Jpn. J. Infect. Dis., 55, 37‑44, 2002

Imyited Review

Discovery oHmmunostimulatory CpG‑DNA and its Application to
Tuberculosis Vaccine Development
Saburo Yamamotot *, Toshiko Yamamotol,2, Yasuhiro Nojimal , Kiyoko Umemori1,

Susan Phalen2, David N･ McMurray2, Etsuro Kuramoto3, Sumiko Iho4,
Rumiko Takauji4, Yukio Sato5, Takeshi Yamada6, Naoya Ohara6, Sohkichi Matsumoto7,
Yoshitaka Goto8, Kazuhiro Matsuo9 and Tohm TokunagalO
lNational Institute oflnfectious Diseases,
Gakuen 4‑7‑1, Musashimurayama, ITob70 208‑0011,
27Texas A & M Universio7 Health Science Center; College Station, Texas, USA,
3Mitsui Pharmaceuticals, Inc･, Togo, Mobara, Chiba 297‑001 7,
4Fukui Medical Universi07 School ofMidicine,
Matsuoka‑cho, Yoshida, Fukui 91011193,
5Fukushima Medical UniversiO) School ofMedicine,
Hikarigaoka, Fukushima 960‑ 1295,

6Nagasaki UniversioZ School ofDentisky,
Sakamoto, Nagasaki 852‑8523,

7osaka Cio, UniversiO, School ofMedicine,
Asahi, A beno‑ku, Osaka 545‑8585,

8Ml'yazaki UnivezISio?, Faculo; ofAgricuiture,
Gakuen Kibanadai Nishi, Miyazaki 889‑2 I 92,
9Japan Science and Technology Cooperation, AIDS Vaccine P710jec1 0jiPce,
Nonthaburi 11000, Thailand andl0Fukuoka Jo‑Gakuin UniversiO?,

Osa, Minami‑ku, FILkuoka 811‑1313, Japan(Received April 22, 2002)

CONTENTS:
L introduction
2. lmmunostimulatory sequences

3. Bacterial DNA, but not animal or plant DNA, possesses immunostimulatory activity
4. Antitumor activity of immunostimulatory CpG‑DNA
5. 7mmtmostimulatory activity of CpG‑DNA
6. IntraceHular mechanisms of immunostimulatory CpG‑DNA
7. 1mmtlnOStimulatory CpG‑DNA as immunoadjuvants for vaccine development
8･ Therapeutic applications of immunostimulatory CpG‑DNA

SUMMARY: DNA contalnlng an unmethylated CpG motif has a potent immunostimulatory effect on the vertebrate
immune system̲ Because such CpG motifs are relatively common in bacterial DNA, but rare in mammaliananimal and plant DNA,they may be an evolutionary adaptation augmenting innate immunity, most likely inresponse to pathogens that replicatewithin the host cells, such asviruses and intracellular bacteria. Microbial
infection induces innate immunity by trlggerlng Pattern‑recognition systems･ The infected cells produce

proinnammatory cytokines that directly combatmicrobialinvaders and express costimulating surface molecules,
which develop adaptive immunity by inducing distinct T cell differentiation. Bacterial DNA with unmethylated
CpG‑DNA stimulates vertebrate immature immune cells to induce maturation and to produce TNT‑α as well as

Thl ‑type cytokines, IL‑ 1 2 and lFN‑7. Therefore, CpG‑DNAfunctions as an adjuvant for regulating the initiationofTb 1 difFerentiation･ role roles ofimmunostimulatory CpG motifs in DNA vaccine developments andintherapeutic
applications have been discussed.

I. Introdtletion*Corresponding author: Tel: +8 i ‑42‑56 1 ‑0771, Fax: +8 i ‑42‑565‑

Mycobactfrium bovis BCG (BCG) has been used as a

33 1 5, E‑mail: saburo@nih.goJp

viable bactenal vaccine against tuberculosis since the 1920S.

