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Department of Internal Medicine, University of Iowa, Iowa City, IA 52242;
CpG ImmunoPharmaceuticals GmbH, Hilden, Germany;
Loeb Health Research Institute at the Ottawa Hospital, and Faculties of Health Sciences and Medicine, University of Ottawa, Ottawa, Canada;
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Veterans Affairs Medical Center, Iowa City, IA 52246;
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Primate Research Center, Bogor Agricultural University, Bogor, Indonesia;
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Hepatitis Viruses Section, Laboratory of Infectious Diseases, National Institute of Arthritis and Infectious Diseases, National Institutes of Health, Bethesda, MD 20892; and
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CpG ImmunoPharmaceuticals, Inc., Wellesley, MA 02481
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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, and
IFN-ß (8, 9, 10, 11, 12, 13), and stimulates murine dendritic cells
(14, 15) and murine B cells (16, 17, 18). Acting
in synergy with the CpG DNA (which does not directly stimulate highly
purified NK cells), the IL-12 secondarily activates murine NK cells to
secrete IFN-
(10, 13) and to have increased lytic
activity (9). Overall, CpG DNA induces a predominantly Th1
pattern of immune activation. Synthetic oligodeoxynucleotides (ODN)3 containing the optimal murine CpG motif (5'-GACGTT-3') are known to be excellent immune adjuvants in various murine disease models and to drive Th1 immune responses (19, 20, 21, 22, 23, 24, 25). They are comparable or superior to CFA (but without apparent toxicity) and are superior to the standard human adjuvant alum with respect to the induction of Ag-specific humoral and cell-mediated immune responses (19, 20, 21, 22, 25). Murine CpG ODN induce potent anti-tumor immune activity (26) and induce resistance to lethal challenge with L. monocytogenes (27). Our studies also support the use of CpG DNA for the conversion of allergic Th2-type immune responses into nonallergic Th1 responses (28).
Recently, we found that phosphorothioate ODN with the
purine-purine-CG-pyrimidine-pyrimidine formula that had been identified
as the most stimulatory motif in mice show no or only weak activity in
human immune cells (29). We identified a potent human CpG
motif, 5'-GTCGTT-3', by testing phosphodiester ODN for the ability to
stimulate human primary B cells (29) and found that a
phosphodiester ODN with a single copy of the optimal human CpG motif
triggers 
60% of human peripheral blood B cells to proliferate and
express high levels of CD86. We also demonstrated that this ODN, of
sequence 2080, promotes growth, activation, and maturation of human
peripheral blood dendritic cells (30).
To have in vivo clinical utility, ODN must be administered in a form that protects them against nuclease degradation. The native phosphodiester internucleotide linkage can be modified to become highly nuclease resistant via replacement of one of the nonbridging oxygen atoms with a sulfur, which constitutes phosphorothioate ODN. However, a phosphorothioate backbone reduces the affinity of the CpG ODN to a putative CpG binding protein(s) (B. Noll, W. Shen, C. Schetter, M. Wold, and A. M. Krieg, manuscript in preparation). Of note, an ODN containing a single optimal human CpG motif followed by a poly C tail, which is highly active with a phosphodiester backbone, is essentially inactive with a phosphorothioate backbone. In contrast, murine leukocytes are strongly activated by phosphorothioate ODN containing just one optimal murine motif. This argues for differences in the precise mechanism of CpG recognition between human and murine immune cells.
The goal of the present study was to identify the sequence of a human CpG phosphorothioate ODN that would have optimal adjuvant activity in vivo. Because we saw not only quantitative but also qualitative differences in the activities of different CpG ODN in mice, we first screened a panel of CpG and non-CpG control ODN on mouse cells to find in vitro assays with reliable and strong correlation to in vivo adjuvant activity with hepatitis B surface Ag (HBsAg). We then systematically tested a panel of >250 phosphorothioate ODN in corresponding human assays to identify sequences with in vitro immunostimulatory activity. We next examined whether the ODN with the highest activity in these human assays also activate B cell proliferation in chimpanzees and monkeys, and finally, whether they are active as adjuvants with HBsAg in chimpanzees and cynomolgus monkeys in vivo.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Phosphorothioate-modified ODN were purchased from Operon
Technologies (Alameda, CA) and Hybridon Specialty Products (Milford,
MA). The sequences used are provided in Table I
and Fig. 1. ODN were tested for endotoxin using
the LAL-assay (BioWhittaker, Walkersville, MD; lower detection limit,
0.1 endotoxin units /ml). For in vitro assays, ODN were diluted in TE
buffer (10 mM Tris, pH 7.0, and 1 mM EDTA) and stored at -20°C. For
in vivo use, ODN were diluted in PBS (0.1 M PBS, pH 7.3) and stored at
4°C. All dilutions were conducted using pyrogen-free reagents.
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Spleens were removed from 6- to 12-wk-old female BALB/c mice
(The Jackson Laboratory, Bar Harbor, ME), 2 x
106 splenocytes were cultured with 0.2 µM ODN
for 4 h (TNF-) or 24 h (IL-6, IFN-, IL-12), and
cytokines were detected by ELISA as previously described
(16). To evaluate CpG-induced B cell proliferation, spleen
cells were depleted of T cells with anti-Thy-1.2 and complement and
centrifugation over Lympholyte M (Cedarlane Laboratories, Hornby,
Canada), cultured for 44 h with the indicated ODN, and then pulsed
for 4 h with 1 µCi of [3H]thymidine as
described previously (5). To examine NK cell lytic
activity, murine spleen cells were depleted of B cells using magnetic
beads coated with goat anti-mouse Ig as previously detailed
(31). Cells were cultured at 5 x
106/well in 24-well plates and harvested at
18 h for use as effector cells in a standard 4-h
51Cr release assay against YAC-1 target cells.
