Vaccine 23 (2005) 1326–1334

Schistosoma mansoniinfection reduces the protective efficacy of BCGvaccination against virulentMycobacterium tuberculosis

Daniel Eliasa,b,e, Hannah Akuffoa,∗, Andrzej Pawlowskib, Melles Haileb,Thomas Schonc, Sven Brittond

a Microbiology and Tumor Biology Center, Karolinska Institute, Box 280, 17177 Stockholm, Swedenb The Swedish Institute for Infectious Disease Control, Stockholm, Sweden

c Department of Medical Microbiology, Link¨oping University, SE-58185 Link¨oping, Swedend Department of Medicine, Unit of Infectious Diseases, Karolinska Institute, 17176 Stockholm, Sweden

e Armauer Hansen Research Institute, Box 1005, Addis Ababa, Ethiopia

Received 5 February 2004; accepted 20 September 2004Available online 14 November 2004

Abstract

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We hypothesized that the ability of BCG vaccination to protect againstMycobacterium tuberculosisis less in hosts exposed to chroelminthes infection compared to unexposed individuals. To test this hypothesis we evaluated the efficacy of BCG vaccination ingainstM. tuberculosischallenge inSchistosoma mansonipre-infected mice by analyzing their ability to limit the replication of TB ba

n the lung and liver and the histology of lung sections. The results show that BCG vaccinated mice with priorS. mansoniinfection showignificantly higher number of colony forming units of TB bacilli as well as significant reduction in air exchange area in the lung co controls. In addition, spleen cells fromS. mansoniinfected mice were found to produce significantly less IFN-� and nitric oxide whetimulated in vitro with PPD and several fold higher soluble egg antigen (SEA) and Concanavalin A induced IL-4 and IL-5 secretiogether, our data show thatS. mansoniinfection reduces the protective efficacy of BCG vaccination againstM. tuberculosispossibly byttenuation of protective immune responses to mycobacterial antigens and/or by polarizing the general immune responses to the2004 Published by Elsevier Ltd.

eywords:Tuberculosis; Vaccination; Helminths

. Introduction

Tuberculosis (TB) is one of the major threats to humanealth today. The problem is worsened by the deadly synergyith HIV/AIDS and the emergence of multi-drug resistanttrains ofMycobacterium tuberculosis[1]. The current TBaccine, BCG, was developed in the beginning of the lastentury and it is still the most widely used vaccine in theorld. This vaccine, although proven to be highly effective

n animal models[2] and in humans in certain parts ofhe world, fails to confer protection against pulmonary

∗ Corresponding author. Tel.: +46 8 698 5053; fax: +46 8 698 5656.E-mail addresses:hannah.akuffo@mtc.ki.se, hannah.akuffo@sida.se

H. Akuffo).

tuberculosis in adults in many other parts of the w[3].

A number of explanations have been put forward tocount for the unpredictable outcome of BCG vaccinatThese include varying exposure of the trial population tovironmental mycobacteria[4], genetic or physiologic diffeences among the populations[5], strain variation in BCGpreparation[6] and nutritional differences among the tpopulations. Among these, exposure to environmentalcobacteria is the only explanation with a reasonably stscientific basis[4,5,7]. However, an overview of publisheliterature show that environmental mycobacteria couldcount for only about 40% of the observed variability[8].

Most areas where the vaccine has not performed wecharacterized by high endemic prevalence of worm infec

264-410X/$ – see front matter © 2004 Published by Elsevier Ltd.oi:10.1016/j.vaccine.2004.09.038

D. Elias et al. / Vaccine 23 (2005) 1326–1334 1327

[9,10]. Several investigators have shown[13,17–19,32]thatpersistent helminth infection could affect immune responsesto unrelated antigens[11] and/or the outcome of subse-quent infections[12] or vaccinations[14,15]. To mentiona few,

(i) Schistosomiasis was shown to alter Th1/2 responses tonon-parasite antigens[18].

(ii) Th1 responses to mycobacteria was reported to bereduced by helminth induction of Th2 responses[11].

(iii) Humans infected withS. mansonishow impaired Th1response to tetanus toxoid antigens[14].

(iv) The ability of BCG vaccine to sensitize against TBantigens was found to be impaired in helminth infectedindividuals[16].