In addition, BCG appears to elicit protective cell‑mediatedThisarticleisanJnvitedReviewbasedonalecttlrePreSentedat

immune responses and has greatly contributed to tumor

theHthSymposiumoftheNationallnstituteoflnfectious
Diseases,Tbkyo,21May2001.

immunotherapy (1‑6). Viable BCG has been thought to beeffective irl inducing tumor regression, and the trials have37

(uJul) q一LqOJC･(OuLnL

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4

6

8

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70

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Weeks Qiter tumor cel一 inocuIQtion

Fig. I. AntittlmOr eirects ofMY｣ , RNase digest of MY‑1, and DNase digest of MY‑I on Line‑10 guinea pig hepatoma･ Strain‑2
gulnea pigs Were intradermany lnOCulatedwith1 0(I Line‑ I 0 hepatoma cells lnthe lel川anksI Seven days aRer cell implantation,

ttlmOrS approximately 10 mm in diameter were palpable; and B; MY‑I, C; RNase‑digest of MY‑1, D; DNase‑digest of MY‑1 at
500 LLg m PBS oT A; PBS alone was repeatedly injected into the ttlmOr nodules every other day･ The animals were examined

for ttlmOr growth (Solid lines) and metastasis (dotted lines) twice a week as shown on the vert)cal axis･

cancer (7‑8). Many investigators have also attempted tofragment of the 3'‑terminus of BCG‑A4, is much less thanthat of BCG‑A4a. These results suggest that BCG‑A4 andisolate components of BCG possessing ant血mor activity andBCG‑A4a contain an active sequence. We pointed out that

producing few side effects. Tokunaga et al. flrSt demonstrated

hexamer palindromic sequence is the case and is essential

resulted in the development of BCG therapy agalnSt bladderthat BCG cytoplasm precipitated by streptomycin (SM‑ppt)

for immunostimulation of the cells augmenting the NK cells

Contains a substance that strongly inhibits mouse and guinea( 10). In order to detemine the minimal and essential sequences
responsible for the immunostimulatory activity ofODNs, theplg Syngeneic tumors･ Through alrther purification, a nucleic
acid‑rich fraction possessing Strong antitumor activity but

palindromic sequence, GACGTC, in an active 30‑mer ODN,

producing few adverse effects in experimental animals has been

5 '‑accgat GACGTC gccggt gacggc accac8‑3 '(BCG‑A4a) was

obtained (9‑10). This fraction is designated as MY‑i and is
98% nucleic acids (70% DNA, 28.0% RNA). DNA containedin MY‑1 appears to be essential to the antitumor actlVlty,
because the RNase‑digest of MY‑1 shows higher antittlmOr
activity than MY｣, while the DNase‑digest or MY‑1 has

replaced with each of the 6‑mer palindromic sequences･ Morethan lOO% relative activity was obseⅣed in the eight ODNs

with one of the followlng Palindromic sequences: AACGTT,
AGCGCT, ATCGAT, CGATCG, CGTACG, CGCGCG,
GCGCGC, and TCGCGA. All the potent palindFOmeSincluded

reduced activity (Fig･ 1 )I MY‑ I is uni.que because its component
is mostly nucleic acids and its actlVity is ascribed to DNA･

one. or more CpG motints)･ In contrast, palindromes composed

entirely ofadenine (A) and thymine (T) and thosewith a

No direct cytotoxiclty Of MY‑I has been observed, and it issuggested that the antitumor activity of MY‑l is due to its

unfavorable for the activity (12,13). Krieg et al. also noted

immunostimulatory activity.

the importance of CpG motif as the basic structtlre requiredsequence of either Pu‑Pu‑Pu or Py‑Py‑Py were generally

for B cell activation, and stated that CpG dunucleotide nanked
by two 5'purines (preferably a GpA dtlnuCleotide) and two

2. 1mmunostimulatory sequences

3'pyrimidines (preferably a TpT) is optional ( 14). Ballas etal. pointed out that palindromiC sequences are not always

MY‑I is distributed over a broad range ofmolecular size,

necessary for NK activation (I 5).

and its elution peak corresponds to 45 bases on gel filtration

SeqtlenCeS flanking hexamer palindromes are also

Polumn chromatography (1 1)･ To determine whether the

lmmunOStimtllatory activity of MY‑ 1 is sequence‑dependent,

meaningful. The NK‑augmentlng activity Of ODNs was

13 kinds of 45‑mer slngle‑stranded oligodeoxynucleotide

compared among fotlr types Of single‑stranded 30‑mer(ODN) were synthesized based on having sequence randomly
selectedfrom the known CDNA‑encoding BCG proteins.

