Doctor of Philosophy in Biotechnology

Role of cytokines in eshtablisment and survival of intracellular pathogen in the host

THESIS SUBMITTED FOR THE AWARD OF DEGREE OF Doctor of Philosophy In Biotechnology

By Anzar Abdul Mujeeb

Maulana Azad UnderLibrary, the Aligarh Supervision Muslim of University

PROF. Owais Mohammad

INTERDISCIPLINARY BIOTECHNOLOGY UNIT ALIGARH MUSLIM UNIVERSITY ALIGARH-202002 (INDIA)

2019

Dedicated to My Beloved Family & Friends

Maulana Azad Library, Aligarh Muslim University

Certificate

This is to certify that the thesis entitled “Role of cytokines in establishment and survival of intracellular pathogen in the host” herewith submitted by Mr. Anzar Abdul Mujeeb in fulfilment of the requirements for the degree of Doctor of philosophy in Biotechnology of the Aligarh Muslim University, is an authentic record of the research work carried out by him under my supervision and guidance and that no part, thereof, has been presented before for any other degree.

Prof. Owais Mohammad (Supervisor)

Maulana Azad Library, Aligarh Muslim University

INTERDISCIPLINARY BIOTECHNOLOGY UNIT ALIGARH MUSLIM UNIVERSITY, ALIGARH-202002 (INDIA)

CANDIDATE’S DECLARATION

I, Anzar Abdul Mujeeb, from Interdisciplinary Biotechnology Unit, certify that the work embodied in this Ph.D. thesis is my own bonafide work carried out by me under supervision of Prof. Owais Mohammad at Aligarh Muslim University, Aligarh, India. The matter embodied in this Ph.D. thesis has not been submitted for the award of any other degree.

I declare that I have faithfully acknowledged, given credit to and referred to the research workers wherever their works have been cited in the text and the body of the thesis. I further certify that I have not willfully lifted up some other’s work, para, text, data, result, etc., reported in the journals, books, magazines, reports, dissertations, theses, etc., or available at websites and included them in this Ph.D. thesis and cited as my own work.

Date:…………… Anzar Abdul Mujeeb ……………………………………………………………………….………………………

CERTIFICATE FROM THE SUPERVISOR

This is to certify that the above statement made by the candidate is correct to the best of my knowledge.

Maulana Azad Library, Aligarh Muslim University Prof. Owais Mohammad (Supervisor) Interdisciplinary Biotechnology Unit Aligarh Muslim University, Aligarh-202002 (India)

Prof. AsadUllah Khan (Coordinator)

INTERDISCIPLINARY BIOTECHNOLOGY UNIT ALIGARH MUSLIM UNIVERSITY, ALIGARH-202002 (INDIA)

COURSE WORK/COMPREHENSIVE EXAMINATION/PRE-SUBMISSION SEMINAR COMPLETION CERTIFICATE

This is to certify that Anzar Abdul Mujeeb, from Interdisciplinary Biotechnology Unit has satisfactorily completed the course work/comprehensive examination and pre-submission seminar requirement which is a part of his Ph.D. Programme.

Date: ……………. Prof. AsadUllah Khan (Coordinator)

Maulana Azad Library, Aligarh Muslim University INTERDISCIPLINARY BIOTECHNOLOGY UNIT ALIGARH MUSLIM UNIVERSITY, ALIGARH-202002 (INDIA)

Title of the Thesis: Role of cytokines in establishment and survival

of intracellular pathogen in the host.

Candidate’s Name: Mr. Anzar Abdul Mujeeb

The undersigned hereby assigns to the Aligarh Muslim University, Aligarh copyright that may exist in and for the above thesis submitted for the award of the Ph.D degree.

Anzar Abdul Mujeeb

Maulana Azad Library, Aligarh Muslim University

ACKNOWLEDGMENTS

ALLAH, The most Affectionate, The most Merciful Alhamdulillah, all praises to Almighty ALLAH for giving the strengths and patience and bestowed me with ample patience and perseverance that paved the path to keep me going through thick and thin for the accomplishment of this endeavor.

First and foremost, I am deeply indebted to my supervisor Prof. Owais Mohammad for giving me the opportunity to be a member of his young and enthusiastic team. With due respect, your immense repertoire of your scientific ideas, aptitude and experience has taught me many meaningful lessons related to both science as well as life. I have been extremely lucky to have a supervisor who cared so much about my work and who responded to my questions and queries so promptly. The freedom to work, the acceptance of ideas, the respect for our belief and the delight to humanity were the qualities of your mentorship that has furnished me into a responsible person. “Thank You Sir” for your encouragement, trust and the teaching that enables me to become skillful in terms of both bench work and manuscript writing. Your motivation and disciplines to finish work timely (with deadlines and odd hours) has really helped me to become more active and focused in my research endeavors.

I owe my sincere gratitude to all the faculty members Prof. Asad Ullah Khan, Prof. M. Saleemuddin, Prof. Rizwan Hasan Khan, Prof. Waseem Siddiqi and Dr. Hina Younus of Interdisciplinary Biotechnology Unit for their valuable support and encouragement. Most of the results described in this thesis would not have been obtained without a close collaboration with few laboratories. My special thanks to our collaborators, Dr. U. D. Gupta Maulana(JALMA), Dr. Azad Pushpa Library, Gupta (JALMA). Aligarh Muslim University

It is hard for me to explain the contribution of my seniors cum friends Dr.Shadab Kazmi, a person of charisma and creativity who inspired me, my fellow colleagues, and maintained a healthy environment throughout his stay and I was lucky enough to have performed several experiments under his Nobel guidance, and Dr.Syed Mohd. Faisal, who always was at a hand distance to help whatever the problem, he always had a way out for anything that comes up anytime. They especially deserve a biggest thank. I expand my thanks to Mr. Haris Saeed, and Mr. Khursheed for his guidance and unconditional support during the course of the thesis and would like to warmly thank them for their valuable advice and constructive criticism related to thesis. The long valuable discussions that helped me sort out the technical details of my work. They have always been truly helpful, inspiring and supportive throughout my research.

I am also thankful to the all members of non-teaching staff Mr. Lal khan, Mr. Faisal Maqbool, Mr. Aqtedar Bhai, Mr. Amir, Mr. Nasir, Mr. Pal Singh, Mr. Ramesh and Mr. Wasim. Without their help I could not have procured the required chemicals, access the DISC facility and thereby, not able to complete my experimental work.

Our laboratory is filled with vibrant scholars whose company, ideas, critics and fun time has made my home away feeling lighter. As a family we had shared many good moments and I am thankful to Dr. Ahmar, Dr. Faraz, Dr. Nida, Mr. Mohd Saad, Miss.Nazoora, Miss.Saba, Ms.Ashima for their timely help, humble support and cooperation.

I wish my warm thanks to special people in my life for their love, care, encouragement, understanding and blessings that kept me moving. I am feeling incredibly emotional in acknowledging them (my family) who laid seeds of enthusiasm in me for knowledge and gave meaning to my life... The unfathomable blessings of my Ammi, Mrs. Chand Sultana, My brothers Mr. Umar A.Mujeeb, the person to keep it cool even in most adverse of the situations, Mr. Zafar A.Mujeeb, the most hardworking in the ways only few know, Dr. Athar A.Mujeeb, special mention for keeping it all together since the time I can remember and also for his encouragement, trust and valuable support at all the juncture of my life, Mr. Asghar A.Mujeeb, never backs down from handling any situation that comes his way , and Mr.Absar MaulanaA.Mujeeb, the Azad youngest Library, and avid Aligarh gamer. areMuslim the spiritual University strength with which I have persuaded this doctoral work.

I am indebted to my sisters Mrs. Yumna Aamir, always the stronghold and Mrs.Yusra Taab, the baby girl of the house for the longest time and my brothers-in-law Dr.Ali Aamir, cool down to earth person with strong family values and Mr.Taab Ahmad Samad, the numerical prodigy and enthusiastic biker and explorer. Also, to acknowledge my Nephews Ahmad Umar, Yusuf Umar and cute niece Zuniarah Athar and new comers in family our angel like cutie pie Zaira Athar and the only person among all of them to be infront of our eyes all the time Amaira Aamir. Last but the most important with a special mention goes to my Bhabhi’s Mrs. Nazish Umar, Dr. Sana Athar and the latest addition Mrs.Zohra Asghar for their unconditional love and support.

I am thankful to the funding agency Department of Biotechnology, Govt. of India for the financial support in the form of JRF/SRF during the tenure of my Ph. D. I am also thankful to the Aligarh Muslim University, Aligarh for providing the necessary facilities.

I am grateful to everyone who is not listed above but contributed to my research or my life.

Anzar Abdul Mujeeb

Maulana Azad Library, Aligarh Muslim University

List of Contents

Acknowledgements Abbreviations and symbols List of Figures List of Tables

Chapter 1 Review of literature 1.1 Introduction 1 1.2 Immune System 2 1.3 Infections 16 1.4 Pathogenesis of Brucellosis 32 1.5 Immunotherapies available against intracellular pathogen 46 1.6 The problems of tuberculosis as a human disease 53 1.7 Apoptosis 61

Chapter 2 Evaluation of Immunogenic Potential of Exosome Mediated Delivery of Recombinant rL7/L12 Protein against Experimental Murine Brucellosis 2.1 Introduction 65 Maulana2.2 Materials Azad Library, & Methods Aligarh Muslim University 68 2.3 Results 77 2.4 Discussion 88 2.5 Conclusion 92

Chapter 3 TLR Agonist studded Nanosized formulation of OMP-19 antigen augments prophylactic potential against experimental brucellosis

3.1 Introduction 94 3.2 Materials & Methods 96 3.3 Results 103 3.4 Discussion 112 3.5 Conclusion 117

Chapter 4 TLR agonist mediated immunomodulation augments vaccine potential of BCG against Mycobacterium tuberculosis infection 4.1 Introduction 118 4.2 Materials & Methods 121 4.3 Results 125 4.4 Discussion 136 4.5 Conclusion 140

Chapter 5 Evaluating the immunomodulating potential of CLEC4E agonist trehalose 6,6'- dimycolate (TDM) in enhancing the vaccine potential of BCG against Mycobacterium tuberculosis infection

Maulana Azad Library, Aligarh Muslim University 5.1 Introduction 142 5.2 Materials & Methods 145 5.3 Results 150 5.4 Discussion 162 5.5 Conclusion 167

Bibliography 168

Abbreviations and Symbols

ABBREVIATIONS AND SYMBOLS

ACK Ammonium chloride potassium bicarbonate lysis buffer Ag Antigen ACT Adoptive cell therapy ADC Albumin dextrose catalase ADCC Antbody Mediated Cell Cytotoxicity AIDS Acquired Immunodeficiency Syndrome AIM2 Absent in melanoma 2 AIPC Androgen Independent Prostate Cancer ALL Acute Lymphoblastic Leukemia AMTV Allogeneic macrophage tuberculosis vaccine ANOVA Analysis of variance AP-1 Activator protein-1 APC Antigen presenting cells APC-Cy7 Allophyococyanin-Cyanin7 ASC Apoptosis-associated speck-like protein containing a CARD (caspase recruitment domain) ATCC American Type Culture Collection Atg Autophagy-related Atg16L1 Autophagy-related 16-like 1 ATP Adenosine triphosphate BCA Bicinchoninic acid BCG Bacillus/Bacilli Calmette-Guerin BMDC Bone marrow derived dendritic cells Maulana Azad Library, Aligarh Muslim University BSA Bovine serum albumin BCG Bacille Calmette-Guerin Bcl B-cell lymphoma BCR B cell receptor for antigen BIR Baculovirus inhibitor of apoptosis protein repeat Birc1 BIR-containing 1 BsA Bispecific Aptamer CA125 Cancer antigen 125 CARD Caspase recruitment domain

Abbreviations and Symbols

CARDIAK Card-containing IL (interleukin)-1β converting enzyme-associated kinase; CCR Chemokine receptor CD Cluster of differentiation cDC Conventional dendritic cell CDR Complementarity-Determining Region CFSE Carboxyfluorescein succinimidyl ester CFU Colony Forming Unit Ci Curie CLR C-type lectin receptor CMI Cell mediated immunity cpm, counts per minute CNBr Cyanogen Bromide CPCSEA Control and Supervision of Experiments on Animals Cpm Counts per minute CR Complement receptor CTL CytotoxicT lymphocytes CSF Colony stimulating factor CT Computerized Tomography CTD C-terminal domain CTL Cytotoxic T lymphocyte DC Dendritic cell DCF 2',7'-dichlorofluorescin DCFH-DA 2',7'-dichlorofluorescin diacetate DED Death effector domain DLS Dynamic Light Scattering Maulana Azad Library, Aligarh Muslim University DMEM Dulbecco's modified eagle's medium DMSO Dimethyl Sulphoxide DNA Deoxyribose Nucleic Acid Ds Double-stranded Dtxel Docetaxel DVD Dual Variable Domain ECL Enhanced chemiluminiscence EDA Extracellular domain A EDC 1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide Abbreviations and Symbols

EDTA Ethylenediamine tetra-acetic acid EE Entrapment Efficiency EGFR Epidermal Growth Factor Receptor ELFA Enzyme-linked immunoflourescence Assay ELISA Enzyme linked immuno-sorbent assay EPR Enhanced Permeability and Retention ER Endoplasmic reticulum ERK Extracellular-signal-regulated kinase F9 l Peptide 91-110 from 16 kDa Fab′ Fragment of Antigen Binding FACS Fluorescence-activated cell sorting FADD Fas-associated death-domain GPI Glycosylphosphatidylinositol FBS Fetal bovine serum FDA Food and Drug Administration Fc Fraction crystallizable Fig Figure FISH Fluorescence in-situ hybridization FITC Fluorescent Isothiocyanate FV Variable Fragment G Gram(s) GalA Glucosaminide aminoakyl phosphate GM-CSF Granulocyte macrophage colony stimulating factor H&E Hematoxylin and eosin H2L2 Whole antibody Maulana Azad Library, Aligarh Muslim University H2O2 Hydrogen peroxide H2 SO4 Sulphuric acid HCl Hydrochloride HCV Hepatitis C virus HEPES N-2-hydroxyethylpiperazine-N'-2-ethanesulponic acid HER Human Epidermal Growth Factor Receptor HIV Human Immunodeficiency Virus HLA Human Leukocyte Antigen

Abbreviations and Symbols

HRP Horse radish peroxidase Hr Hour(s) HRP Horseradish Peroxidase i.p Intraperitoneal ICAM Intercellular adhesion molecule ICS Intra-cellular staining IFI Interferon-inducible IFN Interferon (e.g., IFN-y) Ig Immunoglobun IGFR Insulin-ike Growth Factor IL Interleukin IL-7R Interleukin-7 receptor iNOS Inducible NO synthase IPAF IL-1β converting enzyme protease activating factor IRAK IL-1 receptor-associated kinase IRF IFN regulatory factor ISCOMS Immune stimulating complexes IκB Inhibitor of κB JNK C-jun N-terminal kinase kDa Kilo Dalton LB Luria Bertani medium LB1 Aptamer against Listeria InlA LC Loading Capacity LCMV Lymphocytic choriomeningitis virus LD50 50% lethal dose Maulana Azad Library, Aligarh Muslim University LFA Lymphocyte function associated Ig Immunoglobulin LN Lymph node LPS Lipo-polysaccharide LRR Leucine-rich repeat LT Lethal toxin LTA Lipoteichoic acid M Molar M. tb Mycobacterium tuberculosis Abbreviations and Symbols m.Abs Monoclonal antibodies MAB Monoclonal antibodies MAL MyD88-adaptor-like MALP-2 Macrophage-activating lipopeptide MAPK Mitogen-activated protein kinase MAVS Mitochondrial antiviral signalling mBsAbs Monoclonal Bispecific Antibody MCC Matthew's correlation coefficient MCMV Murine cytomegalovirus MD2 Myeloid differentiation protein-2 Mda5 Melanoma differentiation-associated gene 5 MDP Muramyl dipeptide MDR Multi drug-resistant MFI Mean fluorescent intensity Mg Milligrams Min Minutes Ml Millilitre mM Millimolar ng Nanogram MgSO4 Magnesium Sulphate MHC Major Histocompatability Complex miRNA microrna MITA Mediator of IRF3 activation MRI Magnetic Resonance Imaging mRNA Messenger RNA Maulana Azad Library, Aligarh Muslim University MS Mass Spectometric mTOR Mammalian target of rapamycin MTT 3, (4, 5-dimethylthiazol-2-yl) mvAb Monovalent Antibody MyD88 Myeloid differentiation primary response gene 88 Na2CO3 Sodium Carbonate NaCl Sodium chloride NaHCO3 Sodium Bicarbonate

Abbreviations and Symbols

NAIP NLR family, apoptosis inhibitory protein NAIP5 Neuronal apoptosis inhibitor protein 5 NAK NF-κB activating kinase NCI National Cancer Institute NF-κB Nuclear factor κB NHS N-hydroxysuccinimide NIH National Institute of Health NK cell Natural Killer Cell NLR NOD like receptor NLRC NLR family CARD-domain-containing NLRP NLR family pyrin-domain-containing nm Nanometer nM Nanomolar nmol Nanomoles NO Nitric oxide NOD Nucleotide-binding oligomerization domain NP Nanoparticle NPV Negative predictive value OADC Oleic acid albumin dextrose complex ˚C Degrees celcius OD Optical density OPD O-phenylenediamine dihydrochloride OVA Ovalbumin PAGE Polyacrylamide Gel Electrophoresis Pam2Cys S-[2, 3-bis (palmitoyloxy)propyl] cysteine Maulana Azad Library, Aligarh Muslim University PAMP Pathogen-associated molecular pattern; PBMCs Peripheral blood mononuclear cells PBS Phosphate Buffer Saline pBsAbs Polyclonal Antibody PBST Phosphate Buffer Saline conatinig Tween PCR Polymerase Chain Reaction PDB Protein Data Bank pDCs Plasmacytoid dendritic cells PE Phycoerythrin Abbreviations and Symbols

PECs Peritoneal exudates cells PE-Cy5 Phycoerythrin-CyaninS PEG Polyethylene Glycol PET Positron Emission Tomography pH Negativelogarithm of hydrogen ion concentration PIM Phosphatidylinositol mannosides PMSF Phenylmethylsulfonyl fluoride PPD Purified protein derivative PKR Protein kinase receptor PMN Polymorphonuclear cells PMSF Phenylmethylsulfonyl fluoride PRR Pattern-recognition receptor PVA Polyvinyl alcohol RBC Red Blood Cells RES Reticulo Endothelial Cells RNA Ribose Nucleic Acid RNAi RNA interference ROS Reactive Oxygen Species Rpm Rotations per minute RPMI Roswellpark memorial institute s Second SD Standard deviation SDS Sodium dodecyl sulfate siRNA Small interfering RNA SNP Single-nucleotide polymorphism Maulana Azad Library, Aligarh Muslim University Ss Single stranded TAA Tumor-Associated Antigens TAB TAK1-binding protein TAK1 Transforming-growth-factor-β-activated kinase 1 TB Tuberculosis TBK1 Tank-binding kinase 1 Tc Cytotoxic T cell TCA Tricarboxylic acid TCH Thiophene carboxylic hydrazide

Abbreviations and Symbols

TCR T cell receptor TEM Transmission Electron Microscopy TGF Transforming Growth Factor Th T helper TICAM Tir-domain-containing adaptor molecule TIR Toll/IL-1 receptor TIRAP Tir-containing adaptor protein TLR Toll Like Receptor TNF Tumor Necrosis Factor Treg Regulatory T cells Tth T follicular helper TB Tuberculosis U Unit UNICEF United nations children's emergency fund UV Ultra violet v/v Volume/volume VEGF Vascular Endothelial Growth Factor VH Variable Domain of Heavy Chain VL Variable Domain of Light Chain Vol Volume w/v Weight/volume WHO World health organization WT Wild-type x g Centrifugal force equal to gravitational force XDR MaulanaExtensi vAzadely dru Library,g resistant Aligarh Muslim University μl Microliter μM Micromolar µCi Micro Curie µg Microgram µl Microlitre % Percentage °C Degree centigrade

LIST OF FIGURE

FIGURE TITLE Page No

Chapter 1

Figure 1.1 Typical structure of a TLR and DECTIN-1 5

Figure 1.2 Control of TH cell development by TLRs on APC 6

Figure 1.3 Overview of Conventional TLR signaling pathways 8

Figure 1.4 Development of T helper cell subsets 17

Figure 1.5 Network of cellular development and cytokine 22 expression on pathogenic infection through macrophages

Figure 1.6 Generalized response of the immune system upon 29 encountering varied form of pathogens and the mechanisms used by the pathogen to alter immune responses

Figure 1.7 Overview intracellular life cycle of Brucella 34

Figure 1.8 Different sources of Brucella infection in human 38

Figure 1.9 Pathway of exosome formation 52

Figure 1.10 Exosome composition 53 Maulana Azad Library, Aligarh Muslim University Figure 1.11 Various outcomes of infection with M tuberculosis, 58 different T cell populations are involved in protection, and in anti-mycobacterial effector mechanisms of macrophages

Chapter 2

Figure 2.1 Overexpression and purification of ribosomal L7/L12 78 SDS-PAGE

Figure 2.2 Western Blot analysis of recombinant rL7/L12 protein 78

Figure 2.3 Representative TEM image of the as-formed exosomes 79

Figure 2.4 Total antibody response in sera of the mice immunized 80 with various forms of Brucella rL7/L12 protein

Figure 2.5 Analysis of IgG isotype response in Balb/c mice 81 immunized with various forms of Brucella recombinant rL7/L12 protein

Figure 2.6 CD4+ T cell Proliferation response of splenocytes 82 derived from mice immunized with various forms of Brucella recombinant rL7/L12 protein

Figure 2.7 Effect of immunization with various forms of rL7/L12 85 protein on production of various cytokine in Balb/c mice

Figure 2.8 Delayed type cell mediated immune response in mice 86 immunized with various forms of Brucella rL7/L12 protein: DTH response was evaluated by measuring footpad swelling

Figure 2.9 Level of protection against B. abortus 544 infection 87 conferred by immunization with various form of Brucella rL7/L12 protein

Chapter 3

Figure 3.1 MaulanaSDS-PAGE Azad and Library, Immunoblot Aligarh profile ofMuslim the inhouse University 103 purified recombinant unlipidated Omp19

Figure 3.2 Analysis of humoral immune response and isotype 105 distribution in immunized BALB/c mice under defined

conditions

Figure 3.3 Analysis of Th1/Th2 cytokines in splenocyte culture 107 supernatant of immunized BALB/c mice under defined conditions

Figure 3.4 Figure 3.4. T cell proliferation induced by diverse 108 forms of Omp19 under defined conditions

Figure 3.5 Analysis of CD80/CD86 co-stimulatory molecules on 110 antigen presenting cells of the immunized BALB/c mice

Figure 3.6 Analysis of memory marker on CD4+/ CD8+ T cells 111 population in the immunized BALB/c mice

Chapter 4

Figure 4.1 Cytokine profile in the various immunized groups 126

Figure 4.2 T cell proliferation response in various immunized 128 groups

Figure 4.3 Confirmation of Th1/Th2 polarization upon 128 immunization GalA+MΦBCG based vaccine protocol

Figure 4.4 GalA upregulates expression of costimulatory molecules 130 CD80+ on antigen presenting cells

Figure 4.5 GalA upregulates costimulatory molecules CD80+ 131 /CD86+ on antigen presenting cells

Figure 4.6 Splenic CD4+ Proliferation Assay 132 Maulana Azad Library, Aligarh Muslim University Figure 4.7 Splenic CD8+ Proliferation Assay 133

Figure 4.8 Augmentation of long-lasting CD4+ T cell 134 (effector/central) response upon immunization of mice with GalA+MBCG

Chapter 5

Figure 5.1 Cytokine profile in the various treated groups 151

Figure 5.2 Cytokine profile in the various treated groups 152

Figure 5.3 T cell proliferation response in various immunized 153 groups

Figure 5.4 Confirmation of Th1/Th2 polarization expression upon 154 immunization with primed mice macrophages

Figure 5.5 Splenic CD4+ Proliferation Assay 156

Figure 5.6 Splenic CD8+ Proliferation Assay 157

Figure 5.7 ttgM+BCG upregulates costimulatory molecules 159 CD80+ and CD86+ on antigen presenting cells

Figure 5.8 Augmentation of long-lasting CD4+ T cell 160 (effector/central) memory response upon immunization of mice with ttgM+BCG

Figure 5.9 Augmentation of long-lasting CD8+ T cell 161 (effector/central) memory response upon immunization of mice with ttgM+BCG

Maulana Azad Library, Aligarh Muslim University

LIST OF TABLES

Tables Titles Page No. Table 1.1 Table shows specific types of Toll-like receptors and 9 their expression on different cells, the ligands to which TLR binds and regulation by various agents Table 1.2 Table shows specific types of T cells and their 16 association with the MHC complexes through which they express antigens and also various cytokines expressed as effector molecules Table 2.1 . CD8+ T cell proliferation response of splenocytes 83 derived from mice immunized with rL7/L12 loaded exosomes Table 3.1. Protection against B. abortus 544 in immunized 112 BALB/c mice

Maulana Azad Library, Aligarh Muslim University

XXVI

Review of literature

I would picture myself as a virus, or as a cancer cell, for example, and try to sense what it would be like to be either. I would also imagine myself as the immune system, and I would try to reconstruct what I would do as an immune system engaged in combating a virus or cancer cell. When I had played through a series of such scenarios on a particular problem and had acquired new insights, I would design laboratory experiments accordingly… Based upon the results of the experiment, I would then know what question to ask next… When I observed phenomena in the laboratory that I did not understand, I would also ask questions as if interrogating myself: “Why would I do that if I were a virus or a cancer cell, or the immune system?” Before long, this internal dialogue became second nature to me; I found that my mind worked this way all the time.

~ Jonas Salk

1.1 Introduction

Since the time when the earth was inhospitable and lifeless, there has been a constant change in the environment making it suitable for the survival of a whole lot of different species from microorganisms to multicellular organisms. Around 3.5 billion years ago life forms first appeared and have been subsequently changing since then transforming into diversified organisms, which have been continuously evolving (Mayr E et al., 2001). This continuous evolution of life forms and their interaction with each other posts the challenge of peaceful coexistence. Sometimes peaceful coexistence can turn into survival battle for the organisms that constantly invade others in order to survive and propagate themselves. There are various ways for species interaction with each other e.g. mutualism, commensalism and parasitism and establishing different ecological interactions. Parasitism, which involve a detrimental Maulana Azad Library, Aligarh Muslim University relationship between the organism known as the parasite interacting with the other, known as the host, exploiting their machinery for nutrients or their growth and survival. Thus, overall health of the host is negatively influenced by pathogens. We humans have cleverly used these microorganisms to understand their invasion and aversion. Among these the intracellular bacteria has been used to understand the underlying mechanism of infections. The capability of host to evade and resist damage by the pathogens is what we known as the immunity. Consequently, hosts employ defense methods against these pathogens which is performed by the immune system is our most important self-preserving system.

Review of literature

1.2 The immune system

Immune system depends on cells and molecules for immunity. When a pathogen attacks the body the immune system which is an intricately balanced system of cells, cell products, along with the tissues and the organ that they from, all work in harmony for the defense of body against invasions by these invaders. Immune systems have capacity to differentiate between self and the foreign non-self-components and upon recognition eliminates them. If there is a discordance in distinguishing self and non- self then it may lead to autoimmune diseases. Sometimes non-harmful substances like pollens may case an exaggerated or unfitting immune response which we know as allergies. It is known that response by the innate system are rapid as compared to acquired immune responses.

In vertebrates the immune system, can be broadly categorized as: innate and adaptive or acquired system. In general, innate immunity rapidly reacts to a pathogenic invasion, whoever its nonspecific and short-lived. While in the case of adaptive immune responses the time to react to an invasion is longer, however, it is more specific and has the capacity to enhance itself through immunological memory and responds more quickly upon future encounters of similar pathogens. To provide protection against diverse and always evolving pathogens both innate and acquired responses work in harmony. Both the systems use different types of receptors to recognize the pathogens (Flajnik et al., 2004).

1.2.1 The Innate immune system

The part of the immune system that has been evolutionarily conserved and is a vital part of the host defense system is known as the innate system of immune defense. Maulana Azad Library, Aligarh Muslim University Innate immune defenses is the first line of defense for mammals comprising of surfaces that act as physical barriers as is the case with the skin and the mucosal layers of respiratory and gastrointestinal pathways (Gallo RL et al., 2012). In response to damage to tissues, or infection specialized lymphoid and myeloid cells along with some non-hematopoietic cells have the capacity to initiate and activate innate defense mechanisms (Nagasawa M et al., 2018). The response by these cells is rapid but lacks specificity. The innate immune system includes the following two components. First one includes the humoral arm of the immune system which works in conjugation with the complement system consisting of proteins and defensins

Review of literature

(Chaplin DD et al., 2012). These proteins and defensins have the capacity to hinder the spreading of pathogens by making them identifiable as foreign substances through clumping and opsonization so that they can be eliminated (Thaiss CA et al., 2016). Second component known as cellular innate immunity, is carried out by cells called phagocytes that ingest and degrade, or “eat” pathogens (Lim JJ et al., 2017). Innate Immunity was initially considered to be a rapid and non-specific response exemplified by engulfment and digestion of microorganisms and foreign substances by phagocytic cells and acting as antigen presenting cells (APC) to adaptive immune cells. However, after the dawn of Toll-Like Receptors (TLRs), a kind of Pattern recognition receptors (PRRs), has redefined the importance of innate immune system in specifically discriminating between self and non-self (Muñoz-Wolf N et al., 2016). These components are indispensable for effective running of the innate and adaptive immune system.

Pattern-recognition receptors and their functions

For the body to protect against the infection the first thing that the body has to do is to detect the microbial presence. The body looks for molecules that are rarely seen within the human cells and are also specific for the microorganism. These specific molecules unique to the specific organism are known as pathogen-associated molecular patterns or PAMPs (Mourao-Sa D et al., 2013). To recognize these PAMPs, there are germline-encoded receptors which are called as pattern-recognition receptors or PRRs. The germline encoded receptors of the innate immune recognition system has broad specificities to counter the conserved as well as the mutating features of the microorganisms. These receptors can be expressed on surfaces of various cells, or in intracellular compartments and can also be secreted in the Maulana Azad Library, Aligarh Muslim University bloodstream or tissue fluids. The main functions of these PRRs are to opsonize the pathogens, pro-inflammatory signaling pathways activation, activation of the complement system cascades, phagocytosis, apoptosis induction (Medzhitov R et al., 2013). While in case of adaptive immune recognition, which are done through antigen receptors formed by the rearrangements within the gene encoded by the germline and also through the process of somatic recombination creating a repertoire of diverse antigenic receptors (Boehm T et al., 2011). On the surface of T and B lymphocytes these clonally selected pathogen specific receptors are distributed ultimately leading to the creation of immunological memory (Farber DL et al., 2016). So the way the 3

Review of literature two systems deal with diversity in molecules of the pathogens is basically different, with each having its own specific way to counter microbes.

1. Transmembrane PRRs: includes the Toll-like receptors

i. TLRs which recognize viral nucleic acids and several bacterial products, including lipopolysaccharide and lipoteichoic acids TLRs activate tissue- resident macrophages to produce pro-inflammatory cytokines, including tumor-necrosis factor (TNF), interleukin-1β (IL-1β) and IL-6, which coordinate local and systemic inflammatory responses. TNF and IL-1β, in turn, activate the local endothelium to induce vasodilation and increase the permeability of the blood vessel, allowing serum proteins and leukocytes to be recruited to the site of infection. TLRs also induce macrophages to produce antimicrobial proteins and peptides (Creagh EM et al., 2006).

ii. Dectin 1, a transmembrane receptor that binds to β-glucan and is present on dendritic cells and macrophage. This PRR has an important role in antifungal defense and the production of cytokines (Brown GD et al., 2003).

iii. Several cell surface receptors expressed on macrophages function as pattern

recognition receptors that mediate phagocytosis of microorganisms. Macrophage mannose receptor (MMR), macrophage scavenger receptor (MSR) interacts with variety of gram-positive and gram-negative bacteria and fungal pathogens. In addition to recognition of microbial PAMPs, MSR also plays a role in lipid homeostasis by binding and endocytosing acetylatedMaulana low -Azaddensity Library,lipoproteins Aligarh (Janeway MuslimJr CA et al., University 2002).

TLRs and acquired immunity

Acquired immunity is activated with the contact of TLRs with their similar ligands, after the activation of the complex itself. TLRs, obtainable through antigen presenting cells (APCs) recognize the forms linked with pathogens and thus generates acquired immunity (Akira S et al., 2001). Adaptive immune response is heightened when stimulated DCs travel from peripheral tissues to the lymph nodes and exhibit an interaction with T cells. Molecules that stimulate each other (CD80/CD86 and CD40),

Review of literature present on the surface of DCs undergo expression enrichment when TLR is activated. Interface between costimulatory molecules and CD28 along with CD40 ligand on immature CD40+ increase APC activity. After which receptors of T cells relate with antigenic peptides with the help of major histocompatibility complex II (MHC II) present on DCs. The most crucial player involved in the initiation of T cell differentiation is the environment of cytokine which exists in the microenvironment of T-cells (Itano AA et al., 2003,. Krutzik SR et al., 2005)

Figure 1.1 Typical structure of a TLR and DECTIN-1. Codomain of a typical TLR containsMaulana the Leucine Azad rich repeats Library, (LRRs), Aligarh while Toll/ILMuslim-1receptor University domain known as TIR-domain is present on the cytoplasmic face. Dectin-1 which is a type II transmembrane protein expressed by myeloid cells and consists of an extracellular carbohydrate recognition domain (CRD) and a cytoplasmic domain containing an ITAM-like motif. TLRs and T helper cells

Th1 type responses are increased through TLR3, TLR4, TLR7, TLR8 and TLR9, signaling. On contrary Th2 type responses are favored by TLR5, TLR2/1 and TLR2/6 (Van Duin D et al., 2006; Manicassamy S et al., 2009).

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Figure 1.2 Control of TH cell development by TLRs on APC. TLRs induce the production of cytokines such as IL-12 and IL-4 in APCs by recognizing pathogens or their products. These cytokines instruct naive T cells to differentiate into TH1 cells or TH2 cells depending on the cytokine environment.

TLRs induce Th1 responses through the secretion of cytokines IFNα and IL-12p70 from APCs and an elevated Th2 type response is found in MyD88-deficient mice. These deficient mice have impaired TLR signaling. It is Important to note that Th1 cells secrete TNFα, IL-12, and IFNγ and stimulates B cells to enhanced secretion of IgG and IgA antibodies that helps in defense against intracellular pathogens (Rengarajan J et al., 2000). Th2 type differentiation is influenced by IL-10 and IL-4. In general, Th2Maulana cells secrete Azad IL-4, Library,IL-5 and IL Aligarh-10 and stimulate Muslim the productionUniversity of IgG, IgA and specifically IgE antibodies that imparts protection against parasitic infections (Abbas AK et al., 1996; Yazdanbakhsh M et al.,2002). It is noteworthy that Th1 type cytokines restrain the Th2 cells differentiation (Diehl S et al., 2002).

In essence, TLRs activation on APCs also stimulates IL-21 and IL-23 secretion, which impel differentiation of Th17 cells (Ivanov II et al., 2007). Th17 cells have cardinal role in host-defense against pathogens at mucosal sites. Th17 cells produce the cytokines IL-17 and IL-23. These cytokines modulate function of B and T-cell especially of Treg cells (Afzali B et al., 2007). Treg cells play an indispensable role in

Review of literature sustaining peripheral T cell tolerance (Sakaguchi S et al., 2008). TLR activation of APCs especially DCs produce anti-inflammatory cytokines viz. IL-10, which further stimulates proliferation of Treg cell. In turn, Treg cell suppress the inflammatory cytokines IL-12p40, IL-6 and TNFα (Veldhoen M et al., 2006). Recent research also reported that the Treg cells mediated suppression can be elevated or weakened depending on TLR stimulation nature. TLR2, TLR4 and TLR5 stimulation can maximize responses against pathogens and in this way, lessen the severity of damage of the host whereas TLR7, TLR8 and TLR9 decrease Treg functions (Mellor AL et al., 2008). Further, DCs cross present antigen with major histocompatibility complex class I molecules to CD8+ T cells and stimulate cytotoxic T lymphocyte (CTL) response that is important for effective vaccine responses against intracellular pathogens (Den Haan JM et al., 2000). CTL responses have been shown with respect to TLR3 and TLR9 activation (Fujimoto K et al., 2011). Therefore, the nature of a specific infection or diseased state will activate specific TLRs and will be responsible for the different type of T-cell responses.

Toll-like receptor 4

TLR4 was first identified member of the TLR family in mammals (Zhang G et al., 2001). It stimulates inflammatory pathways that are crucial in making it ideal target for therapeutic treatment and adjuvant development. TLR4 recognizes lipopolysaccharide (LPS) from Gram-negative bacteria. TLR4 consist of a recognition subunit MD2 (myeloid differentiation protein-2) and a membrane-bound GPI (glycosylphosphatidylinositol)-anchored CD14 recognizes the LPS. (Chaplin DD., 2006). The activation is further enhanced by another protein known as LBP (LPS- binding protein) (Guha M et al., 2001).Even though LPS is the most Maulana Azad Library, Aligarh Muslim University immunostimulatory TLR4 ligand but its toxicity issue limits its use as vaccine adjuvant. Most importantly, the lipid A an active component of LPS molecule interacts with TLR4 and activates downstream signaling (Netea MG et al., 2002).

Toll-like receptor 2

TLR2 forms functional heterodimers with TLR1, TLR6 and TLR10 and recognizes a structurally diverse range of PAMPS (Kumar H et al., 2011). TLR1/2 heterodimers are receptors for triacyl lipopeptides molecules present in bacteria while TLR6 interacts with TLR2 to recognize diacyl lipopeptides such as M. fermentans

Review of literature macrophage-activating lipopeptide (MALP-2) (Omueti KO et al., 2005; Takeda K et al., 2002). The TLR2 activation level is elevated in presence of CD14 and CD36

(Nilsen NJ et al., 2008). Pam3Cys and Pam2Cys are synthetic triacylated and diacylated lipoproteins for TLR2 activation respectively. Since these synthetic ligands are highly specific for Th2-biased TLR2 activation, therefore, these ligands can be used as adjuvant for TLR2 activation (Spohn R et al., 2004). Moreover, lipopeptides represent the most widely used ligand to target TLR2.

Figure 1.3 Overview of Conventional TLR signaling pathways. TLR’s are either located on the cell surface for ligand recognition and binding TLR2 (TLR2 in association with TLR1 or TLR6), TLR4, TLR5 and TLR11 or are located on the endosome (TLR3, TLR7, and TLR9). Except TLR3, all TLRs signal through MyD88. TLR1, TLR2, TLR4 and TLR6 engage adaptor TIRAM while TLR3 and TLR4 signals throughMaulana TRIF. TLR4 Azad activates Library, both MyAligarhD88-dependent Muslim and University TRIF-dependent pathways. However, TLR4 requires the additional linker adaptor TRAM, which links TIR domain of TLR4 with TRIF in the TRIF-dependent pathways. Both NF-κB and AP-1 control inflammatory responses through the induction of inflammatory cytokines. In the TRIF-dependent pathway, TRIF interacts with RIP1 and TRAF6. Activated TRAF6 and RIP1 activate NFκB and MAPKs. TRIF also interacts with TRAF3 and activates TBK1/IKKi, which mediate phosphorylation of IRF3 and IRF7. Phosphorylated IRF3 dimerizes and translocates into the nucleus to regulate transcription.

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Table 1.1 Table shows specific types of Toll-like receptors and their expression on different cells, the ligands to which TLR binds and regulation by various agents.

Receptor Ligands Cell type Regulation TLR1 Tri-acyl lipopeptides (bacteria Ubiquitous PHA down- and mycobacteria), soluble regulates factors expression on T- cells TLR2 Lipoproteins/ lipopeptides, Neutrophils, Induced by LPS Peptidoglycan, Porins, Dendritic cells and Zymosans, monocytes Glycoinositolphospholipids, Glycolipids, LPS TLR3 Double- stranded RNA(Virus), Dendritic cells, Induced by polyinosine-polycytidylic acid natural killer cells differentiation, (poly (I:C)) reduced upon maturation TLR4 LPS, Taxol, Fusion protein, Macrophages, Enhanced by Envelope proteins, , dendritic cells, inflammatory Polysaccharides of Fibrinogen epithelial cytokines and cells bacterial products, down-regulated by anti- inflammatory cytokines TLR5 Flagellin (bacteria) Monocytes, No alterations immature observed dendritic cells, epithelial cells, NK cells, T cells TLR6 Di-acyl lipopeptides B cells, No alterations (Mycoplasma) monocytes, NK observed cells TLR7 Imidazoquinolines, Single B cells, Highly inducible stranded RNA and plasmacytoid by IL-6 Bropirimine precursors of DC TLR8 Imidazoquinoline Monocytes, Highly inducible Natural killer by Maulana Azad Library, Aligarh Muslimcells, T cells University IFN- LPS TLR9 CpG DNA (bacteria) pDC precursors, B Inducible by IFN- cells, ,LPS Macrophages, Neutrophils, natural killer cells, microglial cells TLR10 Not known B cells, No alterations plasmacytoid precursors of DC TLR11 Profilin. Urinary Tract, No alterations Macrophages TLR12 Not known TLR13 Not known

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1.2.2 Adaptive Immune System

The antibodies and the T-cell receptors are of pivotal importance in the adaptive immune response. The B cell receptors and the T cell receptors mediate the function of recognition by the adaptive immune system. Basically, it is through recombination- activating gene (RAG)-protein mediated somatic recombination which assembles variable and constant fragments leading to formation of genes encoding antigen receptors.

Lymphocytes that express antigen receptors can be categorized into two types: conventional lymphocytes and innate-like lymphocytes. Conventional lymphocytes include conventional T cells (most αβ T cells) and B cells (also called B2 cells). The antigen receptors in case of conventional lymphocytes get assembled at random. On the other hand, innate-like lymphocytes include B1 cells, natural-killer T cells and subsets of γδ T cells. Their antigen receptors don’t get formed entirely at random and hence, their diversity is restricted as compared to that of conventional lymphocytes. Innate-like lymphocytes are specific towards a predefined set of ligands.

Before the encounter to an antigen, conventional lymphocytes keep on circulating in the lymph nodes and the spleen. Antigen Presenting Cells in the peripheral tissues take up the antigen and deliver it to the lymph nodes or spleen through the lymph or blood, respectively. It is here that they get recognized by the conventional lymphocytes. The innate immune system provides the instructions for the differentiation of conventional lymphocytes into an effector cell type and their emplacement to the site of infection with the help of cytokines and chemokine’s, respectively. Maulana Azad Library, Aligarh Muslim University Conventional αβ T cell are categorized into two types: T-helper (TH) cells which possess the co-receptor CD4 and cytotoxic T cells, which possess co-receptor CD8 on their cell surfaces, respectively. During antigen presentation, the peptides bound to major histocompatibility complex (MHC I) get recognized by CD4 bearing T helper (TH) cells whereas the peptides bound to MHC II get recognized by T cytotoxic (TC) cells. Any antigen could easily get recognized by the conventional B cells by binding to their antigenic determinants or epitopes.

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The peritoneal and the pleural cavities are the residential sites for innate-like B cells or B1 cells. They produce IgM type antibodies that are specific for some of the bacterial polysaccharides and self-antigens. There is one more class of MHC molecules known as non-classical MHC molecules or MHC class I molecules that can present bacteria specific ligands ( for instance, bacterial lipids and formylated peptides in case of CD1 and H-2M3 families, respectively) to innate-like T cells that recognize them. Not only this, some of the non-classical MHC molecules may themselves act as ligands to T cell receptors without displaying any other molecule. It is thought that these molecules get enunciated due to the involvement of PRRs on specific cell types, such as mucosal epithelial cells.

Immune homeostasis

The immune homeostasis is always maintained by mutually interacting molecules and cells. Th1 and Th2 cells are in antagonism in most of the models established and both of these subsets are proposed to be regulated by “Regulatory T cells” (Saito S et al., 2010). Th17 is a newly recognized T helper cell type and is assigned to be an inflammatory T cells especially in autoimmune diseases (Bettelli E et al., 2006). On the other hand, the need to explain the control mechanism of overt T-rag responses led to the discoveries of regulatory switches in T-reg cells.

Altogether the immune system is an intellectual edifice a system that co-ordinate and integrate multiple cells and organ system to decide on immunity or tolerance - an outcome of the complex interactions between synergizing and counteracting cellular and molecular components of the immune system.

Immune T cells involved in immune response Maulana Azad Library, Aligarh Muslim University Conventional T Cells

Conventional T cells comprises of the αβ T cell receptor(TCR) CD4+ T-helper which recognizes the processed peptides in the phagolysosomes when they are complexed with MHC class II molecules which starts the cytokine production as well as the differentiation of the B-cells for the production on antibodies (Janeway Jr CA et al.,2001), while CD8+ T-cytotoxic cells recognize the protein-antigen complexed with MHC I molecules, promoting the killing of intracellular pathogens (Andersen MH et al.,2006).There are various subsets of the T cell that regulate immune response

Review of literature to pathogens. During the effector phase of the immune response different t cell subsets are formed which include T helper-1 (Th1) and T-helper-2 (Th2) cells. The cytokine released by the Th1 cells promote the production of proinflammatory cytokines that includes interferon-gamma (IFN-γ) and TNF-α mainly (Domingo- Gonzalez R et al.,2016), whereas Th2 is responsible for humoral immunity by secreting IL-4, IL-5, and IL-13 which induce B-cells for the production of antibodies for the opsonization of the pathogens (Cano RL et al.,2013). Chronically stimulated T-cells are polarized into Th1 or Th2 giving rise to the Th1/Th2 paradigm. IL-12, IL- 18, promote Th1-cell development, whereas IL-4 is a potent stimulus for Th2- cell development.

The unconventional T cells

The unconventional t cell comprises of γδ T cells, NK cells, NKT cells, invariant NKT (iNKT) cells, and mucosal-associated invariant T cells (MAIT) cells (van Wilgenburg B et al.,2018). The main cell that plays a role as a bridge between the innate and adaptive immunity are the γδ T cells and are also important in intracellular infections. The response shown by the γδ T cell to infections can occur both before and after the activation of αβ T cells. The functions of γδ T cells in various immune stages depend on the production of Th1/Th2 cytokines. This has been observed in the case of L. monocytogenes infections (Wu YL et al., 2014). During the advance stage of L. monocytogenes infection, γδ T cell depletion was characterized through liver necrosis, secondary inflammation, and disruption of macrophage homeostasis. These were mediated TNF-α+CD8+ T cells and reduction in the levels of IL-10 and IL-17 produced by γδ T cells. It can be inferred that the role that γδ T cells play in intracellular infections is to regulate the inflammation and consequently pathogen Maulana Azad Library, Aligarh Muslim University elimination (Witter AR et al., 2016).

NK cells are also involved in connecting the innate and adaptive immunity through the cytokine production and their interaction with APCs (Gaudino SJ et al., 2019). The role that NK cells play has been observed in the case of tumors and early viral infections. Reduced levels of IFN-γ produced by the NK cells due to some defects in them have been related to many viral infections. NK cells can also act as regulatory cells during inflammation and can have effect on adaptive immune response despite being redundant under certain condition (Gasteiger G et al., 2014).

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iNKT cells release the cytokines IFN-γ, TNF-α, IL-4, IL-5, IL-13, IL-17, chemokine’s, imparting immunoregulatory functions and promoting cell mediated immunity in case of intracellular pathogenic infection. Differential induction of iNKT subsets takes place during intracellular bacterial infections which leads to different adaptive immune response controlling the infection (Crosby CM et al., 2018).

Both the T cells i.e. conventional and unconventional T cells work in tandem with each other and complement each other to present an immune response against chronic intracellular infections. Mostly the conventional T cells are involved in antigen- specific functions and formation of memory cells through the cell-mediated immunity, while the unconventional T cells show limited TCR diversity but have the capacity to respond very quickly to infectious agents (Pasman L et al., 2017). The whole system of cell-mediated immune responses in tandem are required to overcome pathogenic assault which have evolved themselves to escape these mechanisms of the host to persist inside the host.

