THE DEVELOPMENT OF A CANDIDATE TUBERCULOSIS
DNA VACCINE EXPRESSING MtbS.4 AND Ag858 of
Mycobacterium tuberculosis
by
MARYAM AZLAN
Thesis submitted
infulfillment
of therequirements
for thedegree of
Master ofScience
February 2007
DEDICATIONS
This thesis is specially dedicated
to:My beloved husband, Ahmad Zarizi b. Shaari My
sons,Aiman Haris and Aiman Hakimi My parents, Dr. Azlan and Dr. Kamariah
Thank you for your love, support
andpatience
...May God bless you all..
..ACKNOWLEDGEMENTS
Inthe name of
Allah,
the most Generous and the most Merciful. Allpraise
is due toAllah,
forgiving
meinspiration
and stoutheartednessalong
thisjourney.
During
this researchproject,
there are severalpeople
involveddirectly
orindirectly
whom I wish to
acknowledge
in this section.I would like to thank my
supervisor,
Prof. Norazmi Mohd. Nor for hissupport,
excellent
guidance
andsupervision throughout
the researchproject
and alsoduring
thewriting
of this thesis. I wish to thank him for his trustee and confidence in me to carry out thisproject.
Hisguidance
isgreatly appreciated.
I would like also to thank Assoc. Prof. Dr. Nik Soriani
Yaacob,
Prof. Zainul F.Zainuddin,
Dr. Shaharum Shamsuddin and Dr.Rapeah Suppian
who haveprovided advice, guidance,
comments andhelpful
discussionsduring
thisstudy.
A
special
thanks to my friends andcolleagues
in thelaboratory especially, Teo, Rahimah, Asma, Rafeezul, Boonyin, Kenny, Zila, Ayu, Syam
and K. Rosilawani. Not toforget,
my friends who are nolonger
in this group but havepreviously participated
incontributing
to mywork,
thank you toHalisa,
Dr.Zul,
K.Rozilawati,
K. NikNorliza, Arifin,
Dr. Mohammed Abd. Aziz
Sarhan,
Dr.Fang
Chee Mun andWong
Vic Cern. I would like to thank my friends in ZFZ and S5 group,Eza, Abdah, Suwaibah,
K.Salwana, Ayuni, Nurul,
Zura, Aniek, Tini,
Bad andVenugopal.
My deepest appreciation
will be to myparents especially
my belovedhusband,
Ahmad Zarizi for hisgreatest support. patience,
love andencouragement.
Thank you foralways being
there for me.My appreciation
also goes to my beloved sons, Aiman Haris and AimanHakimi;
their mischievousness hadalways
cheered me. Aspecial
thanks to myparents,
Dr. Azlan and Dr.Kamariah,
my brother and sisters for theirsupport
andguidance during
my work. I would like also to thank myparent in-law, Hj.
Shaari and Pn.Noriah,
mybrothers and sisterin-law for their
understanding
andsupport.
Finally,
I would like to thankpeople
who aredirectly
orindirectly
contributed to mywork,
inparticular,
Mr. Jamaruddin Mat Asan whoprovided
technical assistance in flowcytometry handling,
students and staff ofPPSK,
INFORMM andMicrobiology department.
I cannotmention you all
here,
so Ihope
you could feel mygratitude.
TABLE OF CONTENTS
Page
ACKNOWLEDGEMENTS TABLE OF CONTENTS LIST OF TABLES LIST OF FIGURES
LIST OF ABBREVIATION ABSTRACT
ABSTRAK
iii
v x
xi xii xiv
xv
CHAPTER ONE: LITERATURE REVIEW
1.1
History
of tuberculosis 11.2 Disease burden 2
1.3
Mycobacterium
tuberculosisinfectlon
41.4
Diagnosis
61.5
Symptoms
and treatments 91.6 Immune response
against
TB 101.6.1
Macrophage
121.6.2 Cellular immune response 14
1.6.3 Humoral immune response 19
1.7 BCG - the current vaccine 21
1.7.1
Efficacy
and effectiveness of BCG 221.7.2
Advantages
ofBCG 231.8 Candidate
antigens
of M. tuberculosis 241.8.1 Mtb8.4 24
1.8.2
Ag85B
241.9
Experimantal
vaccinesdeveloped against
TB 251.9.1 DNA vaccine 26
1.9.1.1 Mechanisms of immune stimulation 26
1.9.1.2 Mechanisms of DNA vaccination 28
1.9.1.3
Advantages
ofDNAvaccination 331.9.2 Recombinant BCG
expressing heterologous antigen
361.9.3 Prime-boost
approach
1.1 OObjectives
of thestudy
39 42
CHAPTER TWO: MATERIALS AND METHODS
2.1 Materials
2.1.1
Mycobacteria
and Eschericia coli(E. colt)
strains2.1.2 Plasmids
2.1.3 Chemicals and
reagents
2.1.4 Kits and consumables2.1.5
Antibodies,
enzymes andlaboratory equipment
2.1.6 Mice
2.1.7
Sterilised,
deionised distilled water 2.2Preparation
ofmedia,
buffers and solutions2.2.1 Luria-Bertani
(LB)
broth2.2.2 Luria-Bertani agar
(LA)
2.2.3 7H9 broth 2.2.4 RPMI media
2.2.5
Kanamycin
stock solution(50mg/ml)
2.2.6
Ampicillin
stock solution(50mg/ml)
2.2.7
Magnesium
chloride(MgCI2)
solution(100mM)
2.2.8 Calcium chloride
(CaCI2)
solution(100mM)
2.2.9
Glycerol
solution(80%)
2.2.10Ethanol solution
(70%)
2.2.11
Hydrochloride
solution(HCI)
solution(1 M)
2.2.12 Sodium
hydroxide (NaOH)
solution(3M)
2.2.13
Ethylene
diaminetetraacetic acid(EDTA)
solution(O.5M)
2.2.14 Tris-EDTA
(TE)
buffer2.2.15 Tris-acetate-EDTA
(TAE)
solution2.2.16
