Immune mechanisms of responses to environmental mycobacteria

IMMUNEMECHANISMSOFRESPONSESTO
ENVIRONMENTALMYCOBACTERIA






HOPEIYING
(M.Sc,NUS)






ATHESISSUBMITTED


FORTHEDEGREEOFDOCTORATEOFPHILOSOPHY


DEPARTMENTOFMICROBIOLOGY


NATIONALUNIVERSITYOFSINGAPORE








2011




i
ACKNOWLEDGEMENTS

Funding for this research was received from grants funded by the Ministry of
EducationandMicrobiologyDepartmentVaccine Initiativeawardedto DrSeah
Geok Teng (2006 – 08), and from the National Research Foundation (2008 –
2011)undertheauspicesoftheSingapore‐MITAllianceforResearch and
Technology (SMART) Infectious Disease Interdisciplinary Research Group (ID‐
IRG)whereDrSeahGeokTengandthelateProfessorDavidBSchauer were
investigators.Mygraduatestudieswerefinancedbyascholarshipfundedbythe
MinistryofEducationthroughtheYongLooLinSchoolofMedicine Graduate
Programme,andpartiallybySMART.

Thishasbeenatoughandarduousjourney,andeverythinginthisthesiswould
nothavebeenpossibleifnotforthemany people whohaveso selflesslygiven
mesoundguidance,strongsupportandimmenseencouragementalongtheway.
Iowemydeepestgratitudetomysupervisorandmentor,DrSeahGeokTeng,for
herinvaluable guidanceand advice,her strongsupportin timesofuncertainty
andwhenIwasfarawayinaforeignland,andespeciallyfordevoting her
precioustimetomyprojectamidsthermultiplerolesasanadjunctprofessor,a
practicingdoctorandanewmother,inthelastlegofmyPhD.Ialsoextendmy
sincerethankstomylateco‐supervisor,ProfessorSchauer,forthefine
experienceatMIT,forgivingmemuchaidandadviceduringmystintinhislab

(Sept08‐Jul09),aswellassincereconcernovermywell‐being.Youwillalways
bedeeplyremembered.ItiswithpleasureandheartfeltgratitudethatIgivemy

ii
specialthankstomycolleagues,MrsThong,Wendy,ChaiLian,Irene,Nicola,Tse
Mien, Megan, Puk, Adrienne, Katie and Alex, who have given me much needed
support,especiallyonbadexperimentaldays,andkeptmesaneatonepointor
anotherthroughthecourseofmyPhD.MyspecialthanksalsotoMrYNChan,
MrsKTThong,ArekandSiewChinfortheirtechnicalhelpwithmanaging the
equipmentneeded for thisproject,including the flowcytometersand Luminex
machine.IfeelindebtedtomymanycolleaguesintheSGTlabat NUS, in the
SchauerlabatMIT,aswellasinSMART,particularlyCarmen,Joanne,WeiXing,
Arek,Isadora,SiewChin,HooiLinn,Maggie,DaHai,Lena,YieHou,LanHiong,
RashidiandFarzadforkindlygivingmemuchassistanceandhelp in multiple
waysalongtheway.Last,butnottheleast,Igivemywholeheartedthankstomy
entirefamilyfortheirstrong,unwaveringsupportandunderstandingduringmy
PhDpursuit,especiallytotheloveofmylife–myhusbandAaron,withoutwhom
Iwouldnothavemadeitsofar.




iii
SUMMARY

Human epidemiological studies suggest that poor efficacy of the tuberculosis
(TB) vaccine, Mycobacterium bovis bacilleCalmette‐Guérin(BCG),maybe
because of immuno‐modulatory effects of exposure to environmental
mycobacteria (Env). However, exactlyhowandwhythishappensremains
unclear.ThisstudyexaminedthehypothesisthateffectsofEnvsensitisationare

relatedtoinductionofregulatoryandcytotoxicTcells.Mycobacteriumchelonae
(CHE)sensitisationofBalb/cmicethroughvariousrouteswasusedasthemodel
system.

