguanosines form quaternary structures that enhance uptake,^18,19 possibly by binding to scavenger receptors. These alterations influence the immunostimulatory properties of CpG-ODN. Dependent on the localization and the backbone modification of polyguanosine runs CpG-ODN showed enhanced or diminished immunostimulatory properties.^18,20,21

Most adjuvant formula make use of CpG-ODN with complete or partial PTO backbone modification. The rational is to increase resistance to nucleases and thus to prolong the action of the ODN. Use of PO ODN is limited because of their shorter half-life time and the resulting weaker immunostimulatory potency.¹ Furthermore, a recent report indicates that phosphodiester CpG-ODN might induce a different type of immune response with increased production of interferons.²⁰ In contrast, PTO-ODN induce long-lasting local immune-stimulating effects with sustained interferon-γ (IFN-γ) as well as interleukin-12 (IL-12) production.²⁴ In addition a massive lymphadenopathy has been observed after local administration of PTO-ODN.²⁴ Long-lasting IFN-γ and IL-12 production induces a state of sustained Th1 bias which denotes a potential risk to induce autoimmune immune responses.^25,26 Therefore, it would be of great value to define CpG-ODN that are immunostimulatory and yet avoid local long-lasting undesired effects.

We therefore examined whether sequence modifications of CpG-ODN, especially combinations of CpG-motifs and poly(dG) runs, would influence cellular uptake and immune stimulating activity. Cellular uptake of PTO-ODN was high and independent of the DNA sequence. In contrast, uptake of PO-ODN could be significantly enhanced by adding a poly(dG) run at the 3′ terminus. These sequence modification resulted in a dramatically enhanced biological activity in vitro. Moreover these PO-CpG-ODN were also effective in vivo as shown in an infection model of leishmaniasis. Most importantly, neither lymphadenopathy nor sustained cytokine production were observed. Thus, this class of PO-CpG-ODN represents a promising alternative as adjuvant in therapeutic approaches.

Materials and methods

Media and reagents

RAW 264.7 cells (a kind gift from Dr R. Schumann, Berlin, Germany) were cultured in Clicks/RPMI-1640 supplemented with 5% fetal calf serum (FCS), 50 µm β-mercaptoethanol and antibiotics (penicillin G (100 IU/ml of medium) and streptomycin sulphate (100 IU/ml of medium)). PO, PTO-modified and 5′ fluorescein isothiocyanate (FITC)-labelled ODN were custom synthesized by TIB Molbiol (Berlin, Germany) and MWG Biotech (Munich, Germany). ODN were controlled for similar amounts of FITC-labelling. Nomenclature and sequences of the used ODN are depicted in Table 1. ODN were negative for endotoxin contamination as measured by the Limulus assay (Sigma, Deisenhofen, Germany). Fucoidan and DNA from Escherichia coli were purchased from Sigma.

Table 1. Sequences, characteristics and immunostimulative capacities of studied ODN.

							Cytokine production*
Name	Sequence	Length (bp)	CpG-motif	G-motif†	PTO	PO	IL-12p40 (pg/ml)	TNF-α (ng/ml)
1668-PTO	TCC ATG ACG TTC CTG ATG CT	20	+	Ø	+		1620±24	24·1±1·3
1668-(G)5-PTO	TCC ATG ACG TTC CTG ATG CTG GGG G	25	+	+ 3′	+		402±45	15·8±0·9
1668-AGAGA-PTO	TCC ATG ACG TTC CTG ATG CTA GAG A	25	+	Ø	+		1378±94	26·7±1·1
1668GC-PTO	TCC ATG AGC TTC CTG ATG CT	20	Ø	Ø	+		0	1·5±0·2
(G)5central-PTO	CTC CTA TTG GGG GTT TCC TAT	21	Ø	+ central	+		17±6	0·6±0·1
12p40-PTO	AGC TAT GAC GTT CCA AGG	18	+	Ø	+		106±20	12·8±0·5
1668-PO	TCC ATG ACG TTC CTG ATG CT	20	+	Ø		+	86±14	3·4±0·5
1668-(G)5-PO	TCC ATG ACG TTC CTG ATG CTG GGG G	25	+	+ 3′		+	638±11	17·0±0·6
1668GC-PO	TCC ATG AGC TTC CTG ATG CT	20	Ø	Ø		+	13±0	0·9±0·6
(G)5central-PO	CTC CTA TTG GGG GTT TCC TAT	21	Ø	+ central		+	2±0	0·3±0·1
(G)5–1668-PO	GGG GGT CCA TGA CGT TCC TGA TGC T	25	+	+ 5′		+	372±64	5·7±0·5
1668-ATATA-PO	TCC ATG ACG TTC CTG ATG CTA TAT A	25	+	Ø		+	217±27	7·4±2·0
Medium							0	0·1±0

^*Cytokine production was determined in the culture supernatant by ELISA after stimulation of 1·5×10⁵ cells with 0·3 µm ODN for 24 hr. Values represent mean of quadruplicate±SEM.