(v) Helminth infected animals with a dominant Th2 im-mune profile fail to clear viral infections[17,32].

(vi) People infected with helminths show preferential in-duction of Th2 lymphocyte subsets[19–21].

(vii) Intestinal parasites in domestic animals were reportedto influence post vaccination immunity against viraland bacterial infections[29].

(viii) Deworming of individuals with HIV infection was re-ported to be associated with reduced plasma viral load[31].

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Additional mice were used for preliminary evaluation ofthe effect ofS. mansoniinfection on the replication and im-munogenicity of BCG vaccination—at weeks 3 and 6 post-vaccination.

2.2. Experimental infection

2.2.1. S. mansoniS. mansonicercarieae (Puerto Rican strain) were obtained

from department of Parasitology, the Swedish Institute for In-fectious Diseases control (SMI) (Stockholm, Sweden). Micewere anesthetized by injecting 100�l of 8.6 mg/ml pento-barbitalnatrium intraperitonially. One hundred microliters oftap water, containing 30S.mansonicercariae was placed on ashaved skin area on the abdomen and were left for 20 min foractive infection to occur. The presence of the infection waschecked repeatedly by direct microscopy of stool samplesduring the course of the experiments.

2.2.2. BCG vaccinationLyophilizedM. bovisBCG, strain 1331 (Statens Serum

Institute, Copenhagen, Denmark) was suspended in the dilu-ent provided with the preparation and 100�l of the suspen-sion containing 2× 105 colony forming units (CFU) of BCGbacilli was injected subcutaneously into the abdominal re-g rei oc pli-c

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Based on these findings we hypothesized that one oeasons why BCG has such a poor impact on TB epidlogy in resource poor countries in the tropics is immodulation by persistent helminth infections. To test thisothesis we compared the protective efficacy of BCG vaation againstM. tuberculosischallenge in mice pre-infecteithS. mansoniand controls. And for the first time we repnegative effect ofS. mansoniinfection on BCG-induce

rotection against challenge with virulentM. tuberculosisinmouse model.

. Materials and methods

.1. Animals

Sixty female C57/bl mice were obtained from B &niversal (Stockholm, Sweden). The animals were m

ained at the animal facility of the Swedish Institutenfectious Diseases Control (SMI) (Stockholm, Swedll animals were 8–10 weeks of age at the beginnin

he experiments and were kept in cages with unlimood and water supply. The animals were divided intoroups. One group was infected with 30 cercariae ofS. manoni and the other group was kept uninfected and useontrol. Both groups were further subdivided into two sroups and one subgroup in each group was BCG vated (Fig. 1). AfterM. tuberculosischallenge animals weoused in cages contained within a laminar flow safetylosure.

ion 8 weeks afterS. mansoniinfection. Sixteen mice wenjected with 2× 105 CFU of BCG bacilli intravenously theck the immunogenicity and ability of the vaccine to reate inS. mansoniinfected mice.

.2.3. M. tuberculosis infectionM. tuberculosisH37Rv Erdman strain was obtained fro

he American Type Culture Collection (ATCC). The bactere cultured on Lowenstein Jensen medium for 3–4 wnd then propagated in Middlebrook 7H9 medium (Diweden) for 3 weeks and aliquots of the bacilli wereended in Middlebrook 7H9 medium containing 10% grol at 108 bacilli/ml and frozen at−70◦C. On the day o

he challenge, frozen aliquots of the bacteria were thand washed twice in PBS. After the last wash, the baial pellet was diluted to 106 bacilli/ml in 0.05% Tween0 (Sigma-Aldrich, Sweden) in normal saline. The susion was briefly sonicated for 10 s and 100�l containing 105

acilli were inoculated into the tail vein 6 weeks post Baccination. The infection dose was confirmed by plahe bacterial suspension on Middlebrook 7H11 agar mend counting the number of colony forming units perolume.

.3. Determination of M. tuberculosis CFU in organs

To evaluate the effect ofS.mansoniinfection on the levef protection achieved by BCG vaccination, mice were c

enged 6 weeks after BCG vaccination withM. tuberculosisrdman strain through the intravenous route.

1328 D. Elias et al. / Vaccine 23 (2005) 1326–1334

Fig. 1. Study protocol.

