INFECTION AND IMMUNITY, Feb. 2002, p. 672–678 Vol. 70, No. 2
0019-9567/02/$04.00⫹0 DOI: 10.1128/IAI.70.2.672–678.2002
Copy igh © 2002, Ame ican Socie y o Mic obiology. All Righ s Rese ed.
Failu e o he Mycobac e ium bo is BCG Vaccine: Some Species o
En i onmen al Mycobac e ia Block Mul iplica ion o BCG and
Induc ion o P o ec i e Immuni y o Tube culosis
Lise B and ,
1
† Joana Feino Cunha,
2
Anja Wein eich Olsen,
1
Ben Chilima,
3,4
Penny Hi sch,
4
Rui Appelbe g,
2
and Pe e Ande sen
1
*
Depa men o TB Immunology, S a ens Se um Ins i u , Copenhagen, Denma k
1
; Labo a o y o Mic obiology and Immunology
o In ec ion, Ins i u e o Molecula and Cell Biology, Uni e si y o Po o, Po ugal
2
; and Depa men o In ec ious
and T opical Diseases, London School o Hygiene and T opical Medicine, London,
3
and Depa men o
Soil Science, Ins i u e o A able C ops Resea ch-Ro hams ed, He o dshi e,
4
Uni ed Kingdom
Recei ed 15 Augus 2001/Re u ned o modi ica ion 5 Oc obe 2001/Accep ed 7 No embe 2001
The e icacy o Mycobac e ium bo is bacillus Calme e-Gué in (BCG) accine agains pulmona y ube culosis
(TB) a ies eno mously in di e en popula ions. The p e ailing hypo hesis a ibu es his a ia ion o in e -
ac ions be ween he accine and mycobac e ia common in he en i onmen , bu he p ecise mechanism has so
a no been cla i ied. Ou s udy demons a es ha p io exposu e o li e en i onmen al mycobac e ia can
esul in a b oad immune esponse ha is ecalled apidly a e BCG accina ion and con ols he mul ipli-
ca ion o he accine. In hese sensi ized mice, BCG elici s only a ansien immune esponse wi h a low
equency o mycobac e ium-speci ic cells and no p o ec i e immuni y agains TB. In con as , he e icacy o
TB subuni accines was una ec ed by p io exposu e o en i onmen al mycobac e ia. Six di e en isola es
om soil and spu um samples om Ka onga dis ic in No he n Malawi (a egion in which BCG accina ion
has no e ec agains pulmona y TB) we e in es iga ed in he mouse model, and wo s ains o he Mycobac-
e ium a ium complex we e ound o block BCG ac i i y comple ely.
Tube culosis (TB) is one o he mos p e alen causes o
dea h om in ec ious diseases in he wo ld. As is he case o
many in acellula pa hogens, cell-media ed immuni y plays an
impo an ole in hos p o ec ion agains TB (25, 29). In pa -
icula , gamma in e e on (IFN-␥)-sec e ing T cells ha e been
shown o be impo an o he p o ec i e immune esponse
(17). The only accine cu en ly a ailable agains TB is he
a enua ed Mycobac e ium bo is s ain bacillus Calme e-
Gué in (BCG). The e icacy o his accine a ies om 0 o
80% in di e en popula ions, wi h a consis en ly low e icacy in
many opical egions o he wo ld whe e he accine is mos
needed (15, 16, 35, 38). The eason o he ailu e o BCG in
some popula ions has been a subjec o deba e since he 1950s,
and many di e en hypo heses ha e been sugges ed o explain
he obse ed a ia ion. Some in es iga o s ha e sugges ed ha
di e ences in he s ain o BCG (23), he age a accina ion
(40), o me hodological di e ences a e impo an ac o s o
he a ia ion epo ed (8). The mos widely accep ed hypo h-
esis ela es he e icacy o BCG o geog aphic loca ion, wi h
low o nonde ec able le els o p o ec ion agains pulmona y
TB seen in opical egions such as A ica and India, whe e
exposu e o non ube culous mycobac e ia is common (15).
One excep ion om his gene al ule is he consis en high
e icacy when BCG is used o accina e newbo ns. Neona al
accina ion wi h BCG impa s p o ec ion agains he child-
hood mani es a ions o TB (in pa icula , meningi is) (1, 9, 24),
bu he e icacy wanes o e a pe iod o 10 o 15 yea s, and
he e o e i does no p e en agains he la e b eakdown wi h
pulmona y TB in he adul popula ion in he hi d wo ld (37).
The e is con incing e idence ha exposu e o labo a o y
animals o en i onmen al mycobac e ia can p o ide some p o-
ec ion agains in ec ion wi h M. ube culosis (7, 14, 20, 30, 33).
The in luence o such c oss p o ec ion on he e icacy o sub-
sequen BCG accina ion is no ye cla i ied, bu based on
animal expe imen s, i has been sugges ed ha he p o ec ion
p o ided by en i onmen al mycobac e ia may pa ly mask he
e ec o a subsequen BCG accina ion (33, 42) o ha en i-
onmen al mycobac e ia ha e a di ec an agonis ic in luence
on subsequen BCG accina ion (34, 36). Ou s udy demon-
s a es ha p io sensi iza ion wi h en i onmen al mycobac e-
ia can inhibi BCG mul iplica ion and he eby p e en he
induc ion o an e icien BCG-media ed immune esponse and
p o ec ion agains TB challenge. In e es ingly, di e en species
isola ed om soil and spu um in Ka onga, Malawi, an a ea in
which BCG has been shown o p o ide no p o ec ion agains
TB (22), di e ed in hei abili y o inhibi BCG mul iplica ion.
In con as , a TB subuni accine had he same p o ec i e
e ec in nai e and sensi ized animals.