^†G-motif: run of five guanosines.

Uptake assay with FITC-labelled ODN

To analyse the uptake of ODN, 3×10⁵ macrophages (RAW 264.7) were either preincubated at 4° or 37° for 10 min in Clicks/RPMI. Cells were then incubated with FITC-labelled ODN in the indicated concentrations for various intervals at either 4° or 37°. Then cells were washed once in phosphate-buffered saline (PBS)/2% FCS followed by an acid wash with 0·1 m glycin-HCl/0·1 m NaCl (pH 3·0) for 5 min to remove unbound and cell surface bound ODN.²⁷ Subsequently, cells were washed two times in PBS/2% FCS and then fixed in PBS/1% paraformaldehyde. Flow cytometric analyses were performed using a Partec PAS flow cytometer (Dako, Germany). Cells were gated on forward and side scatter and mean fluorescence activity (MFI) was measured. ΔMFI (MFI_37° − MFI_4°) was calculated as parameter of internalized FITC-ODN. In some experiments cells were put on glass slides and examined by microscopy.

In competition experiments cells were preequilibrated with the indicated concentrations of unlabelled ODN, fucoidan or DNA from E. coli for 15 min (again at either 4° or 37°), and then FITC-labelled ODN was added at a fixed concentration for further 45 min.

In pulse-chase experiments cells were first loaded with FITC-labelled ODN for 60 min followed by wash cycles with PBS/2% FCS and 0·1 m glycin-HCl/0·1 m NaCl (pH 3·0), as described above. Then cells were either incubated at 37° to determine the active exclusion from the cells or at 4° as control. After the indicated incubation period cells were treated as described and measured by flow cytometry. Dead cells were excluded from analyses by gating out according to propidium iodide staining.

Determination of cytokine secretion

To examine the immunostimulatory capacities of various ODN cytokine induction on RAW 264.7 cells was tested. In general, 1·5×10⁵ cells/well were stimulated with ODN in 96-well plates for 24 hr. Cytokine levels in culture supernatants were determined using commercially available enzyme-linked immunosorbent assay (ELISA) kits for tumour necrosis factor-α (TNF-α) and IL-12p40 according to the manufacturer's instructions (BD Biosciences, Heidelberg, Germany). Each value represents mean of duplicate values. IFN-γ production from lymph node preparations was determined by sandwich ELISA with matched-pair monoclonal antibodies (BD Biosciences) and recombinant IFN-γ as standard.

Long-term in vivo effects of ODN

To determine the long-lasting effects of different ODN on size and cytokine production of local draining lymph nodes ODN were injected at 10 nmol/footpad s.c. in 50 µl PBS in the right hind footpad of female BALB/c mice. After 6 and 11 days ipsi- and contralateral popliteal lymph nodes were aseptically removed into PBS/2% FCS. Lymph nodes were minced through a mesh, cells were counted and 3×10⁵ cells/well were put in culture in Clicks/RPMI-1640 supplemented with 10% FCS, 50 µm β-mercaptoethanol and antibiotics (penicillin G (100 IU/ml of medium) and streptomycin sulphate (100 IU/ml of medium)) without additional stimulus. After 24 h IL-12p40 and IFN-γ were determined as described above.