Th1 cells

Th1 cells produce hallmark cytokine, interferon (IFN-γ). It plays important role in the eradication of intracellular pathogens along with promoting IgG2a class switching on B cells in mice. Th1 helper cells differentiation along with cytokine receptors through STAT1 is initiated by TCR signaling. Th1 helper cells are activated by Type I and II IFNs and IL-27 through their receptors, which are present on naïve CD4+ T cells. STAT1 signaling results in upregulation of expression of transcription factor T-bet, notable regulator of Th1 differentiation. T-bet aggravates expression of IFN-γ and stimulates the IL-12 receptor chain, conversely suppressing GATA-3 and other Th2 differentiationMaulana factors. Azad The Library, generation Aligarhof IL-12R Muslimon developing University Th1 cells leads to IL- 12 signaling via STAT4. IFN- γ is produced from newly matured Th1 effector cells. Moreover, IL-12 induced IL-18 receptor synergizes with it to enhance IFN-γ production from Th1 effector cells. IL-23 generally acts on the multiplication of memory T cells and continues the development of Th1.To get rid of the intracellular pathogens, such as those of Listeria genus and Mycobacteria effective cellular immunity achieved from pertinent induction of Th1 cells is required. However, over activation of Th1 cells results in autoimmune inflammatory diseases, including Experimental autoimmune encephalitis (EAE), rheumatoid arthritis (RA), and allergic

Review of literature disorders, for example contact hypersensitivity or delayed-type hypersensitivities (DTH).

Th2 cells

Major cytokines of Th2 cells include IL-5, IL-4 and IL-13. Promoting activation of innate cells and also helping B cells in IgG1 and IgE class switching. Th2 cells starts developing through TCR signaling along with IL-4 receptor through STAT6. Their signals upregulate expression of primary regulator of Th2 differentiation, GATA-3. GATA-3 up regulates its own expression, along with altering epigenetic regulation of Th2 cytokine gene cluster (for IL-4, IL-5 and IL-13). Simultaneously, GATA-3 suppresses dominant factors of Th1 polarization, which includes IL-12R and STAT4 to block Th1 development (Dong C et al., 2001). Furthermore, IL-4 is also responsible for negatively regulating Th17 effector cells (Korn T et al., 2007).

Mostly, Th lineage producing cytokines promote their own lineage and on the other hand inhibit the propagation of other Th lineages. So, Th2 cells producing IL-4 can support further Th2 development and inhibit Th1 and Th17 developments (Korn T et al., 2007). Humoral immunity promoted by Th2 effector cells is important for controlling infections caused due to extracellular pathogens (Casadevall A et al., 2006).

T helper17 cells

Th17 cells are independent subset of T helper cells and have different differentiation factors as well as transcription factors. In 2006, researchers found out that combination of immunoregulatory cytokine TGF-β and cytokine IL-6 is required to induce IL-17 Maulana in naive T Azad cells. Moreover,Library, Aligarh IL-23-cultured Muslim T cells University mainly express inflammatory cytokines including, IL-6, IL-17 and TNF-α. TGF-β and IL-6 were recognized as the differentiation factors for Th17 cells. Surprisingly, it was seen that two cytokines which have opposite effects work together to induce differentiation of Th17 cells. This suggests that signaling molecules and transcription factors involved downstream of the TGF-β and IL-6 receptors work together. IL-4 and IFN-γ actively suppress Th17 development, while neutralization of IFN-γ and IL-4 is important for Th17 development. Therefore, Th17 cells symbolize a distinct lineage of CD4+ T cells (Korn T et al., 2009).

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Treg Regulatory T cells (Treg)

The immune system is highly institutionalized in discriminating between self and non-self- antigens. With the discovery of Treg, principal mechanism of self-tolerance and how autoimmune diseases occur became topic of growing interest. Tregs are naturally occurring subset of CD4+CD25+ regulatory T cells, and are mainly involved in suppressing activation and expansion of self-reactive T cells. Inducible CD4+ Treg cells are of two types: Tr1, IL-10-secreting T regulatory type 1 cells and Th3 which are TGF- secreting T helper type 3 cells. Tr1 cells are efficient in controlling chronic intestinal inflammation while, Th3 cells are responsible for maintaining tolerance of dietary antigen (Mittrücker HW et al., 2004).

CD4+CD25+ regulatory T cells (nTreg) nTreg cells forms 5-10% part of the total peripheral CD4+ T cell population produced in the thymus during ontogeny. They suppress wide range of immune cells of both adaptive and innate immune systems, including effector CD8+ T cells, CD4+CD25- T cells, NK cells, B cells, and macrophages. nTreg cells are anergic and proliferate feebly and produce cytokines in response to TCR stimulation. nTreg cells population in IL-2, IL-2R, or IL-2R deficient mice is reduced in peripheral circulation, suggesting that IL-2/IL-2R pathway is important in the development, function, and expansion of nTreg cells in vivo. Specific markers which are exclusively expressed by nTreg cells are still not known. CD25 is a component of the IL-2 receptor and is the most widely recognized and useful marker of nTregs. However, glucocorticoid-induced tumor necrosis factor receptor family related gene (GITR), CD40, CD28 and OX40 and cytotoxic T lymphocyte-associated antigen are other cell surfaceMaulana markers present Azad on Library, nTregs. Moreover, Aligarh new Muslim molecule University markers like lymphocyte activation gene-3 (LAG-3), and programmed death receptor 1 are being identified. Specific transcription factor Foxp3 differentiates nTreg cells as a distinct lineage from other T cells. Foxp3 is mainly expressed in CD4+CD25+ T cells population while no expression of Foxp3 is observed in activated CD4+CD25- T cells, Th1 cells, Th2 cells, and Th17 cells. Expression of Foxp3 in CD4+CD25- T cells can convert them into to CD4+CD25+ T cells with similar properties to nTreg (Horwitz DA et al., 2008).

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Table 1.2 Table shows specific types of T cells and their association with the MHC complexes through which they express antigens and also various cytokines expressed as effector molecules.

Lymphocyte Antigen Transcription Secreted effector Mechanism subset presentation factors molecules Th1 MHC class T-bet, STAT4, Activation of IL-2, IFN-γ, II STAT1 macrophages by, IFNγ, TNF-α, upregulation of iNOS lymphotoxin-α and ROI, proliferation of CTL Th2 MHC class GATA3, Stimulate B cells, IL-4, IL-5, IL-9 II STAT5 antibody production, IL-13 STAT6 antibody class switching Th17 MHC class RORγt, Recruitment, activation IL-17A, IL-17F II STAT3 and migration of IL-21, IL-22, neutrophils CCL20 Tfh MHC class Bcl6, c-MAF Provides help to B cells IL-10, IL-21 II for their transformation to plasma cells and memory B cells Tregs MHC class FOXP3, Immunosuppression IL-10, TGF-β, II SMAD, and tolerance IL-35 STAT5

1.3 Infections

Pathogens such as viruses, bacteria, fungi and other parasites can be classified as extracellular or intracellular from immunopathological point of view (Mogensen TH et al., 1993). Upon encountering these pathogens acute infections develops which is followed by clinical manifestations. These infections are eliminated within a few days when they occur in a healthy host as the pathogens encounter apt immune response (Hopper et al., 2012). However, there are still pathogens that can evade the host immune systemMaulana of healthy Azad individuals Library, and can Aligarh cause persistent Muslim infections University lying latent inside the host for years or even lifelong (Janeway Jr CA et al., 2001). In such persistent cases the pathogen cannot be cleared quickly by the host and the pathogen associated products for its survival are produced that includes proteins synthesised by the incorporation of pathogen genome inside the host cells and using of cellular as well as their own machinery for extended periods keeping the production of pathogens continuous. (Eisenreich W et al., 2019) Intracellular pathogens can be divided on the basis of how much of the host they use:

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1- Facultative intracellular pathogens: they are capable of living both inside as well as within the host cells. (Ismail N et al., 2002)E.g. Listeria monocytogenes, Salmonella typhi etc. 2- Obligate intracellular pathogens: they are entirely dependent on intracellular resources and cannot reproduce outside the host cells (Leon-Sicairos N et al., 2015) E.g. Viruses, bacteria including Chlamydia and Rickettsia 3- Persistent pathogens: the host cells are invaded when the pathogen gain an advantage over the host cell. These pathogens do not have to counter the humoral response of the immune system and can only be countered with cellular immune response. While inside the cell these pathogens overcome the adverse conditions within the cells when they encounter lysosomal enzymes (Newton HJ et al., 2010). E.g. Legionella pneumophila.

Maulana Azad Library, Aligarh Muslim University

Figure 1.4 Development of T helper cell subsets. Naive Th cells under the influence of different cytokines can differentiate to various types of effector cells including Th1, Th2, Th17. T-bet STAT-1,and STAT-4 are involved in the development of th1while GATA3,STAT5 AND STAT6 are involved in Th2 development. Th17 cells are highly heterogeneous and produce various types of other cytokines in addition to IL-17, including the Th1 and Th2 marker cytokines IFN-γ and IL-4.

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The knowledge of these strategies employed by the pathogens for evading the host immune system has helped us to deal with them in a more efficient way and for the development of therapeutics and more impactful vaccines. Moreover, it is of prime importance that knowing the interactions that pathogens have with the host for their survival leads to mortality because of the diseases. In the current review, we try to focus on the mechanisms induced by the host for protection against these pathogenic infections along with the strategies that pathogen employs to overcome these challenges.

1.3.1 Defense mechanisms of Host against pathogenic Infections

The astonishingly complex Immune system consists of diverse integration of cells to regulate the two arms of immune system i.e. innate and adaptive (Nicholson LB et al., 2016). When a cell is infected by intracellular pathogens it alters the nature of the host cells, changes the integration of immune cells leading to changes in the protection provided by these cells to the host (Nicholson LB et al., 2016; Chattopadhyay PK et al., 2018). Both innate and the adaptive system are adaptively interlinked and the innate system regulates or activates adaptive responses in a controlled fashion (Alexander KL et al., 2014).

The first line of defense against the pathogens in the host is constituted by the innate immune system. The innate responses are non-specific and cover a broad range of pathogens (John CM et al., 2017). The pathogens carry conserved repetitive sequences or molecules which are known as pathogen associated molecular patterns (PAMPs) (Zipfel C et al., 2009). These PAMPs are recognized through conserved germline encoded pattern recognition receptors (PRRs) (Suresh R et al., 2013). Damage-associatedMaulana molecular Azad patterns Library, (DAMPs) Aligarh present Muslim in theUniversity host are also recognized by the PRRs (Roh JS et al., 2018). PRRs includes the following categories: (1) Toll-like receptors(TLR)( Takeda K et al., 2005), (2) nucleotide- binding oligomerization domain(NOD) (Inohara N et al., 2003),(3) C-type lectin-like receptors(CLRs) (Geijtenbeek TB et al., 2009), (4) Retinoic acid-inducible gene(RIGs) ( Binder M et al., 2011) and recently found Absent in melanoma (AIM) like receptors(ALRs) ( Li Y et al., 2017). Immune system employs complement proteins, immune phagocytotic cells i.e. monocytes, macrophages and neutrophils along with natural killer cells to counter the infections (Storey M et al., 2008). These

Review of literature cells act as the initial step for countering the pathogens and they do not induce any immunological memory for encountering the same or similar pathogen in the future (Sánchez-Ramón S et al., 2018).

The adaptive immunity arm of the immune system comprises of two mechanistic arms: 1) cell mediated 2) and humoral (Chaplin DD et al., 2010). Adaptive immune system shows extreme specificity to the target antigens. The main feature of the adaptive immune system are the effector systems based mainly on the antigen specific receptors as a result of receptor rearrangement mechanisms that leads to expression of receptors on the surface of B- and T- lymphocytes (Bonilla et al., 2010). Such rearrangement of germ line encoded elements leads to formation of millions of varying receptors each having the potential to recognize different antigen (Jackson KJ et al., 2013). These rearrangement mechanisms include the somatic hypermutation to form the B cell receptors and V(D)J recombination occurring in both T cell and B cell receptors (Alberts B et al., 2007). Unlike the innate arm of the immune system the adaptive immune system creates immunological memory and also regulate the immune homeostasis (Cooper MD et al., 2006). These trained cells create a repertoire of memory cells and lead to enhanced and quick response upon encountering the same class of pathogen for a second time (Mayer A et al., 2019). Upon secondary infection more inflammatory mediators are produced and mechanisms independent of T and B cells responses have increased potential of protection (Cronkite DA et al., 2018).

However recent developments in the ever-growing complexity of the immune system has revealed that innate immune cells can also from memory from previous encounters to pathogens or antigens.

Cell mediatedMaulana immunity Azad Library, Aligarh Muslim University

Cell mediated immunity depends on the various T cells which respond to the antigens presented by the antigen protein complex through MHC class I molecules on the infected cells (Vivier E et al., 2011). All immune responses are coordinated by the two major group of lymphocytes i.e. lymphocytes which mature in the thymus and B lymphocytes which mature in the secondary lymphoid tissues. The path these cells follow for development and activation are almost similar in nature but the effect that they show when there is a pathogen attack is vastly different. On activation, T cells differentiate into different subsets and carry effector functions (Zhu et al., 2011).

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Intracellular processing of pathogens

The recognition and internalization of various pathogens by professional phagocytes (macrophages, neutrophils or dendritic cells) recognized through PRRs or through opsonization by antibodies leads to a cascade of events that leads to formation of phagosomes (Joller N et al., 2011). This formation of phagosome further leads to changes in acidity and influx of more proteases, GTPases and more acid hydrolases transforming the phagosome from early phagosome to highly acidic formation of late phagolysosome formation. The killing of pathogens inside the phagolysosome is mainly due to the acidic conditions present within the phagosome along with production of reactive oxygen species and also nitrogen intermediates (Joller N et al., 2011). This degradation of pathogens by lysosomes slices microbial proteins into antigenic peptides which can be presented on the APC in association with MHC II subsequently leading to CD4 activation.

Antimicrobial proteins are also produced such as lactoferrin which hinder in the iron metabolism. Various ions such as Fe2+, Zn2+, and Mn2+ from the phagosome lumen associated with the membrane proteins act as bactericidal agents when extruded under the influence of resistance-associated macrophage protein 1 (Flannagan RS et al., 2009).

Proinflammatory Cytokines

The main Proinflammatory Cytokines that play a crucial role in the host response against the pathogens involve:

1. IFN-γ: IFN-γ is one of the most important cytokines in cases of intracellular infections Maulana which primarily Azad coordinates Library, Aligarh distinct cellular Muslim events University and signalling cascades which result in increased immune surveillance and enhanced immune functions. The shifting from innate response to adaptive response is also coordinated through IFN-γ by enhancing production of IL-12 and IL-18 production and enhancing Th1-type response. The expressions levels of MHC molecules (both MHC I and MHC II) are upregulated through IFN-γ which in turn promotes induction of cell-mediated immunity and activation of Th1 cells (Hamza T et al., 2010).

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2. TNF-α: Macrophages are also activated through TNF-α and the strategies used by them for killing of pathogens which include inducible nitric oxide synthase (iNOS), ROS, and autophagy. In case of TB TNF-α plays an important role in the formation of granuloma and therefore containing the disease. Various studies involving M.tb, L. monocytogenes and using KO mouse models lacking the TNF- α receptor has demonstrated the importance of this cytokine in bacterial infections as well as in case of viruses such as HIV and HSV (Jamaati H et al., 2008). 3. Other interleukins that are involved include (IL-1α, IL-2, IL-6, IL-8, IL-12, and IL-18): During intracellular infections such as Mycobacterium, leishmania, Listeria, and viral infection like HIV cytokines IL-1α, IL-6, and IL-8 play an important role the activation of macrophage. The induction of IFN-γ from NK cells is initiated by IL-1α mediated through IL-12 during intracellular infections. The stimulation of cytotoxic T cells development into immunological memory cells depends on the cytokine IL-2 during intracellular infections (Perera PY et al., 2012). Regulatory T cells (Tregs) which has an important role in correct T cell responses and hosts immune response to microbes, depends on IL-2 which plays an important role in the regulating the maturation of Tregs, a temporary reduction in Tregs is seen in case of IL-2 unavailability. Both the cytokines IL-12 and IL-18 regulates IFN-γ production which acts as bridge between innate and adaptive immunity. The maturation and release of IL-18 is prompted through caspase-1 which itself is activated through a multiprotein oligomer known as inflammasome. IFN-γ levels are further increased from the immune T cells, NK cells and macrophages by the action of cytokines IL-12 and IL-18. These cytokines are activated through the inflammatory caspases which are produced through the inflammasomes (Perera PY et al., 2012). Maulana Azad Library, Aligarh Muslim University The main stay of the Humoral arm of the immune system are antibodies produced by B cells matured in to plasma cells. Both the antibodies and the B cells impact the immune either as response or in case of protection against many intracellular pathogens (Maglione PJ et al., 2009). The main difference between the cell-mediated immunity and the humoral immune response is that antibodies can block the antigenic target of the t cells by either binding on them or through opsonization (Achkar JM et al., 2015).

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Figure 1.5 Network of cellular development and cytokine expression on pathogenic infection through macrophages. Expression of cytokines on pathogenic invasion on macrophages leads release of various cytokines which are involved in promoting Th1 response as well as suppressing Th2 response.

IgG, IgA, and IgE, are produced from the B cells undergoing class switching and affinity maturation in the germinal centers and help in neutralization, opsonization, and complement activation (Schroeder Jr HW et al., 2010).It was previously thought that Ig’s cannot enter into the cells and therefore do not take part in eliminating intracellular bacterial infections. However, in case of listeria infection it was noticed that anti-listeriolysinMaulana O antibody Azad hasLibrary, potential Aligarh to neutralize Muslim listeriolysin University toxin hence protecting host from infection (Seveau S et al., 2014). Along with the antigen specificity that antibodies show there is another important function that are mediated through different Fc variable functions which have given rise to both pro- and anti- inflammatory cytokine production which in turn increase the microbial clearance by activation of complement (Ricklin D et al., 2010). These properties of antibodies arise from their binding to stimulatory and inhibitory Fc receptors on the cells. In this respect , FcRs are thought to be main player in protective responses against intracellular pathogens which are mainly manifested through oxidative burst, antibody

Review of literature dependent cellular cytotoxicity(ADCC) for infections with M. tuberculosis, L. monocytogenes (Luukinen H et al., 2018). In case of Ehrlichia muris infection, humoral immune response independent of T-cells has been demonstrated (Bitsaktsis C et al., 2007). In chronic intracellular infections, the same antibody may be proinflammatory or anti-inflammatory depending on the host and the stage of infection. It thus appears that the protection mediated by antibodies cannot be defined solely by molecular structure and glycosylation of antibodies but also depends on components of host as well as the pathogen and the stage of infection (Muñoz-Carrillo JL et al., 2018).

1.3.2 Mechanisms used by pathogens to overcome host immune responses

Despite the presence of a robust immune system, many intracellular pathogens can cause infection in the host. Sometimes the infection can be asymptomatic i.e., pathogen can remain dormant but when favorable conditions arise it can be reactivated, thereby causing a risk for the host (Kaufmann SH et al., 2012). Persistent infection can be of two types- (a) When the pathogens are not completely removed from the host, but the adaptive immune system keeps a check on them e.g. M. tuberculosis and S. enterica (Belon C et al., 2105) (b) When the pathogens resides in the host but do not induce adaptive immune responses, instead they establish infection when the host is immunocompromised e.g., Neisseria (F Bhavsar AP et al., 2007). Thus, there is always an intimate crosstalk between the host and the pathogen and the complexity of persistent infections is determined by the balance between the immune response of host and the pathogen counter-defense. Microbial pathogens tend to subvert the immune response of host. General mechanisms of such response can be divided into two broad groups (a) evasion of immune recognition in host – this can be Maulana Azad Library, Aligarh Muslim University done by changing the microbial surfaces, antigenic variation, or secretion of immunomodulators, and (b) suppression and modulation of the immune response in host e.g. evasion of phagocytosis and complement system; inhibition of apoptosis; resistance to host effector mechanisms; or inducing inappropriate immune response such as immunosuppression and T-reg induction. Some of the key mechanisms, used by pathogens for prolonged survival are described below.

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Evasion of Host Immune Recognition

Surface Immunomodulation

First interaction between the pathogen and host occurs on the external surface of microbial pathogens. Many pathogens secrete immunomodulators from infected cells in order to avoid immune detection (Ribet D et al., 2015). Immunomodulators can be in the form of proteins and toxins, receptors and inhibitors, or their own surface molecules/ligands in a modified form (Gokhale et al., 2011). On the other hand, some viruses mimic the structure and function of host cell receptors in order to avoid detection e.g., herpes and poxviruses (encode more than 40 viral proteins, hijacking transmembrane G-protein coupled-receptor) (Crow MS et al., 2016).

Bacterial pathogens can alter TLR agonists (such as lipid A, flagella, and peptidoglycan) on their surfaces. Some can avoid processing of peptidoglycan-derived muropeptides and their detection by the cytosolic receptors, NOD1 and NOD2 proteins (Irazoki O et al., 2019).

Secretion of Immunomodulators

Persistent bacterial pathogens interfere with apoptosis of the host cells by delivering virulence factors such as toxins and effectors via a secretion system. Out of seven such secretion systems the most widely studied are: Type III (T3SS) (used by Chlamydia trachomatis and Salmonella typhimurium) (Dai W et al., 2012) and; Type IV secretion systems (T4SS) (used by Legionella and Brucella) (Voth DE et al., 2012).

M. tuberculosis uses a specialized Esx secretion systems (ESX-1, ESX-3, and ESX- Maulana Azad Library, Aligarh Muslim University 5), for delivering major T cell antigens (ESAT-6 and CFP-10) in host (Wong KW et al., 2017). Similarly, Ess system of Staphylococcus aureus (Ess allows it to persist, establish staphylococcal abscesses, and evade the host immune response) (Anderson M et al., 2017) and the Yuk/Yue system of Bacillus subtilis (Yuk/Yue mediates YukE protein secretion and is homologous to Ess proteins of S. aureus) have been identified (Huppert LA et al., 2014). Viral immunomodulators mimicking host molecules (cytokines, chemokine’s, interferons, and complement and inflammatory cascades) can be probable therapeutic targets.

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Antigen Variation

Antigenic variation is another classical method to avoid immune responses especially adaptive immune responses. Neisseria is the best example, which has three antigenically or phase-variable major surface determinants (Grohmann E et al., 2018) (a) Opacity (Opa) outer membrane proteins governing bacterial adhesion and uptake into host cells (Sadarangani M et al., 2011), (b) Lipooligosaccharide (LOS), present in the outer membrane, involved in host interactions and, (c) Type IV pilus (Tfp), involved in cellular adherence (Coureuil M et al., 2012). Upto 11 antigenically different Opa proteins and 12 recognized LOS immunotypes that are turned on and off independently and exhibit multiple combinations (Anderson MT et al., 2013). Tfp antigenic variation relies on a programmed homologous recombination system to express antigenically distinct peptide sequences. A protozoan parasite Trypanosoma brucei contains Variant surface glycoprotein (VSG) in the blood and tissues of its mammalian host, but during an infection, some parasites switch their VSG into a new and antigenically distinct variant, resulting in parasitemia in the infected host (Mugnier MR et al., 2016). RNA viruses show antigenic drift and shift to cause antigenic variation (e.g. HCV, HIV, and influenza virus). DNA viruses, (both single and double-stranded) can selectively escape from the host’s immune system by exhibiting mutations (Ernst JD et al., 2017).

1.3.3 Subverting of the host immune defense and hiding form the host

Pathogens have the capacity to interfere with most of the immune responses elicited by the host. One the microbes reach inside the phagocytes they can manage to manipulate the effector responses being shown by the host in numerous ways such as phagolysosomeMaulana escape Azad and Library, modifying Aligarh their surrounding Muslim into University favorable habitat. These are carried out generally in three mechanisms:

1) Escaping the phagosome: listeria and rickettsia use this strategy to inhibit further downstream activation of immune system. Listeria uses its degrading enzymes such as phospholipases, listeriolysin and ActA to breakdown the phagosome and escape into the cytosol and therefore is known as the escape specialist (Uribe- Querol E et al., 2017). 2) Phagosome lysosome fusion inhibition: various pathogens use this mechanism by preventing the fusion. Salmonella utilizes this in a very efficient manner. Type

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III Secretion Systems in salmonella known as Spi/Ssa exports SpiC protein into the cytoplasm that prevent the fusion of phagosomes to lysosomes. The other such organism that uses this mechanism efficiently in mycobacterium tuberculosis which uses different proteins such as kinases, SapM, ZmpA and lipoarabinomannan. They reduce phosphatidylinositol 3-phosphate from immature phagosomes and stop the formation of phagolysosomes (Foley SL et al., 2013). 3) Survival within the phagolysosomes: salmonella also has the capacity to reside within the phagolysosomes by using its Pleiotropic Two-Component Regulatory System known as PhoP-PhoQ which controls its virulence through the Mg2+ ion regulated transcription of phosphatases (Le Sage V. et al., 2009).

In case of viruses which prevent their lysis by phagocytes by preventing the iNOS induction. INOS production depends on the transcription factors NF-κB and STAT. Various viral proteins have been identified which inhibit the production of NF-κB. However, it is to be noted here that in certain cases the virus maintains a dynamic balance between NF-κB activation and suppression to prolong their stay within the cells during the latent phase e.g. HSV (Landskron G et al., 2014).

Modulation or Suppression of Host Immune Responses

Subversion of Innate Immune Receptors

The immune system uses the innate immune receptors PRR for the recognition of pathogens. for evasion these pathogens inhibit the signaling pathways of these receptors. RNA Viral pathogens which replicate within the cytoplasm are recognized thorough the cytoplasmic PRRs, RIG-1 and MDA5, viruses inhibit these for their establishment (Kell AM et al., 2015). While in case of DNA viruses which replicate Maulana Azad Library, Aligarh Muslim University within the nucleus or cytoplasm are detected by the factors IFI16 or cGAS (Chan YK et al., 2106). These viruses have evolved themselves to prevent their exposure to these receptors by keeping themselves inside the viral capsid e.g. HIV-1(Blood GA et al., 2016).

In cases of bacterial infection, it is seldom seen that they inhibit PRR signaling cascades. However, in case of Yersinia pestis it is seen that the antigen LcrV hijacks the TLR 2/6 pathway which stimulates IL-10(Grabowski B et al., 2017). In cases of other bacterial infections intracellular signaling cascades are the target. These involve

Review of literature the MAPK signaling pathways and NF-κB pathway. Other strategies used by the pathogens involve the mimicking of intermediates of signaling pathways operative within in the cells. In case of Helicobacter pylori which releases proteins which mimics immunoreceptor tyrosine-based inhibitory motif (ITIM) whose function is to suppress immune (Krachler AM et al., 2011). For development of vaccines these knowledge’s can be used in improving the host immunity against these infections.

Regulation of autophagy by cytokines

A wide range of intracellular bacteria can directly be eliminated by autophagy and it is through it that the endogenously expressed antigens get processed and presented via MHC I and MHC II molecules. Autophagy could be induced by so many cytokines, as well as TLR and NOD-like receptor (NLR) ligands. Thus, it can be said that autophagy has arisen as an efficient mechanism of the immune response to combat the pathogens (Harris J., 2011). The role of cytokines in regulating the autophagy can be understood from the action of macrophages against M. tuberculosis (Harris J et al., 2007). It has been established that the host immune response against M. tuberculosis is Th1 biased. When macrophages get activated in presence of IFN-ϒ, with or without LPS, they boost the maturation of mycobacteria-containing phagosomes. IFN-ϒ induces GTPase Irgm1 (formerly LRG-47, IRGM in humans) which plays a vital role in immune response against M. tuberculosis. It has been recently established that when human and murine macrophages get activated with IFN-ϒ in an Irgm1/IRGM- dependent manner, it induces autophagy (Singh SB et al., 2006). Macrophages infected with mycobacteria upon treatment with IFN-ϒ lead to increased killing of bacilli, intracellularly and uptake of mycobacteria containing phagosomes by autophagosomes which are finally destined to join the lysosomes (Parihar SP et al., Maulana Azad Library, Aligarh Muslim University 2013).

However, it still remains a puzzle that how IFN-γ-induced phagosome maturation could be a diagnostic for autophagy in M. tuberculosis-infected macrophages. Studies have shown that upon using the avirulent H37Ra strain of M. tuberculosis to infect macrophages, it could induce autophagy in murine macrophages (Freeman S et al., 2006). Reports show that the mycobacteria-containing phagosomes are LC3-negative. Moreover, the co-localization of BCG with LC3 is seen upon the activation of macrophages with IFN-ϒ (Gutierrez MG et al., 2004). When Beclin 1 was silenced in

Review of literature murine macrophages using siRNA, the effect of IFN-γ on the maturation of BCG- containing phagosomes was withdrawn which directs us to the conclusion that it is an autophagy dependent process (Jagannath C et al., 2009).

IFNγ-augmented phagosome maturation got nullified by the TNF blockers like infliximab and adalimumab (monoclonal antibodies against TNF-a) and etanercept (soluble TNF receptor) in BCG or M. tuberculosis-infected human macrophages directing us to the conclusion that IFNγ-generated phagosome maturation relies on TNF-α (Bruns H et al., 2009). There are numerous studies that throw light on the importance of TNF-α in the activation of autophagy in various cells, including human T lymphoblastic leukemic cells, human vascular smooth cells, human and murine macrophages, and rat epithelial cells (Harris J., 2011). It has been found in murine macrophages that CD40 ligation together with TNF α signaling enhances autophagic expulsion of Toxoplasma gondii (Subauste CS., 2009). TNF α is efficient in upregulating the expression of autophagic genes i.e. LC3 and Beclin1 as has been studied in human atherosclerotic vascular smooth cells. This action involves the c-Jun N-terminal kinases (JNKs) pathway and the inhibition of Akt activation (O’Leary MF et al., 2009). In MCF-7 cell line, TNF-α induced autophagy relies on the ERK1/2 pathway, while in Ewing sarcoma cells, NF-κβ activation has inhibitory effect on TNF-α-induced autophagy which is dependent on the generation of reactive oxygen species (Harris J et al., 2009). Studies on murine model intestinal epithelial cells have shown TNF α to be responsible for mitochondrial dysfunction which further involves increased mitochondrial ROS decreased oxygen consumption and decreased mitochondrial membrane potential, which ultimately boosts autophagy of mitochondria (mitophagy) (Suematsu N et al., 2003). Maulana Azad Library, Aligarh Muslim University Th1 cytokines act as the activators of autophagy whereas Th2 cytokines (like IL-4 and IL-13) act as inhibitors to it (Harris J et al., 2011). It was found that on infection with M. tuberculosis, Th2 cytokines inhibited IFN-γ directed autophagosome formation which resulted in decreased phagosome maturation and increased survival of the intracellular bacteria (Weiss G et al., 2015). Akt signaling and signaling through STAT 6 are responsible for this. In case of HT-29 human epithelial cells, it was found that IL 13 could potently inhibit starvation induced autophagy and this is dependent on the Akt pathway. Thus, we can conclude that in case of M. tuberculosis infection, autophagy is an effector of Th1/Th2 polarization (Harris J et al., 2011). It was

Review of literature reported that in murine macrophages, IL-10 was found to inhibit starvation-induced autophagy via the Akt pathway and rapamycin-induced autophagy via both Akt and STAT3 pathways. IL 10 also inhibits LPS-induced autophagy but, the signaling pathway is yet to be discovered. Thus, IL 10 has a great contribution in the regulation of autophagy (Harris J et al., 2011).

In some carcinomas, TGF β has shown growth inhibitory effects and could potently induce autophagosome formation and boosted autophagy in MDA-MB-231 mammary carcinoma cells and enhanced expression of Beclin-1, Atg5 and Atg7 mRNA in human hepatoma cells (Kiyono K et al., 2009). In addition to it, the CC chemokine CCL2 (monocyte chemoattractant protein-1) and IL-6 both have the tendency to activate autophagy and induce antiapoptotic proteins in human macrophages (Kung HJ et al., 2011).

Maulana Azad Library, Aligarh Muslim University

Figure 1.6 Generalized response of the immune system upon encountering varied form of pathogens and the mechanisms used by the pathogen to alter immune responses: Involvement of TLR, NLR, RLR upon encountering pathogens and the subsequent downstream activation of transcription factors for the counter attack on the pathogens.

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Evasion of Autophagy

During the process of autophagy, which is a trafficking pathway, the lysosomes are delivered with certain cytoplasmic components for degradation and sequestering. It is one of the main components which plays a vital role in the intracellular surveillance of products that are to be degraded or recycled within the cytoplasmic system along with any foreign pathogens which have gained entry within the cytoplasm. However, certain bacteria and viruses have developed systems during the process of evolution that can subvert such responses for persisting within the cells. In case of L. monocytogenes certain proteins such as ActA and internalin K help in evading the autophagic recognition (Cossart P et al., 2011). In case of viruses’ selective autophagy takes place in cases of HSV-1 and influenza A virus as proteins TRIM regulate autophagy through the TRIM23-TBK1-p62 axis (Thakur A et al., 2019). While in cases of RNA viruses which replicate within the nucleus proteins such as Tat, Vpu and Nef are used to inhibit the maturation of autophagosomes, HIV (Nardacci R et al., 2017).

Inhibition of Complement Proteins

The complement system has a vital status in recognizing bacteria, opsonizing them so that phagocytes can engulf them. The complement system can also directly kill the bacteria through their proteins. However, pathogenic bacteria have evolved strategies to evade them and cannot be killed by the immune system. Certain bacteria target the classical or lectin pathway of complementation and secrete proteins that bind to C4BP of the complement pathway. Binding of these proteins prevents the cofactor function in the cleavage of C3b/C4b thus rendering the complement inactivated Neisseria uses two kinds of porinMaulana molecules, Azad Por1A Library, and Por1B, Aligarh that binds Muslim complement University component C4BP (Avirutnan P et al., 2011). Certain viruses such as HIV, and human lymphoma virus type I incorporate have evolved to such an extent that their envelope contains complement inhibitor proteins DAF, MCP, and CD59 while others contain homologues of complement inhibitors (Agrawal P et al., 2017).

Inhibition of Cytokines and Chemokines

Another strategy to escape host immune response in viral infection is by altering the cytokines production. The cytokines and chemokine’s involved in the immune

Review of literature response include type I and II interferons, TNFs, and IL-1, and chemokine’s. Certain viruses such as herpes and poxviruses have evolved to produce surface proteins that mimics cytokine and cytokine receptors. While others produce their own analogue chemokine’s or receptors that can modulate the whole network of chemokine’s (Christiaansen A et al., 2015).

Inhibition of Adaptive Immune Responses

Adaptive immune responses play a crucial role in clearance of bacterial and viral infections. However, Survival mechanism have been developed by tenacious pathogens to offset adaptive immune response. Expression of viral proteins can block either NK-cell receptor function, cytokine release or MHC-I homologs thereby counteracting the first line of cellular defense in viral infections i.e. NK-cells. Host mediated CD 4+ T cell control is forfeited by infected cells (in case of M. tuberculosis) by export of antigen for uptake and presentation by uninfected bystander cells, thereby bringing a reduction in MHC class II antigen presentation by infected cells. Secretion that degrade immunoglobulins by the secretion of IgA protease in case of N. meningitidis has been documented also (De Pelsmaeker S et al., 2018). In HIV, HSV, HPV, HCMV, and adenovirus workers have reported viral interference by proteasome cleavage, translocation through the transporters connected with antigen processing, and presentation through MHC class I as well as MHC class II (McCarthy MK et al., 2015). Host mediated antigen specific immune response can be restricted by exploiting immune checkpoint inhibitors. E.g. S aureus evades immune activation by modulating PD-ligand 1. Blocking immune checkpoint can be an innovative strategy for managing chronic infections which at present lack effective therapies or vaccines (Goldmann O et al., 2018). Maulana Azad Library, Aligarh Muslim University Suppression of Cell Death

In order to evade immune system of the host, pathogens can cause cell death either by apoptosis, pyroptosis, necrosis/necroptosis, or NETosis. Viruses can use different antiapoptotic mechanisms like inhibition of multiple caspases and TNF induced apoptosis, inactivation of p53, and homologs of Bcl-2 to target cellular proteins (FLIP, caspase inhibitor, selenoproteins, ligands of the TNF family, Bcl-2, and p53) involved in the control of apoptosis(Nichols DB et al., 2017). Bacteria can produce toxins e.g. Listeria; secrete effector proteins and T3SS e.g. Salmonella and

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Yersinia; block proapoptotic proteins Bax and Bak or activate caspase-3 e.g. Chlamydia infection (Coelho C et al., 2017). M. tuberculosis infects macrophages and then induces necrosis (caspase-independent pathological cell death, triggering inflammation and resulting in extensive tissue damage) in order to avoid immune response (Man SM et al., 2017). A type of necrosis i.e. Necroptosis depends on activation of the necrosome (a protein complex in which receptor-interacting protein kinase 3 (RIPK3) is activated). Necroptosis is observed in case of Vaccinia, influenza, and HSV-1 (Dara L et al., 2018). Pyroptosis which is mediated by gasdermin is a highly inflammatory form of programmed cell death. It requires the activation of caspase-1 in inflammasomes. Pyroptotic death of macrophages and dendritic cells is one of the key mechanisms for intracellular pathogens survival in host cell (Bergsbaken T et al., 2009).

1.4 Pathogenesis of Brucellosis

Host–Microbial Interactions

Smooth Brucellae gain entry into the host cells following interaction with cell surface micro domains known as lipid rafts. These lipid rafts contain significant amounts of glycosyl-phosphatidyl-inositol anchored proteins, glyco-sphingolipids and cholesterol and are believed to play a significant role not only for internalization, but also for intracellular replication of Brucellae. Investigation of the cellular interactions following internalization within macrophages has demonstrated that Brucellae reside within the acidified phagosome. Indeed, the acidification of this environment is an important stimulus for expression of virulence genes of the virB operon, essential for expression of components needed for assembly of a type IV secretion system required for intracellulaMaulanar trafficking. Azad Library, Aligarh Muslim University

Once inside the host cell phagosome, and after being subjected to the initial oxidative burst, the internalized Brucella commence their subversion of the host phagosome, termed as “brucellosome” through mechanisms as yet not fully elucidated. What is known is that the products of virB operon interact with the endoplasmic reticulum in a way that the pH becomes neutral, nitrate ions may be utilized for anaerobic respiration and the Brucellae undergo regulated multiplication

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Interaction with placental trophoblasts suggests that the ability to acquire iron is vital as the Brucellae enter their acute replicative stage with placental disruption resulting in fetal loss or birth of weak and infected offspring. It is interesting to note that iron acquisition is altered in the attenuated vaccine strain of B. melitensis, Rev1 when compared with the virulent strain. 16M Erythritol is believed to be important for determining tissue tropism for B. abortus. Indeed, Brucella uses this sugar alcohol in preference over alternatives and mutants unable to utilize erythritol are severely attenuated in ruminant hosts. Considerable literature suggests that Brucella metabolizing erythritol have a heightened requirement for iron, scavenged through siderophores such as 2, 3-dihydroxybenzoic acid or brucebactin. This may be linked with the requirement for effective iron acquisition for virulence in ruminant hosts.

Molecular Mechanisms of Brucella Pathogenesis

The availability of the genomic sequences of B. melitensis, B. suis and B. abortus have provided an opportunity to identify the virulence mechanisms of Brucella at the molecular level that have previously proven highly elusive. The recent release of the Brucella genome sequences verified the absence of “classical” virulence factors, as well as virulence or pathogenicity islands commonly found in other intracellular pathogens (Foulongne V et al., 2000; Paulsen et al., 2005; He Y et al., 2012). On the other hand, some potential sequences for virulence involved in adhesion, invasion, immune evasion, adaptation and intracellular trafficking during the course of Brucella infection were discovered.

To be a successful infectious agent, Brucella requires four steps, like other pathogenic intracellular bacteria: adherence, invasion, establishment and dissemination within the host (KoMaulana J et al., Azad2003).The Library, bacterial Aligarh factors andMuslim cellular University receptors implicated in attachment and entry of B. abortus into host cells have not yet been fully identified. Thus, the internalization process involve widespread ligand(s) since B. abortus is able to invade a broad range of mammalian cell lines, including the murine fibroblasts NIH3T3, the green monkey kidney vero cells, the human epitheloid cell line Hela and Madin Darby Bovine Kidney (MDBK) cells (Detilleux et al., 1990; Pizarro-Cerda et al., 1998). It was observed that opsonized Brucella internalized in macrophages and monocytes via complement and Fc receptors, whereas found non-opsonized Brucella exploits lectin or fibronectin receptors, in addition to other unknown receptors to

Review of literature penetrate mammary gland macrophage (Harmon et al., 1988; Campbell et al., 1994). In HeLa cells, it seems that Brucella is ingested through a mechanism, which involves a discrete recruitment of actin filaments at the binding site (Guzman-Verri et al., 2001).

Figure 1.7 Overview intracellular life cycle of Brucella: The life cycle of Brucella within host cells. During infection, Brucella first invade the host cells (1), form Brucella-containing vacuoles (BCVs) (2), and undergo fusion with the lysosome in a controlled manner (3). In this step, about 90% of the Brucella are degraded, and the remaining 10% survive (4). Then, the BCV traffic to and reach the endoplasmic reticulum (ER)Maulana (5), and establishAzad Library, the replicative Aligarh site ( 6Muslim). After ERUniversity replication, the Brucella traffic toward the autophagy-like vacuoles (7), survive within these compartments (8), and finally, leave the host cells to promote cell-to-cell spreading (9). The following molecular events involved in these steps are potentially targeted by T4SS: excluding markers of the late endosome or lysosome, acquiring ER markers, interacting with secretory pathways, acquiring markers for autophagosomes, resisting the harsh intracellular environment, and regulating the activation of vital immune pathways.

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Brucella genome analysis has implicated a variety of genes in the course of attachment and uptake of Brucella in mammalian cells. Three proteins containing a domain homologous to auto transporters, such as the Salmonella typhimurium fibronectin binding protein hsdA, were identified that may serve as putative adhesions mediating attachment before phagocytosis. Additional putative adhesins related to the Bartonella Pap31/HbpA proteins and homologues of the Bartonella bacilliformis invasion associated proteins IalA and IalB are present in Brucella (Paulsen et al., 2002). However, future study is necessary before giving any conclusive remarks about the relationship between newly identified gene and entry of Brucella into the host cell.

The chronic nature of brucellosis is likely to be multifactorial, utilizing both the ability of Brucella to evade immune detection and adaptation to intracellular survival inside both phagocytic and non-phagocytic cells. This strategy involves the inhibition of the phagosomal-lysosomal maturation pathway and the deviation of intracellular trafficking, which allows the bacterium to reach its endoplasmic reticulum-derived replicative niche (Starr T et al., 2012).

Canning et al. (1986) suggested that an important determinant of Brucella virulence is the production of adenine and guanine monophosphate, which inhibit phagolysosome fusion, degranulation and activation of the myeloperoxidase-halide system and production of tumor necrosis factor. The production of these inhibitors is prevented in pure mutants, which are substantially attenuated in consequence. Lower biological activity of Brucella lipopolysaccharides (LPS) probably plays a substantial role in intracellular survival.

B. abortusMaulana has twoAzad component Library, regulatory Aligarh systems Muslim consisting University of the Brucella virulence-related regulatory (BvrR) and sensory (BvrS) proteins which are highly similar to the two component regulatory systems ChvG-ChvI of A. tumefaciens and equivalent to Salmonella PhoP-PhoQ and Bordetella bronchoseptica BvgA-BvgS systems. Insertional bvrR or bvrS mutants showed reduced multiplication and minimal persistence in mouse spleen in vivo and displayed lower level of invasiveness and replication in macrophages and HeLA cells in vitro, suggesting the inability to inhibit lysosome fusion and defect in intracellular trafficking of brucella containing phagosome (Sola-Landa et al., 1998).

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The most recent significant breakthrough in Brucella pathogenesis research is the application of signature tagged mutagenesis (STM), where mutants were tested in phagocytic and non-phagocytic cells (Foulongne et al., 2000) as well as acute and chronic (Lestrate et al., 2000) stages of infection in mice. Many Brucella attenuated mutants have been identified and are being characterized individually. B. abortus cydB transposon mutants, attenuated in vitro and in vivo, are defective in cytochrome bd oxidase, a high-oxygen-affinity terminal oxidase used by Escherichia coli under microaerobic condition (Endley et al., 2001). Compared to that of wild-type, Brucella cyd mutants are susceptible to reactive oxygen intermediates (ROIs) in macrophages. However, the specific mechanisms of ROI susceptibility in cydB mutants have not been investigated.

1.4.1 Epidemiology of Brucellosis

Knowledge of the genome of Brucella spp. helped to simplify many experimental approaches to investigative techniques. A variety of predicted virulence factors were identified whose experimental characterization may enhance our understanding of Brucella pathogenesis. Finally, the response of B. abortus to its host environments, featuring low pH and nitrous oxide, as well as its ability to evade the host immune response, will provide a major challenge for future studies. The term brucellosis is applied to a group of closely related infectious diseases, all caused by Gram-negative bacterial pathogens in the genus Brucella. Manifestation of the disease may range from abortion in the cow and to orchitis or epididymitis in the bull. Characteristically, all Brucella species establish persistent infection in the reticuloendothelial system of the natural host species. Plans to eliminate ovine, caprine and bovine brucellosis by the European Union were expected to receive over half of the total European Maulana Azad Library, Aligarh Muslim University Commission funding for animal diseases control measures in 1997 implies the importance of this disease.

Zoonotic Importance of Brucellosis

Human brucellosis is a notifiable disease in many countries, but real incidence has been estimated to be between 10 and 25 times higher than the reported figures due to inaccuracy of official figures, which do not fully reflect the number of people infected each year. Because of inaccurate diagnosis, cases very often remain unrecognized and are thus treated as other diseases or as "fever of unknown origin". Animal brucellosis

Review of literature also possess a barrier to trade of animals and animal products and could seriously impair socio-economic development of livestock owners the most vulnerable fraction of many rural populations. The most frequent symptoms of human brucellosis are fever, chills or shaking rigors, malaise, generalized aches and pains all over the body, joint and low back pain, anorexia, easy tiredness and general weakness (Balagnaur AS et al., 2007; Dossey BM, et al., 1998; Madkour, 1989). Man almost always receives the infection from infected animal; transmission from man to man rarely occurs (Stolte M, 1994) (Figure 1.8). Brucellosis in humans and animals is increasing in certain parts of the world, especially in developing areas of the Mediterranean region, Middle East, Western Asia and parts of Africa and Latin America. In Mediterranean and Middle East countries the annual incidence of brucellosis in people varies from less than 1 up to 78 cases per 100 000; however, over 550 cases have been reported from confined endemic areas where no animal control measures are applied. Up to 77 cases per 100 000 people have been reported from certain communities of south European countries in which animal control measures are mandatory.

From a recent survey in a randomly selected human population of a country of the Arabic Peninsula, serological evidence of exposure to Brucella has been found to be close to 20%, with more than 2% of these having active disease. Similar figures may be expected from most countries in which the disease is endemic in the animal population. Higher seroprevalence of brucellosis should also be expected in occupationally exposed groups.

1.4.2 Virulence Factors of Brucella

Identification and characterization of antigens that modulate host immune system is primaryMaulana due to the Azad development Library, of Aligarh a diagnostic Muslim test or University a vaccine. A substantial number of antigenic components of Brucella have been characterized. As is the case for other bacterial pathogens, several cellular components contribute to the survival and virulence of the Brucella. Research concerning the virulence factors expressed by the Brucellae has focused primarily on the structural components of the outer membrane. Components are included as virulence factors on the basis of the following classification (Gray GL et al., 1984):

 Components that when inactivated by mutation markedly decrease virulence, 37

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 Components that mimic pathological effects when administered in purified form  Components that elicit a partially protective immune response.

Figure 1.8 Different sources of Brucella infection in human

In contrast to other Gram-negative pathogens, the outer surface of the Brucella does not have complex structures, such as pilli or fimbriae, nor does it have capsular material. The outer membrane contains only two components that have been identified as virulence factors: the lipopolysaccharide (LPS) and the outer membrane proteins (OMPs) (Razzaq MS et al., 2014). Maulana Azad Library, Aligarh Muslim University Surface antigens

Lipopolysaccharides (LPSs)

The LPS was founded to be a major immunodominant antigen of Brucella (Nielson et al., 1988; Berman et al., 1992). Of the potential virulence factors expressed by the Brucellae, the LPSs are the best defined (Moreno CA et al., 1984). Virulence is associated with the smooth colony morphotype, designated S, which is known to contain the full complement of Brucella LPS. The rough colony morphotype, designated R, arises by dissociation from the smooth form and its accumulation is

Review of literature typically observed in vitro. Dissociation is stimulated by changes in environmental conditions, including different nitrogen sources or exposure to D-alanine (Goodlow et al., 1952). The R morphotypes are characterized by their opaque appearance and lusterless colony morphology (Bea JE, et al., 1999). In general, for most Brucella spp, smooth strains are virulent and rough and mucoid strains are not. The LPS of smooth strains consists of lipid A, a core region containing glucose, mannose, quinovosamine and 2-keto-3-deoxyoctulosonic acid (KDO) and an O chain composed of a homopolymer of about 100 residues of N-formylated perosamine (Caroff et al., 1984).