One unit was defined as the number of cells needed to effect 30%
specific lysis. ODN 1758 was also induced NK activity when tested at a
higher concentration.
Immunization of mice against HBsAg and evaluation of the humoral response
Groups of 6- to 8-wk-old female BALB/c mice (n =
5 or 10; Charles River, Montreal, Canada) were immunized against HBsAg
as previously described (21). In brief, each mouse
received a single i.m. injection of 50 µl of PBS containing 1 µg of
recombinant HBsAg (Medix Biotech, Foster City, CA) and 10 µg of CpG
ODN or non-CpG control ODN (see Table I
for sequences) as a sole
adjuvant or combined with alum (Alhydrogel "85," Superfos
Biosector, Vedbaek, Denmark; 25 mg of Al3+/mg of
HBsAg). Control mice were immunized with HBsAg without adjuvant or with
alum. Plasma was recovered from mice at various times after
immunization, and Abs specific to HBsAg (anti-HBs) were quantified
by end-point dilution ELISA (in triplicate) as described previously
(21). End-point titers were defined as the highest plasma
dilution that resulted in an absorbance value
(OD450) 2 times higher than that of nonimmune
plasma with a cut-off value of 0.05.
Isolation of primate PBMC and cell culture
PBMC were isolated from peripheral blood of healthy volunteers, chimpanzees, or rhesus or cynomolgus monkeys by Ficoll-Hypaque density gradient centrifugation (Histopaque-1077, Sigma, St. Louis, MO) as previously described (32). Cells were suspended in RPMI 1640 culture medium supplemented with 10% (v/v) heat-inactivated (56°C, 1 h) FCS (HyClone, Logan, UT), 1.5 mM L-glutamine, 100 U/ml penicillin, and 100 µg/ml streptomycin (all from Life Technologies, Grand Island, NY; complete medium). Cells (final concentration, 1 x 106 cells/ml) were cultured in complete medium in a 5% CO2 humidified incubator at 37°C. ODN and LPS (from Salmonella typhimurium; Sigma) or anti-IgM were used as stimuli. For measurement of human NK lytic activity, PBMC were incubated at 5 x 106/well in 24-well plates. Cultures were harvested after 24 h, and cells were used as effectors in a standard 4-h 51Cr release assay against K562 target cells as previously described (9, 31). For B cell proliferation, 1 µCi of [3H]thymidine was added 18 h before harvest, and the amount of [3H]thymidine incorporation was determined by scintillation counting on day 5. The SDs of the triplicate wells were <5%.
Flow cytometry on primate PBMC
Surface Ags on primate PBMC were stained as previously described
(33). Monoclonal Abs to CD3 (UCHT1), CD14 (M5E2), CD19
(B43), CD56 (B159), CD69 (FN50), and CD86 (2331 (FUN-1)) were purchased
from PharMingen (San Diego, CA). IgG1,
(MOPC-21) and IgG2b,
(30) were used to control for nonspecific staining. NK
cells were identified by CD56 expression on CD3-, CD14-, and
CD19-negative cells, whereas B cells were identified by expression of
CD19. Flow cytometric data from 10,000 cells/sample were acquired on a
FACScan (Becton Dickinson Immunocytometry Systems, San Jose, CA). The
viability of cells within the forward/side scatter gate used for
analysis was examined by propidium iodide staining (2 µg/ml) and
was > 98%. Data were analyzed using the computer program FlowJo
(version 2.5.1, Tree Star, Stanford, CA).
Immunization of chimpanzees and cynomolgus monkeys against HBsAg and evaluation of the humoral response
Fourteen cynomolgus monkeys (2.0–3.5 kg) were immunized with a
pediatric dose of Engerix-B (SmithKline Beecham Biologicals, Rixensart,
Belgium) containing 10 µg of HBsAg adsorbed to alum (25 mg of
Al3+/mg of HBsAg). This was administered alone
(n = 5) or combined with CpG ODN 1968
(n = 5; 500 µg) or CpG ODN 2006 (n =
4; 150 µg; see Fig. 2 for sequences).
Four chimpanzees (10–20 kg) were immunized in the same fashion, with
two receiving control vaccine (Engerix-B only) and two receiving
experimental vaccine (Engerix-B plus 1 mg of CpG ODN 2006). All
vaccines were administered i.m. in the right anterior thigh in a total
volume of 1 ml. Monkeys were maintained in the animal facility of the
Primate Research Center (Bogor, Indonesia), and chimpanzees were housed
at Bioqual (Rockville, MD). Animals were monitored daily by animal care
specialists. No symptoms of general ill health or local adverse
reactions at the injection site were noted. Plasma was recovered by
i.v. puncture before and at various times after immunization and was
stored frozen (-20°C) until assayed for Abs. Anti-HBs Abs were
detected using a commercial ELISA kit (Monolisa Anti-HBs,
Sanofi-Pasteur, Montreal, Canada), and titers were expressed in
milliinternational units per milliliter based on comparison with World
Health Organization-defined standards (Monolisa Anti-HBs Standards,
Sanofi-Pasteur).