home‑01igomers with a potent palindromic sequence, such

as AACGTT, CGATCG, GACGTC, or ATCGAT at the center

Interestingly, six of the 1 3 0DNs were found to induce strong

position (13). No activity was fわund in the hom0‑01igomers

immunostimulatory activity tO atlgment murine NK cellwithout a palindromicヲe9uePce･ Olig0‑guanylate (oligo‑G)

activity, while the others were not (1 I ). The activityofspleen

showed the highest actlVlty Irrespective of the palindromic

cells is elevated remarkably by BCG‑A4, One of the active

sequence included, but oligo‑adenylate (oligo‑A) and oligo‑

ODNs, while the cells incubatedwith BCG‑A2, One of thecytidylate (oligo‑C)with the palindrome produced only

inactive ODNs, show no significant change in activity. BCG‑

marginal activity･ This was also true of the hom0‑01igomers

A4a, a 30‑mer ODN with a sequence beginnlng什om the 5'‑

including the GACGTC palindrome. ln contrast, no activity wasterminus ofBCG‑A4, also activates NK cells as well as BCG‑

found in a 30‑mer oligo‑G containing an impotent palindrome･A4 (Table 1). The activity ofBCG‑A4b, which is a 30‑mer

These results indicate the independent and cooperative

38

Table 1. The sequence ofODNs used･a‑es

proteins

N慧:de

Base

sequencelmmuna:ts.I:;Jlatoq

BCG‑A1

327‑371

AACGAGGGGC ATGACCCGGT

GCGGGGCTTCTTGCACTCGG

CATAG

BCG‑A2694‑738AAAAGAAGTG GGGTGCCCCC

ACGATCACCA

ACGATGGTGT

GTCCA

TCC ATCGCC A AGGAGATCGA

GCTGGAGGATCCGTACGAGA

AGATC

813‑857

ACCGATGACG TCGCCGGTGACGGCACCACG

ACGGCCACCGTG CTG

BCG̲A4a (Antigen‑A) 813̲842

ACCGATGACG TCGCCGGTGACGGCACCACG

BCG ‑A3

BCG‑A47 3 5 ‑7 79

64KDaりGGTGACGGCACCACG

ACGGCCACCG

TGCTG

TATGCGGTTC GACAAGGGCTACATC TCGGG

GTACTTCGTG

ACCGA

1552‑1596

ACGAGACCAC CATCGTCGAG

GGCGCCGGTG

ACACCG ACGC

CATCG

BCG‑A7

1962‑2006

GCCGAGAAGG TGCGCAACCTGCCGGCTGG CC ACGGACTGA

ACGCT

BCG‑A82371‑2415

ACCGAGAACA GCCACGCAGT

CGTGTAG GCA

ACCTTTGGCC

GCTGT

BCG‑A4b828‑857

BCG‑A5

1 145‑1 1 89

BCG‑A6≡諾二"M三MPB702一
BCG‑α 1

三‑.4.5̲455

α‑Aniigenn

一一十+十一一十十一≡≡三言cA:CATcGGGT :GG冒cGTCGCTCGGGGTG喜TGGGCGGTCGGTGcfAA :AcCGGcCcGAGACcAGGc CGCAAcAGTG
348‑392

CGACTACAAC GGCTGGGATA TCAACACCCC GGCGTTCGAG TGGTA■I 64KDa: 64KDa heat shock protein (Antigen‑A), 243 Ibロ:I MPB70: MPB70 proteln, 492bp
3) a‑Antlgen, I 165bp

effects of the palindromic sequence and the extra‑palindromic

same conclusion was drawn

sequence on the activity of ODNs.
The effects ofdle numbers and locations of the palindromic
sequences were also investigated. Among the 30‑mer oligo‑
G nucleotides each contalnmg a Varylng number ofAACGTT

genes is methylated at the 5‑position on the cytosine rlmg,
and that this accounts for most of the methylcytosine in the

BiFd described that between 60 and 90% ofCpGinvertebrate

vertebrate genome (18)̲ His hypothesis may in part explain
the differences in the immunostimulatory activities among

palindromes, an ODN with one palindrome showed thestrongest activity. The ODN with AACGTT at the 5'‑end orDNAs &om different sources.

the 3'‑end showed slightly stronger activity than those with it

In 1994, we fわund that certain hexamer ODNs alone canin the center, althoughthe actlVlty Was more innuenced byinduce IFN when encapsulated in liposome (19). Using thisthe number of palindromes rather than by the locations.

