• Tiada Hasil Ditemukan

STUDIES ON IMMUNE EXHAUSTION AND SENESCENCE OF CERTAIN CD8+ T-CELL PHENOTYPES IN HIV-TB CO-INFECTED INDIVIDUALS

N/A
N/A
Protected

Academic year: 2022

Share "STUDIES ON IMMUNE EXHAUSTION AND SENESCENCE OF CERTAIN CD8+ T-CELL PHENOTYPES IN HIV-TB CO-INFECTED INDIVIDUALS"

Copied!
177
0
0

Tekspenuh

(1)of M al. ay. a. STUDIES ON IMMUNE EXHAUSTION AND SENESCENCE OF CERTAIN CD8+ T-CELL PHENOTYPES IN HIV-TB CO-INFECTED INDIVIDUALS. U. ni. ve. rs i. ty. ALIREZA SAEIDI. FACULTY OF MEDICINE UNIVERSITY OF MALAYA KUALA LUMPUR. 2017.

(2) M al. ay a. STUDIES ON IMMUNE EXHAUSTION AND SENESCENCE OF CERTAIN CD8+ T-CELL PHENOTYPES IN HIV-TB CO-INFECTED INDIVIDUALS. of. ALIREZA SAEIDI. U. ni. ve. rs i. ty. THESIS SUBMITTED IN FULFILMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY. FACULTY OF MEDICINE UNIVERSITY OF MALAYA KUALA LUMPUR. 2017.

(3) UNIVERSITY OF MALAYA ORIGINAL LITERARY WORK DECLARATION. Name of Candidate: ALIREZA SAEIDI Registration/Matric No: MHA 120008 Name of Degree: Doctor of Philosophy Title of Thesis:. in HIV-TB Co-Infected Individuals Field of Study: Immunology. M al. I do solemnly and sincerely declare that:. ay a. Studies on Immune Exhaustion and Senescence of Certain CD8+ T-cell Phenotypes. U. ni. ve. rs i. ty. of. (1) I am the sole author/writer of this Work; (2) This Work is original; (3) Any use of any work in which copyright exists was done by way of fair dealing and for permitted purposes and any excerpt or extract from, or reference to or reproduction of any copyright work has been disclosed expressly and sufficiently and the title of the Work and its authorship have been acknowledged in this Work; (4) I do not have any actual knowledge nor do I ought reasonably to know that the making of this work constitutes an infringement of any copyright work; (5) I hereby assign all and every rights in the copyright to this Work to the University of Malaya (“UM”), who henceforth shall be owner of the copyright in this Work and that any reproduction or use in any form or by any means whatsoever is prohibited without the written consent of UM having been first had and obtained; (6) I am fully aware that if in the course of making this Work I have infringed any copyright whether intentionally or otherwise, I may be subject to legal action or any other action as may be determined by UM. Candidate’s Signature. Date:. Subscribed and solemnly declared before, Witness’s Signature. Date:. Name: Designation:. ii.

(4) ABSTRACT The syndemic relationship between human immunodeficiency virus (HIV) and tuberculosis (TB) has inflicted a significant disease burden worldwide. Mycobacterium tuberculosis (MTB) and HIV appear to impact the pathogenesis of each other leading to eventual and rapid immune deterioration, the key feature of acquired immunodeficiency syndrome (AIDS). Hence, in depth investigations are required for the improved. ay a. understanding of the complex interaction between the two pathogens. Several hypotheses have been proposed on the exacerbation of TB by HIV and vice versa. We hypothesized. M al. immunosenescence, chronic immune activation and immune exhaustion as potential mechanisms that inflict deterioration of T-cell responses in HIV-TB co-infection. Our investigations showed that increased plasma viremia and decreased CD4:CD8 T-cell ratio. of. observed in HIV-TB co-infected subjects were significantly associated with CD57 levels and markers consistent to late-senescence of CD8+ T cells. Up-regulation of CD57 was. ty. associated with loss of CD27 and CD28 on CD8+ T cells, besides the diminished. rs i. expression of IL-7Rα, CD27 and CD28 levels on CD57+CD4+ T cells. We also observed increase of CD38 on CD8+ T cells. Increased HIV viremia, decreased T-cell counts and. ve. CD4:CD8 T-cell ratio were directly associated with elevated levels of CD38 on T cells.. ni. Functional deterioration of antigen-specific CD8+ T cells was also clearly evident as. U. intracellular perforin, granzyme B and IFN-γ levels were relatively decreased in HIV-TB co-infected subjects. Given that HIV-TB co-infection results in proliferative senescence, chronic immune activation, and functional insufficiency of CD8+ T cells, we also studied the role of a special subset of CD8+ T cells called TCR Vα7.2+CD161++CD8+ mucosalassociated invariant T (MAIT) cells among anti-retroviral therapy (ART)/anti-TB therapy (ATT) treatment-naïve HIV-TB co-infected, ART/ATT-treated HIV-TB co-infected, ART naïve HIV-infected, ART-treated HIV-infected patients, and HIV negative healthy controls (HCs). Our data revealed that the frequency of MAIT cells was severely depleted. iii.

(5) in HIV-infected as well as HIV-TB co-infected patients. Further, PD-1 expression on MAIT cells was significantly increased in HIV-infected and HIV-TB co-infected patients. The frequency of MAIT cells did not show any significant increase despite the initiation of ART and/or ATT. Majority of the MAIT cells in HCs showed a significant increase in CCR6 expression as compared to HIV-TB co-infection. No marked difference was seen with the expression of CCR5 and CD103 among the study groups. Decrease of CCR6. ay a. expression appears to explain why HIV-infected patients display weakened mucosal immune responses. Functional longitudinal investigations on MAIT cells and the signaling events underlying the up-regulation of co-inhibitory molecules using blocking. M al. experiments may be required to develop novel intervention strategies to harness. U. ni. ve. rs i. ty. of. protective immune responses in HIV-TB co-infected individuals.. iv.

(6) ABSTRAK. Hubungan sindemik antara virus human immunodeficiency (HIV) dan tuberculosis (TB) telah mangakibatkan beban penyakit yang ketara di seluruh dunia. Mycobacterium tuberculosis dan HIV memberi kesan kepada pathogenesis antara satu sama lain sehingga kemerosotan immun yang cepat. Oleh itu, siasatan yang mendalam adalah diperlukan. ay a. untuk meningkatkan pemahaman tentang perhubungan antara dua patogen. Beberapa hipotesis telah dicadangkan atas eksaserbasi TB disebabkan oleh HIV dan sebaliknya. Kami hipotesiskan imunosenesen, imun aktivasi kronik dan keletihan imun adalah. M al. mekanisma-mekanisma yang berpotensi untuk mengakibatkan kemerosotan respons selT dalam jangkitan bersama HIV-TB. Siasatan-siasatan Kam kami menunjukkan bahawa peningkatan virus dalam plasma dan penurunan nisbah sel-T CD4:CD8 diperhatikan. of. dalam subjek-subjek berjangkitan HIV-TB adalah berkaitan secara ketara dengan tahap. ty. CD57 dan tanda-tanda konsisten dengan senses lewat untuk sel-T CD8+. Regulasi meningkat dalam CD57 adalah berkaitan dengan kehilangan CD27 dan CD28 pada sel-T. rs i. CD8+, selain itu kehilangan ekspresi IL7Rα, tahap CD27 dan CD28 pada sel-T. ve. CD57+CD4+. Kami juga mendapati kenaikan CD38 pada sel-T CD8+. Peningkatan viremia HIV, penurunan bilangan sel-T dan nisbah CD4:CD8 sel-T adalah berkaitab. ni. secara langsung dengan peningkatan tahap CD38 atas sel-T. Kemerosotan fungsi untuk. U. sel-T yang antigen-spesifik adalah juga jelas dibuktikan dengan tahap-tahap perforin intracellular, granzyme B dan IIFN-γ di mana ia menurun dalam subjek-subjek yang dijangkiti HIV-TB. Diberi bahawa jangkitan bersama HIV-TB menyebabkan senesens proliferatif, imun aktivasi kronik, dan ketidakcukupi fungsi sel-T, kami juga mengkaji peranan. untuk. sejenis. subset. khas. untuk. sel-T. yang. dipanggil. “TCR. Vα7.2+CD161++CD8+ mucosal-associated invariant T (MAIT) cells” antara pesakit HIV-TB yang naïve anti-retroviral terapi )ART) / anti-TB terapi (ATT), pesakit HIV-TB. v.

(7) yang telah diubati dengan ART/ATT, pesakit HIV sahaja yang naïve ART, pesakit HIV sahaja yang telah diubati dengan ART, serta control yang HIV negative (HCs). Data kami mendedahkan bahawa frekuensi sel-sel MAIT adalah amat kekurangan dalam pesakit HIV dan pesakit jangkitan bersma HIV-TB. Tambahan lagi, ekspresi PD-1 pada sel MAIT adalah meningkat pada pesakit dijangkiti HIV dan pesakit HIV-TB. Frekuensi sel MAIT tidak meningkat ketara selepas permulaan ART dan / atau ATT. Kebanyakan sel. ay a. MAIT dalam HCs menunjukkan peningkatan ketara dalam ekspresi CCR6 berbanding dengan pesakit HIV-TB. Tiada perbezaan dinampak dengan ekspresi CCR5 dan CD103 antara kumpulan-kumpulan kajian. Pengurangan ekspresi CCR6 menerangkan kenapa. M al. pesakit-pesakit HIV mempunyai imun respons mucosal yang lemah. Siasatan fungsi secara longitudinal atas sel MAIT dan signal berasaskan regulasi-naik untuk molekulmolekul ko-inhibitori dengan eksperimen halangan mungkin diperlukan untuk mencari. of. strategi intervensi novel bagi memanfaatkan ketahanan imun respons bagi individu-. U. ni. ve. rs i. ty. individu yang dijangkiti HIV-TB.. vi.