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Our previous studies showed that the precise bases on the 5' and
3' sides of a CpG dinucleotide within a CpG motif have a major impact
on the level of immune activation of a synthetic ODN, but it has been
unclear whether different CpG motifs might display different immune
effects. To evaluate this possibility, we tested a panel of CpG ODN for
the ability to induce NK lytic activity and B cell proliferation and to
stimulate synthesis of TNF-, IL-6, IFN-, and IL-12 in murine
spleen cells (Table I). Immunostimulatory activity of ODN without CpG
motifs (ODN 1982 and ODN 1983; Table I) was negative or weak compared
with that of CpG ODN. Consistent with our earlier findings
(5) ODN with nonoptimal CpG motifs (ODN 1628 and ODN 1758)
were less active than ODN containing CpG motifs flanked by two 5'
purines and two 3' pyrimidines (ODN 1760, ODN 1826, and ODN 1841).
Within these ODN, ODN 1826 containing two optimal murine CpG motifs
(5'-GACGTT-3') had the highest activity for five of six measured end
points. Except for ODN 1628, all ODN showed a generally similar pattern
of activity (NK cell-mediated lysis, B cell proliferation, IL-12, IL-6,
TNF-, and IFN-). Of note, ODN 1628, which was unique in this
panel for containing two G-rich regions, showed preferential induction
of IFN- synthesis but relatively low stimulation of the other
activities.
Identification of in vitro assays that correlate with in vivo adjuvant activity
Because adjuvant activity is an in vivo end point, we were interested in identifying in vitro assays that would predict the adjuvant activity of a CpG ODN in vivo. The same ODN used for in vitro end points therefore were tested for their adjuvant activity to immunize mice against HBsAg. This was conducted both with ODN alone and with ODN combined with alum, since earlier studies had shown strong synergy for CpG ODN and alum adjuvants (21).
BALB/c mice immunized with HBsAg without adjuvant attained only low
titers of anti-HBs by 4 wk, and this was not affected by addition
of non-CpG control ODN. In contrast, addition of CpG ODN raised
anti-HBs titers 5- to 40-fold depending on the sequence used (Table I). When alum was added, titers of anti-HBs were 6 times higher
than those with HBsAg alone. Nevertheless, the various ODN combined
with alum gave similar levels of augmentation relative to alum alone,
as was found with the nonalum formulations relative to no adjuvant.
Specifically, non-CpG ODN had no effect, and the various CpG ODN
augmented these titers 2- to 36-fold (Table I). Results obtained with
the different ODN alone correlated very strongly (r =
0.96) with those obtained using the same ODN plus alum. When linear
regression was performed, a very high degree of correlation was found
between certain in vitro assays and in vivo augmentation of
anti-HBs titers. Of all the in vitro end points examined, the
induction of NK lytic activity showed the best correlation to in vivo
adjuvant activity (without alum, r = 0.98; with alum,
r = 0.95; p < 0.0001). A good
correlation regarding adjuvant activity was also obtained for B cell
stimulation (r = 0.84 and 0.7) as well as secretion of
TNF- (r = 0.9 and 0.88), IL-12 (r =
0.88 and 0.86), and IL-6 (r = 0.85 and 0.91; Table I).
The one in vitro assay that did not correlate well with the in vivo
results was IFN- secretion (r = 0.57 and 0.68; Table I). This was due to the preferential IFN--inducing activity of ODN
1628, which alone among the ODN in this panel contained G-rich regions.
These data demonstrate that in vitro assays for NKlytic activity, B
cell activation, and production of TNF-, IL-6, and IL-12 provide
valuable information in vitro to predict the adjuvant activity of a
given ODN in vivo.
Screening of a phosphorothioate ODN panel to activate human NK cells
In previous studies we found that synthesis of inflammatory
cytokines by human PBMC is induced by extremely low amounts of
endotoxin (induced TNF- secretion is detectable with just 6 pg/ml
endotoxin, 2 logs more sensitive than murine immune cells)
(34). In contrast, activation of human B cells and
induction of human NK cell lytic activity with endotoxin are low even
at high endotoxin concentrations. Based on these results we selected
activation of NK cells (lytic activity and CD69 expression) and B cells
(proliferation and CD86 expression) as the most highly specific and
reproducible assays with low intersubject variability and used these
assays for in vitro screening of a pool of ODN.
First we studied the effect of phosphorothioate ODN containing various
combinations and permutations of CpG motifs on NK cell-mediated lysis
of target cells. For clarity and ease of presentation, only data with
selected representative CpG and control ODN are shown. Human PBMC were
incubated with different phosphorothioate ODN (6 µg/ml) for 24 h
and tested for their ability to lyse 51Cr-labeled
K562 cells. ODN without CpG motifs (ODN 1982 and ODN 2010; Fig. 1),
runs of CpGs, ODN with nonoptimal CpG motifs, ODN containing only one
6-mer CpG motif (either 5'-GACGTT-3' or 5'-GTCGTT-3' underlined for
clarity), and ODN containing two of these motifs without a TpC on the
5' end of the ODN failed to induce NK lytic activity substantially
above background. Examples of such nonactive ODN with CpG motifs are
ODN 1781 (5'-ACCATGGACGTTCTGTTTCCCCTC-3'), ODN 1823
(5'-GCATGACGTTGAGCT-3'), and ODN 1829
(5'-ATGACGTTCCTGACGTT-3'; not
shown in figure). ODN with two 6-mer CpG motifs (either 5'-GACGTT-3' or
5'-GTCGTT-3') in combination with a TpC at the 5' end of the ODN (ODN
1840,
5'-TCCATGTCGTTCCTGTCGTT-3';
ODN 1851,
5'-TCCTGACGTTCCTGACGTT-3'; not
shown in figure) or with at least three 6-mer motifs without a TpC at
the 5' end (ODN 2013; Fig. 2) show intermediate activity. High activity
was found when the 5' TpC directly preceded a 6-mer human CpG motif
(5'-TCGTCGTT-3') and was followed by two 6-mer motifs (ODN
2005, ODN 2006, and ODN 2007). The best results were obtained when the
6-mer CpG motifs were separated from each other and from the 5' 8-mer
CpG motif by TpT (ODN 2006).