Infection

Eight- to 10-week-old female BALB/c mice were purchased from Harlan-Winkelmann (Borchen, Germany). Leishmania major promastigotes (strain MHOM/IL/81/FE/BNI; a kind gift from Dr W. Solbach, University of Lübeck, Germany) were grown in Clicks/RPMI supplemented with 10% FCS on Novy-Nicolle-MacNeal-agar and washed twice in PBS before infection. Stationary phase promastigotes (1×10⁶) were injected into the right hind footpad of BALB/c mice in PBS concomitant with 10 nmol ODN. The lesion size was measured weekly with a metric calliper, and the uninfected footpad served as an internal control. The percentage increase of footpad swelling was calculated from both values. Mice were bled 7 weeks after infection and the Leishmania-specific antibody titres in the sera were determined by ELISA, as previously described.¹⁰

Immunizations

Six-week-old female C57BL/6 mice were immunized with 5 nmol ODN and 300 µg ovalbumin in PBS s.c. in each hind footpad. Mice were boostered at day 14 and bled at day 24. Ovalbumin specific serum antibodies were detected by isotype-specific antibody ELISA.⁷

Results

Cellular uptake of ODN by murine macrophages is rapid, dose and temperature dependent

To study the mechanism of cellular uptake of ODN by murine macrophages we measured the increase of MFI in RAW 264.7 cells after incubation with FITC-labelled ODN. To exclude cell surface-bound but not internalized ODN from the analysis an acidic wash was performed which removed unbound ODN.²⁷ Cellular uptake was measured at 4° and 37°. The difference of MFI_{4° – 37°} was calculated as measure of internalized FITC-ODN. As shown in Fig. 1(a) cellular uptake was dose dependent and not saturable within the incubation time of 45 min. In addition uptake of the PTO ODN 1668-PTO was much higher in comparison to the PO counterpart 1668-PO. Uptake of 1668-PTO was measurable at concentrations of 10–30 nm while uptake of 1668-PO was observed at concentrations higher than 0·3 µm.

Figure 1.

Uptake of FITC-labelled ODN by RAW 264.7 cells. (a) RAW 264.7 cells were incubated with the indicated concentrations of FITC-labelled ODN either at 4° or at 37° for 45 min (compare Table 1 for sequences). Then, cells were washed and fixed as described in materials and methods and mean fluorescence intensity (MFI) was determined by flow cytometry. (b) RAW 264.7 cells were incubated with 0·5 µm FITC-labelled ODN 1668-PTO and 1 µm FITC-labelled ODN 1668-PO for various time periods at either 4° or 37°, MFI was measured and ΔMFI_{37° − 4°} was calculated. The results of one of three experiments are displayed. (c) RAW 264.7 cells were incubated with 1 µm FITC-labelled ODN 1668-PTO for 1 hr. Cells were treated as in (a), put on glass slides and photographed. Left panel: incubation at 4°, right panel: incubation at 37° and additional counter staining with propidium iodide.

Because initiation of cellular signals after stimulation with CpG-ODN occurs within the first minutes¹⁴ we examined the kinetics of cellular uptake of FITC-labelled ODN (Fig. 1b). Uptake could be measured as early as 1 min after addition of the ODN, thus being very rapid. There were no significant differences in the uptake kinetics of PTO and PO ODN, thus excluding that low uptake of PO ODN was a result of slower uptake rates. Cellular uptake of ODN increased steadily reaching plateau values between 6 and 12 hr after addition of the ODN (data not shown). Because previous studies suggested that ODN are endocytosed into acidic vesicles^14,28 we also examined the cellular localization of FITC-labelled ODN. Accumulation of fluorescence showed a speckled pattern within the cytoplasm (Fig. 1c), which only occurred after incubation at 37°. First occurrence of fluorescence was in compartments below the plasma membrane. Because of the acidic wash the plasma membrane showed very little staining. Exclusion of this wash step resulted in a strong binding of PTO ODN to the cell surface (data not shown). No significant staining of the nucleus was observed even after hours.

PTO-modified ODN are taken up equally and independent of CpG sequence motifs

When we tested various ODN (sequences and properties are shown in Table 1) for their cellular uptake relative to the reference ODN 1668-PTO, we found that PTO ODN were taken up equally yet independent of CpG-motifs (Fig. 2a, b) (compare CpG-ODN 1668-AGAGA-PTO or 12p40-PTO with the non-CpG-ODN 1668GC-PTO). It has been reported that poly(dG) motifs can enhance cellular uptake of PO ODN.¹⁸ To examine whether this holds also true for PTO ODN we tested poly(dG) motifs either at the 3′ end or in a central position of an ODN. However cellular uptake of phosphothioate ODN was not changed significantly (Fig. 2a, b; (G)5central-PTO, 1668-(G)5-PTO). Thus, uptake of PTO ODN is very efficient yet independent of the DNA sequence (CpG-motif).

Figure 2.