liposome system, We leaned that methylation of the cytosine

The ability of immunostimulatory ODNs to induce IFN isofAACGTT sequence resulted in a significant decrease in its

also associatedwith their whole base length. Ten kinds of 1 2‑

activity (20). We also found that the treatment ofE. colt DNA

to 30‑mer ODNs having an AACGTT palindromic sequencewith CpG methylase reduced the IFN‑inducing activity with

were examined, and the ODNs of 18 0r more bases in lengtha lapse in incubation time, while the E. coli DNA treatedinduced the immunostimulatory activity proportionally to thewith buffer alone for the same period and under the samebase length, with a maximum at 22‑30 bases ( 16)･

conditions retained the immunostimulatory activity (S.
Yamamoto, unpublished data). Next, we synthesized a ヨo‑

mer ODN, 01igo‑B, 5'‑gggggg gggggg AACGTT gggggg

3. Bacterial DNA, but not animal or plant DNA,

gggggg‑3'sequence, and its analogue in which cytosine waspossesses immunostimtllatory activity

replaced with methylated cytosine; the methylated Oligo‑B

In 1984, we fotlnd that DNA‑rich fraction from various

gave only marginal activity in vitro, while the Oligo‑B showed

species of bacteria exhibitthe immunostimulatory activity

strong actlVlty. Further, antitumor effects of ODNs was

similar to that ofMYll, but that DNA extracted from calf

examined. 01igo‑B completely inhibited ttlmOr growth after

thymus and salmon testis does not (I 7). These results were

up to six repeated inoculations, while methylated Oligo‑B
did not. We also surveyed theincidence &equency of the potent

difficult to explain at that time. ln addition to the DNA kac‑tions tested thus far, 19 kinds ofDNA samples were found topalindromic sequences in some of the CDNA sequences ofI 7 species. The summedincidences of the potent palindromic

stimulate NK activityand to induce IFN production in mousespleen cells. Each of the DNA samples from Mycrococcus
lysodeikticaLS, Mycobacterium bovis BCG, Escherichia coit',
and Mycoplasma pneumoniae strongly augmented NK

sequences in all orthe CDNA sequences舟om vertebrates andplants were less than 1.0 in 1 ,000 base‑pal一s, While those舟om

most of the bacterium,viruses, and silkworm were larger thanactivity and induced IFN. The biological activity of the DNA
sample from CioStrZ.dL.um PerfrizlgenS Was relatively low, but

I.0. The incidence of particular 8‑, 1 0‑ or 1 2‑mer palindromic
sequences, showing stronger immunostimulatory activity than

was statistically significant. ln contrast, all of the DNA samples
from 1 0 different species of vertebrate, Salmon testis, herring

partictllar 6‑mer palindrome, however, was not taken into
account. These seqtlenCeS Ofpalindrome having an unmethylated

sperm, trout liver, chicken liver, frog liver, human placenta,

CpG core arewidely observed in DNAs not only from BCG,

calfthymus, mtlrine liver, rabbit liver, and porcine liver, andbtlt also舟om bacterium and invertebrate animals, and are rarely

kom four species of plant, rice, tomato, spinach, and parsley
exhibited no activity (Fig. 2). Although the data is not shown
here, we tested several other DNA samples･ All of thepresent in vertebrate and plant DNAs. Therefore, palindromic
sequenceswith CpG motifs are foreign DNAsfor mammalian

DNA from virtlSeS and vertebrates as well as bacterium

why ODNs with these sequences exhibit immunogenicity inexhibited inmunostimulatory activi ty, b上lt that舟om vertebrates,

murine and human immtlne Systems.

immtlnOCOmPetent Cells, and this may be one of the reasons

including fish and mammals, showing no activity, and the39

Mbov/s BCG
E co/('
a /ysode肋us

M pneumonJbe
αperfnhgens

salmon testis
he汀ing sperm
trout liver

chicken ljver
frog liverhuman placenta
caげthymus

murine liver

rabbk liver
porcine liver
rice

tomatospinachparsley

medium
40

30

20

10

0

NK ACTJVITY (%)

20

40

60

80

100

120

140

JFN TITER (lU/mJ)