(8) ACKNOWLEDGEMENTS. ay a. At the outset, I would like to express my sincere and deepest appreciation to my supervisors, Assoc. Prof. Shankar Esaki Muthu, and Prof. Adeeba Kamarulzaman, who have given me such an incredible opportunity in venturing scientific research along at the University of Malaya. I also register my gratitude to all our Research and Clinical Collaborators, Prof. Marie Larsson, Department of Clinical and Experimental Medicine, Linkoping University, Sweden; Prof. Shamala Devi Sekaran, Department of Medical Microbiology, University of Malaya; Dr. Vijayakumar Velu, Emory Vaccine Center, USA; Dr. Karlhans Fru Che, Karolinska Institutet, Solna, Sweden, Dr. Rada Ellegård, Linköping University, Sweden, and Dr. Sasheela Ponnampalavaanar, University of Malaya Medical Center, Malaysia for their prompt supports with my research. Without all your guidance and constant encouragements, this thesis seldom would have been possible!. M al. I would like to thank my colleagues from Shankar’s Lab, Mr. Barathan Muttiah, Mr. Chong Yee Kien and Ms. Vicky Lau Tien Tien for their supports with the experiments. Special thanks are due to Mr. Yong Yien Kong and Dr. Tan Hong Yien for their kind efforts in assisting me along the identification and recruitment of patients as well as collection of samples. Not forgetting, I would like to register my special thanks to Dr. Keivan Zandi and my fellow postgraduate colleagues, Mr. Pouya Hassandarvish and Mr. Ehsan Moghaddam for their constant supports.. rs i. ty. of. I would like to place my sincere gratitude to all the study participants, clinical, paraclinical and laboratory staff of University of Malaya Medical Center for their assistance with patient recruitment, specimen collection and cooperation. Ms. See Hui Shien for antibodies, reagents and supports with optimization of BD FACSCanto™ II at the Independent Testing Laboratory (ITL) of the University of Malaya Medical Center.. U. ni. ve. I would like to acknowledge the humongous and generous funding support provided for my research by the University of Malaya Research Grant (UMRG) RG448-12HTM of the Health and Translational Medicine Research Cluster to Dr. Esaki Muthu Shankar, and UM.C/625/1/HIR/MoHE/MED/014 to Prof. Adeeba Kamarulzaman by the High Impact Research (HIR) of the University of Malaya. I also acknowledge grant supports from AI52731: the Swedish Research Council, the Swedish Physicians against AIDS Research Foundation, the Swedish International Development Cooperation Agency; SIDA SARC, VINNMER for Vinnova, Linköping University Hospital Research Fund, CALF and the Swedish Society of Medicine to Prof. Marie Larsson. Special and last, but not the least, my immense gratitude to my beloved parents, Mrs. Azam Sadat Sadeghi Makki and Mr. Javad Saeidi for their continued advice that positioned me on the path of excellence. I am very much sanctified for having blessed with them who have always supported me with all my decisions in life. I dedicate my work to my loving parents and thank them for having blessed me with tonnes of affection. I would not have reached this stage if it weren’t for their concerns and enthusiastic thoughtfulness. Alireza Saeidi 27-04-2017. vii.

(9) TABLE OF CONTENTS. ORIGINAL LITERARY WORK DECLARATION ...................................................... ii ABSTRACT ................................................................................................................ iii ABSTRAK ................................................................................................................... v ACKNOWLEDGEMENTS ........................................................................................ vii TABLE OF CONTENTS ........................................................................................... viii. ay a. LIST OF FIGURES .................................................................................................... xii LIST OF TABLES ...................................................................................................... xv. M al. LIST OF SYMBOLDS AND ABBREVIATIONS ..................................................... xvi LIST OF APPENDICES ............................................................................................ xxi CHAPTER 1: INTRODUCTION............................................................................... 1. of. CHAPTER 2: BACKGROUND AND REVIEW OF LITERATURE ...................... 3 Epidemiology of HIV and Tuberculosis ............................................................... 3. 2.2. The Epidemiology of HIV and Tuberculosis Co-infection in Malaysia ................. 4. 2.3. Human Immunodeficiency Virus (HIV) ............................................................... 7. rs i. ty. 2.1. 2.3.1 Discovery and Epidemiology ...................................................................... 7. ve. 2.3.2 Virology ..................................................................................................... 8. ni. 2.3.3 Pathogenesis ............................................................................................. 10. U. 2.3.4 HIV-1 Disease Progression ....................................................................... 13 2.3.5 Treatment ................................................................................................. 16. 2.4. Mycobacterium Tuberculosis ............................................................................. 18 2.4.1 Discovery and Epidemiology .................................................................... 18 2.4.2 Bacteriology ............................................................................................. 19 2.4.3 Transmission and Pathogenesis ................................................................. 19 2.4.4 Tuberculosis Disease Progression ............................................................. 20 2.4.5 Treatment ................................................................................................. 22. viii.

(10) 2.5. T Cells and Immune Responses ......................................................................... 23 2.5.1 T Cell Co-Stimulatory and Co-Inhibitory Pathways .................................. 26. 2.6. T-cell Exhaustion ............................................................................................... 28 2.6.1 Loss of Effector Functions During T-Cell Exhaustion ............................... 29. 2.7. Unraveling The Complexity Of T-cell Co-stimulation in HIV Infection ............. 31. 2.8. T Cells in HIV Immunopathogenesis ................................................................. 32. ay a. 2.8.1 HIV-TB Co-Infection and Immunopathogenesis ....................................... 34 2.8.2 Role of HIV in the Exacerbation of MTB Infection ................................... 34 2.8.3 The Impact of TB on the Exacerbation of HIV-1 Infection ........................ 37 Immunosenescence ............................................................................................ 38. M al. 2.9. 2.9.1 Immunosensescence and Chronic Immune Activation............................... 39 2.9.2 Immunosenescence and HIV-TB Co-Infection .......................................... 40. of. 2.9.3 CD38 and HLA-DR Immune Activation Markers in 41. ty. HIV-TB Co-infection .................................................................................... rs i. 2.9.4 Role of CD57 and CD127 in Cellular Immune Senescence ....................... 42 2.10 Exhaustion, Anergy and Senescence .................................................................. 44. ve. 2.11 MAIT Cells ....................................................................................................... 45 2.11.1 MAIT Cell Phenotype ............................................................................ 46. ni. 2.11.2 MAIT Cell Activation ............................................................................ 47. U. 2.11.3 MAIT Cell Cytotoxicity ......................................................................... 49 2.11.4 Anti-Bacterial and Anti-Fungal Properties of MAIT Cells ...................... 50 2.11.5 MAIT Cells in MTB Infection................................................................ 52 2.11.6 MAIT Cells in HIV Infection ................................................................. 53. 2.12 Key Research Questions and Aims..................................................................... 62 CHAPTER 3: MATERIAL AND METHODS ........................................................ 63 3.1. Ethical Approval Statement ............................................................................... 63. ix.

(11) Study Population................................................................................................ 63. 3.3. Isolation of Peripheral Blood Mononuclear Cells ............................................... 64. 3.4. T-cell Immunophenotyping ................................................................................ 65. 3.5. Primary Cell Culture .......................................................................................... 66. 3.6. T-cell Stimulation .............................................................................................. 66. 3.7. T-cell Immunophenotyping ................................................................................ 66. 3.8. Intracellular Staining .......................................................................................... 67. 3.9. Mait Cell Immunophenotyping .......................................................................... 68. ay a. 3.2. 3.10 Flow Cytometry ................................................................................................. 68. M al. 3.11 Statistical Analysis............................................................................................. 69 CHAPTER 4: RESULT ............................................................................................ 70 4.1. Plasma HIV Viral Loads were Higher and CD4/CD8. T cells of both HIV-infected and HIV-TB Co-infected Individuals. ty. 4.2. of. T-cell Ratios were Significantly Lower in HIV-TB Co-infected Subjects ........... 70. 4.3. rs i. Showed Comparable Accentuation in CD38 and HLA-Dr Levels ...................... 73 CD8+ T-cell Subsets of HIV-TB Co-infected Patients. ve. Showed Significantly Increased Levels of CD57 and Exhibited Altered Expression of Phenotypic Markers ........................................ 79. HIV-TB Co-infection Led to Proliferative Senescence. ni. 4.4. U. by Accentuating the Frequency of Late-differentiated CD8+ T Cells with Concurrent Loss of CD27 and CD28 Levels, Potentially Leading to Diminished Cytokine Secretion Attributes ............................................................................................ 85. 4.5. Close Association was Observed between Markers of Chronic Immune Activation with Differentiation and Senescence Markers in T Cells of HIV-infected and HIV-TB Co-infected Subjects ................................ 93. x.