Differences in cellular uptake of modified ODN. (a) RAW 264.7 cells were incubated with various FITC-labelled ODN for 45 min. Cellular uptake as measured by ΔMFI_{37° – 4°} of PTO as well as PO ODN was determined relative to the reference ODN 1668-PTO. (b) Dose–response curves for some of the ODNs of (a) are given in detail.

Uptake of PO ODN is low in comparison to PTO counterparts but can be enhanced by poly(dG)-motifs

Next we examined the uptake of a panel of FITC-labelled PO ODN in comparison to reference ODN 1668-PTO. 1668-PO which is the PO counterpart of 1668-PTO was taken up very poorly (Fig. 2a, b). Uptake was only 6% (mean) in comparison to the PTO-modified ODN. Again uptake was independent of CpG-motifs as 1668GC-PO which lacks the central CpG-motif was taken up equally (4%). However, introducing a poly(dG) motif with a run of five guanosines (GGGGG) enhanced the uptake of phosphodiester ODN significantly (Fig. 2a, b; 1668-(G)5-PO, (G)5central-PO, (G)5–1668-PO). The position of the poly(dG) motif influenced the degree of augmentation of uptake slightly ((G)5–1668-PO [5′]: 20% (G)5central-PO [central]: 47%, 1668-(G)5-PO [3′]: 28%), yet differences were not significant. Improved uptake was not caused by the increased length of the ODN because a control ODN (1668-ATATA-PO) with the same length but no poly(dG) motif showed no enhancement in cellular uptake (data not shown).

Uptake of PTO ODN dominates that of PO ODN

To confirm differences in cellular uptake of PO and PTO ODN and to determine whether these ODN bind to the same receptor(s) we performed competition experiments. First we tested competition for uptake of labelled PTO ODN 1668-PTO (Fig. 3a). Unlabelled 1668-PTO and 1668GC-PTO decreased uptake in concentrations higher than 3 µm which was a 30-fold molar excess. In comparison 1668-(G)5-PTO and (G)5central-PTO which contained the poly(dG)-motif were able to compete at concentrations of 0·3 µm and 1 µm, respectively. Thus, poly(dG)-containing ODN have a higher affinity in competition studies although they did not show a marked enhancement in cellular uptake (Fig. 2).

Figure 3.

Competition of uptake of FITC-labelled PTO or PO ODN by different modified ODN, bacterial DNA and SR-A ligands. (a, b) RAW 264.7 cells were preincubated with the indicated, unlabelled competitors for 15 min. Then, FITC-labelled ODN 1668-PTO was added in a concentration of 100 nm for 45 min. Cellular uptake was determined by flow cytometry and is expressed in relation to uptake without competitor. (c) The same experiments as in (a) were performed with 3 µm FITC-labelled PO ODN 1668-PO as reporter.

In contrast, PO ODN were not effective as competitors in concentrations up to 10 µm. Surprisingly DNA from E. coli (Fig. 3b) was able to compete for uptake of 1668-PTO (maximal reduction to 52%) which could indicate a common receptor for PO and PTO ODN.

To further address this question we performed competition studies with PO ODN 1668-PO as reporter ODN. While PTO ODN and the poly(dG) containing PO ODN 1668-(G)5-PO inhibited uptake of 1668-PO even at low concentrations of 30 nm (Fig. 3c), unlabelled 1668-PO failed to compete in concentrations up to 30 µm (10-fold molar excess) corroborating the poor cellular uptake determined in Fig. 2(a). Higher ODN concentrations were not possible because of assay limitations. Because PTO ODN compete for uptake of PO ODN, the results argue for a common mechanism of uptake for ODN. Apparently the underlying structure binds different ODN with different affinities (poly(dG)-PTO>PTO>poly(dG)-PO>PO). Interestingly, fucoidan, a known ligand of the scavenger receptor A (SR-A) was able to inhibit the uptake of PTO and PO ODN (Fig. 3b, and data not shown). This could support previous reports suggesting that the SR-A on macrophages can bind ODN.²⁹

Differences in uptake of PTO and PO ODN are not due to differences in ODN stability or intracellular retention

Because complete PTO-modified ODN display enhanced nuclease resistance we next verified that decreased uptake of PO-ODN was not caused by lower stability of the ODN. Because nuclease activity could be derived from FCS included in the culture media we measured cellular uptake in serum-free culture medium. However, serum did not alter the uptake of either PTO- or PO-ODN (Fig. 4a).

