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*
Laboratory of Mycobacteria, Division of Bacterial Products,
Laboratory of Enteric and Sexually Transmitted Diseases, Division of Bacterial Products, and
Retroviral Immunology Section, Laboratory of Retrovirology, Division of Viral Products, Center for Biologics Evaluation and Research, Rockville, MD 20852
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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, IL-12, TNF-
, and IL-6, which
are important in the control of intracellular bacterial infection.
Here, we show that bacterial DNA, as well as synthetic oligonucleotides
containing CpG motifs, induce protection against large lethal doses of
Francisella tularensis live vaccine strain (LVS) and
Listeria monocytogenes. Methylation of DNA at CpG
dinucleotides or inversion of the motif abolished this protection.
Surprisingly, DNA-mediated protection was highly dependent on
lymphocytes, particularly B cells, as well as the production of
IFN-. Optimal protection was elicited 2–3 days after inoculation
with DNA and persisted for up to 2 wk. Further, animals surviving
lethal challenge developed pathogen-specific secondary immunity. These
findings indicate that host innate immune responses to bacterial DNA
may contribute to the induction of protective immunity to bacteria and
the subsequent development of memory. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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, IL-12, IL-6, IL-18, and TNF- 3, 4, 5, 6, 7 . Further, CpG
motifs may facilitate the development of Ag-specific immunity by
initiating an innate, Ag-nonspecific inflammatory response at the site
of vaccination 8, 9 .
The rapid induction of an innate immune response, including the
production of Th1-related cytokines, is critical in controlling the
early spread of intracellular pathogens. Nonspecific bacterial
stimulation of NK cells and macrophages induces production of IFN-,
TNF-, and IL-12 10, 11, 12, 13, 14 , providing early resistance to infection
with intracellular bacteria such as Listeria monocytogenes
14 , Francisella tularensis 15 , and mycobacteria 16 .
This innate, lymphocyte-independent immune response apparently permits
time for the development of a specific, T cell-dependent immune
response that ultimately controls infection and clears the pathogen. In
addition to traditional innate immunity, we recently described a second
type of nonspecific innate protective immunity that is dependent on
lymphocytes. Normal mice, but not lymphocyte-deficient scid
mice or B cell knockout (KO)3
mice, given a sublethal infection with F. tularensis live
vaccine strain (LVS) survive a strong lethal challenge with LVS given
only 2–3 days later 17, 18 . F. tularensis is a highly
virulent pathogen in humans, and the LVS strain of F.
tularensis, while attenuated for people, retains full virulence
for rodents; murine infection in LVS is histopathologically quite
similar to that in humans 19 . Thus, this bacterium has been studied
as a model intracellular pathogen 20, 21, 22 . Early protection, which is
also demonstrable in L. monocytogenes infection in mice
23 , is nonspecific and requires IFN- and lymphocytes, particularly
B cells. However, the bacterial determinants that stimulate either
inflammatory or lymphocyte-dependent innate immune responses are poorly
understood.
Recognizing that the cell types and cytokines important in an early protective immune response to LVS and L. monocytogenes were the same as those stimulated by bacterial DNA, we hypothesized that host recognition of bacterial DNA containing CpG motifs contributes significantly to the stimulation of innate protective immunity. Here, we show that treatment of mice with either bacterial chromosomal DNA or oligonucleotide DNA, containing unmethylated CpG motifs that stimulate Th1-associated cytokine production, induces lymphocyte-dependent protection against lethal challenge with virulent F. tularensis and L. monocytogenes.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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F. tularensis LVS (ATCC 29684; American Type Culture Collection, Manassas, VA) was cultured on a modified Mueller-Hinton (MH) agar plate or in modified MH broth (Difco Laboratories, Detroit, MI) as previously described 17, 21, 24 . L. monocytogenes strain EGD (ATCC 15313) was a gift from Dr. William Schwan (Pathogenesis) and was cultured in brain-heart infusion broth or plates (Difco). Aliquots (1 ml) of bacteria were frozen in broth alone at -70°C, periodically thawed for use, and viable bacteria were quantified by plating serial dilutions on MH agar plates.
Animals
Specific pathogen-free, male BALB/cByJ mice were purchased from
The Jackson Laboratory (Bar Harbor, ME) and were used at 6–16 wk of
age. Male BALB/c.scid mice, as well as Igh6- (B
cell KO) and GKO (IFN- KO) mice on a C57BL/6J background, were also
purchased from The Jackson Laboratory. Male BALB/c-nu/+,
BALB/c.nu/nu, C3H/HeN, and C3H/HeJ mice were purchased from
the Biological Resources Branch, Frederick Cancer Research and
Development Center, National Cancer Institute (Frederick, MD). All mice
were housed in sterile microisolator cages in a barrier environment in
the CBER specific pathogen-free animal facility. All materials used in
mouse inoculations, including bacteria, were diluted in PBS
(BioWhittaker, Walkersville, MD) containing <0.1 ng/ml of endotoxin.