Heat‐killedCHE intra‐peritonealsensitisationinducedCD4
+
Tcellswhichlysed
BCG‐infected macrophages in vitro. The cytotoxicity was dependent on IFN‐,
perforinandFasL.Sensitisationwithanunrelatedbacteriumfailed to induce
cytotoxicity,thereforepriming ofT cellscross‐reactive withBCG, andnot non‐
specific inflammation, underlies the cytotoxicity. Sensitised mice had reduced
BCGviabilityinthelungsuponsubsequentinhalationchallenge;thiscanexplain
thereducedBCG‐inducedprotection.

BothIFN‐γandIL‐10wereincreasedinthelungsofCHE‐sensitisedmice,relative
tonaïvemice,afterBCGlungchallenge.Althoughthefrequencyofsystemic
CD4
+
CD25
+
cellswasunremarkableafterCHEsensitisation,adoptivetransferof
these Tregs to naïve mice followed by BCG challenge led to reduced lung
lymphocyte recruitment, reduced lung IL‐2 and increased systemic IL‐10

iv
production. This suggests functional suppression of local BCG responses by
CD4
+
CD25
+

TregsfromCHE‐sensitisedmice.

Memory responses after transient CHE lung colonisation led to increases in
TregsweeksafternoliveCHEwasrecoverable.Differentdosesof inhaledCHE
exposure were tested – higher doses induced stronger Treg responses and
weaker BCG‐specific IFN‐γ responses. Subsequent experiments used repeated
lowdoseliveintra‐trachealCHEexposuretomimicnaturalhumaninhalational
exposure, followed by subcutaneous BCG vaccination. Systemic IL‐10, mainly
produced by CD4
+
CD25
‐
FoxP3
+
 inducible Tregs, was increased and associated
withreducedfrequencyofIFN‐γproducingmemorycellsrecognising a BCG‐
specific epitope. Thus, adaptive Tregs also have a role in suppressing BCG‐
specificinflammationinCHE‐sensitisedmice.

To explore if post‐BCG CHE exposure had similar effects, BCG vaccination of
weanling mice was followed by low dose CHE intra‐tracheal exposures. This
mainlyinducednaturalTregs,withminimalIL‐10induction.Suppression of
inflammatory cell recruitment in the lungs to subsequent BCG lung challenge
was noted, associated with reduced lung chemokines, in spite ofelevated
systemicIFN‐γresponses.Therateofinflammatorycellrecruitmenttothelung
early in TB infection is increasingly recognised as the critical determinant of
effective immunity, more than systemic IFN‐γ responses. Thus, CHE exposure
evenafterBCGvaccinationcansuppressMycobacterium‐specificimmunity.



v
These two mechanisms proposed for effects of CHE exposure on BCG‐induced
immunityarenovel.Thisworkisalsothefirsttoprovideamechanistic
explanationforhowEnvexposuremodulatesanexistingBCGvaccineresponse.
This accounts for observations of lack of BCG‐induced protection in humans
livinginEnv‐prevalentareas,andsuggestshowprospectivecandidate TB
vaccines could be screened to avoid problems of BCG vaccine. It also explains
why even early neonatal BCG vaccination fails to provide long‐lasting effects
againstadultpulmonaryTB,withimplicationsforitscontinueduse.

Publicationsarisingfromthisthesis
Journalpapers:
1) Peiying,Ho,LinZhang,XingWeiandGeokTengSeah(2009).
MycobacteriumchelonaesensitisationinducesCD4
+
‐mediatedcytotoxicity
againstBCG.EurJImmunol39(7):1841‐9
2) Peiying, Ho, Xing Wei and Geok Teng Seah (2010). Regulatory T cells
induced by Mycobacterium chelonae sensitization influence murine
responsestobacilleCalmette‐Guérin.JLeukocBiol88:1073‐80.
ThisarticlewasfeaturedintheFrontlineScienceSectionofJLBas"Leading
EdgeResearch"withadedicatededitorialandpressrelease