Figure 4.

Influence of serum nucleases and exclusion mechanisms on ODN uptake. (a) RAW 264.7 cells were incubated with ODN 1668-PTO (boxes) and 1668-(G)5-PO (triangles) in the indicated concentrations for 45 min. Incubations were performed in medium containing 5% FCS (filled symbols) or in medium without FCS (open symbols). Cellular uptake was determined by flowcytometry as ΔMFI_{37° – 4°}. (b), RAW 264.7 cells were loaded with either 1 µm FITC-labelled 1668-(G)5-PTO (left panel) or 1 µm FITC-labelled 1668-(G)5-PO (right panel) for 60 min. Cells were washed and exclusion of ODN was monitored as decrease in MFI at 37° (triangles). As control cells were left at 4° after initial loading and MFI was measured at the end of the experiment (circles).

It has been reported that uptake of ODN is accompanied with active inside out transport of ODN in parallel.²³ Therefore we performed pulse-chase experiments to examine whether differences in ODN exclusion would underlie the different loading efficiencies of PTO- and PO-ODN. After initial loading of FITC-labelled ODN for 1 hr, reduction of MFI in the cells was measured. Exclusion of ODN was an active process as decrease in MFI was only marginal at 4°. Only slight differences in the exclusion from cells for 1668-(G)5-PTO in comparison to 1668-(G)5-PO were obtained (Fig. 4b). Reduction to 50% of the initial loading MFI was seen after approximately 13 min (PO-ODN) compared to 9 min (PTO-ODN), nevertheless differences were not significant. Therefore, differences in loading do not seem to be a result of ODN stability or different exclusion rates.

PO CpG-ODN containing a poly(dG) motif at the 3′ terminus display improved cellular uptake and enhanced immunostimulatory activity in vitro

To investigate whether enhanced uptake of PO-CpG-ODN containing an additional G-motif correlates with enhanced immunostimulation, we stimulated RAW 264.7 macrophage cells with a panel of different ODN and measured the resulting cytokine production. The results are shown in Table 1. As has been reported earlier ODN 1668-PTO showed marked induction of IL-12p40 as well as TNF-α while the PO counterpart, 1668-PO, was only marginally effective. In contrast, 1668-(G)5-PO which contained the central CpG-motif as well as a-3′ poly(dG) motif showed a significant enhanced cytokine induction in comparison to 1668-PO and was the most effective PO ODN. In contrast, (G)5central-PO which contains a central G-motif but lacks the CpG-motif failed to induce any cytokines. Surprisingly, (G)5-1668-PO which has a 5′ poly(dG) and a central CpG-motif induced cytokines only slightly more efficient than 1668-PO although cellular uptake was enhanced (Fig. 2). The properties of 1668-(G)5-PO were not caused by the increased ODN length, because the ODN 1668-ATATA-PO, which also is 25 bp long, was not effective.

Unexpectedly, introducing the G-motif at 3′ terminus in a PTO-CpG-ODN (1668-(G)5-PTO) decreased the capacity to induce cytokines markedly. Thus, a paradoxical pattern became apparent: 3′ poly(dG) extensions improve the immunostimulatory potential of PO CpG-ODN but not that of PTO CpG-ODN.

PO ODN lack local lymphadenopathy and prolonged local cytokine production after injection compared to PTO ODN

Long-lasting effects of PTO-CpG-ODN have been described such as local lymphadenopathy and prolonged spontaneous cytokine production (IL-12p40 and IFN-γ). We next tested whether long-lasting effects also would occur with immunostimulatory G-rich PO ODN (Fig. 5). All PTO modified ODN (1668-PTO, 1668-(G)5-PTO, 1668-GC-PTO) showed a marked local lymphadenopathy at day 6 after injection while none of the PO ODN (1668-PO, 1668-(G)5-PO) displayed an increase in lymph node cellularity. Interestingly even the non-stimulatory PTO-ODN 1668GC-PTO led to an increase in lymph node size thus proving that initial lymphadenopathy is a function of the PTO backbone rather than of the presence of a CpG-motif. Contralateral lymph nodes did not increase in size. At day 11 after injection lymphadenopathy reached its maximum (35×10⁶ cells/lymph node) for the stimulatory ODN 1668-PTO. Lymph nodes of the lower stimulatory (Table 1) PTO-ODN 1668-(G5)-PTO and of the non-stimulatory PTO-ODN 1668-GC-PTO decreased in size in comparison to day 6. Again, neither the PO-ODN 1668-PO nor the PO-ODN 1668-(G)5-PO displayed lymphadenopathy. In addition, the PTO-CpG-ODN 1668-(G)5-PTO and 1668-PTO showed elevated spontaneous IL-12p40 levels in the local lymph node up to 11 days post-injection, while PO-ODN were inactive in this respect. Similar results were obtained for IFN-γ (data not shown).