At 3, 6, and 9 weeks postM. tuberculosischallenge,the number of CFU of tuberculosis bacilli in the liver andlung were evaluated as described previously[30]. Briefly,animals were sacrificed and organs were aseptically isolatedand placed in tubes containing 5 ml dilution solution (salinecontaining 0.05% Tween 80). The organs were homogenizedusing polytron PT 1200 (Kinematica AG, Switzerland)and serial 10-fold dilutions were plated on Middlebrook7H11 agar medium containing 2-thiophene-carboylic acidhydrazide (TCH) (Sigma-Aldrich, Stockholm, Sweden) toselectively inhibit the growth of residual BCG in the testorgans. Plates were incubated at 37◦C and 5% CO2 untilcolonies were large enough to be counted (3–4 weeks).

The CFU of BCG bacilli in the lungs were determined inessentially the same way except that the medium containedno TCH.M. tuberculosisCFU analysis was repeated at weeks6 and 9 postM. tuberculosischallenge with five animals ineach group at both time points. Results were expressed asmean CFU per organ.

2.4. Histology

Lungs of mice were fixed in 10% buffered formalinand embedded in paraffin. Sections made from paraffinblocks were stained with hematoxylin and eosin. The sec-tions were examined by a pathologist well experiencedin the histopathology of mycobacterial diseases but with-out prior knowledge of the experimental groups. Ten con-secutive lung sections were examined for each animalin the two groups and a representative photograph wastaken.

2.5. Spleen cell culture

Single cell suspensions from individual spleens from fiveanimals in each group were prepared by teasing the or-gans loose into complete RPMI [RPMI 1640 supplementedwith 10% fetal calf serum, 20 mM HEPES buffer (Sigma-Aldrich, Stockholm, Sweden), 0.05 mM 2-mercaptoethanol

D. Elias et al. / Vaccine 23 (2005) 1326–1334 1329

(Life Technologies Paisley, London, UK), 1 mM glutamineand 1% penicillin/streptomycin (Sigma-Aldrich, Stockholm,Sweden)]. Red cells were lysed using red blood cell lysingbuffer (Sigma-Aldrich, Stockholm, Sweden). For nitrite as-say, the cell culture was made in IMDM medium sup-plemented with 10% fetal calf serum, 20 mM HEPESbuffer (Sigma-Aldrich, Stockholm, Sweden), 0.05 mM 2-mercaptoethanol (Life Technologies Paisley, UK), 1 mMglutamine and 1% penicillin/streptomycin (Sigma-Aldrich,Stockholm, Sweden). The suspension was washed twice andthe cell concentration was adjusted to 2× 106/ml. Spleencells were cultured at a concentration of 4× 105/well intriplicate wells of 96-well flat bottom Nunc culture platesin the absence or presence of 5�g/ml Concanavalin A(Sigma-Aldrich, London, UK) or 6.5�g/ml purified proteinderivative ofM. tuberculosis(PPD) (Statens Serum Insti-tute, Copenhagen, Denmark) or 15�g/ml soluble egg antigen(SEA). SEA was prepared as described by Carter and Col-ley [33]. IL-4 and IL-5 assays were performed using frozenspleenocytes. Culture supernatants were collected at days 2,3, 4 and 5 for assays of IL-4, IFN-�, nitrite and IL-5, respec-tively.

2.6. Measurement of cytokine production by ELISA

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3. Results

3.1. Chronic S. mansoni infection reduces BCG-inducedprotection against intravenous M. tuberculosis challenge

To evaluate whether chronicS. mansoniinfection couldimpair BCG-induced protection againstM. tuberculosischal-lenge, mice infected with low doseS. mansonifor 8 weekswere vaccinated with BCG through the subcutaneous routeand 6 weeks later, challenged withM. tuberculosisthroughthe intravenous route. The number of CFU ofM. tubercu-losis bacilli in the lungs and liver were then determined atweeks 3, 6 and 9, post challenge. In the lung, at week 3 postM. tuberculosischallenge, animals BCG vaccinated in thepresence ofS. mansoniinfection showed significantly lowerCFU. At week 6, however, the trend was reversed and theS.mansoniinfected group showed a tendency for higher CFUand at week 9 there was a statistically significant higher num-ber of TB bacilli in theS. mansoni-infected BCG vaccinatedgroup (p< 0.05) compared to BCG vaccinatedS. mansoniuninfected controls (Fig. 2a).