Loading dye
solution2.2.17 DNAmarker
2.2.18 Ethidium bromide solution
2.2.19
Isopropyl-D-thiogalactopyranoside (IPTG) (1 M)
44 44 44 44 48 48 48
48 52 52 52 52 53 53 53 53 54 54 54 54
54 55 55 55 55 55
2.2.20 Tris-base
(1.5M) containing
0.4% 50S 562.2.21 Tris-Hel
(1.5M) containing
0.4% 50S 562.2.22
Resolving
buffer 562.2.23
Stacking
buffer 562.2.24Ammonium
persulfate
solution(20%)
562.2.25 SOS-PAGE
running
buffer 572.2.26
Sample
buffer 572.2.27 Coomassie blue solution 57
2.2.28
Destaining
solution 572.2.29 Towbin transferbuffer 58
2.2.30
Blocking
solution(5%)
582.2..31
Phosphate
buffered saline(PBS) (10X)
582.2.32 PBS-Tween 20
(PBS-T20)
582.2.33
Washing
buffer(Buffer C)
582.2.34 Elution buffer
(Buffer
D & BufferE)
592.2.35
Dialysis
buffer 592.2.36
[melhyl-3H] Thymidine
solution 592.2.37
Trypan
blue solution(0.4%)
592.2.38
Staining
bufferfor flowcytometry
602.2.39 Ammonium chlorideI
potassium (ACK) lysis
solution 602.2.40
Coating
bufferforELISA 602.2.41
Blocking
buffer for ELISA 602.2.42 ABTS substrate 61
2.2.43
Stop
solution 612.3 Methods
2.3.1
Preparation
of E. colicompetent
cells 612.3.2 Transformationof
competent
E. coli cells 622.3.3
Glycerol
stock of E. coli 622.3.4 BeG and recombinant BCG
(rBCG)
culture 632.3.5
Preparation
forPolymerase
chain reaction(PCR)
2.3.5.1
Preparation of oligonucleotides working
solution 632.3.5.2
Preparation
ofprimer working
solution 642.3.5.3
Preparation
of 'master mix' for PCR 642.3.6
Assembly
PCR 642.3.7 DNAagarose
gel electrophoresis
2.3.7.1
Preparation
of agarosegel
2.3.7.2
Separation
of DNA onagarosegel elctrophoresis
2.3.8 Extraction of
plasmid
DNA2.3.9 DNA extraction from agarose
gel
2.3.10 DNA
purification
2.3.11 Plasmid extraction for removal ofendotoxin 2.3.12 Restriction enzyme
(RE) digestion
2.3.13 Quantification of DNA 2.3.14 DNA
Ligation
2.3.15 Protein
analysis
2.3.15.1
Expression
of Mtb8.4in E. coli2.3.15.2
Preparation
ofresolving gel (10%)
2..3.15.3
Preparation
ofstacking gel (4.5%)
2.3.15.4 Sodium
dodecyl sulphate-polyacrylamide gel elctrophoresis (SOS-PAGE)
65 65 65 66 67 67 68 69 70 70 70 70 71 71 72
2.3.15.5Western
blotting
2.3.15.6
Quantification
ofprotein
concentration 2.3.15.7Purification of6XHis-tagged protein
2.3.15.8
Dialysis
ofpurified protein
2.3.16
Immunogenicity
studies2.3.16.1 Immunization
procedure
2.3.16.2 Collection ofsera
2.3.16.3
Splenocyte preparation
2.3.16.4 Cell culture
2.3.16.5Cell surface and intracellular
cytokine
assay 2.3.16.6 Proliferation assay2.3.16.7
Enzyme-linked
immunosorbent assay(ELISA)
72 73 74 74
75 77 77 78 78 79 80
CHAPTER THREE: RESULTS
3.1 Introduction
3.2 Construction ofthe DNA
vaccine, pNMN023
3.3 Purification of Mtb8.4
81 84 84
3.4
Immunogenicity
studies3.4.1 DNA vaccine
3.4.1.1 Proliferation assay of mice
splenocytes
immunized with DNA vaccine89 89 89
3.4.1.2
Antibody
response ofmice immunized with DNA vaccine89
3.4.1.3 Detection ofintracellular
cytokines produced by CD4+
91T cells and
CDS+
T cells fromsplenocytes
ofmice immunizedwith
pNMN023
3.4.2 Prime-boost
approach
1013.4.2.1
Antibody
response of mice immunized with theprime-
101boost
approach
3.4.2.2 Detection ofintracellular
cytokines produced by CD4+
101T cells and
CDS+
T cells fromsplenocytes
of mice immunized with theprime-boost approach
CHAPTER FOUR: GENERAL DISCUSSION 109
BIBLIOGRAPHY 119
APPENDICES 139
LIST OF TABLES
Page
Table 1.1
Comparative analysis
of various vaccineformulations 34Table 1.2 rBCG as vaccine candidates 37
Table 2.1 List of
general
chemicals andreagents
45Table 2.2 List ofkits and consumables 47
Table 2.3 List ofantibodies 49
Table 2.4 Listofenzymes 50
Table 2.5 List of
equipment
51Table 2.6 Immunization schedules of the DNA vaccine and the 76
prime-boost approach
Table 3.1 List of
peptides
for stimulation ofsplenocytes
90 Table 3.2 Classification ofresponses based onthe increase in 95the
percentage
of cellsexpressing
selectedintracellular
cytokines by
flowcytometry
Table 3.3
Summary
of thepercentage
of cellsexpressing
100cytokines
as determinedby
flowcytometry
and theSIof cells in the
proliferation
assayTable3.4
Summary
of thepercentage
ofcellsexpressing
106selected
cytokines following
immunizations with theprime-boost approach
LIST OF FIGURES
Page
Figure
1.1 Tuberculosis notificationrates,
2004 3Figure
1.2 Outcomes associated with exposureto M. tuberculosis 5Figure
1.3 AFB smear 7Figure
1.4 Fourstages
ofpulmonary
TB 11Figure
1.5CD4+
Thelper lymphocyte
subsets uponactivation
15Figure
1.6Pathways
associated with MHC class I 27Figure
