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PROTEOLIPOSOMES FROM Mycobacterium bovis

BCG AND Mycobacterium smegmatis AS NEW

VACCINE CANDIDATES AGAINST

TUBERCULOSIS

RAMLAH BINTI KADIR

UNIVERSITI SAINS MALAYSIA

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PROTEOLIPOSOMES FROM

Mycobacterium bovis

BCG

AND

Mycobacterium smegmatis

AS NEW VACCINE

CANDIDATES AGAINST TUBERCULOSIS

by

RAMLAH BINTI KADIR

Thesis submitted in fulfillment of the requirements

for the degree of

Doctor of Philosophy

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ACKNOWLEDGEMENTS

In the name of Allah, the Most Generous and the Most Merciful.

First and foremost, I would like to express my gratitude and appreciation to Allah SWT, the Most Merciful and Most Compasionate for granting me the opportunity, health, strength and motivation for the completion of my PhD. I would like to thank people around me who have motivated, inspired and encouraged me to reach my goal. Special thanks to:

Prof Dr Norazmi Mohd Nor, my main supervisor. I feel highly indebted to you for your scholarly guidance and generosity in assisting me in the beginning of the research and throughout this study. I feel intensely grateful for your support through this journey in completing my study. Thank you also for always keeping your door open.

I wish to acknowledge Associate Professor Dr Rapeah binti Suppian, my co-supervisor, for her encouragement and support throughout this study. I will always remember your sweet smile.

I also wish to acknowledge the generous support of husben and wife, Prof Dr Armando Acosta, my co-supervisor and Prof Maria Elena Sarmiento, my field supervisor for their advice, comments, support, as well as for the knowledge they have given and their enthusiasm in assisting me in the preparation of this thesis. I also like to thank Dr Wan Amir Nizam Wan Ahmad for his advice on statistical analyses.

I would like to record my appreciation to my sponsor Ministry of Education and Universiti Sains Malaysia for awarding me a Fellowship under Skim Latihan Akademik. My thanks are also extended to the Ministry of Science, Technology and Innovation, Malaysia, the Ministry of Education, the LRGS Grant and the Ministry of Science and Technology, Cuba.

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Last but not least I owe my greatest gratitude to my beloved husband Mohd Zuhardie bin Haji Ramli for his patience, endurance, understanding, spiritual, and material support in accompanying me in this journey to realize this endeavor. Thank you for always being by my side.

Also my deepest appreciation to my late father, Haji Kadir bin Jusoh for his support until he meet the Greatest Creator, Allah SWT and special thanks to my parents: a very handsome father Haji Ramli bin Ab Rahman and both my gorgeous mother; Hajah Timah binti Abas and Hajah Nik Kelthom binti Nik Ismail for their understanding and support. My sincere thanks also go to my siblings, especially to my second brother, Hanafi bin Kadir who always support and encourage me to grab any opportunity to be a ‗somebody‘ in my family. Thanks for always encouraging and being with me.

Finally my appreciation is extended to my colleagues in the vaccine and immunology group, PPSK postgraduate students and staff in the Molecular Biology Labaratory, Pusat Pengajian Sains Kesihatan, Animal Research and Service Centre, Universiti Sains Malaysia (ARASC) for all their support and encouragement. Thanks for being so helpful. I would especially like to thank my dear friend, Dr Rohimah Mohamud for her great encouragement and support throughout my study.

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iv TABLE OF CONTENTS ACKNOWLEDGEMENTS ... ii TABLE OF CONTENTS ... iv LIST OF TABLES ... x LIST OF FIGURES ... xi

LIST OF SYMBOLS AND ABBREVIATIONS ... xiv

ABSTRAK … ... xvi ABSTRACT .. ... xix CHAPTER 1 INTRODUCTION ... 1 1.1 Tuberculosis (TB) ... 1 1.1.1 History of TB ... 1 1.1.2 TB incidence ... 2 1.1.3 Mycobacterium tuberculosis ... 5

1.1.4 Transmission and pathogenesis ... 7

1.1.5 Symptoms of TB ... 9

1.1.6 TB in children ... 10

1.1.7 TB in Malaysia ... 11

1.1.8 Risk factors ... 12

1.1.8(a) Human Immunodeficiency Virus (HIV) ... 12

1.1.8(b) Non-communicable diseases (NCDs) ... 13

1.1.9 Immune response against Mtb ... 14

1.1.9(a) Innate immune response ... 14

1.1.9(b) Adaptive immunity ... 17

1.1.9(bi) Antibody responses against Mtb ... 17

1.1.9(bii) Cellular responses against Mtb ... 19

1.1.10 Control of TB ... 21

1.1.10(a) Diagnosis ... 21

1.1.10(b) Treatment ... 23

1.1.11 Vaccine ... 25

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v

1.1.11(b) BCG vaccine ... 26

1.1.12 TB vaccine development ... 28

1.1.12(a) Live vaccines ... 30

1.1.12(b) Viral vectored vaccines ... 32

1.1.12(c) Adjuvanted subunit vaccines ... 34

1.1.12(d) Whole-cell or fragmented mycobacteria vaccine candidates ... 36

1.1.13 Bioinformatics tools for vaccine development ... 39

1.1.14 Mycobacterial Proteoliposomes (PLs) ... 42

1.1.15 Objectives ... 43

1.1.15(a) General objective ... 43

1.1.15(b) Specific objectives ... 43

CHAPTER 2 MATERIALS AND METHODS ... 44

2.1 Materials ... 44

2.1.2 Antibodies ... 44

2.1.3 Antigens ... 44

2.1.4 Chemicals and reagents ... 44

2.1.5 Kits and consumables ... 47

2.1.7 Sterilized, deionized distilled water ... 48

2.1.8 Media ... 48

2.1.8(a) 7H9 broth ... 48

2.1.8(b) 7H11 agar ... 48

2.1.8(c) RPMI 1640 medium ... 48

2.1.9 Buffers ... 49

2.1.9(a) ABTS substrate for ELISA ... 49

2.1.9(b) Assay diluents ... 49

2.1.9(c) Blocking buffer for ELISA ... 49

2.1.9(d) Blocking buffer for Western blotting ... 49

2.1.9(e) Calcium chloride (CaCl2) solution (100 mM) ... 50

2.1.9(f) Coating buffer for ELISA ... 50

2.1.9(g) Coating buffer for extracellular cytokine ELISA ... 50

2.1.9(h) Resolving buffer for SDS-PAGE ... 50

2.1.9(i) Running buffer for SDS-PAGE ... 51

2.1.9(j) Sample buffer for SDS-PAGE ... 51

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2.1.9(l) Towbin transfer buffer ... 51

2.1.9(m) Tris-EDTA buffer for production of PLs ... 52

2.1.10 Solutions ... 52

2.1.10(a) Ammonium persulfate (AP) solution (20%) ... 52

2.1.10(b) Capture antibody: Purified anti-mouse IFN-solution ... 52

2.1.10(c) Capture antibody: Purified anti-mouse IL-2, IL-4 and TNF-... 52

2.1.10(d) Coomassie blue solution ... 52

2.1.10(e) Destaining solution ... 53

2.1.10(f) Detection solution for Western blotting ... 53

2.1.10(g) Ethanol solution (70%) ... 53

2.1.10(h) EDTA solution (0.5 M) ... 53

2.1.10(i) Human IFN-conjugate solution ... 53

2.1.10(j) Human IFN- standard solution ... 54

2.1.10(k) Loading dye solution ... 55

2.1.10(l) NaOH solution (3 M) ... 55

2.1.10(m) PBS solution ... 55

2.1.10(n) Glycerol solution ... 56

2.1.10(o) Standard solution for extracellular cytokine ELISA ... 56

2.1.10(p) Stop solution for ELISA ... 57

2.1.10(q) Tris-base solution (1.5 M) containing 0.4% SDS ... 57

2.1.10(r) Tris-HCl solution (1.5 M) containing 0.4% SDS ... 57

2.1.10(s) Trypan blue solution (0.4%) ... 58

2.1.11 Mouse strain ... 58

2.1.12 Laboratory equipment ... 59

2.2 Research Methodology ... 60

2.4.1 Bioinformatics study ... 61

2.4.1 (a) Identification and selection of in-vivo expressed genes of Mtb ... 61

2.4.1(b) Searching for identical epitopes on Ms and BCG ... 61

2.4.1(c) Prediction of subcellular localization of proteins from Ms and BCG ... 61