Mean time to death (MTD) was calculated by arithmetic mean ± SD
for all mice within a group that died; surviving mice were not included
in this calculation. The statistical significance of differences in MTD
were assessed using Student’s t test. Experiments
enumerating numbers of CFUs in organs of various mice were performed as
follows: Mice were treated and infected as indicated, and spleens,
livers, lungs, and peritoneal cells (using 7–8 ml of PBS to lavage the
peritoneal cavity) were removed aseptically. The organs were emulsified
in a Stomacher (Seward, London, U.K.) in 5–10 ml of sterile
PBS, and appropriate dilutions of organ homogenates or peritoneal cells
were plated on MH plates. Results are expressed as the mean ± the
SD of the mean for groups of three to four mice.
Bacterial DNA preparations
Bacterial DNA was prepared from late log phase cultures of LVS as previously described 25 . Briefly, bacteria were frozen and thawed repeatedly, then treated with 200 µg/ml of RNase, 10 mg/ml of lysozyme (in 50 mM Tris (pH 8.0) and 1 mM EDTA), 20 mg/ml of proteinase K, and 10% sarcosyl. After 1 h at 37°C, when bacterial lysis was visible, DNA was recovered through repeated extraction with phenol-chloroform-isoamyl alcohol and then precipitated with sodium acetate and ethanol. Alternatively, bacteria were treated and lysed as described and applied to a cesium chloride gradient. After centrifugation, the genomic DNA band was recovered and dialyzed exhaustively against low-endotoxin Tris-EDTA (pH 8.0) and then low-endotoxin PBS. All DNA preparations were cut with EcoRI for verification of a characteristic banding pattern on agarose gels and were tested for endotoxin content by chromogenic Limulus amebocyte lysate (LAL) assay (through the courtesy of Dr. Donald Hochstein, Division of Product Quality Control, Center for Biologics Evaluation and Research (CBER), Food and Drug Administration (FDA)) and for protein content using the Pierce (Rockford, IL) bicinchoninic acid (BCA) protein assay kit. LAL activity was always 1 pg/µg of DNA or less, and protein was always 10 ng/µg DNA or less. As indicated, DNA preparations were treated with either SssI CpG methylase (New England Biolabs, Boston, MA) according to package insert instructions. Successful methylation of cytosine residues within the dinucleotide sequence 5'... CG... 3' was confirmed by attempting to digest methylated DNA with the restriction enzyme HpaI, which cuts only at unmethylated CpG dinucleotides, and with its isoschizomer MspI, which can cut DNA when the C residue of a CpG dinucleotide is methylated. Male human placental (HP) DNA was purchased from Sigma (St. Louis, MO). Oligonucleotides were synthesized in the CBER core facility and tested upon reconstitution for endotoxin as above; all contained <0.1 EU/ml at a concentration of 1 mg/ml or greater. The sequences of the oligonucleotides used were: #1 (see 26 , TCT CCC AGC GTG CGC CAT; Me-#1, the latter sequence with methylated C's at positions 9, 13, and 15; #2 (#A2 in 5 , GCT AGA CGT TAG CGT; #2', GCT AGA GCT TAG GCT; #3, TCA ACG TTG A; and #3', TCA AGC TTG A.
Enzyme-linked immunospot (ELISPOT) assay
Numbers of cytokine-secreting spleen cells, after 8 h of in vitro stimulation with the indicated DNA preparations, were determined by ELISPOT as previously described 27 . Briefly, microtiter plates were coated with primary anti-cytokine Abs, blocked, and serial dilutions of single spleen cell suspensions incubated for 8 h at 37°C. Cytokine-secreting spots were detected by the addition of secondary biotinylated anti-cytokine Abs, followed by avidin-conjugated alkaline phosphatase and BCIP/NBT solution (Kirkegaard and Perry, Gaithersburg, MD).
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The ability of genomic DNA from pathogenic bacteria to protect
against lethal bacterial infection was studied, first using F.
tularensis LVS as a model pathogen. High m.w. genomic DNA (>25
kb) was purified from F. tularensis LVS by traditional
methods 25 and contained low or no detectable levels of endotoxin
(<1 pg/µg of DNA) and protein (<10 ng/µg of DNA). Male BALB/cByJ
mice were treated with 0.01–20 µg of LVS DNA, and then challenged
with 103 LD50 of LVS 3 days later. All
mice treated with 0.5 µg of LVS DNA or greater survived, while
PBS-treated controls died within an average of 6 days (Table I
). LVS DNA further purified by cesium
chloride equilibrium centrifugation was equally protective (Table I).