Differences in uptake of smooth and rough organisms have been demonstrated in experiments using guinea pig and bovine monocytes. LPS plays a role in enhancing intracellular survival. Experiments performed by Frost, (1972) indicate that in vivo grown, smooth organisms may have an additional membrane component that increases survival and virulence. The mouse model system has been investigated extensively with respect to the protective immunity induced by antibodies directed against LPS. The protective immunity elicited by LPS in the cow has not been clearly established. Winter et al. (1988) have demonstrated effective protection in the mouse model using a combination of O-polysaccharide covalently linked to Brucella porin. Under identical conditions, porin linked to R-LPS did not stimulate a significant level of protection. Montaraz et al. (1986) were able to demonstrate effective immunization by passive transfer of monoclonal antibodies directed against the O-antigen, while monoclonal antibodies directed against a number of outer membrane proteins induced no significant protection.

Outer membrane proteins (OMPs)

The majorMaulana fraction Azad of cell Library, wall associated Aligarh protein Muslim antigens University have been shown to be outer membrane proteins (OMPs). The major OMPs of Brucella spp. were initially identified in the early 1980s (Verstreate and Winter, 1984) and characterized as potential immunogenic and protective antigens. The OMPs have been classified as virulence factors according to category 3 described by Gallart Palau XR (2016). They were classified according to their apparent molecular mass as 36-38 kDa OMPs or group 2 porin proteins and 31-34 and 25-27 kDa OMPs that belong to group 3 proteins. Little variation among species has been observed in the OMP profiles (Davies RL et al., 2003). All three groups of proteins appear to be recognized by the

Review of literature bovine immune system during the course of infection. Protective immunity stimulated by OMPs has been documented for several Gram-negative organisms (Yang G, 1984; Isibasi et al., 1988). Protection stimulated by Brucella OMPs is still a matter of debate, and despite considerable experimental effort, is far from resolved. The doses employed have either been insufficient to produce an immune response or presentation of the antigen in the killed vaccines does not mimic the situation obtained with a living vaccine strain.

In vivo antigens

Evidences that Brucella cells grown in vivo exhibited differences from the cells grown in culture media has been has been presented. Proteins related from acquisitions are most common ones, heat shock proteins, chaperones, analogs to groEL, Hsp62 (Lin and Ficht et al., 1995) and Htr protein (Tatum et al., 1992).

Intracellular antigens

Various immunogenic antigens have been reported from time to time in this organism. They are Cu-Zn Superoxide dis-mutase, a 30 kDa protein, BA 14K, an 18 kDa cytoplasmic protein, glyceraldehydes-3-phosphate-dehydrogenase, B. melitensis ribosome recycling factor (RRF)-homologous protein (CP24) (Chirhart-Gilleland et al., 1998; Goldbaum et al., 2000; Cassataro et al., 2002;). Denoel et al., (1997) purified a 39-kDa protein from Brucellergen by anion exchange chromatography on mono Q sepharose fast flow column HR5/5. The antigen was immunoreactive and did not cross react with any other bacteria.

Cytosolic antigens Maulana Azad Library, Aligarh Muslim University The cell surface antigens predominantly stimulating the humoral arm of immune response. Identification and characterization of cytosolic antigens which are usually processed and presented via MHC to CD4+ and CD8+ T cells are important in any effort to develop a prophylactic agent against Brucellosis. Mallick AI (2007) separated SDS extracted B. abortus S-19 antigens to several bands ranging from molecular weights of 6 kDa to 87 kDa by two dimensional immunoblot. They also partially sequenced n-terminus of a12 kDa protein which gave maximum T cell proliferation. Bhongbhibhat et al. (1970) prepared an antigen, Brucellein, by cold hypertonic saline (2.7%) precipitation. Mallick AI (2007) showed Brucellein

Review of literature contained significant amount of nucleic acids material along with this protein. Bachrach et al. (1994) identified and sequenced 12 kDa protein from Brucellein and found that the sequence matches with the 12 kDa proteins as reported by Oliveira SC et al., (1994). Later the same group identified the 12 kDa protein as L7/L12 50S ribosomal protein and reported the nucleotide sequence of L7/L12 gene in B. melitensis (Bachrach et al., 1994). Oliveria and Splitter (1994) showed that E. coli expressed L7/L12 protein protected mice against B. abortus infection. Kurar E and Splitter GA. (1997) concluded that L7/L12 DNA vaccine construct elicit immune responses in mice against B. abortus. The B. abortus L7/L12 protein was also expressed in Lactobacillus lacti for the development of foods grade vaccine (Ribeiro et al., 2002).

Cytosolic L7/L12 ribosomal protein

The rp1JL operon encodes the L10 and L7/L12 proteins, essential for ribosomal function and protein synthesis (Oliveria et al., 1994). The rp1JL-rpoB operon (1-1099 bases) encodes 4 proteins. The first open reading frame (1-129 bases) encodes rp1J gene. The gene product is ribosomal protein L10. Bases 280-654 encodes rp1L gene. The product of this gene is the L7/L12 ribosomal protein. The bases 900-1009 encodes rpoB gene. The protein product of this gene is RNA polymerase beta subunit.

Structure and function of the L7/L12 ribosomal protein

Recent crystallography study of the L7/L12 protein have been led to the unraveling of the structure and the organization of the protein within the ribosome (Sanyal et al., 2002) the L7/L12 protein is associated with the large subunit of the ribosome. L12 protein is associated with the 50S ribosomal subunit whereas L7 is an acetylated form Maulana Azad Library, Aligarh Muslim University of the L12 protein. This acetylation occurs in the N-Terminus of the L12, which become L7. Both L7 and L12 protein form dimmers with each other. Four molecules of this acidic protein are present in each ribosome and they exist as two dimmers. Being the only protein present in each ribosome in more than one copy, L7/L12 may become very abundant in multiplying bacteria. Translation elongation is a dynamic process in which mRNA and tRNA move successfully on the ribosome .It is stimulated by the action of two elongation factor EF-Tu and EF-G at a common site of the ribosome termed as “GTPase center” and by coupled GTP hydrolysis. Ribosomal protein L7/L12 is an important component of this functional center and

Review of literature constitutes a part of the so-called transnational engine as investigated mainly in Escherichia coli L7/L12 has unique properties whose functional significance is not clear yet i.e. there are 4 copies in the ribosome and these molecules are flexible in the 50S ribosome subunit. There are four copies (2 homodimers) of L7/L12 bind to anther protein L10 and form a stable complex L10.L7/L12. This complex then binds to the highly conserved RNA domain around position 1070 of 23S rRNA. The speed and the error orate of the translation process depends on this protein the L7/L12 is required for maximal rate of the synthesis as well as for minimal missense error frequency (Pasqualetti P et al., 2009).

Toxins and other factors

To date, no concrete evidence exists that toxins similar to those produced by other organisms (i.e., Vibrio chloera, Corynebacterium diphtheriae, E. coli, Shigella dysenteriae, Clostridium botulinum, C. tetani) are produced by the Brucellae. A toxin has been postulated to exist due to the severe effects of the disease on the central and autonomic nervous systems. Endotoxin, consisting of LPS, has been suggested as the causative factor. The Brucellae also produce catalase. Gee JM et al., 2004) determined that catalase activities decrease with changes from smooth to rough morphology. Although the biochemical advantage of expressing catalase in Brucella has not been identified definitively, it may not only protect the organisms from peroxide, but could also be important in maintaining oxygen tension required for survival of the organism (McCullough et al., 1970).

1.4.3 Immunology and Protection against Brucella

Brucellosis frequently progresses towards a chronic state involving different organs. Maulana Azad Library, Aligarh Muslim University Once the organism has transgressed the mucous membranes or the skin, it tends to localize in the local lymph nodes and then a systemic dissemination occurs affecting organs of the mononuclear phagocytic system such as spleen and liver. Classic experiments by Mackaness (1964) and Mackaness GB. 1968 showed the role of cellular immunity and the fundamental part played by activated macrophages in resistance to, and in protection against, intracellular pathogens such as Mycobacterium sp., Salmonella sp., Listeria monocytogenes and Brucella sp.

Review of literature

Innate immunity provides first line of defence against non-self-pathogens in a non- specific manner. These are mediated by clonal selection of specific lymphocytes; these responses are rapid and independent of antigen. In case of Brucella infection, the utmost roles of the innate immune system are to reduce the initial number of infected bacteria without their memorization and also provide the condition for generating Th1 immune response in the host. Innate immune system work in coordination with their different components to control Brucella infection in the host. Complement system comprises of plasma proteins that interact with bound antibodies or bacterial surfaces to opsonize or directly kill Brucella by forming a membrane attack complex (MAC).

The bactericidal functions of macrophages comprise of production of reactive oxygen intermediates (ROIs) and reactive nitrogen intermediates (RNIs) that are induced by gamma interferon (IFN-γ) and tumour necrosis factor (TNF-). Jiang et al. (1993) reported the role of ROIs and RNIs in controlling the load of B. abortus in murine macrophage infection and concluded that ROIs play a seminal role than RNIs in Brucella killing in vitro. In contrast, Superoxide dismutase (SOD) and nitrate reductase (NarG) of Brucella detoxify ROIs and RNIs activity respectively and help brucella reside inside macrophages (Tatum et al., 1992; Kohler et al., 2003).

Murine susceptibility to Leishmania, Salmonella and Mycobacterium is regulated by natural resistance associated macrophage protein 1 (Nramp1) gene. Nramp1 endorse host with natural heritable resistance against intracellular pathogen. Barthel et al. (2001) transacted bovine Nramp1 gene into murine RAW264.7 macrophage cells and observed that bovine Nramp1 stable transfectants of RAW264.7 cells control B. abortus infection better than do parental cell lines in vitro, implying that natural Maulana Azad Library, Aligarh Muslim University heritable resistance plays a major role in bovine brucellosis.

Adaptive immunity

Adaptive immune responses are critical for providing the memory function that is the main player in vaccination. Cytokines play important role in providing protection against brucellosis. Functions of the adaptive immune response in brucellosis can be classified into three mechanisms. First, IFN-g produced by CD4+, CD8+ and gdT cells activates the bactericidal function in macrophage to hamper the intracellular survival of Brucella. Second, cytotoxicity of CD8+ and gdT-cells kills the infected 43

Review of literature macrophages. Third, Th1 type antibody isotypes such as IgG2a and IgG3 opsonize the pathogen to facilitate phagocytosis.

Humoral Immune Responses

Various constituents of Brucella cells are capable of inducing specific antibodies in their hosts (Ko J et al., 2003; Moreno et al., 2002). The classic reports include agglutinins, precipitins; complement fixing and blocking antibodies (Glenchur et al., 1963; Wilkinson et al., 1966). Several research groups have shown that in naturally acquired and experimental brucellosis there is an early marked increase of agglutinating antibodies in the serum. This IgM antibody may be detected one week after onset of infection and reaches a peak level four weeks later, at the same time when IgG antibody also rises to its peaks level. In contrast, IgG agglutinating globulin has appeared later. Although it is found mixed with IgM, the IgM agglutinin level always higher than IgG agglutinin level. A third category of agglutinating globulin, IgA appears later in a significant concentration in the serum. IgG agglutinating antibody has shorter half-life than IgM and its titre is much lower than IgM after one year of treatment. IgE levels of antibody to Brucella have not been studied extensively, but the work of Escande and Serre (1982) indicates that IgE levels may be significantly high in brucellosis patients and vaccinated subjects. According to available evidence, the IgE anti-Brucella antibodies appear somewhat after the IgM antibodies and a little earlier than IgG.

Multiple precipitating antibodies such as IgG globulin have been reported to be usually detectable within 3-4 months of severe or prolonged active Brucella infection (Godfroid J et al., 2010). Anti-Brucella complement fixing activity is mainly shown by IgG, and toMaulana some extent Azad by IgM Library, and but itAligarh is not found Muslim in IgA University(Heremans et al., 1963). Several studies reported that blocking antibodies (Auwaerter PG, 1999) appear later in the course of infection than agglutinating antibodies (Zinneman et al., 1959). These are thermo stable, divalent and found as IgG and IgA fractions (Young EJ., 1991). Of note, both IgG and IgA have been reported to act as agglutinating or blocking antibodies and they show prolonged and sustained response against brucellosis infection. However, a quantitative assessment of the relative amounts contributed by each globulin to the blocking effect cannot be made (Wilkinson et al., 1966). (Glenchur et al., 1963; Kerr, 1967; Coombs et al., 1981). In spite of all

Review of literature the development in the study of immunoglobulins, structure, sequential production and persistence after antigenic stimulation, this knowledge is limited to the brucellosis diagnosis. As a result of this, these findings are mainly used for the development of supplemental serological tests and in research it is limited to the identification of cell fractions and the antibody classes per se. Due to this little is known about the understanding the biological role of this complex antibody response. Various serum passive-transfer experiments corroborated the significance of humoral immunity in murine brucellosis. Araya et al., (1990) and Winter et al. (1989) showed that passive transfer of sera containing anti-LPS antibodies to mice could protect against challenge with virulent B. abortus. IgG2a and IgG3 are dominant antibody isotypes detected from the infected mice similar to the natural host, implying that Th1 response to Brucella infection occurred (Elzer et al., 1998; Elzer et al., 2002). Opsonization, probably coupled with enhancement of intracellular killing, is regarded as the principal protective role of antibody against Brucella infection.

Cell-Mediated Immune Responses

In essence, antibodies play cardinal role in restricting Brucella infection but the development of a solid cell-mediated immune response is fundamental for the resolution of infection caused by various intracellular pathogens viz. Brucella sp., Mycobacterium sp. and Salmonella sp. T lymphocyte sub-populations (Cantor and Boyse, 1975) has permitted the study of the individual response towards the different Brucella antigens. Many attempts have been made to identify the antigen(s) responsible for the protective immunity as well as the one responsible for the chronic state. Studies using cell transfer assays have unequivocally established that T helper Maulana Azad Library, Aligarh Muslim University cells are responsible for the response that confers protection (Kauffmann et al., 1979). T helper cells exert their protective effect by activation of macrophages, which enhances the bactericidal capacity through the production of IFN-γ. Regulation of T helper cell activity is controlled by soluble factors produced by suppressor macrophages and T suppressor cells. All experimental evidences show that the cell mediated immune response is necessary to mount and sustain antigen-specific response against Brucella infection in order to protect host.

Review of literature

The crucial role of T-cells in imparting immunity against Brucella is secretion of IFN- γ for the activation of macrophages and cytotoxic T-lymphocyte as well as IgG2a and IgG3 isotype switching. The significance of CD4+ and/or CD8+ T-cells in Brucella immunity has been controversial. Data obtained in b-/- and b2 m-/- mice infected with B. abortus strain 19 suggest that CD8+ T cells comprised the predominant protective population by lysing autologous infected macrophages (Oliveira and Splitter, 1994). Data from MHC class I-deficient mice as well as wild-type-mice indicate that Th-1 type cytokines such as IFN-g and IL-2 by CD8+ T-cells has an important function in controlling brucellosis (Oliveira and Splitter et al., 1994). However, CD4+ T-cells are predominant in number during brucella infection, and these cells exert their effect via cytokine secretion.

During Brucella infection several cytokines such as IFN-γ, TNF-, IL-2, IL-10, IL-12 and IL-18 control the intracellular growth of Brucella strains within macrophages, whereas IL-1a, IL-4, IL-6 and GM-CSF do not have clear effects (Golding et al., 2001). Furthermore, IL-2, IL-10, IL-18 and IL-12 that influence the acquired cellular resistance and specifically contribute to control the Brucella multiplication seem to work via the IFN-γ dependent pathway. The significance of IL-12 in brucellosis has been well described. However, the mechanisms of IL-12 induction in those cells have not been elucidated. IL-18, possesses broad and potent immunomodulatory properties (Dinarello et al., 1998). Pivotal role in the development of immunity against pathogens, by acting on Th1 cell differentiation, cell mediated cytotoxicity, and inflammation via IFN-γ production. Many studies documented its protective role against intracellular pathogens viz. Salmonella enterica serovar, Typhimurium, Mycobacterium tuberculosis, Shigella flexneri, Chlamydia trachomatis; Biet et al., 2002). Maulana Azad Library, Aligarh Muslim University

1.5 Immunotherapies available against intracellular pathogen

Live vaccines

Conventional live vaccines are derived from avirulent mutants, which can grow in host, however fail to invoke disease. The mutations are generally induced by exposure of the organism with mutagens, chemicals, heat or passage in vitro. Following these mutational events, avirulent mutants are selected and tested in vivo. In most of the cases, multiple mutations are induced and that led to the development

Review of literature of attenuated form of pathogen although it is often unknown what constellations of genes are altered to achieve attenuation. Attenuated pathogens have less possibilities to revert back to pathogenic form and cause disease in the host. Moreover, sometimes the attenuation happens to be due to a single-point mutation and chances of reversion to virulence form high Example of this was observed in case of oral polio vaccines when virus gets virulent upon administration (Minor et al.,1986). This reversion can result to full blown disease, especially to the underlying immunosuppressive conditions.

The most widely used vaccine for the prevention of bovine brucellosis is live B. abortus strain 19 (Nicoletti et al., 1990). This was first developed in 1930 and was originally isolated from milk of a Jersey cow as a virulent strain in 1923. Being kept in laboratory at room temperature for long period it was found to become attenuated. This strain was able to induce protective immunity in cattle. Efficacy of this vaccine depend on various factors viz. age, dose and route of administration and prevalence of brucellosis in vaccinated herds. Strain-19 vaccine suffers from safety concerns and some other drawbacks like

 Cross-reaction with natural infection during sero-diagnosis,

 Does not prevent completely udder infection,

 S-19 is pathogenic to human (Smith and Ficth, 1990, Nicoletti, 1990),

 In pregnant animal this vaccine strain induces abortion (Corbel, 1997) and

 Long term persistence of antibodies in serum hinders the sero-diagnosis of brucellosis among diseased and vaccinated animals.

AnotherMaulana live vaccine Azad is B. Library,abortus strain Aligarh 45/20, a Muslimrough derivative University of Brucella abortus smooth strain 45/20, was able to protect guinea pigs and cattle from Brucella infection (McEwen, 1940). Unfortunately, when used as a live vaccine, strain 45/20 was not stable and tended to revert to the smooth, virulent form, thereby defeating the purpose of using rough strains, usually associated with attenuation and inability to induce positive, diagnostic serology. Furthermore, it has also reported that this vaccine gives unpredictable serological effects and the occurrence of several local reactions at the site of vaccine injection in some animals. This pitfall eventually prompted the discontinuation of Strain 45/20 vaccination (Schurig et al., 2002). Brucella abortus

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RB51, a rough attenuated strain, was originally derived from a rifampicin-resistant mutant of B. abortus strain 2308 (Schurig et al., 1991), has replaced S19 strain as a vaccine candidate in some developed countries. Strain RB51 is very stable, it has no or highly reduced abortifacient characteristics (Schurig et al., 1991; Palmer et al., 1997). It has certain advantages over S-19 like

 Vaccine is safe until 7th month of pregnancy

 Able to differentiate vaccinated and naturally infected animals by both serological and molecular techniques like PCR and pulse field electrophoresis.

 The organism is cleared swiftly from the host and the longevity of the protection is about 1 year.

 Protective efficacy and immunity induced by strain RB51 is similar to or better than that induced by strain 19.

Although strain RB51 has an excellent record of stability, it is still infectious to humans and the exact nature of the mutations is not known.

Killed vaccines/antigenic fractions

A wide variety of preparations have been tested in this area including killed field isolates but with the exception of B. abortus strain 45/20, practical acceptance of these preparations has limited. There are various reasons such as production, costs, poor protection, unacceptable local reactions and serological problems played a role in vaccine failure. Antigenic fractions extracted from Brucella have been tested, particularly in association with a variety of adjuvants. Preparations used have included whole killed cells (Blasco et al., 1993), outer membrane protein, soluble and Maulana Azad Library, Aligarh Muslim University insoluble residues of sodium dodecyl sulfate (SDS) extracts of cell envelopes (Winter and Rowe, 1983; Dzata et al., 1991) and periplasmic proteins and salt extractable proteins (Tabatabai et al., 1992). There is limited success in achieving immunity with some of these preparations in laboratory animals.

Antigen Delivery Systems

Adjuvants are designed to boost the immune response. At times antigens fail to induce a desirable immune response as they do not reach the local lymph nodes. In such situation, adjuvants are co-administered to initiate a desirable immune response. This

Review of literature enhancement of immune response may be due to several ways: increase cellular infiltration into the injection site by adjuvant, or delivery of antigen to the local lymph nodes. Once antigen is taken up by APS’s they undergo maturation and then migrate to lymph nodes, presenting antigens to naïve T cells. Thus, the particulate adjuvants, or ‘antigen delivery systems’ e.g. microparticles, emulsions, liposomes, ISCOMS etc. promote uptake of the antigen by APC at the injection site. For the development of vaccine antigens, selection of adjuvants depends on the type of immune response required for protective immunity. In many infectious diseases, protective immunity hasn’t yet been defined, so selection of adjuvants is rather factual.

1.5.1 Lipid Particles as Antigen Delivery Vehicles

Liposomes are phospholipid vesicles which have been evaluated both as adjuvants and as delivery systems for antigens and adjuvants. Liposomes have been commonly used in complex formulations, often including monophosphoryl lipid A, which makes it difficult to determine the contribution of the liposome to the overall adjuvant effect. Immunopotentiating reconstituted influenza virosomes are unilamellar liposomes comprising mainly phosphatidylcholine, with influenza haemagglutinin intercalated into the membrane. The use of viral membrane proteins in the formation of virosomes offers the opportunity to exploit the targeting and fusogenic properties of the native viral membrane proteins, perhaps resulting in effective delivery of entrapped antigens into the cytosol for CTL induction. An alternative approach to vaccine delivery which may have some advantages over traditional liposomes has been described using ‘archaeosomes’, which are vesicles prepared from the polar lipids of Archaeobacteria (Benvegnu T et al., 2009). In some studies, archaeosomes have been shown to be more potent than liposomes. Liposomes which are spherical vesicles having at least Maulana Azad Library, Aligarh Muslim University one lipid bilayer have been used both as adjuvants and in delivery systems for adjuvants and antigens.

Immunostimulatory fractions from Quil A

As opposed to immunostimulatory adjuvants, several groups have supported the use of particulate adjuvants, or antigen delivery systems. Particulate adjuvants like microparticles and ISCOMs have dimensions comparable to the pathogens, but in order to enhance the level of response, immunostimulatory adjuvants can also be

Review of literature included in particulate delivery systems. Potent immunostimulator adjuvants, when added into delivery systems may limit adverse events by restricting the systemic circulation of the adjuvant.

Microspheres

Microsphere based delivery system is widely explored in many immunological studies. Poly-lactide co- glycolide (PLGA) microspheres, composed of spherical polymeric matrix have been used for controlled delivery system of peptides, native and synthetic proteins and also nucleic acids. Since many years, PLG has been used as suture material in humans because of its biodegradable and biocompatible nature, however recently it was shown that encapsulation of antigen in PLG microparticles has high adjuvant effect and the immunogenicity was comparable to Freund’s adjuvant. Moreover, recent reports also suggest that microparticles also exert an adjuvant effect for the induction of CMI and have high potential as an adjuvant for DNA vaccines (Lima KM et al., 2004). When microparticles are injected intramuscular, they are uptake up by DC, macrophages and local lymph nodes. Entrapped antigen is released slowly by microparticles making them significant for the development of single dose vaccine.

Immune Stimulating Complex (ISCOMs)

Immune Stimulating Complex is composed of the saponin mixture Quil A, cholesterol and phospholipids. It is a spherical, open cage like micellar assembly of about 40 nm size. Amphiphilic antigens like membrane proteins are present in ISCOM and they interact via hydrophobic interactions with the components of ISCOM. Several reports suggest that ISCOM based vaccines have the ability to induce CTL in both mice and Maulana Azad Library, Aligarh Muslim University non-human primates. Which may be important in the protection / recovery from viral as well as other intracellular pathogens. They boost both cellular mediated and humoral immune responses. Immune response generated by ISCOM can include activation of APC, increased expression of MHC II on APCs, induction of cytokine, especially IL-2 and IFN-γ, CD4+T-cell responses and CD8+ CTL responses. This broad range of immune response makes them ideal for vaccine as it can also provide protection / recovery from viral as well as other intracellular pathogens. Anti- influenza vaccine for horses is a licensed ISCOM vaccine. Some particulate antigen delivery systems employed for vaccine development are as follows.

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Niosomes

Niosomes, also known as non-ionic surfactant vesicles, are small unilamellar vesicles made from non-ionic surfactants. They show immunogenicity comparable to Complete Freund’s adjuvant and stability higher than phospholipid liposomes. Their ability to act as adjuvants has been proved by administering it with different antigens and peptides like, BSA, a soluble T. gondii antigen, a synthetic measles peptide, etc (Conacher M et al., 2014). These results suggested that the immune responses particularly activated by NISV are of the Th1- type which are useful for vaccines directed against intracellular pathogens, requiring cell mediated immune responses. Low toxicity and enhanced stability makes NISV as excellent candidates for vaccine adjuvants.

Cochleates

Cochleates are stable phospholipid–calcium precipitates, composed of simple, naturally occurring phosphatidylserine (PS), cholesterol and calcium. Cochleates have shown strong, long-lasting, antibody responses. Cochleates, which can be administered via oral, intranasal and intramuscular routes are simple, safe and highly efficacious for in vivo delivery of proteins, peptides and DNA. Cochleates were not even challenged with live viruses following oral or intra muscular administration; therefore, they can be potentially used with subunit vaccines.

Archaeosome

Archaeobacterial lipid liposomes constitute Archaeosomes, which shows enhanced stability against extreme pH, oxidation, temperature etc. They even show enhanced cell mediatedMaulana and humoralAzad Library, immune response Aligarh as comparedMuslim toUniversity conventional liposomes and Freund’s adjuvants.

Liposomes

Liposome, the spherical shaped lipid bilayer acts as an alternative and universal adjuvant system. They have been established as immune-adjuvants as they potentiate both cells mediated and humoral immune responses. Liposomal immune adjuvants accumulate in lymph node, from where they slowly release encapsulated antigenic components.

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1.5.2 Exosomes

Exosomes are small, single-membrane, secreted organelles of ∼30 to ∼200 nm in diameter having similar topology to that of the cell and enriched with selected proteins, lipids, nucleic acids, and glycoconjugates. Exosomes are created from the budding of the plasma as well as endosome membrane and contain an array of membrane-associated, oligomeric protein complexes along with displaying noticeable molecular heterogeneity. Exosome show diverse functions such as remodeling the extracellular matrix and transmitting signals and molecules to other cells (Su SA et al., 2017). This intracellular vesicle plays some critical roles in many human health and diseases, including development, immunity, tissue homeostasis, cancer, and neurodegenerative diseases. The secretion of exosomes is a process in which multivesicular bodies fuse with the cell membrane. All cells that contain multivesicular endocytic compartments could theoretically secrete exosomes. It is already clear that they serve to remove obsolete membrane proteins and act as messages of inter-cellular communication. Exosome formation and secretion includes budding at the limiting endosomal membrane, fusion of endocytic compartments with the cell surface, and final exosome secretion. The internal vesicles of MVBs are the precursors of exosomes.

Maulana Azad Library, Aligarh Muslim University

Figure 1.9 Pathway of exosome formation. Cells release exosomes via two mechanisms. The classic pathway involves the formation of intraluminal vesicles (ILVs) within MVBs. In turn, the membrane of MVB fuses with the plasma membrane, resulting in the release of ILVs. When secreted, ILVs are called exosomes.

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Viruses acquire the exosome biogenesis pathways for both assembling of the infectious particles as well as for establishing host permissiveness. On the basis of these and other properties, exosomes are being developed as therapeutic agents in multiple disease models. The putative dual roles of exosomes: 1. elimination of obsolete proteins during cell maturation, and 2. regulation of intercellular communication

Figure 1.10 Exosome composition: Exosomes carry a wide array of molecules including proteins, DNAs, mRNAs, and miRNAs, depending on a variety of factors including the cell type from which the exosome originates, the state of health of the host, and extracellular stimuli. The contents of exosomes can be transferred from origin Maulana cells to target Azad cells, Library, resulting Aligarh in an elaborate Muslim intercellular University communication network.

1.6 The problems of tuberculosis as a human disease

In spite of being treatable to a great degree, tuberculosis (TB) is still a potent cause of death worldwide except in developed countries. In 2010, the number of recent cases was 9 million and mortality reached up to 1.4 million, despite all the efforts made to keep this global threat to human health, under check. Robert Kotch was awarded by the Nobel Prize for its discovery in 1882. Pulmonary TB is responsible for 70% of cases. However, other than the lungs it can cause extra-pulmonary disease by

Review of literature attacking areas like lymph nodes, bone, and meninges. New drug-resistant forms of M. tuberculosis, less-sensitive and delayed diagnostic procedures, have weakened the control of the global TB epidemic. Additionally, the complex nature and incomplete characterization of immune response to M. tuberculosis has affected the advancement of new tests, vaccines, and treatments (Nunes-Alves C et al., 2014). CD4+ T cells and proinflammatory cytokines like IL-12, IFN-γ and TNF are crucial in controlling M. tuberculosis infection in human and experimental murine models, however, it’s still not known why some people are at a greater risk of developing the infection as compared to others. Recently, the frequency and profiling of CD4, CD8, and γδ T cells did not correlate with protection against TB, in newborns that were vaccinated with Bacillus Calmette-Guerin (BCG). The major components (Like IFN-γ production by CD4+ T cells) of the immune mechanism against M. tuberculosis may not render the immune equivalents of protection against disease. Thus, a correlation for protection, is lacking which is required to help predict the outcomes of infection and to monitor vaccine efficacy. The infection is a cumulative effect of all the exchanges between the environment, the host, and the pathogen. Presence and absence of risk factors like HIV infection, immunodeficiency, diabetes mellitus, overpopulation, lack of proper nutrition, and general poverty complicates the disease etiology further. By exploiting the host and the causal bacterial factors that influence M. tuberculosis exposure outcomes, researchers are of the view that certain groups of host and M. tuberculosis genotypes are at a greater risk of evolving into active TB.

The classical tuberculin skin test (TST), generally used to diagnose latent TB, M. tuberculosis infection or exposure is carried out by analyzing the host response to the bacterial antigen. An extract containing M. tuberculosis antigens, is injected intradermally Maulana (tuberculin, Azad a glycerin Library, extract Aligarh of M. tuberculosis Muslim orUniversity a commercially purified protein derivative, PPD) and the subsequent induration in the skin, developed due to a delayed-type hypersensitivity reaction, is measured in millimeters. A widespread method, TST has its drawbacks too. The test is often negative in those individuals who are at a risk of progression to active form of disease: the young, the elderly, and the immunosuppressed. PPD, made from filtrate of M. tuberculosis culture, contains over 200 antigens that are found in both, M. bovis BCG vaccine and environmental nontuberculous mycobacteria. Together with limited specificity and high false positive rates, the method of TST is obsolete and underproductive.

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In order to overcome this problem, recently many technically advanced assays that make use of specific M. tuberculosis antigens predominantly early secretory antigen target-6 (ESAT-6) and culture filtrate protein-10 (CFP-10)) have been introduced. Quantification of IFN-γ production by blood cells can then be employed for evaluation of reactivity to M. tuberculosis through methods like enzyme-linked immunoassay after whole blood incubation (Qiagen) or by the enzyme-linked immunospot technique, before incubation (TSPOT. TBTM, Oxford Immunotec, Oxford, UK). In spite of such sensitive and specific tests, there is a need for those which can spot the difference between hidden and active TB disease. Moreover, assessment methods that could predict the progression of TB infection in latent individuals or in those who have subclinical disease is warranted. Preventive measures and therapy could then be applied which would eliminate the risk of toxic and lengthy treatment procedures.

1.6.1 Human tuberculosis and experimental animal models

As discussed above the pathology of human TB is suggestive of its heterogenous nature and shows a range of lesions that imitate stages of latent and active TB. Mouse models have been continuously used for studying the immune response to TB infection. However, the commonly used mouse strains, such as C57BL/6 or BALB/c mice does not model the human pathology seen in this disease, perfectly. (Rivera J et al., 2008) Some of the drawbacks are poor arrangement and sole cellular granulomas, absence of fibrosis or hypoxia, a relatively high and controlled bacterial counts and ultimate death of mice with progressive infection. Thus, the model is incapable of demonstrating the range of latency to active disease seen in humans. TB disease in guinea pigs, rabbits, and mice resulting from aerosol infection with low-dose virulent Maulana Azad Library, Aligarh Muslim University M. tuberculosis exhibits many of the important features of human TB. The usage of inbred knockout mice (of genes IL- 12, IFN-γ, and TNF-α) as well as of mice lacking CD4+ T cells, shows that these immune factors govern M. tuberculosis infection in the mouse as well as in humans, without which HIV-infected individuals succumb rapidly to TB disease. Regulation of mycobacterial infection by host immune factors is discussed in more detail later in this review.

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Orchestration of the Host Immune Response to M. tuberculosis results in the formation of granulomas

Granulomas play a multifunctional role depending on the stage of the disease by behaving either in a protective manner for the host or by promoting infection. They have been reported to even cause tissue pathology. Ramakrishnan and colleagues, with the help of a zebrafish model of TB, showed that virulent intracellular mycobacterium cause the conscription of macrophages to early granulomas which being highly motile tend to leave these granulomas after becoming infected. This suggests that mycobacteria make use of the host to facilitate the spread of infection. The TB model of zebrafish help to investigate the very early response to mycobacterial infection although they lack lungs or an adaptive immune system. Such a mechanism may also be seen in patients of HIV which also lack an adequate adaptive immune response.

1.6.2 Mycobacterium tuberculosis infection

The spread of M. tuberculosis is caused mainly due to airborne droplet nuclei, which are produced during coughing by an infected individual. However, the dose of M. tuberculosis required for the manifestation of the infection or active disease is still debatable along with the status of mycobacteria in these droplets. Mycobacterial early infection in host is responded by an incursion of phagocytic cells, resident alveolar macrophages and employed neutrophils. The bacilli are thought to be phagocytized by the alveolar macrophages and taken up by the neutrophils and dendritic cells after the establishment of infection in the airways and lung parenchyma. The first line of defense is shown by macrophages and neutrophils by expression of antimicrobial peptides, functionalMaulana in the Azad initial Library, immune response.Aligarh Extrinsic Muslim innat Universitye and adaptive immune factors govern macrophage and neutrophil activation to restrict and/or kill the pathogen.

The succeeding immune response towards clearance or control of the pathogen is stimulated by macrophages and neutrophils after infection of M. tuberculosis, causing latent infection to persist or manifestation of active disease (Nunes-Alves C et al., 2014).

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An array of scavenger receptors, complement receptors and the mannose receptor help M. tuberculosis to gain entry into the macrophage. The internalization of M. tuberculosis, cause formation of phagosome around the phagocytized bacillus, shown by experiments done with murine peritoneal macrophages. Autophagy, a process of sequestration of cytoplasmic components like organelles and intracellular pathogens in an auto-phagosome and delivery to the lysosome for degradation, influence the fate of M. tuberculosis. IFN-γ for example, activates autophagy and cause phagosome maturation, increased acidification and M. tuberculosis killing.

Viable and virulent M. tuberculosis bacilli form a niche for survival by preventing phagolysosomal fusion and averting acidification of the phagosomal compartment and in doing so adapt to the intracellular environment of the macrophage. Blockade of Phagolysosomal fusion is inhibited by opsonization of the bacilli prior to infection. There are many mechanisms through which macrophages can eliminate mycobacteria, if properly activated. With the help of genetic and immunological tools available it has been possible to separate early stage of aerosol infection with M. tuberculosis in mouse model along with the phases of cellular and molecular innate and adaptive immune responses which provide protection against or the development of disease. In order to fully understand the factors that are responsible for pathogenesis in TB or its protection, it is important to compare data between different experimental models and human disease. The initiation of T cell immunity in the mediastinal lymph nodes leads the dissemination of M. tuberculosis in the mouse and is found to occur earlier in resistant C57BL/6 mice than in susceptible C3H mice (O'Garra A et al., 2013). This leads to an earlier immune response in the C57BL/6 mice. This finding is suggestive of timely adaptive immune response, which is stringently controlled by host genome. Maulana Azad Library, Aligarh Muslim University Macrophage defense against M. tuberculosis infection via induction of apoptosis

The bacilli spread from cell to cell is caused by necrosis or lysis of infected macrophages. However, at times infected macrophages may undergo apoptotic cell death with intact cell membrane which is helpful in reducing pathogen viability and increasing immunity.

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Figure 1.11 Various outcomes of infection with M tuberculosis, different T cell populations are involved in protection, and in anti-mycobacterial effector mechanisms of macrophages. Firstly, the scheme depicts the different outcomes of tuberculosis in healthy and immunocompromised individuals. Secondly, the figure shows anti-mycobacterial effector mechanisms of activated macrophages. Thirdly, the figure shows the different T cell populations and T cell effector mechanisms in the controlling the disease.

The anti-microbial mechanism of apoptosis was first seen in alveolar macrophages of humans, where weakened mycobacterial strains, including M. tuberculosis H37Ra, showed reduced viability in macrophages which underwent apoptosis. Besides, another mechanism that was seen to potentially curb the spread of infection was induction of apoptosis in neighboring macrophages after infection of M. tuberculosis H37Ra. Interestingly,Maulana virulent Azad strains Library, of M. tuberculosis Aligarh Muslimhave developed University a mechanism of inhibiting apoptosis by which they could grow intracellularly and progressively in these cells. The above observation has been confirmed by attenuation of the pro- apoptotic M. tuberculosis secA2 and nuoG deletion mutants upon infection, in vivo (Hmama Z et al., 2015). Apoptosis of infected macrophages was seen to be magnified by inactivation of secA2 gene in M. tuberculosis, which reduced the secretion mycobacterial superoxide dismutase. In in-vivo conditions it was seen that strains of mycobacteria with secA2 deletion heightened antigen-specific CD8+T cell priming.

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When mice and guinea pigs were vaccinated with a secA2 mutant an increase in CD4+T cell response and resistance to Mycobacterial challenge.

It has been demonstrated by workers that infected macrophages are engulfed by healthy macrophages through efferocytosis, a process known as housekeeping function of macrophages. The mycobacteria, doubly coated with macrophages is then delivered to the lysosome and subsequently killed. lipid mediator’s eicosanoids, prostaglandin E2 (PGE2) (proapoptotic), and lipoxin A4 (LXA4) (pronecrotic), regulate this type of cell death which follows M. tuberculosis infection. This control decides the consequence of infection. However, induction of LXA4 and inhibition of PGE2 production by virulent strains of mycobacteria helps M. tuberculosis in eluding the innate defense mechanism by initiating necrosis and inhibiting macrophage apoptosis which ultimately results in the spread of the infection (Aporta A et al., 2012)

Infected macrophages of mice(able to synthesize LXA4) lacking 5-lipoxygenase (Alox5−/− mice) show a greater tendency of apoptosis while macrophages of mice deficient in prostaglandin E synthase (Ptges−/− mice) are highly susceptible to cell death after infection by avirulent strains. They even show sensitivity to aerosol infection by virulent strains. Infection of M. tuberculosis prevents macrophage apoptosis and averts DCs from cross- presenting M. tuberculosis antigens, by activating 5-lipoxygenase pathway. Hence, T cell immunity is impended.

Cytokines in adaptive immunity to M. tuberculosis IL-12, IFN-γ, and the Th1 axis are requisite for protective anti-mycobacterial responses

Secretion of IFN-γ by CD4+ Th1 cells is the chief immune mechanism against Maulana Azad Library, Aligarh Muslim University mycobacterial infection. Although IFN-γ is also secreted by antigen- specific CD8+ T cells, natural killer (NK) cells, γδ T cells, and CD1-restricted T cells but they cannot replace CD4+ T cells. Unable to control low dose of M. tuberculosis infection, in the absence of IFN-γ, mice tend to manifest progressive tissue destruction. It is connected with uncontrolled bacterial replication.

IL-12 (p40/p35), secreted by M. tuberculosis activated DCs, as part of TLR- dependent mechanisms determines the initiation of protective IFN-γ T cell responses against primary M. tuberculosis infection and mice which lack IL-12p40, cannot

Review of literature regulate the growth of the bacterial infection. In order to regulate chronic infection, IL-12p70 production is crucial for prolonged and constant IFN-γ Th1 responses in the lungs.

Recent studies have brought into light the different role played by p40 and p35 subunits of IL-12 in the control of mycobacterial infection. In comparison to p35−/− mice, mice deficient of the p40 subunit were more defenseless against M. tuberculosis infection besides showing increased bacterial growth, increased transience and lessened IFN-γ T cell response. The observation indicated the protective role of IL- 12p40. IL-23p19 subsidizes to protective immunity which is reason why p40−/− mice are more sensitive to mycobacterial infection than p35−/− mice. The expression of IL- 23p19 occurs early in M. tuberculosis infection. Functional IL-23 is formed by the association of IL-23p19 with IL-12p40. No decrease in IFN-γ-specific T cells or IFN- γ mRNA in IL-23p19−/− mice and effective control of infection was seen.

On the other hand, decrease in IL-17 m RNA and CD4+ T cells which secrete IL-17 was seen in lungs. It is doubtful whether Th17 responses perform any protective role since IL-23p19 is not involved in the regulation of M. tuberculosis infection. Although IL-23 can generate cells that secrete IFN-γ at the time of M. tuberculosis infection in place of IL-12p70, however it’s not enough to control the infection. Hence, despite its compensatory role IL-23 is not crucial for infection control.

Control of inflammation and CD4+ T Cells is necessary to inhibit host damage during M. tuberculosis infection

In the condition of microbial infection, unimpeded TNF and IFN-γ secretion can be problematic to the host. Mechanisms arbitrated by Foxp3+ regulatory T cells and IL- Maulana Azad Library, Aligarh Muslim University 10 exist which can avert immunopathology. The stability between these regulators and TNF/IFN-γ is crucial for is the deciding factor for eradication of M. tuberculosis with minimum pathology.

Mycobacterial regulation can be hampered if there is extreme inflammation or immune response. PD-1, a cell surface receptor of T and B cells, is documented as an important controller of responses which are specific to pathogens (Chen L, 2004). It binds with its ligands (PD-L1 and PD-L2) and gives a negative signal for proliferation and cytokine production. T cells of TB patients express PD-1 while PD-L1 expression

Review of literature was induced on T cells stimulated with sonicated H37Rv M. tuberculosis. In the whole blood of patients of TB (active TB) PDL-1 is found to be overexpressed, mainly caused due to expression by neutrophils.

In cells of TB patients in vitro, antigen-specific IFN-γ responses were heightened by antibodies that blocked PD-1/PD-L1/PD-L2. Hence, PD-1 can cause chronic infection as seen in case of viral infections. It has been seen that if PD-1 is deleted (PD-1−/− mice) in M. tuberculosis aerosol–infected mice, the number of CD4+ T cells specific for M. tuberculosis increases but interestingly, infection susceptibility was amplified where it was observed that enlarged bacterial load and pathology was augmented by CD4+ T cells themselves. Despite the regulatory role of CD4+ T cells in infection of M. tuberculosis, the vital role of PD-1 signaling points towards an exceptionally controlled immune response which cannot be overlooked.

1.7 Apoptosis

A genetically governed process of “programmed” cell death, apoptosis takes place along the process of growth and ageing. Furthermore, it is the governing mechanism of homeostasis, whereby it controls the population of cell in tissues. Apoptosis in immune reactions acts as a defense mechanism or when disease damage the cells. Apoptotic cell death also occurs in cancer chemotherapy and irradiation, through a pathway that is reliant on p53. Cells expressing Fas or TNF receptors cause apoptosis via ligand binding of ligand and cross-linking of protein whereas cells that follow the pathway of default death block it by a hormone or growth factor (Rastogi RP and Sinha RP. 2010). The synchronized process of apoptosis is energy consuming and encompasses initiation of a class of cysteine proteases called “caspases” which further lead toMaulana cell death. Azad Library, Aligarh Muslim University

Molecular mechanisms of apoptosis signaling pathways

An extremely complicated process, apoptosis mainly occurs through two important pathways:

I. The pathway involving death receptor or the extrinsic pathway. II. The pathway involving mitochondria or the intrinsic pathway.

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Another path way exists which comprises of toxicity mediated through T cells and kill perforin-granzyme based cell death. This pathway in known to trigger apoptosis either by granzyme B or granzyme A. When caspase-3 is cleaved it starts the pathway which cause DNA fragmentation, breakdown of the cell’s structural elements, cross linking of proteins, followed by formation of apoptotic bodies, expression of ligands for phagocytic cells and ultimately uptake by phagocytic cells. Single stranded DNA damage activates granzyme A pathway which operates in a caspase-independent manner.

1.7.1 Extrinsic apoptosis pathway

Transmembrane interactions regulated through receptors make up the extrinsic signaling pathway. The pathway consists of death receptors that are affiliated to the superfamily of tumor necrosis factor (TNF) receptor gene.

Comparably extracellular domains which are cysteine-rich are shared by members of the TNF receptor family and also comprise of “death domain”, a cytoplasmic domain of about 80 amino acids. FasL/FasR, TNF-α/TNFR1, Apo3L/DR3, Apo2L/DR4 and Apo2L/DR5, make up the ligands and corresponding death receptors.

The events of extrinsic pathway are best modeled by FasL/FasR and TNF- α/TNFR1.Binding of grouped receptors with homologous trimeric ligand takes place. Death domains of cytoplasmic adapter proteins bind with the receptors, after ligand binding. Binding of the adapter protein FADD results after binding of Fas ligand to Fas receptor. TRADD is bound with conscription of FADD and RIP, when TNF ligand binds to TNF receptor. Death-inducing signaling complex (DISC), is formed by dimerizing death effector domain when FADD combines with procaspase-8. Maulana Azad Library, Aligarh Muslim University Consequently, procaspase 8 is activated with which the execution phase of apoptosis starts.

1.7.2 Intrinsic apoptosis Pathway

Intrinsic apoptosis is mitochondrial-stimulated pathway involving a varied array of stimuli which are not mediated by any receptor. It produces intracellular signals which act directly on targets in the cell. The stimuli may operate positively or negatively. In case of negative signals certain growth factors, hormones and cytokines are absent, which might prevent the suppression of death programs, ultimately

Review of literature causing apoptosis. Radiation, hypoxia, toxins, hyperthermia, viral infections and free radicals are some of the positive stimuli.

Inner mitochondrial membrane is transformed by these signals which cause opening of the mitochondrial permeability transition (MPT) pore and alters the mitochondrial membrane potential. Normally sequestered pro-apoptotic proteins are released in the cytosol from the space between the membrane. Among the pro-apoptotic proteins (cytochrome c, Smac/DIABLO, and the serine protease HtrA2/Omi.), cytochrome c binds and activates Apaf-1 as well as procaspase-9, thereby forming an “apoptosome”.

Caspase-9 is triggered by the accumulated procaspase-9. Apoptosis is triggered by inhibition of IAP (inhibitors of apoptosis proteins) by Smac/DIABLO and HtrA2/Omi (Fumarola C et al., 2004).

AIF, endnuclease G and CAD belong to the second group of pro-apoptotic proteins which are freed from the mitochondria during apoptosis after the cell has committed to die.

The process is as follows:

i. Step 1 condensation: translocation of AIF to the nucleus and DNA fragmentation (~50–300 kb pieces) followed by condensation of peripheral nuclear chromatin. ii. Step 2 condensation: production of oligonucleosomal DNA fragments from nuclear chromatin by endonuclease G. Endonuclease G and AIF act independent of caspase. Oligonucleosomal DNA fragments and progressive Maulanacondensation Azad of chromatin Library, occurs Aligarh through CADMuslim which University gets cleaved by caspase- 3 on relocating to the nucleus from mitochondria.

Members of the Bcl-2 family of proteins control and regulate these apoptotic mitochondrial events. Regulation of Bcl-2 family proteins is brought by p53, however the mechanism is still not clear. Mitochondrial membrane permeability is guided by the Bcl-2 family of proteins and could neither pro-apoptotic or anti-apoptotic. Bcl-2, Bcl-x, Bcl-XL, Bcl-XS, Bcl-w, BAG are some of the anti-apoptotic proteins while Bcl-10, Bax, Bak, Bid, Bad, Bim, Bik, and Blk are pro-apoptotic proteins. Bcl-2 family of proteins function by managing the release of cytochrome c from the

Review of literature mitochondria by altering its membrane permeability. Cell death can also be instigated by the heterodimerization of Bad and Bcl-Xl or Bcl-2 where their protective effect is compromised. If not requisitioned by Bad, both Bcl-2 and Bcl-Xl cause inhibition of cytochrome c release. Workers have shown that apoptotic death is inhibited by Bcl-2 and Bcl-XL through the control of activation of caspase proteases. Activation of procaspase-9 is prevented when a protein called “Aven” binds both Bcl-Xl and Apaf- 1. Evidence exist for down-regulation of either Bcl-2 or Bcl-Xl if either of them is overexpressed.