2.4.1(d) Population coverage calculation ... 62

2.4.2 Production of PLs ... 63

2.4.2(a) Culture of mycobacteria ... 63

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2.4.2(aii) M. smegmatis (Ms) mc2 155 culture ... 63

2.4.2(b) Production of PLBCG and PLMs ... 63

2.4.3 Characterization of PLs ... 64

2.4.3(a) Transmission electron microscopy ... 64

2.4.3(b) Size exclusion chromatography ... 64

2.4.3(c) Sodium Dedocyl Sulphate-Polyacrylamide Gel Electrophoresis ... 65

(SDS PAGE) ... 65

2.4.4 Antigenicity study in humans ... 66

2.4.4(a) Recognition of PLs by human sera. ELISA ... 66

2.4.4(b) Recognition of PLs proteins by human sera. Western blot ... 67

2.4.4(c) IFN-γ production in human whole blood upon stimulation with PLs ... 68

2.4.5 Immunogenicity and cross reactivity studies ... 70

2.4.5(a) Blood collection ... 70

2.4.5(b) IgG and IgG subclasses response against PLs/Mtb antigens. ELISA ... 71

2.4.5(c) IgG response against PLs/Mtb-SCWP antigens. Western blot ... 71

2.4.5(d) Lymphocyte culture ... 72

2.4.5(e) Extracellular cytokine profile of stimulated splenocytes from immunized mice ... 73

2.4.5(f) Determination of DTH response ... 74

2.4.6 Statistical analysis ... 75

CHAPTER 3 BIOINFORMATICS STUDY ... 76

3.1 Identification and selection of in vivo expressed Mtb genes ... 76

3.1.1 Identification of identical epitopes of Mtb proteins on BCG and Ms ... 77

3.1.2 Calculation of population coverage in Malay and Cuban populations ... 79

3.2 Discussion ... 81

CHAPTER 4 CHARACTERIZATION OF PLs ... 85

4.1 Transmission electron microscopy (TEM) ... 86

4.2 Size exclusion chromatography (SEC) ... 87

4.3 Sodium Dedocyl Sulphate-Polyacrylamide Gel Electrophoresis (SDS PAGE) ... 89

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4.4 Antigenicity study in humans ... 91

4.4.1 Recognition of PLs by human sera. ELISA ... 91

4.4.2 Recognition of PLs proteins by human sera. Western blot... 93

4.4.3 IFN-γ production in human whole blood upon stimulation with PLs ... 96

4.5 Discussion ... 98

CHAPTER 5 IMMUNOGENICITY STUDY ... 103

5.1 IgG and IgG subclasses response against PLs. ELISA ... 103

5.1.1 IgG and IgG subclasses response against PLBCG ... 104

5.1.2 IgG and IgG subclasses response against PLMs ... 109

5.2 IgG response against PLs. Western blot ... 113

5.2.1 IgG response against PLBCG ... 113

5.2.2 IgG response against PLMs ... 115

5.3 Extracellular cytokine profile of PLs stimulated splenocytes from immunized mice ... 117

5.3.1 Extracellular cytokine profile of PLBCG stimulated splenocytes from immunized mice ... 117

5.3.2 Extracellular cytokine profile of PLMs stimulated splenocytes from immunized mice ... 122

5.4 DTH response in PLs-immunized mice ... 126

5.4.1 DTH response in PLBCG-AL-immunized mice ... 126

5.4.2 DTH response in PLMs-AL-immunized mice ... 127

5.5 Discussion ... 128

CHAPTER 6 CROSS REACTIVITY STUDY ... 135

6.1 IgG and IgG subclasses response against Mtb antigens. ELISA ... 136

6.1.1 IgG and IgG subclasses response against Mtb antigens (Mtb-CFP, Mtb-CW and Mtb-SCWP) in mice immunized with different PLBCG formulations ... 136

6.1.2 IgG and IgG subclasses response against Mtb antigens (Mtb-CFP, Mtb-CW and Mtb-SCWP) in mice immunized with different PLMs formulations ... 140

6.2 IgG response against Mtb antigens. Western blot ... 143

6.2.1 IgG response against Mtb-SCWP in mice immunized with different PLBCG formulations ... 143

6.2.2 IgG response against Mtb-SCWP in mice immunized with different PLMs formulations ... 145

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6.3 Extracellular cytokine profile of Mtb antigens stimulated splenocytes

from PLs immunized mice ... 147

6.3.1 Extracellular cytokine profile of Mtb antigens (Mtb-CFP, Mtb-CW and Mtb-SCWP) stimulated splenocytes from mice immunized with different PLBCG formulations ... 147

6.3.2 Extracellular cytokine profile of Mtb antigens (Mtb-CFP, Mtb-CW and Mtb-SCWP) stimulated splenocytes from mice immunized with different PLMs formulations ... 152

6.4 DTH response to Mtb antigen (Mtb-WCL) in PLMs-AL-immunized mice ... 156

6.5 Discussion ... 157

CHAPTER 7 SUMMARY AND CONCLUSION ... 159

7.1 Summary ... 159

7.2 Conclusion ... 161

7.3 Recommendations ... 163

REFERENCES ... 164

APPENDICES ... 198

APPENDIX A: List of 38 Mtb genes highly expressed in vivo in humans and animals (mice and rabbits) at different stages of infection ... 198

APPENDIX B: Animal ethics approval ... 201

APPENDIX C: Home page of Bioinformatics tool ... 203

APPENDIX D: Standard curve of cytokines ... 208

APPENDIX E: Poster Presentation ... 212

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LIST OF TABLES

Table 1.1: Global clinical TB vaccine pipeline ... 30

Table1.2: Comparison of conventional and genomic approaches to vaccine development………....41

Table 2.1: Antibodies... 44

Table 2.2: Chemicals and reagents.. ... 44

Table 2.3: Kits and consumables ... 47

Table 2.4: Conjugate preparation ... 54

Table 2.5: Laboratory equipment ... 59

Table 2.6: Experimental groups ... 70

Table 3.1: BCG genes containing identical epitopes to the Mtb proteins expressed in vivo………...78

Table 3.2: Ms genes containing identical epitopes to the Mtb proteins expressed in vivo………...79

Table 3.3: Population coverage calculation of BCG and Ms identical T-cell epitopes potentially included in PLBCG and PLMs….…………...80

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LIST OF FIGURES

Figure 1.1: TB incidence rates in 2012 ... 4

Figure 1.2: TB incidence in top-ten countries, 2012 ... 4

Figure 1.3: Scanning electron microscope of Mtb ... 6

Figure 1.4: Schematic representation of the mycobacterial cell wall ... 6

Figure 1.5: Summary of TB transmission and pathogenesis ... 8

Figure 1.6: The choronolgy of Mtb infection ... 9

Figure 2.1: Preparation of human IFN- standard ... 55

Figure 2.2: Preparation of IFN-LIL-and TNF-standard ... 57

Figure 2.3: Flow chart showing an overview of the research methodology ... 60

Figure 2.4: Flow chart of bioinformatics analysis ... 62

Figure 4.1: Transmission electron microscopy of PLs.. ... 86

Figure 4.2: Chromatographic profile of PLBCG.. ... 87

Figure 4.3: Chromatographic profile of PLMs.. ... 88

Figure 4.4: SDS-PAGE analysis of PLBCG. ... 89

Figure 4.5: SDS-PAGE analysis of PLMs. ... 90

Figure 4.6: Recognition of PLBCG by human IgG ... 92

Figure 4.7: Recognition of PLMs by human IgG ... 92

Figure 4.8: Recognition of PLBCG proteins by human sera. ... 94

Figure 4.9: Recognition of PLMs proteins by human sera. ... 95

Figure 4.10: IFN-γ production in human whole blood upon stimulation with PLBCG ………..…...96