In contrast, protection was not observed in mice treated with HP DNA
(Table I). Protection was not dependent on the route of administration,
as mice given 5 µg of LVS DNA i.m., i.p., or intradermally were well
protected (Table I and data not shown). The magnitude of the protective
effect was evaluated by challenging mice treated with 5 µg of
purified LVS DNA with increasing numbers of F. tularensis
LVS. As seen in Table I, 100% of the mice survived challenge with
106 LD50 of LVS bacteria. The time course of
protection was examined by treating mice with 5 µg of LVS DNA on day
0 and challenging 3, 7, 10, 14, 21, and 28 days later; mice given
genomic DNA 3–14 days before challenge were well protected, while
those challenged at later time points were partially or completely
succumbed to infection (data not shown).
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). These findings suggest that DNA
containing unmethylated CpG motifs play a critical role in the
protection conferred by bacterial DNA. However, we could not exclude
the possibility that other entities contribute to the protection
observed with LVS genomic DNA, particularly since methylation of LVS
DNA did not completely abolish protection (Table II). The well known
mitogen LPS was of particular interest. To test the potential
contribution of traditional endotoxin in these preparations, both
LPS-responsive C3H/HeN and LPS-hyporesponsive C3H/HeJ mice were treated
with cesium chloride-purified LVS DNA. Although both C3H
substrains are equally quite susceptible to LVS infection, with the
LD50 approaching a single bacterium 21 , both were fully
protected against lethal challenge (Table III). This indicates that traditional
endotoxin activity was not responsible for the protection induced by
the bacterial DNA preparation.
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To further examine the contribution of DNA alone to protection
against lethal infection, we tested whether synthetic oligonucleotides
expressing CpG motifs could mimic the activity of bacterial DNA;
oligonucleotides were phosphorothioate-modified to increase their
resistance to endogenous nucleases. Mice treated with CpG-containing
oligo #1 survived lethal challenge with either LVS or L.
monocytogenes, whereas mice treated with an oligonucleotide of the
same sequence in which all CpG dinucleotides were methylated (Me-#1)
did not survive challenge (Fig. 1A). Further, mice given a
mixture of two other CpG-containing oligos, #2 and #3, survived LVS or
L. monocytogenes challenge 3 days later (Fig. 1B), while mice treated with oligos #2' and #3', in which
the respective CpG dinucleotides were inverted to GpC dinucleotides,
all succumbed to challenge. In all experiments, no significant
protection was conferred by treatment of mice with mammalian (HP) DNA.
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). However, protection was not strictly
dependent on the route of administration, as mice given 20 µg of
oligo #1 DNA i.p. or intradermally were well protected, while those
given oligonucleotide DNA i.m. were less well protected (Table IV and
data not shown). The magnitude of the protective effect was evaluated
by challenging mice treated with 20 µg of oligo #1 DNA with
increasing numbers of F. tularensis LVS. As seen in Table I
,
mice readily survived challenge with 104-105
LD50 of LVS bacteria.
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A). However, improved
survival was observed on day 1 when the challenge dose was lowered to
1000 LD50 (Fig. 2B). As noted in studies using
chromosomal DNA, protection with oligonucleotide DNA lasted about 2 wk
(Fig. 2C). Taken together, these experiments indicate that
protection improves over the first few days after exposure to
CpG-containing DNA, then wanes over time.
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) on day 1 after challenge in oligo
#1-treated mice compared with control mice. By day 4, however, there
were significantly lower (p < 0.001) numbers
of bacteria in both sites of oligo #1-treated mice compared with
Me-oligo #1 mice, although numbers of bacteria in all mice increased
compared with numbers observed on day 1. By day 7, numbers of bacteria
in the peritoneal cells and spleens of oligo #1-treated mice had
peaked, and they decreased thereafter until bacterial clearance was
complete by day 24. Thus, bacteria readily disseminated and replicated
in oligo #1-treated mice, but replication was eventually controlled and
bacteria cleared over a 3-wk period. Similar trends were found in
livers and lungs of the same mice (data not shown).
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The immunostimulatory effects of genomic chromosomal LVS DNA and
CpG-containing oligonucleotides were examined in vitro. Spleen cells
from normal BALB/cByJ mice, which are predominantly lymphocytes, were
incubated with DNA for 8 h and then assayed for cytokine
production in vitro. Similar to previous results 3, 4, 5, 6, 7 , LVS genomic
DNA and all three oligonucleotides (nos. 1, 2, and 3) stimulated
significant in vitro production of IFN-, IL-12, and IL-6; methylated
LVS genomic DNA, methylated oligo #1, inverted oligos #2' and #3', and
mammalian HP DNA did not (Fig. 4). No DNA
preparation stimulated IL-4 production. Since GKO mice given LVS DNA do
not survive LVS challenge (Table V), this
cytokine clearly contributes to DNA-mediated protection and resolution
of LVS challenge.