Conferencepresentations:
1) Peiying,Ho,MeganMcBee,GeokTengSeahandDavidSchauer.M.
chelonae exposure post‐BCG vaccination suppresses BCG‐specific
responses. Poster presentation. International Congress of Mucosal
Immunology,July2009,Boston,USA.

vi

2) Effects of environmental mycobacteria post‐BCG vaccination. Oral
Presentation. Singapore MIT Alliance for Research and Technology
(SMART)Infectious Diseases Inter‐disciplinary Research Group (ID‐IRG)
Workshop,January2010,Singapore.Wonbestoralpresentationaward.
3) Peiying,Ho,Carmen,Low,Joanne,Kang,TseMienTan,NicolaLeungand
Geok Teng Seah. Mycobacterium chelonae sensitisation prior to BCG
vaccination induces regulatory T cells that suppress IFN‐ responses.
Poster presentation. SMART ID‐IRG Annual Workshop July 2010,
Singapore.

vii
TABLEOFCONTENTS
ACKNOWLEDGEMENTS i
SUMMARY iii
LISTOFFIGURES xvi
ABBREVIATIONS xviii
CHAPTER1–INTRODUCTION 1
1.1Theglobaltuberculosissituation 1
1.2Immuneresponsesofenvironmentalmycobacteriaexposureandeffects
onBCGvaccination 2
1.3Objectivesandscopeofproject 3
CHAPTER2–LITERATUREREVIEW 5
2.1Epidemiologyoftuberculosis(TB) 5
2.1.1Clinicaltuberculosis 5
2.1.2Bacterium‐hostimmuneinteractions 6
2.2ImmuneresponsestoTB:celltypesandtheirfunctions 7
2.2.1CD4cells 7
2.2.2CD8cells 9
2.2.3Tcells 10
2.2.4Naturalkiller(NK)cells 11

2.2.5Othercells 11
2.2.5.1CD1‐restrictedTcells 11
2.2.5.2Bcells
12
2.2.5.3Antigenpresentingcells 12

viii
2.3Relevancetovaccinedevelopment 13
2.4BCGasavaccine 13
2.4.1MeasuringBCGresponses 14
2.4.2BCGprotectiveefficacy 16
2.4.2.1Humantrials 16
2.4.2.2Experimentalmodels 17
2.4.3RoutesofBCGadministration 18
2.5Cell‐mediatedimmuneresponseswithBCGvaccinationandimmune
correlatesofprotection 19
2.5.1Thelpertype1CD4
+
responseandIFN‐responses 19
2.5.2CD8
+
Tcells 20
2.5.3RegulatoryTcell(Treg)responses 21
2.6ProblemswithBCGandnovelstrategiestoreplaceorimproveBCGasaTB
vaccine 22
2.7Environmentalmycobacteria(Env) 25
2.7.1ClassificationofEnv 26
2.7.2M.chelonae(CHE) 27
2.8ImmuneresponsestoEnv 27
2.9EffectsofenvironmentalmycobacteriaexposureonBCGvaccination 29

2.10RegulatoryTcells(Tregs) 32
2.10.1NaturalTregs(nTregs) 33
2.10.2NaturalTregsinTB 35
2.10.3AdaptiveorinducibleTregs(iTregs) 36
2.10.4IL‐10inTBdisease 37

ix
CHAPTER3–MycobacteriumchelonaesensitisationinducesCD4
+
‐mediated
cytotoxicityagainstBCG 38
3.1INTRODUCTION 38
3.2MATERIALSANDMETHODS 40
3.2.1Mice… 40
3.2.2Bacteria 40
3.2.3Preparationofheat‐killedandlivebacterialcultures 40
3.2.4MurineimmunisationandliveBCGchallenge 41
3.2.5Isolationofmurineperitonealmacrophages 42
3.2.6Isolationofmurinesplenocytesandlungtissue 42
3.2.7TrypanBlueexclusionassay 43
3.2.8Positivecellselectionusingmagneticbeads 43
3.2.9CytokineanalysisbyELISA 44
3.2.10Cytotoxicityassay 45
3.2.11Cytotoxicityassayexperimentalset‐up 45
3.2.12Flowcytometry 47
3.2.13Statisticalanalysis 47
3.3RESULTS 49
3.3.1Cellsubsetsinvolvedincytotoxicity 49
3.3.2Mediatorsofcytotoxicity 53
3.3.3Specificityofcytotoxicresponses 53