Fig. 2. Augmentabon ofNK activity and IFN induction by the DNA samples舟077】 VanOuS Sources. Mouse spleen cells (1 × 107/m))

were incubatedwith 10 LLg/ml of each of the DNA samples for 20 h, and centrialged･ The cell fiactions were used for NK
assay, and the supematants fわr IFN assay･

4. AntittlmOr activity of immtmostimlllatory CpG‑DNA●α1

The antitumor activity of each ODN was examined. A cellsuspension of lMC tumor cells was mixed,with each test▲

吉r^OJ6Jo∈コこOuO!l!q三ut
8 6 4 02 0 0 0.A3 /A4Aa4

sample contalnlng 100 leg Ofthe ODN, and themixtures

BCGIDNA

were intrademally inoculated into (BALB/c x DBA/2)Fl..!6A7(CDF I )mice.Asa control, saline alone instead of test sample
was used. At 35 days after inoculation, allmice were kiued

M3

and the tumors were resected for weighing.When tumor cells
weremixedwithBCG‑A4, tumor growth was markedly
suppressed, but BCG‑A2 did not significantly affect the tumor
growth (21)̲ Next, the effects of repeated injections of the
ODNs into established ttlmOrS Were examined. IMC ttlmOrcells (5 × 105) were inoculated intradermally into CDF lmice.
(U

The ODN test samples (loo leg each) dissolved in O･l ml ofsaline were lnjeCted into the tumor lesion twice a week舟om

the 5th day of tumor inoculation. At 35 days after the tumor

甘ん

義.1｡併‑一計

NK eell activity (㌔)inoculation, all the mice were killed and the tumors were

Fig. 3. Correlation between antlttlmOr act)Vlty and NK‑augment)ng
activity induced by ODNs. AntittlmOT activity ofODNs was assessed

resected fb∫ weighingJt was fわund that six of the 1 1 ODNs

tested, i.e., BCG‑A3, BCG‑A4, BCG‑A6, BCG‑A7, BCG‑

as the growth lnhibitlOn OfIMC tumors in CDFlmiee･ MotlSe Spleen

M3, and BCG‑α 1 , sign捕cant)yinhibited tumor growth, whilecells were incubated wLth or without ODN (50 LLg/m)) for 20 h, andthe others did not (Fig‑ 3). The antitumor activity of thethe cells were assayed for NK activity,

active ODNs co汀elated weH with the NK‑augmenting and

IFN‑inducing activity (2 1 ). lt is noteworthy that the inactive

ODNs, BCG‑Al, BCG‑A2, BCG‑A5, and BCG‑A8, do notcontain a hexameric palindrome, while the active ODNs do

phosphate‑buffered saline (PBS) alone, tumors grew progres‑sively; on the 35th day, the mean tumor weight was 3.82 j=possess stlCh a sequence, I.e., GACGTC, GGCGCC, or

I.55 g‑ ln the groups given a DNA fraction from either of the

TGCGCA. The exceptlOn is that an inactive ODN BCG‑M I,
contains an overlapping palindromic sequence ( GGGCCCGGG).
To test the invivo effects of the bacterial DNA fraction,

bacteria species, tumor growth was significantly inhibited.

mice bearing lntradermaHMC tumors were treatedwith

0.45 g, with 90% growth inhibition. Seven of eight mice were
cured in this group. The DNA fraction from all of the other

For instance, the mean tumor weight in the mice glVen the

DNA fraction from Slreptomyces aureofaciens was 0.39 j=

intralesional injections of the DNA &action. 7n the group glVen

40

bacteria was also effective and showed more than 85% tumor‑

for IFN or other cytokine induction, or whether the ODNs

growth inhibition. On the other hand, little effect was observedin the DNA &aetion &om the vertebrate cells. The DNA &ac‑

are Just trapped with the receptor and tbell taken up bytion from salmon testis showed moderate tumor‑growth

nuclear apparatus･ Several studies have demonstrated that

inhibition, but no mouse was cured. Although data are notmodified low density lipoprotein (LDL) receptors, also called

endocytosis mechanisms to reactwith the cytoplasmic orshown here, the bacterial DNA丘action digested with DNase

scavenger receptors, are expressed on macrophages (26)･

completely lost the ant血mor activity; digestion with RNase

Oxidized LDL is rapidly taken up by cultured macrophages

had no innuence.

via scavenger receptor‑mediated endocytosis, resulting lnfoam‑cell formation. Scavenger receptors bind not only

modiaed LDL, but also various negatively charged compounds.