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, while those using mice treated with
oligonucleotide DNA are shown in Fig. 6.
Neither lymphocyte-deficient BALB/c.scid mice nor B cell KO
(Igh6-) mice treated with either genomic DNA or oligo #1
DNA survived lethal challenge any longer than control mice. In
contrast, most T cell-deficient BALB/c.nu/nu mice, or TCR KO
mice deficient in both ß and 
T cells, treated with DNA
survived lethal challenge for 2–3 wk longer than control mice; these
mice eventually succumbed to challenge after about 3–4 wk. Overall,
82% of B cell KO mice given LVS genomic DNA (13 of 16 mice in 3
separate experiments) and 100% of B cell KO mice given 20 µg of
oligo #5991 (19 of 20 mice in 4 separate experiments) died within a
week following 100-1000 LD50 challenge, indicating that B
cells are critical in DNA-mediated protection. On the other hand, 44%
of TCR KO mice given 20 µg of oligo #5991 (7 of 16 mice in 3 separate
experiments) died within a week following 100-1000 LD50
challenge, suggesting a lesser, but nonetheless significant,
contribution of T cells.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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and
the activity of lymphocytes, particularly B cells.
These results confirm and extend other recent observations concerning
the ability of bacterial DNA to modulate infection by intracellular
pathogens. Mice treated with CpG-containing oligonucleotides limited
primary infection and resisted secondary infection with
Leishmania major, as measured by footpad swelling;
resistance to infection was correlated with the induction of
Th1-related cytokines, particularly IL-12 30 . In contrast to the
results presented here, Zimmermann et al. 30 further demonstrated
that oligonucleotides protected susceptible mice from
Leishmania infection when administered several weeks after
infection. However, this difference may simply be due to the slower
proliferation of Leishmania parasites compared with
bacteria, permitting time for intervention. In another recent study,
mice treated with Escherichia coli DNA or unmethylated
oligonucleotide of the same sequence used for some of these studies
(oligo #1) produced a sustained serum IL-12 (but not IFN-) response;
further, DNA-treated mice infected with L. monocytogenes
2–4 days after treatment subsequently had much lower bacterial burdens
in spleens and livers 31 . On the other hand, in some circumstances
bacterial DNA appears to promote shock in vivo, probably through
production of TNF- 32, 33 . Here, we show that host response to
genomic DNA from a pathogenic bacterium results in long-term survival
of a frank lethal infection with the homologous bacterium. Further, for
the first time, we define the cellular basis of protection induced by
DNA containing CpG motifs, with the surprising result that lymphocytes
are required with an apparently Ag-nonspecific activity. The structural
basis of the protection induced by genomic DNA was defined by using
oligonucleotide DNA containing unmethylated CpG dinucleotides, which
also protected against lethality in two different intracellular
bacterial model systems. Preliminary data also suggests that
oligonucleotide DNA does not protect against infection with two
extracellular bacterial pathogens, enterohemorrhagic E. coli
and Yersinia enterocolitica (data not shown).
While it is difficult to precisely compare the dose responses of protection induced by genomic DNA and oligonucleotide DNA on a molar basis, they seem to be roughly comparable. This was also true in previous studies comparing bacillus Calmette-Guérin DNA and a synthetic oligonucleotide in terms of their capacity to stimulate NK cell activity and IFN production 7 . To date, LVS DNA has been found to be rather A-T rich but completely unmethylated 34, 35, 36 , which may mean that it has a rather high number of immunostimulatory GpC motifs in non-A-T regions. Our preparations of genomic DNA may also contain other entities that contribute to protection and/or that synergize with CpG-containing DNA to promote protection. The most obvious of these is LPS, derived either from the LVS bacteria themselves or the broth in which the bacteria are grown, which could copurify with DNA. Francisella is a Gram-negative bacterium with its own LPS. However, LVS LPS apparently lacks endotoxic activity as well as stimulatory activity for murine B cells and macrophages 37, 38 , does not react in LAL assays 37 , and is difficult to detect by silver staining or other physicochemical methods 39 . Here, genomic DNA was prepared using endotoxin-controlled reagents and was tested for presence of endotoxin by LAL; all preparations were less than 1 pg/µg of DNA. These data, as well as results using C3H/HeJ endotoxin-hyporesponsive mice and the dose response studies, strongly indicated that protection was not due to simple endotoxin contamination. However, we cannot exclude the possibility that LVS LPS, which is difficult to detect, contributes to protection observed with genomic DNA. Indeed, we have recently found that mice treated with purified LVS LPS are protected against lethal LVS challenge as well (although we cannot exclude the possibility that the LPS preparation is not itself contaminated with LVS DNA).