1.7 Mechanismsof DNA vaccination 29Figure
1.8 Prime-boost vaccinationstrategies
40Figure
1.9 Flowchart of thestudy
43Figure
3.1 Amino acid sequence ofTB 1.0fragment
82Figure
3.2pVAX1
Plasmid map 83Figure
3.3Agarose gel electrophoresis
ofassembly
PCR 85Figure
3.4 Schematicdiagram
of the construction ofpNMN023
86Figure
3.5pPROExHTa™
andpNMN022 plasmid
map 87Figure
3.6 50S-PAGE and Western blotanalyses
88Figure
3.7 Mean 00 of total serumIgG
in mice(DNA vaccine)
92Figure
3.8 Mean 00 ofIgG
subclasses in mice(DNA vaccine)
93Figure
3.9Examples
offlowcytometry profiles
94Figure
3.10Percentage
ofC04'"
andCD8'" expressing
IL-2(DNA
97vaccine)
Figure
3.11Percentage
ofC04+
andC08+ expressing
IL-4(DNA
98vaccine)
Figure
3.12Percentage
ofCD4+
andCDS+ expressing IFN-r (DNA
99vaccine)
Figure 3.13
Mean 00 of total serumIgG
in mice(Prime-boost)
102Figure
3.14 • Mean 00 ofIgG
subclasses in mice(Prime-boost)
104LIST OF ABREVIATIONS
AFB
af3 Ag8S
APes BCGJ32-m
CMI CFU CTL ddH20 DTH DCs DNA ER
y8
HIV HLA IFN IL i.m
Lp
kOa KO LB MHC mAbs MOR-TB NAA NK
Nramp
0.0
Acid fast bacillus
Alpha
betaAntigen
85Antigen presenting
cellsBacille Calmette Guerin
Beta-2-microglobulin
Cell mediated
immunity Colony forming
unitCytotoxic
TIym phocyte
Deionised distilled water
Delayed type hypersensitivity
Dendritic cells
Deoxyribonucleic
acidEndoplasmic
reticulumGammadelta
Human
Immunodeficiency
VirusHuman
leukocyte antigen
Interferon Interleukin Intramuscular
Intraperitoneal
kilodalton Knock-out Luria-bertani
Major histocompatibility complex
Monoclonal antibodies Multi
drug
resistant TBNucleic acid
amplification
Natural killer
Natural-resistance-associated
macrophage protein
Optical density
PBMC
Peripheral
blood mononuclear cell PCRPolymerase
chain reactionPPD Purified
protein
derivativerBCG Recombinant bacille Calmette Guerin RNI Reactive
nitrogen
intermediates ROIReactive
oxygen intermediatesRD Region
ofdifference
RE Restriction enzyme
SIV Simian
immunodeficiency
virusSI Stimulation index
Th T
helper
TAP
Transporter
associatedprotein
TB Tuberculosis
TNF Tumornecrosisfactor
UV Ultraviolet
WHO World Health
Organization
THE DEVELOPMENT OF A CANDIDATE TUBERCULOSIS DNA VACCINE EXPRESSING MtbS.4
andAgSS8
ofMycobacterium tuberculosis
ABSTRACT
Tuberculosis
(TB)
is still one ofthemajor
healthproblems
worldwide. Theonly
TB vaccinecurrently
available is an attenuated strain ofMycobacterium bovis,
bacille ealmette Guerin(BeG). However,
theefficacy
of BeG vaccine continues to be debated.Therefore,
a moreeffective
vaccineagainst
TB isurgently
needed. DNA vaccination is a newapproach
tothecontrol of infectious
agents.
In thisstudy,
a DNA vaccineencoding
the candidate TBantigens
Mtb8.4 andAg85B
wasdeveloped using assembly
peR. Balb/c mice wereimmunized
intramuscularly
with 50 JAg of the DNAvaccine, pNMN023, containing
the twoantigens.
in eachhindleg. Reactivity against
theAg85B peptides,
P1 and P3 as well asMtb8.4 showed a consistent Th1
type
of immune responseby
virtue of the increasedexpression
ofIL-2, IFN-y
andIgG2a. Splenocytes
from immunized micewere also found toproliferate
moreaggressively
when stimulated with theantigens compared
to the vectoralone. In order to
improve
the vaccineefficacy,
apreliminary prime-boost approach
wasused.
Priming
withpNMN023
andboosting
with recombinant BeG(rBeG)
in Balb/c micewas carried out. Flow
cytometric
intracellularcytokine analyses
ofsplenocytes
from miceimmunized with the DNA-rBeG
prime-boost regime
showed that bothCD4+
andCD8+
Tcells showed an increase in IL-2 and
IFN-y production following
stimulation with eitherantigens
atsignificantly higher
levels than those immunized with rBeG-DNAprime-boost.
In
conclusion,
the data obtained from thisstudy suggest
that DNA vaccination in combination with theprime-boost approach provide
apotential strategy
fordeveloping
acandidate vaccine
against
TB.PEMBANGUNAN CALON
DNAVAKSIN
TERHADAPTUBERKULOSIS YANG MENGEKSPRESKAN
MtbS.4 danAgS5B DARIPADA
Mycobacterium
tuberculosisABSTRAK
Tuberkulosis
(TB) merupakan
salah satupenyakit
utama di dunia.Satu-satunya
vaksin TByang
terdapat pada
masa ini ialah strain yang telah dilemahkaniaitu, Mycobacterium
bovisbacille Calmette-Guerin
(BCG). Bagaimanapun,
keberkesanan BCG masihdiperdebatkan.
Oleh
itu,
vaksin yang lebih efektifterhadap
TBsangat diperlukan.
Vaksin DNAmerupakan
salah satu cara untuk
mengawal ejen
infeksi. Di dalamkajian ini,
vaksin DNA yangmengkodkan antigen
TBiaitu, antigen
Mtb8.4 danantigen AgS5B
telahdibangunkan menggunakan
kaedah PCRhimpunan.