5. Immunostimulatory activity of CpG‑DNA

We have recently found that the binding of some ODNs to

Profile ofcytokine production by mouse BM cells stimulated

macrophages is inhibited by the presence of antagonists forwith CpG‑DNA was studied･ BALB/c BM Cells wereinctlbatedthe scavenger receptor, and also that acetyl‑LDL binding towith 50 LLg/miCpG‑DNA at a concentration of 1 X 107 cells/

the scavenger receptor on mouse macropbages is inhibited by

ml for 20‑24 h in a 24‑well plate. The culture supematants

仙is ODN (27). We also obseⅣed that mouse bone ma汀OW Cells

were haⅣested, and the cytokines were measured by the&om scavenger‑receptor knockout animals can be stimulated

ELISA technique. Both AAC‑30 and Oligo‑B induced IL‑1 α ,with CpG‑ODNs. These results suggest that scavenger

lFN‑γ, IL6, and TNF‑α to a similar extent, however,production of them. To confirm which cytokines contribute toreceptors are required for the binding of the ODN to cells,
but not for the immunostimulatory activity of the ODN. The
immunostimulatory efFects of CpG ODN do not appear toaugmentation of NK activity in mouse BM cells a洗er stimula‑

be mediated through a cell‑surface receptor but rather withmethylation of cytosine in the CpG motif partially reduced

tion with ODNs, the anti‑cytokiTle antibodies were added,

specific, as yet unidentified, intracellular binding pfOteins･

respectively, to the cultures ofBM cellswith ODNs. Mono‑clonal antibodies against mouse lL6, iL‑12, TNF‑a, andThereafter, a number of intracellular signaling pathways
are activated, resulting ln Sequence‑specific generation of

IFN‑T and polyclonalantibodies agalnSt mouse lFN‑α

intracellular reactive oxygen species (ROS), the activation

and IL‑18 Were tested. The treatment of cultures with anti‑

ofNF‑FC B, andthe induction of MAP kinases･ There is a rapid

TNF‑α, anti‑ill 8 or anti‑IFN‑γ antibody significantly

enhancement of nuclear transcription (15 to 30 min) aRer

reduced the NK activity, while that with anti‑lL12 or anti‑

exposure to CpG ODN, with the subsequent expression of a

IFN‑α did not･ Thetype ofIFN produced by mouse BM cells

large number of early‑response genes, prot0‑oncogenes, andstimulated with ODNs was detemined by the netltralizing
effect ofanti‑IFN antibodies on the bioassay of lFN, and by

predominantly Thトpromotlng CytOkines. More recently, lt

EuSA technique.Asa result, a large amount ofIFN‑a andhas been reported that the TolHike receptor (TLR) family is
responsible for recognlt10n Ofmicrobial cell wall components.

a small amount ofIFN‑β were found in the culture恥id of

The involvement ofTLR9 in the recognltlOn Ofunmethylatedmouse BM cells. Anti‑IFN‑γ antibody had no in恥ence on

CpG‑DNA has been shownthrough the generation ofTLR9lFN levels; significant levels of IFN‑ r were, however, also

knockoutmice (28). We havealso confirmedthatthe immtno‑

detected in the ctllttlre nuid. ln order to clarifythe cell typestimulatory ODNs fail to induce IFN and augment NK Cells

targeted by immmostimulatory CpG‑DNA for lFN‑ α produc‑

after inctlbation with mouse BM cells or spleen cells &om

tion, We separated human PBC by the magnetic cell‑sorting

TLR9 knockout mice. However, it remains unknown bow

method, and stimtllated each subset with potent ODNs, show‑
ing thaHhe CD31CD64‑CD4dim cells produce a large amountoflFN‑a. A fraction of the blood dendritic cells, the CD3‑
CDl lb‑CD16‑CD4+ cells,also were found to produce a large
amount of IFN‑a in response to the ODNs, suggesting that

TLRs recognize invading pathogens under physiologicalimmature cells in the monocyte/dendritic cell lineage ofof IFN regulatory factor (IRF)‑7, and also mentioned thathuman peripheral blood mononuclear cell (PBMC) produce