Maulana Azad Library, Aligarh Muslim University

Chapter 2: Exosome mediated delivery of recombinant rL7/L12

Evaluation of immunogenic potential of exosome mediated delivery of recombinant rl7/l12 protein against experimental murine brucellosis

2.1 Introduction

Exosomes, the naturally occurring extracellular vesicles, with structural analogy to ‘liposomes’ are generally formed by inward budding of the inner endosomal membrane. The reverse budding of the endosome membrane may lead to formation of multivesicular bodies (EL Andaloussi S et al., 2013). The as-formed multi-vesicular bodies (MBV) can fuse with the plasma membrane of the parent cells to exocytose nanosized vesicles (exosomes) in the surrounding. The dimension of the exosomes ranges from 30 to 150 nm. With lipid bilayer exterior, the exosomes may harbour both transmembrane and cytosolic proteins (Cai J et al., 2016). They may also accommodate miRNAs, mRNAs, long non-coding RNAs and DNA etc. (Cai J et al., 2016). There are several reports that glorify exosomes as a natural communication means between adjacent and distant cells. Exosomes actively participate in various biological activities in vivo and play noteworthy roles in various pathological conditions such as cancer, infectious and neurodegenerative diseases etc. (Isola AL et al., 2016). The plasticity of the exosomal composition under altering physiological stimuli and pathological states render them a perfect biomarker for a specific disease. Among various immune cell populations, dendritic cells have been reported to profusely produce and secrete exosomes (Robbins PD et al., 2014). Besides endosome related proteins viz. Hsc70 and Gi2 etc.; the exosomes secreted from dendritic cells can harbor MHC and costimulatory molecules as well (Robbins PD et al., 2014,Maulana Shenoda AzadBB et al., Library, 2016). S imilarly,Aligarh several Muslim B-cell University lines have been reported to secrete exosomes loaded with MHC class-II, co-stimulatory and adhesion molecules. It is tempting to speculate that that such vesicle could directly stimulate CD4 T-cell clones. However, in spite of harbouring MHC II/costimulatory molecules, exosomes stimulate T cell clones and T cell lines feebly and fail to activate naïve T cells. This could be attributed to low T cell receptor cross linking capacity (cf. naïve T cells) and small size and above all membrane composition of the as-formed exosomes. However, dendritic cells can maneuver exosomes for their ability to activate T lymphocytes (Raposo G et al., 1996). Thus, the peptide-pulsed dendritic

Chapter 2: Exosome mediated delivery of recombinant rL7/L12 cells (DCs) derived exosome-based vaccines have been widely employed in priming of tumor-specific CTLs (Zitvogel L et al.,1998). Besides evoking CTL response against tumor, exosomes-based vaccine can evoke specific immune response against Epstein-Barr virus, cytomegalovirus and influenza virus in the host (Admyre C et al., 2006). Exosome can also supplement cancer chemotherapy by involving tumor associated antigens to recruit cancer specific immune response in the host (Shao Y et al., 2016).

Beside cancer, the exosome can also evoke immune responses against bacterial pathogens as well. Exosomes can harbour bacterial antigens and evoke immune responses. For example, macrophages infected with Mycobacterium bovis BCG-have been reported to release mycobacterial antigen bearing EVs that promote protective T-cell immunity in mice (Giri PK et al., 2008). HIV Gag protein bearing exosome can induce desirable anti-HIV immunity in the host (Booth AM et al., 2006). Similarly, CMV gB protein loaded exosome stimulate memory CD4 T cells against CMV in the host (Walker JD et al., 2016). Porcine respiratory and reproductive syndrome virus (PRRSV) specific antigens derived exosomes have been reported in both viremic (V) and non-viremic (NV) pigs (Montaner S et al.,2014). The exosome derived viral proteins demonstrate same level of antigenic properties similar to the antigenic activity contained in the commercially available vaccine (Porcilis PRRSV vaccine, Intervet, Boxmeer, The Netherlands). Interestingly, a strong correlation has been established between circulation of viral antigens through the exosomes in the serum of the host with no pathogen load detected in peripheral circulation (Montaner S et al., 2014, Gutzeit C et al., 2014)

Many EVs, including exosomes have been reported to share physical and chemical Maulana Azad Library, Aligarh Muslim University characteristics as well as their biogenesis pathways with those of retroviruses (Nolte-'t Hoen E et al., 2016, Cepko C et al., 2001). It is a puzzling observation that why exosome associated with PRRSV viral RNA dependent RNA polymerase and nucleocapsid protein are circulating in the serum of the host without the ongoing viral replication. It is tempting to speculate that exosomes associated viral proteins act as immune modulators to establish the viral infection by immune evasion mechanisms (Alenquer M et al., 2015). It has been reported that the viral RNA dependent RNA polymerase and helicase increase the virulence of the atypical highly pathogenic HP- PRRSV (Li Y et al.,2014). However, it is unclear that how they could contribute to

Chapter 2: Exosome mediated delivery of recombinant rL7/L12 pathogenicity in the host. The N protein and three non-structural (Nsps) PRRSV proteins play important role in type I IFN suppression and modulation of the NF-κB pathway (Sun Y et al., 2012). More elaborated efforts are required to understand the encapsidation of viral proteins in exosomes and their role as a novel vaccine approach.

Dendritic cells have also been reported to release exosomes (Qazi et al.,2009). On the other hand, the exogenous exosomes can be avidly taken up by the dendritic cells as well. In fact, both endogenous as well as exogenous exosomes behave like a potent immune regulator and considered relevant for the design of vaccine adjuvants and therapeutic intervention strategies to modulate host immune responses (Qazi et al., 2009). Since their discovery, a range of therapeutic applications have been attributed to exosome-based delivery system. Several earlier reports have highlighted that exosomes may play a critical role in some pathophysiological situations.

Both bone marrow derived mast cells (BMMC) as well as their commercially available cell lines have the ability to constitutively secrete membrane vesicles or exosomes that have the capacity to induce B and T lymphocyte proliferation and cytokine production (Skokos et al., 2001). In contrast to exosomes derived from various cell types including B lymphocytes and macrophages, only BMMC and peritoneal mast cell-derived exosomes have the potential to induce B and T lymphocyte activation in the host. Moreover, when dendritic cell-derived exosomes were pulsed with tumor-derived peptides, they were able to eradicate or suppress growth of established murine tumours in a T cell-dependent manner (Zitvogel et al.,1998).

VaccinesMaulana and associated Azad adjuvants Library, can Aligarh manipulate Muslim both target University as effector components of host immune system to impart protection against various infectious diseases. The phenotype of effector CD4+ T helper and CD8+ T cytotoxic be influenced by cytokine milieu, the differential expression of co-stimulatory molecules, antigen dose, differences in affinity at the TCR-peptide-MHC interface and adjuvants (Blazar BR et al.,1997) In general, vaccine delivery system in conjunction with suitable adjuvants has the potential to maneuver host T helper cells in a specific fashion (Kahlon R et al.,2003) The generated T-helper cells (Th) can regulate elicitation of cytotoxic T cell (CTL) and B cell responses in the host. The cytokines, secreted by T helper cells, can

Chapter 2: Exosome mediated delivery of recombinant rL7/L12 induce class switching that leads to induction of antibody with exclusive isotype. Cytotoxic T cells, the effector arm of the cell-mediated immune response, kills cells infected with intracellular pathogens. They also work towards surveillance mechanisms against cancer cells. In previous studies, several research groups demonstrated that exosome mediated delivery of antigen can elicit both cells mediated as well as humoral immunity simultaneously (Skokos et al.,2001).

DC-derived exosomes accumulate both MHC class II and MHC class I molecules on their surface. Previous investigations have shown the biologic significance of exosomes in different areas of research, such as tumor and transplantation immunology, (Théry C et al., 2002) vaccine therapy against infection, (Kuate et al.,2007; Beauvillain et al., 2007) as well as a biomarker for diagnostic purposes (Zhou et al., 2006). Several studies have demonstrated that DC-derived exosomes when pulsed with desirable antigen can elicit activation of antigen specific T cells. (Colino and Snapper 2006; Thery et al.,2002). Keeping into consideration the intracellular abode of Brucella species, the desirable immunity should be able to activate both humoral as well as cell mediated immunity in the host. Incidentally, exosomes-based antigen delivery system reckoned to activate both arms of the host immune system to eliminate Brucella infection from systemic circulation.

2.2 Materials and methodology

Agarose, ammonium per sulphate, acrylamide, isopropyl thiogalactosepyranoside (IPTG), X-gal, lysozyme, sodium dodecylsulphate, diamino-benzidine, Nitro- cellulose membrane (0.45 µm pore size) TEMED, glycine, ethidium bromide, proteinase K, RNase, Triton X-100, PMSF, bovine serum albumin and antibiotics (ampicillin andMaulana kanamycin) Azad were Library, purchased Aligarh from Sigma, Muslim St. louis, University USA). Foetal bovine serum was obtained from Life Technologies (GrandIsland, NY, USA). Rabbit anti-mouse IgG HRPO conjugate was purchased from Sigma-Aldrich, USA. Potassium chloride, disodium hydrogen orthophosphate, potassium dihydrogen orthophosphate, hydrogen peroxide, certrimide, CTAB and glucose were from Qualigens (India, Mumbai). Glycerol, sodium nitrite and orthophosphoric acid were from Merck, (India, Mumbai.). Sodium carbonate, sodium bicarbonate, isopropanol, phenol, chloroform, ethylene diamine tetra-acetic acid, boric acid, glacial acetic acid, isoamyl alcohol, sodium hydroxide, sodium acetate, sodium dihydrogen phosphate,

Chapter 2: Exosome mediated delivery of recombinant rL7/L12 magnesium sulphate was procured from SRL India, Mumbai. [3H]-thymidine and [51Cr] sodium-chromate were bought from Bhabha Atomic Research Center, Mumbai, India. Monoclonal anti-mouse CD4 and CD8 and their isotype control were from Sigma Immuno Chemicals, St Luis, USA. Sandwich ELISA kit (BD Biosciences, San Diego, Calif) was used to estimate mouse Gamma-Interferon level. Bicinchoninic acid protein assay Kit was from Pierce Chemical (CI, USA). Cholesterol (Chol) was purchased from Centron Research Laboratory, Bombay, India.

Enzymes

The restriction endonuclease, DNA modifying enymes-T4 DNA ligase, Taq DNA polymerase, dNTPs solution and buffers for these enzymes were procured from Ambion. Restriction enzymes and DNA marker supplied from MBI Fermentas (Germany).

Oligonucleotide primers

Oligonucleotide primers were designed from published sequence of B. abortus (Acc No. L19101). The primers BA1 (Forward) and BA2 (Reverse) both had linner sequences at their 5' ends.

Restriction site for Sall in the forward primer and Pstlin the reverse primer was incorporated for directional cloning. The nucleotide sequences of these primers were as follows:

Primer Forward/ Sequence

Code Reverse Maulana Azad Library, Aligarh Muslim University BA1 Forward 5'- CGC CGT CGA CAT GGC TGA TCT CGC AAA GATC - 3'

BA2 Reverse 5'- GCC CCT GCA GTC TAG ATT ACT TGA GTT CAA CCT TGGC-3'

Molecular weight marker

Medium range Protein molecular weight marker, Prestain molecular weight marker (Sigma) and 100 bp DNA ladder were obtained from MBI Fermentas (USA).

Chapter 2: Exosome mediated delivery of recombinant rL7/L12

Bacterial strains and clones

Brucella.S-19 and B. abortus 544 were obtained from Division Biological Standardization, IVRI, Izatnagar. pQEL7 clone was kind gift from Dr. Pallab Chaudhury, GEB Lab, Division of Animal Biotechnology, IVRI, India.

Experimental animals

Healthy female Balb/c mice, weighing 20 ±2 g, (4 to 6 weeks age) were obtained from Animal House Facility, CDRI, Lucknow. The animals were housed in Biosaftey level (BSL)-3 facility having free access to the pellet diet and water ad libitum. A set of two New Zealand White rabbits was procured from Institute’s Animal facility for raising hyperimmune sera. The experimental protocol was approved by the Institutional Animals Ethics Committee (Registration No. CPSEA 332).

Culture of B. abortus & E. coli

B. abortus 544 was grown and maintained at 37 °C on Tryptose phosphate agar for 48 hrs at 5% CO2. E. coli cells were grown in Luria Bertani (LB) medium at 37 °C with shaking at 150-200 rpm. For the preparation of LB plates, 1.5 % agar powder was added to LB medium prior to autoclaving. E.coli DH5 cells containing pTZ57R/T cloning vector were grown in presence of ampicillin 50 µg/ml while E. coli M15 cell containing pQE-31 vector were grown in presence of ampicillin 50 µg/ml and kanamycin 20µg/ml. Frozen stock of E. coli were prepared by resuspending mid-log cells in fresh LB medium supplemented with 20 % glycerol and store at -70°C.

Isolation of chromosomal DNA from B. abortus

B. abortus 544Maulana was grown Azad in Tryptose Library, phosphate Aligarh agar Muslim slant at 37University °C. Cells were harvested by pelleting them at 6000x g for 10 min. the pellet was suspended in TE buffer and washed thrice with 1X TE. After resuspending in 3.6ml of 1X TE containing lysozyme (10 mg/ml) 300 µl, 400 µl of 10 % SDS and 25µl of proteinase K (20 mg/ml) was incubated at 37 ºC for 90 min. Further 1ml of 5M NaCl was added and 800 µl CTAB/NaCl solution was added. The suspension was again incubated at 65 ºC for 15 min. The cell lysate was extracted once with phenol/ chloroform/ isoamyl alcohol (25:24:1) and centrifuged at 13000 g for 20 min and finally with chloroform: isoamylalcohol (24:1). The aqueous phase containing genomic DNA was

Chapter 2: Exosome mediated delivery of recombinant rL7/L12 precipitated with 0.6 volume of isopropanol at room temperature and spooled out with glass rod. The DNA was washed with 80 % ethanol, dried and redissolved in 200 µl of TE buffer. Finally, RNase (100 µg/ml) treatment for 1 h at 37 ºC followed by phenol: chloroform extraction and ethanol precipitation were carried out. The DNA pellet was washed with prechilled 70% alcohol and dissolved in water. The purity of DNA was determined spectrophotometricaly (OD260/280) while quantitative estimation was performed by using the following formula, Concentration of dsDNA (µg/ml) was calculated as: A260 X 50 X dilution factor.

Purification of recombinant rL7/L12 protein

Polyhistidine tagged (6xHis tag) fusion protein was purified under denaturation conditions by metal chelate affinity chromatography (Qiagen Protocol), with certain modifications introduced in the purification parameter.

Briefly, pellet obtained from 50 ml suspension of induced culture was resuspended in 4 ml of lysis buffer (buffer B) containing imidazole (20 mM), 1M NaCl, glycerol (20 %) and Triton X 100 (25 %). The incubation mixture was held at room temperature for 1 hour with intermittent mixing. The cell debris was removed by centrifugation at 10,000 g for 10 min. The supernatant was incubated with 1 ml of Ni+-NTA resin for 40 min and packed into column. Subsequently, the column was washed thoroughly with wash buffer (buffer C). The fusion protein was eluted in 0.5 ml x 4X with the elution buffer (buffer E). Five elutes were collected. The elutes were dialyzed against PBS to remove urea. The elutes were checked by SDS-PAGE as described earlier.

SDS-PAGE analysis of the protein

ExpressedMaulana recombinant Azad rL7/L12 Library, protein Aligarh was analysed Muslim by SDSUniversity-PAGE using method of Laemmli (1970), in a vertical mini-gel electrophoresis apparatus. To execute resolution the protein sample was run in 15 % resolving gel having a stacking gel of 5% concentration. The resolving gel was poured between the glass and over layered with water-saturated n-butanol. After gel polymerization, surface of the gel was washed with MilliQ water and 5 % stacking gel was added and comb was inserted carefully. After polymerization, the comb was removed and the wells were flushed with the electrode buffer. The eluted fractions were mixed with 2X Laemmli sample buffer (1:1), boiled for 5 min, cooled and loaded in the wells. Molecular weight

Chapter 2: Exosome mediated delivery of recombinant rL7/L12 marker proteins were prepared following the manufacturers protocol and loaded in one well.

Electrophoresis was carried out at a constant current of 18 mA, until the tracking dye reached the bottom of the gel. The gel was taken out from the plates and stained with 0.25 % (W/V) Coomassie brilliant blue R-250 prepared in methanol, glacial acetic acid and water (45:10:45) and destained with destaining solution (20 parts methanol : 10 parts glacial acetic acid and 70 parts distilled water).

Estimation of recombinant protein concentration

The concentration of recombinant rL7/L12 protein was estimated by BCA protein estimation kit as per the method described earlier.

Raising hyperimmune sera against recombinant rL7/L12 protein

Two New Zealand white rabbits (8-10 weeks old) were immunized to raise antibody against the recombinant rL7/L12 protein. Rabbits were inoculated subcutaneously with 100µg of immunogen in (0.5ml PBS, pH to 7.4) with equal volume of Freund’s complete adjuvant (CFA). The subsequent boosters of 50 µg immunogen were given subcutaneous at 2 weeks intervals. The rabbits were bled one week after third booster and sera were separated. The sera were stored at -20 °C till further use.

Western blot analysis of rL7 specific hyperimmune rabbit sera

Western blotting was carried out according to the method of Towbin et al., (1979). Purified r-L7/L12 protein was loaded on 15% SDS-PAGE and electro blotted on to the nitrocellulose paper (NCP) at 2 mAmp/cm2 constant current for an hour, the transfer was carriedMaulana out in Azad semi-dry Library, electro blot Aligarh apparatus Muslim containing University transfer buffer. The membrane was blocked with 2 % Bovine serum albumin for 4 hrs at 4 C and washed five times with PBS-T. The membrane was then cut into strips and incubated with (1:100) dilution of pre-immune sera in plastic bags on rocker for 2h at 4 C. The strips was washed with PBS-T and incubated with 1:1000 dilution of HRPO conjugated goat- antirabbit IgG antibody for 1 h on rocker at 4 C. The strips was finally washed three times with PBS-T and developed with Diaminobenzidine (10 mg/6 ml of D.W.) substrate in the presence of 30 % H2O2. After the optimum color was developed, the NCP was washed with excess water to stop the reaction.

Chapter 2: Exosome mediated delivery of recombinant rL7/L12

Generation of DCs

Bone marrow derived dendritic cell (BMDCs) were generated following published procedure as standardized in our lab (Morelli et al.,2001). BM cells were isolated from femurs of Balb/c mice. Erythrocytes were depleted by treatment with hypotonic lysis. Erythroid cells, natural killer (NK) cells, T and B lymphocytes and granulocytes were removed by incubation with mAbs (TER-119, CD3, B220, NK-1.1, Gr1, and IAb; BD PharMingen, San Diego, CA) followed by rabbit complement (Cedarlane, Hornby, Ontario, Canada). BM cells were cultured with RPMI-1640 (Life Technologies, Grand Island, NY), supplemented with 10% volume/volume fetal calf serum (FCS), glutamine, nonessential amino acids, sodium pyruvate, HEPES (N-2- hydroxyethylpiperazine-N-2-ethanesulfonic acid), 2-ME, and antibiotics. Finally, the incubation medium was supplemented mGM-CSF and mIL-4 (1000 U/mL) to facilitate growth of BMDCs.

Preparation of exosomes from DC culture supernatants

We isolated bone marrow–dendritic cells (BMDCs) from Balb/C mice following published procedure. On day 4, conditioned culture medium was replaced by fresh medium that was supplemented with cytokines and 10% volume/volume exosome- free FCS obtained by overnight ultracentrifugation (100 000g). The supernatant of cultured DC cells was collected on days 6 and 8 and centrifuged at 4°C at 300g (10 minutes), 1 200g (20 minutes), 10 000g (30 minutes), and 100 000g (60 minutes) (Raposo et al.,1996). Finally, the exosomes were washed in phosphate-buffered saline (PBS) and pelleted by ultracentrifugation (100 000g) for 2 hours. The amount of protein in the exosome preparation was assessed by BCA Assay (Sigma). Maulana Azad Library, Aligarh Muslim University Sucrose gradient

To obtain pure population, the exosomes were layered on a linear sucrose gradient (0.25-2 mM sucrose and 20 mM N-2-hydroxyethylpiperazine-N-2-ethanesulfonic acid/NaOH, pH 7.4; Sigma-Aldrich). The gradients were centrifuged for 21 hours at 79 000g at 4°C. Eighteen fractions were collected, and the density was determined by refraction index measurements.

Chapter 2: Exosome mediated delivery of recombinant rL7/L12

Preparation of exosomes

Direct loading of exosomes with L7 antigen was achieved using the acid elution method as described by Hsu et al. Exosomes were mixed with 0.2 M sodium acetate at pH 5.2, with antigen at the concentration 10 µg/mL. The pH of the suspension was neutralized to pH 7.0 with 2M Tris-HCl (2.6% of total volume; Bio-Rad) of pH 11 and incubated at room temperature to allow reassembly of MHCII/antigen on exosomes. Unbound antigen was removed by filtering through 100-kDa UltrafreeBiomax filters (Millipore). The same amount of antigen as loaded on exosomes was filtered in parallel and the fraction above the filter was used as control for the removal of free form of antigen.

Immunization

The immunological studies were performed in inbred female Balb/C mice. Each group was consisted of fifteen animals. Various groups of mice were injected separately, through subcutaneous route, with a total three doses (immunization on day 0, 21, and 28) of exosome entrapped r L7/L12 [pre-treatment with Pam2cys] (Exo- L7-Pam2Cys), exosome entrapped rL7 protein (Exo-L7), Incomplete Freund’s adjuvant emulsified rL7 protein (IFA-L7), rL7/L12 protein alone (free form), sham exosomes (no antigen). Positive control group (vaccinal control) was immunized subcutaneously on day 0 with B. abortus S-19 (5X105 CFU/mice) while another set of animals (negative control) received only saline. Animals were immunized with priming dose 50 g/animal recombinant protein and boosted with 30 g/kg animal subsequently on day 21 and 28.

Collection of sera Maulana Azad Library, Aligarh Muslim University Serum was collected at weekly interval. Route of blood collection was retro orbital plexus of eye with the help of glass capillaries (size 90mm X 1mm). The volume of blood collected from individual mouse ranged from 100-200 µl at one time and kept in separate micro centrifuge tube having any anticoagulant. The blood was allowed to clot at 4 C for 1 to 2 hrs. After that the tube was kept at RT. Serum was retracted and collected in micro centrifuge tube and centrifuged at 10000x g to remove the lysed RBCs. Supernatant collected and kept at -20C for future use.

Chapter 2: Exosome mediated delivery of recombinant rL7/L12

Determination of antigen-specific antibody by dot immunobinding assay

The production of antigen-specific antibodies in sera of the animals immunized with various vaccine formulations were determined by dot immunobinding assay as per published protocol (Batra et al., 1989). Briefly, optimum concentration of rL7/L12 protein was blotted on nitrocellulose membrane with slot blot apparatus. The NC membrane was dried and blocked with 3 % BSA for 2 hr at 37 0C. After proper washing, the strip was incubated with antisera collected from immunized mice of different group at the dilution of 1:100. After usual washing, the membrane was incubated with 1:1000 dilution of HRP conjugated rabbit anti-mice antibody for 1hr on rocker at 4 oC. Finally, strips were developed with diaminobenzidine (10mg/6ml

D.W.) in presence of 20 l of 30 % H2O2.

Determination of antigen-specific immunoglobulin and their isotypes by indirect ELISA

Sera of the immunized mice were collected at regular interval to monitor the presence of antibodies by indirect ELISA method as described earlier.

CD4+T lymphocyte proliferation assay

One week after last immunization, four mice from each group were sacrificed and their spleens were removed under aseptic conditions. CD4+ and CD8+ T cells were prepared as described elsewhere (Mishell et al., 1980). CD8+ T cells were used as effector cells for CTL while CD4+ T cells were used in proliferation assay. CD4+T lymphocyte proliferation assay was performed as per the method described in the previous section. Maulana Azad Library, Aligarh Muslim University CD8+T lymphocyte response

Target cells

Balb/c mice were injected with thioglycolate broth. On day 4, the macrophages were isolated from the peritoneal exudate cells (PEC) by adherence on petri plates. The peritoneal macrophages were washed three times with HBSS and incubated at 37 C for 3 to 4 hrs with either free, exosome encapsulated forms of rL7/L12. The cells were again washed three times to remove residual antigen present in the supernatant.

Chapter 2: Exosome mediated delivery of recombinant rL7/L12

This was followed by incubation with 51Cr (100 Ci/2 x107 cells) for 45-60 min at 37 C. The cells were finally washed with RPMI solution and were used as target cells.

Cytotoxicity assay

The 51Cr-labelled macrophages (5x103/well) were used as target cells. The antigen primed target cells were incubated with CD8+ T cells (effector cells isolated from the spleen of the four mice, were pooled, and used for assay at an effector to target (E/T) ratios of 10:1 to 20:1. The target and effector cells were incubated at 37 C for 6 hrs, after completion of incubation period, the cells were pelleted at 3,000x g for 15 min at 5 C and the amount of released 51Cr was determined by measuring the radioactivity in the supernatant. The experiments were performed three times and the error bars represent standard deviation of the means from three different experiments. Total 51Cr release was calculated by treating an aliquot of the target cells with Triton X-100 (10 % final concentration). The spontaneous release of 51Cr in the supernatant was determined by incubating the labelled macrophages for 6 h. Amount of auto-release was subtracted from the total release to determine the extent of macrophage lysis. In most of the experiments, the auto-release was less than 25 %. The percent specific release was calculated as the (mean sample cpm-mean spontaneous cpm/mean maximum cpm-mean spontaneous cpm) x 100 %.

Detection of Gamma-Interferon (IFN-) level

Gamma interferon (IFN-) was estimated by antigen capture ELISA using BD Biosciences assay kit. Spleen cells from various immunized groups of animals were cultured and at concentration of 2x106 and then stimulated with recombinant protein (5 µg/well) inMaulana RPMI-1690 Azad containing Library, 10 % FAligarhCS. After Muslim48 hours of University incubation at 37 0 0 C in humidified atmosphere (5 % CO2) the plate was centrifuged at 400xg at 40 C and the supernatant were aliquoted. Gamma interferon was detected in triplicate by Enzyme linked immunosorbent assay using microplate, coated with capture antibody (anti mouse IFN-). After proper washing wells were blocked for 1-hour room temperature with 250 l of PBS (pH 7.0) containing 10 % FCA per well. Cell culture supernatant were plated in 1:10 dilution in blocking buffer and incubated for 2h at room temperature. The plates were washed five times with PBS–0.05 % Tween 20. Biotinylated Anti mouse IFN- and Avidin horseradish peroxidase conjugate were

Chapter 2: Exosome mediated delivery of recombinant rL7/L12 used according to the manufacturer’s instruction and evaluated by TMB substrate. The plates were then read at 450 nm within 390 min of stopping solution with 2N

H2SO4. All assays were performed in triplicate. The concentration of IFN- in the culture supernatants was calculated for each experimental group using a linear- regression equation obtained from the absorbance values of the standards.

Protection study against Brucella abortus 544 challenge

One week after last immunization, the mice belonging to various groups were challenged with a virulent culture of B. abortus544 (2X105 CFU / mice) intra- peritoneally in 0.2 ml of saline solution. After 7, 15- and 30-days post challenge, four mice from each group were sacrificed and spleens were processed as per the method described in previous section.

Statistical Analysis

The data were analyzed by one-way analysis of variance (ANOVA) following Student’s t test. P value of less than < 0.05 and < 0.01 were considered statistically significant.

2.3 Results

Purification of the ribosomal L7/L12

The recombinant rL7/L12 was expressed in E.coli. The expressed protien was purified employing Ni-NTA column. To determine the purity, antigenicity and specificity of purified recombinant rL7/L12 protein, Western Blot analysis was performed using antigen-specific hyperimmune rabbit sera, according to published procedure (TowbinMaulana et al., 1979). Azad Briefly, Library, purified Aligarh and dialyzed Muslim recombinant University rL7/L12 protein was run with prestain marker on 15% SDS-PAGE and subsequently transferred to Nitrocellulose membrane using semi-dry Electro blot assembly. After usual blocking and washing the membrane was incubated with (1:100) dilution of test sera in plastic bags on rocker for 2 hr at 4oC. The strips were washed with PBS-Tween and incubated with 1:1000 dilution of HRPO conjugated rabbit anti mice antibody for 1hr on rocker at 4oC and finally developed with diaminobenzidine in presence of 20l of

30% H2O2.

Chapter 2: Exosome mediated delivery of recombinant rL7/L12

Figure 2.1 Overexpression and purification of ribosomal L7/L12. SDS-PAGE showing expression of recombinant rL7/L12 protein and steps involved in its purification. Various lanes show the proteins at various stages of expression lane1; molecular weight markers, lane2; uninduced cell lysate, lane3; induced cell lysate, lane4, soluble fraction after sonication.lane5; Flow through. Lane6; wash1,lane7; wash2, lane8; partially purified ribosomal L7/L12 protein eluted from Ni-NTA column, and protein after purification from Superdex 200HR column, respectively, lane 9; rL7/L12.

Maulana Azad Library, Aligarh Muslim University

Figure.2.2 Western Blot analysis of recombinant rL7/L12 protein immunostained with anti-rL7/L12 sera raised in rabbit. Lane 1 Prestained protein marker; Lane 2 Probing presence of recombinant rL7/L12 protein as probed by rabbit anti-rL7/L12 antibodies.

Chapter 2: Exosome mediated delivery of recombinant rL7/L12

Figure 2.3 Representative TEM image of the as-formed exosomes TEM images of exosomes derived from BMDC. Exosomes were negatively stained with 2% uracyl acetate after removing the extra moisture. Cup-shaped structures, with 30–50 nm size were identified as exosomes.

Immunization with recombinant rL7/L12 evoke humoral immune response in the immunized animals

The vaccine potential of various rL7/L12 based vaccine was assessed on the basis of their potential to evoke antigen specific humoral response in the immunized animals. The sera of the immunized mice were analyzed for the presence antigen specific antibodies at various time intervals. There was non-significant antibody titre in the sera (1:200 dilution) in groups of mice that were immunized with free form of rL7 or its physical mixture with exosomes on day 14 post immunization. In contrast, Pam2Cys pre-treatment followed by administration of rL7 loaded exosome resulted in elevated antibody titre in the plasma of immunized animals (Figure 2.4). On day 21 post immunization, significant increase (A492≈1.110±0.180) in the antibody titre was observedMaulana in the animals Azad whichLibrary, were Aligarh primed withMuslim Exo-L7 University-Pam2Cys form of the antigen. The animals immunized with Exo-L7 showed elevated level of antibody titre

(A492≈0.880±0.210; P<0.05). The animals immunized with IFA-L7 showed relatively less antibody titre (A492≈0.480±0.095; P<0.001). On day 60-day post first immunization, the antibody titre was found to be further elevated in the animals immunized with Exo-L7-Pam2Cys mice (A492≈1.88±0.210), while there was slightly less titre (A492≈1.650±0.220) in the animals immunized with Exo-L7. Significantly less antibody response was evoked in S-19 (A492≈0.920±0.140) or IFA-

L7(A492≈0.840±0.120) immunized animals(P < 0.05).

Chapter 2: Exosome mediated delivery of recombinant rL7/L12

We determined level of IgG1 and IgG2a antibody isotypes in the sera of animals on various time points post first immunization. As shown in the Figure 2.5[A], there was higher IgG1 (A492≈1.120) and IgG2a (A492≈1.34) antibody isotypes level in the Exo- L7-Pam2Cys group. The mice immunized with Exo-L7 showed comparatively less antibody isotypes abundance (IgG1, A492≈0.980; IgG2a, A492≈1.18) (P<0.05). Among the two isotypes, the IgG2a level was more profused as compared to IgG1 counterpart in both Exo-L7/L12-Pam2Cys and Exo-L7/L12 immunized group of animals.

On day 45 post immunization, the animals administered with Exo-L7-Pam2Cys maintained significantly higher titre of IgG1 (A492 ≈ 1.48) and IgG2a (A492 ≈ 1.65) titre as compared with Exo-L7 (IgG1, A492≈1.32; IgG2a, A492 ≈1.46) immunized group (P<0.05, Exo-L7-Pam2Cys Vs Exo-L7) (Figure 2.5[B]). Both free L7 or its physical mixture with sham exosome failed to induce significantly higher level of IgG1 and IgG2a titre in the serum on day 45 days post first immunization. There was relatively less IgG1/IgG2atitre in these two control groups even on day 60 post immunization (Figure 2.5[C]).

Maulana Azad Library, Aligarh Muslim University

Figure 2.4 Total antibody response in sera of the mice immunized with various forms of Brucella rL7/L12 protein. The antibody titre in various immunized groups on days 14, 21, 30 and 45,60 (post first immunization) was determined by indirect ELISA method as described in materials and methods. The level of IgG was expressed as absorbance (A492) of the coloured complex developed in the immunosorbent assay. Each bar represents the mean of three determinations SD. P values: Control vs Ex0-L7<0.001; S-19 vs Ex0-L7<0.01; Ex0-L7-Pam2cys vs Ex0- L7< 0.05; IFA-L7 vs Ex0-L7< 0.01; Free-L7 vs Ex0-L7<0.001; Exo+FreeL7 vs Ex0- L7<0.001; Sham EL vs Ex0-L7<0.001

Chapter 2: Exosome mediated delivery of recombinant rL7/L12

Figure 2.5. Analysis of IgG isotype response in Balb/c mice immunized with various forms of Brucella recombinant rL7/L12 protein. The sera (1:100 dilution), isolated from various experimental groups on days [A]30,[B] 45 and [C] 60 post immunizations, were analyzed by indirect ELISA as described in materials and methods. The level of IgG isotypes was expressed as absorbance (A492) of the coloured complex developed in the immunosorbent assay. Each bar represents the mean of three determinations SD.P-values (on days 45 post immunization): Control vs Exo-L7-Pam-2-Cys<0.001; S-19 vs Exo-L7-Pam-2-Cys <0.01; Exo-L7-Pam-2-Cys vs Exo-L7<0.5; IFA-L7 vs Exo-L7-Pam-2-Cys <0.01; F-L7 vs Exo-L7-Pam-2-Cys <0.001;Maulana EL+L7 vs AzadExo-L7 -Library,Pam-2-Cys Aligarh <0.001; Sham Muslim EL vs UniversityExo- L7<0.001. CD4+- T lymphocyte proliferation response

The prophylactic potential of various in-house prepared vaccine was assessed on the basis of T cell proliferation. The cell proliferation was determined on the basis of 3H- thymidine incorporation in the proliferating T lymphocytes. The extent of proliferation was expressed as stimulation index (S.I.) showing the ratio of T lymphocyte proliferation in experimental versus control (RPMI only) group of animals induced at a particular concentration of antigen (0.1 µg/well). The

Chapter 2: Exosome mediated delivery of recombinant rL7/L12 proliferation of T lymphocytes in various immunized animals was found to depend on dose of antigen. It was observed that cell proliferation increases exponentially with increase in concentration of recombinant rL7 protein. On day 30 post immunization the immunization with Exo-L7 resulted in robust CD4+ T-cell proliferation (S.I. value: 9.20±0.98) as compared with the free L7 (S.I. value: 3.6±0.80; P<0.01) as well as S- 19 (S.I. value: 8.7±1.2 P<0.05) or IFA-L7 (S.I. value: 4.5±0.60) immunized mice (P<0.05) (Figure 2.6). The priming with Pam2Cys resulted in further increase in the T cell proliferation (S. I. value 10.50±0.90; P<0.01).Splenocytes collected from free antigen injected mice (F-L7) or sham exosomes (no antigen; Sham EL) or sham exosomes + rL7 (physical mixture) or animal immunized with PBS only failed to elicit detectable proliferative response against rL7antigen.

Maulana Azad Library, Aligarh Muslim University Figure. 2.6 CD4+ T cell Proliferation response of splenocytes derived from mice immunized with various forms of Brucella recombinant rL7/L12 protein:CD4+ T cell Proliferation response of splenocytes derived from control and immunized mice vaccinated with Exo-L7-Pam2cys, exosome entrapped rL7/L12 protein (Exo-L7), IFA emulsified rL7 and free form of rL7 protein. CD4+ T lymphocytes were co- stimulated with increasing concentration of purified rL7/L12 protein. After 72 h, [3H]-thymidine was added and its incorporation was measured after 16 h incubation by liquid scintillation spectroscopy. Saline vs Exo-L7-Pam-2-Cys<0.001; S-19 vs Exo-L7-Pam-2-Cys <0.01; Exo-L7-Pam-2-Cys vs Exo-L7<0.5; IFA-L7 vs Exo-L7- Pam-2-Cys <0.01; F-L7 vs Exo-L7-Pam-2-Cys <0.001; EL+L7 vs Exo-L7-Pam-2- Cys <0.001; Sham EL vs Exo- L7<0.001. 82

Chapter 2: Exosome mediated delivery of recombinant rL7/L12

The stimulation index (S.I.) was calculated by counting mean cpm values of stimulated culture/mean cpm values of unstimulated culture. Each bar represents the mean of three determinations SD. There was further augmentation in T cell proliferation in both Exo-L7 (S. I. Value 12.8 0.80) and Pam2Cys-Exo-L7 (S. I. Value 14. 80.60) immunized group on day 45 post first immunization (Figure 2.6).

CD8+ T lymphocyte response

Considering the fact that Brucella is an intracellular pathogen, its elimination could be executed by induction of pathogen specific CTLs in the immunized animals. Working on the same strategy, we assessed in-housed L7 based vaccine for their potential to evoke CTL response. The efficacy of generated CTLs was assessed on the basis of their ability to induce target cell lysis. The cytotoxicity assay suggested that immunization with rL7 entrapped in exosome generated cytotoxic T cells in the immunized animals. We observed significant increase target cell lysis upon increase in effector cells versus target cells ratio. We established CD8+phenotype of the as- generated effector cells by pretreatment with anti-CD8 antibodies. The antibody pretreatment abolished CTL response, while anti-CD4 antibodies did not abolish the target cell lysis (Table 2.1).

Table 2.1 . CD8+ T cell proliferation response of splenocytes derived from mice immunized with rL7/L12 loaded exosomes

Percent Chromium (51Cr) Release Exo-L7-Pam2cys Exo-L7

Effector: Target Ratio Effector: Target Ratio Pretreatment

Maulana 10:1Azad Library,20:1 Aligarh Muslim10:1 University20:1

Anti CD8+BR 7.2±1.2 8.4±0.90 6.4±0.80 7.5±0.85 Complement Anti CD4+BR 24.60±1.80 35.80±2.4 20.80±1.20 27.60±2.10 Complement BR 26.80±2.20 37.80±1.80 22.40±1.20 30.50±1.60 Complement No 27.80±2.20 39.50±2.30 23.80±1.20 33.40±2.20 pretreatment

Chapter 2: Exosome mediated delivery of recombinant rL7/L12

Potential of exosome-based antigen delivery system to evoke Th1 cytokines in the immunized host

The splenocytes belonging to various immunized groups were cultured with matching form of the antigen. Next, we evaluated potential of various forms of L7 based antigen delivery system to induce cytokine IFN-γ in the immunized animals. On day 35, the Exo-L7-Pam2Cys combination was found to express high level of IFN-γ (2.23±0.140 ng/ml), while Exo-L7 induced 1.99±0.14 ng/ml of IFN-γ (Figure 2.7[A]). The level of IFN-γ was found to further increase (4.14±0.340 ng/ml) in the group immunized with Exo-L7-Pam2Cys based formulation on day 60 post immunization (Figure 2.7[A]). The level of cytokine IFN-γ was also found to reach 3.6±0.08 in Exo-L7 immunized animals. Animals immunized with free form of antigen or sham exosome + rL7/L12 (physical mixture; Exo+ L7/L12) did not elicit significant level of IFN-γ (P<0.001). The control animals inoculated either with saline or sham exosome (no antigen) also failed to induce Significant cytokine IFN-γ. Similar were the trends in other cytokines IL-2 (Figure 2.7[B]) and also in the case of IL-12 (Figure 2.7[C]).

Induction of delayed type hypersensitivity reaction as a function of cell mediated immunity

We also assessed potential of various rL7/L12 formulations to induce cell mediated immunity in the immunized animals. The increase in footpad thickness was used as a parameter to assess DTH response in the immunized animals (Figure2.8). Briefly, various exosome-based forms of rL7/L12 protein was injected intradermally in right footpad of each mouse belonging to various matching immunized groups. The left foot pad was Maulana inoculated withAzad same Library, volume ofAligarh PBS and Muslim considered University as control. The swelling of foot pad was considered as a measure of induced CMI response. Footpad thickness after intradermal injection of rL7/L12 increased exponentially, attained maxima at specific time period. The mice immunized with Exo-L7-Pam2Cys underwent significant (P<0.01) rise in footpad thickness as compared with the other control groups. The Exo-L7 immunized mice also showed nearly same level of increase in footpad thickness at 24hrs post immunization, while in S-19 vaccinated mice, maximum increase in footpad thickness was observed at 48 hrs. The change in footpad thickness, observed in the mice belonging to control (PBS only) or those

Chapter 2: Exosome mediated delivery of recombinant rL7/L12 immunized with free antigen (F-L7) or sham exosomes (no antigen; Sham exo) or sham exosome + rL7/L12 (physical mixture; (P value < 0.001) was not significant.

Figure 2.7. Effect of immunization with various forms of rL7/L12 protein on production of various cytokine in Balb/c mice: The level of expressed cytokines was determined in the supernatant of spleen cell culture stimulated with matching form of antigen at various time points. The concentration of in the culture supernatant was calculated for each experimental group using linear regression equation obtained from the absorbance value (A450) of standards IFN-γ and expressed in ng/ml. [A]IFN- γ, [B] ILMaulana-2 and [C] Azad IL-12 Each Library, bar represents Aligarh the Muslimmean of three University determinations  S.D. Control vs Exo-L7-Pam-2-Cys<0.001; S-19 vs Exo-L7-Pam-2-Cys <0.01; Exo-L7- Pam-2-Cys vs Exo-L7<0.5; IFA-L7 vs Exo-L7-Pam-2-Cys <0.01; F-L7 vs Exo-L7- Pam-2-Cys <0.001; EL+L7 vs Exo-L7-Pam-2-Cys <0.001; Sham EL vs Exo- L7<0.001.

Chapter 2: Exosome mediated delivery of recombinant rL7/L12

Figure 2.8. Delayed type cell mediated immune response in mice immunized with various forms of Brucella rL7/L12 protein: DTH response was evaluated by measuring footpad swelling in the groups immunized with Saline only, S-19, Exo- L7-Pam2Cys, Exo-L7, IFA-L7, Free-L7, Exo+free L7, Sham Exo. DTH value (in mm) was expressed as the absolute increase in footpad thickness. p<0.001, p<0.001, p<0.0001 and p>0.05 represented as (*) (**), (***) and (NS) respectively.

Prophylactic potential of Exo-L7 based antigen against B.abortus544 challenge

The prophylactic potential of various recombinant rL7 bearing exosome vaccines was assessed on the basis of their ability to eliminate bacterial burden (B. abortus544) in spleens of immunized mice. The immunized mice were challenged with B. abortus544 infection. The animals were sacrificed on day 7, 15 and 30 post challenge with infection. The protection efficacy was represented as the log10 value of bacterial load to assess extent of protections. On day 7 post challenge with infection, the Maulana Azad Library, Aligarh Muslim University bacterial burden in unimmunized animals augmented upto log 5.21, while it was log 4.85 in Exo-L7-Pam2Cys immunized mice. The bacterial burden was log 4.90 in Exo- L7 immunized animals. On the other hand, immunization with IFA-L7 (log 5.10) or free form of rL7 (bacterial burden, log 5.20) imparted less degree of protection. The S19 immunized mice showed best protection (bacterial burden, log 5.20) (Figure 2.8).

On day 15 post challenge of the infection, mice immunized with Exo-L7-Pam2Cys and Exo-L7 protein showed significant (P<0.05) decline in bacterial load in the

Chapter 2: Exosome mediated delivery of recombinant rL7/L12 spleen. There was less reduction in bacterial load (from log 5.20 to log 5.10) in the animals immunized with free-L7 form of the vaccine. In contrast, S-19 vaccine showed remarkable decrease in splenic bacterial burden (from log 4.73 to log 3.91 units) (Figure 2.8).

On 30 days post infection, splenic burden of B. abortus 544 in Exo-L7-Pam2Cys vaccinated mice reduced to log 2.84, which was comparable to the protection efficiency conferred by S-19 (log 2.65) vaccine. The splenic B. abortus 544 burden in Exo-L7 group was log 3.10. The control immunized groups viz. IFA emulsified antigen, sham exo (no antigen) or physical mixture of sham exosome + rL7 as well as free form of the antigen failed to induce protection against B. abortus 544 infection (Figure 2.8). The protection data explicitly suggest that exosome encapsulated antigen evoke significant protection against Brucella infection in Balb/c mice. However, the golden standard S-19 vaccine was most successful in imparting protection against B. abortus 544.

Maulana Azad Library, Aligarh Muslim University

Figure. 2.9: Level of protection against B. abortus 544 infection conferred by immunization with various form of Brucella rL7/L12 protein. The mean CFU/spleen (log10) in four mice from each group was calculated on days 7, 15 and 30 post challenge with infection. Each line represents the mean of three determinations ± SD.

Chapter 2: Exosome mediated delivery of recombinant rL7/L12

2.4 Discussion

Inspite of availability of effective vaccines against brucellosis, the scientific community is still on surge of developing an ideal vaccine against this important disease. The presently available less efficient killed and whole cell-based vaccines against brucellosis fail to fulfil most of the criteria required in a model vaccine. Inspite of being attenuated, it is less likely to rule out that whole cell-based vaccine revert its virulence and inflict disease or persistent infection in the immunized animals. Moreover, live cell based vaccine could be pathogenic for humans as well. Infact, an ideal vaccine should be able to provide a strong type 1 T helper immune response in the host. Beside inducing a strong and long-lasting protection against systemic and uterine infection, it should not induce antibodies that interfere with the serological tests employed in the diagnosis of infected cattle

Being completely inert with predetermined composition, the recombinant subunit vaccines have numerous advantages. The co-production of subunit vaccine can be better controlled with much higher homogenecity. Keeping into consideration the fact that the success of vaccine depends on the selection of right antigen(s), adjuvant(s) and delivery system(s), we have tried to tailor a novel exosome-based vaccine against brucellosis. The efficacy of the as-developed vaccine was further enhanced by priming the host innate immune system with suitable TLR-2 agonist.

It is difficult to formulate an effective prophylactic strategy against Brucella species as the life style of the pathogen can be circumvented by induction of complex immune responses in the host. Considering the limited utility of antibody in killing of the facultative intracellular bacterium, protection against B. abortus can be acquired through cell-mediatedMaulana immunity Azad (CMI)Library, mainly. Aligarh Infact, Muslimin a manner University similar to other intracellular infections, brucellosis can be suppressed by generation of potent cytotoxic T lymphocytes in association with induction of a Th1 type immune response. The pathogen specific CTLs, induced in the immunized animals, can successfully kill B. abortus harboring inflicted cells.

The generation of pathogen specific CTLs is made plausible by processing of antigen in proteasome machinery of target cells followed by co-presentation of generated peptide along with MHC-I molecules. Most of the subunit-based vaccines failed to generate pathogen specific CTLs as it is difficult to deliver exogenous antigen to

Chapter 2: Exosome mediated delivery of recombinant rL7/L12 cytosol of the target cells. The development of an effective vaccines against Brucella infection implies both identification of major immunodominant antigen and an adequate delivery or adjuvant system to elicit appropriate immune response. In this regard, exosome-based antigen delivery system not only activate host dendritic cells, however, enforce cross presentation of pathogen specific peptide to activate CTLs in the immunized animals.

Several proteins have been tried as candidate antigen to impart effective immunity against brucellosis. The T cell based rL7/L12 protein upon supplementation with suitable adjuvant is likely to evoke appropriate immune response and can tender a suitable alternate to currently available live vaccine.

The employed recombinant rL7/L12 was tailored to possess histidine tag to facilitate its purification employing Ni-NTA column chromatography. The interaction of six- mer His tag acquire conformation that facilitates its interaction with the Nickel- nitrilo-acetic acid (Ni-NTA). The protein was allowed to incubate with Ni-NTA Agarose at pH 8.0. The undesirable cellular protein as well as nucleic acids were removed by manipulating pH conditions of the column. Finally, the elution was executed at pH 4.5. Addition of NaCl, Triton X-100 and glycerol removes most of the proteins linked with the 6X histidine tagged protein. Imidazole helps in removal of non-specific low affinity binding of the background proteins. Β-mercaptoehtanol helps in reduction of disulfide bonds that might be formed between the contaminating agent and recombinant protein. The purified rL7/L12 protein was dialyzed and characterized by SDS-PAGE. The recombinant rL7/L12 proteins had nearly 99% purity.

