Failure of the Mycobacterium bovis BCG vaccine: Some species of environmental mycobacteria block multiplication of BCG and induction of protective immunity to tuberculosis

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. 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