Expression of the activation marker CD69 is rapidly up-regulated on the
surface of NK cells subsequent to stimulation. To confirm the
results from the NK cell lysis assay, PBMC were incubated for 18 h
with ODN (2 µg/ml). CD69 expression was examined on CD56-positive NK
cells (CD3, CD14, and CD19 negative). Although induction of CD69
expression was less sequence restricted than stimulation of NK cell
functional activity, control ODN (ODN 1982, ODN 2116, ODN 2117, and ODN
2010) showed only low activity similar to background levels (Fig. 2).
ODN with two human CpG motifs separated by 5'-TTTT-3' (ODN 1965) or
four human CpG motifs without spacing (ODN 2013) were relatively more
active at inducing CD69 expression (Fig. 2, left panel) than
at stimulating NK cell lytic activity (Fig. 1). Optimal NK cell
functional activity as well as CD69 expression were obtained with ODNs
containing a TpC dinucleotide preceding the human CpG motif and
additional human motifs within the sequence (ODN 2006 and ODN 2007;
Fig. 2, left panel).
Activity of phosphorothioate ODN for stimulating human B cells
In preliminary experiments we found that the percentage of
proliferating B cells (5- (and 6-)carboxyfluorescein diacetate
succinimidyl ester (CFSE) assay, see Materials and Methods)
correlated with the surface expression of the costimulatory CD86 on B
cells, as measured by flow cytometry. Thus, we used CD86 expression on
B cells to screen a panel of ODN for immunostimulatory activity. PBMC
were incubated with 0.6 µg/ml ODN. Expression of CD86 (mean
fluorescence intensity) was examined on CD19-positive B cells (Fig. 2, right panel). A poly C ODN (ODN 2017) or ODN without CpG
dinucleotides (ODN 1982) failed to stimulate human B cells under these
experimental conditions. A phosphorothioate ODN (ODN 2116) with one
optimal human CpG motif preceded by a TpC (5'-TCGTCGTT-3') was inactive
(Fig. 2, right panel). The presence of one human 6-mer CpG
motif (5'-GTCGTT-3') had no activating effect (not shown). Two of these
CpG motifs within the sequence showed no (ODN 1960 and ODN 2016) or
intermediate (ODN 1965) activity dependent on the sequence context. If
the ODN was composed of three or four copies of this motif (ODN 2012,
ODN 2013, and ODN 2014), intermediate activity on B cells could be
detected. The combination of the human 8-mer CpG motif on the 5' end of
the ODN with two 6-mer CpG motifs (ODN 2005, ODN 2006, ODN 2007, ODN
2102, and ODN 2103) led to a considerable increase in the ability of
the ODN to stimulate B cells. The spacing between the single motifs was
critical. The separation of CpG motifs by TpT was preferable (ODN 2006)
compared with that of unseparated CpG motifs (ODN 2005; also compare
ODN 1965 to ODN 1960). The human 6-mer CpG motif (5'-GTCGTT-3') was
better than the optimal mouse 6-mer CpG motif (5'-GACGTT-3') when
combined with the human 8-mer CpG motif on the 5' end (ODN 2006 vs. ODN
2102 and ODN 2103). A (TCG)poly ODN was inactive
or only weakly active, as were ODN containing CpG dinucleotides flanked
by guanines or other CpG dinucleotides (ODN 2010; Fig. 2). Taken
together, the findings for NK cells and B cells showed consistently
that of the ODN tested, ODN 2006 has the highest immunostimulatory
activity on human immune cells.
Comparative analysis of potency of CpG phosphorothioate ODNs in different primates
Different CpG motifs are optimal to activate murine and human
immune cells. Furthermore, the number and location of CpG motifs within
an active phosphorothioate ODN are different in mice and humans. We
were interested to know whether CpG phosphorothioate ODN show similar
activity among different species of primates. We compared a panel of
CpG ODN for their ability to induce B cell proliferation in humans,
chimpanzees, and rhesus or cynomolgus monkeys. The capability of ODN to
stimulate human B cell proliferation (Table II) correlated well with their ability to
induce CD86 expression on B cells (Fig. 2, right panel). ODN
2006, which showed the highest activity in human B cells and NK cells,
was also the most active in stimulating chimpanzee and rhesus monkey B
cell proliferation (Table II). ODN 1968 and ODN 2006 gave the highest
activation of cynomolgus monkey B cells in vitro (stimulation index, 25
and 29, respectively, at 6 µg ODN/ml). Surprisingly, CpG ODN 2007,
which displayed similarly high activity as the optimal ODN 2006 in
human cells, did not stimulate rhesus monkey or chimpanzee B cell
proliferation, and ODN 1968 showed low activity. CpG ODN originally
identified with high activity in mice (ODN 1760 and ODN 1826) showed
little activity in monkeys (Table II).