(12) 4.6. HIV Mono- and HIV/Mtb Co-infections Led to Significant Depletion of CD161++CD8+ T Cells ............................................... 98. 4.7. CD161++ MAIT Cell Frequencies Did not Recover during ART and ART/ATT Therapy .......................................................................... 103. 4.8. PD-1 Expressing CD161++ MAIT Cells Represented an Important Cellular Phenotype in HIV. 4.9. ay a. Mono- and HIV-TB Co-infections .................................................................... 103 MAIT Cells Expressed Significantly Lower Levels of. CCR6 during HIV-TB Co-infection.................................................................. 105. M al. 4.10 Attrition of TCR vα7.2+CD8+ MAIT Cells. Co-expressing CD161 was Noticed among HIV-infected Patients .................... 107 4.11 Surface Expression of PD-1 on CD161++/MAIT Cells. of. was Inversely Proportional to the Frequency of MAIT. ty. Cells in the Peripheral Blood ........................................................................... 109. rs i. CHAPTER 5: DISCUSSION .................................................................................. 110 CHAPTER 6: CONCLUSIONS ............................................................................. 119. ve. REFERENCES ....................................................................................................... 121 LIST OF PUBLICATIONS AND PAPERS PRESENTED ................................... 154. U. ni. APPENDIX ............................................................................................................. 159. xi.

(13) LIST OF FIGURES Figure 2.1: Reported HIV and AIDS-related Deaths, Malaysia (1986 –2014) ................ 6 Figure 2.2: New TB, HIV and Prevalence of TB-HIV Co-infection, Malaysia (1999-2014)................................................................................. 6 Figure 2.3: Global Distribution of HIV-1 Subtypes and Recombinants .......................... 7. ay a. Figure 2.4: Structure of HIV-1 ...................................................................................... 8 Figure 2.5: HIV Life Cycle ......................................................................................... 10 Figure 2.6: Typical Course of HIV-1 Infection ............................................................ 12. M al. Figure 2.7: Granule-mediated Cytotoxicity through Immunological Synapse .............. 26 Figure 2.8: Hierarchical T-Cell Exhaustion during Chronic Infection .......................... 31. of. Figure 2.9: Infection with HIV Facilitates the Upregulation of. Inhibitory Molecules in T Cells ................................................................ 32. ty. Figure 2.10: Proposed Model of HIV/Mycobacterium tuberculosis Co-infection ......... 36. rs i. Figure 2.11: Potential MAIT Cell Response to Bacterial Infected Cells ....................... 49. ve. Figure 2.12: Phenotype of Exhausted MAIT Cells ...................................................... 60 Figure 2.13: Proposed Mechanism of Long-term Activation of. ni. MAIT Cells and Their Subsequent Exhaustion during HIV Infection ...... 61. U. Figure 4.1: HLA-DR and CD38 Expression Profiles in CD8+ T-Cell Subsets of HIV-TB Co-Infected Group. .................................................... 74. Figure 4.2: HLA-DR and CD38 Expression Profiles in CD4+ T-Cell Subsets of HIV-TB Co-Infected Group. .................................................... 75 Figure 4.3: Immune Activation Co-expression Profile within CD4+ and CD8+ T-Cell Subsets of HIV-TB Co-infected Groups ............. 76 Figure 4.4: Expression of CD57 in both T-cell Subsets of. xii.

(14) HIV-TB Co-infected Subjects ................................................................... 81 Figure 4.5: Differential Expression of CD27, CD28, and CD127 in CD57- and CD57+ CD8+ T-cell Subsets .............................................. 82 Figure 4.6: Differential Expression of CD27, CD28, and CD127 in CD57- and CD57+ CD4+ T-cell Subsets .................................. 83 Figure 4.7: CD127 Expression Levels in T-cell Subsets of HIV-infected Groups ........ 84. ay a. Figure 4.8: Altered Distribution of Differentiated CD8+ T-cell. Populations in HIV-Infected Groups .......................................................... 87. M al. Figure 4.9: Comparative Distribution of Differentiated CD4+ T-cell Populations ........ 88 Figure 4.10: CD57 Expression Followed the Differentiation. Phases, Irrespective of Differentiation Stages.......................................... 89. of. Figure 4.11: Senescence of CD8+ T-cell Subsets with Altered. ty. Distribution of the Sub-populations in HIV-infected Groups ................... 90. rs i. Figure 4.12: Distribution of Senescent Sub-populations of CD4+ T Cells within the Study Groups ................................................... 91. ve. Figure 4.13: Intracellular Secretion Profile of. ni. Granzyme B, Perforin, CD57, and IFN-γ Following. U. Stimulation with HIV Gag p24 by CD8+ T cells ..................................... 92. Figure 4.14: Gating Strategy for Analyzing CD161 Expression on MAIT Cells .......... 99 Figure 4.15: Phenotypic Properties of CD161++CD8+ T Cells ................................. 100 Figure 4.16: Percentage of CD8+ T cells Expressing CD161++ across the Different Study Groups ........................................................ 101 Figure 4.17: Correlation Analysis of Peripheral MAIT Cell Frequency and Plasma Viral Load or CD4+ T-cell Counts .................... 102. xiii.

(15) Figure 4.18: Expression Levels of different Markers by CD161++CD8+ T-cell Subsets in the Study Population. ....................... 104 Figure 4.19: Differential Expression Levels of Inhibitory and Mucosal Homing Markers by Peripheral MAIT Cells across the Different study Groups. ........................................................ 106 Figure 4.20: Profile of Expression of CD161 on MAIT Cells .................................... 108. ay a. Figure 4.21: Correlation Analysis between PD-1 Expression. U. ni. ve. rs i. ty. of. M al. and CD161++/MAIT Cell Frequency in the Study Participants ............. 109. xiv.

(16) LIST OF TABLES. Table 4.1: Demographic, Clinical and Laboratory Characteristics of Subjects ............. 71 Table 4.2: Demographic, Clinical and Laboratory Characteristics of Study Participants ....................................................................................... 72 Table 4.3: Correlation Analysis of Clinical HIV Disease. ay a. Progression Parameters in CD8+ T-Cell Subsets......................................... 77 Table 4.4: Correlation Analysis of Clinical HIV Disease. Progression Parameters in CD4+ T-Cell Subsets......................................... 78. M al. Table 4.5: Correlation Analysis of Immune Activation in CD8+ T-cell Subsets .......... 94 Table 4.6: Correlation Analysis of Immune Activation in CD4+ T-cell Subsets .......... 95. of. Table 4.7: Correlation Analysis of Biosignatures of Immunosenescence and Differentiation in CD8+ and CD4+ T-cell Phenotypes. ........................ 96. ty. Table 4.8: Correlation Analysis of CD127 Protein Expression. U. ni. ve. rs i. in CD8+ and CD4+ T-cell Subsets .............................................................. 97. xv.

(17) LIST OF SYMBOLDS AND ABBREVIATIONS. :. Activation-induced cell death. ABC. :. ATP-binding cassette. AML. :. Acute myeloid leukemia. AIDS. :. Acquired immune deficiency syndrome. APCs. :. Antigen-presenting cells. ART. :. Anti-retroviral therapy. ATT. :. Anti-tuberculosis therapy. APC. :. Allophycocyanin. ARV. :. Anti-retroviral. BCG. :. Bacillus Calmette-Guérin. BLIMP-1. :. B lymphocyte-induced maturation protein-1. BTLA. :. B- and T-lymphocyte attenuator. BV421. :. Brilliant violet 421. CCR. :. C-C chemokine receptor. M al. of. ty. rs i. Cluster of differentiation. C. glabrata. :. Candida glabrata. CIA. :. Chronic immune activation. CMV. :. Cytomegalovirus. CTLs. :. Cytotoxic T cells. CTLA-4. :. Cytotoxic T-lymphocyte-associated protein 4. CXCR. :. C-X-C chemokine receptor. Cy5. :. Cyanine 7. DC. :. Dendritic cell. dsDNA. :. Double-stranded DNA. ni. ve. :. U. CD. ay a. AICD. xvi.

(18) :. Dimethyl sulfoxide. ECs. :. Elite controllers. E. coli. :. Escherichia coli. E.faecalis. :. Enterococcus faecalis. ELISA. :. Enzyme-linked immunosorbent assay. FACS. :. Fluorescence-activated cell sorting. FBS. :. Fetal bovine serum. FITS. :. Fluorescein isothiocyanate. F.tularensis. :. Francisella tularensis. GE. :. Granule exocytosis. GAS. :. Group A Streptococcus. GALT. :. Gut-associated lymphoid tissues. GrzB. :. Granzyme B. Gp. :. Glycoprotein. K.pneumoniae. :. M al. of. ty. :. Killer cell lectin-like receptor subfamily G, member 1. :. High active anti-retroviral therapy. ve. HAART. Klebsiella pneumoniae. rs i. KLRG1. ay a. DMSO. :. Herpes virus entry mediator. HBV. :. Hepatitis B virus. HCV. :. Hepatitis C virus. HCs. :. Healthy controls. HIV. :. Human immunodeficiency virus. HIR. :. High Impact Research. HLA. :. Human leukocyte antigen. IDUs. :. Injecting drug users. IFN. :. Interferon. U. ni. HVEM. xvii.