Mencit Balble telah diimunisasi intraototdengan
50 J.lg vaksinDNA, pNMN023,
yangmengandungi
kedua-duaantigen.
Kereaktifanterhadap peptida AgS5B,
P1 dan P3juga
MtbS.4 telahmenunjukkan peningkatan
tindakbalas imunjenis
Th1 yang konsisten melaluipeningkatan pengekspresian IL-2, IFN-y
danIgG2a.
Splenosit
dari mencit yang diimunisasijuga didapati menunjukkan peningkatan gerak
balas
proliferasi apabila dirangsang dengan
kedua-duaantigen.
Untukmeningkatkan
keberkesanan
vaksin, kajian
awalmenggunakan pendekatan 'prime-boost
telahdigunakan. 'Priming dengan pNMN023
dan'boosting dengan
BCG rekombinan(rBCG)
didalam mencit Balble telah
dijalankan.
Analisis intrasel sitokin darisplenosit
mencit yang telah diimunisasidengan
DNA-rBCGmenunjukkan peningkatan
IL-2 danIFN-y
kedua-dua sel TCD4+
danCDS+ apabila dirangsang dengan
kedua-duaantigen berbanding
mencityang diimunisasi
dengan
rBCG-DNA.Sebagai kesimpulan,
datayang diperolehi
darikajian
ini
mencadangkan
bahawa vaksin DNAdigabungkan dengan
kaedah'prime-boost merupakan
salah satu kaedah yangberpotensi
untukmembangunkan
calon vaksin·terhadap
TB.CHAPTER 1
LITERATURE REVIEW
1.1 History of tuberculosis
Tuberculosis
(TB)
in humans is causedby Mycobacterium
tuberculosis while M.bovis causes TB infection in cattle. The
Hippocratic
Collectioncompiled
around 400 � 350B.C. recorded the clinical manifestations and
epidemiologic
features ofphthisis (Greek term),
the tuberculousprocess
in thelungs
wascalled
a'phyma' (Iseman, 2000).
Thefrequency
of unearthed skeletons withapparent
tubercular deformities in ancientEgypt suggests
that the disease was common among thatpopulation.
Evidence of bone lesionssuggestive
ofTB in mummies of North America andEgypt
confirms the ancientimpact
ofthis disease on
early
civilizations(Nerlich
etai., 2000;
Rothschild etai., 2001)
and further confirmedby
the use of molecular-baseddiagnosis
of TB in some ancientEgyption
mummies
(reviewed by Bedeir, 2004).
During
thegolden
age ofIslam,
Ibnu Sina described the clinical features andpathology
ofTB in Arabic
scripts (reviewed by
Madkour etai., 2004).
Thediscovery
ofsimilarly
deformed bones in various Neolithic sites in
Italy, Denmark,
and countries in theMiddle
East also indicates
that
TB was foundthroughout
the worldapproximately 4,000
yearsago.
In the 18th
century, TB
was well established inEurope
and hadspread
toAfrica, Asia, South
America and EasternEurope by
the end of the 19thcentury.
In1882,
Robert Kochdiscovered tubercle
bacillus
as the causativeagent
of TB. In1993,
due to theemergence
.of TB incidenceworldwide,
TB was declared as a'global emergency' by
the World HealthOrganization (WHO)
and a decadelater,
the first international conference on 'TB vaccine for the world' was held in Montreal.1.2 Disease
burden
It is estimated that two billion
people (one
third of theworld'spopulation)
is infected with M. tuberculosis(WHO, 2001),
where 8.8 millionpeople
will show clinical diseases and 1.5 million will die every year(WHO, 2004).
TB also occurs in Southeast Asia with three million new cases every year and aquarter
of a million in EasternEurope (Girard
etai., 2005).
These situations are worsened with the estimation thatonly
40% of new cases ofpulmonary
TB arecurrently
detected(Dye
etal., 2002).
Ifcontrolling
efforts are notaccelerated,
10 million newTB cases areexpected
in 2010(Dye, 2000).
Rising
rates ofdrug-resistant
TB have contributed to worsen treatment outcomes in someregions (Figure 1.1).
The incidence of TB increased in areas withhigh
rates of humanimmu,:,odeficiency
virus(HIV)
infection.Approximately,
14 millionpeople
are co-infected with M. tubercutosis andHIV, including
more than 70% of thoseliving
in someregions
ofsub-Saharan Africa
(WHO, 2004).
An initiative to address the increase of TB disease burden known as
UStop
TB" wascreated in 1998 to ensure that endemic countries are
adequately supported by technically
and
financially
to control TB(Raviglione
&Pio, 2002). Among
thesupports
include the US National Institute forAllergy
and Infectious Diseases(NIAID),
theAeras Global TB VaccineFoundation,
theEuropean
Union Commission andpharmaceutical
manufacturersincluding
GlaxoSmithKline
(GSK)
and IDRI-Corixa(Hewinson, 2005).
w
Notified T8
cases(new and relapse)
per 100000
o
population
0·24 25·49 50·99
-
100ormor.No
report
�
•
•
o
o '.
, ,
o
o
j;
Figure 1.1: Tuberculosis notification rates, 2004. (Adapted from WHO report 2006)
1.3 Mycobacterium tuberculosis infection
M. tuberculosis
belongs
to theMycobacteriaceae family
andActinomycetales
order. Humans are the
only
reservoirs. M. tuberculosis is anaerobic,
non-sporeforming, non-motile, slightly
curved orstraight
rod bacterium of 0.2 - 0.6 X 1.0 - 10 urn inlength.
The cell wall of M. tuberculosis contains
high
content ofcomplex lipids.
One of thecomponents
ismycolyl-arabinogalactan
which acts as ahydrophobic permeability
barrierthat
prevents penetration
of common anilinedyes.