It is alwaysMaulana desirable Azad to employLibrary, cloned Aligarh gene productsMuslim in University development of subunit vaccines as they are free from other Brucella proteins as well as Brucella lipopolysaccharides etc. Immunoblot of recombinant rL7/L12 protein as well as B. abortus native antigen showed strong immunoreactivity with rabbit anti rL7/L12 sera. The interaction with specific antibody suggests that recombinant rL7/L12 expressed in E. coli was identical to that of B. abortus native protein (rL7/L12).

Other antigen delivery system viz. ISCOMs, virosomes, pH sensitive liposomes or fusogenic proteins, employed in development of vaccine cannot be recommended

Chapter 2: Exosome mediated delivery of recombinant rL7/L12 because of cost factor, toxicity as well as elicitation of immunological responses against structural components (protein and carbohydrate) of the delivery vehicles.

We found that immunization with exosomes-encapsulated antigen generates high serum antibody (IgG) titre at various time points as compared to the physical mixture of free L7 with exosomes as such or IFA adjuvanted delivery of the antigen (Figure 2.4). This is certainly an indication of superiority of the exosome-mediated delivery of recombinant protein than other form of the antigen. Interestingly, the magnitude of antibody production was highest in the group of mice primed with Pam2cys followed by administration of Exo-L7 based vaccine. Since the subclass of IgG response is generally regulated by the pattern of cytokines secreted by CD4+helper T cells, we assessed level of both IgG1 and IgG2a isotypes antibody generated against the various preparation of rL7/L12 protein in the immunized animals. Significantly higher IgG1 and IgG2a levels were detected in the sera of the mice immunized with Exo-L7 (Figure 2.5). Critical analysis of the data revealed the predominance of IgG2a over IgG1 in the animals immunized with exosome form of rL7/L12 protein.

The Exo-L7-pam2cys based exosome vaccine ensured higher induction of IgG2a level over IgG1 isotype. Infact, IgG2a isotype of the antibody has been reported to play strategic role in clearance of intracellular pathogens. The IgG2a isotype upon its binding with Fc receptors of the phagocytes activates a broad spectrum of antimicrobial response viz. cytokine synthesis, release of various inflammatory mediators, and generation of reactive oxidant species. The heightened induction of IgG2a isotypes over IgG1 has great deal of correlation with high abundance of IFN- in the animals that were immunized with Exo-L7-pam2cys and exo-L7 forms of the antigens. The T cell antigen Ribosomal L7/L12 employed in the present study has Maulana Azad Library, Aligarh Muslim University intrinsic property to activate T cells. The co-administration of L7/L12 antigen alongwith exosomes does not alter its intrinsic immune attributes. Nevertheless, the data of the present study suggest that exosomes-based delivery system elevate order of magnitude of desirable immune response in the host. The co-administration of Pam-2Cys alongwith exosome further potentiate adjuvant attributes of the as- developed antigen delivery system. The failure of both sham exosome as well as its physical mixture with preformed exosome to activate T lymphocytes clearly support the notion that the developed antigen response is antigen specific and the delivery

Chapter 2: Exosome mediated delivery of recombinant rL7/L12 system just accentuate the elicitation of immunological responses in the host. Keeping into consideration the potential of exosomes to deliver their content in to the cytosol of the APCs, our present study is mainly focused on its possible use as a successful delivery system of recombinant rL7/L12 protein against brucellosis.

In vitro proliferation of CD4+ T lymphocytes collected from Exo-L7-pam2cys immunized mice after their stimulation with the rL7/L12 protein suggests that vaccination with exosome entrapped protein could elicit strong T cell proliferation response. The immunization of animals with Exo-L7 resulted in relatively less lymphoproliferative response as compared to Exo-L7-pam2cys immunized animals (Figure 2.6). Nevertheless, the proliferation responses in both the exosomal formulation were significantly higher than that of S-19 or IFA adjuvanted groups. It seems that because of their particulate nature, could result in higher CD4+ T response but still that level remains less than Exo-L7-pam2cys immunized animals. The results of the present study clearly indicate that rL7/L12 protein antigen could adapt different mechanism to identify different components of immune system and evoke characteristics effector response depending on its delivery mode, for example, rL7/L12 entrapped in exosome elicits antigen specific CTL generation as well. Phenotype analysis of the effector cells were performed by pretreatment of the cells with anti-CD8+ antibodies, before using them for 15Cr release assay. The results revealed that such pretreatment abolishes CTL response, which confirms the CD8+ phenotype of the generated T lymphocytes by exosome mediated delivery of rL7/L12 protein. The type I and type II cytokines have characteristic effects on the development of T helper cell subsets, for example, type I cytokines promotes growth of Th1 subset; while inhibiting the development and activity of Th2 subset, an effect knownMaulana as cross regulation. Azad Library, For instance, Aligarh IFN-γ Muslim (secreted byUniversity the Th1) preferentially inhibits proliferation of the Th2 subset, while IL-4 and IL-10 (secreted by the Th2 subset) down regulate secretion of IL-12, one of the critical cytokines that regulate differentiation of Th1 subset. IFN-γ secreted by the Th1 subset in turn promotes production of IgG2a production by B-cells but inhibits IgG1 and IgE production. The recombinant rL7/L12 ribosomal protein previously shown to be an immunodominant antigen in cattle induced murine CD4+ T cells to express Th1 subset of cytokines. Our results showed that the IFN-γ level was significantly higher (P<0.001) in exosome entrapped protein immunization than the other experimental groups.

Chapter 2: Exosome mediated delivery of recombinant rL7/L12

The genesis and release of exosomes suggest its tendency to fuse with plasma membrane of the target cells. It is also likely that exogenous exosomes once internalized through active phagocytosis can fuse with inner leaflet of phagolysosomal compartment of antigen presenting cells including dendritic cells may facilitate release of the associated antigen in cytosol of the target cells. Once in the cytosol, the exosome-based antigen can undergo class I processing to copresent generated peptides for activation of CD8+ effector T lymphocytes.

The data of the present study suggest that exosomes are internalized efficiently by DCs. Although, targeting of exosomes to DCs depends on the surface display of specific ligands on the exosome that facilitate their interaction with DC, we have not worked out much on the elaborated mechanism involved in this process. The internalized exosomes are sorted into recycling endosomes and then through late endosomes/lysosomes. The proficient antigen presenting DCs cells process and present peptides derived from the internalized exosomes to T cells. Our results demonstrate that allogeneic exosomes, employed in the present study, are efficiently targeted, internalized, and processed by splenic DCs in vivo, and eventually cross presented allopeptides to specific CD4+/CD8+ T cells.

The safety and compatibility of as-formed exosomes can be ascertained by the fact that their incubation with antigen presenting cells did not hamper their physiological activities and they successfully process antigen to express them with appropriate major histocompatibility complexes.

2.5 Conclusion Maulana Azad Library, Aligarh Muslim University The overall immune response elicited against administered exosome based L7 antigen suggest that the proposed antigen delivery system can effectively deliver antigen to both class I and class II processing compartments of antigen presenting cells, thus generating a strong antigen-specific T cells. Such considerations are pertinent in designing exosomes-based vaccines to subdue intracellular pathogen based infectious diseases. In spite of the rapid advances in the strategies for controlling brucellosis, we are not able to develop effective vaccines against brucellosis. The vaccines such as SRB51 and B. suis S-2 have demonstrated some promise to control the disease,

Chapter 2: Exosome mediated delivery of recombinant rL7/L12 however lot of issues have to be settled about their efficacy and safety before their routine use. A great deal of efforts have been executed to establish successful Brucella subunit vaccine across the world. The immunoprophylactic outcome highlighted in the present study conclude that exosome-based delivery system may offer several advantages for its possible use as subunit vaccine against brucellosis.

Maulana Azad Library, Aligarh Muslim University

Chapter 3: TLR Agonist studded Nanosized formulation of rL7/L12

TLR Agonist studded nanosized formulation of Omp-19 antigen augments prophylactic potential against experimental brucellosis

3.1 Introduction

Brucellosis has been considered as a highly infectious and re-emerging disease with great public health and economic repercussion throughout the world (Mallick AI et al.,2007). The etiological agent, Brucella species is a Gram negative facultative intracellular pathogen that gets a hold of more attention because of its zoonotic nature, globally affecting more than 500,000 people on an annual scale (Pappas, G, et al., 2006). Despite elaborate control measures, human brucellosis is widely prevalent in endemic areas and remains a constant challenge in the developing countries (Gwida M et al.,2010).

With the bottle neck where available antibacterial agents have limited scope; fittingly supported in the present circumstances, prophylactic strategies draw a great deal of attention to control this important disease (Pasquevich, K.A et al.,2009). In the current scenario, the only recommended vaccines are live attenuated strains of Brucella (B. abortus S19, B. abortus RB51 and B. abortus R); unfortunately, they have several downsides including safety concerns, interference with diagnosis, prospect of causing human infection and propensity to induce abortion in immunized subjects when administered in pregnant animals (Pasquevich, K.A et al., 2009, Schurig, G.G et al.,2002). Concomitantly, interest has shifted to vaccines based on proteins, peptides or DNA expressing desired Ag that conquers with safety and efficacy. In this context, many immunodominant protein Ags including p39 (A.l- Maulana Azad Library, Aligarh Muslim University marari et al.,2001), Omp-19/16 [2], Cu–Zn SOD [7], L7/L12 (Singha, H et al.,2001;Bachrach, G et al.,1994; Oliveira, et al., S.C) etc. have been characterized in Brucella to engender prophylactic immune response. Although the employment of subcellular entities of pathogens represents attractive and safer alternative, unfortunately, many of these subcellular entities are only partially protective and thereby requires immunopotentiation to elicit desirable protective immune responses upon administration in the host (Alving, C.R et al.,2012). Adjuvants have long been used to potentiate immune response against soluble protein antigens. The inclusion of adjuvant in various immunization protocols provides an effective measure to initiate,

Chapter 3: TLR Agonist studded Nanosized formulation of rL7/L12 direct, and sustain the immunological responses evoked by soluble protein-based candidates (Maisonneuve, C et al., 2014). Nanosized delivery platforms viz. liposomes by mediating a slow and sustained release of the entrapped Ag (depot effect) accelerate induction of improved immune response against the associated Antigens. Besides, facilitating their avid uptake by APCs, liposome based delivery system can also avoid degradation in biological milieu, thereby contributes additionally towards induction of improved immune response (Henriksen-Lacey, M et al.,2010). Along these sides, earlier we and others have provided evidence in support of the concept that liposomes-based protein Ags evokes immunoprotective response against brucellosis (Goel, Det al.,2013).

Although the immunological responses evoked by the soluble protein based Ags could be manipulated by liposomal assemblages; however, there is always a scope to categorically influence the immune response by supplementation of pathogen associated molecular patterns (PAMPs) in the form TLR agonists.

With the better understanding of the innate immune response and their role in orchestrating adaptive immune responses; large body of literature suggests that triggering host immune components through pattern recognition receptors (PRRs) arouse various pivotal events that embodies potentials to modulate/generate specific immune response (Pulendran, B. et al., 2006). Infact, it is generally argued that intrinsic possessions of varied bacterial constituents (PAMPs) in the traditional vaccines bequeath them the appropriate potential to stimulate the host immune responses (Rueckert, C. et al., 2012). Accordingly, various molecular entities either synthetic or naturally acquired especially functionalized TLR agonists have remained the focus of recent adjuvant research endeavours. Signalling through dipalmitoyl-s- Maulana Azad Library, Aligarh Muslim University glyceryl cysteine (Pam2cys), a sparingly soluble synthetic analogue of the lipid component of MALP-2 and a well-recognized agonist of TLR-2 has been known to activate host system especially dendritic cells (DCs).. Along this line of action, Pam2cys plays role towards development of Th1 polarized response (Jackson, D.C et al., 2004); a feature of fundamental importance to combat intracellular pathogens (Cassataro, J et al., 2005 ). Reckoning with the above detailed facts, herein, we investigated the repertoire of immune response influenced upon administration of Brucella Ag along with Pam2cys in nanosized delivery systems. As a candidate Ag, we selected recombinant unlipidated outer membrane protein19 (rU-Omp19) of

Chapter 3: TLR Agonist studded Nanosized formulation of rL7/L12

Brucella, a T cell Ag which has been regarded as immunoprotective against brucellosis in BALB/c mice (Pasquevich KA, et al.,2011). Besides evaluating the immunological responses influenced under adjunctive ambience, we also endeavored to establish their prophylactic potential against B. abortus 544 infection in model animals.

3.2 Materials and Methodology

Chemicals, reagents and Antibodies

All the reagents employed in the study were of the highest purity available. Egg PC, Sepharose–polymyxin B, BCA protein estimation kit were purchased from Sigma- Aldrich (St Louis, USA), cholesterol (Chol) was procured from Centron Research Laboratory (Mumbai, India).

Fluorochrome labelled anti–mouse antibodies (Abs): fluorescein isothiocyanate labeled CD4 (GK 1.5), CD8 (53.67); phycoerythrin labelled CD44 (IM7), CD80 (B7- 1), CD86 (GL-1); PerCP labelled CD62L (MEL-14), and isotype control IgG2a (R35- 95) were purchased from e-Biosciences (San Diego, CA). IgG, IgG1, IgG2a isotype kit; Cytokine detection kits were purchased from BD Biosciences (San Diego, USA). All tissue culture media, reagents and plasticware were procured from BD Biosciences (San Diego, USA). [3H]-thymidine was procured from BARC, Mumbai, India and Pam2Cys from Invivogen(Toulouse,France). Incomplete Freund’s Adjuvant was obtained from Bangalore Genei, India.

Bacterial strain

Brucella abortus 544 and Brucella strain 19 were a kind gift from Division of Maulana Azad Library, Aligarh Muslim University Standardization, Indian Veterinary Research Institute (IVRI), Izatnagar, India.

Cloning, expression and purification of recombinant unlipidated Omp19

Bacterial strain and plasmid

Brucella abortus 544 was cultured on Tryptose phosphate broth (HiMedia, Mumbai,

India) for 48 hours at 10 % CO2 atmospheric pressure. For rU-Omp19 purification, a pET 22b+ expression vector (Novagen, Madison, WI) was used. E. coli JM109 was used as vector host. E.coli BL21 (DE3) (Stratagene, LaJolla, CA) was used for

Chapter 3: TLR Agonist studded Nanosized formulation of rL7/L12 expression of the recombinant protein. Bacterial strains were cultured in Luria Bertani, Miller (HiMedia, Mumbai, India) media having requisite amount of antibiotics according to the standard reference (Sambrook, J.2001).

Primer design

Primers containing NdeI and XhoI restriction sites at the 5’ ends were designed as per the published sequence of rU-Omp19 of Brucella abortus. The primer sequences were as follows: forward p3-5’ CC TGGCCATATGC CAGAGC TCCCG-3’ and reverse p2-5’-AAACTCGAGGCGCGACAGCGTCAC-3’.

Cloning of rU-Omp19 in bacterial expression vector

Cloning, expression and purification of recombinant unlipidated Omp19 was carried following protocol described elsewhere as standardized in our lab (Tibor, A1999). In brief, Brucella abortus DNA encoding rU-Omp19 gene was amplified by PCR. The resulting product was cloned in the aforementioned expression vector giving rise to pET-Omp19 construct with C-terminal 6×His-tag which was subsequently employed for transformation of competent E.coli BL21 (DE3). The rU-Omp19 was expressed following successful induction with IPTG (1mM). The rU-Omp19 was extracted from bacterial cytosol by sonication and purified employing Ni-NTA resin. Moreover, to eliminate interference by LPS, the protein solution was adsorbed with sepharose– polymyxin B. The purified rU-Omp19 was quantitated by BCA and finally analyzed by SDS-PAGE and western blot assay. The protein was aliquoted and stored at -800C until further use.

SDS-PAGE and western blot analysis of rU-Omp19 Maulana Azad Library, Aligarh Muslim University Various protein fractions viz. rU-Omp19, uninduced culture lysate, induced culture lysate was electrophoresed in an electrophoresis apparatus (Biorad, CA, USA) using 15% polyacrylamide gel by applying a constant current of 18mA.The gel was analyzed by silver staining. For western blot experiment, protein fractions corresponding to rU-Omp19 purified protein (PP) was resolved on 15% SDS-PAGE and electroblotted to PVDF membrane (Millipore, MA, USA). Thereafter, the membrane was block with 3% BSA in 20mM phosphate buffered saline (pH 7.4), containing 0.05% Tween-20 (PBST). Subsequently, the membrane was washed with PBST and incubated with the Abs raised in rabbit against rU-Omp19 for 3 hours at

Chapter 3: TLR Agonist studded Nanosized formulation of rL7/L12

37°C. After usual washing step with PBST, the membrane was finally incubated with HRP conjugated secondary Abs for 1 hour at 37°C. The blot was thereafter developed employing enhanced chemiluminescence detection system (GE Biosciences, Pittsburgh, PA).

Preparation of Dried rehydrated vesicles (DRVs)

Liposomes encapsulated rU-Omp19 (Lip-Omp19) was formulated following the published protocol as standardized in our laboratory (Kirby, C. 1984). In brief, egg PC/cholesterol (2:1 molar ratio, total 30 mg) were dissolved in mixture of chloroform and methanol, and reduced to form thin dry film under N2 atmosphere using rotary evaporator. Thereafter, the thin lipidic film was hydrated with normal saline followed 0 by brief sonication at 4 C under N2 atmosphere. The liposomal formulation thus formed was mixed at this stage with an equal volume of rU-Omp19 (10mg/ml) and the suspension was flash frozen and thawed several times and finally lyophilized. The lyophilized mixture was then resuspended with normal saline to obtain dry reconstituted vesicles. Finally, the formulation was centrifuged at 14,000×g and the pellet was further washed with PBS to remove the traces of the unentrapped solute. The protein Ag entrapped in the liposome was estimated by BCA method as detailed in our previous studies (Chauhan, A., 2011). The level of entrapment was around 40- 60%.

Taking account of the fact that proper formulations of adjuvants are paramount in maintaining their stability and activity (Orr, M.T., 2013); we envisage development of liposomal formulation of Pam2cys so as to foster a system with coveted benefits of circumventing solubility issues along with increasing half-life which could provide adequateMaulana signals Azad for modulating Library, the Aligarh immune Muslim system.....For University the preparation of Pam2cys bearing liposomes (Pam2cys-Lip), egg PC/cholesterol/Pam2Cys (2:1:0.4 molar ratio, total 30 mg) were dissolved in mixture of chloroform and methanol and thereafter subjected to same procedure for preparation of lipid vesicles as detailed above, except for mixing with the Ag solution.

Chapter 3: TLR Agonist studded Nanosized formulation of rL7/L12

Experimental Animals and Immunizations procedure

Animals

Inbred female BALB/c mice (4-6 weeks old), weighing 20±2 gm were obtained from the animal house facility of National JALMA Institute for Leprosy and Other Mycobacterial Diseases, Agra (India). After procurement, animals were acclimatized for 7 days under standard husbandry conditions: room temperature (22±3°C), relative humidity (65±10%) and 12-hour light/dark cycle. They had free access to standard dry pellet diet and water ad libitum under strict hygienic conditions. Animals were anesthetized with ketamine (5 mg/kg) and xylazine (4 mg/kg), prior to sacrifice.

Humane endpoints were undertaken for animals which survived at the finale. Animals were administered an anesthetic mixture of ketamine and xylazine through intraperitoneal route and then sacrificed via cervical dislocation.

Ethics Statement

Experiments involving bleeding, injection and sacrifice of animals were executed following the procedures approved by the Institutional Animal Ethics Committee constituted as per the recommendations of Committee for the Purpose of Control and Supervision of Experiments on Animals (CPCSEA), Govt. of India (http://moef.nic.in/division/committee-purpose-control-and-supervision-experiments animalscpcsea- 1). In all experimental procedures, efforts were undertaken to reduce pains of experimental subjects. Our approval ID was 332/CPCSEA, Ministry of Environment and Forest, Govt. of India.

Immunization Maulana Azad Library, Aligarh Muslim University For immunization procedures, various groups of mice were immunized by subcutaneous route with total three doses (immunization on day 0, 21 and 28) of antigenic formulation. Various vaccinated group are as follows: Group 1: Animal immunized with liposome entrapped recombinant unlipidated Omp19(Lip-Omp19); Group 2: Animal immunized with liposome entrapped recombinant unlipidated Omp19(Lip-Omp19) and liposomised Pam2cys (Pam2cys-Lip + Lip-Omp19); Group 3: Animal pre-stimulated with liposomised Pam2Cys in succession followed by routine immunization with antigenic formulation (Pam2cys-Lip [Lip-Omp19]) (prime

Chapter 3: TLR Agonist studded Nanosized formulation of rL7/L12 stimulated group) at the same site. Animals were immunized with priming dose of 50 µg antigenic formulations per animal and boosted with 30 µg of antigenic formulations per animals; whereas 10 µg of Pam2cys were used. Mice vaccinated with Brucella S19 (5X106 CFU/mice), Pam2cys-Lip and media served as control. At stipulated time intervals, animal groups were examined for immunological responses.

Determination of antigen specific total IgG and isotypes by ELISA

The level of Ag specific total IgG and its different isotypes were measured in the sera of the immunized mice by indirect ELIZA method as detailed in our previous studies (Ansari, M.A., et al., 2005). Briefly, polystyrene plates (Maxisorp, Nunc, Roskilde, Denmark) were coated with 100 µl of Omp19 Ag (5µg/ml) in carbonate-bicarbonate buffer (pH 9.5) overnight at 40C. After usual washing and blocking steps, the plates were incubated with log 2 dilutions of test and control sera at 370C.for 2h. After excessive washing of the plates, they were further incubated with 100 µl of (1:5000 dilution of stock) goat antimouse IgG, IgG1 and IgG2a Abs for 1h at 37oC. After the usual washing steps, 100µl of (1:5000 dilution of stock) HRP conjugated rabbit anti- goat Abs were added to each well and the plate was incubated at 370C.for 1h. The plate was washed again before addition of 100µl of substrate solution (6 mg OPD in 0 12 ml of substrate buffer with 5µl of 30% H2O2) and finally incubated at 37 Cfor 40 min. The reaction was terminated by the addition of 50 µl of 7% H2SO4. The absorbance was read at 490 nm with a microtitre plate reader (BioRad, CA, USA).

Splenocyte and T cell isolation

At stipulated time, animals from each group were euthanized and their spleens were aseptically isolated. Single cell suspension of splenocytes was formed following Maulana Azad Library, Aligarh Muslim University protocol described elsewhere (Mallick AI, et al.,2007). Briefly, splenocytes were obtained by maceration of spleen followed by centrifugation at 1400×g for 8 min at 40C. The pellet was thereafter resuspended in ACK lysis buffer (10-15 ml) for 10-15 minutes for the lysis of the RBCs and washed twice with HBSS. CD4+ and CD8+ T cells were isolated using MACS column (Milteyni Biotech, USA). The purity of CD4+ and CD8+ T cells was found to be more than 98% as analysed by FACS using Express Plus software provided by GUAVA, USA. About 2 µl was taken in a haemocytometer and the cells were counted. The cells were counted by using formulae:

100

Chapter 3: TLR Agonist studded Nanosized formulation of rL7/L12

Total No. of cells = 104 X Dilution factor X No. of cells counted × total volume of cell suspension

T lymphocyte proliferation assay

Lymphocyte proliferation assay was executed following published protocol (Owais, M. 2000). In brief, lymphocytes cell population at a density of 4X105/100µl were seeded in each well of 96 well flat bottom microtitre plates and cultured in presence of recombinant unlipidated Omp19, LPS, ConA; wherein LPS, ConA and complete RPMI media served as positive and unstimulated control groups respectively. Cells 0 were cultured for 3 days at 37 C with 10%CO2 in humid atmosphere and pulsed with 0.5Ci [3H]-thymidine/well (3H-Tdr, Specific actitiviy18 Ci/mole, BARC, India) for 16 hour before harvesting the cell with automatic cell harvester (Skartron, Tranby, Norway). [3H]-thymidine incorporation was measured by a standard liquid scintillation spectroscopy with a β-plate counter (WALLAC Oy, Turku, Finland).The stimulation index (S.I.) was calculated as mean cpm values of triplicate culture by following formula:

S. I. = [3H] thymidine counts of experimental group/ [3H] thymidine counts of Un-Stimulated group

Determination of cytokines levels by sandwich ELISA

The culture supernatants of splenocytes were collected after 48 hr of Ag stimulation and examined for the presence of various cytokines employing appropriate purified and biotinylated Ab pairs following manufacturer’s recommended protocols. Briefly, 50μl of the purified capture Abs were adsorbed on polystyrene microtitre plates in carbonate/bicarbonateMaulana buffer Azad (pH 9.5)Library, overnight Aligarh at 40C. PlatesMuslim were Universitywashed five times with PBST and blocked with 5% skimmed milk (prepared in PBST). After usual washing, 50µL of splenocyte culture supernatant (obtained from cultured splenocytes after 48 h) was dispensed in each well for detection of cytokines. After stipulated incubation period, the plates were thoroughly washed and incubated with biotinylated polyclonal goat anti-mouse detection Abs. After washing the plates with PBST, 100 μL of streptavidin-HRP was added to each well, and incubated for 30 min at room temperature. Thereafter, the plates were again washed with PBST (thrice) and developed with tetra methyl benzidine. The absorbance was read at 450 nm with a

101

Chapter 3: TLR Agonist studded Nanosized formulation of rL7/L12 microtitre plate reader (Bio-Rad, CA, USA). Titration curves of recombinant cytokines were used as standards for calculating cytokine concentrations in the samples tested.

Staining for co-stimulatory and memory surface markers

Splenocytes were harvested and stained for flow cytometric analysis as described elsewhere. Briefly, 1x106 splenocytes were washed thrice into FACS buffer (PBS with 1% BSA and 0.1% sodium azide). Thereafter incubated with Fc block (2.4G2) and FITC/PE tagged monoclonal Abs (CD4, CD8, CD44, CD62L, CD80, CD86 and isotype control) for 30 min at 4°C. Following incubation cells were washed, fixed with 1% paraformaldehyde and acquired employing a fluorescence activated cell sorter (GUAVA, USA). The data was analyzed with Express-Plus software. The total number of cells of a definite phenotype was calculated by taking the percentage of a gated cell type, as determined by flow cytometry, and multiplying by the total number of cells obtained per mouse, further divided by factor of hundred (Darrah, P.A et al., 2007)

Protection study against Brucella abortus 544 challenge

Protection experiments were executed as detailed earlier (Singha et al., 2011). In brief, following 21 days after last immunization, animals belonging to various immunized groups were challenged with Brucella abortus 544 (2X105 CFU/mice) by intra peritoneal route. Later on, on day 7 and day 30 following challenge, animals from each group were euthanized and their spleens were aseptically removed. The spleens were homogenized and their dilutions were prepared in TPB and plated. 0 Following incubation at 37 C with 10% CO2, colonies were enumerated and Maulana Azad Library, Aligarh Muslim University delineated as log10 CFU/spleen. For Brucella abortus S19 group, dilutions were prepared on TSA-YT with 0.1% Erythriotol for discriminating S19 and Brucella abortus 544. For histopathological studies, spleens from euthanized animals were aseptically removed and immersion fixed in 10% formalin and processed following our published protocol ( Ansari, M.A et al.,2011).

Statistical Analysis

The data were analyzed by one-way analysis of variance (ANOVA) following Student’s t test.

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3.3 Results

SDS-PAGE and Immunoblot analysis of rU-Omp19

The unlipidated rOmp19 was cloned and expressed in E.coli employing pET22b+ expression vector system and purified by affinity chromatography with Ni-NTA resins. Thereafter, the rU-Omp19 was characterized by SDS-PAGE and Immunoblot assay (Fig. 3.1). SDS-PAGE profile of the rU-Omp19 and cell lysate reveals distinct band corresponding to 19kDa (Fig. 3.1A). Further, to ascertain the immune-dominant properties including purity, antigenicity and specificity of the purified recombinant unlipidated Omp19; they were probed with the hyper immune sera raised against the rU-Omp19. Interestingly, the presence of distinct band formed as a result of specific interaction between anti-rU-Omp19 rabbit Abs and purified protein provides considerable evidence for the purity, antigenicity and specificity of the as purified rU- Omp19 (Fig. 3.1B).

Maulana Azad Library, Aligarh Muslim University

Figure 3.1. SDS-PAGE and Immunoblot profile of the inhouse purified recombinant unlipidated Omp19. SDS-PAGE profile of recombinant unlipidated Omp19 developed with silver staining: Lane 1 corresponds to protein molecular weight marker; Lane 2 corresponds to uninduced culture pellet; Lane 3 corresponds to induced culture pellet; Lanes 4–5 corresponds to puriﬁed unlipidated Omp19 (A). Western blot analysis of the purified unlipidated Omp19 following probing with anti- rU-Omp19 Abs as an evidence of the immunodominance of recombinant unlipidated Omp19 (B).

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Liposomised delivery of rU-Omp19 along with Pam2cys evokes potent humoral immune response with dominant IgG2a isotype

The generation of Ag specific Abs were examined in the sera of the various immunized groups. Apparently, immunization with Lip-Omp19 along with immunomodulator Pam2cys-Lip resulted in generation of higher humoral immune response at day 21 post immunization compared to Lip-Omp19 immunized groups (p<0.05) (Fig. 3.2A). Most importantly, prime stimulation regime i.e. pre- immunization with liposomised Pam2Cys followed by routine immunization with Lip-Omp19 resulted in highest Abs production. As evident from figure 2A, high Ab titre was maintained following day 45 post immunization in these vaccinated groups. Although Lip-Omp19 vaccinated group also further augmented the response; nevertheless, the level of augmentation was lesser compared to those conferred by immunization with Lip-Omp19 along with immunomodulator Pam2cys-Lip (p<0.05). Further, there were no noticeable Ab responses found in Pam2cys-Lip immunized group indicating that the immune response thus evoked was Ag specific.

Moreover, reckoning with the fact that the Th1 skewed immune response are required for prophylaxis against brucellosis, we determined the isotypes (IgG1 and IgG2a) of the developed Abs at both the time intervals. Intriguingly, mice vaccinated with Lip- Omp19 along with Pam2cys-Lip formulations elicited higher IgG2a isotype Abs (Fig. 3.2B), which was statistically significant compared to Lip-Omp19 immunized group (p<0.05). More interestingly, animals immunized under prime stimulation regime resulted in highest increment of IgG2a isotype Abs. Not unlikely the levels were maintained on day 45 post immunization as well. There was no significant distribution of IgG1 and IgG2a isotype in the animals immunized with PBS (data not Maulana Azad Library, Aligarh Muslim University shown). Of note, the higher distribution of IgG2a over IgG1 isotype in the immunized groups is suggestive of skew towards a Th1 type response.

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Figure 3.2. Analysis of humoral immune response and isotype distribution in immunized BALB/c mice under defined conditions. Sera from various experimental groups were collected at regular intervals and the presence of Ag specific Ab responses (A) and isotype distribution (B) were assessed by indirect ELISA method as described in the material and method section. Representative profile of total Ab titres and isotype distribution obtained at day 21 post immunization and day 45 post immunization are delineated. The data represent mean of three determinants ± S.D. and are representative of three different experiments with similar observation. The differences in immunological responses in various vaccinated groups were compared using student’s t test analysis and p < 0.05 was regarded as significant.

Liposomised delivery of rU-Omp19 along with Pam2cys induces Th1 polarized immune response

To further ascertain the Th1/Th2 polarization, the expression of Th1 (IFN-γ, TNF-α, IL-2, IL-12) and Th2 (IL-4, IL-6) cytokines were examined in the splenocyte culture supernatant of immunized animals (Figure. 3.3). It is persuading that immunization protocol involving Lip-Omp19 along with immunomodulator Pam2cys-Lip have predilection to skew the immune response towards Th1. More intriguingly, it was Maulana Azad Library, Aligarh Muslim University found that prime stimulation regime exhibited highest level of Th1-skewing as evidenced by remarkably higher expression of Th1 cytokines in these groups compared to Lip-Omp19 (p<0.01) (Figure.3.3). With regard to cytokine profile, splenocyte culture supernatants from Lip-Omp19 along with immunomodulator Pam2cys-Lip immunized mice induced higher levels of IFN-γ expression, a cytokine playing seminal role in curtailment of intracellular Brucella infection. Excitingly but not unlikely amongst these groups, Pam2cys-Lip [Lip-Omp19] immunized mice induced highest levels of IFN-γ expression (p < 0.01) followed by co-administered

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Chapter 3: TLR Agonist studded Nanosized formulation of rL7/L12 immunized animals (p < 0.05) compared to Lip-Omp19 immunized groups. Expression levels of IL-2, IL-12, TNF-α were also significantly higher in these group of immunized mice compared to Lip-Omp19 vaccinated mice (p < 0.01) (Figure. 3.3). Further, no appreciable levels of IL-6 and IL-4 were detected (Figure. 3.3) in any of the vaccinated groups. Moreover, reckoning with the role played by IL-10 in suppressing Th1 skewed immune response, we determine their expression in the immunized animals. Interesting, their expression were remarkably suppressed in the animals immunized with Lip-Omp19 along with immunomodulator Pam2cys-Lip; nonetheless, their levels were slightly augmented in Lip-Omp19 immunized mice.

Further, it should be noted that splenocytes from various immunized animals produced comparable and significantly higher levels of IFN-γ, TNF-α, IL-2, IL-12, IL-4, IL-6 and IL-10 upon stimulation with LPS (positive control group). On the contrary, no significant levels of either cytokines were detected in the group immunized with sham preparations signifying the immune response generated thereof were Ag specific. Also, no noticeable changes were observed in the negative control (media).This explicitly demonstrates that Pam2cys treatment inclined the immune response towards Th1 type; an immune response desirable for the elimination of intracellular pathogens as also corroborated by other authors (Cassataro, J., et al.,2005).Taken together, these results further substantiate the isotype profiling data.

Liposomised delivery of rU-Omp19 along with Pam2cys induces T cell proliferation in immunized animals

Reckoning with the fact that induction of cellular (Th1 biased) immune response plays a cardinal role in the clearance of intracellular pathogens; lymphocytes proliferationMaulana response Azad was Library, examined Aligarh in the Muslim immunized University groups. To this end, lymphocytes were stimulated with diverse forms of Omp19 to assess their vaccine potentialities. Splenocytes cell viability was around 95%. As evident from the Figure.3.4, immunization with liposomised rU-Omp19 along with Pam2cys-Lip resulted in enhanced rU-Omp19 specific T-cell proliferation. The S. I. value for the same was statistically significant (p < 0.05) compared to Lip-Omp19.

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Figure 3.3. Analysis of Th1/Th2 cytokines in splenocyte culture supernatant of immunized BALB/c mice under defined conditions. Th1/Th2 cytokines were determined in the splenocyte culture supernatant of immunized animals as detailed in the materials and method section; Representative profile of IFN-γ (A), IL-2 (B), IL- 12 (C), TNF-α (D), IL-4 (E), IL-6 (F), IL-10 (G) are delineated. The data represents mean of three determinants ± S.D. and are representative of three different experiments with similar observations. The differences in various vaccinated groups were compared using student’s t test analysis and p < 0.05 were regarded as significant.

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Intriguingly, lymphocytes from animals vaccinated under prime stimulation regime induced enhanced T cell response (S.I. 16.15±0.4) followed by co-administered group (12.8±0.3). Not surprisingly, S19 vaccinated group had S.I. value of 16.58 ±0.45. Lymphocytes isolated from animals immunized with liposomised Pam2cys (sham preparation) or PBS exhibited feeble proliferative response against Omp19 Ag (S.I.<2.5) (p value < 0.001) certainly demonstrating that response is Ag specific. Further, lymphocytes obtained from various immunized animals responded to the stimulant LPS (positive control).

Figure 3.4. T cell proliferation induced by diverse forms of Omp19 under defined conditions. T cells were isolated from spleens of immunized animals and proliferation was assessed by the levels of [3H]-thymidine incorporation as detailed in the materialsMaulana and methodAzad Library,section. The Aligarh data represent Muslim mean University of three determinants ± S.D. and are representative of two different experiments with similar observation. The differences in various vaccinated groups were compared using student’s t test analysis and p < 0.05 were regarded as significant.

Liposomised delivery of rU-Omp19 along with Pam2cys induces expression of co-stimulatory molecules and memory phenotype in immunized animals

Flow cytometric studies were executed to examine the expression of CD80 and CD86 co-stimulatory and memory markers in various immunized groups. Immunophenotyping data clearly depicts that animals immunized with Lip-Omp19

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Chapter 3: TLR Agonist studded Nanosized formulation of rL7/L12 along with Pam2cys exhibited higher expression of both CD80 (B7.1) and CD86 (B7.2) co-stimulatory molecules compared to Lip-Omp19 immunized groups (Figure.3.5). Intriguingly, amongst these, animals vaccinated under prime stimulation regime exhibited highest expression of both CD80 (21%) and CD86 (31%) co- stimulatory molecules compared to co-administered group in which the expression of CD80 and CD86 co-stimulatory molecules accounted for 19% and 24% respectively. Not surprisingly S19 immunized groups showed highest level of expression of both markers CD80 (46%) and CD86 (48%) amongst all the vaccinated groups; whereas PBS exhibited feeble expression accounting 3%, and 2.5% for CD80 and CD86 molecules respectively.

Since induction of T cell population endowed with memory phenotype is fundamental aspects of any vaccination strategy; we further determined the presence of memory marker in these immunized groups. Both CD4+ and CD8+ T cells obtained from animals immunized with liposomised rU-Omp19 along with Pam2cys exhibited typical effector memory (CD44highCD62Llow) as well as central memory phenotype (CD44highCD62Lhigh) although the effector memory phenotype was found to be more pronounced (Figure. 3.6). Concording results were obtained with S19 vaccinated animals as well; nevertheless, the magnitude of expression was higher compared to Pam2-lip and Lip-Omp19 immunized group. On the contrary, Lip-Omp19 immunized groups educed lowest level of memory enhancement on both the cell populations.

Cumulatively, our immunization protocol especially prime stimulation regime induced Th1 biased immune response endowed with memory phenotype that could later respond to Brucella infection and confer improved protection.

Liposomised Maulana delivery of Azad rU-Omp19 Library, along Aligarh with Pam2cys Muslim renders University protection against experimental brucellosis in BALB/c mice

Reckoning with the above observations, we sought to determine whether our immunization protocol could confer protection against Brucella abortus 544 infection. Not surprisingly, we found that mice immunized with Lip-Omp19 along with Pam2cys-Lip harbored lower bacterial burden compared to other immunized animals at various time points.

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Maulana Azad Library, Aligarh Muslim University

Figure 3.5. Analysis of CD80/CD86 co-stimulatory molecules on antigen presenting cells of the immunized BALB/c mice. Mice belonging to various immunized groups were analyzed for expression of co-stimulatory markers CD80 (B7.1) (A) and CD86 (B7.2) (B) by staining of target cells with specific Abs and subsequent analysis by flow cytometry as detailed in materials and methods

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Figure 3.6. Analysis of memory marker on CD4+/ CD8+ T cells population in the immunized BALB/c mice. Mice belonging to various immunized groups were analyzed for expression of CD4+ CD44high CD62Llow/high (upper panel) and CD8+ CD44high CD62Llow/high (lower panel) phenotype on gated T cell population by FACS as detailed in materials and methods. The graphs represents various immunized groups: Saline (A), Lip-Omp19 immunized group (B),Pam2Cys-Lip + Lip-Omp immunized group (C), Pam2Cys-Lip [Lip-Omp] immunized group (D), S19 immunized group (E).

Of note, on day 7 post challenge, Brucella burden in Pam2cys-Lip [Lip-Omp19] immunized mice was log 3.45 units, whereas it was log 4.10 in co-administered group. Interestingly, the level of protection conferred by these groups especially prime stimulated vaccinated group was comparable to that conferred by conventional S19 vaccinated group (log 3.85). (Table 3.1).

Further, on day 30 post challenge, splenic load of Brucella in S19 immunized mice reduced to logMaulana 3.40 unit, which Azad was Library, better to thatAligarh conferred Muslim by immunization University by Lip- Omp19 (log 4.25) group (p<0.05). The animals pre-immunized with liposomised Pam2Cys followed by immunization with Lip-Omp19 could further reduce splenic bacterial load to log 3.65 units; whereas co-administered group had a load of log 3.80 units. These results are suggestive of the fact that liposomal Ag along with the immunomodulator conferred early and enduring protection against Brucella infection in experimental animals akin to protection offered by Brucella abortus S19 vaccination.

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Further, we also analyzed the histological alterations particularly splenomegaly in the immunized mice post bacterial challenge (data not shown). Mice immunized with sham preparation and PBS exhibited evident splenomegaly; whereas spleen size was of the same order in animals from protected groups (mice immunized with Lip- Omp19 along with Pam2cys adjuvant in diverse mode) and healthy groups. Additionally, several foci and discolored superficial surfaces along with granulomas were observed in unimmunized infected groups. On the contrary, there were no evident lesions in protected animals.

TABLE 3.1. Protection against B. abortus 544 in immunized BALB/c mice

Vaccine Log10 CFU of B. abortus 544

DAY 7 DAY 30

PBS Only 5.55±0.15 5.85±0.65

Pam2Cys-Lip (No Ag ) 5.55±0.55 5.25±0.65

Lip-Omp19 4.65±0.35 4.25±0.85

Pam2Cys-Lip + Lip-Omp19 4.10±0.45 3.80±0.45

Pam2Cys-Lip [Lip-Omp19] 3.95±0.30 3.65±0.30

B abortus S19 3.85±0.25 3.40±0.25

Appraisal of protective efficacy engendered by our immunization protocol in BALB/c mice following challenge with B. abortus 544. The mean of CFU in spleen of experimental animals were determined at day 7 and day 30 post-challenge. The data representsMaulana mean of Azad three determinants Library, Aligarh ± S.D. and Muslim are representative University of two different experiments with similar observation. The differences in various vaccinated groups were compared using student’s t test analysis and p < 0.05 were regarded as significant.

3.4 Discussion

Brucellae, the etiological agent of brucellosis, represents considerable burden to the society; efforts are into practice to thwart the repercussions arising due to this emerging pathogen (Nicoletti, P et al.,1990). To this end, the significance of

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Chapter 3: TLR Agonist studded Nanosized formulation of rL7/L12 development of prophylactic measures especially those based on subunit vaccines that provides safer alternatives to live vaccines is well established (Siadat, S.D2012., Schurig GG 2002). Moreover, employment of adjuvants to improve their immunogenic potentialities is also widely recognized (Perrie, Y et al.,2008). Although, the magnitude of immune responses evoked by the soluble protein Ags could be influenced by employment of various nano-sized delivery platforms which bequeath various coveted benefits; nevertheless, there still remains a lacuna to develop adjuvanted subunit vaccine effective in prophylaxis against this important disease and research continues to explore the same. Here in the present study, we hypothesized and provided evidence in support of the concept that the supplementation of Pam2cys to Brucella Ag rU-Omp19 in nanosized particulate delivery system help in categorical stimulation of host immune response.

Immunization with Lip-Omp19 along with the immunomodulator Pam2cys-Lip elicited high Ab titres with development of IgG2a isotype Abs compared to Lip- Omp19 vaccinated groups(Fig. 2); which is indicative of Th1bias immune response, as B cell instructed in IFN-γ cytokine milieu favours development of IgG2a isotype (Champagne, P et al.,2001). The development of IgG2a isotype observed upon Pam2cys supplementation provides an indication of role played by the same in enhancement of IgG2a isotype switching, inturn supporting their role in skewing the Th1:Th2 balance towards Th1 profile(Zeng et al.,2002). The cytokine profiling data further ascertained the predominance of Th1 biased immune response in Pam2cys-Lip adjuvanted Lip-Omp19 immunized animals (Figure 3.3); a phenomenon requisite for efficacious prophylaxis against brucellosis (Goel, D., 2013). Fundamentally, Th1 contender viz. IFN-γ, which activates macrophages and shapes B cell responses for anticipated inhibitionMaulana and Azad clearance Library, of the Aligarh intracellular Muslim pathogens University are broadly considered critical in the battle against Brucella species (Cassataro, J et al.,2005). Interestingly, these components of the immune system have been predominantly augmented in animals immunized with Lip-Omp19 along with the immunomodulators Pam2cys-Lip (Figure.3.3).

Although the comprehensive picture of the protective immunity against Brucella spp. is feebly understood. Nevertheless, in analogy to other intracellular pathogens, orchestrated enrolments of both humoral Ab responses as well as CD4+ and CD8+ T

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Chapter 3: TLR Agonist studded Nanosized formulation of rL7/L12 cells responses corroborate well with prophylaxis against brucellosis; with the latter playing critical role in disease resolution (Cassataro, J. et al., 2005).

Intriguingly, both the cytokine as well as isotype profiling data indicates that the regime have propensity to incline the immune response towards Th1 type. Nevertheless, the aforementioned results were further substantiated and validated by the lympho-proliferation studies. Amongst the diverse vaccination strategy employed, Lip-Omp19 along with immunomodulator Pam2cys-Lip induced enhanced T cell proliferation; and infact, the response was comparable to the standard S-19 vaccine (Figure.3.4). Seemingly, this could probably due to TLR activation phenomena’s which modulates the drift of cytokine milieu and in turn provide the requisite signals for cell proliferations and in fact, it is this effect that plays utmost role in influencing the adjuvant effects of TLRs agonists.

Co-stimulatory molecules play major role in the cross talk between effector and target cells (Lenschow, D.J et al.,1996). Interestingly, animals vaccinated with Lip-Omp19 along with Pam2cys-Lip exhibited significant augmentation of CD80 and CD86 co- stimulatory molecules (Figure. 3.5). Upregulation of co-stimulatory molecules clearly suggests efficient priming of splenocytes after immunization with our vaccine preparations. Further, APCs prime T cells, leading to their activation and subsequent transition into the memory phase, a hallmark of effective vaccination strategy.

In fact, an intriguing feature of any successful vaccination approach is induction of memory responses. Concomitantly, the ability of Lip-Omp19 when administered along with Pam2cys-Lip to evoke memory responses (with prime stimulation regime eliciting more pronounced response) merits their candidature for future exploration. There Maulana was considerably Azad higher Library, percentage Aligarh of both Muslim effector University memory phenotype and central memory phenotype on both T cells populations in these groups of immunized mice (Figure.3.6). While generation of memory responses are basically fundamental aspects of live vaccines; nonetheless, the features that govern generation and maintenance of memory responses remain intriguing. It is argued that a robust primary response may perhaps corroborate with generation of a larger pool of memory T cells; additionally pertinent stimulation of potent APCs may also corroborate with establishment of memory response (Dutton, R.W et al.,1998). Fitting Immunopotentiating interaction with APCs together with pertinent immune

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Chapter 3: TLR Agonist studded Nanosized formulation of rL7/L12 stimulation mediated by Pam2cys-Lip adjuvanted Lip-Omp19 may possibly have facilitated a strong primary response and consequent memory response. Seemingly, a fundamental facet of this prophylactic strategy is generation of functional CD4+/CD8+ T cell responses with memory phenotype.

Finally, the improved and well-orchestrated immune response evoked by the in house developed vaccination strategy prompted us to execute protection studies. Interestingly, the enhanced Th1 response, lymphocyte proliferation, augmented expression of CD80/CD86 co-stimulatory molecules on APCs and induction of CD4+/CD8+ T cells endowed with memory phenotype correlated with the decreased bacterial loads in the Pam2cys-Lip adjuvanted Lip-Omp19 immunized animals (Table 3.1). Interestingly, mice vaccinated under prime-stimulation regime exhibited higher protection at each time points compared to other vaccinated groups. In consonance with the present study, our previous reports suggested that conditioning of the system with tetrapeptide tufstin conferred better therapeutic effects and bestowed successes in combating experimental murine candidiasis as well as aspergillosis following antifungal therapeutic regimen.

Of note, the systemic protective response thus evoked not only conferred protection against B. abortus, but also against other Brucella strains; which anticipate their superiority as broad spectrum vaccine candidate; nevertheless, it has to be established in the final hosts of Brucella spp. Considering the immune activation intricacies, we are indeed of opinion that pleiotropic effects are responsible for the protective efficacy engendered by the Lip-Omp19 in conjunction with the immunomodulator Pam2cys-Lip.