(19) :. Interferon-γ release assay. IL. :. Interleukin. Ig. :. Immunoglobulin. IDO. :. Indoleamine-pyrrole 2,3-dioxygenase. INSTIs. :. Integrase inhibitors. INKT cells. :. Invariant natural killer T cells. ITL. :. Independent Testing Laboratory. LAG-3. :. Lymphocyte activation gene-3. LAM. :. Lipoarabinomannan. LILRB. :. Leukocyte Ig-like receptor B. M al. ay a. IGRA. Listeria monocytogenes. LNTPs. :. Long term non-progressors. LTBI. :. Latent tuberculosis infection. LCMV. :. Lymphocytic choriomeningitis virus. MIP-1β. :. ty. :. Mucosal-associated invariant T. :. Medical Ethics Committee. ve. MEC. Macrophage inflammatory protein-1β. rs i. MAIT. of. L.monocytogenes :. :. Major histocompatibility complex. MR1. :. MHC class I-related. mRNA. :. Messenger RNA. MTB. :. Mycobacterium tuberculosis. MOM. :. Mycobacterial outer membrane. mAbs. :. Monoclonal antibodies. MDR-TB. :. Multi-drug resistant TB. mL. :. Mililiter. NK. :. Natural killer. U. ni. MHC. xviii.

(20) :. Nucleotide reverse transcriptase inhibitors. NNRTIs. :. Non-nucleoside reverse transcriptase inhibitors. OIs. :. Opportunistic infections. PAS. :. Para-aminosalicylic acid. PRR. :. Pattern recognition receptor. PBMC. :. Peripheral blood mononuclear cell. PD-1. :. Programmed cell death protein 1. PE. :. Phycoerythrin. PIs. :. Protease inhibitors. PGE2. :. Prostaglandin E2. PLZF. :. Promyelocytic leukemia zinc finger. PVI. :. Persistent viral infections. PVLs. :. Plasma viral loads. qRT-PCR. :. Quantitative real-time PCR. RORγt. :. M al. of. ty. :. Reverse transcriptase. :. Roswell Park Memorial Institute. ve. RPMI. Retinoic acid-related orphan receptor. rs i. RT. ay a. NRTIs. :. South East Asia. STAT3. :. Signal transducer and activator of transcription 3. S. cerevisiae. :. Saccharomyces cerevisiae. SOCS1. :. Suppressor of cytokine signaling 1. SIV. :. Simian immunodeficiency virus. T cell. :. Thymus-derived lymphocyte. TB. :. Tuberculosis. TCR. :. T cell receptor. TIM-3. :. T-cell Ig mucin-containing domain-3. U. ni. SEA. xix.

(21) :. Thymus-derived helper cell. TLRs. :. Toll-like receptors. TNF-α. :. Tumor necrosis factor-alpha. TRAIL. :. TNF-related apoptosis-inducing ligand. Tregs. :. Regulatory T cells. TST. :. Tuberculin skin test. UMMC. :. University Malaya Medical Centre. UNAIDS. :. Joint United Nations Programme on HIV/AIDS. UMRG. :. University of Malaya Research Grant. VCs. :. Viremic controllers. WHO. :. World Health Organization. U. ni. ve. rs i. ty. of. M al. ay a. Th. xx.

(22) LIST OF APPENDICES. Appendix A: Gating Strategies Used in the Immunophenotyping of T Cells by Flow Cytometry ………………………………………………………………..160 Appendix B: Scatter Plots (Gated on The CD3+ T-Cell Population) Show CoStaining with CD8 and CD161 on Representative Samples from 5 Different Clinical Groups: CPTNs, CPTPs, HVTNs, HVTPs, and. ay a. HCs.…………………………………………………………………………….162 Appendix C: The Zebra Plots Depict the Gating Strategy for the Analysis of. M al. Expression of CD161 on MAIT cells…………………………………………..163. U. ni. ve. rs i. ty. of. Appendix D: List of Presentations……………………………………………..163. xxi.

(23) CHAPTER 1: INTRODUCTION. It has been reported that the syndemic interaction between HIV and TB has imposed a significant disease burden worldwide. A recent 2012 estimate shows that HIV has infected 35.3 million (32.2–38.8 million) individuals, and ~30% of this HIV-positive population are believed to harbor MTB in both latent and active forms (Getahun, Gunneberg, Granich, & Nunn, 2010; UNAIDS, 2013). On the other hand, of the 8.70. ay a. million TB positive cases worldwide, it has been reported that 1.13 million (13%) are also co-infected with HIV (WHO, 2013b). Despite the disparity in pathogenesis and clinical. M al. course of the disease due to HIV-TB co-infection, current investigations suggest that both the pathogens have higher impact on each other, subsequently contributing to accelerated immunological deterioration (Shankar et al., 2014).. of. There are several hypotheses according to current literature addressing how concurrent. ty. HIV infection exacerbates TB disease progression and vice versa in co-infected. rs i. individuals (Diedrich & Flynn, 2011). Therefore, considering the rapid immune deterioration, there is an urgent necessity of subsequent deep investigation to better. ve. understand the involved interaction between these two pathogens at the molecular the. ni. cellular levels.. U. Immunosenescence is associated with functional impairment of immune surveillance attributes which is developed against infectious pathogens (Larsson et al., 2013). Currently, there are growing evidence that suggest differential expression of CD28, CD27, CD127 and CD57 on senescent T cells in PVIs (Mojumdar et al., 2011). Moreover, persistent antigenic stimulation, or CIA culminates in the functional impairment of antigen-specific and -non-specific T cells (Effros, 2004b), supporting the notion that Tcell impairment and proliferation deficits in chronic HIV disease (Mendez-Lagares et al., 2013) are comparable to events occurring generally in aging healthy uninfected. 1.

(24) individuals (Desai & Landay, 2010). Intriguingly, sustained expression of surface markers including ki-67, CD38, HLA-DR, PD-1 and CD69 on HIV-specific CD4+ and CD8+ T cells (W. Cao, Jamieson, Hultin, Hultin, & Detels, 2009) are profoundly reported in chronic HIV disease with both HIV infection and HIV-TB co-infections despite the initiation of ART as a signal for CIA (Feuth et al., 2013). Surface markers like CD38 and HLA-DR also have been identified to have critical roles as indicators of HIV disease. ay a. progression. Increasing body of evidence suggests the potential associate on between CIA and T cell immunosenescence, although it is not still clear whether TB have a significant role in HIV disease progression. We hypothesized that TB may have a supplementary. M al. role in increasing progression of HIV-associated immune deterioration by enhancing immunosenescence and CIA, which leads to deteriorated protective responses. Therefore, we measured and correlated the surrogate markers closely associated with immune. of. activation, immunosenescence, and functional cytolysinse specially in CD8+ T cells of. U. ni. ve. rs i. ty. HIV-TB co-infected, HIV-infected and healthy subjects.. 2.

(25) CHAPTER 2: BACKGROUND AND REVIEW OF LITERATURE 2.1. Epidemiology of HIV and Tuberculosis. Human immunodeficiency virus (HIV) and tuberculosis (TB) co-infection poses immense health challenges and accounts for significant rates of morbidity and mortality world-wide. Global estimates suggest that ~35.3 million people were living with HIV at the end of 2012, of whom ~30% are reported to have been co-infected with. ay a. Mycobacterium tuberculosis (MTB) in both latent and active forms (Getahun et al., 2010; UNAIDS, 2013). On the contrary, of the 8.7 million TB cases worldwide, ~1.13 million (13%) are reportedly co-infected with HIV (Bourhis et al., 2013). The burden of both the. M al. diseases is particularly high in third-world nations especially across South East Asia (SEA) and sub-Saharan Africa, which necessitates prompt and comprehensive disease control strategies (UNAIDS, 2013; WHO, 2013a). HIV-TB co-infection is also a major. of. health problem in Latin America and the Caribbean, as TB is endemic in the region.. ty. According to the WHO, TB-HIV co-infection in 2010 was high in Bahamas, Belize,. rs i. Brazil, Dominican Republic, Guyana, Haiti, Jamaica, Suriname, and Trinidad and Tobago (WHO, 2011).. ve. Although numerous efforts have been undertaken, with integrations from technology. ni. advancements and community supports, we are still in the dawn of deciphering the. U. complex synergistic interaction between HIV and MTB, warranting in-depth investigations to unveil the complex pathophysiological association underlying coinfection. Therefore, the global emergence of HIV-TB pandemic undoubtedly has generated keen interest among biomedical scientists to identify the fundamental immunological mechanisms to design effective therapeutic strategies against HIV-TB coinfection.. 3.

(26) 2.2. The Epidemiology of HIV and Tuberculosis Co-Infection in Malaysia. Over the last 29 years since HIV was first reported, Malaysia has experienced tremendous advances in HIV prevention, diagnosis, and treatment. As a result, there has been a significant decline in the number of new cases by close to half from 28.4 cases per 100,000 individuals in 2002 to 11.7 cases per 100,000 individuals in 2014 (UNAIDS, 2015).. ay a. Estimates suggest that Malaysia has ~91,8484 people living with HIV by end of 2014. During this period, the national surveillance system has reported a cumulative of 105,189. M al. cases, 21,384 cases with AIDS and 17,096 deaths due to HIV/AIDS accounting for a total of 88,093 (96%) people living with HIV (UNAIDS, 2015).. The annual number of new HIV-infected cases has steadily declined from 6,978 reported. of. in 2002 (Figure 2.1). Furthermore, there has also been a steady decline in the number of. ty. AIDS-related deaths reported, which could be attributed to the introduction of first-line. rs i. and second-line anti-retroviral (ARV) treatment. By the end of 2014, there were 21,654 people living with HIV on treatment, which is 51% of the estimated number of people. ve. living with HIV eligible for ARV treatment (42,408) (UNAIDS, 2015).. ni. Mother-to-child transmissions (MTCT) take places during the course of delivery or. U. through breastfeeding. Without any intervention, up to 45% of children born to HIV positive mothers in lower income countries will become infected. This can be declined to less than 2% with strategies to decrease MTCT (Cock et al., 2000; Cooper et al., 2002). Approximately 75% of all pregnant mothers in Malaysia accessed public healthcare between 2007 and 2009. Of these, 98% were screened for HIV and 0.05% were found to be infected with HIV. Amongst those who were screened positive for HIV in 2011, more than half were newly detected cases (Azwa & Khong, 2012; "Global AIDS Response 2012," 2012).. 4.