TB is
spread through
the air from one person to another.Primary
infectionbegins
upon inhalation of 1-10 aerosolized bacilli. The bacteria can settle in thelungs
andbegin
togrow. From
there, they
can movethrough
the blood to otherparts
of thebody,
such asthekidney, spine,
and brain. Thispathogenic mycobacteria
can survive in the hostile habitat of themacrophage,
the main immune cell that attract the bacilli.Following
the infection of M.tuberculosis,
30% of individuals will becomeinfected,
with about 40% of these individualsdevelop primary
active TB while theremaining
60%develop
latent infection(Figure 1.2).
Latentinfection is described as a clinical
syndrome
thatoccurs afteran individual has beenexposed
to M. tuberculosis.During
thatparticular stage,
the immune response has beengenerated
to control thepathogen
and force it into a dormantstage.
Individuals with latent TB do not transmit the disease. After years ofdormancy,
thisorganism
may start toreplicate, leading
to reactivation of infection and clinical disease. Individual who islatently infected,
candevelop
active disease via eitherendogenous
reactivation ofthe latent bacillior exogenous reinfection with a second
mycobacterial
strain.Approximately,
2 - 23% ofimmunocompetent patients
with latent TB will reactivate at a laterdate,
whilepatients
withHIV
develop
reactivation of TB at a rate of 5- 10% per year(Figure 1.2)
due toprogressive
depletion
anddysfunction
of themacrophage (Goletti
etaI., 1996).
VI
No infection
/ 70%
Exposure Primary active TB
(close contacts)
/
40%
� Infection
30% "" latent IB /
60%
-.
Continued latent TB
Reactivation TB 2-23% per lifetime
Reactivation TB HIV infection
-�,.,5-10% per year
Figure 1.2:
Outcomesassociated with exposure
toM.
tuberculosis.(Adapted
fromParrish
etaI., 1998).
The
capacity
to limit theproliferation
of tubercle bacilli withinmacrophage
resideslargely
with
CD4+ T-helper (Th) lymphocytes. Despite
HIVpatients,
the reactivation of thepathogen
will mostprobably
occur inpeople
withimmunosuppression
due to age, corticosteroids and malnutrition(Flynn, 2004).
The
pathogenicity
of theorganism
is determinedby
itsability
to escape the host immune response as well aseliciting delayed type hypersensitivity (DTH).
DTH is used as ageneral category
to describe all thosehypersensitivity
reactions that take more than 12 hours todevelop,
which involve cell-mediated immune(CMI)
reactions rather than humoral immune reactions. DTH skintesting
or Mantoux reaction is carried out to determineprevious
exposure to TBby injection
of tuberculin into the skin of an individual in whomprevious
infection with themycobacterium
had induced a state of eM!. The reaction is characterizedby erythema
and induration which appearsonly
after several hours andreaches a maximum at 24 - 48 hours.
1.4 Diagnosis
The most common method used to
diagnose
TB isby
smearmicroscopy
or known asAcid-fast bacillus
(AFB)
shown inFigure
1.3 which is the mostpopular. rapid
andinexpensive
method.However,
thereliability
of this method ishighly dependent
on theexperience
of thelaboratory personnel
and on the number oforganisms present
in thespecimen.
Another method known as the current'gold
standard' isby
culture whether onsolid or
liquid
media.One ofthe latest
technologies
used todiagnose
TB isby
nucleic acidamplification (NAA)
based assays. NAA refers to a
technique
in which the nucleic acid(DNA
orRNA)
ofanFigu
re 1.3: AFB smearorganism
isamplified by
as much as 40 orders ofmagnitude,
after which aprobe
detects atarget
sequence of DNA or RNAunique
to aparticular organism. Compared
to smear andculture
technique, sensitivity
andspecificity
of NAA areusually
veryhigh (Pfyffer, 1999)
and can detect as few as 10
organisms
in 1 ml of clinicalsample (Schluger
&Rom, 1995).
NAA method can also reduce the
diagnostic
time from weeks todays. Currently.
two NAAmethods are available
commercially,
the EnhancedMycobacterium
tuberculosis Direct Test(Gen-Probef)
and theArnplicor" Mycobacterium
tuberculosis Test(Roche Diagnostic Systems) (reviewed by
Soini &Musser, 2001).
Bothproducts
have beenapproved by
theFood and
Drug
Administration USA(FDA)
in 1999 for direct detection of M. tuberculosis from clinicalspecimens (CDC, 2000).
The NAA test can enhancediagnostic speed,
butcould not
replace
AFB smear or culture because the test cannotdistinguish
between live and deadorganisms.
Inaddition,
NAA testrequire complex equipment
as well ashighly
technical staff.
Therefore,
clinicians shouldinterpret
the NAA test results based on theclinical situation and the test should be
performed
at therequest
of the clinician(Soini
&Musser, 2001).
Besides the
AFB,
culture and NAA methods of TBdiagnosis, susceptibility testing
is one ofthe available alternatives if the culture remains
positive
over alonger period
of time.Drug susceptibility testing
ismandatory
on initial isolates of M. tuberculosis and relatedspecies
from all
patients. Susceptibility testing
is conducted to monitor apossible development
ofdrug
resistance. Conventional method fordrug susceptibility
isby testing
on solid media(Middlebrook
7H 11 orLJ).
Another recent method ofdrug susceptibility testing
isby
radiometricliquid
culturesystem (BACTEC)
whichprovides
a vialcontaining
a substance[para-nitro-alpha-acatylamine-hydroxypropiophenone (NAP)],
whichselectively
suppress thegrowth
of M. tuberculosiscomplex species. Among
members of the M. tuberculosiscomplex
are M.tuberculosis,
M.bovis,
M. africanum and M. microtiI. Each member of theM. tuberculosis
complex
ispathogenic.
If a subculture from the initial vial fails to demonstrategrowth
in theNAP,
that ispresumptive
evidence for aspecies
within M.tuberculosis
complex.
1.5
Symptoms and
treatmentsThe
symptoms
of TBdepend
on the site where the bacteria aregrowing
whether inpulmonary
orextrapulmonary.
In thelungs, symptoms
such ascoughing
for 3 weeks orlonger, pain
in the chest andcoughing
out blood orsputum
are very common.Only
activeTB
patients
will show some otherpossible symptoms
which are; weakness orfatigue, weight loss, fever, sweating
atnight
and reducedappetite.