CpG‑DNA induces p38 MAPK‑dependent phosphorylation

IFN‑α in response to immtlnOStimtllatory CpG‑DNA

ofSTATl in a manner independent oflFN‑α/β, which may(Yamamoto, T. et al., unptlblished data). More recently, theresult in ISGF3 formation increaslng the transcrlPt10n Of the

CD4+CD3‑CD 1 1 c‑type2 dendritic cell precursors (pre‑DC2)
known as plasmacytoid DC precursors (PDC) (22) were

IRF‑7 gene, leading to 7FN‑α production in hlman PDC (30).

conditionsI Takeshita et all reported that TLR9 and CpG ODN
are eolocalized in the same endosomes (29). Takauji, R. et al.
demonstrated that unmethylated CpG‑DNA induced IFN‑α

production in PDC is associated with the enhanced expression

ident捕ed as natural IFN‑producing cells in response to

7･ Immunostimulatory CpG‑DNA as immuno‑

viruses, bacteria, and tumor cells (23). Kadowaki et aL also

adjuvants for vaeeilte development

reported that irrmunostimulatory CpG‑DNA prodtlCeS IFN‑α(24)･ Baller et al. demonstrated that CpG‑DNA promotes thelt is generally known that vaccines consisting Ofviable,

survival and maturation ofCDl lc‑CD123+ PDC (25), butattenuated bacterium or virus can induce s打ong and persistent

the mechanisms by which CpG‑DNA induces IFN‑α andimmtmity, but evoke relatively severe adverse effects. ln

stimulates the immune system remain unknown.

contrast, killed vaccines a托relatively safe and induce antibody

responses, btlt hardly ever induce cell‑mediated immunlty.

Efforts have been made to devise effective and safe immuno‑

6･ ⅠれtraCelllllar mechanisms of immllnOSiimulatory

adjuvants for vaccines, includingmicrobial components,

Cp G‑DNA

recombinant proteins thereof, and synthetic peptides. However,

It is well known that DNA binds to the mammalian cell

Only aluminum salts and liposomes have been approved for use

ship. However, it has not been knownwhether the nucleotide

in humans･ Recently, lmmunizationwith antigen‑encoding
plasmid DNA has been found to effectively induce immune

receptor complexes on the cell surface mediate the signal

responses in the encoded fわreign antlgenS. The numbers ofsu血ce in a manner COnSistent wi血a ligand‑receptor relation‑

41

entlmeration ofM･ tuberculosis. The levels of protection wererevealed as the mean logl. CFU ofM. tuberculosis recovered

plasmids encoding immunogens of bacteria, viruses, parasites,
and ttlmOrS areワow under investigation (3 1‑36)･ Many ofthese Can induce Immunity against infection, and clinical hials&om the right lower lung lobes or spleens of guinea pigs

for protection or treatment agalnSt infection of HIV, herpes,

vaccinatedwith either plasmid or BCG. ln the case of theinnuenza, or hepatitis B viruses and against Careinoma are in

DNA vaccines, CpG moをifs in the plasmids may play anprogress i

important role in immtmostimulatory acdvity as anintramolecular

In 1996, Sato et al. reported that plasmid vectors expresslng

adjuvant. 1mmunizationwith the DNA vaccines contalnlng

large amounts of gene product do not necessarily induce

CpG motifs and encoding the mycobacterial protein antlgenS

immune responses in encoded antigens, but those contalnlng

elicited protective responses in comparison with a vector

control or saline. BCG resulted in a much higher level ofshort immunostimulatory ODN do (37); an effective plasmid
was fotmd to conmin two repeats of the palindromic CPG
hexamer 5 '‑AACGTT‑3 'Jmmunostimtllatory ODN was also

protection against myCObacterial infection than the DNA
vaccines. BCG has recently been questioned regarding its ability

reported to stimulate Th 1 respopses (38)･ Many subsequent

to protect against adtllt pulmonary tuberculosis. However,studies showed that immunostlmulatory ODNwithin theplasmid backbones contributes by stimulating an immune

may be tlSefu1 for a booster ofanti‑tuberculosis immunity.