3.3.4EffectofEnvsensitisationonliveBCGinfection 56
3.4DISCUSSION 59
3.4.1CD4
+
TcellsinvolvedinCHE‐mediatedcytotoxicity 59
3.4.2CHE‐inducedcytotoxicitydependentonFasLandperforin 60

x
3.4.3CHE‐inducedcytotoxicityassociatedwithmycobactericidal
activity 61
3.4.4IFN‐dependentCHE‐mediatedcytotoxicity 62
3.4.5PotentialreasonsfordifferentialresponsestoEnvsensitisation 63
3.4.6Conclusion 65
CHAPTER4–EvidenceforregulatoryTcellactivityinMycobacterium
chelonaesensitisedmiceandfunctionalimpactofCD4
+
CD25
+
cellsonBCG
responses 66
4.1INTRODUCTION 66
4.2MATERIALSANDMETHODS 68
4.2.1Mice&Immunisation 68
4.2.2Bronchoalveolarlavage(BAL) 68
4.2.3Cellsortingandadoptivetransfer 68
4.2.4Co‐cultureproliferationsuppressionassay 69
4.2.5ELISA 70
4.2.6Flowcytometry 70
4.2.7Statistics 71
4.3RESULTS 72

4.3.1CHEsensitisationreducesIFN‐γandincreasesIL‐10production
withassociatedreducedlymphocyteactivity 72
4.3.2Reducedlunginflammatorycellsandincreasedlungcytokinesupon
BCGlunginfectionafterCHEsensitisation 73
4.3.3UnremarkablefrequencyofCD4
+
CD25
+
cellswithCHE
sensitisation 77

xi
4.3.4CD4
+
CD25
+
cellsfromsensitisedmicereduceIFN‐γproductionand
proliferationofco‐culturedeffectorcells 78
4.3.5AdoptivetransferofCD4
+
CD25
+
TregsfromCHE‐sensitisedmice
suppressesBCGresponses 82
4.4DISCUSSION 85
4.4.1NaturalandinducedTregswithCHEsensitisation 85
4.4.2UsageofdeadCHEforsensitisation 85
4.4.3QualitativesuppressiveactivityofCD4
+
CD25

+
Tregswithout
quantitativechanges 86
4.4.4Tregsmayaffectnon‐Tcelltypes 88
4.4.5PotentialmechanismsforCHE‐inducedTregactivity 88
4.4.6Conclusion 89
CHAPTER5–DifferentialeffectsofvaryingMycobacteriumchelonae
exposureparametersandeffectsofincreasedIL‐10producingregulatoryT
cellswithlowdoseinhaledexposure 91
5.1INTRODUCTION 91
5.2MATERIALSANDMETHODS 93
5.2.1Bacteriastrains,miceandimmunisationprotocols 93
5.2.2ELISpot 93
5.2.3ELISA 94
5.2.4Flowcytometry 94
5.2.5CytokineSecretionAssay 95
5.2.6Statistics 96
5.3RESULTS 97

xii
5.3.1DifferentialpersistenceofCHEinmurinelungsatdifferentintra‐
nasaldoses 97
5.3.2DoseandtimingofCHEexposureaffectssystemicIFN‐butnotIL‐
10responses 98
5.3.3HighdoseCHEinducedstrongerTregresponsesandweakerIFN‐
responses 100
5.3.4IncreasingcellularrecruitmenttolungovertimeafterCHE
inhalation 100
5.3.5LiveanddeadCHEinducesimilarlevelsofnTregsandIL‐10 103
5.3.6SuppressionofBCG‐specificmemoryIFN‐producingcellsbyCHE