(27) Tuberculosis also remains a significant public health concern in Malaysia with constantly increasing rates of infection reported annually across the country. Individuals infected with HIV are highly vulnerable to acquisition of TB due to weakening of the immune system. As part of its disease control and prevention measures, the Government conducts routine TB-HIV screening for all new inmates in closed settings such as prisons and drug rehabilitation centers, since 2001. From 1990 to 2014, the number of TB-HIV co-. ay a. infection reported nationwide has increased significantly. Without treatment, as with other opportunistic infections (OIs), HIV-TB co-infection would shorten the life of the person infected. In an effort to reduce the morbidity and mortality rates of TB-HIV co-. M al. infection, the government has also initiated isoniazid prophylaxis since 2010 (UNAIDS, 2015).. of. The number of TB cases detected annually has been on the rise constantly and in the last couple of years, new cases reported was more than 24,000 cases; relatively high as. ty. compared to HIV with a ratio of TB/HIV cases of 3:1 in 2000 to 7:1 reported in 2014. In. rs i. 2014, ~24,711 new TB cases were registered in Malaysia with reported TB-HIV coinfection of 5.9%. It was estimated that in 2012, ~23,027 people were infected with TB,. ve. and TB incidence without HIV infection was ~2.4 per 1000 individuals and TB-HIV co-. U. ni. infection was ~8% (Figure 2.2) (UNAIDS, 2015).. 5.

(28) ay a. Figure 2.1: Reported HIV and AIDS-related Deaths, Malaysia (1986 –2014). U. ni. ve. rs i. ty. of. M al. (Adapted from Global AIDS Response Progress Report 2015, Malaysia, Page 13). Figure 2.2: New TB, HIV and Prevalence of TB-HIV Co-infection, Malaysia (19992014) (Adapted from Global AIDS Response Progress Report 2015, Malaysia, Page 27). 6.

(29) 2.3. Human Immunodeficiency Virus (HIV). 2.3.1. Discovery and Epidemiology. HIV-1 was initially identified as the principal cause of AIDS by Luc Montagnier at the Institut Pasteur in 1983. (Barre-Sinoussi et al., 1983), and was later characterized. independently in 1984 by Robert Gallo and Jay A. Levy. Subsequently, HIV-2 was recovered as a milder form of the virus from a West African national in 1986 (Clavel et. ay a. al., 1986). HIV-1 comprises four distinct lineages including M, N, O, and P. The M (major) group is recognized as predominant (90%) and constitutes 11 distinctive subtypes/clades (denoted by AK). The O (outliers) group is limited to western and Central. M al. Africa, whereas group N (Non-M and O) isolated from Cameroon in 1998, appears to be extremely rare (Robertson et al., 2000). Lately, group P has been reported in a Cameroonian female, was found to closely resemble the simian immunodeficiency virus. of. (SIV) seen in primates (Robertson et al., 2000). The HIV-2 subtype is comprised of seven. ty. distinct phylogenetic lineages, and is mainly restricted to West Africa. HIV-2 may be. rs i. categorized as epidemic subtypes (A and B) and non-epidemic subtypes (C-G) (Chen et. U. ni. ve. al., 1997; Lemey et al., 2003).. Figure 2.3: Global Distribution of HIV-1 Subtypes and Recombinants (Hemelaar, Gouws, Ghys, & Osmanov, 2006). 7.

(30) 2.3.2. Virology. HIV-1 is composed of two copies of positive single-stranded RNA (diploid +ve sense ssRNA) with a genome of 9.7 kilobases and is classified under family Retroviridae, subfamily Lentiviridae, and genus Lentivirus. The two ssRNA strands are surrounded by a conical capsid composed of ~2000 units of p24 protein (Chiu et al., 1985; FanalesBelasio, Raimondo, Suligoi, & Butto, 2010). The capsid is enclosed within a lipid. ay a. envelope derived from the cell membrane of the host during viral budding (Fanales-. U. ni. ve. rs i. ty. of. M al. Belasio et al., 2010) (Figure 2.4).. Figure 2.4: Structure of HIV-1 (Adapted from (Abbas, Lichtman, & Pillai, 2007). The envelope contains glycoprotein (gp) spikes integrated into the envelope, and is composed of a trimer of external (gp120) and transmembrane (gp41) subunits (Wyatt et al., 1998). The matrix protein p17 is attached to the interior face of the envelope. Beneath the envelope lies a cone-shaped capsid (composed of p24) that contains two singlestranded positive sense RNA strands (diploid genome), each consisting of ~9.7 kilobases, and tightly bound to nucleocapsid proteins p6 and p7, and viral enzymes (Gelderblom, Ozel, & Pauli, 1989).. 8.

(31) The HIV life cycle begins via gp120 binding to the corresponding CD4 co-receptor on the target cell, and eventually to a chemokine co-receptor; either CCR5 or CXCR4 depending on the viral tropism (i.e. R5 or X4 virus). Subsequently, the gp120 undergoes conformational changes leading to membrane fusion and eventual delivery of viral RNA into the host cell cytoplasm. Here, the viral RNA is transcribed by reverse transcriptase (RT) into ssDNA, and later into double stranded DNA (dsDNA). The dsDNA becomes. ay a. incorporated into the host cell genome by viral integrase, and the integrated DNA is known as a provirus. The proviral DNA could either be replicated as part of the normal cell division (Fanales-Belasio et al., 2010) or could remain latent within cells as viral. M al. reservoirs leading to episodes of infections, especially after interruption of ART (AquaroCalio, et al., 2002; Carter et al., 2010; Collman, Perno, Crowe, Stevenson, & Montaner, 2003; Glass & Johnson, 1996; Nottet et al., 2009; Redel et al., 2010; B. A.. of. Smith et al., 2001; Sonza et al., 2001).. ty. The RNA transcripts are spliced and translated into viral proteins or remain unspliced and. rs i. exported to the cytoplasm, and are packaged into virus particles. Immature viral polypeptides are translated to their corresponding functional forms by viral protease, and. ve. packaged with two full-length ssRNA transcripts. The virus buds with an envelope. ni. derived from host cell membrane (Fanales-Belasio et al., 2010; Gomez & Hope, 2005). U. (Figure 2.5).. 9.

(32) ay a M al. Figure 2.5: HIV Life Cycle. Pathogenesis. rs i. 2.3.3. ty. of. (Adapted from (Abbas et al., 2007). ve. The characteristic feature of HIV-1 infection is the depletion of CD4+ T cells, systemic immune activation and eventual functional immune catastrophe in the host. HIV-1 infects. ni. CD4+ T cells, macrophages/monocytes, dendritic cells (DCs), thymocytes, and. U. microglial cells (Fanales-Belasio et al., 2010; Gomez et al., 2005). HIV is transmitted either by exposure of the virus to oral, rectal, or vaginal mucosa during unprotected sexual intercourse; by use of blood-contaminated injection equipments; by intravascular inoculation through transfusion of contaminated blood products; or vertically during pregnancy, and labor, or via breast-feeding (Suligoi, Raimondo, Fanales-Belasio, & Butto, 2010). The evolution of co-receptor usage by HIV-1 commonly involves a shift from CCR5 (R5 phenotype) to CXCR4 use alone (X4) or a combination of both CCR5 and CXCR4 (i.e. R5X4) during progression to terminal disease (Bjorndal et al., 1997;. 10.

(33) Connor, Sheridan, Ceradini, Choe, & Landau, 1997; Scarlatti et al., 1997; Shankarappa et al., 1999; Tersmette et al., 1988). The use of CXCR4 has been linked to rapid decline in CD4+ T-cell counts and onset of OIs (Berger, 1997). Grivel et al (1999) reported that R5 HIV is highly cytopathic, but only for CCR5+/CD4+ T cells and owing to the fact that these cells comprise only a smaller fraction of CD4+ T cells and their depletion does not significantly alter the total CD4+ T-cell counts. However, the effects of X4 HIV-1 isolate. ay a. is believed to be due to extensive loss of CXCR4+ CD4+ T cells (Grivel & Margolis, 1999).. M al. HIV-1 viral load rises rapidly during the initial weeks of infection (acute period), which is followed by increase in both cellular and humoral responses (Bangham, 2009; Borrow, Lewicki, Hahn, Shaw, & Oldstone, 1994). The appearance of HIV-specific antibodies. of. commonly occurs ~3-4 weeks after infection, and during this period (window period) the infected individuals could potentially transmit the virus to others as virus-specific. ty. antibodies are yet to be available for detection (Butto, Raimondo, Fanales-Belasio, &. rs i. Suligoi, 2010; Butto, Suligoi, Fanales-Belasio, & Raimondo, 2010). Subsequently, the immune system controls viral replication for a considerable duration (5-10 years), which. ve. largely depends on a combination of viral and host factors (Gupta, 1993). The most. ni. common symptom during acute HIV infection is a ‘flu-like’ syndrome characterized by. U. fever, rashes, lymphadenopathy, oral ulcers, weight loss, joint pain, pharyngitis, and malaise (Kahn & Walker, 1998).. HIV-1 persistently replicates in infected cells, and employs direct or indirect strategies to target HIV-specific T cells for elimination. This ultimately leads to steady establishment of viral dominance, decline in immune responses and onset of OIs. Infected individuals with CD4+ T-cell count ≤200cells/mm3 progress more rapidly to AIDS and develop OIs due to viral, bacterial, fungal, parasitic agents, and neoplasms (J. T. Brooks et al., 2009). 11.