Figure 4.11: IFN-γ production in human whole blood upon stimulation with PLMs………..…...97

Figure 5.1: Specific total IgG response to PLBCG in immunized mice ... 105

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Figure 5.3: Specific IgG2a subclass response to PLBCG in immunized

mice…...…….………..…...107

Figure 5.4: Specific IgG2b subclass response to PLBCG in immunized mice…...…….………..…...108

Figure 5.5: Specific Total IgG response to PLMs in immunized mice... 109

Figure 5.6: Specific IgG1 subclass response to PLMs in immunized mice ... 110

Figure 5.7: Specific IgG2a subclass response to PLMs in immunized mice ... 111

Figure 5.8: Specific IgG2b subclass response to PLMs in immunized mice …..112

Figure 5.9: Recognition of PLBCG proteins by sera of mice immunized with PLBCG formulations….………..…...114

Figure 5.10: Recognition of PLMs proteins by sera of mice immunized with PLMs formulations………..…………...116

Figure 5.11: IFN- secretion by splenocytes stimulated with PLBCG.. ... 118

Figure 5.12: IL-2 secretion by splenocytes stimulated with PLBCG.. ... 119

Figure 5.13: IL- secretion by splenocytes stimulated with PLBCG.. ... 120

Figure 5.14: TNF- secretion by splenocytes stimulated with PLBCG.. ... 121

Figure 5.15: IFN- secretion by splenocytes stimulated with PLMs …... ... 122

Figure 5.16: IL- secretion by splenocytes stimulated with PLMs …... ... 123

Figure 5.17: IL- secretion by splenocytes stimulated with PLMs …... ... 124

Figure 5.18: TNF- secretion by splenocytes stimulated with PLMs ….. ... 125

Figure 5.19: DTH response of BALB/c mice inoculated with PLBCG... 126

Figure5.20: DTH response of BALB/c mice inoculated with PLMs ... 127

Figure 6.1: Total IgG response against Mtb antigens (Mtb-CFP, Mtb-CW and Mtb-SCWP) in mice immunized with different PLBCG formulations…...…………...137

Figure 6.2: IgG1 subclass response against Mtb antigens (Mtb-CFP, Mtb-CW and Mtb-SCWP) in mice immunized with different PLBCG formulations ..…..…………...138

Figure 6.3: IgG2a subclass response against Mtb antigens (Mtb-CFP, Mtb-CW and Mtb-SCWP) in mice immunized with different PLBCG formulations………...139

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Figure 6.4: Total IgG response against Mtb antigens (Mtb-CFP, Mtb-CW and Mtb-SCWP) in mice immunized with different PLMs

formulations ………..…………...140 Figure 6.5: IgG1 response against Mtb antigens (Mtb-CFP, Mtb-CW

and Mtb-SCWP) in mice immunized with different PLMs

formulations ………..…………...141 Figure 6.6: IgG2a response against Mtb antigens (Mtb-CFP, Mtb-CW

and Mtb-SCWP) in mice immunized with different PLMs

formulations ………..…………...142 Figure 6.7: Recognition of Mtb-SCWP by sera of mice immunized with

PLBCG formulations …….…………...144 Figure 6.8: Recognition of Mtb-SCWP by sera of mice immunized with

PLMs formulations …….…………...146 Figure 6.9: IFN- secretion by splenocytes stimulated with Mtb antigens

(Mtb-CFP, Mtb-CW and Mtb-SCWP) in mice immunized with different PLBCG formulations...148 Figure 6.10: IL-2 secretion by splenocytes stimulated with Mtb antigens

(Mtb-CFP, Mtb-CW and Mtb-SCWP) in mice immunized with different PLBCG formulations...149 Figure 6.11: IL-4 secretion by splenocytes stimulated with Mtb antigens

(Mtb-CFP, Mtb-CW and Mtb-SCWP) in mice immunized with different PLBCG formulations...150 Figure 6.12: TNF- secretion by splenocytes stimulated with Mtb antigens

(Mtb-CFP, Mtb-CW and Mtb-SCWP) in mice immunized with different PLBCG formulations...151 Figure 6.13: IFN- secretion by splenocytes stimulated with Mtb antigens

(Mtb-CFP, Mtb-CW and Mtb-SCWP) in mice immunized with different PLMs formulations...152 Figure 6.14: IL-2 secretion by splenocytes stimulated with Mtb antigens

(Mtb-CFP, Mtb-CW and Mtb-SCWP) in mice immunized with different PLMs formulations...153 Figure 6.15: IL-4 secretion by splenocytes stimulated with Mtb antigens

(Mtb-CFP, Mtb-CW and Mtb-SCWP) in mice immunized with

different PLMs formulations...154 Figure 6.16: TNF- secretion by splenocytes stimulated with Mtb antigens

(Mtb-CFP, Mtb-CW and Mtb-SCWP) in mice immunized with

different PLMs formulations...155 Figure 6.17: DTH response of BALB/c mice inoculated with Mtb-WCL.………156

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LIST OF SYMBOLS AND ABBREVIATIONS

AIDS Acquired immunodeficiency syndrome

AL Aluminum hydroxide gel

ANOVA One Way Analysis of Variance

AFB Acid fast bacillus

B.C Before Century

BCG Bacille Calmette Guerin

CD Cluster of differentiation

CFU Colony forming unit

CFP Culture filtrate protein

DCs Dendritic cells

DNA Deoxyribonucleic acid

DOC sodium deoxycholate

DTH Delayed type hypersensitivity

ELISA Enzyme Linked Immunosorbent Assay

ESAT-6 6-kDa Early Secreted Antigenic Target

ETH Ethambuthol

 Gamma delta

HIV Human Immunodeficiency Virus

HLA Human Leukocytes Antigen

HRP Horseradish Peroxidase

IFN- Interferon-

gG Immunoglobulin G

IGRAs Interferon-Release Assays

INH Isoniazid

IL Interleukin

kDa kilo Dalton

LTBI Latent TB infection

MDR-TB Multi-drug resistant-Tuberculosis MHC Major histocompatability complex

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Mtb Mycobacterium tuberculosis

Mtb-CFP Culture filtrate protein from Mycobacterium tuberculosis

Mtb-CW Cell wall from Mycobacterium tuberculosis

Mtb-SCWP Soluble cell wall protein from Mycobacterium tuberculosis

Mtb-WCL Whole cell lysate from Mycobacterium tuberculosis

NAATs Nucleic acid amplification Test

NCDs Non-communicable disease

NK Natural killer

NTM Non-Tuberculous Mycobacteria

OD Optical density

PAMPs Pathogen associated molecular patterns

PLs Proteoliposomes

PLBCG Proteoliposomes from BCG

PLMs Proteoliposomes from Mycobacterium smegmatis

PPD Purified protein derivative

PZA Pyrazinamide

QFT-G Quantiferon TB Gold

QFT-GIT Quantiferon TB Gold In Tube

rBCG Recombinant bacilli Calmette Guerin

RD Region of difference

RIF Rifampin

RT Room temperature

SDS PAGE Sodium Dedocyl Sulphate-Polyacrylamide Gel Electrophoresis

SEC Size exclusion chromatography

SEM Standard error mean

TB Tuberculosis

TEM Transmission electron microscopy

Th T helper

TLR Toll-like receptor

TNF Tumor necrosis factor

TST Tuberculin skin test

WHO World Health Organization

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PROTEOLIPOSOMES DARIPADA Mycobacterium bovis BCG DAN