In the liver, animals vaccinated with BCG in the presenceof S. mansoniinfection had significantly higher CFU com-pared to vaccinated animals in theS. mansoniuninfectedgroup at weeks 3 and 6, post challenge. At week 9, however,a -s ticals

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Assessment of cytokine levels were performed onulture supernatants using commercially available ELits (R & D Systems, London, UK) following the manacturer’s instructions in duplicate wells. Concentrationytokines (IL-4, IL-5 and IFN-�) in samples were calculaty a standard curve generated from recombinant cytoR & D systems, London, UK), and results were expressicograms per milliliter. The differences in results betwuplicate wells were consistently less than 10% of the mhe sensitivity of the assay was 2 pg/ml for all cytokieasured.

.7. Nitrite assay for the estimation of nitric oxidenduction

Nitrite levels in 4-day culture supernatants were deined by the method of Green et al. using the Greiss rea

22]. The titer was determined by the standard curve geted by the absorbance of serial dilutions of NaNO2 (Wako,tockholm, Sweden).

.8. Statistical analysis

The data obtained were assessed by analysis of varFU data were assessed by the Student–Newman–Keuhereas data from nitrite and IFN-� assays were assessy the Student’st-test. In all cases, ap value of≤0.05 wasonsidered significant in comparisons of results betweeroups.

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lthough there was a tendency for higher CFU in theS. manoni infected group, the difference did not reach statisignificance (Fig. 2b).

Unvaccinated mice in the control group had over five-igher CFU than BCG vaccinated mice at all time po

ested (p< 0.001).In the unvaccinated group the number of CFU in the lu

as comparable between the two groups of mice. In the

ig. 2. Protective efficacy as assessed by determining kinetics of theation of TB bacilli in the lungs (a) and the liver (b) at weeks 3, 6, aostM. tuberculosischallenge in animals BCG vaccinated in the preser absence ofS. mansoniinfection (*p< 0.05). Results are mean CFU frove mice per experimental group± S.E.M.

1330 D. Elias et al. / Vaccine 23 (2005) 1326–1334

Fig. 3. Kinetics of replication of TB bacilli in the lungs (a) and liver (b) ofanimals in theS. mansoniinfected group and control at weeks 3, 6 and 9postM. tuberculosischallenge in non-BCG vaccinated animals. Results aremean CFU from five mice per experimental group± S.E.M.

animals with priorS. mansoniinfection show a tendency forhigher CFU compared to those withoutS. mansoniinfectionat weeks 6 and 9 post challenge but the difference was notsignificant. However, an initial lower CFU was observed atweek 3 in the lungs of the non-BCG-vaccinated group ofmice withS. mansoniinfection compared to those withoutS.mansoniinfection (Fig. 3).

M. tuberculosisCFU analysis was repeated in a secondset of 20 mice BCG vaccinated in the presence or absence ofS. mansoniinfection at weeks 6 and 9 postM. tuberculosischallenge and the results were reproducible. Five mice wereused per group at both week 6 and 9. In the lung at week 6CFU was 91,346± 15,219 versus 42,617± 6143 inSchisto-somacoinfected and controls, respectively (p< 0.05) and atweek 9 CFU was 512,478± 97,143 versus 133,941± 43,414in the coinfected and controls, respectively (p< 0.01). Inthe liver CFU at week 6 was 361,493± 82,439 versus93,978± 23,994 in the coinfected and controls, respectively(p< 0.01) while at week 9 CFU was 439,126± 101,224 ver-sus 179,652± 95,111 in coinfected and controls, respectively(p< 0.05).

3.2. Effect of S. mansoni on vaccine ‘take’

In preliminary experiments, the multiplication of intra-v8 dt n int -s ep tion

of BCG in the lungs was significantly higher inS. mansoniinfected group compared to controls (32,000± 3900 versus12,000± 4200,p< 0.05). In the liver, BCG replication wasobserved to be higher inSchistosoma-coinfected mice at bothtime points (result not shown). Results were expressed asmean CFU/organ± S.E.M.