The considerationsMaulana that the Azad robust Library, protections Aligarh are conferred Muslim with University live vaccines are suggestive of the fact that persistence of Ags and possessions of molecular clues (PAMPs) are key features requisite for effective vaccination strategy. In view of these facts, the rationale we presently favour to explicate the enhanced immunogenicity of soluble Ags when administered along with immunomodulator in controlled release system is based on the premise that besides the additive effects of liposomal preparation in enhancing the intrinsic immunogenicity of rU-Omp19 Ag mainly by supporting sustained low-level continued perseverance of Ag (depot formation); innate imprinting by Pam2cys working in concordance with the liposomal system and

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Chapter 3: TLR Agonist studded Nanosized formulation of rL7/L12 intrinsic immunogenic Ag might have ensued into better protective immunity. One can argue that conditioning of the system with Pam2cys may plausibly have fostered an environment conducive for the generation of effective protective immune response. This is indeed in consonance with previous studies wherein immunomodulators conferred enhanced potentiation of already ongoing immune response or bestowed additional adjuvate ensuing in induction of better immune response culminating into better protection (Dietrich, J et al.,2007; Rao, M et al., 2011). Deitrich et al., in their study explicitly demonstrated that supplementation of BCG to liposomes-based subunit vaccines evokes a synergistic response ensuing in enhanced protection against subsequent M.tb infection and the reason they favour to explain the enhanced efficacy of the same has been attributed to the ability of BCG to act as adjuvants especially triggering through multiple TLRs. Of note, the BCG based adjuvant or triggering of innate immune responses by BCG has been exploited for other diseases as well including Schistosoma japonicum and Leishmania induced infections (Armijos, R. X. et al.,2004). Further, in consonance with this study, BRANDT et al., demonstrated that co-adjuvantion of DDA adjuvanted ESAT-6 with MPL conferred better protective immune responses compared to DDA adjuvanted ESAT-6 when employed solitarily. They have explicitly shown that the combination of ESAT-6 in DDA were ineffective in engendering immune responses to ESAT-6; whereas upon supplementation of MPL, the ESAT-6/DDA system was efficacious in bestowing protective immune responses. These studies indeed provide support to the finding of the present study.

Nonetheless, the thorough picture for the mechanistic insight of the protective efficacy offered in the present study has not been explicated with evidences; as of yet, the intricaciesMaulana of these Azad phenomena’s Library, areAligarh under constant Muslim vigilance University especially of those conferred under prime stimulation condition and so far remain intriguing. Nevertheless, it is interesting to note that the strategy especially prime stimulation regime conferred better protection against brucellosis; the distinctions offered by co- administered and prime stimulation regime certainly underlie their intrinsic differences to persuade the magnitude of the immune response they corroborate when employed as adjuvanting strategies for vaccines. As more and more is gleaned about their intricacies they could be instrumental in offering better vaccination strategy against brucellosis. Studies from our laboratory are underway to fully elucidate the

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Chapter 3: TLR Agonist studded Nanosized formulation of rL7/L12 mechanisms instrumental in induction of more favorable immune response by these liposomal-adjuvant amalgam especially prime stimulation regimes and to study its applicability with other Antigenic repertoire as well.

3.5 Conclusion

Cumulatively, the present study insinuates that Pam2cys when combined with Brucella Ag in nanosized delivery systems help in categorical stimulation of host immune responses. Recombinant unlipidated outer membrane protein19 (rU-Omp19) of Brucella, a T cell Ag which is regarded as immunoprotective against brucellosis in BALB/c mice is shown to act as a immunoprotective role when used in a liposomised form. Additionally, the study offers important information for formulating successful prophylactic approach against brucellosis which could lead towards newer arsenal of effective protein-based vaccination strategy against brucellosis.

Maulana Azad Library, Aligarh Muslim University

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TLR agonist mediated immunomodulation augments vaccine potential of BCG against Mycobacterium tuberculosis infection

4.1 Introduction

The disease tuberculosis remains one of the major health concerns across the world. Globally the estimated cases of tuberculosis encompass 10.4 million, while around 1.8 million tuberculosis patients succumb to death each year (World Health Organization., 2015). The only available BCG vaccine fails to impart long term protection against tuberculosis. Moreover, the growing trend in the rise of MDR, TDR and XDR strains of Mycobacterium tuberculosis (M.tb) along with the increasing incidences of tuberculosis in patients with HIV co-infection (because of the weakened immune system) has led to the worsened situation (Gandhi NR et al.,2010). With exponential emergence of the development of resistant strains of M.tb, the utility of the presently available antibiotics will be rendered ineffective in the years to come (Gilbert P et al.,2003). WHO predicts uprise in totally resistant strains to presently available antibiotics in the years to come. The evolving resistant strains have developed several mechanisms to evade the actions of most of the available antibiotics (Munita JM et al.,2016). The manipulations executed on the part of M.tb isolates range from mutations in the protein target, to influence upon the efflux systems for not allowing attainment of effective dose inside the bacterial cell etc. (Zhao X et al.,2001). The microbes can also synthesize molecules that render the specific antibiotics ineffective (Walsh CT et al.,2014). Under such circumstances, it has become obligatory to look for alternative methods to deal with issues that may counteMaulanar evolving AzadM.tb Library,infection. Aligarh While Muslim development University of more effective chemotherapeutic approaches that can overpower drug resistant isolates of M.tb looks relatively challenging, it is more pragmatic to opt for development of prophylactic strategies to combat tuberculosis.

M.tb accesses to the prospective host through airway passage and eventually find lung macrophages as a host rescue (Meunier I et al.,2017). Once engulfed by phagocytic cells, M. tb tries to evade the host armoury by manipulating phagolysosome fusion process (Stutz MD et al.,2018). Many a times M.tb increases its survival chances by entering in latent phase. The host immune system that has already sensed presence of

118 Chapter 4: Immunomodulator confer protection against M.tb infection invading M.tb may respond by the formation of granuloma which is comprising of the phagocytic cells that includes dendritic cells, macrophages along with fibroblasts alongwith B and T lymphocytes (Cheung LS et al.,2019). It has been proposed that the Th1 cells play central role in the formation of granuloma. Granuloma restricts spread of bacteria wherein it acts as a barrier to the dissemination, along with downregulation of inflammation in the surrounding tissue (Domingo-Gonzalez R et al., 2016). Besides harbouring antimicrobial factors, the granuloma also creates an oxygen deprived environment to check propagation of invading M.tb (Hatfull GF et al.,2014). Inspite of active role of Th1 cells, the granuloma formation also depends on the signals originated from the phagocytic cells specially the macrophages (Pahari S et al.,2018). In response to M.tb invasion, the activated macrophages express tumour necrosis factor alpha (TNF-) cytokine (Parameswaran N et al.,2010). There seems to be a great deal of correlation between development of the granuloma and TNF- expression in both humans as well as in the mice. The released TNF-, induces co- expression of chemokines in the stimulated macrophages. Further, the TNF- provides chemotaxis stimulus required in the recruitment of derivable immune cell population in the host (Yang T et al., 2018).

In this regard, upregulated expression of Interleukin IL-12 plays a crucial role in macrophage activation alongwith transformation of naive T cells into antigen specific Th1 cells (Jankovic D et al.,2010)

Another cytokine IL-2, produced by the Th1 cells, may propagate development of M.tb specific CTLs. The smart M.tb also manipulates STAT-3/IL-10 praxis to induce pathogen favouring anti-inflammatory ambiance (Abdalla AE et al., 2016). The increased production of IL-10 has been observed to play role in transformation of Maulana Azad Library, Aligarh Muslim University latent M.tb infections to its active form. Further, the cytokines IL-4 inhibits the growth of Th1 and Th17 cells in the infected host. In general, the anti-inflammatory cytokines are considered to be crucial in progression and pathogenesis of M.tb as these can downregulate the production of NO synthase as well (Sasindran SJ, et al.,2011). The inefficiency of BCG vaccine to prevent M.tb infections can also be attributed to the IL-4 production by the dendritic cells in response to BCG-infected monocytes (Kowalewicz-Kulbat M et al., 2018). However, it is premature to suggest that the mechanisms involved in the inhibition of Th1 responses and formation of

119 Chapter 4: Immunomodulator confer protection against M.tb infection granuloma are governed by the same factor. The host cell autophagy plays a protective role against infectious pathogens including M.tb (Gu X, et al., 2017). The cytokines TNF-α and IFN-γ play important role in induction of autophagy in the infected cells. In contrast, pro pathogen cytokines viz. IL-4 and IL-13 help M.tb to establish itself and prevent the formation of granuloma. These cytokines also play role in inhibition of autophagy. By manipulating autophagy related factors, it becomes possible to direct invading M. tb to lysosomes for their degradation and clearance (Mohareer K et al., 2018). Moreover, the antigen presenting ability of APCs (via MHC class I and MHC class II ) to T cells is also increased through the process of autophagy (Munz C et al., 2012).

TLRs have been characterized as one of the most vital components of pattern recognition receptor (PRRs) families in relation to the immune responses elicited by the antigen presenting cells (APCs) (Dowling JK et al.,2016). The binding of specific ligands on TLRs present on the APCs (namely macrophages(MΦ) and dendritic cells) leads to their maturation and the synthesis of inflammatory cytokines along with the transformation of naïve cells into mature T cells for evoking the acquired immunity (Kornete M et al.,2012). The activation of TLRs promotes innate immune responses that inturn ensues in activation of adaptive immune system. The TLRs activation can be manipulated to regulate host immune responses that favours imparting protective immunity against a given pathogen (Thaiss CA et al.,2014). It is important to choose the appropriate adjuvant or delivery platform for making the vaccine program a success. In the current study , we used glucosaminide aminoalkyl phosphates, which is a potent TLR-4 agonist , for its potential to activate monocytes and macrophages. The activated macrophages induce a pro inflammatory ambiance that helps in categorialMaulana activation Azad of IFN Library,- producing Aligarh Th1 cells. Muslim In fact, weUniversity have tried to maneuver existing BCG based vaccine where it was allowed to inflict allo-macrophages. The as- developed BCG harbouring allo-macrophages were used for immunization of the host animals.

The present study was conducted to check whether immunization strategy employing TLR-4 agonist in combination with the BCG vaccine alone or BCG inoculated allo- MΦ generate enduring T cell memory in the host. Mice were primed with the immunomodulator Glucosaminide aminoalkyl phosphates (GalA) before immunization with BCG harbouring allo-macrophages. The infection with BCG

120 Chapter 4: Immunomodulator confer protection against M.tb infection induces apoptosis in the host MΦ . The apoptotic bodies are formed as a process of autophagic activity of macrophages when they are co-incubated with the classical BCG vaccine. To further enhance the potential of BCG vaccine the animals were primed with immunomodulators GalA. Interestingly, TLR-4+BCG treated group demonstrated considerable augmentation in both CD4+ and CD8+ T cell pool, as evidenced by significant improvement in immune response and protection against M.tb. Importantly, the combination showed an improved immunogenicity and protective efficacy along with appropriate memory response and hence can be a potent future vaccination strategy against M.tb and other related diseases caused by intracellular pathogens. In conclusion, the proposed prophylactic approach provides new insight about the adjuvenation potential of TLR-4 and BCG combination and has implications in development of more effective prophylaxis against M.tb.

4.2 Materials and Methodology

Reagents

Mycobacterium culture medium viz. Middlebrook 7H11 medium, albumin, dextrose, oleic acid, Middlebrook 7H9 broth and catalase etc. were purchased from Difco laboratories (Michigan, USA). High capacity cDNA reverse transcription kit and PCR reagents were purchased from Applied Biosystems, Forster city, CA, USA. Fluorescein isothiocyanate (FITC)–conjugated rabbit anti human CD4+ and CD8+, PerCP conjugated rabbit anti human CD62L (MEL-14), phycoerythrin (PE)- conjugated rabbit anti human CD44 (IM7), anti-CD80 (B7-1), anti-CD86 (GL1), from BD-Biosciences. ELISA based Kits used for the estimation of various cytokines in plasma as well as in culture supernatants were also procured from BD Biosciences. GlucosaminideMaulana aminoalkyl Azad phosphates Library, (GalA) Aligarh were Muslim synthesized University as previously described by Johnson DA et al.1999. Stock solutions (1 mg/ml) were made in 0.2% triethanolamine (Sigma) (pH 7). For intravenous administration, GalA were injected into the lateral tail vein (BALB/c mice) or retro-orbital venous plexus in a volume of 100 l. The rest of the chemical reagents were purchased from Sigma Aldrich and some sourced locally. Tissue culture grade plastic-ware was purchased from Eppendorf, Hamburg, Germany.

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Ethics Statement

All experimental procedures involving animals, blood, infected cell lines and Mycobacterium tuberculosis strains were approved by the Institutional Animal Ethics Committees of JALMA, Agra. All the animal’s studies were performed under BSL-2 cabinet hood and BSL-3 animal facilities, in accordance with CPCSEA (Committee for the purpose of control and supervision of experiments on animals, Govt. of India) norms. The study protocol was approved by CPCSEA, Govt. of India (CPCSEA/332). All animals were observed for signs of illness, weight loss, injury, or abnormal behaviour by animal house staff twice on week days and only once on weekends. Humane painless handling of all experimental animals includes proper maintenance of living conditions and minimization of distress. The CPCSEA mandates, as formulated by institutional ethical committee, were considered throughout the experimental set up while handling the animals to minimize suffering of animals. Experimental animals were frequently managed and cared throughout the commencement of the study following acceptable standard mandates as specified by ethical committee approved by CPCSEA, Govt. of India.

The animals, survived after conclusion of the experiment were euthanized following humane endpoint mandates of the institutional animal ethics committee (CPCSEA, Govt. of India). The infected MΦ were treated with antibiotics followed by autoclaving to kill residual M.tb pathogens. The euthanized animals were autoclaved followed by incineration as per the recommended guidelines.

Mycobacterial strains and its culture

M. tuberculosis strains were grown in Middlebrook 7H9 broth supplemented with OADCMaulana and also contained Azad Library, 0.2% glycerol Aligarh and 0.05% Muslim Tween University-80 at 37°C in 5% CO2. For macrophage infections, recovered bacteria were plated on Middlebrook 7H11 agar plate that contained 10% oleic acid-albumin-dextrose-catalase. All experimental procedures involving live M. tuberculosis were performed under standard biosafety level 3 (BSL-3) laboratory practices while of M. smegmatis were performed in BSL-2 laboratory practices. Bacillus Calmette–Guérin (BCG) Mycobacterium bovis, Pasteur strain (TMC 1011, ATCC) was grown in Dubos base (without addition of glycerol) with 10% supplement, 5% bovine serum albumin (BSA), 2% dextrose (Sigma), and 0.05% Tween 20 on an orbital shaker at 37°C for 2 weeks before use. BCG was

122 Chapter 4: Immunomodulator confer protection against M.tb infection diluted with 1× PBS to 3×108bacteria/mL, estimated using McFarland standards (Sigma-Aldrich, St. Louis, MO) and confirmed by plating dilutions onto 7H11 agar plates (Remel, Lenesa, KS). Plates were incubated at 37°C for 3–4 weeks, and colonies were enumerated.

Immunization and Immunotherapy Schedule

Priming of animal with immunomodulator GalA : Various group of animals were pre- treated with liposomised preparation of GalA for three times on days -3 to -1. On day 0 animals were inoculated with macrophages harbouring BCG (gggM+BCG). The animals belonging to second group were immunized with BCG infected MΦ only (M+BCG). The groups of animals that were inoculated with only BCG or PBS served as experimental control groups.

Establishment of Infection and Determination of Residual Mycobacterial Loads

Two weeks post immunization, the mice belonging to various experimental and control groups were challenged with virulent M.tbH37Rv through aerosol route. A bacterial suspension (10ml) corresponding to 5 × 107 bacteria/ml was dispensed in nebulizer unit of the Aerosol Inhalation Exposure System (Glas-Col, USA). To evaluate the protective efficacy of vaccines, we determined the bacterial load in lungs and spleen of experimental animals at 2 weeks post immunization. The challenged animals were also sacrificed on 4- and 8-weeks post immunization. At stipulated time intervals, a minimum of 4 animals from each group were euthanized; their spleen and lungs were removed aseptically and homogenized in 7H9 media. Four different dilutions of prepared homogenate were plated onto 7H11 agar plates supplemented with OADC. Thiophene carboxylic acid hydrazide (TCH) at concentration of 2 mg/ml was added to Maulanainhibit the growthAzad ofLibrary, BCG in immunizedAligarh Muslim groups. All University the plates were incubated for 3–4 weeks at 37 °C in CO2 incubator (Galaxy 180S, Eppendorf,

Hamburg, Germany) with a constant supply of 5% CO2.After incubation, colonies were counted to calculate the bacterial load. Bacterial loads were interpreted and expressed as mean log10 CFU/g of lungs and spleen tissue of infected animal.

Isolation of splenocytes and its culture

The animals belonging to various experimental immunized groups were sacrificed by cervical dislocation. Splenocyte cell suspension was prepared as described previously

123 Chapter 4: Immunomodulator confer protection against M.tb infection

(Tufail S et al., 2015). Briefly, spleens were aseptically isolated from experimental animals, macerated and single cell suspension was treated with cold water for the lysis of RBCs. Subsequently, cell suspension was centrifuged at 1200g for 10 min, and the cell pellet was washed twice with 20mM PBS and re-suspended in RPMI 1640 medium containing 10% fetal bovine serum and 0.1% antimycotic cocktail. About 10 l of the cell suspension was taken in a haemocytometer to execute counting of the cells. The CD4+ and CD8+ T cell population was were purified using MACS column (Miltenyi Biotec, USA). The purity of CD4+ and CD8+ T cells was found to be more than 98% as analysed by BD verse flow cytometer using BD verse software provided by BD, USA. The cells were re-suspended in media for further immunological experimentation.

Lymphocyte proliferation assay

Lymphocyte proliferation assay was performed as described elsewhere (Tufail S et al., 2015). In brief, lymphocytes were isolated from the spleens of various groups of immunized mice, and cultured in triplicate at 2×105 cells per well with graded doses (1–50µg/well) of GalA in 200µL of RPMI 1640 medium supplemented with 10% fetal bovine serum in 96-well flat bottom plates. In the next set, splenocytes from various experimental immunized groups were incubated with a known amount of corresponding matching formulations of GalA. Splenocytes incubated with the medium alone were used as the control. After 60 h, the plates were pulsed with 0.5 Ci/well of [3H]-thymidine. After 12 h, the plates were harvested using filler-mate cell harvester (PerkinElmer Life Sciences), and the incorporated [3H]-thymidine was measured by a Top Count scintillation counter (PerkinElmer Life Sciences) and expressed as counts per minutes (CPM). Maulana Azad Library, Aligarh Muslim University Phenotyping analysis of T Lymphocytes and Mononuclear Phagocytes employing flow cytometry

Ex vivo re-stimulated splenic lymphocytes/phagocytes or PECs (unstimulated) were harvested and stained appropriate markers for flow cytometric analysis following a protocol provided by BD Biosciences. Briefly, 1×106 splenocytes were washed twice with FACS staining buffer (PBS with 1% BSA and 0.1% sodium azide). Cells were incubated with Fc block (2.4G2) or with FITC/PE/PerCP tagged monoclonal antibodies against , CD4+, CD8+, CD44, CD62L,for 30 minutes at 4 ⁰C. After

124 Chapter 4: Immunomodulator confer protection against M.tb infection washing, the cells were fixed with 4.0% paraformaldehyde (PFA). The flow cytometry data was acquired using FACS Aria-II platform with FACS Diva software (BD Biosciences). A minimum of 10,000 events were recorded for each sample. Data were further analysed with FlowJo (Treestar Inc.) software. The definite phenotype analysis [CD4+CD44highCD62Llow/high (CD4 Tmem), CD8+CD44highCD62Llow/high (CD8 Tmem) was deduced as percentage of the gated cell population, employing flow cytometry.

Statistical Analysis: Results were expressed as the mean ± SEM and data were analysed by means of one-way analysis of variance (ANOVA) and two-way ANOVA to assess the differences among various groups. Statistical calculations were performed with the help of Graph-Pad prism version 6.0, GraphPad software Inc. San Diego, California, USA. Significance was indicated as *** for p values <0.001; ** for p values < 0.01 and * for p values < 0.05. Data shown are representative of at least triplicate experiments. A value of p < 0.05 was considered to be statistically significant.

4.3 Results

TLR agonist GalA modulates pro-inflammatory response in the primed host

Pro-inflammatory cytokines that are secreted from immune cells, such as T cells and macrophages, orchestrate inflammatory signals that accentuate protective immunity against intracellular pathogens. Pro-inflammatory cytokines are predominantly involved in the upregulation of immune responses, including activation of macrophages, induction of apoptosis, and recruitment of additional immune cells. In contrast, anti-inflammatory cytokines secreted from immune cells, such as regulatory T cells and someMaulana macrophages, Azad Library, suppress inflammation Aligarh Muslim and evoke University (pro-pathogen) ambience in the host. However, excess pro-inflammatory response can sometime lead to inflammation and tissue damage in the host. In order to achieve desirable prophylactic immune response, a dynamic balance between pro- and anti- inflammatory mediators must exist and evolve over time.

The induction of immune responses towards the Th1 responses can be assumed on the basis of priming of animals with TLR4-agonists and the autophagic bodies of the macrophages generated upon its co-culture with BCG. Splenocytes from primed animals were harvested two weeks after the completion of priming and vaccination

125 Chapter 4: Immunomodulator confer protection against M.tb infection which was followed by challenge with the M.tb. Subsequently, the splenocytes were cultured to assess profile of signature anti-inflammatory as well as pro-inflammatory cytokines employing sandwich ELISA . The level of pro-inflammatory cytokines viz. IFN-γ (Figure 4.1[A]) and IL-12 (Figure 4.1[B]) was found to be elevated in animals which have been primed with GalA followed by vaccination with BCG harboring MΦ. The cytokine, IL-12, a pro-inflammatory molecule, is produced primarily by professional antigen-presenting cells (APCs), including monocytes/macrophages and dendritic cells (DCs). The master cytokine primarily activates natural killer (NK) cells on one hand and also induces differentiation of naïve CD4 + T lymphocytes to interferon-gamma (IFN-γ) producing T helper 1 (Th1) effector cells.

FigureMaulana 4.1 Cytokine Azad profile Library, in the Aligarh various immunized Muslim University groups. GalA stimulated infected macrophages produced significantly higher level of [A] IFN-γ, [B] IL-12 and [C] IL-2 in the immunized mice. Infected macrophages along with the adjuvant has been reported to promote Th1 polarization and suppress the Th2 type cytokine including. Statistical significance was determined as described in materials and methods. The data were analyzed with the Student’s t test and are shown as the means (± S.D.) of 3 independent experiments. P-value :p<0.01, p<0.001, p<0.0001 and p>0.05 represented as (*) (**), (***) and (NS) respectively.

The anti-inflammatory response is generally mediated by IL-4 and IL-10 cytokines. Immunized animals were assessed for their potential to produce pro- and anti- inflammatory cytokines at 4- and 8-weeks PC. The expression level of IL-12, was

126 Chapter 4: Immunomodulator confer protection against M.tb infection found to be significantly elevated in the GalAM+BCG immunized group both at 4- and 8-weeks PC. The expression of IFN-γ, the major effector molecule of Th1 type immunity, was also found to be significantly upregulated at both week 4 and week 8 PC. The TLR-4 agonist-based priming ensued in profuse induction of IFN-γ in the GalAM+BCG group, as compared with BCG alone. More importantly, the priming with GalA followed by vaccination with BCG harbouring macrophages-based immunization resulted in profound expression of pro-inflammatory cytokines as compared to M+BCG (no GalA priming) immunization group.

GalA priming based immunization protocol evoke robust lymphocyte proliferation in the immunized animals

To assess vaccine potential of various formulations of BCG, the proliferation of lymphocytes was measured in the immunized animals. The lymphocytes were isolated from the spleen of immunized mice at various time points. The proliferation of lymphocytes was found to be grossly depending on dose of the antigen (Figure4.2[A]). In the case of both BCG harbouring macrophages with or without GalA priming, the lymphocyte proliferation rate was significantly higher as compared to the group of animals that was immunized with BCG only (Figure4.2[A]). Mice belonging to the negative control (PBS) did not induce significant proliferation of lymphocytes. We also determined lymphocyte proliferation response at various time point viz. 2-weeks, 4-weeks and 8-weeks post-immunization using fixed dose of antigen (50ug). Among various immunized groups, mice primed with the immunomodulator GalA maintained a higher proliferative response at post-challenge, as compared to the free form of antigen (Figure 4.2[B]) .

GalA primedMaulana mice upon Azadimmunization Library, with Aligarh BCG evokes Muslim predominantly University IgG2a and IgG2b type antibodies

To determine the antibody isotype switching, we estimated level of IgG1 and IgG2a antibodies in the serum of immunized animals at various time points. The GalA+MBCG treated mice showed skewed upregulation of IgG2a antibody response as compared to IgG1 isotype (Figure 4.3).

127 Chapter 4: Immunomodulator confer protection against M.tb infection

Figure 4.2 T cell proliferation response in various immunized groups. (A) To determine the effect of increasing amount of GalA on proliferation of T lymphocytes; splenocytes,isolated from various groups of immunized BALB/c mice at two weeks post immunization time point, were co-cultured in the presence of increasing amounts (1.56 to 50 μg) of GalA in flat-bottomed 96 well plates. After 72 h, [3H]-thymidine was added to each well and its incorporation in multiplying cells was measured after 16 h incubation with liquid scintillation counting. The accumulation of 3H-thymidine in proliferating cells was determined and denoted in term of counting per minute (CPM) values of stimulated cultures to represent Ag specific stimulation. (B) Proliferation of specific T lymphocytes isolated from immunized animals at 2-weeks, 4 weeks and 8-weeks post immunization upon stimulation with a fixed amount (50μg) of PPD. Data represents the mean of three determinants ± S.D.

Maulana Azad Library, Aligarh Muslim University

Figure 4.3 Confirmation of Th1/Th2 polarization upon immunization GalA+MΦBCG based vaccine protocol. The sera of immunized animals were analysed for the evaluation of IgG2a:IgG1 isotypes by sandwich ELISA method. The data represent mean of three determinants ± S.D. and are representative of two different experiments with similar observation. Statistical significance was determined as described in materials and methods. P-value :p<0.01, p<0.001, p<0.0001 and p>0.05 represented as (*) (**), (***) and (NS) respectively.

128 Chapter 4: Immunomodulator confer protection against M.tb infection

As shown in Figure, the ratio of IgG2a to IgG1 in the BCG immunized group at post- challenge time points did not increase significantly. The ratio of IgG2a to IgG1 in GalA+MBCG immunized animals was found to be statistically significant at any time points when compared with the BCG. Furthermore, this ratio was further significantly upregulated in the group of mice primed with GalA at post challenge time points. The increased IgG2a titers at post challenge clearly correlate with increase in Th1 cytokine level which was further boosted upon challenge with M.tb and helps in the clearance of the pathogen.

Upregulation of Costimulatory Molecules on APC upon co-stimulation with GalA and BCG

M.tb is considered as one of the most tenacious pathogens in the history of mankind. The pathogen employs a strategy to survive where it downregulates the expression of co-stimulatory molecules CD80+, CD86+, and CD40 on the APCs. To assess the expression levels of various costimulatory molecules in response to various immunization protocols, we monitored the expression profile of co-stimulatory markers on antigen presenting cells belonging to various immunized groups. Co- stimulation is required for eliciting T-cell mediated response as its absence leads to weakened T-cell activation anergy. Interestingly, immunization protocol involving GalA formulation was successful in up-regulating expression of CD80+ (B7-1) (Figure 4.4 ) and CD86+ (B7-2) (Figure 4.5) co-stimulatory molecules as compared to immunization with BCG alone, on post challenge with M.tb infection. The expression level of CD80+ (B7-1) and CD86+ (B7-2) was more pronounced in primed animals as compared to BCG as such and also in the cases of macrophages harbouring BCG group of animals. Maulana Azad Library, Aligarh Muslim University GalA +MBCG induces enduring CD4+ and CD8+ T cell response in the host

A successful immunization protocol has potential to strengthen expansion of polyfunctional T lymphocytes in the immunized animals. We found profound upregulation in induction of antigen specific CD4+ T cells in the animals that were immunized with BCG harbouring macrophages. The priming of the animals with GalA prior to immunization with BCG harbouring macrophages boosted CD4+ T cells population several folds as compared to M+BCG (no GalA priming) as well as

129 Chapter 4: Immunomodulator confer protection against M.tb infection

BCG only immunized group (P-value). The intracellular pathogens are eliminated by active involvement of cytotoxic CTLs in the immunized animals. The immunization with M+BCG combination resulted in upregulation of CD8+ T cells in the immunized animals( percent CD8+ Tcell~ 170.8). The CD8+ T cells population was found to be elevated upto 28% in the group of animals where immunization with M+BCG combination was preceded with priming of animals with GalA for three consecutive days(day -3 to day-1)( p>0.05).

Maulana Azad Library, Aligarh Muslim University

Figure 4.4 GalA upregulates expression of costimulatory molecules CD80+ on antigen presenting cells. Target cells were isolated following method as described in methodology section on 4th week post challenge with M.tb infection. The level of co- stimulatory molecules CD80+ (B7-1) was determined by staining of target cells with specific antibodies and subsequent analysis by flow cytometry . CD86+ molecule has also been depicted in the form of bar diagram. Statistical significance was determined as described in materials and methods. P-value :p<0.01, p<0.001, p<0.0001 and p>0.05 represented as (*) (**), (***) and (NS) respectively.

130 Chapter 4: Immunomodulator confer protection against M.tb infection

Figure 4.5 GalA upregulates costimulatory molecules CD80+ /CD86+ on antigen presenting cells. Target cells were isolated following method as described in methodology section on 4th week post challenge with M.tb infection. Co-stimulatory molecules CD80+ (B7-1) was determined by staining of target cells with specific antibodies andMaulana subsequently Azad analysed Library, by flow Aligarh cytometry. Muslim The lower University panel depicts expression level in the form of bar diagrams. Statistical significance was determined as described in materials and methods. P-value :p<0.01, p<0.001, p<0.0001 and p>0.05 represented as (*) (**), (***) and (NS) respectively.

Memory response is required for long-term protection against infection. To evaluate the vaccine potential of GalA primed mice with BCG, to induce memory response , we isolated spleens from the various experimental and control animals and were evaluated at 2-weeks post immunization and also at 4- and 8-weeks post immunization. It has been observed that population of CD4+ (Figure 4.6) and CD8+ (Figure 4.7) T lymphocytes having central memory marker (CD44high and CD62Lhigh)

131 Chapter 4: Immunomodulator confer protection against M.tb infection

(Figure 4.8,4.9) and effector memory marker (CD44high and CD62Llow) (Figure 4.8,4.9) on the gated population of CD4+ and CD8+ cells increased significantly in animals primed with GalA and given BCG as compared to the free form of BCG and also macrophages having engulfed BCG as shown in Figure. Interestingly, pre- immunization with GalA further induces the population of CD4+/CD8+ T- lymphocytes with central and effector memory markers.

Maulana Azad Library, Aligarh Muslim University

Figure 4.6 Splenic CD4+ Proliferation Assay The recall response of T-helper cells was assayed following the vaccination. vaccine induced a strong antigen-specific proliferation of CD4+ splenocytes in response to GalA+MBCG. The maximum CD4+ cell proliferation for GalA+MBCG was higher (P < 0.05) than for BCG (P < 0.05) than for M+BCG . These data indicate that coformulation of GalA+MBCG significantly enhances the CD4+ helper T-cell response in comparison to either BCG alone. P-value :p<0.01, p<0.001, p<0.0001 and p>0.05 represented as (*) (**), (***) and (NS) respectively.

132 Chapter 4: Immunomodulator confer protection against M.tb infection

Figure 4.7 Splenic CD8+ Proliferation Assay The recall response of T-helper cells was assayed Maulana following the Azad boost Library, vaccination. Aligarh vaccine Muslim induced University a strong antigen- specific proliferation of CD4+ splenocytes in response to GalA+MBCG. The maximum CD8+ cell proliferation for GalA+MBCG was higher (P < 0.05) than for BCG (P < 0.05) than for M+BCG. These data indicate that coformulation of GalA+MBCG significantly enhances the CD8+ helper T-cell response in comparison to either BCG alone. P-value :p<0.01, p<0.001, p<0.0001 and p>0.05 represented as (*) (**), (***) and (NS) respectively.

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Maulana Azad Library, Aligarh Muslim University Figure 4.8 Augmentation of long-lasting CD4+ T cell (effector/central) response upon immunization of mice with GalA+MBCG . After 2 weeks post challenge (4-weeks post immunization), the splenocytes were isolated from various immunized groups and analysed for the presence of CD44high, CD62Llow/high on gated CD4+ T cells by FACS. Group [p [A] represent BCG [B] PBS [C] M+BCG [D] GalA+MBCG . The data were analysed with the Student’s t test and are shown as the means (± S.D.) of 2 independent experiments. P-value :p<0.01, p<0.001, p<0.0001 and p>0.05 represented as (*) (**), (***) and (NS) respectively.

134 Chapter 4: Immunomodulator confer protection against M.tb infection

Maulana Azad Library, Aligarh Muslim University Figure 4.9 Augmentation of long-lasting CD8+ T cell (effector/central) response upon immunization of mice with GalA+MBCG . After a week post challenge (4- weeks post immunization), the splenocytes were isolated from various immunized groups and analysed for the presence of CD44high, CD62Llow/high on gated CD8+ T cells by FACS. Group [A] represent BCG [B] PBS [C] M+BCG [D] GalA+MBCG . The data were analysed with the Student’s t test and are shown as the means (± S.D.) of 2 independent experiments. P-value :p<0.01, p<0.001, p<0.0001 and p>0.05 represented as (*) (**), (***) and (NS) respectively.

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4.4 Discussion

Dendritic cells, circulating in the peripheral tissues, play important role to evoke robust and sustainable adaptive immune response against infectious diseases. DCs engulf the pathogens and process the associated antigens followed by their presentation along with MHC molecules (Lewis JS et al., 2014). The pathogen primed DCs migrate to the lymphoid organs to activate the antigen-specific T-cells. Immature DCs have also been reported to interact directly with the M.tb followed by their maturation to activate the immune response. It has also been reported that the DCs play a central role in the development of immunologic memory against M.tb (Schorey JS 2015).

The activated DCs also modulate release pattern of various chemokines and cytokines. Once released, such cytokines recruit various immune cells and direct them to act against the invading pathogen (Galli SJ 2011). One of the most important actions of DCs is to keep monitoring the tissue microenvironment and also to interact with the cells undergoing apoptosis for the maintenance of the self-antigen tolerance (Munn DH,2016). The interaction with the self-antigens does not result in maturation of DCs that inturn does not elicit a T-cell response in the host. On the other hand, if the cell undergoes apoptosis due to some bacterial or viral infections, the DCs are alerted and carry out the process of antigen presentation through both the MHC class I and II pathway (Mueller DL. 2010).

M.tb exploits the short life of polymorphonuclear neutrophils (PMN) by inducing their apoptosis. This leads to activation of macrophages mediated pro-inflammatory response. Host evolves macrophage activation as a strategy to circumvents all the possibleMaulana strategies Azad to counter Library, the bacterial Aligarh invasion Muslim (Andersson University H, 2014). In this regard, M are modulated to express various proinflammatory cytokines including TNF-. TNF- has a multifaceted effect and enhances the intracellular killing of the M.tb and also modulate the formation of granuloma. However, it is important that TNF- should be produced in sufficient amounts as its low-level leads to disruptive immunopathology (Cavalcanti YV et al., 2012).

CD4+ T cells are crucial component of the host immunity and exerts an effector response against M.tb infections. The Th1 subpopulation of CD4+ T Cell is a major

136 Chapter 4: Immunomodulator confer protection against M.tb infection source of IFN-, which is the most vital cytokine to evoke effective immune response against intracellular pathogens. IFN- mediated activation of macrophages leads to activation of NF-kappa-, which regulates the transcription as well as release of pro- inflammatory cytokines in the host (Shen H et al., 2018).

BCG has been the main stay in offering protection against TB in children in the developing countries . However, the protective efficiency of BCG is relatively variable in case of adult subjects. It is tempting to speculate that adjuvant mediated modulation of host immunity can be beneficial strategy to improve efficacy of BCG against M.tb infection.

GalA, a synthetic immune modulator has the capacity to modulate the T cell helper responses necessary in the protection against M.tb. Previous studies have shown that structure analogs of GalA have the capability to promote cytokines necessary in the differentiation and activation of the immune response towards a particular T cell subset (Zhu J 2010). A vaccination strategy that evoke Th1 response in conjugation with pro-inflammatory cytokines can successfully suppress M.tb pathogen.

GalA and other TLR-4 agonists augments Th1 based immune responses in the host. Previous studies have shown involvement of T-bet, as well as IL-12 in signaling activity leading to expression of IFN- cytokine. The data of the present study suggests that priming of the animals with GalA facilitates induction of type I IFNs. IFN is are required for the optimization of the innate immune response that includes rapid lymphocyte proliferation. The master cytokine,IL-12, skews Th1 responses and upregulate production of IFN-γ (Figure.4.1). However, its relevance in in-vivo system depends on the nature of infection and employed immunization protocol. GalA and its Maulana analogs modulate Azad both Library, MyD88 Aligarh and TRIF Muslim signaling University and alter the Th1 responses along with production of IL-12 by the activated macrophages.

The vaccine strategy employed in the present study involves immunization of animals with BCG harbouring macrophages. It is known that interaction with macrophage derived apoptotic bodies induce maturation of DCs. We immunized the animals with BCG harbouring macrophages to increase the vaccine potential of BCG. The infected macrophages undergo autophagy mediated apoptosis. The as-formed apoptotic bodies

137 Chapter 4: Immunomodulator confer protection against M.tb infection avidly taken up by the DCs, that mediate class I processing and presentation of involved antigens.

The priming of animals with TLR-4 specific immunomodulator followed by immunization with BCG harbouring macrophage evoke robust immune response in the host. The classical “T-cell response” paradigm provides the framework for modulating the development of CD4+ T-cell memory. The T-cell response is comprised of three phases, which begin when mature naïve CD4+ T cells are activated by specific antigen in conjugation of appropriate costimulatory signals. Activation is followed by rapid clonal proliferation and differentiation that result in promulgation of functional effector CD4+ T cells in the expansion phase(Munz C et al., 2012). Memory CD4+ T cells are maintained in greater numbers than naïve cells and may persist for extended time period. The phases can be repeated upon antigenic rechallenge, thereby incite memory cells to undergo a second expansion phase that is remarkably more rapid than the primary expansion phase (Meunier I et al.,2017). The second phase helps in induction of secondary effector cells with enhanced functionality. In general, central memory persists after rapid clearance of acute infections, and is more effective in controlling secondary infections involving intracellular pathogens. On the other hand, the effector memory has been reported to be induced by chronic infections. The immunization protocol employed in the present study resulted in heightened expansion of both CD4+ and CD8+ T lymphocytes. The observed expansion of T-cell response post priming with GalA is suggestive of the continued low-level presentation of Ag to both the CD4+ and CD8+ T cells by APCs at later time points. This also predicts an Ag-depot effect offered by GalA+MBCG based immunization. This in turn results in production of a balanced central and effectorMaulana memory in Azad the host Library, (Figure 4.8) Aligarh. Muslim University

Naïve T cell activation takes place through the Ag specific TCR and co-stimulatory molecules signalling(Meunier I et al.,2017). For example, the interaction of CD28 on T cells with the costimulatory molecules CD80+, CD86+ on APCs has been well characterized. We observed that priming of animals with GalA before exposure with BCG (GalA+MBCG) ensues in activation of both M and dendritic cells. The activated APCs display upregulated copies of co-stimulatory molecules viz. CD80+/CD86+ on their surface (Figure 4.4, 4.5). In contrast to unstimulated

138 Chapter 4: Immunomodulator confer protection against M.tb infection splenocytes that express low levels of costimulatory molecules. TLR-4 stimulated splenocytes expresses increased level of costimulatory molecules on their surface. Such modulation resulted in effective APC function to induce T cell immunity. Pre- immunization with GalA induces a variety of signals by the induction of Th1 dependent manner which further enhance the costimulatory molecules in the GalA+MBCG immunized group of animals.

There was a substantially greater percentage of effector (CD44highCD62Llow) as well as central memory phenotype (CD44highCD62Lhigh) induced on both CD4+ and CD8+T cells belonging to animals immunized with M+BCG (Figure 4.8,4.9). The memory response further upregulated when immunized animals were primed with GalA before exposure to M+BCG based vaccine.

T cell proliferation studies further support Th1 bias of the delivery system as GalA+MBCG immunization protocol induced robust T cell proliferation in dose dependent manner. Among various forms, GalA+MBCG induced consistently increasing T cell proliferation as compared to BCG only based immunization. The present study demonstrates that priming of animals with TLR agonist before immunization with BCG can be successfully employed to achieve effective protection against intracellular pathogen Mycobacteria tuberculosis.

The generation of acquired immunity begins with the recognition of a series of events that ultimately prime T cell differentiation. It eventually helps in establishment of adaptive immune responses in the host. The present study provides a clear picture that pre-immunization with TLR-4 agonist followed by immunization with the M+BCG augment IL-2 (Figure 4.1[B]) and IFN- cytokine (Figure 4.1[A]). The cytokine Maulana Azad Library, Aligarh Muslim University profile present within a specific microenvironment regulate T-cell activation and differentiation. Recognition of pathogen-associated patterns via TLRs directs signalling pathways to modulate splenocytes in specific fashion.

Initially, it was thought that CD4+ T cells are of less importance as compared to CD8+ Tcells in relation to the M.tb infection. However, recent reports suggest that CD4+ Tcells play a crucial role in containment of pathogenesis of the disease. The antigen processing of the intracellular pathogens like M.tb can occur either through the cytosolic or endo-phagosomal compartment-based degradation. Cytokines like IL-2,

139 Chapter 4: Immunomodulator confer protection against M.tb infection

IFN-γ, and TNF-, that play a critical role in in inhibiting the M.tb infection have also been shown to be produced by CD8+ cells. Interestingly, CD8+ T cells kill the M.tb infected cells through the perforins, granzymes pathway. They can also induce target cell apoptosis through Fas-Fas ligand interaction. Our results show that priming with GalA followed by immunization with BCG induce a larger CD8+ Tcell repertoire in comparison to immunization with BCG only or M+BCG combination with no prior priming.

The GalA primed animals when subsequently immunized with M+BCG were successful in inducing higher IgG2a response as compared to IgG1. The higher IgG2a level was maintained post challenge with M.tb. infection in the group of animals that were immunized with GalA+MBCG (Figure 4.3). Th1 cytokine IFN-gamma can modulate class switching to B cells in favor of IgG2a isotype production. The immunization with BCG only resulted in balanced production of both IgG1 and IgG2a isotypes. However, BCG bearing M based immunization evokes immune response in favor of IgG2a isotype antibody mainly. The IgG2a/IgG1 response skewed in favor of IgG2a when M+BCG immunization protocol was preceded by GalA pretreatment of the immunized animals.

In case of BCG immunized group, IgG2a∶IgG1 ratio was almost equal but it decreased at 8th-week post immunization (Figure 4.3). Both cytokine, profile as well as antibody isotype data, suggest that pre-immunized with TLR4 agonist followed by BCG has bias towards Th1 response, a requisite for effective immunogenic and protective efficacy against M.tb.

Finally, we can infer that immunization with BCG harbouring allogenic macrophages Maulana Azad Library, Aligarh Muslim University induce robust immune response against M.tb infection. The efficacy of M+BCG based vaccine can be further improved by prior priming of experimental animals with TLR-4 agonist. The strategy has potential to subdue other intracellular pathogen as well.

4.5 Conclusion

In the present study, we explored the effect of TLR agonist to mediate immunomodulation in augmenting the vaccine potential of BCG against the intracellular pathogen Mycobacterium tuberculosis infection. Animals primed with

140 Chapter 4: Immunomodulator confer protection against M.tb infection

Glucosaminide aminoalkyl phosphates(, and immunized with Macrophages with or without BCG, have the capability to promote cytokines necessary in the differentiation and activation of the immune response towards a particular T cell subset., We envisaged to develop a vaccination strategy that would evoke Th1 response in conjugation with pro-inflammatory cytokines which can successfully suppress the M.tb pathogen. It has been established that priming of animals with an immunomodulator was successful in increasing the pro-inflammatory response as well as downregulating the anti-inflammatory responses. The result of the present study suggested that priming of animals with immunomodulator induce robust lymphocyte proliferation, enhanced Th-1 biased response (IL-12, IFN-γ) and production of antibody predominantly of IgG2a isotype response. The elevated expression of co-stimulatory markers (CD80 and CD86) on both antigen presenting cells and T lymphocytes, memory markers (CD44high, CD62Llow/high) on CD4+ and CD8+ memory T cells and reduction in the M.tb burden in the immunized group. Therefore, immunomodulation based immunization strategy open new vistas in the field of immuno-prophylaxis and provide the basis for the treatment of infection caused by other intracellular pathogens such as Listeria monocytogenes, Candida albicans, Salmonella species.

Maulana Azad Library, Aligarh Muslim University

141 Chapter 5: Immunomodulator trehalose 6,6'-dimycolate as vaccine against M.tb

Evaluating the immunomodulating potential of CLEC4E agonist trehalose 6,6'-dimycolate (TDM) in enhancing the vaccine potential of BCG against Mycobacterium tuberculosis infection

5.1 Introduction

Mycobacterium tuberculosis, the pathogen responsible for the pathogenesis of lung tuberculosis, has the property to modulate anti-inflammatory cytokines of the host to facilitate its safe survival (Smith I et al., 2003). This prehistoric disease, prevalent all over the world, inflicted 10 million patients in 2015 according to the WHO, along with killing of 1.8 million people due to infection. Inspite of existence of the traditional vaccine and widely used chemotherapy program the M.tb pathogen was successful in establishing itself in the human beings. This is mainly because of the rise of multi drug resistant (MDR) strains of the M.tb (Niemann S et al.,2009). The infection usually occurs when the aerosolized bacteria reach the lower respiratory tract along with the evasion of innate immune system of the host. This eventuality leads to the formation of granulomas which is the main characteristic disease lesion that ultimately transform to secondary tuberculosis (Peddireddy V et al., 2017). Since the infection occurs in the lungs, the alveolar macrophages are the first to be get inflicted. The progression of the disease depends on the response elicited by residual alveolar macrophages (Redente EF et al., 2010). Mononuclear phagocytes restrict replication of invading M.tb, much before involvement of specialized T cells to combat invading pathogens (Kaufmann SH et al., 2001). Successful restriction of M.tb within macrophages depends largely on fine tuning between pro- and anti- inflammatoryMaulana cytokines Azad (Etna Library, MP et Aligarh al., 2014). Muslim The infected University macrophages produce tumor necrosis factor (TNF-) and other chemokines which act as a chemical stimulus employed for the involvement of other immune cells at the site of infection. This eventually leads to formation of primary granulomas (Cilfone NA et al.,2013). Post formation of granuloma, the adaptive immune response come into action with accompanying translocation of dendritic cells to the lymph nodes (Yoneyama H, et al., 2001). Dendritic cells (DCs) are important subset of myeloid lineage that play a crucial role in clearance of intracellular infections (Mildner A et al., 2014). Although, dendritic cells themselves do not eliminate M.tb, they are crucial to mount an

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Chapter 5: Immunomodulator trehalose 6,6'-dimycolate as vaccine against M.tb effective anti-mycobacterial response by efficiently processing and presenting mycobacterial antigens to effector T cells (Shafiani S et al.,2010). Once the dendritic cells are activated they starts to produce an innate essential cytokine interleukin-12 (IL-12) which plays an important role in immunity against M.tb (Herrera MT et al.,2009). IL-12 regulates the differentiation of CD4+ T cells into T helper 1 cells which produce IFN-γ (Agnello D et al., 2003). The role of IFN-γ in progression of M.tb infections is well established. IFN-γ modulates the site of inflammation alongwith activation of macrophages for their microbicidal activity. This eventually led to the process of autophagy (Arango Duque G et al., 2014). Autophagy had great deal of correlation to infection by bacteria, viruses and parasites. It has been shown that autophagy regulate inflammation and offers a protective tactic against various pathogens including M. tb (Choi AM et al., 2013). It can be modulated by various cytokines. For example, it is stimulated by pro-inflammatory cytokines such as TNF-α and IFN-γ, and inhibited by Th2 cytokines IL-4 and the anti-inflammatory cytokine IL-10 (Ghadimi D et al., 2010). Primary survival trick of pathogenic M. tb is its tendency to subvert phagocytic pathway inside host cells to avoid its fusion with lysosomes (Deen NS et al., 2013). Autophagy targets M. tb antigens to lysosomes for their degradation and clearance (Eskelinen EL et al., 2009). Further, autophagy heightens the antigen presenting ability of APCs via MHC class I and MHC class II to T cells (Comber JD et al., 2011).