Endotoxin was not a consideration in studies using oligonucleotides, which conclusively demonstrate that mice treated with unmethylated CpG-containing DNA survive subsequent lethal challenge with two different intracellular pathogens, F. tularensis LVS and L. monocytogenes. Protection is clearly dependent on CpG motifs, as methylation of the cytosine residues or inversion of the CpG dinucleotide to GpC abolished the protective activity of the oligonucleotides. Oligonucleotide mediated protection against a 1000 LD50 lethal challenge apparently required 1–2 days to develop and was active for about 10 days, but waned thereafter. This time course, coupled with the time course of bacterial growth, strongly suggests that protection is the result of activation of cells of the innate immune system. Somewhat surprisingly, this nonspecific protection was dependent on the presence of lymphocytes, particularly B cells and, to a lesser degree, T cells; scid mice and B cell KO mice treated with DNA were unable to survive lethal challenge for any longer than control mice, and TCR-deficient mice as well as nude mice were somewhat compromised in their ability to survive 100 LD50. It should be noted that, as previously shown, T cell-deficient mice eventually succumb to any LVS infection due to the absence of long-term T cell-dependent immunity 24 . This is reminiscent of our previous results in that normal mice, but not lymphocyte-deficient or B cell-deficient mice, given a sublethal infection of F. tularensis LVS survive a secondary lethal LVS challenge of over 10,000 LD50 given 3 days later 17, 18, 24 . Similar early protection that is also strongly lymphocyte-dependent operates in L. monocytogenes infection 23 . Since sublethal infection with either LVS or L. monocytogenes protects against heterologous lethal challenge with either bacteria, this early protection is nonspecific; however, the bacterial determinants that stimulate this lymphocyte-dependent innate immune response have not been defined. The results presented here imply that lymphocytes, in addition to macrophages and NK cells, contribute significantly to innate immunity. Further, early protective immunity may, at least in part, involve host response to the bacterial DNA released early during the course of infection. These results also provide physiologic evidence in support of the hypothesis that CpG suppression and/or CpG methylation were evolutionarily conserved because these modifications improved the immune system’s capacity to recognize and respond to pathogenic bacteria 6 .
Since treatment of mice with DNA converted a lethal challenge to a
sublethal challenge, it was likely that long-term specific protective
immunity developed. This was indeed the case, as evidenced by the
ability of mice that survived a lethal LVS infection to later survive a
second lethal LVS challenge, but not a lethal L.
monocytogenes challenge, and vice versa. The stimulation of innate
immunity by bacterial DNA, permitting time for development of an
Ag-specific immune response and/or improving the overall quality of the
Ag-specific immune response, is no doubt part of the adjuvant activity
of bacterial DNA previously described for the development of Th1 immune
responses and DNA vaccine-mediated immune responses 9, 28 . Since the
CpG-containing DNA used here primarily stimulates Th1-related
cytokines, we predicted that protection would involve at least some of
these cytokines. Indeed, GKO mice treated with DNA did not survive
lethal LVS challenge, and preliminary evidence suggests that the same
is true for IL-12 KO mice (K. L. Elkins and S. Colombini, unpublished
data). Thus, in addition to B cells, at least IFN- and
probably IL-12 are required for successful DNA-mediated protection.
Taken together, these results indicate that bacterial DNA containing unmethylated CpG motifs induces protective immunity against lethal intracellular infection and that this protection is dependent on the nonspecific activity of lymphocytes as well as the production of immunoregulatory cytokines. Under physiological conditions, we propose that bacterial DNA released during intracellular bacterial infection may provide at least part of the stimulus for innate immunity. Further, synthetic CpG-containing oligonucleotides may be of clinical benefit in stimulation of an innate immune response, or in combination with vaccines, to enhance the development of long-term protective immunity.
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Acknowledgments |
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Footnotes |
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2 Address correspondence and reprint requests to Dr. Karen L. Elkins, Laboratory of Mycobacteria, 1401 Rockville Pike, HFM 431, Rockville, MD 20852. E-mail address:
3 Abbreviations used in this paper: KO, knockout; LVS, live vaccine strain of F. tularensis; MH, Mueller-Hinton; GKO, IFN- knockout; ELISPOT, enzyme-linked immunospot; HP, human placental; LAL, Limulus amebocyte lysate; MTD, mean time to death.