3.3. S. mansoni infection reduces BCG-inducedprotection against lung damage caused by M.tuberculosis infection

The levels of cellular accumulation and organization(granulomatous response) as well as reduction in air exchangeareas caused by inflammatory responses againstM. tubercu-losisinfection were compared between mice BCG vaccinatedin the presence ofS.mansoniinfection and controls. At week9, post challenge, the lungs ofS. mansonipre-infected miceshowed extensive cellular recruitment into the lungs and re-duction of air spaces (Fig. 4a). Moreover, the granulomaswere less organized and diffuse with no sharp demarcation

Fig. 4. Photomicrographs of lungs of mice BCG vaccinated in the presenceor absence ofS.mansoniinfection. The lungs were examined at week 9, postchallenge. Sections were stained with hematoxylin and eosin (a and b). Tenconsecutive sections for each of the five mice were stained and examinedin each of the experimental groups. (a) A section from miceSchistosomainfected prior to vaccination and (b) a section from the control group. Thefigures shown are representatives for each group of mice.

enously injected BCG in mice infected withS. mansoniforweeks was tested.S.mansoniinfection transiently inhibite

he replication of BCG up to 3 weeks post BCG injectiohe lungs (2283± 1275 in theS. mansoniinfected group verus 12,750± 3140 in the controls,p< 0.05). At a later timoint however (week 6 post BCG injection), the replica

D. Elias et al. / Vaccine 23 (2005) 1326–1334 1331

from the surrounding tissue. In contrast, in mice withoutS.mansoniinfection, the granulomas remained small, organizedand distinct from the majority of the tissue that appeared nor-mal (Fig. 4b).

3.4. Comparison of the immunogenicity of BCG in S.mansoni infected and control mice before challenge withM. tuberculosis

Mice infected withS. mansonifor 8 weeks (n= 4) anduninfected control (n= 4) were BCG vaccinated and 6 weekslater spleen cells obtained were cultured in the presenceof PPD or ConA. Supernatants collected on day 3 wereanalyzed for IFN-� content. The result indicated thatS.mansonico-infected group of mice produce significantly lessPPD induced IFN-� compared to controls (547± 106 versus1052± 169, p< 0.05) whereas ConA induces comparablelevels of IFN-� in the two groups of mice (2889± 235versus 3212± 549, p> 0.05). Results are expressedin pg/ml.

3.5. S. mansoni infection affects in vitro PPD inducedIFN-� production

To determine whether the differences between BCG vac-c lsi nv ed atw di ectedfrw on-c utt eek9 fer-e ta nots

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Fig. 5. Systemic immune responses as assessed from IFN-� levels after invitro restimulation of spleen cells from mice BCG vaccinated in the pres-ence ofS. mansoniinfection and challenged withM. tuberculosis. Thisanalysis was made at weeks 6 (A) and 9 (B), post challenge. Results aremean IFN-� levels (in picograms per liter) from five mice per experimentalgroup± S.E.M. (*p= 0.05, ** p= 0.01).

groups of unvaccinated animals was not significant at bothtime points tested (data not shown).

3.7. S. mansoni infected mice produce significantlyhigher SEA and ConA induced IL-4 and IL-5 secretioncompared to controls

To test if theS. mansoniinduced impairment in Th1 re-sponse was associated with altered background immunity, we

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inated animals inS. mansoniinfected group and contron controllingM. tuberculosisinfection was reflected in iitro PPD stimulated Th1 responses, spleen cells obtaineeks 6 and 9 postM. tuberculosischallenge were culture

n the presence of PPD or ConA. The supernatants collrom day 3 cultures were assayed for IFN-� content. Theesults showed that PPD induced in vitro IFN-� productionas significantly lower in BCG vaccinated animals with comitantS. mansoniinfection compared to control mice. Bhis difference did not reach statistical significance at w

(Fig. 5). In the unvaccinated animals, however, the difnces were not significant at both time points tested (dahown).