MATERIALS AND METHODS
Animals. These s udies we e pe o med wi h pa hogen- ee 6- o 12-week-old
CBA/J and C57BL/6J emale mice, pu chased om Bomhol egaa d, Ry, Den-
ma k, o , in some o he expe imen s, pu chased om Ha lan UK, L d., Bel on,
England, o Ha lan In e auna Ibé ica, Ba celona, Spain.
Bac e ia. Mycobac e ium a ium (ATCC 15769), Mycobac e ium sc o ulaceum
(ATCC 19275), and Mycobac e ium accae (ATCC 15483) we e g own in 7H9 b o h
un il he mid-log phase o he bac e ial g ow h. Mycobac e ium ube culosis (Edman)
was g own a 37°C on Löwens ein-Jensen medium o in suspension in modi ied
Sau on medium en iched wi h 0.5% sodium py u a e and 0.5% glucose. In p epa-
* Co esponding au ho . Mailing add ess: S a ens Se um Ins i u ,
Depa men o TB Immunology, A ille i ej 5, 2300 Copenhagen S,
Denma k. Phone: 45 32683480. Fax: 45 32683035. E-mail: [email p o ec ed].
† P esen add ess: Depa men o Mic obiology, Colo ado S a e
Uni e si y, Fo Collins, CO 80523..
672
a ion o immuniza ion o mice, ozen aliquo s o he bac e ial s ains we e hawed
and sonica ed o 5 min wi h a B anson 2210 ul asoni ie , and he iabili y o each
s ain was enume a ed on 7H11 pla es. Mycobac e ium o ui um (S78/2) and M.
o ui um (S160/5) a e soil isola es om he no h and sou h o Ka onga, Malawi,
espec i ely. We used s anda d decon amina ion o samples wi h 4% sodium hy-
d oxide and cul u e a 37°C on nu ien aga -based medium o isola e he o ganisms
om soil. M. o ui um (Sp2001), Mycobac e ium chelonae (Sp2015), and wo s ains
o he M. a ium complex (Sp1891) and (Sp2011) we e spu um isola es om dono s
wi h suspec ed TB in he Ka onga dis ic . The o ganisms we e isola ed wi h acidi-
ied Löwens ein-Jensen medium in Malawi, and hei iden i y was con i med a he
Mycobac e ium Re e ence Uni , Dulwich, Uni ed Kingdom, by s anda d biochem-
ical iden i ica ion es s o mycobac e ia. F ozen aliquo s o hese s ains we e p e-
pa ed o animal inocula ion as desc ibed abo e.
Sensi iza ion wi h en i onmen al mycobac e ia. Mice we e immunized subcu-
aneously (s.c.) in he back h ee imes a 2-week in e als wi h 2 ⫻10
6
CFU o each
o h ee ATCC s ains o en i onmen al mycobac e ia (M. a ium,M. sc o ulaceum,
and M. accae). To clea he emaining mycobac e ia, sensi iza ion was ollowed, 3
weeks a e he las inocula ion, by 1 mon h o ea men wi h i ampin (Sigma; 100
mg/li e ), e hambu ol (Sigma; 200 mg/li e ), and cla i h omycin (Abbo Labo a o-
ies, Solna, Sweden; 200 mg/li e ) added o he d inking wa e .
To assess i ulence o he s ains isola ed om Ka onga, Malawi, mice we e
in ec ed wi h 10
5
CFU o each en i onmen al mycobac e ial s ain in a olume
o 0.2 ml o phospha e-bu e ed saline by in a enous (i. .) injec ion ia a la e al
ail ein. A he app op ia e ime poin s, mice we e killed ( ou in each g oup),
and he o gans we e emo ed o bac e ial enume a ion. Whole o gans we e
homogenized in a 0.04% Tween 80 (Sigma) solu ion in dis illed wa e , se ial
10- old dilu ions we e pla ed on Middleb ook 7H10 medium a 37°C, and he
numbe s o CFU we e de e mined.
Vaccina ions. A single dose o BCG Danish 1331 (5 ⫻10
4
CFU) was injec ed
s.c. a he base o he ail. The e we e no signi ican di e ences in he p o ec ion
ob ained wi h doses anging om 5 ⫻10
4
o 10
7
BCG bac e ia ( esul s no
shown). In one expe imen , an i. dose o 5 ⫻10
6
CFU o BCG was used o
de e mine g ow h o BCG in nai e e sus sensi ized mice. Fo subuni accina-
ion, he mice we e immunized s.c. h ee imes a 2-week in e als wi h 10 ␮g
(pe dose) o ei he ESAT-6 o he Ag85B–ESAT-6 usion p o ein emulsi ied in
dioc adecylammonium b omide (DDA; 250 ␮g/dose; Eas man Kodak, Inc.,
Roches e , N.Y.) plus 25 ␮g o monophospho yl lipid A (MPL; Co ixa, Hamil-
on, Mon .) as desc ibed ecen ly (5).
M. ube culosis in ec ions. Animals we e in ec ed wi h app oxima ely 100 CFU
o M. ube culosis (Edman) pe lung by he ae osol ou e in a Glas-Col inhala ion
exposu e sys em. The mice we e sac i iced 6 weeks a e in ec ion, and bac e ial
numbe s in he lung and spleen we e de e mined as desc ibed be o e (5).
The p o ec i e e ec o BCG o subuni accina ion was exp essed as he log
10
educ ion o he bac e ial coun s compa ed o ha in he un accina ed con ol
mice. All esul s a e based on i e o six animals pe g oup.
Mycobac e ial an igens. A c ude BCG an igen p epa a ion (BCG Ag) was
p oduced as an ammonium sul a e-p ecipi a ed cul u e il a e om cul u es a
week 6 as desc ibed in e e ence 2. In one o he expe imen s (see Fig. 4), he
BCG esponses o an ammonium sul a e-p ecipi a ed ex ac o he cell wall we e
measu ed as desc ibed elsewhe e (31). These wo p epa a ions we e ound o
gi e simila esponses in i o.
P o ein concen a ion was quan i ied by he Mic o bicinchoninic acid me hod
(Pie ce, Rock o d, Ill.).