exposurebeforeBCGvaccination 103
5.3.7SystemicincreaseinIL‐10producingcellswithCHEsensitisation
beforeBCGvaccination 108
5.3.8DifferentialphenotypeofIL‐10producingcellsinBCGvaccinated
micewithandwithoutCHEsensitisation 109
5.3.9ExpansionofnTregswithCHEsensitisationbeforeBCG
vaccination 109
5.4DISCUSSION 111
5.4.1HigherCHEdosesinducemorepro‐andanti‐inflammatory
responses 111
5.4.2CHEinducedresponsesnotdependentonviabilityofCHE
sensitisation 112
5.4.3Implicationsofdose‐dependentimmuneinductionwithCHE
exposure 113

xiii
5.4.4RoleofIL‐10inCHE‐mediatedsuppressionofBCGvaccine
response 114
5.4.5Conclusion 115
CHAPTER6–MycobacteriumchelonaeexposureafterBCGvaccination
reduceslocalinflammatorycellrecruitmentdespiteincreasingsystemic
BCG‐specificresponses 117
6.1INTRODUCTION 117
6.2MATERIALSANDMETHODS 119
6.2.1Mice… 119
6.2.2Immunisation 119
6.2.3ELISpot 120
6.2.4CytokineMultiplexArray 120
6.2.5Flowcytometry 121
6.2.6Statistics 122

6.3RESULTS 123
6.3.1Early,butnotlate,lungCHEexposureincreasedsystemicBCG‐
specificIFN‐responses 123
6.3.2BothIFN‐andIL‐10productionreducedatlate,relativetoearly
CHEexposure 125
6.3.3LungCHEexposuredidnotalterproportionsofCD4
+
Tregs 125
6.3.4Post‐vaccinationlateCHEexposureincreasedsystemicBCG‐specific
IFN‐responsesuponsecondaryBCGchallenge 126
6.3.5LateCHEexposureincreasedsystemicCD4
+
regulatoryTcellsin
vaccinatedmiceafterBCGchallenge 127

xi
v
6.3.6CHEexposurehadnoeffectonCD44
hi
CD62L
lo
memorycell
populations 130
6.3.7CHEexposurereducedlunginflammatoryinfiltrationupon
secondaryBCGexposure 132
6.3.8ReducedlevelsoflungcytokinesandchemokinesafterBCG
challengeinCHEexposedmice 134
6.4DISCUSSION 137
6.4.1MinimaleffectoforalCHE 137
6.4.2Cross‐reactiveboostingofsystemicIFN‐notsuppressedby

increasednTregfrequencyinCHEexposedmicepost‐BCG
vaccination 138
6.4.3ContrastwithM.vaccaemodelofIL‐10mediatedlungsuppressive
effects 139
6.4.4nTregexpansionwithCHEexposureafterBCGvaccinationexplains
suppressedlunginflammation 140
6.4.5DifferentialTregresponsesprimedwithpre‐versuspost‐BCG
vaccinationCHEexposure 141
6.4.6Conclusion 142
CHAPTER7–CONCLUSIONANDFUTUREWORK .144
7.1Keyfindingsandtheirimplications 144
7.2Limitations&futurework 146
7.2.1ExploringantigenspecificityofTregs 146
7.2.2EffectsofIL‐10–directroleinsuppression? 147
7.2.3EffectsonTh17andpolyfunctionalTh1cells 148
7.2.4SuppressiveeffectsofotherEnvspecies 148

x
v
7.2.5Improvingtheresearchmodel 149
7.2.6ImmunecorrelatesofBCGprotection 150
REFERENCES 152
APPENDIX 176

xvi
LISTOFFIGURES
Fig.3.1:SplenocytesofCHE‐sensitisedmicearecytotoxictoBCG‐infectedcells
uponrestimulationwithCHEorBCG 51