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To evaluate whether CpG ODN with strong in vitro stimulatory
effects on primate cells had detectable adjuvant activity in vivo,
cynomolgus monkeys and chimpanzees were immunized with Engerix B, which
comprises HBsAg adsorbed to alum, alone or with added ODN 1968 (500
µg) or ODN 2006 (1 mg), respectively. The results in the cynomolgus
monkeys and chimpanzees cannot be directly compared because different
CpG ODN were used. Nevertheless, compared with controls not receiving
CpG ODN, anti-HBs titers at 4 wk postprime and 2 wk postboost were
66- and 16-fold higher, respectively, in the monkeys, and 15- and
3-fold higher in the chimpanzees (Table III). Thus, a clear adjuvant effect of
CpG ODN was seen, and this was particularly striking after a single
immunization. Because the number of animals studied is small, the
differences seen are qualitative rather than quantitative.
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Discussion |
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10 mIU/ml). In contrast, control chimpanzees receiving
vaccine without ODN 2006 had anti-HBs near or below protective
levels. In humans, the commercial vaccine, which contains alum as an
adjuvant, is typically given in three doses. Although the majority of
people respond well to two doses, the third dose ensures a very high
(>90%) seroprotection rate. The human stimulatory ODN 2006 shows weaker activity in mice compared with the highly active murine CpG ODN 1826 (our unpublished observations), supporting the concept of species specificity of CpG DNA recognition by immune cells. Although ODN 2006 was active in vitro in all primates tested, other CpG ODN, such as ODN 2007, had relatively high activity in human immune cells but no or a weaker effect in chimpanzees and rhesus monkeys. This argues for differences in the CpG recognition mechanism even within primates. Because bacterial DNA provides a large pool of CpG motifs, the evolutionary pressure to conserve the recognition mechanism for one single specific CpG motif is low. In contrast, the loss of binding of a transcription factor to its specific DNA motif would abolish its function. For the recognition of the presence of bacterial DNA, it, rather, makes evolutionary sense that different species develop their own optimized bacterial DNA recognition mechanism based on the bacterial environment to which they are exposed.
The good predictive value of B cell activation for in vivo vaccine
adjuvant activity is most likely linked to the central role of B cells
in the establishment of a specific immune response. Polyclonal
proliferation of B cells (induced by CpG ODN) increases the likelihood
of an Ag-specific B cell/Th cell match. Furthermore, enhanced
expression of the costimulatory molecule CD86 on polyclonally expanded
B cells activates Ag-specific Th cells. B cells also increase their
CD40 expression in response to CpG ODN (29), improving the
capability of CD40 ligand-expressing activated Th cells to stimulate B
cells. Increased ICAM-1 synthesis on B cells facilitates the
cell-to-cell contact (29). Thus, the activation status of
polyclonal B cells plays a critical role during the initiation of a
specific Ab response. The contribution of NK cell activity to the
establishment of specific Abs is less obvious, and so the strong
correlation between NK cell activation and in vivo adjuvant activity
that was observed in Table I was unexpected. NK cells are part of the
innate immune system and as such are involved in the first line of
defense against pathogens. Most likely, the cytokine pattern produced
by NK cells upon activation is closely related to the initiation of a
specific immune response. Overall, IFN- secretion did not correlate
well with in vivo adjuvant activity, but it remains possible that this
may have contributed to the adjuvant activity of ODN 1628, especially
because this ODN failed to induce any detectable NK lytic activity. The
use of dendritic cell activation for the screening of CpG ODN may add
valuable information in future studies and allow for the identification
of CpG ODN other than or even more potent than ODN 2006.
As there is a 2-log higher endotoxin sensitivity of human than mouse
primary monocytes, extreme caution is required to avoid endotoxin
contamination of CpG DNA used for testing in the human system
(34). Because TNF-, IL-6, and IL-12 are produced by
human monocytes in response to even low amounts of endotoxin, their
value for high throughput in vitro screening assays is limited. On the
other hand, human B cells and NK cells show only minor activation by
endotoxin and thus are far more useful in testing for CpG DNA
immunostimulatory activity.
Stimulation of cellular function in either NK or B cells (i.e., lytic activity, proliferation) requires a stronger CpG ODN than the induction of activation markers at their surface (CD69, CD86). For both cell types, the use of cell surface activation markers showed a higher nonspecific background, attributable to the phosphorothioate backbone, compared with the functional assays. This high sensitivity of surface markers requires the use of low ODN concentrations for optimal discrimination between ODN of similar activity. Thus, the use of surface markers allows the comparison of ODN with weak activity, while functional assays are preferred for comparing ODN with high activity. It is of note that the optimal ODN concentrations for stimulating B cells and NK cells differ. Although 0.6 µg/ml ODN is maximal to stimulate B cells, optimal NK cell activation may require 6 µg/ml ODN. Both B cell activation and NK cell functional activity are measured within freshly isolated PBMC. We found earlier that highly purified human primary B cells are activated by CpG DNA (29). The existence of a direct effect of CpG DNA on NK cells is less clear, and a secondary mechanism mediated by another cell type within PBMC might contribute to CpG-induced functional activity of NK cells.