.6. S. mansoni infection reduces in vitro PPD inducedroduction of nitric oxide

To evaluate whether the differences between BCG vated animals inS. mansoniinfected group and controls

FN-� secretion were associated with impaired in vitroric oxide secretion. Spleen cells obtained at weeks 6

postM. tuberculosischallenge were cultured for 4 daith PPD and supernatant collected was assayed for the

rite content using the modified Greiss reaction[22]. As aositive control, cells were stimulated with ConA. Theult indicated thatS. mansoniinfection causes statisticaignificant (p< 0.05) reduction in PPD-induced secretionitric oxide in BCG vaccinated animals at both time po

ested (Fig. 6). The difference in ConA-induced nitrite secion was significant only at week 9. However, the differen PPD or ConA-induced nitric oxide production by the t

ig. 6. Systemic immune responses as assessed from nitrite levelsitro restimulation of spleen cells from mice BCG vaccinated in the pnce ofS. mansoniinfection and challenged withM. tuberculosis. Thisnalysis was made at weeks 6 (a) and 9 (b), post challenge. Resuean nitrite levels (in micromoles per liter) from five mice per experimeroup± S.E.M.

1332 D. Elias et al. / Vaccine 23 (2005) 1326–1334

investigated the in vitro PPD, SEA or ConA-induced Th2 cy-tokines (IL-4 and IL-5) at the time of BCG vaccination,M.tuberculosischallenge and 9 weeks post challenge. The re-sults show that animals withS.mansoniinfection were foundto produce significantly higher SEA and ConA-induced IL-4and IL-5 levels at both time points tested. However, the lev-els of PPD-induced secretion of both cytokines were low andthe differences if any observed between the groups were notsignificant at both time points tested (Fig. 7).

4. Discussion

Our previous report indicated that BCG vaccination inhelminth infected individuals failed to significantly improvein vitro T cell responses to TB antigens, whereas when thesame vaccine was given to helminth free individuals, it sig-nificantly improved in vitro T cell responses to TB antigens[16]. Whether this failure of BCG to significantly improvein vitro TB antigen specific responses was a reflection of re-duced efficacy against TB is a problem that remains to beaddressed.

As a first step to address this question, we investigatedwhether chronicS. mansoniinfection could reduce the ef-ficacy of BCG vaccination in protecting againstM. tuber-c s byap ei instT atedwo eriall aredt

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ulosischallenge in a murine system. We evaluated thinalyzing the number of CFU ofM. tuberculosisbacilli, lungathology caused byM. tuberculosisinfection as well as th

n vitro nitric oxide and Th1/Th2 cytokine response agaB antigens. Our findings demonstrate that mice vaccinith BCG in the presence of chronicS. mansoniinfectionbtain significantly less protection (contain higher bact

oads, and more inflammatory response in the lung) compo controls withoutS. mansoniinfection.

Several reports have shown that helminth infectionensitization to worm antigens could influence immuneponses to unrelated antigens including the mycobacntigen mixture, PPD[11,13,17–19,32]. Moreover, Malhotrand co-workers have shown in humans that prenatal seation to helminth antigens biases T cell immunity induy subsequent vaccination with BCG towards a Th2 typeubsequent reduction in TB antigen induced Th1 respo15]. But there is no conclusive evidence showing that selminth-induced changes in in vitro immune response

he reflections of altered susceptibility toM. tuberculosis.ere we show that chronicS. mansoniinfection in the miceignificantly reduce the efficacy of BCG vaccination in pecting against parenteral challenge with virulentM. tuber-ulosis.

ig. 7. Systemic Th2 immune responses as assessed from IL-4 and ILls after in vitro restimulation of spleen cells at the time of BCG vaccina

ime ofM. tuberculosischallenge as well as 9 weeks postM. tuberculosishallenge. Results are mean cytokine levels (in picograms per liter)ve mice per experimental group± S.E.M. (*p< 0.01, ** p< 0.001).

D. Elias et al. / Vaccine 23 (2005) 1326–1334 1333

It has been repeatedly shown thatS. mansoniinfectionin mice and man is associated with enhanced Th2 responseswith elevated secretion of IL-4, IL-5 and IL-10 in responseto in vivo and in vitro challenge with worm and non wormantigens[11–14]. On the other hand, infections with intracel-lular pathogens are primarily controlled by Th1 type immuneresponses[23–25].