Recombinan ESAT-6 was p oduced as desc ibed p e iously (18). The LPS
con en was below 0.3 ng/␮g o p o ein and had no in luence on cellula ac i i y.
The usion p o ein Ag85B–ESAT-6 was p oduced as desc ibed ecen ly (26). The
p o eins we e kep a ⫺80°C un il use.
Lymphocy e cul u es. Lymphocy es om spleens and blood we e isola ed and
cul u ed as desc ibed p e iously (5). B ie ly, cells om indi idual mice we e
cul u ed in mic o i e wells (96 well; Nunc, Roskilde, Denma k) con aining 2 ⫻
10
5
cells in a olume o 200 ␮l o RPMI 1640 supplemen ed wi h 5 ⫻10
⫺5
M
2-me cap oe hanol, penicillin-s ep omycin, 1 mM glu amine, and 5% ( ol/ ol)
e al cal se um. Based on p e ious dose- esponse in es iga ions, BCG Ag and
ESAT-6 we e each used a 5 ␮g/ml in he cul u es. Phy ohemagglu inin a a
concen a ion o 1 ␮g/ml was used in all expe imen s as a posi i e con ol o cell
iabili y. IFN-␥, in e leukin 4 (IL-4), and IL-5 we e de ec ed in 72-h cul u e
supe na an s by duplica e enzyme-linked immunoso ben assay (ELISA).
Enzyme-linked immunospo (ELISPOT) analyses we e conduc ed wi h cells
om indi idual mice o , when blood was analyzed, wi h cells pooled om g oups
o mice, as desc ibed in e e ence 6. The de ec ion le el was 10 spo s.
S a is ical me hods. Because all o he da a show a no mal dis ibu ion, he
assessmen o expe imen s was ca ied ou by analysis o a iance. Di e ences
be ween means we e assessed by Dunne ’s es (Tables 1 and 2) o S uden ’s
es (see Fig. 2 and 4). A P alue o ⬍0.05 was conside ed signi ican .
RESULTS
The mul iplica ion o BCG is inhibi ed in mice sensi ized
wi h ce ain en i onmen al mycobac e ia. We inocula ed
CBA/J mice s.c. h ee imes a 2-week in e als wi h a mix u e
TABLE 1. Sensi iza ion wi h en i onmen al mycobac e ia
blocks he p o ec i e e ec o BCG
G oup o mice
a
Resul in
b
:
Spleen Lung
CFU
c
Log
10
esis ance
d
CFU Log
10
esis ance
Nai e 4.44 ⫾0.13 6.34 ⫾0.11
BCG 3.76 ⫾0.16 0.68* 5.21 ⫾0.08 1.13*
Sensi iza ion 4.36 ⫾0.17 0.08 6.14 ⫾0.11 0.20
Sensi iza ion ⫹BCG 4.33 ⫾0.17 0.11 6.25 ⫾0.06 0.09
a
Nai e o sensi ized mice we e BCG accina ed (5 ⫻10
4
CFU) ollowed by
ae osol challenge wi h i ulen M. ube culosis.
b
The expe imen was epea ed wice wi h simila esul s.
c
Bac e ial numbe s de e mined by g ow h o indi idual whole-o gan homog-
ena es 6 weeks pos in ec ion.
d
P o ec i e e ec exp essed as he log
10
educ ion in bac e ial loads compa ed
o hose o nai e mice. Bac e ial numbe s signi ican ly di e en (P⬍0.05) om
hose seen in nai e mice a e indica ed by an as e isk.
TABLE 2. Bac e ial numbe s in o gans o nai e and sensi ized
mice a e accina ion and ae osol challenge
wi h i ulen M. ube culosis
Vaccine g oup
a
Resul in:
Lung Spleen
Log
10
CFU
b
Log
10
esis ance
c
Log
10
CFU
Log
10
esis ance
Exp 1
Nai e
Con ol 6.36 ⫾0.08 4.71 ⫾0.05
BCG 5.83 ⫾0.06 0.53* 4.12 ⫾0.12 0.59*
DDA-MPL 6.34 ⫾0.09 4.94 ⫾0.12
ESAT-6 5.76 ⫾0.09 0.60* 4.39 ⫾0.09 0.32*
Sensi ized
Con ol 6.18 ⫾0.08 4.82 ⫾0.16
BCG 6.27 ⫾0.07 ⬍0.05 4.79 ⫾0.05 ⬍0.05
DDA-MPL 6.39 ⫾0.05 4.73 ⫾0.11
ESAT-6 5.74 ⫾0.16 0.44* 4.43 ⫾0.05 0.39
Exp 2
Nai e
Con ol 6.88 ⫾0.12 5.10 ⫾0.18
DDA-MPL 7.19 ⫾0.05 5.48 ⫾0.11
Ag85B–ESAT-6 6.03 ⫾0.12 0.85* 4.40 ⫾0.08 0.70*
Sensi ized
Con ol 6.30 ⫾0.08 4.39 ⫾0.09
DDA-MPL 6.49 ⫾0.05 4.27 ⫾0.08
Ag85B–ESAT-6 5.37 ⫾0.14 0.93* 3.89 ⫾0.07 0.50*
a
Nai e o sensi ized mice we e immunized s.c. wi h BCG o injec ed h ee
imes wi h a subuni accine emulsi ied in DDA-MPL.
b
Bac e ial numbe s a e gi en as log
10
CFU o M. ube culosis isola ed om he
lung and spleen 6 weeks a e ae osol challenge wi h i ulen M. ube culosis.
c
P o ec i e e ec s o he wo accines a e exp essed as log
10
educ ions in
bac e ial numbe s compa ed o hose in un accina ed con ol mice. Bac e ial
numbe s signi ican ly di e en om hose seen in con ol mice a e indica ed by
an as e isk.