Mycobacterium smegmatis SEBAGAI CALON-CALON VAKSIN YANG

BAHARU TERHADAP TUBERCULOSIS

ABSTRAK

Tuberculosis (TB) adalah disebabkan oleh Mycobacterium tuberculosis (Mtb). Penyakit ini adalah jangkitan yang boleh membawa maut dan ia merupakan salah satu daripada punca utama kematian dan penyakit di dunia. Bakteria ini biasanya menyerang paru-paru dan anggota badan yang lain seperti otak, buah pinggang dan tulang belakang. Bacille Calmette-Gúerin (BCG) merupakan vaksin yang digunakan secara meluas di seluruh dunia dan ia merupakan satu-satunya vaksin yang digunakan untuk mencegah TB. Kajian menunjukkan BCG dapat melindungi atau sekurang-kurangnya mencegah TB sistemik yang teruk pada kanak-kanak, terutamanya meningitis TB. Walau bagaimanapun, BCG kurang atau tidak dapat memberi perlindungan terhadap penyakit TB pulmonari pada orang dewasa. Maka, calon vaksin yang lebih efektif berbanding BCG adalah sangat diperlukan untuk mencegah penyakit TB serta penularannya. Dengan mengambil kira persamaan genomik and antigenik di antara Mtb dan bakteria tidak patogenik seperti BCG dan

Mycobacterium smegmatis (Ms), kami menghasilkan proteoliposomes (PLs) daripada BCG dan Ms sebagai calon vaksin terhadap penyakit TB. Kajian bioinformatik yang telah dilakukan dan keputusannya menunjukkan kewujudan epitope-epitop sel T dan B di dalam PLs daripada BCG dan Ms yang serupa dengan yang dihasilkan secara in vivo oleh Mtb semasa peringkat jangkitan. Keputusan penentuan antigen leukosit manusia (HLA) telah menunjukkan epitop-epitop ini

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memberikan peratusan yang terbaik dalam populasi Melayu dan Cuba. Oleh sebab itu, PLs daripada BCG (PLBCG) dan Ms (PLMs) telah dihasilkan untuk menilai keimunogenan dan tindak balas reaktif silang terhadap antigen-antigen daripada Mtb. PLs ini telah disahkan berbentuk sfera dan mempunyai sifat yang sama dari segi saiz dan bentuk di bawah pemerhatian mikroskop elektron dan kajian kromatografi. Pada peringkat seterusnya, kajian antigenisiti terhadap PLBCG and PLMs di dalam manusia telah dilakukan. Keputusan menunjukkan individu-individu yang sihat telah menghasilkan IFN-γ, yang mungkin disebabkan oleh imunisasi BCG dan pendedahan kepada mikrobakteria persekitaran. Sebaliknya, pesakit TB menunjukkan tindak balas penghasilan IFN-γ yang rendah terhadap PLs, ini berkemungkinan disebabkan oleh kelemahan sistem imun dan faktor-faktor genetik. Tambahan pula, keputusan esei imunoserapan berkaitan enzim (ELISA) dan analisa pemblotan Western, menunjukkan sera daripada pesakit TB dapat bertindak balas dengan protein-protein yang terdapat di dalam PLs. Ini menunjukkan bahawa PLs mengandungi antigen-antigen yang diekspres secara in vivo ketika peringkat jangkitan TB yang aktif. Kajian haiwan pula menunjukkan bahawa mencit yang diimunisasi PLBCG yang digabungkan dengan adjuvan alum (AL) telah menghasilkan tindak balas imun humoral yang spesifik dengan mecetuskan tindak balas campuran Th1 dan Th2 serta tindak balas selular yang spesifik secara in vivo

melalui tindak balas hipersensitiviti jenis lewat (DTH) yang positif terhadap PLBCG. Imunisasi mencit dengan kombinasi PLBCG dan Montanide ISA 51 VG (MT) telah mencetuskan jenis tindak balas Th2. Kumpulan mencit yang diimunisasi PLMs dengan kombinasi AL telah menghasilkan tindak balas antibodi yang spesifik dengan mencetuskan bentuk tindak balas Th2. Splenosit daripada mencit PLMs tanpa adjuvan telah menghasilkan tindak balas Th2 dalam penghasilan sitokin secara in

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vitro. Kedua-dua kumpulan mencit PLBCG dan PLMs diadjuvankan dengan alum dan kombinasi PLBCG dengan adjuvan MT telah menghasilkan tindak balas reaktif silang humoral terhadap antigen-antigen Mtb serta menghasilkan bentuk tidak balas Th2 dan gagal menghasilkan sitokin terhadap antigen daripada Mtb. Mencit yang diimunisasikan dengan PLMs diadjuvankan dengan AL telah menghasilkan tindak balas reaktif silang selular yang signifikan secara in vivo terhadap lisat keseluruhan sel daripada Mtb (Mtb-WCL). Pengenalpastian protein-protein daripada PLs dan Mtb oleh kumpulan mencit-mencit ini adalah berbeza bergantung kepada jenis adjuvan yang digunakan. Secara kesimpulannya, keputusan-keputusan yang diperolehi daripada kajian ini menyokong pembangunan keupayaan perlindungan PLs terhadap jangkitan Mtb di dalam model haiwan pada masa depan.

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PROTEOLIPOSOMES FROM Mycobacterium bovis BCG AND Mycobacterium

smegmatis AS NEW VACCINE CANDIDATES AGAINST TUBERCULOSIS

ABSTRACT

Tuberculosis (TB) is caused by Mycobacterium tuberculosis (Mtb). It is an infection that can be fatal and one of the leading causes of death and illness in the world. The bacteria typi cally attack the lungs, but can also attack other parts of the body such brain, kidney and spine. Bacille Calmette-Gúerin (BCG) is the world‘s most widely used vaccine and is the only vaccine available against TB. Studies show that BCG can protect against, or at least ameliorate, severe forms of systemic TB in children, particularly meningitis. Nevertheless, it seems to be of low or no protective value against pulmonary TB in adults. Thus, candidate vaccines more potent than BCG are desperately needed to protect against TB disease and transmission. Considering the genomic and antigenic homology between Mtb and non-pathogenic BCG and

Mycobacterium smegmatis (Ms), we decided to evaluate the potential of proteoliposomes (PLs) from BCG and Ms as vaccine candidates against TB. Bioinformatics studies predicted the presence of T and B cell epitopes in PLs from BCG and Ms which are expressed in vivo by Mtb during infection. Determination of human leukocyte antigen (HLA) have shown these epitopes provide the best percentage in the population of Malay and Cuba. Therefore, PLs from BCG (PLBCG) and Ms (PLMs) were prepared to evaluate the immunogenicity and cross-reactivity with Mtb antigens. These PLs were confirmed to be spherical and homogenous in their size and shape under electron microscopy and chromatographic study. We subsequently, evaluated the antigenicity of PLBCG and PLMs in humans. We showed that healthy individuals produced IFN-γ in the presence of PLs which

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could be explained due to BCG vaccination and contact with environmental mycobacteria. In contrast, the lower response of IFN- of TB patients could be explained by immunodepression and genetic factors/susceptability. In addition, ELISA and Western blot analyses showed that the PLs are recognized by sera from TB patients, which suggested the presence of Mtb antigens expressed in vivo during active infection in PLBCG and PLMs. Animal studies demonstrated that immunization with PLBCG adjuvanted with alum (AL) induced a specific humoral immune response with elicitation of a mixed Th1/ Th2 pattern and stimulation of specific cellular immune response in vivo with positive delayed type hypersensitivity (DTH) response against PLBCG. Immunization with PLBCG combined with Montanide ISA 51 VG (MT) elicited a humoral Th2 immune response against PLBCG. Mice immunized with PLMs adjuvanted with AL showed a specific antibody response against PLMs with elicitation of Th2 pattern. Splenocytes from PLMs- immunized mice produced a Th2 pattern of cytokine response in vitro against PLMs. Mice immunized with both PLBCG and PLMs adjuvanted with AL and PLBCG with MT induced a humoral cross-reactive response against Mtb antigens with elicitation of a Th2 pattern but failed to produce cytokine response against Mtb antigens. Mice immunized with PLMs adjuvanted with AL produced cellular cross-reactive responses in vivo with significant increase of DTH response against whole cell lysate from Mtb (Mtb-WCL). The pattern of recognition of proteins from PLs and Mtb by immunized animals differs depending of the adjuvant used. In conclusion, the results obtained in the current study support the future evaluation of the protective capability of these formulations in challenge experiments with Mtb in animal models.