It has been shown that some phosphorothioate ODN can induce human B cell proliferation (35, 36). Liang et al. found that the 6-mer 5'-TCGTCG-3' at the 5' end is critical for the activity of an ODN to stimulate human B cells at low concentrations, but that a (TCG)n ODN did not have higher activity. Poly T, A, C, or G ODN or ODN with a random sequence were not active in their assays. These results are in agreement with our findings. In addition, we demonstrate that the activity of an ODN is markedly enhanced if the 5'-TCGTCG-3' is followed by TpT. This transforms the 5'-TCGTCG-3' into a human CpG motif preceded by a TpC. This 8-mer motif followed by a poly C tail shows maximal activity if used as a phosphodiester ODN. but not as a phosphorothioate ODN (29). If a 5' end 5'-TCGTCG-3' is followed by another human CpG motif within the sequence, the activity is also increased. However, ODNs with three or four copies of the human CpG motif (5'-GTCGTT-3') without the 5'-TCGTCGTT-3' motif at the 5' end showed only low activity. This is consistent with the study by Liang et al. (36), who tested ODNs with several copies of GACGTT, TGACGTT, or TGACGTC that were not particularly potent. Consequently, combining the 5'-TCGTCGTT-3' located at the 5' end with additional 5'-GTCGTT-3' motifs gave the best results in our study.
An important question is whether the immune effects of CpG motifs may
be modified by the presence of other types of sequence motifs. To date,
only two distinct immune effects of ODN sequences could be clearly
identified: the effects due to CpG motifs and the effects due to G-rich
motifs. We found a surprisingly poor correlation between ODN that
induce strong IFN- secretion and those that are strong adjuvants.
This poor correlation was largely due to the effects of ODN 1628, which
was relatively weak as an adjuvant and at inducing secretion of most of
the other cytokines, yet induced high level production of IFN-. A
distinguishing feature of ODN 1628 is the presence of two G-rich
regions, or poly G motifs, one of which has four Gs in a row, and the
other of which has six Gs. Such poly G sequences show immunostimulatory
effects that are distinct from CpG-mediated effects. For example, the
level of IFN induction by a CpG ODN can be enhanced by poly G sequences
at the ends of the same ODN (37). On the other hand, an
ODN containing poly G sequences alone can block induction of IFN
secretion by another ODN with a CpG motif (37). Poly G ODN
can also block the production of IFN- induced by the mitogens Con A,
bacterial DNA, or the combination of PMA and the calcium ionophore
A23187 (44). This inhibition was only seen with the
phosphorothioate backbone. Of note, poly G-rich ODN can also block the
downstream effects of IFN- (38, 39). Also, we
previously showed that the effects of poly G depend on the ODN
backbone; poly G motifs increase the NK activity of chimeric ODN
(phosphorothioate linkages on both ends, phosphodiester ODN in the
middle), but reduce the NK activity of phosphorothioate ODN
(9). Further studies will be required to determine whether
the IFN- response to the poly G ODN 1628 is IL-12 independent and to
identify the producing cell type.
Alum (e.g., Al2O3) was developed >75 yr ago and is still the only adjuvant approved for human use in most countries. Alum induces a Th2-type rather than a Th1-type immune response, appears to interfere with the development of cell-mediated immunity, and blocks activation of CD8+ CTL (40). We showed earlier that CpG ODN induce a predominant IgG2a Ab response (Th1-like) to HBsAg in mice and, when the two adjuvants are used together, can even overcome the Th2 bias (IgG1) of alum for both Ab isotype and CTL responses (21). Furthermore, we found that the CpG ODN could induce HBsAg-specific CTL in young mice, in which a Th1 response normally is difficult to obtain (22, 41). Besides a shift toward a Th1 immune response, CpG ODN have the advantage over alum that it could be used as an adjuvant with live attenuated or multivalent vaccines that cannot be mixed with alum. In situations where it is necessary to overcome non- or hyporesponsiveness, the synergistic effect of CpG ODN and alum may be useful.
In preclinical studies antisense phosphorothioate ODN that are designed
to inhibit target protein synthesis have been found to be safe at doses
>100 mg/kg. In clinical antisense studies, phosphorothioate ODN have
been used in doses up to 12 mg/kg with little drug-related toxicity
(42, 43). This is 30 times higher than the dose of 10
µg/mouse used in the present study (0.36 mg/kg). Phosphorothioate ODN
can be produced on a large scale under good manufacturing practices
conditions at a cost of approximately $200/g (42). Thus,
the use of CpG ODN as adjuvant could significantly lower the cost of
vaccination when repeated doses are normally required to induce a
protective immunity (for example, with hepatitis B).
In conclusion, our study defines a CpG phosphorothioate ODN with high activity on human immune cells and with excellent adjuvant characteristics in chimpanzees. The design of this compound is based on the optimal human CpG motif and additional features that are essential if a phosphorothioate backbone is used. As a drug, ODN 2006 is inexpensive and easy to manufacture and is a candidate ODN for human clinical trials as an adjuvant for immunotherapy of cancer, infectious diseases, and allergy.
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Acknowledgments |
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Footnotes |
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2 Address correspondence and reprint requests to Dr. Arthur M. Krieg, Department of Internal Medicine, University of Iowa, 540 EMRB, Iowa City, IA 52242. E-mail address:
3 Abbreviations used in this paper: ODN, oligodeoxynucleotides; HBsAg, hepatitis B surface Ag.
Received for publication September 15, 1999. Accepted for publication November 19, 1999.