VOL. 70, 2002 INTERACTIONS OF BCG WITH ENVIRONMENTAL MYCOBACTERIA 673
o he mycobac e ial s ains M. a ium, M. sc o ulaceum, and M.
accae. These species ha e epea edly been isola ed om soil
and wa e samples in opical egions (21). Th ee weeks pos i-
nocula ion, a low bu signi ican mycobac e ium-speci ic ecall
esponse was measu ed in he spleen, wi h de ec able le els o
IFN-␥ elease in esponse o BCG Ag. (1.26 ⫾0.01 ng/ml)
(da a no shown). The BCG Ag p epa a ion ga e no IFN-␥
elease (⬍0.05 ng/ml) om splenocy es isola ed om nai e
mice. No IL-4 o IL-5 was de ec ed in any o he supe na an s.
Th ee weeks a e he las inocula ion wi h en i onmen al my-
cobac e ia, we subjec ed he mice o 4 weeks o chemo he apy
o clea emaining li e mycobac e ia. A e he end o chemo-
he apy ea men , no en i onmen al mycobac e ia we e de-
ec ed in any o he a ge o gans (li e , spleen, and lymph
nodes).
We inocula ed g oups o sensi ized and age-ma ched nai e
CBA/J mice i. . 1 week a e he end o chemo he apy ea -
men wi h 5 ⫻10
6
BCG and moni o ed he g ow h in he
spleen and li e o e ime. Sensi iza ion wi h en i onmen al
mycobac e ia esul ed in inhibi ion o he ini ial mul iplica ion
o BCG in he spleen and li e (Fig. 1A). In nai e mice, he
ini ial mul iplica ion o BCG esul ed in 10- o 30- old mo e
bac e ia in he spleen pos inocula ion han in sensi ized mice.
A di e ence was also seen a e a con en ional s.c. accina ion,
al hough he bac e ial numbe s we e a lowe le els (da a no
shown). Simila da a we e ob ained wi h C57BL/6J mice, which
a e mo e suscep ible o BCG (11). In his s ain, la ge di e -
ences in BCG numbe s we e ound be ween sensi ized and
nonsensi ized mice (Fig. 1B).
Immune esponses induced by BCG accina ion in sensi-
ized and nai e mice. We con inued by in es iga ing he im-
mune esponse induced by BCG in sensi ized and age-ma ched
nai e con ol CBA/J mice. ELISPOT was used o moni o
equencies o BCG-speci ic T cells be o e and 3, 5, 8, and 11
weeks a e he s.c. accina ion wi h BCG (Fig. 2). Be o e BCG
accina ion, no mycobac e ium-speci ic IFN-␥-p oducing T
cells we e de ec ed in any o he mice. Th ee weeks a e BCG
inocula ion, he numbe o BCG-speci ic IFN-␥-p oducing
cells in he d aining lymph nodes had inc eased and eached
he same le el in sensi ized and in nai e mice (Fig. 2A). The
esponse in sensi ized mice was, howe e , ansien , and om
5 weeks a e BCG inocula ion and onwa ds, a highe e-
quency o mycobac e ium-speci ic cells was ound in nai e ac-
cina ed mice. A he e mina ion o he expe imen (week 11),
a 10- imes-highe equency o BCG-speci ic T cells was
ound in he nai e accina ed g oup han in he sensi ized
accina ed g oup (P⫽0.032). A simila dynamic de elopmen
o esponses was ound in he blood, al hough i was delayed so
FIG. 1. BCG mul iplica ion is inhibi ed in mice p e iously sensi-
ized wi h en i onmen al mycobac e ia. (A) CBA/J mice. (B) C57BL/6J
mice. The g ow h o BCG was compa ed in nai e mice (open symbols)
and in sensi ized mice (solid symbols). The da a shown a e he means
o BCG CFU ⫾s anda d e o s. Fo bo h g oups, i e animals we e
sac i iced o each ime poin . The expe imen was epea ed wice wi h
simila esul s.
FIG. 2. In luence o p e ious sensi iza ion wi h en i onmen al my-
cobac e ia on he BCG-speci ic immune esponses. BCG was admin-
is e ed s.c., and he equencies o IFN-␥-p oducing cells isola ed om
he d aining lymph nodes (A) and he blood (B) in nai e mice (open
symbols) and sensi ized mice (solid symbols) we e de ec ed by he
ELISPOT assay pos accina ion a e in i o s imula ion wi h BCG-
Ag. The da a p esen ed he e ep esen he loga i hmic mean o esul s
ob ained om lymph node cells om h ee indi idual mice pe g oup
⫾s anda d e o s. The esponses in he blood we e analyzed on cells
pooled om h ee animals o each ime-poin . A pilo expe imen
conduc ed on weeks 2, 4, and 6 suppo ed he o e all di e ence in he
esponse p o iles o he wo g oups o animals.
674 BRANDT ET AL. INFECT.IMMUN.
ha highe equencies o speci ic T cells we e ound om
week 8 onwa ds in nai e accina ed mice (Fig. 2B). A no ime
poin a e accina ion was IL-4 o IL-5 de ec ed in he supe -
na an s o he s imula ed cul u es ( esul s no shown).
Sensi iza ion wi h en i onmen al mycobac e ia blocks he
p o ec i e e ec o BCG, bu no a TB subuni accine. We
con inued by accina ing sensi ized and nai e age-ma ched
con ol CBA/J mice 4 o 5 weeks a e he end o chemo he -
apy- ea men , ollowed 2 mon hs la e by an ae osol challenge
wi h M. ube culosis. The mice we e killed 6 weeks pos -TB
in ec ion, and M. ube culosis CFU we e enume a ed in he
lungs and spleens. The BCG accine impa ed app eciable
p o ec ion o nai e mice agains he TB challenge, wi h signi -
ican ly educed bac e ial numbe s in he o gans (0.68 o 1.13
log
10
educ ion; Table 1). Sensi iza ion wi h en i onmen al
mycobac e ia on i s own, o ollowed by BCG accina ion,
ailed o induce a s a is ically signi ican le el o p o ec ion
agains TB (Table 1).