Post development of the immune cells against a given pathogen, they move to the site of infection (cf. lungs) under the chemotactic stimulus. This induction of immune response regulate the course of pathogenesis (Chen K et al., 2013). The formed granuloma containing all the cells varying form the macrophages infected by M.tb to the cells that haveMaulana arrived Azadat the siteLibrary, of infection Aligarh and transformed Muslim intoUniversity different cells such as giant langhans (formed under granulomatous condition) cells, epithelioid cells and also foamy macrophages (Kumar SN et al., 2013). The infected macrophages develop a fibrous structure around them bacterial spreading (Turner OC et al., 2003). This causes a physical barrier between the infected macrophages and the surrounding lymphocytes that have the capacity to kill the infected cells and also downmodulate the potential of antibiotics to penetrate and kill the cells (Eisele NA et al., 2011). The granulomas can persist for lifetime of the individual, however, in cases of some individual there are chances of reoccurrence of the infection. On histopathological

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Chapter 5: Immunomodulator trehalose 6,6'-dimycolate as vaccine against M.tb analysis of such infection there exists a characteristic lipid arena along with macrophages that appear foamy in the alveoli and walls of alveoli (Hunter RL et al.,2007). It has been found that that more CD8+ cells are seen in comparison to CD4+ cells in such infections. It is speculated that during re-occurrence of the infection the accumulated antigens may contain glycolipids which have the capacity to start tissue necrosis leading to the formation of cavities (Reece ST et al.,2019). Various lymphocytes such as CD4+, CD8+, CD20+, as well as T-reg cells line the cavity wall (Vieyra-Lobato MR et al.,2018). The chemicals and other substances released by the innate as well as cell mediated responses in response to the bacterial infection leads to tissue damage, wasting and ultimately leads to death (Lima VM et al.,2001). However, in retrospect it has to be noted that these responses not only kill the bacteria however also stop their spread. The establishment to the disease depends on the evasion of the host defence system by the M.tb (Tiwari BM et al.,2012). The pathogen can remain as latent infection for long periods of time by evading the host immune system (Monack DM et al., 2004). However, the fine-tuned balance regarding the protective effect as well as the detrimental effects of the immune response can be increased in such a way that the protective efficiency of the immune system increases by use of certain components of the M.tb. Many components of the M.tb have been isolated to access their role in pathogenesis and progression of the disease.

The cord factor or trehalose 6,6'-dimycolate (TDM), is basically a component of the external cell wall layer of M.tb (Guidry TV et al.,2007). TDM has the innate property to induce formation of granulomas when injected in mice or rabbit models (Yamagami H et al.,2001). TDM triggers activation of protective immune reaction against the pathogenesis of mycobacterial infections. Keeping this fact into consideration,Maulana TDM Azad has beenLibrary, perceived Aligarh as pathogen Muslim-associated University molecular pattern (PAMP) which is thought to be involved by immune system in sensing the danger signal. More importantly, TDM has also been recognized as a virulence factor of M.tb (Agger EM et al.,2016). The role of TLRs has been studied in relation to TDM, it was reported that murine macrophages that are TLR2 TLR4−/− showed a relatively weak TDM response indicating that these may be involved in the signalling (Kawai T et al.,2011).

Keeping above facts into consideration, we investigated the role of TDM to modulate BCG vaccine potential against M.tb infection. We found that mice primed with TDM

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Chapter 5: Immunomodulator trehalose 6,6'-dimycolate as vaccine against M.tb showed an enhanced production of inﬂammatory cytokines with increased antigen presentation. The immunization protocol involving BCG harbouring macrophages leads to formation of apoptotic bodies due to the process of autophagy. Interestingly, TDM+BCG treated group demonstrated considerable augmentation in both CD4+ and CD8+ T cell. Importantly, the combination showed an augmented immunogenicity and protective efficacy and hence can be a potent future vaccination strategy against M.tb and other diseases caused by intracellular pathogens. In conclusion, our study provides new insight about the mechanism of action of TDM with BCG and has implications for developing more effective treatment of M.tb.

5.2 Materials and Methods

Reagents

Mycobacterium culture medium viz. Middlebrook 7H11 medium, albumin, dextrose, oleic acid, Middlebrook 7H9 broth and catalase etc. were purchased from Difco laboratories (Michigan, USA). High capacity cDNA reverse transcription kit and PCR reagents were purchased from Applied Biosystems, Forster city, CA, USA. Fluorescein isothiocyanate (FITC)–conjugated rabbit anti human CD4 and CD8, PerCP conjugated rabbit anti human CD62L (MEL-14), phycoerythrin (PE)- conjugated rabbit anti human CD44 (IM7), anti-CD80 (B7-1), anti-CD86 (GL1), were obtained from BD-Biosciences. Kits used for the estimation of various cytokines by using ELISA in plasma as well as in culture supernatants were also procured from BD Biosciences. The rest of the chemical reagents were purchased from Sigma Aldrich and some sourced locally. Tissue culture grade plastic-ware was purchased from Eppendorf, Hamburg, Germany. Maulana Azad Library, Aligarh Muslim University Ethics Statement

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Chapter 5: Immunomodulator trehalose 6,6'-dimycolate as vaccine against M.tb behaviour by animal house staff twice on week days and only once on weekends. Humane painless handling of all experimental animals includes proper maintenance of living conditions and minimization of distress. The CPCSEA mandates, as formulated by institutional ethical committee, were considered throughout the experimental set up while handling the animals to minimize suffering of animals. Experimental animals were frequently managed and cared throughout the commencement of the study following acceptable standard mandates as specified by ethical committee approved by CPCSEA, Govt. of India. Human blood was collected from healthy volunteers. The informed consent was obtained from all volunteers. The animals, cells, infected macrophages survived after conclusion of the experiment were euthanized following humane endpoint mandates of the institutional animal ethics committee (CPCSEA, Govt. of India). The euthanized animals were autoclaved followed by incineration as per the recommended guidelines.

Mycobacterial strains and Mycobacterium bovis BCG and its culture:

M. tuberculosis H37Rv strains and M. smegmatis were grown in Middlebrook 7H9 broth supplemented with OADC and also contained 0.2% glycerol and 0.05% Tween- 80 at 37°C in 5% CO2. For macrophage infections, recovered bacteria were plated on Middlebrook 7H11 agar plate that contained 10% oleic acid-albumin-dextrose- catalase. The Bacille Calmette–Guerin (Danish) and M.tbH37Rv strains were kindly provided by Director, National JALMA Institute for Leprosy and Other Mycobacterial Diseases (NJIL and OMD), Agra, India. M.tb was cultured in Middlebrook 7H9 broth containing 0.2% glycerol and 0.05% Tween-80 supplemented with 10% OADC at 37 °C as a shaking culture. M. bovis BCG was cultured in Middlebrook 7H9 broth containing 0.02% Tween-80 supplemented with 10% OADC Maulana Azad Library, Aligarh Muslim University at 37 °C with continuous shaking. The M.tb H37Rv used in the study was passaged through mice on regular basis to ascertain its virulence. Bacterial viability was determined by culturing on Middlebrook 7H11 medium supplemented with OADC and counting the number of colony forming units (CFUs). All experimental procedures involving live M. tuberculosis were performed under standard biosafety level 3 (BSL-3) laboratory practices while of M. smegmatis were performed in BSL-2 laboratory practices.

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TDM synthesis: Preparation of TDM

Semisynthetic strategies capitalize on the ability to purify native mycolic acids from M.tb and then couple them to benzyl- or silyl-protected trehalose by common esterification techniques. The mycolyl β-hydroxyl is an additional complication and must be protected to prevent dehydration. The 2,2′-, 3,3′-, and 4,4′- protected trehaloses can be obtained by utilizing either selective silyl protection or selective mesylation. Once the 6- and 6′-hydroxyl groups are freed, a variety of esters can be coupled to form TDM analogues. Synthesis of a corynebacterial TDM analogue. The lipid was synthesized starting from a simple epoxide. The final DCC coupling and deprotection yielded the native TDM as well as three stereoisomers.

Preparation of TDM encapsulated Liposomes:

The TDM bearing liposomes were prepared using E. coli lipid by following the published procedure as standardized in our lab (Mallick AI et al., 2007). Briefly, E. coli lipids (total lipid 20 mg) were reduced to thin dry film under N2 atmosphere. The film was hydrated, sonicated using bath sonicator for 1 hr at 4˚C. The liposomes thus formed were mixed at this stage with an equal volume of TDM (50µg/ml stock). The mixture was frozen and thawed (3 cycles) very quickly and then lyophilized. The lyophilized liposomes thus obtained was rehydrated with distilled water (120µl) and finally re-constituted with PBS. The re-constituted liposomes were centrifuged at 14,000×g and the pellet thus obtained was further washed to remove the traces of the un-entrapped solute.

Immunization and Immunotherapy Schedule

Priming of animalMaulana with immunomodulator Azad Library, TDM Aligarh and glucopyranosyl Muslim University lipid adjuvant: Various group of animals were pre-treated with liposomised preparation of TDM for 2 days on -3 to -2 day and on -1day with glucopyranosyl lipid adjuvant(ttgM+BCG). On day 0 animals were inoculated with macrophages harboring BCG. The animals belonging to second group were immunized with BCG infected MΦ. The groups of animals that were inoculated with only BCG or PBS served as experimental control groups.

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Establishment of Infection and Determination of Residual Mycobacterial Loads

Post priming , mice from various experimental and control groups were challenged with virulent M.tbH37Rv through aerosol route. A bacterial suspension corresponding 5 × 107bacteria/ml in 10 ml normal saline was added to the nebulizer unit of the Aerosol Inhalation Exposure System (Glas-Col, USA). To evaluate the protective efficacy of vaccines, we determined the bacterial load in lungs and spleen of experimental animals at 4- and 8-weeks post M.tb aerosol challenge. At stipulated time intervals, a minimum of 4 animals from each group were euthanized; their spleen and lungs were removed aseptically and homogenized in 7H9 media. Four different dilutions of prepared homogenate were plated onto 7H11 agar plates supplemented with OADC. Thiophene carboxylic acid hydrazide (TCH) at concentration of 2 mg/ml was added to inhibit the growth of BCG in immunized groups. All the plates were incubated for 3–4 weeks at 37 °C in CO2 incubator (Galaxy 180S, Eppendorf,

Isolation of splenocytes and its culture

Various experimental immunized groups were taken from animal house and sacrificed by cervical dislocation. Splenocyte cell suspension was prepared as described previously (Tufail S et al., 2015). Briefly, spleens were aseptically isolated from experimental animals, macerated and single cell suspension was treated with cold water for the lysis of RBCs. The cell suspension was then centrifuged at 1200g for 10 min, and the cell pellet was washed twice with 20mM PBS and re-suspended in RPMI 1640 mediumMaulana containing Azad Library, 10% fetal Aligarh bovine serum Muslim and 0.1% University antimycotic cocktail. About 10 l of the cell suspension was taken in a hemocytometer and the cells were counted. Further CD4 and CD8 T cells were purified using MACS column (Miltenyi Biotec, USA), purity of CD4 and CD8 T cells were found to be more than 98% as analyzed by BD verse flow cytometer using BD verse software provided by BD, USA. The cells were then re-suspended in media.

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Lymphocyte proliferation assay

Lymphocyte proliferation assay was performed as described elsewhere (Tufail S et al., 2015). In brief, lymphocytes were isolated from the spleens of various groups of immunized mice, and cultured in triplicate at 2×105 cells per well with graded doses (1–32µg/well) of TDM in 200µL of RPMI 1640 medium supplemented with 10% fetal bovine serum in 96-well flat bottom plates. In the next set, splenocytes from various experimental immunized groups were incubated with a known amount of corresponding matching formulations of TDM. Splenocytes incubated with the medium alone were used as the control. After 60 h, the plates were pulsed with 0.5 Ci/well of [3H]-thymidine. After 12 h, the plates were harvested using filler-mate cell harvester (PerkinElmer Life Sciences), and the incorporated [3H]-thymidine was measured by a Top Count scintillation counter (PerkinElmer Life Sciences) and expressed as counts per minutes (CPM).

Cytokine Assay: Assessment of Antigen Induced Cytokine Profile

Both pro (IFN-γ, IL-12, IL-6) and anti (IL-4, IL-10) inflammatory cytokines induced in ex vivo re-stimulated splenocyte culture supernatants (from various experimental and control groups) were estimated using OptEIA sandwich ELISA kits (BD Biosciences). Briefly, 100 μL of the purified capture antibodies were adsorbed overnight on polystyrene micro-titer plates (Maxisorp, Thermo Scientific) at 4 °C in the kit recommended coating buffer. Plates were washed five times with PBST and blocked with 1% BSA. After washing, 100 μL of the supernatant (isolated from cultured splenocytes after 24 h) was dispensed in each well. After incubation for the time stipulated, the plates were thoroughly washed and incubated with respective biotinylated antiMaulana-mouse detection Azad Library,antibody. Afterward, Aligarh theMuslim plates wereUniversity washed three times with PBST. Subsequently, 100 μL of streptavidin-HRP conjugate was added to each well and plate was incubated for 30 min at room temperature (RT). The plates were again washed three times with PBST and finally colored complex was developed with tetra methyl benzidine (TMB). The absorbance was read at 450 nm with a micro- titer ELISA plate reader (Bio-Rad).

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Flow Cytometric Phenotyping of T Lymphocytes and Mononuclear Phagocytes

Ex vivo re-stimulated splenic lymphocytes/phagocytes or PECs (unstimulated) were harvested and stained for flow cytometric analysis following a protocol provided by BD Biosciences. Briefly, 1×106 splenocytes were washed twice with FACS staining buffer (PBS with 1% BSA and 0.1% sodium azide). Cells were incubated with Fc block (2.4G2) or with FITC/PE/PerCP tagged monoclonal antibodies against , CD4, CD8, CD44, CD62L, CD11b, F4/80, andLy6cfor 30 minutes at 4 ⁰C. After washing, cells were fixed with 4.0% paraformaldehyde (PFA). The flow cytometry data was acquired using FACS Aria-II platform with FACS Diva software (BD Biosciences) and minimum of 10 000 events were recorded for each sample. Data were further analyzed with FlowJo (Treestar Inc.) software. The cells of a definite phenotype [CD4+CD44highCD62Llow/high (CD4 Tmem), CD8+CD44highCD62Llow/high (CD8 Tmem) CD11b+F4/80+SSSlowLy6clow/high (Macrophages), were deduced as percentage of the gated cell population, as determined by flow cytometry.

Statistical Analysis: Results were expressed as the mean ± SEM and data were analyzed by means of one-way analysis of variance (ANOVA) and two-way ANOVA to assess the differences among various groups. Statistical calculations were performed with the help of Graph-Pad prism version 6.0, GraphPad software Inc. San Diego, California, USA. Significance was indicated as *** for p values <0.001; ** for p values < 0.01 and * for p values < 0.05. Data shown are representative of at least triplicate experiments. A value of p < 0.05 was considered to be statistically significant.

5.3 Results Maulana Azad Library, Aligarh Muslim University Priming with CLEC4E agonists TDM elicits an enhanced Th1 response in the immunized animals

The various cytokine levels reveal that priming leads to biasing of the immune system towards the Th1 response. Splenocytes from primed animals were harvested two weeks from the completion of priming and vaccination which was followed by challenge with the M.tb were subsequently cultured to assess the cytokine profile for the signature anti-inflammatory cytokines as well as pro-inflammatory cytokines employing sandwich ELISA . The level of pro-inflammatory cytokines viz. IFN-γ

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(Figure 5.1[A]) and IL-12(Figure 5.1[B]) was found to be elevated in animals which have been primed with TDM and vaccinated with BCG (Figure 5.1). Once IL-12 directs the differentiation into Th-1 cells, which starts to produce IFN-, hence the levels of IFN- also starts to increase driving the cell towards further synthesis of IFN-. This inhibits the disease progression and further leads to maturation of macrophages. The anti-inflammatory effect of priming was also evident in terms of expression level of IL-4 cytokines(Figure 5.1[D]).

Figure 5.1 Cytokine profile in the various treated groups. Priming of animals with TDM and vaccination with BCG produce significant higher levels of pro- inflammatory cytokines [A] IFN-γ, [B] IL-12 and [C] IL-2 promoting the TH1 polarization and suppression of Th2 type cytokine anti-inflammatory [D] IL-4. Statistical significanceMaulana was Azad determined Library, as described Aligarh in ma Muslimterials and University methods. p<0.01 and p<0.001 represented as (**) and (***) respectively.

The level of IL-4 cytokines was found to be significantly reduced in case of primed animals. This cytokine analysis directs towards the development of more Th1 response. Immunized animals were assessed for their potential to produce signature pro- and anti-inflammatory cytokines at 4- and 8-weeks PC. The expression level of IL-12, the master cytokine of the Th1 cell-mediated immunity, was found significantly elevated in the ttgM+BCG group both at 4- and 8-weeks post- immunization. So, from this analysis of the after effects of the priming of animals

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Chapter 5: Immunomodulator trehalose 6,6'-dimycolate as vaccine against M.tb with TDM followed by vaccination with macrophages harboring BCG revels that the pro-inflammatory mediators are induced and inhibition of production of anti- inflammatory cytokines.

Assessment of the lymphocyte proliferation potential of TDM

Various combinations of the formulations were assessed to ascertain their role in as vaccine. The lymphocytes were isolated at various time points from the spleen of immunized mice. As shown in Figure 5.3 the proliferation of lymphocytes was observed in dose-dependent pattern. In the case of both ttgM+BCG, gggM+BCG , the lymphocyte proliferation rate was significantly higher than BCG alone . while the mice serving as negative control (saline) did not induce sufficient level of lymphocytes. The histograms reveal the lymphocyte proliferation is response to TDM (50μg) at different time points, viz, 2-week post-immunization and 2-week post- challenge with infection. Among various forms of antigens, ttgM+BCG maintained a higher proliferative response at post-immunization as well as post-challenge, when compared to BCG at post-immunization as well as post-challenge.

Maulana Azad Library, Aligarh Muslim University

Figure 5.2 Cytokine profile in the various treated groups. Priming of animals with TDM and vaccination with BCG produce significant higher levels of pro- inflammatory cytokines [A] IL-1, [B] IL-6 and also [C] Nitric oxide promoting the TH1 polarization and suppression of Th2 type cytokine anti-inflammatory [D] TNF- . Statistical significance was determined as described in materials and methods. p<0.01 and p<0.001 represented as (**) and (***) respectively.

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Isotyping profile showed TDM primed mice vaccinated with BCG evokes IgG2a and IgG2b type antibodies predominantly

To determine the antibody isotype switching, we evaluated IgG1, IgG2a, and IgG2b antibody response in the serum of immunized animals at various time points. As shown in figure 5.4, at two weeks post-priming and immunization the ratio of IgG2a to IgG1 was assessed which seemed to be low in comparison to the ratio post- challenge at 4 and 8 weeks. However, the ratio of IgG2a to IgG1 in mice primed with immunomodulator TDM along with immunization with macrophage harboring BCG animals was observed statistically significant at any time points when compared with only BCG immunized groups . Furthermore, this ratio was further significantly upregulated for primed mice at post-challenge. The increased IgG2a titers at post- challenge clearly supported the increase in Th1 cytokine level which was further boosted upon challenge with M.tb and helps in the clearance of the pathogen.

Figure 5.3 T cell proliferation response in various immunized groups. (A) To determine the effect of amount of TDM on proliferation of T lymphocytes; splenocytes, isolated from various groups of immunized BALB/c mice at two weeks post-immunization time point, were co-cultured in the presence of increasing amounts 3 (1.56 to 50 μg)Maulana of TDM inAzad flat-bottomed Library, 96 Aligarhwell plates. Muslim After 72 Universityh, [ H]-thymidine was added to each well and its incorporation in multiplying cells was measured after 16 h incubation with liquid scintillation counting. The accumulation of 3H, thymidine was determined in proliferating cells and denoted in term of counting per minute (CPM) values of stimulated cultures to represent Ag specific stimulation. (B) Proliferation of specific T lymphocytes isolated from immunized animals at two-week post-immunization and also at 4-weeks and 8-weeks post-immunization upon stimulation with a fixed amount (50μg) of TDM. Data represents the mean of three determinants ± S.D. P-value :p<0.01, p<0.001, p<0.0001 and p>0.05 represented as (*) (**), (***) and (NS) respectively.

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TDM priming followed by vaccination with M+BCG ensued in enduring CD4+and CD8+ T cell response

Induction of functionally superior polyfunctional T cells following immunization and/or infection is an important feature of T cell mediated immunity. Polyfunctional T cells are known as important anti-pathogen effectors indispensable for immunity and protection against M.tb and other infectious agents. In this study, antigen specific CD4+ T cells, induced in various immunized and primed groups, were assessed ex vivo for simultaneous expression at 2 as well as 4 weeks post-immunization. Interestingly, TDM primed animals followed by M+BCG (i.e. ttgM+BCG) administration displayed significantly high frequencies of CD4+,and CD8+ cells when compared with BCG alone at 2 weeks post-immunization. The levels of CD4+ , and CD8+ in TDM plus M+BCG administered animals were even higher in comparison with the group only given immunomodulator at this time point. Moreover, the frequencies of CD4+ induced in saline, BCG administered animals were almost identical at 2 weeks PC. Nevertheless, the level of induced CD4+ ,CD8+ cells and was still higher in primed animals, as compared with all other groups.

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Figure 5.4 Confirmation of Th1/Th2 polarization expression upon immunization with primed mice macrophages . The sera of immunized animals were analyzed for the evaluation of IgG2a: IgG1 isotypes by sandwich ELISA method. The data represent mean of three determinants ± S.D. and are representative of two different experiments with similar observation. Statistical significance was determined as described in materials and methods. P-value :p<0.01, p<0.001, p<0.0001 and p>0.05 represented as (*) and (**) respectively.

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Figure 5.5 Splenic CD4+ Proliferation Assay. The recall response of T-helper cells was assayed following various vaccination protocol. Vaccination with ttgM+BCG induced a strong antigen-specific proliferation of CD4+ splenocytes. Elevated CD4+ Tcell proliferation was observed in ttgM+BCG as compared to BCG alone as well as in gggM+BCG group (P < 0.05). The data indicate that ttgM+BCG immunization protocol significantly enhances the CD4+ helper T-cell response as compared to either BCG alone group. P-value :p<0.01, p<0.001, p<0.0001 and p>0.05 represented as (*) (**), (***) and (NS) respectively.

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Figure 5.6 Splenic CD8+ Proliferation Assay The recall response of T-helper cells was assessed following the vaccination. ttgM+BCG induced a strong antigen- specific proliferation of CD8+ splenocytes. Elevated CD8+ Tcell proliferation was observed in ttgM+BCG as compared to BCG alone or ggg M+BCG groups (P < 0.05). The data indicate that ttgM+BCG immunization protocol significantly enhances the CD8+ helper T-cell response as compared to either BCG alone group. P- value :p<0.01, p<0.001, p<0.0001 and p>0.05 represented as (*) (**), (***) and (NS) respectively.

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TDM priming induces enhanced expression of co-stimulatory molecules :

We further evaluated the effect of employed immunotherapeutic strategy for its potential to evoke T cell memory late till 8 weeks PC. We examined splenic T cells from immunized animals for expression of established T cell memory markers viz. CD44 and CD62L. Co-stimulation are required for eliciting T-cell mediated response as its absence leads to weakened T-cell anergy. Interestingly, immunization protocol involving ttgM+BCG was successful in up-regulating expression of CD80 (B7-1) and CD86 (B7-2) co-stimulatory molecules when compared with BCG alone , on post-challenge with M.tb infection (Figure 5.7, 5.8). The expression level of CD80 (B7-1) and CD86 (B7-2) was more pronounced in animals primed with TDM as compared to BCG alone and also in the animals immunized with BCG harbouring M.

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Figure 5.7 ttgM+BCG upregulates costimulatory molecules CD80+ and CD86+ on antigen presenting cells. Target cells were isolated following method as described in methodology section on 4th week post-immunization with M.tb infection. Co- stimulatory molecules CD80+ (B7-1) was determined by staining of target cells with specific antibodies and subsequently analyzed by flow cytometry . The lower set of figures depicts the expression level of CD86+ cells. Statistical significance was determined as described in materials and methods. P-value :p<0.01, p<0.001, p<0.0001 and p>0.05 represented as (*) (**), (***) and (NS) respectively. ttgM+BCG induces enduring CD4+ and CD8+ T cell response:

Splenocytes isolated from various experimental and control animals were evaluated 2- week PC for the presence of central (CD44highCD62Lhigh) as well as effector (CD44highCD62Llow) memory phenotype on both CD4+ and CD8+ T cells. Memory response is required for long-term protection against infection. To evaluate the vaccine potential of TDM primed mice, to induce memory response , we have stained the lymphocyte for memory marker (CD44) present on the surface of T cells and compared the response among various immunized groups. After 2 weeks post- Maulana Azad Library, Aligarh Muslim University challenge, both CD4+ and CD8+ T lymphocytes were isolated from various immunized mice groups. It has been observed that population of CD4+ and CD8+ T lymphocytes having central memory marker (CD44high and CD62Lhigh) and effector memory marker (CD44high and CD62Llow) on the gated population of CD4+ and CD8+ cells increased significantly in animals primed with TDM and vaccinated with BCG as compared to the free form of BCG and also macrophages harboring BCG as shown in Figure 5.8,5.9. Interestingly, pre-immunization with TDM further induces the population of CD4+/CD8+ T-lymphocytes with central and effector memory markers.

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Figure 5.8 Augmentation of long-lasting CD4+ T cell (effector/central) memory response upon immunization of mice with ttgM+BCG. After 2-week post- challenge, the splenocytes were isolated from various immunized groups and analyzed for the presence of CD44high, CD62Llow/high on gated CD4+ T cells by FACS. Group [A] represent group vaccinated with BCG [B] PBS and [C] gggBCG [D]gggM+BCG [E] ttgM+BCG . The data were analyzed with the Student’s t test and are shown as the means (± S.D.) of 2 independent experiments. p<0.01 and p<0.001 represented as (**) and (***) respectively.

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Figure 5.9 Augmentation of long-lasting CD8+ T cell (effector/central) memory response upon immunization of mice with ttgM+BCG. After a week post- challenge, the splenocytes were isolated from various immunized groups and analyzed for the presence of CD44high, CD62Llow/high on gated CD8+ T cells by FACS. Group [A] represent group vaccinated with BCG [B] PBS and [C] gggBCG [D]gggM+BCG [E] ttgM+BCG . The data were analyzed with the Student’s t test and are shown as the means (± S.D.) of 2 independent experiments. p<0.01 and p<0.001 represented as (**) and (***) respectively.

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5.4 Discussion

Mycobacterium tuberculosis inflicts nearly one third of the global population, where 5-10% of the affected individuals show symptoms of active TB. The available anti- TB drugs fail to cure newly emerging isolates of drug resistant strains(Cilfone NA et al.,2013). The prevailing situation demands either development of new drug regimen with no serious side effects or prophylactic means to curb the invading pathogen during the initial invasion stage. Various existing prophylactic strategies that employ M.tb specific T cell targeting fail to manifest desirable immune response. This encourages us to opt for newer approaches that circumvent both APCs and specific effector T-cells in the host.

We and others have emphatically demonstrated that immunomodulator mediated activation of host’s innate immunity successfully suppress certain classes of intracellular pathogens. Incidentally, M.tb adopts intracellular parasitism as a strategy to avoid antibody onslaught. It also subverts inflicted host macrophages towards anti- inflammatory AAM (or M2) phenotype to subdue killing potential of macrophages. Macrophages express several classes of membrane bound recognition molecules to sense array of pathogen related patterns (PAMPS) (Niemann S et al.,2009). Besides TLR and NLR several c-type lectin domain family member (CLEC4E) also actively patriciate in recognition of range of pathogen linked agonist molecules. Earlier reports have indicated involvement of CLEC4E in recognition of Candida albicans and Fonsecaea monophora related molecules by host macrophages. The cord factor component of M.tb such as trehalose-6,6-dimycolate also interact with macrophage through CLEC4E receptor. In a manner similar to other agonists of macrophage-based receptors, TDM acts an important virulent factor of M.tb and evoke inflammatory Maulana Azad Library, Aligarh Muslim University response in the host (Guidry TV et al.,2007)..

Toll like receptors especially TLR-2, TLR-4 and TLR-9 recognize various M.tb associated molecules. The agonists induce pro-inflammatory response in the host.

In order to thrive against hostile host ambience, M.tb and other intracellular pathogens skew pro-inflammatory M2 macrophage prepondence. However, the anti- inflammatory praxis can be countered by activation of pro inflammatory M1 (CAM) macrophages(Kaufmann SH et al., 2001). In this regard, macrophages activation employing PAMP analogs has been well authenticated.

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The role of autophagy in imparting protection against invading intracellular pathogen is well documented. Autophagy facilitate lysosomal degradation of pathogens derived antigens followed by their subsequent co-presentation along with MHC molecules and eventually leads to apoptosis of the infected cells.

Dendritic cells, not only actively participate in engulfment of invading pathogens, rather they also facilitate processing and presentation of pathogen associated antigen for appropriate activation of effector T lymphocytes. While vigil anting the tissue microenvironment post pathogen invasion, the DCs also actively interact with cells undergoing apoptosis. The generated apoptotic bodies are avidly taken up by the dendritic cells.

In general, besides functioning as active professional antigen presenting cells , macrophages play often crucial first line defense against range of pathogens. Infact, pro-inflammatory macrophages (M1 and CAMs) with classical phenotype play crucial role in efficient clearance of invading intracellular pathogens. Both macrophages and dendritic cells have been implicated to evoke effective T cell-based effector response in the host. Interestingly, M1 type macrophages are equipped with several innate immune receptors such as TLR,CLR, NLR, and RLRs etc. the activation of the macrophages through one or other above specified receptors bolster killing of the invading pathogens. Recently, CLEC4E receptor has been exploited to ensure development of effective immune response in the host.

It can be hypothesized that modulation of the host immune system (activation of macrophages) through small sized agonists may prime macrophages for killing of invading pathogens such as M.tb. We found that pretreatment with immunomodulator viz. TDM causesMaulana macrophages Azad to Library, enhance productionAligarh Muslimof cytokines University viz IL-6, IL-2, TNF-, IL-1b etc. it also causes upregulation of surface molecules such as CD40, CDS86, and H2/MHC-II etc.

The priming of the animals with combined immunomodulator therapy was also upregulation of iNOS. This led to led to increased production of NO and other reactive oxygen species by activated macrophages.

In general, both CD4+ T helper cell population and CD8+T cytotoxic cells are crucial in killing of the M.tb. We observed that pre-treatment with immunomodulator boost

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Chapter 5: Immunomodulator trehalose 6,6'-dimycolate as vaccine against M.tb augmented proliferation of total T lymphocytes in the immunized mice .Although BCG only group also showed significant T cell proliferation, however immunization with BCG infected Macrophages induced more robust T cell proliferation as compared to BCG only immunization group (Figure 5.5 and 5.6).

Interestingly pre-treatment with combination of immunomodulators evoked expanded pool of Th1 and Th17 cell subpopulation in the immunized animals. On the other hand, the cell population in both immunomodulator primed MΦ+BCG and MΦ+BCG (no priming) groups of animals. Both Th1 and Th17 cells play pivotal role in imparting protection against M.tb. In contrast Th2 cells are pro M.tb and support their survival in the host.

Besides active participation of Macrophages in killing of invading M.tb, the effector immune response is rather more important in successful eradication of intracellular M.tb. The effector response is primarily governed by cytokine pool expressed by various subpopulation of T helper cells, the Th1 cytokines such as TFN-gamma and IL-2 mediate not only T helper and t cytotoxic cells for pro-host anti M.tb response, however IFN-γ can activate Macrophages cell population as well. Inspite of the fact that M.tb skew invaded APCs towards M2 state, the immunization with MΦ+BCG and more profoundly MΦ+BCG supplemented with immunomodulator priming down-regulated IL-10 production in the immunized mice at 2-week post- immunization stage. Interestingly immunization supplemented with immunomodulator pre-treatment was successful in maintaining the level of IL-1, IL-6 and TNF- in the animals. This in turn suggest that pre-treatment was successful in maintaining Macrophages in M1 state. Simultaneous induction of pro-inflammatory cytokines induced by both APCs as well as effector T cells. Interestingly both Maulana Azad Library, Aligarh Muslim University MΦ+BCG and immunomodulator primed MΦ+BCG groups were successful in down regulating pro M.tb anti-inflammatory IL-4 and IL-10 cytokines.

Next, we assessed cytokine profile at post-challenge with M.tb infection (4- and 8- weeks post immunization). There was significant upregulation in the level of IFN-γ. IL-2 and IL-12 in the MΦ+BCG and immunomodulator primed MΦ+BCG group. The BCG only group showed upregulation of IL-4 cytokine as well. This could be attributed to the combined effect of the presence of virulent M.tbH37Rv and BCG in the immunized animals. In contrast, both MΦ+BCG and immunomodulator primed

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MΦ+BCG and immunomodulator primed MΦ+BCG group of animals were successful in containment of prompt IL-10 and IL-4 cytokines. The immunomodulator mediated fine tuning between involved pro and anti- inflammatory signaling network worked against invading M.tb pathogen(Kaufmann SH et al., 2001).

It is always desirable that successful immunization protocol should induce poly functional T lymphocytes in the primed animal. To our surprise both MΦ+BCG and immunomodulator primed MΦ+BCG groups evoked poly functional IFN-γ/TNF- and CD4+ T cells in the immunized animals at 2-week post-immunization. The population of polyfunctional CD4+ lymphocytes was maintained even at 4- and 8- weeks post-immunization stage. Long persisting poly functional T lymphocytes are crucial attribute of protective immunity against M.tb infection. The skewed expression of the Th1 subpopulation over Th2 helper T cells was further substantiated by isotype switching data of antibodies generated in the immunized animals. The immunization of animals with BCG only resulted in balanced IgG1/IgG2a response in the immunized animals. The animals that received MΦ+BCG with or without priming with immunomodulator pre-treatment evoked prepondence of IgG2A isotype over IgG1 level (p>value,0.001).

The proinflammatory cytokine IFN-γ is considered to educate B cells for expression of IgG2a isotype in the immunized animals. The virulent M.tb however try to subdue IFN-γ producing T helper lymphocytes by manipulating host immune machinery in more than one way. This may be the possible cause that we observed less IgG2a isotype in BCG only as wells as non-immunized animals.

Bedside incitingMaulana active effector Azad response Library, in theAligarh immunized Muslim host, a University desirable vaccine protocol is ought to induce long lasting memory response as well. To this end, we determined expression of CD62L and Cd44 on various T cell population evoked in the response to various immunization protocols employed in the present study.

We found that immunization with MΦ+BCG as well as MΦ+BCG supplemented with immunomodulator priming led to proliferation of long-lasting T cells with effector (CD44high CD62 Low) and central (CD44high CD62high)) phenotype. The frequency of both CD4+ and CD8+ central memory was further augmented in both the

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Chapter 5: Immunomodulator trehalose 6,6'-dimycolate as vaccine against M.tb immunized groups. The induction of central memory T cell during chronic phase of infection might help in increasing the efficacy of in house developed M.tb vaccines.

The decline in effector memory T cells at late time point may be helpful in resisting excess effector response. Role of TL4 agonist in boosting various M.tb immunization strategies in well established. The concomitant usage of TLR4 and Clec4e agonists evoke desirable protective immune response in the host. The immunomodulator TDM has been reported to phosphorylate SYK through involvement of FCER1G/FcRs gamma involvement. This in turn results in CARD9 mediated activation of RELA/NFκB(Agger EM et al.,2016). the observed upregulation of proinflammatory cytokines viz. IL-6, TNF- and IL-1 beta can be correlated with TDM mediated activation of macrophages(Yamagami H et al.,2001).

The pro inflammatory cytokine IL-6 plays important role in differentiation of Th17 cells. More importantly, the cytokine IL-6 has been reported to play significant role in modulating innate immune response of the host against intracellular pathogens (Etna MP et al., 2014. The immunomodulator pre-treatment mediated upregulation in expression of IL-6 is likely to upregulate production of Th17 cells. The cytokine Il-6 plays important role in containment of proinflammatory cytokines such as IL10 and Il-4 etc.

IL-6 as well as IL-10 employ STAT3 signaling for their cellular expression. However, in the presence of pathogen such as M.tb, IL-6 expression is negatively regulated by SOCS3. Infact, STAT3 interacts with gp130 component of IL-6 receptor that may result in transient engagement of IL-6 with STAT3. On the contrary, STAT3 mediated expression of IL-10 is constitutive and last for longer duration. We also assessed effect ofMaulana immunomodulator Azad Library, pre-treatment Aligarh on expression Muslim of ILUniversity-1β cytokine, which is a NLRP3 or AIM2 inflammasome assembly product of macrophages (Etna MP et al., 2014. We found profound increase in level of IL-1β in both TLR agonist alone or TLR/CLEC4E agonist.

We also probed level of pro-inflammatory cytokines viz. IL-10 and Il-4 in the immunized groups. The pre-treatment with either TLR agonist alone, or in combination with TDM, resulted in down regulation of both IL-4 and IL-10 at two- week post-immunization time point. However, the level of IL-10 was found to increase at post-challenge with M.tb stage (4 and 8 weeks post-immunization stage). 166

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The expression of IL-10 cytokine depends on abundance of M2 type macrophages in the host. The significantly low level of IL-10 at week 2 post-immunization time point in the pre-treatment (TLR/CLEC4E agonist) group can be used as evidence regarding abrogation (or at least less abundance) of M2 type macrophages in the treated animals. The resurrection of the two proinflammatory cytokines can be correlated with M.tb pathogen mediated praxis of STAT3 in the animals. Nevertheless, the pre- treatment with immunomodulator was successful in curtailing IL4/IL-10 expression even post exposure with virulent M.tb infection.

5.5 Conclusion

The data of the present study suggest that priming of animals with combination of TLR/CLEC4E agonist prior to immunization with BCG can modulate prophylactic attributes of the BCG. It is confirmed that present study suggested that priming of animals with TLR/CLEC4E induce robust lymphocyte proliferation, enhanced Th-1 biased response (IL-12, IFN-γ) and production of antibody predominantly of IgG2a isotype response. The elevated expression of co-stimulatory markers (CD80 and CD86) on both antigen presenting cells and T lymphocytes, memory markers (CD44high, CD62Llow/high) on CD4+ and CD8+ memory T cells and reduction in the M.tb burden in the immunized group. Therefore, The approach paves new vistas in development of effective vaccines against M.tb and other intracellular pathogens such as Listeria monocytogenes, Candida albicans, Salmonella species.

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Bibliography

A.l-marari, A., Tibor, A., Mertens, P.D., Bolle, X., Michel, P., Godefroid, J., et al. 2001. Protection of BALB/c mice against Brucellaabortus544 challenge by vaccination with bacterioferritin or p39 recombinant protein with CPG oligonucleotides as adjuvants. Infect Immun. 69:4816–22.

Abdalla AE, Lambert N, Duan X, Xie J. Interleukin-10 family and tuberculosis: an old story renewed. International journal of biological sciences. 2016;12(6):710.

Abdelsadik A, Trad A. Toll-like receptors on the fork roads between innate and adaptive immunity. Human immunology. 2011 Dec 1;72(12):1188-93.

Achkar JM, Chan J, Casadevall A. B cells and antibodies in the defense against M ycobacterium tuberculosis infection. Immunological reviews. 2015 Mar;264(1):167-81.

Admyre C, Johansson SM, Paulie S, et al. Direct exosome stimulation of peripheral human T cells detected by ELISPOT. Eur J Immunol 2006;36:1772-81. 10.1002/eji.200535615

Agger EM. Novel adjuvant formulations for delivery of anti-tuberculosis vaccine candidates. Advanced drug delivery reviews. 2016 Jul 1;102:73-82.

Agnello D, Lankford CS, Bream J, Morinobu A, Gadina M, O'Shea JJ, Frucht DM. Cytokines and transcription factors that regulate T helper cell differentiation: new players and new insights. Journal of clinical immunology. 2003 May 1;23(3):147-61.

Agrawal P, Nawadkar R, Ojha H, Kumar J, Sahu A. Complement evasion strategies of viruses: an overview. Frontiers in microbiology. 2017 Jun 16;8:1117.

Akira S, Takeda K, Kaisho T. Toll-like receptors: critical proteins linking innate and acquired immunity. Nature immunology. 2001 Aug;2(8):675. Maulana Azad Library, Aligarh Muslim University Akira S, Uematsu S, Takeuchi O. Pathogen recognition and innate immunity. Cell. 2006 Feb 24;124(4):783-801.

Alberts B, Johnson A, Lewis J, Raff M, Roberts K, Walter P. The generation of antibody diversity. InMolecular Biology of the Cell. 4th edition 2002. Garland Science.

Alcami A, Koszinowski UH. Viral mechanisms of immune evasion. Trends in microbiology. 2000 Sep 1;8(9):410-8.

Alcami A. Viral mimicry of cytokines, chemokines and their receptors. Nature Reviews Immunology. 2003 Jan;3(1):36.

168

Bibliography

Alenquer M, Amorim MJ. Exosome Biogenesis, Regulation, and Function in Viral Infection. Viruses2015;7:5066-83. 10.3390/v7092862

Alexander KL, Targan SR, Elson III CO. Microbiota activation and regulation of innate and adaptive immunity. Immunological reviews. 2014 Jul;260(1):206- 20.

Alving, C.R., Peachman, K.K., Rao, M., Reed, S.G. 2012. Adjuvants for human vaccines. CurrOpinImmunol. 24(3):310-5.

Ambagala AP, Solheim JC, Srikumaran S. Viral interference with MHC class I antigen presentation pathway: the battle continues. Veterinary immunology and immunopathology. 2005 Aug 15;107(1-2):1-5.

Andersen MH, Schrama D, thor Straten P, Becker JC. Cytotoxic T cells. Journal of Investigative Dermatology. 2006 Jan 1;126(1):32-41.

Anderson M, Ohr RJ, Aly KA, Nocadello S, Kim HK, Schneewind CE, Schneewind O, Missiakas D. EssE promotes Staphylococcus aureus ESS-dependent protein secretion to modify host immune responses during infection. Journal of bacteriology. 2017 Jan 1;199(1):e00527-16.

Anderson MT, Seifert HS. Phase variation leads to the misidentification of a Neisseria gonorrhoeae virulence gene. PloS one. 2013 Aug 16;8(8):e72183.

Andersson H, Andersson B, Eklund D, Ngoh E, Persson A, Svensson K, Lerm M, Blomgran R, Stendahl O. Apoptotic neutrophils augment the inflammatory response to Mycobacterium tuberculosis infection in human macrophages. PloS one. 2014 Jul 7;9(7):e101514.

Ansari MA, Zia Q, Kazmi S, Ahmad E, Azhar A, Johnson KE, Zubair S, Owais M. Efficacy of Cell Wall‐Deficient Spheroplasts Against Experimental Murine Listeriosis. Scandinavian journal of immunology. 2015 Jul;82(1):10-24.

Ansari, M.A., Zubair, S., Mahmood, A., Gupta, P., Khan, A.A., Gupta, U.D., Arora, A., Owais,M.RDMaulana antigen Azad based Library, nanovaccine Aligarh imparts Muslim long term University protection by i nducing memory response against experimentalmurine tuberculosis. PLoS One. 2011;6:e22889.

Aporta A, Arbues A, Aguilo JI, Monzon M, Badiola JJ, De Martino A, Ferrer N, Marinova D, Anel A, Martin C, Pardo J. Attenuated Mycobacterium tuberculosis SO2 vaccine candidate is unable to induce cell death. PloS one. 2012 Sep 19;7(9):e45213.

Arango Duque G, Descoteaux A. Macrophage cytokines: involvement in immunity and infectious diseases. Frontiers in immunology. 2014 Oct 7;5:491.

169

Bibliography

Armijos, R. X., M. M. Weigel, M. Calvopina, A. Hidalgo, W. Cevallos, and J. Correa. 2004. Safety, immunogenecity, and efficacy of an autoclaved Leishmaniaamazonensisvaccine plus BCG adjuvant against New World cutaneous leishmaniasis. Vaccine 22: 1320–1326.

Auwaerter PG. Infectious mononucleosis in middle age. Jama. 1999 Feb 3;281(5):454-9.

Avirutnan P, Hauhart RE, Somnuke P, Blom AM, Diamond MS, Atkinson JP. Binding of flavivirus nonstructural protein NS1 to C4b binding protein modulates complement activation. The Journal of Immunology. 2011 Jul 1;187(1):424-33.

Bachrach SM. Population analysis and electron densities from quantum mechanics. Reviews in computational chemistry. 1994 Jan 1:171-228.

Bachrach, G., Banai, M., Bradstein, S., Hoida G., Genizi, A., Bercovier, H. 1994. Brucellaribosomal protien L7/L12 is a major component in antigenicity of brucellin INRA for delayed-type hypersensityvity in Brucellasensitized guinea pigs. Infect Immun. 62:5361.

Balagnaur AS. Efficacy of homeopathic treatment in case of brucellosis with sero type changes.2007 (Doctoral dissertation, RGUHS).

Baron C. Antivirulence drugs to target bacterial secretion systems. Current opinion in microbiology. 2010 Feb 1;13(1):100-5.

Basset C, Holton J, O’Mahony R, Roitt I. Innate immunity and pathogen–host interaction. Vaccine. 2003 Jun 1;21:S12-23.

Batra RC, Liu DS. Adiabatic shear banding in plane strain problems. Journal of applied mechanics. 1989 Sep 1;56(3):527-34.

Bea JE. Generation of baculovirus-Brucella abortus heat shock protein recombinants; mice immune responses against the recombinants, and B. abortus superoxide Maulanadismutase andAzad L7/L12 Library, recombina Aligarhnt proteins, Muslim 1999 University (Doctoral dissertation, Virginia Tech).

Beauvillain C, Ruiz S, Guiton R, Bout D, Dimier-Poisson I. A vaccine based on exosomes secreted by a dendritic cell line confers protection against T. gondii infection in syngeneic and allogeneic mice. Microbes and infection. 2007 Nov 1;9(14-15):1614-22.

Belon C, Soscia C, Bernut A, Laubier A, Bleves S, Blanc-Potard AB. A macrophage subversion factor is shared by intracellular and extracellular pathogens. PLoS pathogens. 2015 Jun 16;11(6):e1004969.

170

Bibliography

Benvegnu T, Lemiègre L, Cammas-Marion S. New generation of liposomes called archaeosomes based on natural or synthetic archaeal lipids as innovative formulations for drug delivery. Recent patents on drug delivery & formulation. 2009 Nov 1;3(3):206-20.

Bergsbaken T, Fink SL, Cookson BT. Pyroptosis: host cell death and inflammation. Nature Reviews Microbiology. 2009 Feb;7(2):99.

Bertholet S, Ireton GC, Ordway DJ, Windish HP, Pine SO, Kahn M, Phan T, Orme IM, Vedvick TS, Baldwin SL, Coler RN. A defined tuberculosis vaccine candidate boosts BCG and protects against multidrug-resistant Mycobacterium tuberculosis. Science translational medicine. 2010 Oct 13;2(53):53ra74-.

Bhongbhibhat N, Elberg S, Chen TH. Characterization of Brucella skin-test antigens. The Journal of infectious diseases. 1970 Jul 1:70-82.

Biet F, Locht C, Kremer L. Immunoregulatory functions of interleukin 18 and its role in defense against bacterial pathogens. Journal of molecular medicine. 2002 Mar 1;80(3):147-62.

Binder M, Eberle F, Seitz S, Mücke N, Hüber CM, Kiani N, Kaderali L, Lohmann V, Dalpke A, Bartenschlager R. Molecular mechanism of signal perception and integration by the innate immune sensor retinoic acid-inducible gene-I (RIG- I). Journal of Biological Chemistry. 2011 Aug 5;286(31):27278-87.

Bitsaktsis C, Nandi B, Racine R, MacNamara KC, Winslow G. T-cell-independent humoral immunity is sufficient for protection against fatal intracellular ehrlichia infection. Infection and immunity. 2007 Oct 1;75(10):4933-41.

Blazar BR, Korngold R. Recent advances in graft‐versus‐host disease (GVHD) prevention. Immunological reviews. 1997 Jun;157(1):79-109.

Blood GA. Human immunodeficiency virus (HIV). Transfusion Medicine and Hemotherapy. 2016 May;43(3):203.

Boehm T. DesignMaulana principles Azad ofLibrary, adaptive Aligarh immune Muslim systems. University Nature Reviews Immunology. 2011 May;11(5):307.

Boneca IG. The role of peptidoglycan in pathogenesis. Current opinion in microbiology. 2005 Feb 1;8(1):46-53.

Bonilla FA, Oettgen HC. Adaptive immunity. Journal of Allergy and Clinical Immunology. 2010 Feb 1;125(2):S33-40.