(34) (Figure 2.6). HIV-1 also establishes a state of latent infection in certain cell types including resting memory CD4+ T cells (Nottet et al., 2009; Siliciano et al., 2003), DCs (Keele et al., 2008; B. A. Smith et al., 2001), macrophages (AquaroBagnarelli, et al., 2002; Crowe et al., 1990) and monocytes (Sonza et al., 2001; T. Zhu et al., 2002). Moreover, naïve CD4+ T cells, pluripotent progenitors (in the bone marrow), CD4+ T cells and macrophages in seminal fluids have also been described as the reservoirs of HIV. ay a. in patients undergoing ART (Quayle, Xu, Mayer, & Anderson, 1997; Wightman et al., 2010). Besides, latent infection can also establish in the central nervous system (CNS) through resident microglial cells and macrophages migrating into the CNS, which likely. M al. has a significant role in protecting HIV-1 from the effects of ARV (anti-retroviral) drugs (Glass et al., 1996). Treatment intervention with ART in HIV-infected individuals with undetectable viral load leads to the appearance of viral plasma, a phenomenon caused by. U. ni. ve. rs i. ty. of. release of virus from latently-infected cells (Siliciano et al., 2003).. Figure 2.6: Typical Course of HIV-1 Infection (Adapted from (Fauci, Pantaleo, Stanley, & Weissman, 1996). 12.

(35) 2.3.4. HIV-1 Disease Progression. The rate of progression of HIV disease from the time of initial acquisition to the commencement of AIDS varies between individuals, and is dependent on a combination of virus and host factors. With regard to host-dependent factors, certain infected individuals carry a mutation called Δ32 in CCR5, which encodes for a non-functional and truncated protein that is unfit for viral attachment and fusion (Zhang et al., 1998) on target. ay a. cells. Homozygosity for Δ32 mutation (CCR5Δ32/Δ32) has been identified in ~1% of Caucasians, who are protected against HIV-1 infection, and the heterozygous state (CCR5Δ32/WT) is identified in ~10% of Caucasians, in whom it may cause slower. M al. disease progression (de Silva & Stumpf, 2004; O'Brien & Moore, 2000). The Δ32 mutation is found only in European, West Asian, and North African populations. The allele frequency exhibits a north–south cline with frequencies ranging from 16% in. of. northern Europe to 6% in Italy and 4% in Greece (Novembre, Galvani, & Slatkin, 2005).. ty. Research suggests that certain haplotypes of the human leukocyte antigen (HLA) are. rs i. associated with disease progression (Kaul et al., 1999; Rowland-Jones et al., 1998). The genes encoding for HLA molecules are reportedly the most polymorphic and therefore,. ve. depending on the composition of the HLA haplotypes, individuals may react differently. ni. to the same pathogen (Parham & Ohta, 1996). HLA-B is a rapidly evolving and the most. U. polymorphic locus that has been associated with a majority of HLA class I-related disorders and disease progression (Kiepiela et al., 2004; Migueles et al., 2000; Schellens, Kesmir, Miedema, van Baarle, & Borghans, 2008). In Chimpanzees, expression of the HLA-B*27 and HLA-B*57 haplotypes target conserved regions of SIV/HIV and reduces the possibilities of viral evasion (de Groot et al., 2010). Others reported that Caucasians with HLA-B*35 and HLA-Cw*04 alleles showed consistent association with rapid progression to terminal AIDS, whereas HLA B*27, HLA-B*57, and HLA-B*5801 have been linked to lower viral loads and slower progression to terminal disease (Carrington. 13.

(36) & O'Brien, 2003; M. P. Martin & Carrington, 2005). HLA class II haplotypes have also been recognized to play a significant role in HIV disease progression. The association of HLA-DRB1*1303 alleles toward slower disease progression as well as decreased viral loads has been described (Julg et al., 2011). HLA-DRB1*01 is more frequently reported in HIV-1 negative individuals in comparison with positive ones, and the expression of HLA-DRB1*08 has been linked to lower median viral loads (Ndung'u et al., 2005).. ay a. Interestingly, the age at which individuals become infected with HIV-1 appears to significantly impact disease progression whereas rapid disease progression apparently is relatively more common in older HIV-infected individuals (Bacchetti et al., 1988; Moss. M al. et al., 1988).. Host genetic background (specifically in the HLA and CCR5 regions) strongly influences. of. outcome of HIV-1 infection. Understanding what and how additional host genetic, viral and environmental factors contribute to the variation in disease progression can help drive. ty. intervention strategies (McLaren & Carrington, 2015). To date, HLA and CCR5 loci have. rs i. been estimated to explain ~13% of variation in viral-load set point (Fellay et al., 2009). However, an essential question in understanding all complex phenotypes (including HIV-. ve. 1 progression) is what amount of the observed trait variation can be attributed to human. ni. genetic background overall (i.e., heritability) (McLaren et al., 2015). For HIV-1 infection. U. in particular, it is unclear whether disease progression is affected by common genetic variants outside the HLA and CCR5 genes. Estimating the total heritability of disease progression and understanding how it may be distributed across different classes of genetic variants (such as common SNPs, rare variants and copy-number variants) can inform on what types of study may have the highest impact. This concept has been elegantly applied in the context of many complex-trait GWAS, often showing that loci present on genotyping arrays (or robustly inferred through genotype imputation) falling below the strict statistical thresholds can collectively explain much of the trait variability. 14.

(37) not attributable to loci of genome-wide significance (Stahl et al., 2012; Yang et al., 2010). This method measures the correlation between phenotypic similarity and genotypic similarity among unrelated individuals across a large number of common SNPs to quantify narrow-sense heritability (i.e., the heritability explained by additive genetic effects) (Stahl et al., 2012) and can help provide understanding of how regulatory variation functions in common diseases. For example, in a study of 11 common diseases,. measured by GWAS (Gusev et al., 2014).. ay a. SNPs in DNase I–hypersensitive sites explained, on average, 79% of the heritability. M al. Lifestyle and factors such as depression (Burack et al., 1993), smoking (Royce & Winkelstein, 1990) and malnutrition (Moseson et al., 1989) have also been reported to influence disease progression. Moreover, evolutionary changes in HIV-1 have also been. of. directly linked to disease progression (Shankarappa et al., 1999). Different HIV-1 strains exhibit different replicating fitness, which in turn can affect disease progression (Borman,. ty. Paulous, & Clavel, 1996; Ho et al., 1994; Kaleebu et al., 2002). HIV-infected individuals. rs i. harboring attenuated virus strains or mutant forms of viral genes, e.g. vif, nef, vif, vpr or rev, appear to progress slowly as these genes reportedly make the virus less virulent. ve. (Poropatich & Sullivan, 2011). However, some individuals likely resist infection for. ni. indefinite periods of time after viral acquisition. Long term non-progressors (LTNPs) are. U. asymptomatic HIV-infected subjects with stable CD4+ T-cell counts, and who lack disease progression and low or intermediate plasma viral loads (PVLs) for >10 years. LTNPs comprise ~5-15% of the global pool of HIV-1-infected individuals (Y. Cao, Qin, Zhang, Safrit, & Ho, 1995; Madec et al., 2009; Moseson et al., 1989; Pantaleo et al., 1995; Rodes et al., 2004). Elite controllers (ECs) consists of <1% of all HIV-infected subjects, and are able to control viral load to <50 copies/mL for a significantly longer duration (S. G. Deeks & Walker, 2007; Migueles et al., 2000). Viremic controllers (VCs) show a lesser degree of viremic control (but detectable HIV-1 RNA levels (50-2000 copies/mL)) as. 15.

(38) compared to ECs (Emu et al., 2008; B. D. Walker, 2007). The key feature of ECs and VCs is their ability to control viral load although a minor proportion of these individuals would undergo CD4+ T-cell depletion and progress to terminal HIV disease (Blankson, 2010; Okulicz et al., 2009).. 2.3.5. Treatment. Lack of effective cure (and vaccines) against HIV infection poses an immense challenge. ay a. to modern healthcare. However, ART prevents the development of progressive immunosuppression and premature death of infected individuals (G. R. Kaufmann, Bloch,. M al. Zaunders, Smith, & Cooper, 2000; C. J. Smith et al., 2003). The main aim of ART is to impede the rate of disease progression, which is best achieved using effective ART to inhibit virus multiplication achieving a plasma HIV-1 RNA level below detection limits. of. using commercial assays. Moreover, high plasma HIV RNA levels has been reported as a risk factor for HIV transmission, and effective ART can decrease viremia and virus. ty. transmission by >96% (Cohen et al., 2011; Quinn et al., 2000). Modeling studies suggest. rs i. that ART may lead to low prevalence at the community or population levels, and hence, the ART also decreases the risk of virus transmission (Granich, Gilks, Dye, De Cock, &. ve. Williams, 2009).. ni. Current guidelines categorize HIV-infected individuals in particular groups of patients to. U. initiate ART. These groups include subjects who exhibit symptoms of AIDS regardless of PVL or CD4+ T-cell counts, asymptomatic subjects with PVLs ≥100,000 copies/mm3 and CD4+ T-cell counts ≤350 cells/mm3 (Piacenti, 2006). There are six main ARV drug classes; (1) Fusion inhibitors that interfere with the virus’s ability to fuse with the plasma membrane, preventing HIV from entering the target cell, e.g. enfuvirtide (Fuzeon) (2) Nucleotide reverse transcriptase inhibitors (NRTIs) that terminates chain elongation during replication, e.g. tenofovir and adefovir, (3) Non-. 16.