Besidespulmonary TS,
mostextrapulmonary
forms of TBincludes;
TBmeningitis,
tuberculouslymphadenitis, pericardial T8, pleural
TB and disseminated ormiliary
TB.People
withHIV,
infants and young children seem to have an increased risk forextrapulmonary
TB.Containment of TB has been carried out
by
the WHO-recommended"directly
observedtreatment short course"
(DOTS) strategy.
This treatment involves TBpatients
observedtaking
everysingle
dosedrug
for the first 2 month of the 6 to 8 month treatmentregimens.
More than 17 million
patients
benefited from the DOTSstrategy,
but in some cases multidrug
resistant TB(MDR- TB)
occurswhen the treatment isincomplete (Girard
etal., 2005).
MDR-TB is defined as strains of M. tuberculosis resistant to at least isoniazid and
rifampicin,
the two mostpowerful
anti-TBdrugs.
The first documented case of MDR-TBwas in a
lung transplant patient
in 1999(Lee
etal., 2003). Transplant patients
arechronically immunosuppressed
and in thatstudy,
the donatedlungs
were from a recent Chineseimmigrant
who was athigh-risk
forprevious
exposure.Fortunately, fluctuations
and variations of
isoniazid, rifampicin, pyrazinamide
and rifabutin were successful Insaving
thepatient.
1.6 Immune response against TB
Immune
response
involved in TBinfection
iscomplex.
Thecomponents
involved areT
cells
(CD4+
andCDS), cytokines (IFN-,,(, TNF-a,
IL-12 andIL-6)
andmacrophages (Flynn, 2004).
The immune response may alsodiffer
in acute andchronic
infection. Fourstages
ofpulmonary
TB(Figure 1.4)
havebeen
reviewedby
van Crevel et al.(2002).
The firststage
is the
inhalation
of tuberclebacilli.
After anincubation period
of 4 to12 weeks, alveolar macrophage
willingest
thebacilli
anddestroy
them. Thesedepend
on the intrinsicmicrobicidal capacity
of hostphagocytes
aswell
as the virulence factors of theingested mycobacteria.
Mycobacteria which escape
the firststage
will enter the secondstage
where three scenarios could occur. The first scenario is when the host failed to contain thepathogen
and die.
Secondly,
themycobacteria may spread throughout the body
whenthe host
immune response is weak (normally
occurs inimmunocompromised patients) causing active
disease.The third
scenario is whenthe
hostimmune response and the virulence of M.
tuberculosis are balanced and the intracellularbacteria
arecontained
within themacrophage. Macrophage disruption
will attractblood monocytes and
otherinflammatory
cells to the
lungs. Monocytes
willdifferentiate into macrophages
andingest
themycobacteria
but will notdestroy them.
Littletissue damage
occurs atthis stage.
T cellimmunity
willdevelop after
2 to 3 weeksof infection, leading
toproliferation of antigen specific
Tlymphocytes within the early lesions.
Hostimmune system isolates
theprimary
site of
infection by granuloma formation. The granuloma
containslymphocytes including
CD4+
andCDB+
Tcells
as well asB cells.
Inaddition. fibroblasts and
other cells canbe
present
within thegranuloma (Co
etaI., 2004).
Thegranuloma
functionsto limit thespread
..'bollrvo",reaeon(1)
l
cont'alnmenl 01,nfOdlonFirst stage
Secondstage
!lronu!omoform:nion
r-T-h-ir-d-s-.-a-ge----.
1"-
Dorman stage (monthsor cars)"""lIrytutlercu!OSIS
bono·luboreulosis(Pan·.dlS03$O LlIndouzy• sepsis
t
lill0nt M.lubcfcuio$.is
"fectlon
Iialanli sIrongOIIllularImmunily conl(linmenllngr,onuloma
"nmunesuppre$S!on Cmalnuttllion.HIV.aging.
ImmunOSUDOre,s.vedrva.ete,\
Fourthstage
�i
_ak OIIlularImmunity exacerbation.spread
l
�pnmary��
..1Figure
1.4: Fourstages
ofpulmonary
TB(Modified
fromKaufmann
&'Ulrichs, 2003)
of
theinfection by walling off
theorganisms
from the restof the lung, prevents
metastasisof the
infection
andproviding
anenvironment for
theaction
of the immunecomponents (Salgame, 2005).
During
thethird stage
ofpulmonary infection,
theearly
bacilligrowth
willstop (Ulrichs
&Kaufmann, 2003). Solid necrosis
in theprimary
lesions will inhibit extracellulargrowth of mycobacteria
and the infection may become dormant for months oryears. During
thefinal stage, any
disturbance of the balance betweenthe
host andpathogen
afterweakening of
the cellular immune response causes
endogenous
exacerbation which leads toactive
TB.Cavity
formationmay
lead torupture
ofnearby bronchi, causing
the bacilli tospread
toother
parts
of thelungs
or host's organ.1.6.1 Macrophage
Macrophage
has beenidentified
as thekey
immune cellfor
thecontrol of M.
tuberculosis infection. The organism
canmultiply within resting macrophage
butbecome
inhibited when the
macrophage is
activated.Cytokines including IFN-y and TNF-a
andalso
vitamin D
involves
inmacrophage
activation(van Crevel et aI., 2002). Following inhalation
of
mycobacteria droplets,
M.tuberculosis
isengulfed by
alveolarmacrophages.
Theinteraction between macrophages
andmycobacteria involves
avariety
of hostcell receptors including
Fcreceptors (FcR), complement receptors (CR), macrophage
mannose
receptor (MMR)
andalso Toll-like receptor 2 (TLR-2) and TLR-4.