Received for publication September 22, 1998. Accepted for publication November 10, 1998.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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production by stimulation of interleukin-12 and tumor necrosis factor-. Cell. Immunol. 167:72.[Medline]
. Proc. Natl. Acad. Sci. USA 93:2879. in resistance to Mycobacterium tuberculosis infection. J. Exp. Med. 178:2249. receptor. Science 259:1742./ß T cell-independent mechanism of resistance to mycobacteria: bacillus-Calmette-Guerin causes progressive infection in severe combined immunodeficient mice, but not in nude mice or in mice depleted of CD4+ and CD8+ T cells. J. Exp. Med. 176:581. in vivo and increases the toxicity of lipopolysaccharides. J. Immunol. 156:4570.[Abstract]
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B. Flynn, V. Wang, D. L. Sacks, R. A Seder, and D. Verthelyi Prevention and Treatment of Cutaneous Leishmaniasis in Primates by Using Synthetic Type D/A Oligodeoxynucleotides Expressing CpG Motifs Infect. Immun., August 1, 2005; 73(8): 4948 - 4954. [Abstract] [Full Text] [PDF]
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 A. Grajkowski, J. Pedras-Vasconcelos, V. Wang, C. Ausin, S. Hess, D. Verthelyi, and S. L. Beaucage Thermolytic CpG-containing DNA oligonucleotides as potential immunotherapeutic prodrugs Nucleic Acids Res., June 21, 2005; 33(11): 3550 - 3560. [Abstract] [Full Text] [PDF]
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S. Ito, K. J. Ishii, A. Ihata, and D. M. Klinman Contribution of Nitric Oxide to CpG-Mediated Protection against Listeria monocytogenes Infect. Immun., June 1, 2005; 73(6): 3803 - 3805. [Abstract] [Full Text] [PDF]
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S. Ito, K. J. Ishii, M. Gursel, H. Shirotra, A. Ihata, and D. M. Klinman CpG Oligodeoxynucleotides Enhance Neonatal Resistance to Listeria Infection J. Immunol., January 15, 2005; 174(2): 777 - 782. [Abstract] [Full Text] [PDF]
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E. Schlaepfer, A. Audige, B. von Beust, V. Manolova, M. Weber, H. Joller, M. F. Bachmann, T. M. Kundig, and R. F. Speck CpG Oligodeoxynucleotides Block Human Immunodeficiency Virus Type 1 Replication in Human Lymphoid Tissue Infected Ex Vivo J. Virol., November 15, 2004; 78(22): 12344 - 12354. [Abstract] [Full Text] [PDF]
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J. C. Deng, T. A. Moore, M. W. Newstead, X. Zeng, A. M. Krieg, and T. J. Standiford CpG Oligodeoxynucleotides Stimulate Protective Innate Immunity against Pulmonary Klebsiella Infection J. Immunol., October 15, 2004; 173(8): 5148 - 5155. [Abstract] [Full Text] [PDF]
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S. Wongratanacheewin, W. Kespichayawattana, P. Intachote, S. Pichyangkul, R. W. Sermswan, A. M. Krieg, and S. Sirisinha Immunostimulatory CpG Oligodeoxynucleotide Confers Protection in a Murine Model of Infection with Burkholderia pseudomallei Infect. Immun., August 1, 2004; 72(8): 4494 - 4502. [Abstract] [Full Text] [PDF]
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S.-i. Ito, K. J. Ishii, H. Shirota, and D. M. Klinman CpG Oligodeoxynucleotides Improve the Survival of Pregnant and Fetal Mice following Listeria monocytogenes Infection Infect. Immun., June 1, 2004; 72(6): 3543 - 3548. [Abstract] [Full Text] [PDF]
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 A. T. Ludwig, J. M. Moore, Y. Luo, X. Chen, N. A. Saltsgaver, M. A. O'Donnell, and T. S. Griffith Tumor Necrosis Factor-Related Apoptosis-Inducing Ligand: A Novel Mechanism for Bacillus Calmette-Guerin-Induced Antitumor Activity Cancer Res., May 15, 2004; 64(10): 3386 - 3390. [Abstract] [Full Text] [PDF]
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S. Bellocchio, C. Montagnoli, S. Bozza, R. Gaziano, G. Rossi, S. S. Mambula, A. Vecchi, A. Mantovani, S. M. Levitz, and L. Romani The Contribution of the Toll-Like/IL-1 Receptor Superfamily to Innate and Adaptive Immunity to Fungal Pathogens In Vivo J. Immunol., March 1, 2004; 172(5): 3059 - 3069. [Abstract] [Full Text] [PDF]
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Y.-F. Chen, C.-W. Lin, Y.-P. Tsao, and S.-L. Chen Cytotoxic-T-Lymphocyte Human Papillomavirus Type 16 E5 Peptide with CpG-Oligodeoxynucleotide Can Eliminate Tumor Growth in C57BL/6 Mice J. Virol., February 1, 2004; 78(3): 1333 - 1343. [Abstract] [Full Text] [PDF]