Fig.3.2:Effectofcellsubsetenrichmentoncytotoxicactivity. 52


Fig.3.3:RoleofFasL,perforin,IFN‐andIL‐10inCHE‐mediatedcytotoxicity 54

Fig.3.4:CytotoxicityisspecifictoCHEandnotnon‐specific 55

Fig.3.5CellsexpressingperforinorIFN‐ininfectedmouselungs 57

Fig.3.6:CytokineproductionandBCGcountsafterBCGinfectioninvivo 58

Fig.4.1:CytokineproductionbysplenocytesfromCHE‐sensitisedmice 74

Fig.4.2:ResponsetoliveBCGafterCHEsensitisation 76

Fig.4.3:FrequencyofCD4
+
CD25
+
andFoxP3
+
cellsinCHE‐sensitisedmice 77

Fig.4.4:PresenceandfunctionalactivityofCD4
+
CD25
+
regulatoryTcellsfrom
CHE‐sensitisedmice 80

Fig.4.5:ImmuneresponsetoBCGinadoptivetransferrecipientmice 84


Fig.5.1:PersistenceofCHEinthelungsanddosedependentsplenicIFN‐but
notIL‐10responses 99

Fig.5.2:FrequencyofCD4
+
CD25
+
CD3
+
TcellsinthelungsandCD25
+
GITR
+
CD4
+

cellsinthespleensofCHE‐sensitisedmice 101

Fig.5.3:RecruitmentofinflammatorycellstothelungsofCHE‐sensitised
mice 102

Fig.5.4:EffectsofCHEviabilityonTregandcytokineresponses 105

Fig.5.5:ReducedfrequencyofBCGspecificIFN‐producingcellsinvaccinated
micewithpriorCHEsensitisation 106

Fig.5.6:ReducedfrequencyofIFN‐producingepitopespecificmemorycellsin
vaccinatedmicewithpriorCHEsensitisation 107

Fig.5.7:HigherfrequencyofIL‐10producingadaptiveTregsinBCG‐vaccinated

micewithpriorCHEexposure 108

Fig.5.8:IncreasedfrequencyofFoxP3
+
andCD25
+
FoxP3
+
CD4
+
Tregin
vaccinatedmicewithpriorCHEexposureuponBCGstimulation. 110


xvii
Fig.6.1:Intra‐trachealCHEexposurereducesBCG‐specificIFN‐secretingcells
inthespleen 124

Fig.6.2:LateexposuretoCHEincreasesBCG‐specificIFN‐secretingcellsafter
BCGlungchallenge. 128

Fig.6.3:LateCHEexposureincreasesproportionofsystemicCD4
+
Tregspost‐
BCGchallenge 129

Fig.6.4:CHEexposurehadlittleeffectonmemorycellpopulations 131

Fig.6.5:CHEexposuredecreasesTcellandmacrophagerecruitmenttothelungs
uponlungchallengewithBCG 133


Fig.6.6:InflammatorymediatorselevatedinlungtissueuponBCGlungchallenge
aredecreasedbypriorexposuretoCHE 135


xviii
ABBREVIATIONS

Ab   Antibody
BAL   Bronchoalveolarlavage
BCG   bacilleCalmette‐Guérin
BSA   Bovineserumalbumin
CHE   Mycobacteriumchelonae
CTL   CytolyticTlymphocyte
DC   Dendriticcell
DDAO‐SE  dodecyldimethylamineoxide‐succinimidylester
DOTS   Directlyobservedtreatment,short‐course
DTH   Delayedtypehypersensitivity
ELISA   Enzyme‐linkedimmunosorbentassay
ELISpot  Enzyme‐linkedimmunospotassay
Env   Environmentalmycobacteria
FAC   Ferricammoniumcitratesupplement
FBS   Foetalbovineserum
FoxP3   Forkhead‐boxP3
HBSS   Hank’sbalancedsaltsolution
HIV   Humanimmunodeficiencyvirus
IFN‐   Interferongamma
IL   Interleukin
i.p.   Intra‐peritoneal
i.n.   Intra‐nasal