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1 CHAPTER 1 INTRODUCTION 1.1 Tuberculosis (TB)

1.1.1 History of TB

Tuberculosis (TB) is an infectious disease caused by Mycobacterium tuberculosis

(Mtb) that may infect any part of the human body, but most commonly occurs in the lungs. It was recognized in ancient and historical times and may have killed millions of people compared to other microbial pathogen. The earliest cases of TB infection were reported in relics from ancient Egypt, India, and China (Zink et al., 2007). Additionally, evidence of spinal TB features has been identified in Egyptian mummies dating back to 9000 Before Century (B.C) (Hershkovitz et al., 2008). Analysis of 483 pre-Columbian skeletons in Chile showed that about 2% of their community had TB correlated with bony TB (Arriaza et al., 1995). Subsequently, molecular studies have established the presence of Mtb complex DNA in ancient bony specimens (Zink et al., 2007; Hershkovitz et al., 2008; Donoghue et al., 2011). In the Hippocrates era, around 460 B.C, TB was known as phthisis, the most widespread disease during that time. Phthisis attacked young adults between the age of 18 to 35 and is usually fatal (Frith, 2014). It is believed that phthisis was a hereditary disease but not an infectious disease because the infection commonly happened across the entire family (Daniel, 2000; Frith, 2014).

In the 18th century TB, known as ‗White plague‘, became an epidemic in Europe and resulted in millions of deaths. Then, in the early 19th century, TB is named Scrofula and was also known as ―king‘s evil‖ because they believed that the kings of England

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and France could cure scrofula simply by touching those affected (Wheeler, 2003; Frith, 2014). Generally, scrofula is an infection in the lymph nodes, known as lymphadenitis and it is the term used for lymphadenopathy of the neck. It can be caused by Mtb or atypical mycobacterial (Amir, 2010; Cruz-Knight and Blake-Gumbs, 2013).

In March 24, 1882, tubercle bacilli, the causative organism for TB, were discovered by Robert Koch (Sakula, 1982; Lawn and Zumla, 2011) using methylene blue staining (Sakula, 1982). After the discovery, the Sanatorium era begun and in 1982 the first World TB Day was announced by WHO.

1.1.2 TB incidence

Globally, TB is the second infectious disease that caused the most death in human after Human Immunodeficiency Virus (HIV)/ Acquired immunodeficiency syndrome (AIDS). In 2013, approximatey 9 million people were infected with TB. The incidence was observed in both developing and industrialized countries. It has reported by WHO that an estimated 45% of TB mortality rate and 41% of prevalence rate have declined since 1990 to 2013 (WHO, 2014). This means that the world is on track in reducing TB incidence and the trend needs to be sustained to achieve the TB-related United Nations‘ Millenium Development Goals (MDG) to narrow down the spread of the TB disease by 2015 (WHO, 2013).

TB incidence has declined in 6 regions in the world where Europe is the fastest region to show the decline at approximately 6.5% per year (Figure 1.1). Eastern Mediterranean and South-East Asia showed the slowest decline compared to other

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regions, at less than 1% and 2% per year respectively (WHO, 2013). In 2012, almost 8.6 million TB cases were reported worldwide at a rate of 122 cases per 100 000 population (WHO, 2013) (Figure 1.1). Asia (58%) showed the highest number of TB cases compared to other regions, followed by Africa (27%). The highest TB cases in Asia were reported in India, followed by China, South Africa, Indonesia and Pakistan (Figure 1.2). The other 4 regions showed smaller number of TB cases; 8% in Eastern Mediterranean, 4% in Europe and 3% in the Americas.

In 2012, the incidence of TB increased significantly in areas with high rate of HIV infection (1.0–1.2 million), particularly in Africa (37%) (WHO, 2013). Additional factors also contributed to the high TB burden including Multi-drug resistant-Tuberculosis (MDR-TB), Extensively Drug-Resistant Tuberculosis (XDR-TB), homelessness and poverty, increasing number of refugees, reduction of governmental support in TB prevention and lack of treatment programs (WHO, 2013). Another reason for the rising rate of TB cases is due to the high rate of immigration from countries with a high TB incidence (Abubakar et al., 2011; Pareek et al., 2011).

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Figure 1.1: TB incidence rates in 2012. (WHO Global Tuberculosis Report, 2013)

Figure 1.2: TB incidence in top-ten countries, 2012 (WHO Global Tuberculosis Report, 2013)

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5 1.1.3 Mycobacterium tuberculosis

Mycobacterium tuberculosis (Mtb) is a species from the Mycobacteriaceae family and belongs to a distinctive genus of Mycobacterium (Figure 1.3). It is a pathogenic bacterium and the causative agent for TB which was discovered by Robert Koch in 1882. It has a complex cell wall structure which hyropobic and waxy; composed of proteins intermingled in a matrix of peptidoglycan, rich mycolic acid, lipids and carbohydrates (Brennan et al., 2003) (Figure 1.4). They are Gram-positive aerobic bacteria, non-motile, non-spore forming, straight or slightly curved rod-shaped microbes, 1-4 m in length and between 0.3-0.6 m in diameter (Iseman, 2000; Lawn and Zumla, 2011). Mtbcontains granules and vacuoles and grows very slowly with a doubling time of 18-24 hours.

The genome of Mtb is 4,411,529 base pairs (bp) with 3924 predicted protein-coding sequences. The Mtb genome was sequenced and published in 1998 (Cole et al., 1998; Ismael et al., 2004). Mtb has a high content of guanine (G) and cytosine (C)in its DNA (65.6 %). The high GC content may be one of the survival strategies employed by the bacterium, since stability of DNA increases directly with number of GC bonds (Cole et al., 1998).

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Figure 1.3: Scanning electron microscope of Mtb (under 15549x magnification) (Centers for Disease Dontrol and Prevention, CDC).

Figure 1.4: Schematic representation of the mycobacterial cell wall (Gokhale et al., 2007).

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7 1.1.4 Transmission and pathogenesis

Airborne transmission is the most frequent route of Mtb infection (Figure 1.5). Although, it can be transmited through the gut, genitourinary tract and eye, or via compromised skin. Mtb is expelled through the airway by a person who have pulmonary or laryngeal TB and is carried in droplets in the air (McNerney et al., 2012), called droplet nuclei (1-5 m in diameter) which are very light and can remain in air for several hours depending on the environment (McNerney et al., 2012). These bacteria traverse the mouth or nasal passages, upper respiratory tract and bronchi to reach the alveoli of the lungs. Once the smaller droplet nuclei reach the alveoli, the organisms are taken up by alveolar macrophages; some of them are killed and a few multiply in the alveoli, which then enter the bloodstream and spread throughout the body (Russell et al., 2010; Orme, 2014) (Figure 1.5). Mtb will reach any part of the body and rapidly develop into either active disease or remain dormant without causing any symptoms (Schluger and Rom, 1998; Frieden and Driver, 2003). Infection does not necessarily lead to TB disease; in fact, it was reported in several studies, that only 3–10% of immunocompetent individuals that are infected will develop the disease during their life-time (Lawn and Zumla, 2011; Zumla, 2011), while more than 90% of infected subjects sustain infection in a subclinical stage known as latent TB infection (LTBI), in which the pathogen remains in a quiesce nt state (McNerney et al., 2012). LTBI may turn to TB disease by a re-activation process (Figure 1.6). A person with LTBI has a 5-10% chance of developing active TB in their lifetime during the first 5 years after becoming infected (Harries, and Dye,2006; WHO, 2015). Re-activation starts when the immune system becomes weakened and the dormant pathogen become active to cause TB disease.

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Figure 1.5: Summary of TB transmission and pathogenesis (Nunes-Alves et al., 2014)

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Figure 1.6: The choronolgy of Mtb infection (Young et al., 2008). 1.1.5 Symptoms of TB

People with LTBI do not have symptoms because they are in inactive state of the disease. People with TB disease may have any or all of the following symptoms; cough, fatigue, loss of appetite, weight loss, fever and night sweats. As the disease progresses, infected individuals may cough up blood (hemoptysis) and develop severe breathing problems. The symptoms of extra-pulmonary TB vary widely depending on which area of the body is infected. Thus, if TB affects the lymph nodes, it can cause swollen glands and a painless red mass, usually at the sides and base of the neck.When bone is infected, the spine is typically involved which causes back pain lasting weeks to months. Sometimes, the disease can progress to the spinal cord, causing paralysis. Mtb can also infect the joints, with slow progressive swelling and pain, usually in a large joint such as the knee.