The reduced ability of BCG vaccination to protect againstM. tuberculosisin S. mansoniinfected animals could be dueto inhibition of BCG induced cellular responses by the Th2immune background caused by on-going worm infection. Toinvestigate this possibility we analysed in vitro PPD, SEAand ConA induced production of Th2 cytokines IL-4 and IL-5. The results showed that spleen cells produced comparablebut low levels of IL-5 in response to PPD in both groupsof animals. However, there were significant differences inSEA and ConA induced secretion of both IL-4 and IL-5,with the S. mansonicoinfected group producing more IL-4 and IL-5 than controls. This indicates the importance ofS. mansoniinduced general immune deviation towards Th2type immunity in reducing the efficacy of BCG vaccinationagainstM. tuberculosisinfection.

It is worth noting however that the effect ofS. mansoniinfection shows variable impact on the ability of the animalsto controlM. tuberculosisinfection in the two organs tested.Id ointd ly ond agesi hereS hus,t tionc

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sinceS. mansoniinfection on its own was shown to impairmultiplication ofM. tuberculosisduring the early stages ofinfection. However, evaluation of the replication of intra-venously injected BCG in the lungs and liver ofS. mansoniinfected mice showed that BCG grows more comfortably inS. mansoniinfected group compared to controls at the timethe mice were challenged (6 weeks post vaccination) withM.tuberculosiswhich would argue against the possibility thatthe effect ofS. mansoniwould be through the inhibition ofthe replication of the vaccine. Despite a more pronouncedmultiplication of the vaccine inS. mansoniinfected mice,the protection it confers against a subsequent challenge withvirulent M. tuberculosisin such animals was significantlylower.

In summary, our findings show that the protective efficacyof BCG vaccination can be significantly reduced by chronicS. mansoniinfection. In addition theS. mansoni-associatedimpairment of BCG induced protection was associated withreduced in vitro PPD induced production of nitric oxide andIFN-� by spleen cells.

It is worth mentioning however that in real life BCG isoffered early in life before exposure to helminth infection.Thus it would be very relevant to examine the effect of postvaccination exposure to worms on BCG induced protectionalthough there is evidence on a negative impact of prena-t tion[

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n the lungs the effect ofS. mansoniinfection in BCG in-uced protection was apparent only at the later time puring the analysis while such effect was apparent earuring the course of TB infection and waned at later st

n the liver. This may be because the liver is the site w. mansoniadult worms reside and lay their eggs and t

he local worm and egg antigen induced immunomodulaould differ from its systemic effects.

Another observation in this study was thatS. mansoniin-ection on its own was shown to give some degree of proion againstM. tuberculosischallenge during the early staf M. tuberculosisinfection. Resistance against intracellu

nfections operates in two stages; the early nonspecifiponses and the late acquired responses[24,25]. The nonspeific immune mechanisms play a major role during the ehases of infection while the acquired cellular responseshown to be the dominant mechanism controlling mycoerial infection at the later stages[27,28].

The observation thatS. mansoniinfection enhances Tesistance at early stages and impairs it at later stagesndicate that the impact ofS. mansoniinfection on TB im-

unity depends on the stage of infection. In other wo. mansoniinfection could have variable effects on earlyate and late acquired responses to mycobacterial infechis assertion is supported by the reports of Ferreiraoworkers who have found that the early stage responycobacterial challenge can be enhanced by injectingith Ascaris suumextract[26].It could be argued that the reduced BCG induced

ection against TB could be the result of inhibition of BCrowth (‘poor vaccine take’) in theS. mansoniinfected mice

al sensitization to worms on the effect of BCG vaccina15].

The aim of this work was to see the role of chronic wonfection on the outcome of BCG vaccination. The findiupport the hypothesis that helminthic infection could hsignificant negative impact on the efficacy of BCG v

ination against TB. If this finding holds true in humat may be of public health importance as it providesdditional explanation why BCG has such a poor imgainst pulmonary TB in the tropical resource poor settegions where helminthic infections are almost unive10].

cknowledgements

We thank Dr. Yohannes Negesse for assistance inxamination of histopathology slides. We are gratefuecilia Thors for providing us withSchistosomaegg anti-en. The study was financed by grants to S.B. & D.E fida/SAREC and the Sequella Global Tuberculosis Fou

ion, USA.

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