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References |
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A. Krug, S. Rothenfusser, S. Selinger, C. Bock, M. Kerkmann, J. Battiany, A. Sarris, T. Giese, D. Speiser, S. Endres, et al. CpG-A Oligonucleotides Induce a Monocyte-Derived Dendritic Cell-Like Phenotype That Preferentially Activates CD8 T Cells J. Immunol., April 1, 2003; 170(7): 3468 - 3477. [Abstract] [Full Text] [PDF]
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C. Nonnenmacher, A. Dalpke, S. Zimmermann, L. Flores-de-Jacoby, R. Mutters, and K. Heeg DNA from Periodontopathogenic Bacteria Is Immunostimulatory for Mouse and Human Immune Cells Infect. Immun., February 1, 2003; 71(2): 850 - 856. [Abstract] [Full Text] [PDF]
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 A. D. Sandler, H. Chihara, G. Kobayashi, X. Zhu, M. A. Miller, D. L. Scott, and A. M. Krieg CpG Oligonucleotides Enhance the Tumor Antigen-specific Immune Response of a Granulocyte Macrophage Colony-stimulating Factor-based Vaccine Strategy in Neuroblastoma Cancer Res., January 15, 2003; 63(2): 394 - 399. [Abstract] [Full Text] [PDF]
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R. Takauji, S. Iho, H. Takatsuka, S. Yamamoto, T. Takahashi, H. Kitagawa, H. Iwasaki, R. Iida, T. Yokochi, and T. Matsuki CpG-DNA-induced IFN-{alpha} production involves p38 MAPK-dependent STAT1 phosphorylation in human plasmacytoid dendritic cell precursors J. Leukoc. Biol., November 1, 2002; 72(5): 1011 - 1019. [Abstract] [Full Text] [PDF]
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D. Yu, E. R. Kandimalla, L. Bhagat, J.-Y. Tang, Y. Cong, J. Tang, and S. Agrawal 'Immunomers'--novel 3'-3'-linked CpG oligodeoxyribonucleotides as potent immunomodulatory agents Nucleic Acids Res., October 15, 2002; 30(20): 4460 - 4469. [Abstract] [Full Text] [PDF]
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A. R. M. Olbrich, S. Schimmer, K. Heeg, K. Schepers, T. N. M. Schumacher, and U. Dittmer Effective Postexposure Treatment of Retrovirus-Induced Disease with Immunostimulatory DNA Containing CpG Motifs J. Virol., October 11, 2002; 76(22): 11397 - 11404. [Abstract] [Full Text] [PDF]
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K. Heckelsmiller, K. Rall, S. Beck, A. Schlamp, J. Seiderer, B. Jahrsdorfer, A. Krug, S. Rothenfusser, S. Endres, and G. Hartmann Peritumoral CpG DNA Elicits a Coordinated Response of CD8 T Cells and Innate Effectors to Cure Established Tumors in a Murine Colon Carcinoma Model J. Immunol., October 1, 2002; 169(7): 3892 - 3899. [Abstract] [Full Text] [PDF]
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E. M. A. van Rooij, H. L. Glansbeek, L. A. T. Hilgers, E. G. te Lintelo, Y. E. de Visser, W. J. A. Boersma, B. L. Haagmans, and A. T. J. Bianchi Protective Antiviral Immune Responses to Pseudorabies Virus Induced by DNA Vaccination Using Dimethyldioctadecylammonium Bromide as an Adjuvant J. Virol., September 11, 2002; 76(20): 10540 - 10545. [Abstract] [Full Text] [PDF]
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B. Jahrsdorfer, R. Jox, L. Muhlenhoff, K. Tschoep, A. Krug, S. Rothenfusser, G. Meinhardt, B. Emmerich, S. Endres, and G. Hartmann Modulation of malignant B cell activation and apoptosis by bcl-2 antisense ODN and immunostimulatory CpG ODN J. Leukoc. Biol., July 1, 2002; 72(1): 83 - 92. [Abstract] [Full Text] [PDF]
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M. Gierynska, U. Kumaraguru, S.-K. Eo, S. Lee, A. Krieg, and B. T. Rouse Induction of CD8 T-Cell-Specific Systemic and Mucosal Immunity against Herpes Simplex Virus with CpG-Peptide Complexes J. Virol., June 5, 2002; 76(13): 6568 - 6576. [Abstract] [Full Text] [PDF]
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V. Hornung, S. Rothenfusser, S. Britsch, A. Krug, B. Jahrsdorfer, T. Giese, S. Endres, and G. Hartmann Quantitative Expression of Toll-Like Receptor 1-10 mRNA in Cellular Subsets of Human Peripheral Blood Mononuclear Cells and Sensitivity to CpG Oligodeoxynucleotides J. Immunol., May 1, 2002; 168(9): 4531 - 4537. [Abstract] [Full Text] [PDF]
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D. Yu, E. R. Kandimalla, Q. Zhao, Y. Cong, and S. Agrawal Immunostimulatory properties of phosphorothioate CpG DNA containing both 3'-5'- and 2'-5'-internucleotide linkages Nucleic Acids Res., April 1, 2002; 30(7): 1613 - 1619. [Abstract] [Full Text] [PDF]
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T.-H. Chuang, J. Lee, L. Kline, J. C. Mathison, and R. J. Ulevitch Toll-like receptor 9 mediates CpG-DNA signaling J. Leukoc. Biol., March 1, 2002; 71(3): 538 - 544. [Abstract] [Full Text] [PDF]
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D. Verthelyi, R. T. Kenney, R. A. Seder, A. A. Gam, B. Friedag, and D. M. Klinman CpG Oligodeoxynucleotides as Vaccine Adjuvants in Primates J. Immunol., February 15, 2002; 168(4): 1659 - 1663. [Abstract] [Full Text] [PDF]