We also asked i a p e ious sensi iza ion wi h en i onmen al
mycobac e ia would in luence p o ec ion induced by a subuni
accine. G oups o nai e and sensi ized CBA/J mice we e
accina ed wi h BCG o injec ed ( h ee imes a 2-week in e -
als) wi h ecen ly de eloped TB subuni accines based on he
immunodominan an igens ESAT-6 and Ag85B mixed wi h a
DDA-MPL adju an emulsion (5, 26). ESAT-6- accina ed an-
imals moun ed a e y s ong ecall immune esponse (5 o 7 ng
o IFN-␥/ml) o he homologous p epa a ion 1 week pos ac-
cina ion in he blood (da a no shown). The p o ec ion ob-
ained by BCG in con ol mice was log 0.53, and as in he
p e ious expe imen , BCG did no p o ec p esensi ized mice
(Table 2, expe imen 1) The ESAT-6 subuni accine, in con-
as , induced a simila deg ee o p o ec ion in bo h nai e and
sensi ized mice. A subuni accine based on a usion p o ein o
Ag85B and ESAT-6 has ecen ly been demons a ed o induce
le els o p o ec ion simila o hose o BCG in he mouse
model (26), and his accine also p o ec ed agains TB chal-
lenge a he same le el in nai e and sensi ized mice (Table 2,
expe imen 2).
Mycobac e ial species isola ed in Ka onga, Malawi, di e in
hei abili y o block BCG ac i i y. We in es iga ed six di e -
en isola es om soil and spu um samples om Ka onga Dis-
ic in No he n Malawi in he mouse model. Th ee o hese
isola es we e yped as M. o ui um, one was a s ain o M.
chelonae, and wo we e classi ied as belonging o he M. a ium
complex (Fig. 3). The g ow h o hese isola es in spleen, li e ,
and lung was in es iga ed wi h C57BL/6J mice o e a pe iod o
30 days. Mos o he isola es we e apidly clea ed o below he
le el o de ec ion, bu he s ains om he M. a ium complex
mul iplied and eached bac e ial numbe s 3 logs abo e hose o
M. chelonae and M. o ui um a e day 14 (Fig. 3). The mice
FIG. 3. G ow h o isola es om Ka onga, Malawi, in he mouse model. To e alua e he i ulence o he isola es, mice we e in ec ed i. . wi h
M. o ui um s ains S160 (open ci cles), S78/2 (solid ci cles), and 2001 (solid iangles); M. chelonae s ain 2015 (open iangles); and M. a ium
complex s ains 1891 and 2011 (solid and open squa es, espec i ely). The mycobac e ial loads we e de e mined in he li e , spleen, and lung a
he ime poin s indica ed. M. chelonae and M. o ui um we e all below he le el o de ec ion om day 14 onwa ds. Da a a e gi en as means wi h
s anda d e o s (n⫽4).
VOL. 70, 2002 INTERACTIONS OF BCG WITH ENVIRONMENTAL MYCOBACTERIA 675
we e ea ed wi h chemo he apy, ollowed by an injec ion o
BCG acco ding o ou s anda d p o ocol. BCG coun s in he
spleen o hese mice we e quan i ied a week 2 pos inocula ion
(Fig. 4A). M. o ui um and M. chelonae did no inhibi he
g ow h o BCG, whe eas bac e ia om he M. a ium complex
educed BCG numbe s by 1 o 1.5 log (P⬍0.01). This di e -
ence co ela ed wi h he immune esponses induced by he
BCG accine. The e was no in luence on he le el o IFN-␥
esponses o BCG Ag by sensi iza ion wi h M. chelonae o M.
o ui um, whe eas he p e ious inocula ion wi h bac e ia om
he M. a ium complex comple ely abla ed BCG immune e-
sponses (Fig. 4B). All s ains, on he o he hand, induced low
and a iable esponses o an igens ex ac ed om he homol-
ogous s ain o en i onmen al mycobac e ia ( esul s no
shown).
DISCUSSION
This s udy demons a es ha animals exposed o ce ain
en i onmen al mycobac e ia aise an immune esponse ha
con ols he mul iplica ion o BCG, he eby cu ailing he ac-
cine-induced immune esponse be o e i is ully de eloped.
The inding is impo an o he long-held discussion on he
ailu e o BCG accina ion agains TB in some pa s o he
wo ld (15, 16, 38). One hypo hesis o explain he ailu e o
BCG was p esen ed in 1966 by Palme and Long, based on
la ge-scale guinea pig expe imen s. They a gued ha because
con ac wi h non ube culous bac e ia o e s some le el o p o-
ec i e immuni y o TB, he p o ec i e e ec o a supe imposed
BCG accine would be masked (33). The p esen s udy con-
i ms he classical obse a ion ha p iming wi h en i onmen al
mycobac e ia p omo es some le els o p o ec i e immuni y o
o he mycobac e ia (7, 10, 14, 33), in his case o BCG. How-
e e , his e ec was no su icien o signi ican ly educe he
g ow h o M. ube culosis, which mul iplied a an almos un-
changed a e in hese sensi ized animals. The di e ence om
he pa ial p o ec ion impa ed by en i onmen al mycobac e ia
in he guinea pig model (14, 33) may be ela ed o he ac ha
he ea lie s udies made no e o o clea he en i onmen al
mycobac e ia by chemo he apy be o e challenge wi h M. ube -
culosis, as well as he di e en gene ic makeup and suscep i-
bili y o mice e sus guinea pigs. The di e ences in hese mod-
els and hei ele ance o human disease a e he subjec o an
ongoing s udy.