xix
i.t.   Intra‐tracheal
iTreg   Inducible(oradaptive)regulatoryTcell
mAb   Monoclonalantibody
MHC   Majorhistocompatibilitycomplex
Mtb   Mycobacteriumtuberculosis
NK   Naturalkillercell
nTreg   NaturalregulatoryTcell
OADC   Oleicacid‐albumin‐dextrose‐catalaseenrichment
PBS   Phosphate‐bufferedsaline
PMA   Phorbolmyristateacetate
PPD   Purifiedproteinderivative(ofM.tuberculosis)
RAG   Recombinationactivatinggene
SD   Standarddeviation
SEM   Standarderrorofthemean
TB   Tuberculosis
TGF‐  Transforminggrowthfactorbeta
Th1   Thelpertype1
Th2   Thelpertype2
TNF‐  Tumournecrosisfactoralpha
Treg   RegulatoryTcell
Tr1   Type1regulatoryTcell
WHO   WorldHealthOrganisation


1
CHAPTER1–INTRODUCTION

1.1Theglobaltuberculosissituation

Tuberculosis (TB) has been declared a global emergency by the World Health
Organisation(WHO)(WHO,2003).It is estimatedthatone‐thirdoftheworld’s
populationisinfectedannually,andin2009therewasanestimated9.4million
cases of TB globally, with an approximate 1.3 million people dying from TB
(WHO,2010).Althoughthereisahighcureratewithappropriateanti‐TBdrugs,
the failure to stem the tide with drugs is related to numerous social, public
health and pharmacological problems, and therefore, effective vaccination
remainstheholygrailinTBeradication.

MembersoftheMycobacteriumtuberculosis(Mtb)complex,whichcomprisesof
Mtb,M.bovis,M.microti,M.africanumandM.canettii,causehumanTB(Cosma,
2003). A highly genetically‐related species, M. bovisbacilleCalmette‐Guérin
(BCG),hasbeenusedasaliveattenuatedvaccineforalmostacentury,andthere
is no clinically available alternative at present. However, the exact immune
mechanisms through which BCG protection is conferred remain unclear. More
importantly,theprotectiveefficacyofBCGagainstadultpulmonaryTB,whichis
themostprevalentformofTB,rangesfrom0‐80%acrossdifferentpartsofthe
world (Fineand Vynnycky,1998).  Strain differences,geographicalfactors and
reinfectionpathways(Smith,2000;Fine,2001a)havebeensuggestedascauses,
butthemostwidelysupportedhypothesisforthefailureofBCG is that prior
exposuretoenvironmentalmycobacteria(Env)affectshowthehostrespondsto

2
the vaccine (Black, 2001a; Brandt, 2002; Buddle, 2002; de Lisle, 2005; Lalor,
2009).However,detailsonhowthishappenshaveyettobefullyelucidated.

1.2Immuneresponsesofenvironmentalmycobacteriaexposureand
effectsonBCGvaccination
Prior exposure to certain Env species blocks the replication ofBCG(Brandt,
2002;Demangel,2005)ormodifiesthenatureofimmunityinduced by BCG

(Young,2007).ThepersistenceoftheEnvspeciesinthehostandtherelatedness
betweentheprimingEnvstrainandBCGhavebeensuggestedasfactors
influencing these effects. Env exposure following BCG vaccination can also
modulate protective immunity generated by BCG in mice (Flaherty, 2006).
Infants with delayed BCG vaccination, allowing time for Env exposure, have
poorerIFN‐γresponsestoBCGantigensaftervaccination(Burl, 2010) and yet
early neonatal vaccinationof thoseliving inareas withhigh prevalence of Env
sensitisation stillresult inpoorer subsequent IFN‐γ responsestoMtbantigens
(Lalor,2009).ThesestudiesprovidehumanepidemiologicalevidencethatEnv‐
inducedimmunomodulationcouldoccurbothbeforeandafterBCGvaccination.

Preliminarystudiesinourlaboratoryonhumanperipheralbloodcellsfoundthat
whenlymphocyteswerestimulatedwitheachofthetencommonestEnvspecies
isolated in Singapore, there was strongest cytolytic activity against autologous
BCG‐infected monocytes with Mycobacterium chelonae(CHE)stimulation,
implyingbest cross‐reactivecytotoxicity (Zhang Lin,MSc thesis).This suggests
that CHE priming had strongest potential to influence BCG responses.This

3
outcome was subsequently replicated in mice after in vivo intra‐peritoneal
sensitisationwithvariousEnvspecies,andagainCHEexposureledtostrongest
cytotoxic responses against BCG‐infected autologous macrophages (Ho, 2009).
ThisledtoourchoiceofusingCHEsensitisationasamodelforEnvexposurein
our murine studies on Env‐induced effects against BCG but in this project,
additionalroutesanddosesofCHEwereused.