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10 1.1.6 TB in children

In 2012, approximately 0.5 million TB cases occurred in children from the 8.6 million TB incidence worldwide. From an estimated 6% of global TB incidence (530 000) in 2012, 8% (74 000) of the incidence caused death among HIV-negative children (WHO, 2013). WHO estimated that TB incidence among children occurs in those younger than 15 years old. Childhood TB appears to be increasing in many parts of the world (Nelson and Wells, 2004b). In many industrialized countries, this situation is associated with new immigration patterns (Nelson and Wells, 2004a; Nelson et al., 2004; Marais, 2014). This trend is associated with many factors including poor economic situation and poor public health infrastructure, rising rates of HIV infection, difficulty of diagnosing childhood TB and the difficulty of diagnosing HIV-associated pulmonary infections in children (Raviglione et al., 2012; WHO, 2013).

An estimated 90% of cases in children progressed to serious TB due to primary infection before the age of 2 within the first 12 months without significant symptoms. Primary infection between the age 2 and 10 rarely progressed to serious TB, and disease progression is associated with significant clinical symptoms. Children older than 3 years who show TB symptoms should be clinically diagnosed before serious disease progression. Frequently, children with primary infection after 10 years of age are more likely to progress to adult-type disease (Centers for Disease Control and Prevention). However, early effective intervention in this group will reduce the burden of caveating disease and associated disease transmission in the community.

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11 1.1.7 TB in Malaysia

TB is the most common infectious disease with the highest number of deaths and remains as a major health problem in Malaysia. Moreover, it is the second biggest killer worldwide after Acquired immunodeficiency syndrome (AIDS) (WHO, 2013). As a middle-income country, the number of estimated TB cases continues to rise with rates of TB incidence and mortality of 82 and 8.5 cases per 100 000 population, respectively. In Malaysia, the number of TB cases detected annually is relatively higher than the reported incidence of HIV. About 9% of TB cases are being diagnosed among people living with HIV/AIDS (WHO, 2011).

The Ministry of Health reported an average of 18 500 TB cases between 2007 and 2010 while in 2011, 20 666 new TB cases were registered (MOH, 2011). Pulmonary TB is the most common infectious disease in Malaysia. However, extra pulmonary TB showed an increase and has spread to the entire community every year. In 2011, 2.7% of TB cases involved children of ≤14 years old, while 12.3% involved those of ≥65 years old. The majority (69.5%) of TB patients are between 21-60 years age.

Among the 15 states, Sabah recorded 143.7 cases/ 100 000 population which is the highest in the country, followed by Wilayah Persekutuan (121.7 cases/ 100 000 population), Sarawak (87.4 cases/ 100 000 population), Pulau Pinang (68.7 cases/ 100 000 population) and Kelantan (55.5 cases/ 100 000 population) (MOH, 2011). Approximately 13.9% of TB cases in Malaysia were discovered among the immigrant population (MOH, 2011).

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TB incidence has not significantly reduced because of the complexities surrounding the diagnosis, management, and treatment of TB, co-infection with communicable disease such as HIV/AIDS and non-communicable diseases (NCDs) (e.g. diabetes mellitus), challenges related to the treatment of drug-resistant Mtb strains, poverty, illiteracy and smoking (MOH, 2012). In Malaysia, the incidence of TB-HIV in 1990 was six cases and it reached to 933 cases in the year 2002 (MOH, 2012). Additionally, it was reported that diabetes mellitus is the leading risk factor for TB in Malaysia. Diabetic patients are three times more likely to develop TB (Dooley and Chaisson, 2009).

1.1.8 Risk factors

1.1.8(a) Human Immunodeficiency Virus (HIV)

WHO has declared that TB is the number two killer after HIV/AIDS. One in ten people infected with TB (but who are not infected with HIV) becomes ill or at least infected with TB once during his lifetime. Immunocompromised individuals such as HIV patients are at greater risk of developing active TB disease, 12 ‒ 20 times higher than normal individuals. In 2012, 8.6 million people were infected with TB while 1.3 million people died from TB: there were 320 000 deaths in HIV-infected people and estimated 170 000 deaths were from MDR-TB (WHO, 2013). Three-quarters of total TB deaths occurred in Africa and Southeast Asia regions, in which South Africa and India accounted for one third of the global TB deaths (Lawn and Zumla, 2011; Zumla, 2013).

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13 1.1.8(b) Non-communicable diseases (NCDs)

While TB has dominated the number of death worldwide, NCDs including diabetes mellitus, diseases related to smoking and alcoholism, chronic respiratory and cardiovascular diseases, and also mental disorders grew concomitantly as associated diseases with increased number of TB case (Marais et al., 2013; Lönnroth et al., 2010a).

NCDs are one of the risk factors for TB, mainly for the development of the disease as early as infection due to the the negative impact of NCDs on the host defense (Sopori, 2002; Wang et al., 2002; Cegielski and McMurray, 2004). Additionally, NCDs may make difficulties in management and treatment of TB, due to clinical challenges among people with diabetes mellitus and behavioral challenges among those who suffer from alcohol use disorders (Rehm et al., 2009; WHO, 2009). Moreover, TB can trigger or aggravate NCDs. For example, TB can worsen glucose control and lead to diabetes mellitus among those at high risk groups (WHO, 2009). TB, although not a classical risk factor for chronic respiratory disease, is one of the main causes of lung sequelae and bronchiectasis. TB also identified as an independent risk factor for chronic respiratory disease in a recent review (Salvi and Barnes, 2009).

Lönnroth et al. (2008) concluded that heavy drinkers consuming ~0.40 g alcohol a day and/or person who have a bad habit of alcohol use have three times risk for getting TB. A person with low to moderate drinking of alcohol has a lower risk to develop TB (Lönnroth et al. (2008). This association seems to be a causal factor for TB progression (Rehm et al., 2009). Additionally, alcohol has been showed a direct

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toxic response on the immune system. Several studies recommend that excessive alcohol consumption can weaken cell-mediated immunity and macrophage functions, which is important for the host response to Mtb infection (Szabo, 1997; Cegielski and McMurray, 2004; Rehm et al., 2009). Moreover, previous studies have demonstrated that heavy alcohol consumption may be one of cause of the malnutrition deficiencies, which can also weaken immunity (Gonzalez-Reimers et al., 2008; Knudsen et al., 2014), which can also weaken immunity (Porretta et al., 2012).

Globally, severe malnutrition in parts of the developing world causes a large increase in the risk of developing active TB (Cegielski and McMurray, 2004; Lönnroth et al., 2010b; Dye et al., 2011; Cegielski et al., 2012), due to its damaging effect on the immune system. Along with overcrowding, poor nutrition may also contribute to the strong link observed between TB and poverty.

1.1.9 Immune response against Mtb 1.1.9(a) Innate immune response

Host immune response plays an important role in the defense against Mtb during primary infection (Cooper et al., 2011). The component of the innate immune system comprised the anatomical barriers, as well as the complement system and several types of innate immune cells, including various leukocytes: macrophage, dendritic cells (DCs), granulocytes and natural killer (NK) cells (Janeway et al., 2001; Quintin

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Infection of Mtb begins with the inhalation of small droplet nuclei containing Mtb into the pulmonary alveoli. When Mtb enters the alveolar macrophage, Mtb are phagocytised by DCs, alveolar type II pneumocytes and monocyte-derived macrophage (Van Crevel et al., 2002; Hossain and Norazmi, 2013; Quintin et al., 2014).