Tha p io sensi iza ion o en i onmen al mycobac e ia in-
e e es in a simila way wi h human BCG accina ion is
FIG. 4. (A) G ow h o BCG in sensi ized and nai e animals. Mice we e in ec ed s.c. wi h 2 ⫻10
6
CFU o ei he M. o ui um,M. chelonae,o
M. a ium o we e le un ea ed. A e chemo he apy, mice we e in ec ed wi h BCG Pas eu . BCG CFU in he spleen a e shown as means wi h
s anda d e o s (n⫽4). S a is ically signi ican di e ences be ween sensi ized (solid ba s) and nai e (open ba s) mice a e indica ed: ⴱ,P⬍0.05;
ⴱⴱ,P⬍0.01, acco ding o S uden ’s es . ND, no done. (B) E ec s o sensi iza ion on he IFN-␥ esponse o BCG. Spleen cells we e pooled om
ou indi idual mice sensi ized wi h he en i onmen al s ains (shaded ba s), nai e mice inocula ed wi h BCG (open ba s), o sensi ized mice
inocula ed wi h BCG (solid ba s). IFN-␥p oduc ion was assessed in he supe na an s o spleen cell cul u es s imula ed in i o wi h BCG Ag and
a e gi en as means wi h s anda d e o s. S a is ically signi ican e ec s o sensi iza ion on he esponse o BCG in ec ion a e labeled: ⴱ,P⬍0.05;
ⴱⴱ,P⬍0.01, acco ding o S uden ’s es .
676 BRANDT ET AL. INFECT.IMMUN.

s ongly sugges ed by a numbe o classical epidemiological
obse a ions: (i) he inding o s ong e icacy o BCG in ials
in which ube culin skin es -posi i e (and he e o e sensi ized)
dono s ha e been igo ously excluded (19); (ii) he consis en
success wi h BCG in neona es accina ed be o e any signi ican
sensi iza ion om en i onmen al mycobac e ia occu s (1, 9,
24); and, (iii) inally, he obse a ion o a lowe a e o skin es
con e sion, much smalle a e age diame e , and apidly wan-
ing esponses a e BCG accina ion in a eas wi h en i onmen-
al sensi iza ion (India and Egyp ), compa ed wi h hose in
a eas wi h minimal en i onmen al exposu e (Denma k) (4,
32). This obse a ion was ecen ly con i med and ex ended by
he obse a ion o only minimal in i o IFN-␥ esponses o
pu i ied p o ein de i a i e (PPD) induced by BCG accina ion
in dono s om Ka onga, Malawi, compa ed o hose om he
Uni ed Kingdom (P. E. Fine and H. Dock ell, pe sonal com-
munica ion). Taken oge he , hese indings a e in ag eemen
wi h he low and ansien immune esponse in he g oup o
animals sensi ized wi h en i onmen al mycobac e ia be o e
accina ion, whe eas he nai e animals de eloped s ong and
sus ained esponses (Fig. 3). Ou expe imen al model is he e-
o e ele an o he many opical egions whe e BCG is no
p o ec i e agains pulmona y TB and whe e he high incidence
o TB indica es ha any pa ial p o ec ion p o ided by expo-
su e o en i onmen al mycobac e ia is insu icien o he p e-
en ion o TB.
Ou main conclusion is ha BCG, as a li e accine, is pa -
icula ly sensi i e o he in luence o p eexis ing immune e-
sponses o an igens sha ed wi h ce ain en i onmen al s ains.
In his ega d, a ecen s udy has demons a ed he c oss-
ecogni ion o a la ge numbe o an igens sha ed be ween M.
a ium and BCG (T. Pais and R. Appelbe g, unpublished e-
sul s). Mul iplica ion is a p econdi ion o he induc ion o
immuni y by BCG and killing o BCG by chemo he apy a e
adminis a ion has been demons a ed o ab oga e subsequen
immuni y comple ely (13, 39). In he p esen s udy, his block-
ing is achie ed by immunological con ol ins ead o chemo-
he apy, bu he ou come in bo h cases is in e e ence wi h he
p o ec i e immune esponse, which would no mally de elop in
esponse o he g owing BCG. The equi emen o BCG mul-
iplica ion can be explained as a simple consequence o dosage,
bu mo e likely is due o he ac ha only li e BCG sec e es
many an igens o impo ance o he induc ion o a p o ec i e
immune esponse (3, 28). In e es ingly, ou da a om he
animal model also sugges ha only en i onmen al s ains,
which a e capable o an ini ial mul iplica ion in he hos , block
he ac i i y o BCG. A de ailed e alua ion o a la ge numbe o
di e en soil isola es om Ka onga, Malawi, and o hei in-
e ac ions wi h BCG is ongoing. In he u u e, in o ma ion on
he geog aphical dis ibu ion o such s ains would be a alu-
able esou ce when ying o unde s and he huge a ia ion in
BCG e icacy in human ials.
This inhibi o y e ec o he en i onmen al mycobac e ia on
he g ow h and ac i i y o BCG p o ides an impo an a gu-
men in he ongoing discussion o li e a enua ed accines
e sus non iable subuni accines agains TB (12, 27, 44). In
compa ison wi h li e a enua ed accines, he p esen s udy
sugges s ha subuni accines may be much less in luenced by
p io con ac wi h en i onmen al mycobac e ia. As men ioned
abo e, neona al BCG accina ion consis en ly impa s p o ec-
ion agains he childhood mani es a ions o TB (mos ly ex-
apulmona y disease), bu as i s e icacy wanes o e a pe iod
o 10 o 15 yea s (37), he adul pulmona y mani es a ions o
TB a e p e en ed nei he by neona al accina ion, by accina-
ion in adolescence a e exposu e o en i onmen al mycobac-
e ia (41), no by a BCG e accina ion s a egy (22, 43). A TB
subuni accine could he e o e ul ill he c i e ion o ha ing
consis en ly high e icacy in di e en popula ions and may ha e
a pa icula ly impo an use o e accina ion o hi d wo ld
child en in adolescence.