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S. Raghavan, J. Nystrom, M. Fredriksson, J. Holmgren, and A. M. Harandi Orally Administered CpG Oligodeoxynucleotide Induces Production of CXC and CC Chemokines in the Gastric Mucosa and Suppresses Bacterial Colonization in a Mouse Model of Helicobacter pylori Infection Infect. Immun., December 1, 2003; 71(12): 7014 - 7022. [Abstract] [Full Text] [PDF]
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J. B. Jorgensen, L.-H. Johansen, K. Steiro, and A. Johansen CpG DNA Induces Protective Antiviral Immune Responses in Atlantic Salmon (Salmo salar L.) J. Virol., November 1, 2003; 77(21): 11471 - 11479. [Abstract] [Full Text] [PDF]
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H. Shao, S. Lei, S. L. Sun, J. Xiang, H. J. Kaplan, and D. Sun CpG-Containing Oligodeoxynucleotide 1826 Converts the Weak Uveitogenic Rat Interphotoreceptor Retinoid-Binding Protein Peptide 1181-1191 into a Strong Uveitogen J. Immunol., November 1, 2003; 171(9): 4780 - 4785. [Abstract] [Full Text] [PDF]
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P. Daftarian, S. Ali, R. Sharan, S. F. Lacey, C. La Rosa, J. Longmate, C. Buck, R. F. Siliciano, and D. J. Diamond Immunization with Th-CTL Fusion Peptide and Cytosine-Phosphate-Guanine DNA in Transgenic HLA-A2 Mice Induces Recognition of HIV-Infected T Cells and Clears Vaccinia Virus Challenge J. Immunol., October 15, 2003; 171(8): 4028 - 4039. [Abstract] [Full Text] [PDF]
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A. A. Ashkar, S. Bauer, W. J. Mitchell, J. Vieira, and K. L. Rosenthal Local Delivery of CpG Oligodeoxynucleotides Induces Rapid Changes in the Genital Mucosa and Inhibits Replication, but Not Entry, of Herpes Simplex Virus Type 2 J. Virol., August 15, 2003; 77(16): 8948 - 8956. [Abstract] [Full Text] [PDF]
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N. B. Ray and A. M. Krieg Oral Pretreatment of Mice with CpG DNA Reduces Susceptibility to Oral or Intraperitoneal Challenge with Virulent Listeria monocytogenes Infect. Immun., August 1, 2003; 71(8): 4398 - 4404. [Abstract] [Full Text] [PDF]
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I. Gursel, M. Gursel, H. Yamada, K. J. Ishii, F. Takeshita, and D. M. Klinman Repetitive Elements in Mammalian Telomeres Suppress Bacterial DNA-Induced Immune Activation J. Immunol., August 1, 2003; 171(3): 1393 - 1400. [Abstract] [Full Text] [PDF]
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D. Verthelyi, M. Gursel, R. T. Kenney, J. D. Lifson, S. Liu, J. Mican, and D. M. Klinman CpG Oligodeoxynucleotides Protect Normal and SIV-Infected Macaques from Leishmania Infection J. Immunol., May 1, 2003; 170(9): 4717 - 4723. [Abstract] [Full Text] [PDF]
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Y. Wang and A. M. Krieg Synergy between CpG- or non-CpG DNA and specific antigen for B cell activation Int. Immunol., February 1, 2003; 15(2): 223 - 231. [Abstract] [Full Text] [PDF]
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W. Jiang, H. J. Baker, and B. F. Smith Mucosal Immunization with Helicobacter, CpG DNA, and Cholera Toxin Is Protective Infect. Immun., January 1, 2003; 71(1): 40 - 46. [Abstract] [Full Text] [PDF]
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A. M. Harandi, K. Eriksson, and J. Holmgren A Protective Role of Locally Administered Immunostimulatory CpG Oligodeoxynucleotide in a Mouse Model of Genital Herpes Infection J. Virol., December 20, 2002; 77(2): 953 - 962. [Abstract] [Full Text] [PDF]
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H. Yamada, I. Gursel, F. Takeshita, J. Conover, K. J. Ishii, M. Gursel, S. Takeshita, and D. M. Klinman Effect of Suppressive DNA on CpG-Induced Immune Activation J. Immunol., November 15, 2002; 169(10): 5590 - 5594. [Abstract] [Full Text] [PDF]
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 L. SFONDRINI, D. BESUSSO, C. RUMIO, M. RODOLFO, S. MENARD, and A. BALSARI Prevention of spontaneous mammary adenocarcinoma in HER-2/neu transgenic mice by foreign DNA FASEB J, November 1, 2002; 16(13): 1749 - 1754. [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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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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Y. He, R. Vemulapalli, and G. G. Schurig Recombinant Ochrobactrum anthropi Expressing Brucella abortus Cu,Zn Superoxide Dismutase Protects Mice against B. abortus Infection Only after Switching of Immune Responses to Th1 Type Infect. Immun., May 1, 2002; 70(5): 2535 - 2543. [Abstract] [Full Text] [PDF]