Macrophage plays multiple roles in TB including antigen processing and presentation,
effector cell function and
alsoapoptosis (Silva
etai., 2001). Apoptosis of phagocytic
cellsmay prevent dissemination of infection
and reducesviability
ofintracellular mycobacteria.
mediated
through
adownregulation
ofbcl-2,
an inhibitor ofprogrammed
cell death. The activatedmacrophage produces
reactive oxygen intermediates(ROls) by
oxidative burst and reactivenitrogen
intermediates(RNls)
via inducible nitric oxidesynthase (iNOS2).
Cooper
et al.(2000) provided
evidence that ROI-mediated control isimportant during early
infectionby
the observation of a 10-foldhigher
bacterial numbers in thelungs
ofp47phox
knockout
(KO) mice, compared
towild-type controls,
after aerosolchallenge
with M.tuberculosis. The
p47phox
is aphagosome
oxidasecomponent
critical for theactivity
orassembly
of the functional oxidase. RNls are the critical effector moleculesagainst
M.tuberculosis in the mouse.
Moreover,
mice deficient in NOS2activity
are verysusceptible
to acute or chronic M. tuberculosis infection
compared
towild-type
mice(MacMicking
etaI., 1997; Scanga
etaI., 2001).
Macrophage
activation also involves natural-resistance-associatedmacrophage protein (Nramp1)
gene and vitamin D.Nramp1
is aninteresting
gene involved inmacrophage
activation and
mycobacterial killing (Blackwell
etai., 2000).
Theprotein
is anintegral
membrane
protein
whichbelongs
to afamily
of metal iontransporters.
These metalions, particularly Fe2+,
are involved inmacrophage
activation andgeneration
of toxicantimicrobial radicals
(Zwilling
etai., 1999). Following phagocytosis, Nramp1
becomespart
of thephagosome. Nramp1
mutant micedisplay
reducedphagosomal
maturation and acidification(Hackam
etaI., 1998).
Macrophage
suppresses thegrowth
of M. tuberculosisby
thehelps
of active metabolite of vitaminD, 1, 25-dihydroxyvitamin
D(Rockett
etai., 1998).
A recentstudy
amongGujarati
.
Hindus,
amainly vegetarian immigrant population
inLondon,
showed that vitamin Ddeficiency
was a risk factor for TB(Wilkinson
etai., 2000). Eventhough
activatedmacrophage
can sometimes kill virulent M. tuberculosis(Sato
etal., 1998)
but it isgenerally
cannoteliminate the infection entirely. Therefore,
othercomponents of
theimmune system including cellular
and humoralimmune responses participate
toeliminate
the mycobacteria.
1.6.2 Cellular Immune Response
Van Crevel
etal. (2002) has discussed three processes that contribute
tothe
initiation ofcellular
immuneresponse against T8; antigen presentation, costimulation and
cytokine production. Antigen presentation involves CD4+
Tcells, CD8+ T cells and unconventional T cells including CD1 and 18
Tcells. In general, CD4+ T cells help to amplify the
host immuneresponse by activating effector
cells andrecruiting additional
immune cells to the
site
ofdisease, whereas CD8+ T cells
areimportant during the latent stage of
TBinfection, which
act ascytotoxic
T cells(CTl) by lysing infected
cells(Schluger
&
Rom, 1998) through production of various cytokines such
asIFN-y
andTNF-a.. Within
aweek
of infection with virulent M. tuberculosis, the number of activated CD4+ and CD8+ T
cells inthe lung-draining lymph
nodesincreases (Feng
etal., 1999; Serbina
etai., 2000).
Basically, CD4+
Thlymphocytes differentiate
fromprecursor ThO cells
underthecontrol of cytokines
such as IL-2and
Il-4into
twofunctionally
distinctsubsets
eithertype
1(Th1)
ortype
2(Th2) cells (Figure 1.5). Th1
secretescytokines such
asIl-2, IFN-y, TNF-a. and
u-12
resulting
inmacrophage activation and induction of CMI. In contrast, Th2
secretesIL-4, Il-5,
Il-6and IL-10 resulting
in theinduction
of humoralimmunity by antibody production.
M.
tuberculosis
residesprimarily in
avacuole within the macrophage resulting in major histocompatibility complex (MHC) Class" presentation of mycobacterial antigens
toCD4+
T
cells. The
HIVepidemic has demonstrated
thatthe loss of CD4+T cells greatly increases
susceptibility of the host to both
acuteand reactivation TB (reviewed by Flynn, 2004).
IL-2
GMyL-3
IFN-y
-
TNF-y f3 IL-1
o
'IL-2
-...
��
IL4INF-y
IL-4
'IL-S
IL-4
IL-6 IL-10
:JsF.IL-3
Figure
1.5:CD4+
Thelper lymphocyte
subsets upon activation.(Adapted from
Cohen etai., 1998).
The other
possible
roles ofCD4+
T cells incontrolling
TB infectioninclude. apoptosis (Keane
etaI
.•1997; 8alcwicz-Sablinska et aI., 1998), conditioning
ofantigen presenting
cells
(APes), help
forB cells andCD8·
T cells andproduction
ofothercytokines. However,
the
inability
of theCD4+
T cells tocompletely
eliminate intracellular bacteriamay
be due to the lack ofrecognition
oractivation ofinfectedmacrophages (Flynn, 2004).
CD8+
T cellsproducing IFN-y probably participate
in the activation ofmacrophages (Caruso
et.ai., 1999; Scanga
etaI., 2000). CD8+
T cellsrecognize antigens presented by
MHC Class I molecules and these
antigens
arefrequently
derived from thecytoplasm
ofthe cells.
However,
M. tuberculosis does not resideprimarily
in thecytoplasm
but invacuoles inside the cells. Studies have
suggested
that the bacilli within the vacuoles may have access to thecytoplasm, perhaps
via a pore in the vacuole's membrane(Teitelbaum
et
al., 1999).
It wassuggested
that CTLkilling
of the bacteriadepends
on theirability
todeliver
potent bactericidal proteins
such asgranulysin
from theirgranules (Silva
etai., 2001). Lysis
oftarget
cellsby CDS+
T cells can occur viaperforin
andgranzymes
or the Fas/FasL(CD95L) pathway resulting
inapoptotic
cell death or release of bacteria from aninfected cell into the
granuloma (Canadayet a/., 2001).