immunizationwith DNA vaccines following BCG vaccination

response in the antigens encoded by the plasmids (39‑44)･

We also examined DNA vaccines including genes encoding

S･ Therapetltic applications of immllmOStimtIIatory

myeobacterialprotein antigens for their ability to preventthe

C pG‑DNA

growth ofbacilli in the lungs and spleens of guinea pigs after

Inmunostimulatory ODN stimulates immune responses topulmonary challenge of virulent M･ tubeyICulosis. Each gene
was inserted into the PACB plasmid舟om cytomegalovinlS

coadministered antigens, for instance, 8‑galactosidase andand expressed under the control of the promoter, followed

HBs antigen (46), the idiotype of the surface lgM of a B

by a polyadenylation site from the bovine growth hormone

lymphoma (47), intracellular pathog?ns such as Listやagene (BGH) (Fig･ 4)･ Incorporation of the intron A andmonotylogenesis (48), Or formalin‑inactlVated inntlenZa virus

polyadenylation site into the plasmidallowed for posttranscnp‑
tional processingwithin the eukaryotic host. Guinea pigs (5animals/group) were immtmized intramuscularly three times(49)･ One of the greatest advantages of immpnostimulatory
ODN as an immunoadjuvant isthat immunostlmulatory ODN

in both quadriceps, at 3‑week intervals. At each vaccination,

the coadministered antigen or to that encoded by the plasmid.

guinea Pigs Were given loo Ltg/qlladriceps of each DNA
vaccine. Negative control animals were Injected with salineor control plasmid vector DNA which lacked a gene insert

foster Thl responses and enhance celトmediated immunity. It

stimulates generation of a Th 1 ‑biased immune response to

It promotes IFN‑ a/B , IFN‑ T, lL‑12, and IL‑18, all of which
is well known that a Thl immune response conveys protec‑

intradermally,with M. bovis BCG Tokyo at a concentration

tion against infectionwith my.Cobacterial infection (50‑5 1 )･
It was reported that immunostlmulatory CpG‑DNAsprotectof 103 bacilli on the first day of the immunization. Six weeks

against infection eitherwith M̲ tuberculosis (52) or M. avium

a免er the third immunizationwith the plasmids, guinea PISS

(53). Because immunostimulatory ODN is known to inducein saline. The positive control group was Injected once,

were infectedwith M･ t乙Lberculosis bythe ptllmonary route

IL12 and TNF‑α, attempts were made to丘nd oligonucleotide

with a low dose (5‑10 CFU) ofM. tuberculosis H37Rv in asequences that differentially activate IL I 2 versus TNF‑aspecial aerosol chamber onglnaHy designed by Dr. Donald

production in antlgen‑preSentlng Cells, and a single‑stranded

W. Smith and colleagues at the University of Wisconsin,

1 8‑mer ODN contalnlng a hexamer ofGACGTT was proposed

Wis., USA, to deliver droplet ntlClei directly to the alveolar

as a prototype for a useRll 0DN without harm魚ll side effects

spaces (45). Five weeks after infection, the animals were(54).

euthanized and their spleens and lungs were obtained for

The dominance of the Th I response generated by immuno‑
stimulatory CpGIDNA may be useful in treating Th2‑typeallergic diseases (48,55). Kline etal. observed that coadministra‑

HI'ndHltion of immunostimulatory CpG‑DNA and Th2‑type response
inducing antigen Can redirect the immune response to Thl‑type and abolish allergic reactions on subsequent antigen

challenge (56). MY‑I was also examined as a treatment ofallergic disease due to its a downregulation of lgE produc‑tion (57).

We also reviewed the discovery or immunostimulatory
DNA and its clinical studies (58).

ACKNOWLEDGMENTS
This work was partly supported by grants &om the Ministryof Health, Labour and Welfare and from the Japan Health
Sciences Foundation

We express our deepest sorrow in the passing OfDr. Tetsuro
Kataoka, who had been a collaborator engaged in CpG‑DNA
Fig, 4. Map ofplasmld PACB. DNA‑encoding mycobacterial secreted

studies and who died on December 6, 2000.

proteln antlgenS Were Cloned into a eukaryotic expression vector.

The authors would also like to thank S. Toyoo for her

Each gene was inserted into the PACB plasmid舟om cytomeg<llovirtlSsecretarial assistance.

(CMV) and expressed under the control of the promoterjollowed
ty polyadenylation by bovine growth hormone gene ( BGH H25).

42

CpG motifs in oligodeoxynucleotides and bacterial DNA.

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