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M. Gursel, D. Verthelyi, I. Gursel, K. J. Ishii, and D. M. Klinman Differential and competitive activation of human immune cells by distinct classes of CpG oligodeoxynucleotide J. Leukoc. Biol., May 1, 2002; 71(5): 813 - 820. [Abstract] [Full Text] [PDF]
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R. Dudani, Y. Chapdelaine, H. van Faassen, D. K. Smith, H. Shen, L. Krishnan, and S. Sad Preexisting Inflammation Due to Mycobacterium bovis BCG Infection Differentially Modulates T-Cell Priming against a Replicating or Nonreplicating Immunogen Infect. Immun., April 1, 2002; 70(4): 1957 - 1964. [Abstract] [Full Text] [PDF]
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N. P. Juffermans, J. C. Leemans, S. Florquin, A. Verbon, A. H. Kolk, P. Speelman, S. J. H. van Deventer, and T. van der Poll CpG Oligodeoxynucleotides Enhance Host Defense during Murine Tuberculosis Infect. Immun., January 1, 2002; 70(1): 147 - 152. [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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 G. Auf, A. F. Carpentier, L. Chen, C. Le Clanche, and J.-Y. Delattre Implication of Macrophages in Tumor Rejection Induced by CpG-oligodeoxynucleotides Without Antigen Clin. Cancer Res., November 1, 2001; 7(11): 3540 - 3543. [Abstract] [Full Text] [PDF]
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T. Hayashi, S. P. Rao, K. Takabayashi, J. H. Van Uden, R. S. Kornbluth, S. M. Baird, M. W. Taylor, D. A. Carson, A. Catanzaro, and E. Raz Enhancement of Innate Immunity against Mycobacterium avium Infection by Immunostimulatory DNA Is Mediated by Indoleamine 2,3-Dioxygenase Infect. Immun., October 1, 2001; 69(10): 6156 - 6164. [Abstract] [Full Text] [PDF]
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I. Gursel, M. Gursel, K. J. Ishii, and D. M. Klinman Sterically Stabilized Cationic Liposomes Improve the Uptake and Immunostimulatory Activity of CpG Oligonucleotides J. Immunol., September 15, 2001; 167(6): 3324 - 3328. [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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A. Al-Mariri, A. Tibor, P. Mertens, X. De Bolle, P. Michel, J. Godefroid, K. Walravens, and J.-J. Letesson Protection of BALB/c Mice against Brucella abortus 544 Challenge by Vaccination with Bacterioferritin or P39 Recombinant Proteins with CpG Oligodeoxynucleotides as Adjuvant Infect. Immun., August 1, 2001; 69(8): 4816 - 4822. [Abstract] [Full Text] [PDF]
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M. Hafner, R. Zawatzky, C. Hirtreiter, W. A. Buurman, B. Echtenacher, T. Hehlgans, and D. N. Mannel Antimetastatic Effect of CpG DNA Mediated by Type I IFN Cancer Res., July 1, 2001; 61(14): 5523 - 5528. [Abstract] [Full Text] [PDF]
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H. Weighardt, C. Feterowski, M. Veit, M. Rump, H. Wagner, and B. Holzmann Increased Resistance Against Acute Polymicrobial Sepsis in Mice Challenged with Immunostimulatory CpG Oligodeoxynucleotides Is Related to an Enhanced Innate Effector Cell Response J. Immunol., October 15, 2000; 165(8): 4537 - 4543. [Abstract] [Full Text] [PDF]
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G. B. Lipford, T. Sparwasser, S. Zimmermann, K. Heeg, and H. Wagner CpG-DNA-Mediated Transient Lymphadenopathy Is Associated with a State of Th1 Predisposition to Antigen-Driven Responses J. Immunol., August 1, 2000; 165(3): 1228 - 1235. [Abstract] [Full Text] [PDF]
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V. C. Dreisbach, S. Cowley, and K. L. Elkins Purified Lipopolysaccharide from Francisella tularensis Live Vaccine Strain (LVS) Induces Protective Immunity against LVS Infection That Requires B Cells and Gamma Interferon Infect. Immun., April 1, 2000; 68(4): 1988 - 1996. [Abstract] [Full Text] [PDF]
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J. Fensterle, L. Grode, J. Hess, and S. H. E. Kaufmann Effective DNA Vaccination Against Listeriosis by Prime/Boost Inoculation with the Gene Gun J. Immunol., October 15, 1999; 163(8): 4510 - 4518. [Abstract] [Full Text] [PDF]
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