Another crucial step in an effective host innate response is the recognition of Mtb or mycobacterial products on macrophages and DCs by toll-like receptor (TLR) (Belvin and Anderson, 1996; Medzhitov et al., 1997; Visintin et al., 2001). In mammals, TLR is an important connection between the innate and adaptive immune systems, and act as linkers for the recruitment and activation of the adaptive immune system (Werling et al., 2003; Akira et al., 2004; Pasare et al., 2005; Kanczkowskial et al., 2007) in host defence against microbial pathogens, particularly Mtb (Kulchavenya, 2013). There are four TLRs involved in recognition of Mtb namely, TLR2, TLR4, TLR9 and possibly TLR8 (Tapping and Tobias, 2003; Bulut et al., 2005; Davila et al., 2008).

The mycobacterial glycolipids, including lipoarabinomannan (LM) and phosphatidylinositol-mannosides (PIM), are the main factors that activate TLR2 (Ferwerda et al., 2005). Several mycobacterial antigens, such as the 19-kilo Dalton (kDa) lipoprotein, activate the innate immune response through TLR2 (Brightbill et al., 1999). In addition, several antigens such Hsp 60, 65, 70 and 38 kDa glycoprotein are reported to activate TLR4 (Jung et al., 2006; Jo et al., 2007; Kleinnijenhuis et al., 2011). A study by Bhatt and Salgame (2007) demonstrated that TLR2 is a central protein for in vitro activation of the innate immune response. In addition, TLR2 was

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shown to influence the maintenance of Th17 cells in the lung of Mtb-infected animals (Teixeira-Coelho et al., 2011) and granuloma formation (Drennan et al., 2004; Teixeira-Coelho et al., 2011), but not bacterial burden (Sugawara et al., 2003; Drennan et al., 2004; Bafica et al., 2005; Teixeira-Coelho et al., 2011). In addition, another important TLR member, TLR8 was suggested to have a potential role in TB-susceptibility in male TB patients and in macrophage immune responses to BCG infection (Davila et al., 2008).

Host recognition of Mtb or mycobacterial products will lead to immune cell activation and production of specific cytokines. IFN- is the central cytokine and plays a key role in the Th1 development leading to host defence against Mtb infection (Belosevic et al., 1989; Sher and Coffman, 1992; Gately et al., 1998). The importance of IFN-was demonstrated by the increased susceptibility to mycobacterial infection of children with IFN-receptor (IFN-R) deficiency (Jouanguy et al., 1996; Jouanguy et al., 1997) and in IFN-R knockout mice (Cooper

et al., 1993; Flynn et al., 1993). In humans, IFN-alone is insufficient to enhance the ability of macrophages to control Mtb infection. Thus, other cytokines such as Tumor Necrosis Factor (TNF)- and Interleukins (IL) are synergistically important in controlling the infection.

TNF- is produced by T cells, macrophages and DCs (Valone et al., 1988; Barnes et al., 1993; Henderson et al., 1997; Ladel et al., 1997; Serbina and Flynn, 1999) and plays a key role in granuloma formation. In TB patients, TNF- is detected at the site of infection (Barnes et al., 1993; Law et al., 1996; Casarini et al., 1999). IL-1β is also detected at the site of infection (Schauf et al., 1993; Law et al., 1996; Bergeron

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et al., 1997). Studies in mice suggested that IL-1β plays an important role in TB eradication (Juffermans et al., 2000; Yamada et al., 2000). In addition, IL-12 is also one of the crucial cytokines in controlling Mtb infection and is secreted following phagocytosis of Mtb by macrophages (Henderson et al., 1997; Ladel et al., 1997). The administration of IL-12 DNA can reduce the bacterial numbers in mice with chronic Mtb infection (Lowrie et al., 1999). Furthermore, IL-12, IL-18 and IL-15 are also important in controlling Mtb infection by increasing the production of IFN-

(Berg et al., 2002; Berg et al., 2003). 1.1.9(b) Adaptive immunity

1.1.9(bi) Antibody responses against Mtb

Humoral immune response is mediated by antibodies that are produced by activated B lymphocytes (plasma cells) and provides the most effective response against extracellular pathogens. These antibodies bind specifically to the antigens and are important elements in protection against infectious diseases (Janeway, 2005). The immune system then neutralizes or eliminates the pathogen from the body through ingestion and degradation of the antibody-antigen complex by phagocytes or by cell lysis (Abbas and Lichtman, 2001).

Recent studies showed that Mtb induced a humoral immune response to various mycobacterial antigens in humans despite being an intracellular pathogen (Steingart

et al., 2009). Intradermal BCG vaccination elicits two classes of antibodies such as IgG and IgM. These antibodies are directed to several mycobacterial antigens such glycolipid lipoarabinomannan (LAM) which is known as the major cell wall antigen

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(Brown et al., 2003; de Valliere etal., 2005). Moreover, a study by de Valliere et al.

(2005) revealed that antibody response shows to boost innate and cell mediated immune responses against mycobacteria.

The humoral immune response against mycobacterial antigens vary depending on the phase of infection (Kunnath-Velayudhan and Gennaro, 2011). For example, in the case of persons with LTBI, they do not have active TB and produce antibody to a different repertoire of Mtb antigens compared to those with active TB. Moreover, animal and human studies have demonstrated that increased antibody titres was directly correlated with the level of mycobacterial burden (Achkar et al., 2010; Kunnath-Velayudhan et al., 2010; Yu et al., 2012). The observation of high antibody titer in active TB patients against mycobacterial antigens has supported the disagreement that these antibodies are not protective. However, similar argument was not used to challenge the perception that immunity against TB is typically based on cell and cytokine mediated, since most of TB patients despite having normal T cell function and producing high IFN- (Okamoto et al., 2005; Cavalcanti et al., 2012). Interestingly, antibody titers can be used as markers for TB, in the same way that increases of IFN- levels indicate the progression of TB (Lin et al., 2009; Diel et al., 2011). Although not all the antibodies induced by Mtb infection are protective, several reports highlight the potential importance of specific antibodies in the defense against Mtb (Glatman-Freedman et al., 2000; Ciurana et al., 2004; Lopez et al., 2009; Olivarez et al., 2009; Acosta et al., 2010; Acosta et al., 2012; Achkar and Casadevall, 2013; Acosta et al., 2013; Alvarez et al., 2013 ).

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19 1.1.9(bii) Cellular responses against Mtb

Cell-mediated immune response is important to control Mtb infection. This response provides a major protective immunity to Mtb rather than the humoral responses (Kaufmann, 1995; Feng et al., 1999). Cluster of differentiation (CD) 4+ T cells are central to the human immune response to Mtb infection (Boom, 1996), and supported by other T cell subsets such as CD8+ cytolytic T cells (CTL) and unconventional T cells and CD1+ T cells) (Kaufmann, 2000; Flynn and Chan, 2001; Kaufmann, 2001; Jung et al., 2005; Kaufmann, 2013).

CD4+ T cells recognize antigens via major histocompatibility complex (MHC) class II molecules (Dorhoi et al., 2012), whereas,CD8+ T cells also contribute to immune protection against TB (Cooper, 2009) by recognition of antigens presented by MHC class I molecules (Dorhoi et al., 2012). In general, CD4+is responsible for directing an immune response by activating effector cells, whereas CD8+ T cellsis the killer cells that lyse infected cells by producing various cytokines such as IFN and TNF-α (Schluger and Rom, 1998). Some authors have reported that T cells such as CD4+, CD8+ and  T cells are important in controlling TB infection in mice and cows (Cassidy et al., 2001; Walravens et al., 2002; Vesosky et al., 2004).

Activated CD4+ T cells can differentiate into either Th1 cells which secrete specific cytokines IFN and TNF-α, or into Th2 cells secrete IL-4, IL-5, IL-10 and IL-13. The activation of Th1 cells by Mtb antigens resulted in cytokine production, mainly IL-2 and IFN(Huygen, 2014; Montagnani et al., 2014These cytokines will activate macrophages and other T cells (CD8+ and  T-cell) to provide strong protection against bacterial infection (Grange et al., 1995; Feng et al., 1999; Flynn

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and Chan, 2001; Raupach and Kaufmann, 2001). Th2 cells play important role in immune responses during Mtb infection by counter-regulating Th1 cells and impairing protective immunity against TB (Romagnani, 2005; Keir et al., 2008; De Libero and Mori, 2005). Th2 cells also contribute to down modulation of the immune response to the pathogen (Kaufmann, 2013) and to TB reactivation (De Libero and Mori, 2005; Keir et al., 2008; Gold et al., 2010; Kaufmann, 2013).