ACKNOWLEDGMENTS
This s udy has been suppo ed by he Danish Resea ch Council and
The Eu opean Commission (con ac no. 18CT970254). Lise B and is
suppo ed by he Facul y o Heal h Science, Uni e si y o Copenha-
gen.
En i onmen al mycobac e ia om Malawi we e isola ed wi hin he
con ex o he Ka onga P e en ion S udy (KPS) wi h he assis ance o
H. Phi i, S. Chagulkuka, and G. Black and we e classi ied by M. Ya es
a he U.K. Mycobac e ium Re e ence labo a o y in Dulwich. The KPS
is coo dina ed by Paul Fine and suppo ed by The Wellcome T us .
Paul Fine is hanked o aluable discussion, ad ice, and help ul
commen s on he manusc ip . We hank Vi a Sko , Lene Rasmussen,
and Tina Le che o excellen echnical assis ance.
REFERENCES
1. Al-Kassimi, F. A., M. S. Al-Hajjaj, I. O. Al-O ainey, and E. A. Bamgboye.
1995. Does he p o ec i e e ec o neona al BCG co ela e wi h accine-
induced ube culin eac ion? Am. J. Respi . C i . Ca e Med. 152:1575–1578.
2. Ande sen, A. B., Z.-L. Yuan, K. Haslø , B. Ve gmann, and J. Bennedsen.
1986. In e species eac i i y o i e monoclonal an ibodies o Mycobac e ium
ube culosis as examined by immunoblo ing and enzyme-linked immunoso -
ben assay. J. Clin. Mic obiol. 23:446–451.
3. Ande sen, P. 1997. Hos esponses and an igens in ol ed in p o ec i e im-
muni y o Mycobac e ium ube culosis. Scand. J. Immunol. 45:115–131.
4. Baily, G. V. 1980. Tube culosis p e en ion ial, Mad as. Indian J. Med. Res.
72:1–74.
5. B and , L., M. Elhay, I. Rosenk ands, E. B. Lindblad, and P. Ande sen.
2000. ESAT-6 subuni accina ion agains Mycobac e ium ube culosis. In ec .
Immun. 68:791–795.
6. B and , L., T. Oe inge , A. Holm, and P. Ande sen. 1996. Key epi opes on
he ESAT-6 an igen ecognized in mice du ing he ecall o p o ec i e im-
muni y o Mycobac e ium ube culosis. J. Immunol. 157:3527–3533.
7. B own, C. A., I. N. B own, and S. Swinbu ne. 1985. The e ec o o al
Mycobac e ium accae on subsequen esponses o mice o BCG sensi iza-
ion. Tube cle 66:251–260.
8. Clemens, J. D., J. J. Chuong, and A. R. Feins ein. 1983. The BCG con o-
e sy. A me hodological and s a is ical eapp aisal. JAMA 249:2362–2369.
9. Coldi z, G. A., C. S. Be key, F. Mos elle , T. F. B ewe , M. E. Wilson, E.
Bu dick, and H. V. Finebe g. 1995. The e icacy o bacillus Calme e-Gue in
accina ion o newbo ns and in an s in he p e en ion o ube culosis: me a-
analyses o he published li e a u e. Pedia ics 96:29–35.
10. Collins, F. M. 1971. Immunogenici y o a ious mycobac e ia and he co -
esponding le els o c oss-p o ec ion de eloped be ween species. In ec .
Immun. 4:688–696.
11. Denis, M., A. Fo ge , M. Pelle ie , R. Tu co e, and E. Skamene. 1986.
Con ol o he Bcg gene o ea ly esis ance in mice o in ec ions wi h BCG
subs ains and a ypical mycobac e ia. Clin. Exp. Immunol. 63:517–525.
12. Dohe y, T. M., and P. Ande sen. 2000. Tube culosis accines: de elopmen-
al wo k and he u u e. Cu . Opin. Pulm. Med. 6:203–208.
13. Dwo ski, M. 1973. E icacy o bacillus Calme e-Gué in and isoniazid- esis-
an bacillus Calme e-Gué in wi h and wi hou isoniazid chemop ophylaxis
om day o accina ion. Am. Re . Respi . Dis. 108:294–300.
14. Edwa ds, M. L., J. M. Good ich, D. Mulle , A. Pollack, J. E. Ziegle , and
D. W. Smi h. 1982. In ec ion wi h Mycobac e ium a ium-in acellula e and
he p o ec i e e ec s o Bacille Calme e-Gue in. J. In ec . Dis. 145:733–741.
15. Fine, P. E. 1989. The BCG s o y: lessons om he pas and implica ions o
he u u e. Re . In ec . Dis. 11(Suppl. 2):S353–S359.
16. Fine, P. E. 1995. Va ia ion in p o ec ion by BCG: implica ions o and o
he e ologous immuni y. Lance 346:1339–1345.
17. Flynn, J. L., J. Chan, K. J. T iebold, D. K. Dal on, T. A. S ewa , and B. R.
Bloom. 1993. An essen ial ole o in e e on gamma in esis ance o Myco-
bac e ium ube culosis in ec ion. J. Exp. Med. 178:2249–2254.
18. Ha boe, M., A. S. Malin, H. S. Dock ell, H. G. Wike , G. Ul und, A. Holm,
M. C. Jø gensen, and P. Ande sen. 1998. B-cell epi opes and quan i ica ion
VOL. 70, 2002 INTERACTIONS OF BCG WITH ENVIRONMENTAL MYCOBACTERIA 677
o he ESAT-6 p o ein o Mycobac e ium ube culosis. In ec . Immun. 66:
717–723.
19. Ha , P. D., and I. Su he land. 1977. BCG and ole bacillus accines in he
p e en ion o ube culosis in adolescence and ea ly adul li e. B . Med. J.
2:293–295.
20. Kamala, T., C. N. Pa amasi an, D. He be , P. Venka esan, and R. P ab-
haka . 1996. Immune esponse & modula ion o immune esponse induced
in he guinea-pigs by Mycobac e ium a ium complex (MAC) & M. o ui um
complex isola es om di e en sou ces in he Sou h Indian BCG ial a ea.