1.3Objectivesandscopeofproject
ThisprojectevaluatedtheimmunemechanismsunderlyinghowexposuretoEnv
affectshostresponsestoBCGvaccine.Therationalewasthatunderstandingwhy
BCG fails will allow development of vaccines that do not have similar pitfalls.

Thisisespeciallypertinentgiventhatseveralvaccinesinthe pipeline are
actuallymodifiedstrainsofBCG(Kaufmann,2010b).

This work investigated the hypotheses that Env sensitisation induced both
regulatory T cells and cytotoxicity mechanisms, which ultimately reduced
immunityinducedbyBCGvaccination.SensitisationofBalb/cmicewithCHEvia
variousrouteswasthemodelforEnvexposureinthisproject.Thebroadaimsof
theprojectwere:
1) InCHE‐sensitisedmice,toanalysetheroleofdifferentcelltypes,
cytokinefactorsandmediatorsofcytotoxicityonthehost’sresponsesto
BCG,andtheirsubsequentimpactonBCGsurvival.
2) ToinvestigatetheroleofCD4
+
CD25
+
regulatoryTcells(Tregs)from
CHE‐sensitisedmiceinsuppressionoftheBCGresponse,through

4
studyingtheimmunomodulatoryeffectsofsuchcellsinvitroandinvivo
uponBCGchallengeafteradoptivetransferintonaïvemice.
3) Toinvestigatehowvariationsonmultiplesensitisationparameters
(dose,timing,in vivopersistenceofCHEandviabilityofCHE)affectTreg
phenotypesand systemic Th1responses induced byCHE sensitisation,
beforeandaftersubsequentBCGvaccination.
4) InamodelofBCGvaccinationofweanlingmicethatmimicshuman
neonatal BCG vaccination, to characterise the influence of post‐
vaccination CHE exposure on local and systemic cytokines, Tregsand
cellularrecruitment,beforeandafterinvivoBCGchallenge.


5
CHAPTER2–LITERATUREREVIEW

2.1Epidemiologyoftuberculosis(TB)
Tuberculosis (TB) in humans is caused by Gram‐negative bacteriacollectively
known as Mycobacterium tuberculosis complex, amongst which the most
prevalentspeciesisMycobacterium tuberculosis(Mtb)(Dye,2006).Thisdisease
hasbeendeclaredaglobalemergencybytheWorldHealthOrganisation(WHO)
since1993.TBcausesanestimated2milliondeathsannually,andone‐thirdof
the world’s population is latently infected, with the potentialforreactivation
(WHO, 2010). Majority of the cases of TB occur in Asia and Africa, with the
highestTBincidenceinIndia,China,SouthAfrica,NigeriaandIndonesia(WHO,
2010).InSingapore,TBisofmoderateendemicity,withnear1500 new cases
eachyear,morethanhalfbeingelderlymales.Avastmajorityofthesecasesare
pulmonary TB and less than 0.5% involve multiple drug resistant(MDR)TB
(MinistryofHealth,2010).

2.1.1Clinicaltuberculosis
Clinically,TBdiseaseexistsintwomainforms–pulmonaryandextrapulmonary
TB,theformerbeingmoreimportantepidemiologicallyasasourceofinfectious
Mtb(Dye,2006).PatientsdiagnosedwithpulmonaryTBtypicallyhaveachronic
cough,feverandweightloss.ExtrapulmonaryTBcaninvolveanyorgan,but
lymphadenitis and pleuritis with effusion are most common, while miliary
disease and meningitis are the most fatal forms. The spread of TB infection
occurs through the inhalation of airborne droplets of respiratory secretions