There are two major lysis mechanisms of CD8+ T cells in protecting against Mtb. Firstly, CD8+T cells expressed perforin and granzymes to lyse the infected cells. In humans, CD8+T cells produce granulysin that is toxic to Mtb which lyse the infected cells (Stenger et al., 1997; Stenger et al., 1998). Secondly, CD8+ T cells lyses the target cells via the Fas/FasL pathway (Clark et al., 1995). In addition to killing of infected cells, CD8+ T cells mediate anti-microbial effector function by secreting related cytokines such IFN and TNF-α (Herbein, 1997). he T cells and CD1+ restricted T cells also produce IFNwhich activate anti-mycobacterial activities in macrophages and inhibit the mycobacterial growth (Raupach and Kaufmann, 2001) which is supported by TNF-α. Tsukaguchi et al. (1995) demonstrated that  T cells have the capacity to display cytotoxicity and produce IFN-in response to Mtb infected macrophages. A study performed by D‘Souza et al. (1997) reported that  T cells contribute in early stages of TB infection. As the disease progress, apoptosis mediated by Fas/FasL clears the majority of Mtb and causes reactivation of  T cells (Li et al., 1998). Raja (2004) suggested that T cells are important in patients with LTBI (Flynn and Chan. 2001; North and Jung, 2004). Studies performed in a primate model demonstrated that the number and activity of T cells increased during primary mycobacterial infection and after BCG vaccination (Barnes et al., 1992).

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The CD1 family (group I CD1 molecules: CD1a, CD1b, CD1c and group II CD1 molecule; CD1d) is the least common T cell subset in human peripheral blood and the lungs. CD1 plays a significant role in protection against microbial pathogens like Mtb that contain unique lipids on their cell walls or membranes (Brigl et al., 2004; Cohen, et al., 2009). In humans, CD1a, b, and c have been associated with effective immune response to leprosy and mycobacterial infections. Brigl et al. (2004) have shown that CD1 expressions are down-regulated following exposure to live Mtb.

1.1.10 Control of TB 1.1.10(a) Diagnosis

The standard TB diagnosis in clinical examination isroutinely performed by acid fast bacillus, followed by culture on solid or liquid media for confirmation (Aber et al., 1980) as recommended by WHO guidelines (WHO, 2010). The result of Mtb culture can be obtained up to 8 weeks and in 10‒20 % of cases the bacillus is not successfully cultured (Pottumarthy etal., 1999). If the patient has pulmonary TB by radiographs but no diagnostic sputum sample, then bronchoscopy should be considered to confirm the diagnosis. Rapid and sensitive tools for diagnosis are required due to the increased incidence of TB worldwide particularly in the HIV endemic regions and the length of time required for the classical tests. Currently, rapid molecular tests are being used for the detection of pulmonary TB (Vassall et al., 2011; Boyle et al., 2014).

There are several new techniques being developed including fluorescent microscopy which used similar principle as conventional light microscopy. This technique

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required an ultraviolet light source such as a high-pressure mercury vapour lamp and an acid-fast fluorochrome dye for staining (Steingart et al., 2006). The bacilli are then visibly seen as fluorescent green rods. On the other hand, the most rapid liquid culture system used in laboratory is BACTEC 460. The use of BACTEC for culture can decrease the detection time when culturing using solid media (1‒2 months to 1‒2 weeks) (Enarson and Ait-Khaled, 2003). Safety issues concerning the radioactivity of BACTEC and its cost, however, prevents its widespread application.

The most recent and advanced technologies to diagnose TB are Nucleic acid amplification tests (NAATs) and Interferon-gamma (IFN-) assay (IGRAs). NAATs are used following microscopy to distinguish Mtb from other smear-positive mycobacteria. The technology is able to detect small amounts of genetic material (DNA or RNA target sequences) from Mtb due to repetitive amplification of the target sequences (Dinnes et al., 2007). If no target organism is present, there will be no amplification. The most common technique used is polymerase chain reaction (PCR) (Ramachandran and Paramasivan, 2003) and a new technique recently developed is Xpert MTB/RIF. On the other hand, IGRAs has been developed to replace the tuberculin skin test (TST) for TB detection of either active TB or latent TB. The assay is based on purified protein derivative (PPD) and region of difference (RD)-1 specific antigens are able to discriminate between Mtb and Mycobacterium avium intracellular complex infection (Streeton et al., 1998). Two types of valuable commercial IGRAs: Quantiferon-TB Gold and the newer version of Quantiferon-TB Gold In Tube (QFT-GIT), based on ELISA measure the amount of IFN-produced in response to specific Mtb antigens [QFT-G: Early secreted Antigenic Target

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(ESAT)-6 and CFP-10, QFT-GIT: ESAT-6, CFP-10 TB7.7]; and enzyme-linked immunospot - based T-SPOT.

The assays measure the number of peripheral mononuclear cells that produce

IFN-after stimulation with ESAT-6 and CFP-10. The antigens in IGRAs are absent in most of non-tuberculous mycobacteria (NTM) and BCG strain (Anderson et al., 2000; Mahairas et al., 1996; Harboe et al., 1996). Although, IGRAs cannot distinguish between active TB and LTBI (Herrera etal., 2010), IGRAs results are not confounded by BCG vaccination and less likely to be confounded by exposure to NTM (Lange et al., 2009). Thus, IGRAs shows advantages over the TST in identifying human immune response to Mtb, particularly in population with high NTM exposure and general vaccination.

1.1.10(b) Treatment

TB can be treated by chemotherapy and this approach started in the late 1940s with the introduction of streptomycin. The objective of this approach is to eradicate all tubercle bacteria from the patient and to prevent secondary resistant bacteria. Subsequently, several drugs were introduced including p-aminosalicylic acid (PAS) (1946), isoniazid (INH) (1952), cycloserine (1955), kanamycin (1957), rifampin (RIF) (1965), ethionamide (1966), ethambuthol (ETH) (1968) and pyrazinamide (PZA) (1970) (Friedman, 2000). The usual standard drug regiment short course chemotherapy consists of 2 months of INH, RIF, PZA and ETH, 4 months of INH and RIF. The drugs must be taken either daily or 3 times per week and they have to be given under direct observation (DOTs) by health-care staff as recommended by WHO (WHO, 2010).

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Regular drug treatment is a pre-requisite for treatment success. However, under certain circumstances, the doctor may have modified the particular needs of individual patients. The great majority of patients can be considered as non-infectious after 2 weeks of effective drug treatment. If the treatment is taken irregularly, there may be serious consequences: the Mtb may become resistant to the drugs which can cause MDR-TB, where TB cannot be cured or TB disease can spread to others leading to death.

ETH is added if MDR-TB is suspected (American Thoracic Society, 1993). ETH inhibits the synthesis of arabinogalactan and interrupts the mycobacterial cell wall synthesis (Mikusova et al., 1995). INH is important in inhibiting the synthesis of mycolic acid (Petrini and Hoffner, 1999). PZA plays an important role in the intensive phase of the bacteria. PZA produced specific enzyme to inhibit the fatty acid synthesis and affects the mycobacterial energy production for survival (Mitchinson et al, 2005). RIF is an important agent in the treatment of TB and has bactericidal properties against both intracellular and extracellular Mtb (Friedman et al., 2000). In the late 1970s, TB drug development stopped as it was assumed that the effective drug and the combination with BCG vaccination programs would lead to the prevention of TB as a public health threat. Unfortunately, not all TB patients received an effective control of TB treatment in certain areas (Gandhi et al., 2006; Tadesse et al., 2011). Nachega and Chaisson (2003) reported that this phenomenon resulted in the MDR-TB defined as Mtb that is resistant to at least two drugs, INH and RIF. Therefore, an effective surveillance is very important in reducing the potential for MDR-TB, and this is one of the essential factors in gaining control of the global TB problem.

References

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