Figure 5.

Local lymphadenopathy and prolonged cytokine production by lymphnodes after injection of PTO ODNs. (a) Mice were injected with either PTO or PO ODN in the right hind footpad. After the indicated time popliteal lymphnodes were extracted and cells were counted (n=3, mean±SD). The experiment was repeated twice with similar results. (b) Lymph node cells of day 6 and 11 after local ODN application were harvested and cultured for 24 hr without additional stimuli. IL-12p40 was determined in the culture supernatant of ipsi- and contralateral lymphnode cells.

Poly(dG) containing PO ODN 1668-(G)5-PO is effective in vivo

PTO CpG-ODN have been shown to induce a strong Th1-mediated immunity in infectious diseases. We next tested whether poly(dG) containing PO-CpG-ODN were also effective in a model of murine leishmaniasis. Mice were injected with 1×10⁶ stationary phase L. major promastigotes subcutaneously concomitant with various ODN in the right hind footpad. The increase of the size of the developing lesion was recorded weekly. While PO-CpG-ODN (1668-PO) like PTO-GpC-ODN (1668GC-PTO) failed to protect mice from lethal infections (Fig. 6a), mice treated with the poly(dG)motif PO-CpG-ODN (1668-(G)5-PO) showed only a weak and transient footpad swelling. After 7 weeks the mice cured the infection, as did the PTO-CpG-ODN (1668-PTO) treated group.

Figure 6.

Poly(dG) modified PO CpG-ODN induce resistance to L. major infection in susceptible BALB/c mice. (a) Mice were injected with 1×10⁶ promastigotes into the right hind footpad concomitant with PO-ODN (open symbols) or PTO-ODN (closed symbols). The mean percent increase (plus SD) of footpad thickness is given (four mice per group). (b) After 7 week, mice were killed and sera were recovered and analysed for L. major-specific antibodies of the IgG1 (open bars) and IgG2a (closed bars) isotype. The OD (490 nm) is shown for a serum dilution of 1 : 200.

To confirm, that indeed a Th1 immune response was induced in these mice, sera from animals were collected (week 7) and analysed for L. major-specific immunoglobulin isotypes. The GpC-PTO-ODN (1668GC-PTO) as well as the CpG-PO-ODN (1668-PO) predominantly induced immunoglobulin G1 (IgG1) antibodies corroborating an induced Th2 immune response. In contrast, poly(dG) PO-CpG-ODN (1668-(G)5-PO) and PTO-CpG-ODN (1668-PTO) led to Leishmania-specific antibodies of the IgG2a isotype (Fig. 6b). Cytokine production profiles after specific restimulation in vitro further confirmed the Th1 phenotype of the immune response (data not shown).

Similar data were obtained when mice were immunized with ovalbumin together with PO-CpG-ODN containing a poly(dG) run as adjuvant (Fig. 7). Without adjuvant mice elicited an immune response which was almost exclusively composed of antigen specific antibodies of the IgG1 isotype. Application of ODN 1668-PO also produced only antibodies of the IgG1 isotype. After coadministration of the PTO-ODN 1668-PTO the levels of IgG2a increased dramatically while IgG1 was decreased. Addition of the G-motif at the 3′ terminus of a PO-CpG-ODN (1668-(G)5-PO) significantly increased IgG2a responses and decreased the IgG1 response. Nevertheless, immune repsonses induced by 1668-(G)5-PO were still lower as compared to the PTO ODN 1668-PTO in regard of antibody response.

Figure 7.

The poly(dG) motif at the 3′ terminus of PO CpG-ODN is necessary to induce a Th1 immune response in vivo. C57BL/6 mice were immunized with 300 µg ovalbumin together with or without 5 nmol of different ODNs in each hind footpad. Mice were boostered 2 weeks after the first immunization and blood was collected 10 days later. Ovalbumin-specific IgG responses were determined by isotype-specific ELISA. Bars represent group mean (n=3) of OD values with vertical lines indicating the standard deviation. Statistics were performed using Student's t-test.