Theimportance
ofCDS+
T cells inTB was
reported by
Behar et al.(1999).
whenJ32-microglobulin (J32-m)
andtransporter
associated
protein (TAP1)
KOmice,
which cannotgenerate C08+
Tcells,
were infected with M.tuberculosis
and resulted in an exacerbated course ofinfection.
As mentioned
earlier,
unconventional T cells such as CD 1 andyB
T cells alsoplaya
role inhost
defense against mycobacterial infection.
Both cellsproduce type
1cytokines,
mostimportantly IFN-y
which activatesanti-mycobacterial
activities inmacrophages (Raupach
&Kaufmann, 2001). CD1-restricted aJ3 T-Iymphocytes
arethought
to be activatedby
mycobacterial lipids (Agger
&Andersen, 2002).
The CD1family
consists ofantigen
presenting
molecules encodedby
genes located outside of the MHC. CD1 genes are conserved among mammalianspecies
and areexpressed
on the surface of the cells involved inantigen presentation, notably
dendritic cells(DCs).
The CD1system
is involved in activation of CM) responseagainst mycobacterial
infection. It is the least common T cell subset in humanperipheral
blood andlung.
Inhumans,
most of these T cells express neither CD4 nor CD8 and are referred to asdouble-negative (ON)
cells. Inmice,
C01drestricted natural killer
(NK)
T cells are activatedby mycobacterial
cell wallcomponents
and are involved in
early granuloma
formation(Apostolou
etal., 1999).
Meanwhile, yS
T cells arelarge granular lymphocytes,
non-MHC restricted that candevelop
a dendriticmorphology
inlymphoid
tissues and function as CTL. Unconventionaly8
T cells are activatedby
smallphosphorylated
metabolites(Agger
&Andersen, 2002).
Itwas
suggested
thaty8
T cells mayplaya
role inearly
immune responseagainst
TB and isan
important part
of theprotective immunity
inpatients
with latent infection(reviewed by Raja, 2004).
The second process that leads to the initiation of cellular
immunity
isby
costimulation.Antigen presentation only
leads to T cell stimulation in the presence of severalcostimulatory signals.
The most well knowncostimulatory signals
for T cell stimulation are 8-7.1(C080)
and 8-7.2(C08S).
These molecules areexpressed
onmacrophages
andDCs and bind to CD28 and to CTLA-4 on T cells. In the absence of proper
costlmulatory signals, antigen presentation
may lead to an increasedapoptosis
of Tcells (Hirsch
etai.,
1999 &
2001).
Finally,
theproduction
ofcytokines
may also contribute to the initiation of cellularimmunity
in TB infection. Several
cytokines produced by
activatedmacrophages
and DCs areessential for stimulation of T
lymphocytes.
These includeIFN-y, TNF-a., IL-4, IL-12,
IL-1Band IL-15.
IFN-y
isproduced by
T cells fromhealthy purified protein
derivativepositive (PPD+) subjects
as well asthose with active TB.IFN-y
isimportant
to activatemacrophage
as well as TNF-a. that
synergize
withIFN-y
to induceantimycobacterial
effects. Individualslacking receptors
forIFN-y
sufferfromrecurrent,
sometimes lethalmycobacterial
infections(Holland
etai., 199B).
There arethreepossible
cellsresponsible
fornonspecific production
of
IFN-y
as reviewedby
van Crevel et al.(2002). First,
beforeadaptive
T cellimmunity
hasfully developed,
NK cells may be the mainproducer
ofIFN-y,
either in response to IL-12 and IL-18(Iho
etai., 1999)
ordirectly by
exposure tomycobacterial oligodeoxynucleotides (Garcia
etaI., 1999). Second, lung macrophages
were found toproduce IFN-y
in M.tuberculosis-infected mice
(Wang
etaI., 1999). Third,
theyo
T cells and CD1-restricted T cells mayproduce IFN-y during early
infection.Besides
IFN-y,
stimulation ofmonocytes, macrophages
and DCs(Henderson
etet., 1997)
with
mycobacteria
ormycobacterial products
induce theproduction
of TNF-a.. TNF-a.plays
a role in
granuloma formation,
inducesmacrophage
activation and hasimmunoregulatory properties (Orme
&Cooper, 1999;
Tsenova etal., 1999).
In addition toTNF-a.,
IL-12 has acrucial role in the induction of
IFN-y production (O'Neill
&Greene, 1998).
IL-12 isproduced mainly by phagocytic
cells. InTB,
IL-12 has been detected inlung infiltrates,
inpleurisy,
ingranulomas
and inlymphadenitis (reviewed by
van Crevel etaI., 2002).
Theexpression
ofIL-12
receptors
is also increased atthe site of disease(Zhang
etai., 1999). Together
withIL-12,
IL-18 and IL-15 seem to beimportant
in theIFN-y
axis(O'Neill
&Greene, 1998).
IL-18 KO mice was found to be
highly susceptible
to M. tuberculosis(Sugawara
etai., 1999)
and in mice infected with M.
leprae,
resistance is correlated with ahigher expression
of IL�18.
Moreover,
M. tuberculosis-mediatedproduction
of IL-18by peripheral
bloodmononuclear cells
(PBMC)
is reduced in TBpatients
and this reduction may beresponsible
for reducedIFN-y production (Vankayalapati
etai., 2000).
Another
cytokine
that have been studiedregarding
TB infection is IL-4. Inhibition of IL-4production
did not seem topromote
cellularimmunity.
IL-4-/- micedisplayed
normalinstead of increased
susceptibility
tomycobacteria
in twostudies, suggesting
that IL-4 may be a consequence rather than the cause of TBdevelopment (Erb
etaI., 1998; North, 1998).
1.6.3
Humoral
ImmuneResponse
Researchers
argued
about the role of antibodies in host defenseagainst
M.tuberculosis which was believed that intracellular
pathogens
cannot be reachedby
antibodies.
However,
intracellularpathogens
are found in the extracellular spaceprior
totheir