(39) nucleoside reverse transcriptase inhibitors (NNRTIs) that act via non-competitive binding to a hydrophobic pocket located in the vicinity of the RT enzyme, e.g. nevirapine, efavirenz, and delavirdine, (4) Integrase inhibitors (INSTIs) that block the HIV enzyme integrase so that the virus will be unable to integrate its genetic material into the host cell DNA, e.g. elvitegravir and dolutegravir (Tivicay), (5) Protease inhibitors (PIs), which block HIV-1 proteases to prevent assembly of new viral particles, e.g. nelfinavir and. ay a. saquinavir and (6) Chemokine receptor antagonists, e.g. maraviroc (Arts & Hazuda, 2012; E. D. Deeks, 2014; Lu & Chen, 2010; Savarino, 2006). The CCR5 receptor antagonists are amongst the first drugs that can affect HIV indirectly by preventing viral attachment. M al. to the host cell via blockade of CCR5 receptor (Baba, 2006; Emmelkamp & Rockstroh, 2007).. of. Malaysia is an upper middle-income country that, despite a decade-old policy of government-subsidized ART, remains one of the few Asian countries where HIV. ty. incidence and mortality are increasing and fewer than half (41.6%) of all clinically. rs i. eligible PLH receive ART (Enrico G. Ferro, 2017). Although the HIV epidemic in Malaysia is driven primarily by PWID, less than 5% of HIV infected PWID receives ART. ve. (Louisa Degenhardta, 2014). Despite evidence-based guidelines in Malaysia. ni. recommending ART for all PLH with CD4+ T-cell counts ≤350 cells/μL in 2010 (WHO,. U. 2010), stigma against key populations may exacerbate disparities in ART access precisely when physicians are deciding whether to initiate treatment. In Malaysia, physicians may be less likely to prescribe ART for PLH who inject drugs or use alcohol, were recently released from prison, or lack social support, even at extremely low CD4+ T-cell counts. Although the reasons for this are unclear, unsubstantiated concerns about ART adherence and explicit discrimination may both be factors. Interventions to educate physicians and monitor their prescribing behaviors to ensure that they conform to evidence-based. 17.

(40) treatment guidelines may increase ART use and reduce health disparities in key HIVinfected populations in Malaysia (Enrico G. Ferro, 2017).. 2.4 2.4.1. Mycobacterium tuberculosis Discovery and Epidemiology. TB had been described in ancient texts by Hippocrates and Galen (Donoghue, 2009), a term that originated from Latinate tuberculum, meaning “root vegetable” and was first. ay a. used by Johann Lukas Schönlein, a German scientist in 1830 (Ligon, 2002). TB is caused by a pathogenic bacteria under the Family Mycobacteriaceae, called Mycobacterium. M al. tuberculosis (MTB), one of the members that comprises a genetically-related group known as MTB complex (Forrellad et al., 2013). MTB is regarded as the first facultative intracellular pathogen that causes TB by Robert Koch, a German bacteriologist in 1882. of. (Koch, 1882). The complex lipid-rich cell wall of MTB consists of peptidoglycan, arabinogalactanmycolate and lipoarabinomannan (LAM) as well as free lipids, and. rs i. ty. scattered proteins (Madison, 2001).. Pulmonary TB mainly accounts for ~70% of primary active TB-infected individuals, and. ve. is the most common form of TB. However, it can be spread to other organs leading to extra-pulmonary TB (Burman & Jones, 2003; WHO, 2009). Reports suggest that >2. ni. billion people are infected with MTB, which remains in a quiescent state normally. U. identified as latent TB infection (LTBI), showing no pathognomonic symptoms of active TB (WHO, 2013a). According to a recent WHO report, ~9 million people are actively infected with TB and ~1.5 million succumbed to TB in 2013 (WHO, 2013a).. The immune system plays a significant role in controlling latent MTB from causing disease manifestations due to active MTB infection. This inevitably provides a key implication on the immune system in controlling MTB from causing disease. However, ~5-10% of latent TB can revert to active TB when the immune system fails to subdue. 18.

(41) bacterial persistence, leading to reactivation or post-primary TB (Vynnycky & Fine, 2000), where MTB likely disseminates to extra-pulmonary sites, viz., the lymph nodes, liver, bone, and the meninges (Harisinghani et al., 2000; S. K. Sharma & Mohan, 2004).. 2.4.2. Bacteriology. MTB has been classified as an acid-fast bacteria, and stains poorly with crystal violet owing to its complex cell wall (Ducati, Ruffino-Netto, Basso, & Santos, 2006). MTB is. ay a. rod-shaped and is classically regarded as non-sporulating although others claim the observation of spores in aged bacterial cultures (Ghosh et al., 2009). Moreover, it neither. M al. possesses a flagellum nor a capsule. It is also resistant to decolorization by acids due to its waxy cell wall. MTB has a doubling time of ~24h, and hence it replicates very slowly. The bacterium measures ~0.5µm in diameter and 1-4µm in length, and is a strictly. of. intracellular pathogen (Ducati et al., 2006).. ty. The cell envelope is a distinctive characteristic of the organisms belonging to the Genus. rs i. Mycobacterium. The mycobacterial cell envelope is made of three major entities covalently linked to each other (peptidoglycan, arabinogalactan, and mycolic acids) and. ve. LAM. The outermost layer, the mycobacterial outer membrane (MOM), is composed of a lipid bilayer structure (Favrot & Ronning, 2012). The cell wall of MTB consists of three. ni. key components: mycolic acids, cord factors, and Wax-D. The cell wall is one of the key. U. factors for virulence. The mycolate molecules are of primary interest due to the functional qualities unique to mycobacteria (Rajni, Rao, & Meena, 2011).. 2.4.3. Transmission and Pathogenesis. TB is an airborne disease, and the primary site of involvement is usually the lungs although infection can be established in other organs such as lymph nodes or pleura leading to extra-pulmonary TB. Moreover, MTB infection can be disseminated throughout the body leading to systemic or miliary TB (Lawn & Zumla, 2011).. 19.

(42) Mycobacteria usually enter the host via inhalation of droplet nuclei expectorated by patients with active pulmonary TB (Ahmad, 2011). Majority of the infected individuals contain mycobacteria at the latent or sub-clinical state while ~10% of infected cases develop active disease (Pieters, 2008; Young & Dye, 2006). There are several factors that control the risk of infection such as bacterial load, infectiousness of source case, ,host genome background, proximity of contact and host immune status (Cobat, Orlova,. Kurepina, Fallows, & Kreiswirth, 2008).. ay a. Barrera, & Schurr, 2013; Di Pietrantonio et al., 2010; Hill et al., 2004; Mathema,. M al. Inhaled droplet nuclei overcome the bronchial defense mechanisms due to their small size and penetrate into the terminal alveoli where they are engulfed by resident macrophages (Ahmad, 2011). In an immunocompetent host, alveolar macrophages ingest M.. of. tuberculosis often leading to their destruction that depends on factors such as bacterial resistance and host genetic factors (van Crevel, Ottenhoff, & van der Meer, 2002). If. ty. macrophage activity is inefficient to eradicate the bacteria inhaled within droplet nuclei,. rs i. M. tuberculosis multiplies within the macrophage logarithmically until the cell bursts (Repasy et al., 2013). Bystander macrophages migrate to the site and engulf the released. ve. bacilli, and the cycle continues. Moreover, the bacilli can be disseminated from the site. ni. of initial infection via lymphohematogenous route to other body parts (Ahmad, 2011).. U. Between three and eight weeks post-infection, the host develops specific cellular immunity that culminates in the migration of antigen-specific T cells to activate macrophages at the site of infection (van Crevel et al., 2002).. 2.4.4. Tuberculosis Disease Progression. The early phases of TB disease progression are reliant on certain factors such as age and immunologic responses, and hence the disease commonly occurs in young children and immunocompromised individuals. A progressive Ghon focus, disseminated (miliary) and. 20.

(43) also CNS infections occurs within 2-6 months after initial infection in infants and individuals with other predisposing conditions (Daley et al., 1992).. Early disease symptoms consist of complicated lymph node disease, pleural disease and peripheral lymphadenitis after 4-12 months of infection (Del Mundo & Soriano, 1966). In severely immunocompromised individuals (e.g. those with advanced HIV or AIDS), early disease may reveal intra-thoracic adenopathy (Burman et al., 2003; Post, Wood, &. ay a. Pillay, 1995). Rarely, newly infected individuals who are 10 years of age with adult-type pulmonary or other types of extra-pulmonary TB may progress during the first 24 months. M al. of infection (Burman et al., 2003).. LTBI is a state of persistent immune response to stimulation by MTB antigens where LTB-infected individuals do not show any symptoms of TB, and are seldom infectious.. of. However, these individuals are reportedly at risk for development of active TB and ~10%. ty. of the cases may become infectious by reactivation (Lamberti et al., 2014). LTBI is. rs i. diagnosed using a positive tuberculin skin test (TST) or interferon-γ (IFN- γ) release assay. ve. (IGRA) in the absence of active disease (Trajman, Steffen, & Menzies, 2013).. Recurrence of TB can be due to re-growth of the same strain of MTB that caused the. ni. previous TB episode, also known as relapse, or re-infection through a different strain. U. (Millet et al., 2013). Re-infection of immunocompetent hosts occurs 18 months or more after initial infection, and has a much lower risk of disease progression. In this case, it has been estimated that there is a 21% of risk for disease progression from an initial infection to the onset of TB disease (Houk, Baker, Sorensen, & Kent, 1968). On the other hand, in severely immunocompromised hosts, re-infection and initial infection appears to pose a similar high risk regardless of the time of re-infection.. 21.