Indian J. Med. Res. 103:201–211.
21. Kamala, T., C. N. Pa amasi an, D. He be , P. Venka esan, and R. P ab-
haka . 1994. Isola ion and iden i ica ion o en i onmen al mycobac e ia in
he Mycobac e ium bo is BCG ial a ea o Sou h India. Appl. En i on.
Mic obiol. 60:2180–2183.
22. Ka onga P e en ion T ial G oup. 1996. Randomised con olled ial o
single BCG, epea ed BCG, o combined BCG and killed Mycobac e ium
lep ae accine o p e en ion o lep osy and ube culosis in Malawi. Lance
348:17–24.
23. Lag ande ie, M. R., A. M. Balazuc, E. De iaud, C. D. Lecle c, and M.
Gheo ghiu. 1996. Compa ison o immune esponses o mice immunized wi h
i e di e en Mycobac e ium bo is BCG accine s ains. In ec . Immun. 64:
1–9.
24. Miceli, I., I. N. de Kan o , D. Colaiaco o, G. Pelu o, I. Cu illo, R. Go a, R.
Bo a, S. Hom, and H. G. en Dam. 1988. E alua ion o he e ec i eness o
BCG accina ion using he case-con ol me hod in Buenos Ai es, A gen ina.
In . J. Epidemiol. 17:629–634.
25. No h, R. J. 1973. Impo ance o hymus-de i ed lymphocy es in cell-medi-
a ed immuni y o in ec ion. Cell Immunol. 7:166–176.
26. Olsen, A. W., L. A. H. an Pinx e en, L. M. Okkels, P. B. Rasmussen, and P.
Ande sen. 2001. P o ec ion o mice wi h a ube culosis subuni accine based
on a usion p o ein o an igen 85B and ESAT-6. In ec . Immun. 69:2773–
2778.
27. O me, I. M. 1997. P og ess in he de elopmen o new accines agains
ube culosis. In . J. Tube c. Lung Dis. 1:95–100.
28. O me, I. M., P. Ande sen, and W. H. Bloom. 1993. T cell esponse o
Mycobac e ium ube culosis. J. In ec . Dis. 167:1481–1497.
29. O me, I. M., E. S. Mille , A. D. Robe s, S. K. Fu ney, J. P. G i in, K. M.
Dobos, D. Chi, B. Ri oi e, and P. J. B ennan. 1992. T lymphocy es media ing
p o ec ion and cellula cy olysis du ing he cou se o Mycobac e ium ube -
culosis in ec ion. E idence o di e en kine ics and ecogni ion o a wide
spec um o p o ein an igens. J. Immunol. 148:189–196.
30. O me, I. M., A. R. Robe s, and F. M. Collins. 1986. Lack o e idence o a
educ ion in he e icacy o subcu aneous BCG accina ion in mice in ec ed
wi h non ube culous mycobac e ia. Tube cle 67:41–46.
31. Pais, T. F., R. A. Sil a, B. Smedegaa d, R. Appelbe g, and P. Ande sen. 1998.
Analysis o T cells ec ui ed du ing delayed- ype hype sensi i i y o pu i ied
p o ein de i a i e (PPD) e sus challenge wi h ube culosis in ec ion. Im-
munology 95:69–75.
32. Palme , C. E. 1952. BCG accina ion and ube culin alle gy. Lance May
10:935–941.
33. Palme , C. E., and M. W. Long. 1966. E ec s o in ec ion wi h a ypical
mycobac e ia on BCG accina ion and ube culosis. Am. Re . Respi . Dis.
94:553–568.
34. Rook, G. A., G. M. Bah , and J. L. S an o d. 1981. The e ec o wo dis inc
o ms o cell-media ed esponse o mycobac e ia on he p o ec i e e icacy o
BCG. Tube cle 62:63–68.
35. Smi h, D. W., E. H. Wiegeshaus, and M. L. Edwa ds. 1988. The p o ec i e
e ec s o BCG accina ion agains ube culosis, p. 341–370. In M. Bendinelli
and H. F iedman (ed.), Mycobac e ium ube culosis. Plenum Publishing Co -
po a ion, New Yo k, N.Y.
36. S an o d, J. L., M. J. Shield, and G. A. Rook. 1981. How en i onmen al
mycobac e ia may p ede e mine he p o ec i e e icacy o BCG. Tube cle
62:55–62.
37. S e ne, J. A., L. C. Rod igues, and I. N. Guedes. 1998. Does he e icacy o
BCG decline wi h ime since accina ion? In . J. Tube c. Lung Dis. 2:200–
207.
38. en-Dam, H. G. 1984. Resea ch on BCG accina ion. Ad . Tube c. Res.
21:79–106.
39. Toyoha a, M., S. Kudoh, and Y. Obayashi. 1959. S udies on he e ec o
isoniazid upon he an i ube culous immuni y induced by BCG accina ion.
Tube cle 40:184–191.
40. T ipa hy, S. P. 1983. The case o BCG. Ann. N. Y. Acad. Med. Sci. 19:11–
21.
41. Tube culosis Resea ch Cen e (ICMR). 1999. Fi een yea ollow up o ial
o BCG accines in sou h India o ube culosis p e en ion, Chennai. Indian
J. Med. Res. 110:56–69.
42. Weisz eile , J. C., and V. Ka asse a. 1981. Mixed mycobac e ial in ec ions.
Re . In ec . Dis. 3:1081–1083.
43. Wo ld Heal h O ganiza ion. 1995. W.H.O. news and ac i i ies. W.H.O. Bull.
OMS 73:805–807.
44. Young, D. B. 2000. Cu en ube culosis accine de elopmen . Clin. In ec .
Dis. 30:S254–S256.
Edi o : S. H. E. Kau mann
678 BRANDT ET AL. INFECT.IMMUN.