Taken together, the results show that the poly(dG) motif of PO CpG-ODN is necessary and essential for immunostimulatory activity as well as for induction of Th1 immunity in vivo.

Discussion

DNA containing unmethylated CpG-motifs possesses marked immunostimulatory capacity.^1,2 Implications for vaccination, immunotherapy, gene and antisense therapy have been recognized and now are studied extensively.^11,30–32 Although in DNA vaccination CpG-DNA effects are exerted by native double-stranded DNA, most other applications use single stranded short (∼20 bp) oligonucleotides. In order to preserve ODN from the attack of nucleases, the backbone of ODN is usually protected by introduction of a PTO modification. Nuclease-sensitive PO ODN are only weak in inducing immunostimulation, especially when tested in vivo. However, PTO modification of ODN might induce undesired side effects such as long-lasting activation and toxicity.²⁴ Therefore, it is intriguing to improve the efficacy of PO ODN.

Cellular uptake of CpG-DNA is considered a prerequisite for their immune function. Although this holds true for their activity on macrophages, DCs and murine B cells, at least for induction of some functions of human B-cell uptake of ODN is dispensable,³³ as sepharose-bound ODN still induce B-cell proliferation. Furthermore, it is known that the PTO backbone itself can exert biological functions on B cells independently of CpG-motifs¹⁷ stressing the importance of sorting out CpG-sequence-dependent from backbone-mediated effects. Sester et al.¹⁶ have reported that in contrast to PO-ODN PTO-ODN failed to down-regulate colony stimulating factor-1 receptor (CSF-1R) from primary macrophages, indicating that differences in backbone modifications not only influence stability but also signal qualities.

Cellular uptake of ODN in RAW 264.7 macrophages was rapid, dose and temperature dependent (Fig. 1), thereby confirming earlier reports.^16,22,23 However, in contrast to uptake of ODN in human B cells,¹⁷ we did not observe a saturation in uptake for the tested concentration range on macrophages. Furthermore, PTO-ODN were taken up with much greater efficacy than PO-ODN, yet uptake was independent of CpG-motifs. Addition of a poly(dG) motif which is comprised of five guanosines in a run to ODNs significantly enhanced cellular uptake of PO-ODN but not of PTO-ODN. These results corroborate previous reports showing that PTO backbone modification drastically influences the affinity for cell membrane binding and uptake in mouse spleen cells²² and that poly(dG) runs on PO-ODN enhance uptake on dendritic cells.¹⁸ The optimal length of the G-runs were found to be six guanosines;¹⁸ however, in our experiments runs of five guanosines were sufficient. The effects were specific to guanosines as other extensions did not show similar effects. The position of the poly(dG) motif within the ODN's sequence influenced uptake enhancement slightly. Central poly(dG) runs were most effective to increase uptake of PO-ODNs, while additon at the 5′ or 3′ terminus showed less enhancement of uptake. The higher amounts of exonucleases that degrade from the ends of DNA strains to endonucleases might account for that observation. Furthermore, poly(dG) runs are known to form base-quartet-stabilized four-stranded helices called tetraplex,^19,34 which can bind to type I scavenger receptors and thus improve cellular uptake. Lee et al.¹⁸ reported a correlation between tetraplex formation of poly(dG) containing CpG-ODN and IL-12 induction. Tetraplex formation was dependent on poly(dG) position, which could explain the slight differences in uptake of either 5′, 3′ or central poly(dG) containing PO-ODN which we have observed in our experiments.

Although fluid-phase mediated endocytosis has been suggested to play a role in cellular uptake of ODNs³⁵ competition studies indicate that receptors might exist. Indeed nucleic-acid binding proteins on the surface of lymphocytes have been described.³⁶ Scavenger type A receptor (SR-A) was implicated in cellular uptake of PTO-ODN because known ligands of SR-A competed for ODN uptake.^18,37 Underlining these results we now show here that uptake of PTO as well as PO-ODN can be competed by fucoidan (Fig. 3). Furthermore we show that PTO-ODN compete for uptake of PTO as well as PO-ODN, which argues for one shared receptor system. This is further strengthened by our observation that bacterial DNA competes for uptake of PTO-ODN (Fig. 3). However, because PTO-modification conveys binding affinity to many proteins, it is unclear whether binding of PTO-ODN to certain proteins is really linked to structures involved in uptake. In fact, an uptake function for ODN has been attributed to many distinct cell surface structures: surface immunoglobulins,³⁸ human leucocyte antigen (HLA) class II molecules³⁹ and Mac-1 (CD11b/CD18).³⁷ However, Mac-1^−/− knockout mice showed no alterations in responses to CpG-ODN.⁴⁰ Hence, the biological importance of the various proposed receptors still remain elusive.