(44) Post-primary (re-activation), also known as adult type or secondary TB, occurs in individuals who have developed immunity to primary TB (Hunter, 2011). The term "postprimary" is preferred to as "re-activation" when referring to clinical diagnosis, because strongly distinguishing recurrence from an antecedent infection is impossible in most cases. Approximately, 10% of all infected individuals are likely to experience reactivation, and the risk is highest within the first 2 years or during periods of. 2.4.5. ay a. immunosuppression (Barnes, Lakey, & Burman, 2002; Washington & Miller, 1998).. Treatment. M al. According to WHO reports, it has been estimated that the mortality rate due to TB without drug treatment is ~66%. Appropriate anti-TB therapy (ATT) leads to rapid reduction in bacterial loads in the lungs, which also break the chain of transmission (Tiemersma, van. of. der Werf, Borgdorff, Williams, & Nagelkerke, 2011; WHO, 2013a). In November 1944, the first TB patient was successfully treated with streptomycin discovered by Selman. ty. Waksman, and soon after with para-aminosalicylic acid (PAS) launched in 1949, and. rs i. subsequently with isoniazid (INH) that became available in 1952 (Jawahar, 2004). These discoveries were major breakthroughs, and the drugs substantially improved the. ve. prognosis of patients with active TB. Later, the discovery of rifampin in the late 1960s. ni. and subsequent rediscovery of the anti-mycobacterial activity of pyrazinamide were. U. significant milestones in anti-TB therapeutics (Jawahar, 2004; van Ingen et al., 2011). The typical “short” treatment (initial phase) of MTB involves four antibiotics viz., rifampin, INH, ethambutol and pyrazinamide for two months followed by rifampin together with INH (continuation phase) for an additional period of four months (Ramachandran & Swaminathan, 2012).. There are currently two types of drug-resistant MTB strains including the multi-drugresistant TB (MDR-TB) and extensively drug-resistant TB (XDR-TB) (Fauci & Group,. 22.

(45) 2008). MDR-TB is resistant to at least two of the four first-line drugs and XDR-TB is resistant to INH, fluoroquinolone, rifampin and at least one of the three second-line drugs (Migliori et al., 2009). Increasing numbers of MDR-TB, but also XDR and totally drugresistant cases threaten TB control globally (Gandhi et al., 2010; Zumla et al., 2012). To overcome drug resistance, WHO has implemented a program called directly observed treatment, short-course (DOTS) (Davies, 2003), which facilitates cooperation between. ay a. physicians, health-care workers, and primary health care agencies by enabling patients to receive their TB drugs daily, under direct medical supervision.. M al. Malaysian rates of multidrug resistant- (MDR-) TB have been estimated at 0.3% in 2005 and 1.3% in 2011 (RashidAli et al., 2015). In unpublished data from the Sabah state reference laboratory, 16 MDR-TB cases occurred in 2011, representing 2.1% of isolates. of. submitted that year (RashidAli et al., 2015). Isoniazid resistance (INH-R) is reported in approximately 4% of M. tuberculosis in western Malaysia (Jalleh, Kuppusamy, Soshila,. T cells and Immune Responses. ve. 2.5. rs i. eastern Malaysia. ty. Aziah, & Faridza, 1993; RashidAli et al., 2015), but rates have not been published from. Thymus is a generative lymphoid organ, and is the site of attainment of functional. ni. maturation for T cells where they are selected based on major histocompatibility complex. U. (MHC) class restrictions. Thymocytes that respond to MHC class II are positively selected as CD4+ T cells destined to provide helper functions (T helper cells), whereas those responding to MHC class I are selected as CD8+ cytotoxic T cells (CTLs) destined to mediate cytolytic functions (Huseby, Kappler, & Marrack, 2008). T cells with receptors that can recognize self-antigens are negatively selected (Sebzda et al., 1999). Positively selected naïve T cells re-circulate between the lymph nodes and peripheral circulation are primed into effector T cells (Harty, Tvinnereim, & White, 2000; Pantaleo & Koup, 2004).. 23.

(46) Upon receipt of appropriate stimulatory (signal 1) and co-stimulatory signals (signal 2) from interaction with antigen-presenting cells (APCs) (viz., macrophages and DCs) in the regional lymph node (or spleen), the naïve T cells with specific receptors proliferate and expand via clonal expansion (Arens & Schoenberger, 2010). As a result, interleukin-2 (IL-2) is released that serves as a T-cell growth and differentiation factor, stimulates the expansion of virus-specific T cells (Watts, 2010). The expanded T cell clone differentiate. ay a. into distinct effector T-cells, driven by a microenvironment that primarily consists of polarizing cytokines produced from APCs or other cells in the vicinity of the immunological synapse (Kulpa et al., 2013). Later, the effector T cells migrate to infected. M al. sites under the influence of chemokines present in the host microenvironment to control further dissemination of pathogens (Murphy, 2011).. of. Several different subsets of CD4+ T-cell phenotypes have been identified based on their distinct and identical cytokine secretion profiles, surface chemokine markers and. ty. functionalities. There are at least four diverse CD4+ T-cell subsets viz., Th1, Th2, Th17. rs i. and Tregs, which have been extensively described (O'Shea & Paul, 2010; J. Zhu & Paul, 2008). Th1 cells mount an immune response upon encountering viruses and intracellular. ve. pathogens. The Th1 subsets secrete cytokines involved in inflammatory responses, such. ni. as IL-2, IL-18, IL-12, IFN-γ and tumor necrosis factor-alpha (TNF-α). Studies have. U. revealed that IFN-γ and IL-12-induced amplification of Th1 cells enhances the ability of the host to mount anti-viral immune responses via cytokine secretion and leukocyte recruitment (Viallard et al., 1999; Watford, Moriguchi, Morinobu, & O'Shea, 2003). This provides a platform to augment intracellular microbial killing abilities by effector cells of innate immunity and to trigger high-affinity antibody production by B cell-derived plasma cells (Chaplin, 2010).. 24.

(47) CD8+ T cells are key effector T cells key to clearance of viruses, intracellular bacteria and tumors (Russell & Ley, 2002). However, effector CD8+ T cells remain as the wellrecognized CTLs in humans, although CD4+ CTLs have also been reported previously (Marshall & Swain, 2011). CTLs are believed to destroy target cells via either noncytotoxic or cytotoxic mechanisms. The cytotoxic machinery operates mainly by two principal routes that include the Fas–Fas ligand (FasL) pathway and granule exocytosis. ay a. (GE) (Smyth & Trapani, 1998; Trapani, 1998). In the Fas–FasL pathway, FasL is upregulated on CD8+ T cells further to activation by a target cell (Rouvier, Luciani, & Golstein, 1993). The subsequent engagement of FasL with Fas results in the activation of. M al. caspase cascade ultimately leading to apoptosis of target cells (He & Ostergaard, 2007; Kischkel et al., 1995). The GE pathway involves the release of cytotoxic granules held within specialized secretory lysosomes by effector cells into the immunological synapse. of. to specifically destroy viral-infected target cells (Grakoui et al., 1999; Stinchcombe,. ty. Bossi, Booth, & Griffiths, 2001). Cytotoxic granules contain many proteins, especially. rs i. perforin and serine proteases called granzymes that are located in the proteoglycan matrix (Lieberman, 2003; Russell et al., 2002). Perforin allows granzymes to access their. ve. substrates although there are several views on how exactly the synergy occurs between the various effector molecules (Bade et al., 2005). It’s rapid up-regulation and granule-. ni. independent transport to the immunologic synapse has also been defined as a novel. U. mechanism of CTL cytotoxicity (Makedonas et al., 2009).. 25.

Rujukan

DOKUMEN BERKAITAN

1) To investigate the role of apoptosis in ex vivo cultured PBMCs of chronic HCV- infected patients. 2) To study the in vitro expression of senescence and activation markers on

i) Poster presentation title “Systematic Review: Predictors of mortality in HIV- associated tuberculosis”, in the International Health Conference IIUM 2011, 7-8 December 2011,

Objectives: This study was proposed to model TB/HIV co-infection based on associated factors of anti TB treatment compliance and associated factors of TB

We further compared the differentially expressed immune related genes caused by WSSV or Vibrio infection with with a particular focus on the role of immune

Some people develop active TB disease soon after becoming infected, before their immune system can fight the TB bacteria.. Other people may get sick later, when their immune

During the multivariate analysis analysis using Cox proportional hazards regression model; ethnicity, number of opportunistic infections, anti-retroviral therapy, total white

Advancements in antiretroviral treatment (ART) and shift of HIV as a chronic infection, presents challenges, including adverse drug reactions (ADRs) affecting adherence and

In general, CD4 + T cells help to amplify the host immune response by activating effector cells and recruiting additional immune cells to the site of disease, whereas CD8 + T