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T. Decker, S. Hipp, R. J. Kreitman, I. Pastan, C. Peschel, and T. Licht Sensitization of B-cell chronic lymphocytic leukemia cells to recombinant immunotoxin by immunostimulatory phosphorothioate oligodeoxynucleotides Blood, February 15, 2002; 99(4): 1320 - 1326. [Abstract] [Full Text] [PDF]
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Z. K. Ballas, A. M. Krieg, T. Warren, W. Rasmussen, H. L. Davis, M. Waldschmidt, and G. J. Weiner Divergent Therapeutic and Immunologic Effects of Oligodeoxynucleotides with Distinct CpG Motifs J. Immunol., November 1, 2001; 167(9): 4878 - 4886. [Abstract] [Full Text] [PDF]
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F. Takeshita, C. A. Leifer, I. Gursel, K. J. Ishii, S. Takeshita, M. Gursel, and D. M. Klinman Cutting Edge: Role of Toll-Like Receptor 9 in CpG DNA-Induced Activation of Human Cells J. Immunol., October 1, 2001; 167(7): 3555 - 3558. [Abstract] [Full Text] [PDF]
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 B. Mui, S. G. Raney, S. C. Semple, and M. J. Hope Immune Stimulation by a CpG-Containing Oligodeoxynucleotide Is Enhanced When Encapsulated and Delivered in Lipid Particles J. Pharmacol. Exp. Ther., September 1, 2001; 298(3): 1185 - 1192. [Abstract] [Full Text] [PDF]
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M. Schnurr, P. Galambos, C. Scholz, F. Then, M. Dauer, S. Endres, and A. Eigler Tumor Cell Lysate-pulsed Human Dendritic Cells Induce a T-Cell Response against Pancreatic Carcinoma Cells: an in Vitro Model for the Assessment of Tumor Vaccines Cancer Res., September 1, 2001; 61(17): 6445 - 6450. [Abstract] [Full Text] [PDF]
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B. E. Britigan, T. S. Lewis, M. Waldschmidt, M. L. McCormick, and A. M. Krieg Lactoferrin Binds CpG-Containing Oligonucleotides and Inhibits Their Immunostimulatory Effects on Human B Cells J. Immunol., September 1, 2001; 167(5): 2921 - 2928. [Abstract] [Full Text] [PDF]
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C. Neufert, R. K. Pai, E. H. Noss, M. Berger, W. H. Boom, and C. V. Harding Mycobacterium tuberculosis 19-kDa Lipoprotein Promotes Neutrophil Activation J. Immunol., August 1, 2001; 167(3): 1542 - 1549. [Abstract] [Full Text] [PDF]
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T. D. Crabtree, L. Jin, D. P. Raymond, S. J. Pelletier, C. W. Houlgrave, T. G. Gleason, T. L. Pruett, and R. G. Sawyer Preexposure of Murine Macrophages to CpG Oligonucleotide Results in a Biphasic Tumor Necrosis Factor Alpha Response to Subsequent Lipopolysaccharide Challenge Infect. Immun., April 1, 2001; 69(4): 2123 - 2129. [Abstract] [Full Text] [PDF]
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N. Kadowaki, S. Antonenko, and Y.-J. Liu Distinct CpG DNA and Polyinosinic-Polycytidylic Acid Double-Stranded RNA, Respectively, Stimulate CD11c- Type 2 Dendritic Cell Precursors and CD11c+ Dendritic Cells to Produce Type I IFN J. Immunol., February 15, 2001; 166(4): 2291 - 2295. [Abstract] [Full Text] [PDF]
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D. Verthelyi, K. J. Ishii, M. Gursel, F. Takeshita, and D. M. Klinman Human Peripheral Blood Cells Differentially Recognize and Respond to Two Distinct CpG Motifs J. Immunol., February 15, 2001; 166(4): 2372 - 2377. [Abstract] [Full Text] [PDF]
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B. Jahrsdörfer, G. Hartmann, E. Racila, W. Jackson, L. Mühlenhoff, G. Meinhardt, S. Endres, B. K. Link, A. M. Krieg, and G. J. Weiner CpG DNA increases primary malignant B cell expression of costimulatory molecules and target antigens J. Leukoc. Biol., January 1, 2001; 69(1): 81 - 88. [Abstract] [Full Text]
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G. J. Weiner The immunobiology and clinical potential of immunostimulatory CpG oligodeoxynucleotides J. Leukoc. Biol., October 1, 2000; 68(4): 455 - 463. [Abstract] [Full Text]
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S. W. Lee, M. K. Song, K. H. Baek, Y. Park, J. K. Kim, C. H. Lee, H.-K. Cheong, C. Cheong, and Y. C. Sung Effects of a Hexameric Deoxyriboguanosine Run Conjugation into CpG Oligodeoxynucleotides on Their Immunostimulatory Potentials J. Immunol., October 1, 2000; 165(7): 3631 - 3639. [Abstract] [Full Text] [PDF]
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S. Bauer, C. J. Kirschning, H. Hacker, V. Redecke, S. Hausmann, S. Akira, H. Wagner, and G. B. Lipford Human TLR9 confers responsiveness to bacterial DNA via species-specific CpG motif recognition PNAS, July 31, 2001; 98(16): 9237 - 9242. [Abstract] [Full Text] [PDF]
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