Improving uptake of PO-ODN by a poly(dG) motif at the 3′ terminus also enhanced the immunological function to induce TNF-α as well as IL-12p40 on macrophages. Poly(dG)-containing PO-ODN were only effective with the G-motif at the 3′ terminus not at the 5′ position. Surprisingly, addition of the G-motif at the 3′ terminus of a PTO-ODN abrogated the immunostimulatory capacity although this PTO-ODN was still taken up effectively. This superficial paradox could be resolved by a concept of a two-step mechanism of signal induction by CpG-ODN. In a first step ODN have to be taken up in endosomal compartments. This step is independent of the presence of CpG-motifs yet is influenced by backbone modifications and poly(dG) motifs which both enhance uptake. In a second step a TLR9-dependent recognition of CpG-motifs takes place resulting in triggering of signal cascades such as nuclear factor κB and mitogen-activated protein kinase activation.^14,41 For this second step it has to be assumed that poly(dG) motifs disturb or even inhibit signal transduction. Indeed poly(dG) containing PTO-non-CpG-ODN inhibit signalling of CpG-ODN independent of competition of cellular uptake (unpublished results). However, PO poly(dG) motifs at the 3′ terminus of a PO-ODN can be degraded by nucleases, thus allowing signal transduction. In contrast, G-motifs of PTO-ODN are stable and thus signal initiation does not take place. However, it should be stressed that poly(dG) motif containing ODN might induce immune activity independent of CpG-motifs and TLR9 triggering. ODN containing poly(dG) motifs have been reported to costimulate T cells⁴² and activate lytic activity as well as IFN-γ production by natural killer (NK) cells.⁴³ In a combinatory approach Verthelyi et al.²⁰ divide ODN into two classes. ‘D’-ODN, which work with poly(dG) tails, especially induce IFN-γ and activate NK cells, while ‘K’-ODN, which are PTO-modified induce B-cell proliferation and IL-6 on human monocytes and B cells. Thus, the B-cell immune response is improved by PTO-ODN.

Multiple studies in vaccination^10,44–46 as well as in therapeutic approaches in Th2-associated disorders^10,47 using PTO-CpG-ODN proved their effectiveness because of their strong Th1 biasing properties. However, concerns due to undesired side effects were also raised. PTO-ODN might be toxic as shown in antisense approaches⁴⁸ and PTO-CpG-ODN might cause a lymphadenopathy after local immunization,⁷ which is associated with a sustained long lasting hyperreactivity and Th1 polarization. Although long-lasting effects could be beneficial under certain conditions (e.g. cytotoxic T lymphocyte induction to antigen or protection of mice from lethal challenge with Leishmania major, Francisella tularensis or Listeria monocytogenes^10,49,50 up to 35 days after injection of PTO-CpG-ODN) sustained Th1 hyperreactivity also might induce undesired autoimmune reactions. Segal et al. reported that PTO-CpG-ODN were able to aid activation of autoreactive encephalitogenic T cells in vivo.²⁵

We here show that in contrast to PTO-CpG-ODN, poly(dG)-PO-ODN were effective in vivo yet showed no long-lasting in vivo effects. In leishmaniasis the protective effect of poly(dG)-PO-CpG-ODN was comparable to that of PTO-CpG-ODN.

In summary, PO-ODN containing a central CpG-motif as well as a poly(dG) motif at the 3′ terminus are immunostimulative and bias Th1 immune responses in vivo. Probably, enhanced immunoreactivity is caused by an increased uptake via the guanosine-rich runs. However, in contrast to PTO-CpG-ODN undesired side effects such as long-lasting hyperreactivity, hypercellularity and sustained cytokine production are not observed. Thus, this class of ODN is a promising option in vaccination procedures as well as other therapeutic approaches using CpG-ODN and provides new insights in the initial steps of CpG-ODN induced signalling.

Abbreviations

APC

antigen-presenting cell

G

guanosine

ODN

oligodeoxynucleotide

PO

phosphodiester

PTO

phosphothioate