Booth AM, Fang Y, Fallon JK, et al. Exosomes and HIV Gag bud from endosome- like domains of the T cell plasma membrane. J Cell Biol 2006;172:923-35.

171

Bibliography

Bozzano F, Marras F, De Maria A. Immunology of tuberculosis. Mediterranean journal of hematology and infectious diseases. 2014;6(1).

Braden BC, Velikovsky CA, Cauerhff AA, Polikarpov I, Goldbaum FA. Divergence in macromolecular assembly: X-ray crystallographic structure analysis of lumazine synthase from Brucella abortus. Journal of molecular biology. 2000 Apr 14;297(5):1031-6.

Brightbill, H.D., Libraty, D.H., Krutzik, S.R., et al. 1999. Host defense mechanisms triggered by microbial lipoproteins through Toll-like receptors. Science. 285:732–6.

Brown GD, Herre J, Williams DL, Willment JA, Marshall AS, Gordon S. Dectin-1 mediates the biological effects of β-glucans. Journal of Experimental medicine. 2003 May 5;197(9):1119-24.

Bruns H, Meinken C, Schauenberg P, Härter G, Kern P, Modlin RL, Antoni C, Stenger S. Anti-TNF immunotherapy reduces CD8+ T cell–mediated antimicrobial activity against Mycobacterium tuberculosis in humans. The Journal of clinical investigation. 2009 May 1;119(5):1167-77.

Cai J, Wu G, Jose PA, et al. Functional transferred DNA within extracellular vesicles. Exp Cell Res2016;349:179-83.

Campbell SB. Hard-to-manage preschool boys: Externalizing behavior, social competence, and family context at two-year followup. Journal of abnormal child psychology. 1994 Apr 1;22(2):147-66.

Canning EU, Lom J. The microsporidia of vertebrates. Academic Press (London) Ltd.; 1986.

Cano RL, Lopera HD. Introduction to T and B lymphocytes. InAutoimmunity: From Bench to Bedside [Internet] 2013 Jul 18. El Rosario University Press.

Cantor H, Boyse EA. Functional subclasses of T-lymphocytes bearing different Ly Maulanaantigens. I. Azad The generation Library, ofAligarh functionally Muslim distinct University T-cell subclasses is a differentiative process independent of antigen. Journal of Experimental Medicine. 1975 Jun 1;141(6):1376-89.

Caroff M, Bundle DR, Perry MB, Cherwonogrodzky JW, Duncan JR. Antigenic S- type lipopolysaccharide of Brucella abortus 1119-3. Infection and immunity. 1984 Nov 1;46(2):384-8.

Casadevall A, Pirofski LA. A reappraisal of humoral immunity based on mechanisms of antibody‐mediated protection against intracellular pathogens. Advances in immunology. 2006 Jan 1;91:1-44.

172

Bibliography

Cassataro, J., Velikovsky, C.A., de la Barrera, S., Estein, S.M., Bruno, L., Bowden, R., Pasquevich, K.A., Fossati, C.A., Giambartolomei, G.H. 2005. A DNA vaccine coding forthe Brucella outer membrane protein 31 confers protection against B.melitensis an d B. ovis infection by eliciting a specific cytotoxic response. Infect Immun. 73(10):6537-46.

Cavalcanti YV, Brelaz MC, Neves JK, Ferraz JC, Pereira VR. Role of TNF-alpha, IFN-gamma, and IL-10 in the development of pulmonary tuberculosis. Pulmonary medicine. 2012;2012.

Celli J. Surviving inside a macrophage: the many ways of Brucella. Research in microbiology. 2006 Mar 1;157(2):93-8.

Cepko C. Large-scale preparation and concentration of retrovirus stocks. Curr Protoc Mol Biol 2001;Chapter 9:Unit9.12.

Champagne, P., Ogg, G.S., King, A.S., Knabenhans, C., Ellefsen, K., Nobile, M., Appay, V., Rizzardi, G.P., Fleury, S., Lipp, M., Förster, R., Rowland- Jones, S., Sékaly, R.P., McMichael, A.J., Pantaleo, G. 2001. Skewed maturation of memory HIV-specific CD8 T lymphocytes. Nature. 410(6824):106-11.

Chan YK, Gack MU. Viral evasion of intracellular DNA and RNA sensing. Nature Reviews Microbiology. 2016 Jun;14(6):360.

Chaplin DD. Overview of the immune response. Journal of Allergy and Clinical Immunology. 2010 Feb 1;125(2):S3-23.

Chattopadhyay PK, Roederer M, Bolton DL. A deadly dance: the choreography of host–pathogen interactions, as revealed by single-cell technologies. Nature communications. 2018 Nov 6;9(1):4638. Maulana Azad Library, Aligarh Muslim University Chauhan, A., Swaleha, Z., Ahmad, N., Farazuddin, M., Vasco, A., Abida, M., MohammadO.2011.Escheriosome mediated cytosolic delivery of Candida albicans cytosolic proteins induces enhancedcytotoxic T lymphoc yte response and protective immunity. Vaccine. 29(33):5424-33.

Chen K, Kolls JK. T Cell–mediated host immune defenses in the lung. Annual review of immunology. 2013 Mar 21;31:605-33.

Chen L, Lei L, Chang X, Li Z, Lu C, Zhang X, Wu Y, Yeh IT, Zhong G. Mice deficient in MyD88 develop a Th2-dominant response and severe pathology in the upper genital tract following Chlamydia muridarum infection. The Journal of Immunology. 2010 Mar 1;184(5):2602-10.

173

Bibliography

Chen L. Co-inhibitory molecules of the B7–CD28 family in the control of T-cell immunity. Nature Reviews Immunology. 2004 May;4(5):336.

Cheung LS, Srikrishna G, Bishai WR. Role of Myeloid-Derived Suppressor Cells and Regulatory T-Cells in the Tuberculous Granuloma. InTuberculosis Host- Pathogen Interactions 2019 (pp. 63-93). Springer, Cham.

Chirhart-Gilleland RL, Kovach ME, Elzer PH, Jennings SR, Roop RM. Identification and characterization of a 14-kilodalton Brucella abortus protein reactive with antibodies from naturally and experimentally infected hosts and T lymphocytes from experimentally infected BALB/c mice. Infection and immunity. 1998 Aug 1;66(8):4000-3.

Choi AM, Ryter SW, Levine B. Autophagy in human health and disease. New England Journal of Medicine. 2013 Feb 14;368(7):651-62.

Christiaansen A, Varga SM, Spencer JV. Viral manipulation of the host immune response. Current opinion in immunology. 2015 Oct 1;36:54-60.

Cilfone NA, Perry CR, Kirschner DE, Linderman JJ. Multi-scale modeling predicts a balance of tumor necrosis factor-α and interleukin-10 controls the granuloma environment during Mycobacterium tuberculosis infection. PloS one. 2013 Jul 15;8(7):e68680.

Coelho C, Brown L, Maryam M, Burnet MC, Kyle JE, Heyman HM, Prados-Rosales R, Lauvau G, Nakayasu ES, Brady NR, Brady A. Listeria monocytogenes virulence factors are secreted in biologically active Extracellular Vesicles. bioRxiv. 2017 Jan 1:210906.

Colino J, Snapper CM. Exosomes from bone marrow dendritic cells pulsed with diphtheria toxoid preferentially induce type 1 antigen-specific IgG responses in naive recipients in the absence of free antigen. The Journal of Immunology. 2006 Sep 15;177(6):3757-62.

Comber JD, Robinson TM, Siciliano NA, Snook AE, Eisenlohr LC. Functional Maulanamacroautophagy Azad induction Library, by Aligarh influenza Muslim A virus withoutUniversity a contribution to MHC-class II restricted presentation. Journal of Virology. 2011 Apr 27:JVI- 02122.

Conacher M, Alexander J, Brewer JM. Niosomes as immunological adjuvants. Synthetic Surfactant Vesicles: Niosomes and Other Non-Phospholipid Vesicular Systems. 2014 Apr 21:204.

Cooper MD, Alder MN. The evolution of adaptive immune systems. Cell. 2006 Feb 24;124(4):815-22.

174

Bibliography

Cossart P. Illuminating the landscape of host–pathogen interactions with the bacterium Listeria monocytogenes. Proceedings of the National Academy of Sciences. 2011 Dec 6;108(49):19484-91.

Coureuil M, Join-Lambert O, Lécuyer H, Bourdoulous S, Marullo S, Nassif X. Mechanism of meningeal invasion by Neisseria meningitidis. Virulence. 2012 Mar 1;3(2):164-72.

Creagh EM, O’Neill LA. TLRs, NLRs and RLRs: a trinity of pathogen sensors that co-operate in innate immunity. Trends in immunology. 2006 Aug 1;27(8):352- 7.

Cronkite DA, Strutt TM. The regulation of inflammation by innate and adaptive lymphocytes. Journal of immunology research. 2018;2018.

Crosby CM, Kronenberg M. Tissue-specific functions of invariant natural killer T cells. Nature Reviews Immunology. 2018 Sep;18(9):559.

Crow MS, Lum KK, Sheng X, Song B, Cristea IM. Diverse mechanisms evolved by DNA viruses to inhibit early host defenses. Critical reviews in biochemistry and molecular biology. 2016 Nov 1;51(6):452-81

D'Elios MM, Benagiano M, Della Bella C, Amedei A. T-cell response to bacterial agents. The Journal of Infection in Developing Countries. 2011 Aug 2;5(09):640-5.

Dai W, Li Z. Conserved type III secretion system exerts important roles in Chlamydia trachomatis. International journal of clinical and experimental pathology. 2014;7(9):5404.

Dara L. The receptor interacting protein kinases in the liver. InSeminars in liver disease 2018 Feb (Vol. 38, No. 01, pp. 073-086). Thieme Medical Publishers.

Darrah, P.A., Patel, D.T., De Luca, P.M., Lindsay, R.W., Davey, D.F., et al. 2007. Multifunctional TH1 cells define a correlate of vaccine-mediated protection againstMaulana Leishmania Azad major. Library,Nat Med. 13: Aligarh 843–850. Muslim University Davies RL, MacCorquodale R, Caffrey B. Diversity of avian Pasteurella multocida strains based on capsular PCR typing and variation of the OmpA and OmpH outer membrane proteins. Veterinary microbiology. 2003 Feb 2;91(2-3):169- 82.

De Pelsmaeker S, Romero N, Vitale M, Favoreel HW. Herpesvirus evasion of natural killer cells. Journal of virology. 2018 Jun 1;92(11):e02105-17.

Deen NS, Huang SJ, Gong L, Kwok T, Devenish RJ. The impact of autophagic processes on the intracellular fate of Helicobacter pylori: more tricks from an enigmatic pathogen?. Autophagy. 2013 May 6;9(5):639-52.

175

Bibliography

DePaolo RW, Tang F, Kim I, Han M, Levin N, Ciletti N, Lin A, Anderson D, Schneewind O, Jabri B. Toll-like receptor 6 drives differentiation of tolerogenic dendritic cells and contributes to LcrV-mediated plague pathogenesis. Cell host & microbe. 2008 Oct 16;4(4):350-61.\

Detilleux PG, Deyoe BL, Cheville NF. Penetration and intracellular growth of Brucella abortus in nonphagocytic cells in vitro. Infection and immunity. 1990 Jul 1;58(7):2320-8.

Dick G, editor. Immunological aspects of infectious diseases. Springer Science & Business Media; 2012 Dec 6.

Dietrich, J., Billeskov, R., Doherty, T.M. and Anderson, P. 2007.Synergistic Effect of Bacillus Calmette Guerin and a Tuberculosis Subunit Vaccine in Cationic Liposomes: Increased Immunogenicity and Protection. J Immunol. 178:3721- 3730.

DiGiuseppe Champion PA, Cox JS. Protein secretion systems in Mycobacteria. Cellular microbiology. 2007 Jun;9(6):1376-84.

Dinarello CA. Interleukin-1, interleukin-1 receptors and interleukin-1 receptor antagonist. International reviews of immunology. 1998 Jan 1;16(5-6):457-99.

Domingo-Gonzalez R, Prince O, Cooper A, Khader S. Cytokines and chemokines in Mycobacterium tuberculosis infection. Microbiology spectrum. 2016 Oct;4(5).

Dong C, Flavell RA. Th1 and Th2 cells. Current opinion in hematology. 2001 Jan 1;8(1):47-51.

Dossey BM. Florence Nightingale: Her Crimean fever and chronic illness. Journal of Holistic Nursing. 1998 Jun;16(2):168-96.

Dowling JK, Mansell A. Toll‐like receptors: the swiss army knife of immunity and vaccine development. Clinical & translational immunology. 2016 May;5(5):e85.

Dutton,Maulana R.W., Bradley, Azad L.M., Library, Swain, S.L.Aligarh 1998. TMuslim cell memory. University Annu Rev Immunol. 16:201-23.

Edmonds MD, Cloeckaert A, Hagius SD, Samartino LE, Fulton WT, Walker JV, Enright FM, Booth NJ, Elzer PH. Pathogenicity and protective activity in pregnant goats of a Brucella melitensis Δomp25 deletion mutant. Research in veterinary science. 2002 Jun 1;72(3):235-9.

Eisele NA, Anderson DM. Host defense and the airway epithelium: frontline responses that protect against bacterial invasion and pneumonia. Journal of pathogens. 2011;2011.

176

Bibliography

Eisenreich W, Rudel T, Heesemann J, Goebel W. How Viral and Intracellular Bacterial Pathogens Reprogram the Metabolism of Host Cells to Allow Their Intracellular Replication. Frontiers in cellular and infection microbiology. 2019;9.

EL Andaloussi S , Mäger I, Breakefield XO, et al. Extracellular vesicles: biology and emerging therapeutic opportunities. Nat Rev Drug Discov 2013;12:347-57.

Elzer PH, Enright FM, Colby L, Hagius SD, Walker JV, Fatemi MB, Kopec JD, Beal JV, Schurig GG. Protection against infection and abortion induced by virulent challenge exposure after oral vaccination of cattle with Brucella abortus strain RB51. American journal of veterinary research. 1998 Dec;59(12):1575-8.

Endley S, McMurray D, Ficht TA. Interruption of the cydB Locus inBrucella abortus Attenuates Intracellular Survival and Virulence in the Mouse Model of Infection. Journal of bacteriology. 2001 Apr 15;183(8):2454-62.

Engel LD, Pasquinelli KL, Leone SA, Moncla BJ, Nielson KD, Rabinovitch PS. Abnormal lymphocyte profiles and leukotriene B4 status in a patient with Crohn's disease and severe periodontitis. Journal of periodontology. 1988 Dec 1;59(12):841-7.

Enright FM, Araya LN, Elzer PH, Rowe GE, Winter AJ. Comparative histopathology in BALB/c mice infected with virulent and attenuated strains of Brucella abortus. Veterinary immunology and immunopathology. 1990 Oct 1;26(2):171-82.

Eriksson EM, Jackson DC. Recent advances with TLR2-targeting lipopeptide-based vaccines. Current Protein and Peptide Science. 2007 Aug 1;8(4):412-7.

Ernst JD. Antigenic variation and immune escape in the MTBC. InStrain Variation in the Mycobacterium tuberculosis Complex: Its Role in Biology, Epidemiology and Control 2017 (pp. 171-190). Springer, Cham.

Escande A, Serre A. IgE anti-brucella antibodies in the course of human brucellosis and afterMaulana specific Azad vaccination. Library, International Aligarh Muslim Archives University of Allergy and Immunology. 1982;68(2):172-5.

Eskelinen EL, Saftig P. Autophagy: a lysosomal degradation pathway with a central role in health and disease. Biochimica et Biophysica Acta (BBA)-Molecular Cell Research. 2009 Apr 1;1793(4):664-73.

Etna MP, Giacomini E, Severa M, Coccia EM. Pro-and anti-inflammatory cytokines in tuberculosis: a two-edged sword in TB pathogenesis. InSeminars in immunology 2014 Dec 1 (Vol. 26, No. 6, pp. 543-551). Academic Press.

F Bhavsar AP, Guttman JA, Finlay BB. Manipulation of host-cell pathways by bacterial pathogens. Nature. 2007 Oct 17;449(7164):827.

177

Bibliography

Farber DL, Netea MG, Radbruch A, Rajewsky K, Zinkernagel RM. Immunological memory: lessons from the past and a look to the future. Nature Reviews Immunology. 2016 Feb;16(2):124.

Finlay BB, McFadden G. Anti-immunology: evasion of the host immune system by bacterial and viral pathogens. cell. 2006 Feb 24;124(4):767-82.

Flajnik MF, Du Pasquier L. Evolution of innate and adaptive immunity: can we draw a line?. Trends in immunology. 2004 Dec 1;25(12):640-4.

Flannagan RS, Cosío G, Grinstein S. Antimicrobial mechanisms of phagocytes and bacterial evasion strategies. Nature Reviews Microbiology. 2009 May;7(5):355.

Foley SL, Johnson TJ, Ricke SC, Nayak R, Danzeisen J. Salmonella pathogenicity and host adaptation in chicken-associated serovars. Microbiol. Mol. Biol. Rev.. 2013 Dec 1;77(4):582-607.

Foulongne V, Bourg G, Cazevieille C, Michaux-Charachon S, O'Callaghan D. Identification of Brucella suis genes affecting intracellular survival in an in vitro human macrophage infection model by signature-tagged transposon mutagenesis. Infection and immunity. 2000 Mar 1;68(3):1297-303.

Fox, C.B., Baldwin, S.L., Sivananthan, S.J., Lucas, E., Lin, S., Phan, T., Moon, J.J., Vedvick, T.S., Reed, S.G., Coler, R.N. 2013. Adjuvant formulation structure and composition are critical for the development of an effective vaccineagainst tuberculosis. J Control Release. 172(1):190-200.

Freeman S, Post FA, Bekker LG, Harbacheuski R, Steyn LM, Ryffel B, Connell ND, Kreiswirth BN, Kaplan G. Mycobacterium tuberculosis H37Ra and H37Rv differential growth and cytokine/chemokine induction in murine macrophages in vitro. Journal of interferon & cytokine research. 2006 Jan 1;26(1):27-33.

Frost OL. An algorithm for linearly constrained adaptive array processing. MaulanaProceedings Azad of the IEEE.Library, 1972 AligarhAug;60(8):926 Muslim-35. University

Fumarola C, Guidotti GG. Stress-induced apoptosis: toward a symmetry with receptor-mediated cell death. Apoptosis. 2004 Jan 1;9(1):77-82.

Gallart Palau XR. Synaptic frailty and mitochondrial dysfunction in familial amyotrophic lateral sclerosis, 2016 (Doctoral dissertation, Universitat de Lleida).

Galli SJ, Borregaard N, Wynn TA. Phenotypic and functional plasticity of cells of innate immunity: macrophages, mast cells and neutrophils. Nature immunology. 2011 Nov;12(11):1035.

178

Bibliography

Gallo RL, Hooper LV. Epithelial antimicrobial defence of the skin and intestine. Nature Reviews Immunology. 2012 Jul;12(7):503.

Gandhi NR, Nunn P, Dheda K, Schaaf HS, Zignol M, Van Soolingen D, Jensen P, Bayona J. Multidrug-resistant and extensively drug-resistant tuberculosis: a threat to global control of tuberculosis. The Lancet. 2010 May 22;375(9728):1830-43.

García-del Portillo F, Calvo E, D'Orazio V, Pucciarelli MG. Association of ActA to peptidoglycan revealed by cell wall proteomics of intracellular Listeria monocytogenes. Journal of Biological Chemistry. 2011 Oct 7;286(40):34675- 89.

García-del Portillo F, Pucciarelli MG. Remodeling of the Listeria monocytogenes cell wall inside eukaryotic cells. Communicative & integrative biology. 2012 Mar 1;5(2):160-2.

Gasteiger G, Rudensky AY. Interactions between innate and adaptive lymphocytes. Nature Reviews Immunology. 2014 Sep;14(9):631.

Gaudino SJ, Kumar P. Cross-talk between antigen presenting cells and T cells impacts intestinal homeostasis, bacterial infections, and tumorigenesis. Frontiers in immunology. 2019;10.

Gee JM, Kovach ME, Grippe VK, Hagius S, Walker JV, Elzer PH, Roop II RM. Role of catalase in the virulence of Brucella melitensis in pregnant goats. Veterinary microbiology. 2004 Aug 19;102(1-2):111-5.

Geijtenbeek TB, Gringhuis SI. Signalling through C-type lectin receptors: shaping immune responses. Nature Reviews Immunology. 2009 Jul;9(7):465.

Gengenbacher M, Kaufmann SH. Mycobacterium tuberculosis: success through dormancy. FEMS microbiology reviews. 2012 May 1;36(3):514-32.

Ghadimi D, de Vrese M, Heller KJ, Schrezenmeir J. Lactic acid bacteria enhance autophagicMaulana ability Azadof mononuclear Library, phagocytes Aligarh by Muslim increasing University Th1 autophagy- promoting cytokine (IFN-γ) and nitric oxide (NO) levels and reducing Th2 autophagy-restraining cytokines (IL-4 and IL-13) in response to Mycobacterium tuberculosis antigen. International immunopharmacology. 2010 Jun 1;10(6):694-706.

Ghosh, T.K., Mickelson, D.J., Fink, J., et al. 2006. Toll-like receptor (TLR) 2-9 agonists-induced cytokines and chemokines. I. Comparison with T cell receptor-induced responses. Cell Immunol. 243:48–57.

Gilbert P, McBain AJ. Potential impact of increased use of biocides in consumer products on prevalence of antibiotic resistance. Clinical microbiology reviews. 2003 Apr 1;16(2):189-208.

179

Bibliography

Gill SR, Fouts DE, Archer GL, Mongodin EF, DeBoy RT, Ravel J, Paulsen IT, Kolonay JF, Brinkac L, Beanan M, Dodson RJ. Insights on evolution of virulence and resistance from the complete genome analysis of an early methicillin-resistant Staphylococcus aureus strain and a biofilm-producing methicillin-resistant Staphylococcus epidermidis strain. Journal of bacteriology. 2005 Apr 1;187(7):2426-38.

Giri PK, Schorey JS. Exosomes derived from M. Bovis BCG infected macrophages activate antigen-specific CD4+ and CD8+ T cells in vitro and in vivo. PLoS One 2008;3:e2461. 10.1371/journal.pone.0002461

Glenchur H, Seal US, Zinneman HH, Hall WH. Antigenicity of some Brucella melitensis cell fractions. Journal of bacteriology. 1963 Feb 1;85(2):363-8.

Godfroid J, Nielsen K, Saegerman C. Diagnosis of brucellosis in livestock and wildlife. Croatian medical journal. 2010 Aug 15;51(4):296-305.

Goel, D., Rajendran, V., Ghosh, P.C., Bhatnagar, R. 2013. Cell mediated immune response after challenge in Omp25 liposome immunize d mice contributes to protection against virulent Brucella abortus 544. Vaccine. 31(8):1231-7.

Gokhale AS, Satyanarayanajois S. Peptides and peptidomimetics as immunomodulators. Immunotherapy. 2014 Jun;6(6):755-74.

Golding JS. Southern African herbaria and Red Data Lists. Taxon. 2001 May;50(2):593-602.

Goldmann O, Medina E. Staphylococcus aureus strategies to evade the host acquired immune response. International Journal of Medical Microbiology. 2018 Aug 1;308(6):625-30.

Goodlow RJ, Tucker L, Braun W, Mika LA. Effect of the isomeric configuration of the source of nitrogen on changes in population and metabolism in cultures of Brucella. Journal of bacteriology. 1952 May;63(5):681. Maulana Azad Library, Aligarh Muslim University Gorbea CM, Marchand P, Jiang W, Copeland NG, Gilbert DJ, Jenkins NA, Bond JS. Cloning, expression, and chromosomal localization of the mouse meprin beta subunit. Journal of Biological Chemistry. 1993 Oct 5;268(28):21035-43.

Gorvel JP, Moreno E. Brucella intracellular life: from invasion to intracellular replication. Veterinary microbiology. 2002 Dec 20;90(1-4):281-97.

Grabowski B, Schmidt MA, Rüter C. Immunomodulatory Yersinia outer proteins (Yops)–

Gray GL, Smith DH, Baldridge JS, Harkins RN, Vasil ML, Chen EY, Heyneker HL. Cloning, nucleotide sequence, and expression in Escherichia coli of the

180

Bibliography

exotoxin A structural gene of Pseudomonas aeruginosa. Proceedings of the National Academy of Sciences. 1984 May 1;81(9):2645-9.

Greendyke RM, Kanter DR, Schuster DB, Verstreate S, Wootton J. Propranolol treatment of assaultive patients with organic brain disease: A double-blind crossover, placebo-controlled study. Journal of Nervous and Mental Disease. 1986 May.

Grohmann E, Christie PJ, Waksman G, Backert S. Type IV secretion in Gram‐negative and Gram‐positive bacteria. Molecular microbiology. 2018 Feb 1;107(4):455-71.

Groisman EA. The pleiotropic two-component regulatory system PhoP-PhoQ. Journal of bacteriology. 2001 Mar 15;183(6):1835-42.

Gu X, Gao Y, Mu DG, Fu EQ. MiR-23a-5p modulates mycobacterial survival and autophagy during Mycobacterium tuberculosis infection through TLR2/MyD88/NF-κB pathway by targeting TLR2. Experimental cell research. 2017 May 15;354(2):71-7.

Guidry TV, Hunter Jr RL, Actor JK. Mycobacterial glycolipid trehalose 6, 6′- dimycolate-induced hypersensitive granulomas: contribution of CD4+ lymphocytes. Microbiology (Reading, England). 2007 Oct;153(Pt 10):3360.

Gutierrez MG, Master SS, Singh SB, Taylor GA, Colombo MI, Deretic V. Autophagy is a defense mechanism inhibiting BCG and Mycobacterium tuberculosis survival in infected macrophages. Cell. 2004 Dec 17;119(6):753-66.

Gutzeit C, Nagy N, Gentile M, et al. Exosomes derived from Burkitt's lymphoma cell lines induce proliferation, differentiation, and class-switch recombination in B cells. J Immunol 2014;192:5852-62. 10.4049/jimmunol.1302068

Guzmán-Verri C, Chaves-Olarte E, von Eichel-Streiber C, López-Goñi I, Thelestam M, Arvidson S, Gorvel JP, Moreno E. GTPases of the Rho Subfamily Are Required for Brucella abortus Internalization in Nonprofessional Phagocytes DIRECTMaulana ACTIVATION Azad OFLibrary, Cdc42. Aligarh Journal of Muslim Biological University Chemistry. 2001 Nov 30;276(48):44435-43.

Gwida M, A.I., Dahouk, S., Melzer, F., Rösler, U., Neubauer, H., Tomaso, H. 2010. Brucellosis -regionally emerging zoonotic disease? Croat Med J. 51(4):289- 95.

Hamza T, Barnett JB, Li B. Interleukin 12 a key immunoregulatory cytokine in infection applications. International journal of molecular sciences. 2010 Mar;11(3):789-806.

Harmon R, Gilmore G. The nature of the Galactic Bulge. InComets to Cosmology 1988 (pp. 105-108). Springer, Berlin, Heidelberg.

181

Bibliography

Harokopakis, E., Childers, N.K., Michalek, S.M., Zhang, S.S., Tomasi, M. 1995. Conjugation of cholera toxin or its B subunit to liposomes for targeted delivery of antigens. J Immunol Methods. 185(1):31-42.

Harris J, De Haro SA, Master SS, Keane J, Roberts EA, Delgado M, Deretic V. T helper 2 cytokines inhibit autophagic control of intracellular Mycobacterium tuberculosis. Immunity. 2007 Sep 21;27(3):505-17.

Harris J, Master SS, De Haro SA, Delgado M, Roberts EA, Hope JC, Keane J, Deretic V. Th1–Th2 polarisation and autophagy in the control of intracellular mycobacteria by macrophages. Veterinary immunology and immunopathology. 2009 Mar 15;128(1-3):37-43.

Harris J. Autophagy and cytokines. Cytokine. 2011 Nov 1;56(2):140-4.

Hasko, G. And Szabo, C. 1999. IL-12 as a therapeutic target for pharmacological modulation in immune-mediated and inflammatory diseases: regulation of T helper 1/T helper 2 responses. Br J Pharmacol. 127(6):1295-304.

Hatfull GF, Jacobs Jr WR, editors. Molecular genetics of mycobacteria. American Society for Microbiology Press; 2014 Sep 10.

He Y. Analyses of Brucella pathogenesis, host immunity, and vaccine targets using systems biology and bioinformatics. Frontiers in cellular and infection microbiology. 2012 Feb 1;2:2.

Henriksen-Lacey, M., Bramwell, V.W., Christensen, D., Agger, E.M., Andersen, P., Perrie, Y. 2010. Liposomes based on dimethyldioctadecylammonium promote a depot effect and enhance immunogenicity of soluble antigen. J Control Release. 142(2):180-6.

Herrera MT, Torres M, Nevels D, Perez-Redondo CN, Ellner JJ, Sada E, Schwander SK. Compartmentalized bronchoalveolar IFN-γ and IL-12 response in human pulmonary tuberculosis. Tuberculosis. 2009 Jan 1;89(1):38-47. Maulana Azad Library, Aligarh Muslim University Hill DJ, Griffiths NJ, Borodina E, Virji M. Cellular and molecular biology of Neisseria meningitidis colonization and invasive disease. Clinical science. 2010 Feb 9;118(9):547-64.

Hmama Z, Peña‐Díaz S, Joseph S, Av‐Gay Y. Immunoevasion and immunosuppression of the macrophage by M ycobacterium tuberculosis. Immunological reviews. 2015 Mar;264(1):220-32.

Hooper LV, Littman DR, Macpherson AJ. Interactions between the microbiota and the immune system. Science. 2012 Jun 8;336(6086):1268-73.

182

Bibliography

Hornef MW, Wick MJ, Rhen M, Normark S. Bacterial strategies for overcoming host innate and adaptive immune responses. Nature immunology. 2002 Nov;3(11):1033.

Horwitz DA, Zheng SG, Gray JD. Natural and TGF-β–induced Foxp3+ CD4+ CD25+ regulatory T cells are not mirror images of each other. Trends in immunology. 2008 Sep 1;29(9):429-35.

Hsu C, Morohashi Y, Yoshimura SI, Manrique-Hoyos N, Jung S, Lauterbach MA, Bakhti M, Grønborg M, Möbius W, Rhee J, Barr FA. Regulation of exosome secretion by Rab35 and its GTPase-activating proteins TBC1D10A–C. The Journal of cell biology. 2010 Apr 19;189(2):223-32.

Hsueh IP, Lee MM, Hsieh CL. Psychometric characteristics of the Barthel activities of daily living index in stroke patients. Journal of the Formosan Medical Association. 2001 Aug 1;100(8):526-32.

Hunter RL, Jagannath C, Actor JK. Pathology of postprimary tuberculosis in humans and mice: contradiction of long-held beliefs. Tuberculosis. 2007 Jul 1;87(4):267-78.

Huppert LA, Ramsdell TL, Chase MR, Sarracino DA, Fortune SM, Burton BM. The ESX system in Bacillus subtilis mediates protein secretion. PloS one. 2014 May 5;9(5):e96267.

Inohara N, Nuñez G. Cell death and immunity: NODs: intracellular proteins involved in inflammation and apoptosis. Nature Reviews Immunology. 2003 May;3(5):371.

Irazoki O, Hernandez S, Cava F. Peptidoglycan muropeptides: release, perception and functions as signalling molecules. Frontiers in microbiology. 2019;10:500.

Isibasi A, Ortiz V, Vargas M, Paniagua J, Gonzalez C, Moreno J, Kumate J. Protection against Salmonella typhi infection in mice after immunization with outer membrane proteins isolated from Salmonella typhi 9, 12, d, Vi. Infection and immunity.Maulana 1988 Azad Nov 1;56(11):2953 Library, Aligarh-9. Muslim University

Ismail N, Olano JP, Feng HM, Walker DH. Current status of immune mechanisms of killing of intracellular microorganims. FEMS microbiology letters. 2002 Feb 1;207(2):111-20.

Isola AL, Chen S. Exosomes: The Messengers of Health and Disease. Curr Neuropharmacol 2016. [Epub ahead of print]. 10.2174/1570159X14666160825160421

Itano AA, Jenkins MK. Antigen presentation to naive CD4 T cells in the lymph node. Nature immunology. 2003 Aug;4(8):733.

183

Bibliography

Jackson KJ, Kidd MJ, Wang Y, Collins AM. The shape of the lymphocyte receptor repertoire: lessons from the B cell receptor. Frontiers in immunology. 2013 Sep 2;4:263.

Jackson, D.C., Lau, Y.F., Le T., et al. 2004. A totally synthetic vaccine of generic structure that targets Toll-like receptor 2 on DCs and promotes antibody or cytotoxic T cell responses. Proc Natl AcadSci USA.101:15440–5.

Jagannath C, Lindsey DR, Dhandayuthapani S, Xu Y, Hunter Jr RL, Eissa NT. Autophagy enhances the efficacy of BCG vaccine by increasing peptide presentation in mouse dendritic cells. Nature medicine. 2009 Mar;15(3):267.

Jamaati H, Mortaz E, Pajouhi Z, Folkerts G, Movassaghi M, Moloudizargari M, Adcock IM, Garssen J. Nitric oxide in the pathogenesis and treatment of tuberculosis. Frontiers in microbiology. 2017 Oct 12;8:2008.

Janeway Jr CA, Medzhitov R. Innate immune recognition. Annual review of immunology. 2002 Apr;20(1):197-216.

Jankovic D, Kugler DG, Sher A. IL-10 production by CD4+ effector T cells: a mechanism for self-regulation. Mucosal immunology. 2010 May;3(3):239.

Jensen S, Thomsen AR. Sensing of RNA viruses: a review of innate immune receptors involved in recognizing RNA virus invasion. Journal of virology. 2012 Mar 15;86(6):2900-10.

John CM, Phillips NJ, Stein DC, Jarvis GA. Innate immune response to lipooligosaccharide: pivotal regulator of the pathobiology of invasive Neisseria meningitidis infections. Pathogens and disease. 2017 Apr 5;75(3):ftx030.

Joller N, Weber SS, Oxenius A. Antibody–Fc receptor interactions in protection against intracellular pathogens. European journal of immunology. 2011 Apr;41(4):889-97.

KagimotoMaulana K, Waterman Azad MR,Library, Kagimoto Aligarh M, Ferreira Muslim P, SimpsonUniversity ER, Winter JS. Identification of a common molecular basis for combined 17α-hydroxylase/17, 20-lyase deficiency in two Mennonite families. Human genetics. 1989 Jun 1;82(3):285-6.

Kahlon R, Hu Y, Orteu CH, Kifayet A, Trudeau JD, Tan R, Dutz JP. Optimization of epicutaneous immunization for the induction of CTL. Vaccine. 2003 Jun 20;21(21-22):2890-9.

Kaufmann SH, Dorhoi A. Molecular determinants in phagocyte-bacteria interactions. Immunity. 2016 Mar 15;44(3):476-91.

184

Bibliography

Kaufmann SH. How can immunology contribute to the control of tuberculosis?. Nature Reviews Immunology. 2001 Oct;1(1):20.

Kawai T, Akira S. Toll-like receptors and their crosstalk with other innate receptors in infection and immunity. Immunity. 2011 May 27;34(5):637-50.

Kell AM, Gale Jr M. RIG-I in RNA virus recognition. Virology. 2015 May 1;479:110-21.

Kerr WE. Multiple alleles and genetic load in bees. Journal of Apicultural Research. 1967 Jan 1;6(2):61-4.

Kim YK, Oliveria M. Magnetic properties of reactively sputtered Fe1− x O and Fe3O4 thin films. Journal of applied physics. 1994 Jan 1;75(1):431-7.

Kirby, C. and Gregoriadis, G. 1984. Dehydration–rehydration vesicles: a simple method for high yield drug entrapment. Biotechnology.2:979–84.

Kiyono K, Suzuki HI, Matsuyama H, Morishita Y, Komuro A, Kano MR, Sugimoto K, Miyazono K. Autophagy Is Activated by TGF-β and Potentiates TGF-β– Mediated Growth Inhibition in Human Hepatocellular Carcinoma Cells. Cancer research. 2009 Dec 1;69(23):8844-52.

Ko J, Splitter GA. Molecular host-pathogen interaction in brucellosis: current understanding and future approaches to vaccine development for mice and humans. Clinical microbiology reviews. 2003 Jan 1;16(1):65-78.

Köhler B, Hills A, Blatt MR. Control of guard cell ion channels by hydrogen peroxide and abscisic acid indicates their action through alternate signaling pathways. Plant physiology. 2003 Feb 1;131(2):385-8.

Korn T, Bettelli E, Oukka M, Kuchroo VK. IL-17 and Th17 Cells. Annual review of Maulana Azad Library, Aligarh Muslim University immunology. 2009 Apr 23;27:485-517.

Korn T, Oukka M, Kuchroo V, Bettelli E. Th17 cells: effector T cells with inflammatory properties. InSeminars in immunology 2007 Dec 1 (Vol. 19, No. 6, pp. 362-371). Academic Press.

Kornete M, Piccirillo CA. Functional crosstalk between dendritic cells and Foxp3+ regulatory T cells in the maintenance of immune tolerance. Frontiers in immunology. 2012 Jun 22;3:165.

Kotwal GJ, Kulkarni AP. Antigenic variation in microbial evasion of immune responses. e LS. 2001 May 30.

185

Bibliography

Kowalewicz-Kulbat M, Szpakowski P, Locht C, Biet F, Kaplonek P, Krawczyk KT, Pestel J, Rudnicka W. Tuberculin skin test reaction is related to memory, but not naive CD4+ T cell responses to mycobacterial stimuli in BCG-vaccinated young adults. Vaccine. 2018 Jul 16;36(30):4566-77.

Krachler AM, Woolery AR, Orth K. Manipulation of kinase signaling by bacterial pathogens. J Cell Biol. 2011 Dec 26;195(7):1083-92.

Krishnan, L., Gurnani, K., Dicaire, C.J., van Faassen, H., Zafer, A., Kirschning, C.J., Sad, S., Sprott, G.D. 2007. Rapid clonal expansion and prolonged maintenance of memory CD8+ T cells of the effector(CD44highCD62Llow)and central (CD44highCD62Lhigh) phenot ype byan archaeosome adjuvantindependent of TLR2. J Immunol. 178(4):2396-406.

Krutzik SR, Tan B, Li H, Ochoa MT, Liu PT, Sharfstein SE, Graeber TG, Sieling PA, Liu YJ, Rea TH, Bloom BR. TLR activation triggers the rapid differentiation of monocytes into macrophages and dendritic cells. Nature medicine. 2005 Jun;11(6):653.

Kuate S, Cinatl J, Doerr HW, Überla K. Exosomal vaccines containing the S protein of the SARS coronavirus induce high levels of neutralizing antibodies. Virology. 2007 May 25;362(1):26-37.

Kumar SN, Prasad TS, Narayan PA, Muruganandhan J. Granuloma with langhans giant cells: An overview. Journal of oral and maxillofacial pathology: JOMFP. 2013 Sep;17(3):420.

Kung HJ. Targeting tyrosine kinases and autophagy in prostate cancer. Hormones and Cancer. 2011 Feb 1;2(1):38-46.

Kupsch EM, Knepper B, Kuroki T, Heuer I, Meyer TF. Variable opacity (Opa) outer membrane proteins account for the cell tropisms displayed by Neisseria gonorrhoeae for human leukocytes and epithelial cells. The EMBO journal. 1993 Feb 1;12(2):641-50. Maulana Azad Library, Aligarh Muslim University Kurar E, Splitter GA. Nucleic acid vaccination of Brucella abortus ribosomal L7L12 gene elicits immune response. Vaccine. 1997 Dec 1;15(17-18):1851-7.

Lambert WJ, Levin PS, Berman J. Changes in the structure of a New England (USA) kelp bed: the effects of an introduced species?. Marine ecology progress series. Oldendorf. 1992 Nov 1;88(2):303-7.

Landskron G, De la Fuente M, Thuwajit P, Thuwajit C, Hermoso MA. Chronic inflammation and cytokines in the tumor microenvironment. Journal of immunology research. 2014;2014.

186

Bibliography

Le Sage V. Ligand Sensing and Signal Transduction by the Two-component System PhoP/PhoQ (Doctoral dissertation, McGill University Library).

Lebeer S, Vanderleyden J, De Keersmaecker SC. Host interactions of probiotic bacterial surface molecules: comparison with commensals and pathogens. Nature Reviews Microbiology. 2010 Mar;8(3):171.

Lenschow, D.J., Walunas, T.L., Bluestone, J.A 1996. CD28/B7 system of T cell costimulation. Annu Rev Immunol. 14: 233–258.

Leon-Sicairos N, Reyes-Cortes R, Guadrón-Llanos AM, Madueña-Molina J, Leon- Sicairos C, Canizalez-Román A. Strategies of intracellular pathogens for obtaining iron from the environment. BioMed Research International. 2015;2015.

Lestrate P, Delrue RM, Danese I, Didembourg C, Taminiau B, Mertens P, De Bolle X, Tibor A, Tang CM, Letesson JJ. Identification and characterization of in vivo attenuated mutants of Brucella melitensis. Molecular microbiology. 2000 Nov;38(3):543-51.

Letesson JJ, Tibor A, Van Eynde G, Wansard V, Weynants V, Denoel P, Saman E. Humoral immune responses of Brucella-infected cattle, sheep, and goats to eight purified recombinant Brucella proteins in an indirect enzyme-linked immunosorbent assay. Clin. Diagn. Lab. Immunol.. 1997 Sep 1;4(5):556-64.

Lewis JS, Roy K, Keselowsky BG. Materials that harness and modulate the immune system. MRS bulletin. 2014 Jan;39(1):25-34.

Li JB, Lee DS, Madrenas J. Evolving bacterial envelopes and plasticity of TLR2- dependent responses: basic research and translational opportunities. Frontiers in immunology. 2013 Oct 28;4:347.

Li Y, Li Y, Cao X, Jin X, Jin T. Pattern recognition receptors in zebrafish provide functional and evolutionary insight into innate immune signaling pathways. Cellular & molecular immunology. 2017 Jan;14(1):80. Maulana Azad Library, Aligarh Muslim University Li Y, Zhou L, Zhang J, et al. Nsp9 and Nsp10 contribute to the fatal virulence of highly pathogenic porcine reproductive and respiratory syndrome virus emerging in China. PLoS Pathog 2014;10:e1004216. 10.1371/journal.ppat.1004216

Lim JJ, Grinstein S, Roth Z. Diversity and versatility of phagocytosis: roles in innate immunity, tissue remodeling, and homeostasis. Frontiers in cellular and infection microbiology. 2017 May 23;7:191.

Lima KM, dos Santos SA, Rodrigues Jr JM, Silva CL. Vaccine adjuvant: it makes the difference. Vaccine. 2004 Jun 23;22(19):2374-9.

187

Bibliography

Lima VM, Bonato VL, Lima KM, Dos Santos SA, Dos Santos RR, Gonçalves ED, Faccioli LH, Brandão IT, Rodrigues-Junior JM, Silva CL. Role of trehalose dimycolate in recruitment of cells and modulation of production of cytokines and NO in tuberculosis. Infection and immunity. 2001 Sep 1;69(9):5305-12.

Lin J, Ficht TA. Protein synthesis in Brucella abortus induced during macrophage infection. Infection and immunity. 1995 Apr 1;63(4):1409-14.

Lin PL, Myers A, Smith LK, Bigbee C, Bigbee M, Fuhrman C, Grieser H, Chiosea I, Voitenek NN, Capuano SV, Klein E. Tumor necrosis factor neutralization results in disseminated disease in acute and latent Mycobacterium tuberculosis infection with normal granuloma structure in a cynomolgus macaque model. Arthritis & Rheumatism: Official Journal of the American College of Rheumatology. 2010 Feb;62(2):340-50.

Linkermann A, Green DR. Necroptosis. New England Journal of Medicine. 2014 Jan 30;370(5):455-65.

Lodoen MB, Lanier LL. Viral modulation of NK cell immunity. Nature Reviews Microbiology. 2005 Jan;3(1):59.

Lorenz E. TLR2 and TLR4 expression during bacterial infections. Current pharmaceutical design. 2006 Nov 1;12(32):4185-93.

Luukinen H, Hammarén MM, Vanha-aho LM, Svorjova A, Kantanen L, Järvinen S, Luukinen BV, Dufour E, Rämet M, Hytönen VP, Parikka M. Priming of innate antimycobacterial immunity by heat-killed Listeria monocytogenes induces sterilizing response in the adult zebrafish tuberculosis model. Disease models & mechanisms. 2018 Jan 1;11(1):dmm031658.

Mackaness GB. 1968 The immunology of antituberculous immunity.

Mackaness GB. The immunological basis of acquired cellular resistance. Journal of Experimental Medicine. 1964 Jul 1;120(1):105-20.

MaglioneMaulana PJ, Chan Azad J. Library, How B Aligarh cells shape Muslim the immune University response against Mycobacterium tuberculosis. European journal of immunology. 2009 Mar;39(3):676-86.

Maisonneuve, C., Bertholet, S., Philpott, D.J., De Gregorio, E. 2014. Unleashing the potential of NOD- and Toll-like agonists as vaccine adjuvants. Proc Natl AcadSci USA. 111(34):12294-9.

Mallick AI, Singha H, Chaudhuri P, Nadeem, A., Khan, S.A., Dar, K.A., Owais, M. 2007. Liposomised recombinant ribosomal L7/L12 protein protects BALB/c mice ag ainst Brucella abortus 544 infection.Vaccine. 25(18):3692-704.

188

Bibliography

Mallick AI. Fusogenic Liposome Based Vaccines against some Infectious Diseases, 2007 (Doctoral dissertation, Aligarh Muslim University).

Man SM, Karki R, Kanneganti TD. Molecular mechanisms and functions of pyroptosis, inflammatory caspases and inflammasomes in infectious diseases. Immunological reviews. 2017 May;277(1):61-75.

Mayer A, Balasubramanian V, Walczak AM, Mora T. How a well-adapting immune system remembers. Proceedings of the National Academy of Sciences. 2019 Apr 30;116(18):8815-23.

Mayr E. What evolution is. Science Masters Series; 2001.

McCarthy MK, Weinberg JB. The immunoproteasome and viral infection: a complex regulator of inflammation. Frontiers in microbiology. 2015 Jan 29;6:21.

Medzhitov R. Pattern recognition theory and the launch of modern innate immunity. The Journal of Immunology. 2013 Nov 1;191(9):4473-4.

Meunier I, Kaufmann E, Downey J, Divangahi M. Unravelling the networks dictating host resistance versus tolerance during pulmonary infections. Cell and tissue research. 2017 Mar 1;367(3):525-36.

Mildner A, Jung S. Development and function of dendritic cell subsets. Immunity. 2014 May 15;40(5):642-56.

Minor PD, Ferguson M, Evans DM, Almond JW, Icenogle JP. Antigenic structure of polioviruses of serotypes 1, 2 and 3. Journal of General Virology. 1986 Jul 1;67(7):1283-91.

Mitchell R. A novel GABA receptor modulates stimulus-induced glutamate release from cortico-striatal terminals. European journal of pharmacology. 1980 Oct 3;67(1):119-22.

Mittrücker HW, Kaufmann SH. Mini‐review: regulatory T cells and infection: suppression revisited. European journal of immunology. 2004 Feb;34(2):306- Maulana Azad Library, Aligarh Muslim University 12.

Mogensen TH. Pathogen recognition and inflammatory signaling in innate immune defenses. Clinical microbiology reviews. 2009 Apr 1;22(2):240-73.

Mohareer K, Asalla S, Banerjee S. Cell death at the cross roads of host-pathogen interaction in Mycobacterium tuberculosis infection. Tuberculosis. 2018 Dec 1;113:99-121.

Monack DM, Mueller A, Falkow S. Persistent bacterial infections: the interface of the pathogen and the host immune system. Nature Reviews Microbiology. 2004 Sep;2(9):747.

189

Bibliography

Montaner S, Galiano A, Trelis M, et al. The Role of Extracellular Vesicles in Modulating the Host Immune Response during Parasitic Infections. Front Immunol 2014;5:433. 10.3389/fimmu.2014.00433

Montaraz JA, Winter AJ. Comparison of living and nonliving vaccines for Brucella abortus in BALB/c mice. Infection and immunity. 1986 Aug 1;53(2):245-51.

Moreno CA, Jara F. Ecological studies on fish fauna associated with Macrocystis pyrifera belts in the south of Fueguian Islands, Chile. Marine ecology progress series. Oldendorf. 1984 Jan 1;15(1):99-107.

Morse MA, Garst J, Osada T, Khan S, Hobeika A, Clay TM, Valente N, Shreeniwas R, Sutton MA, Delcayre A, Hsu DH. A phase I study of dexosome immunotherapy in patients with advanced non-small cell lung cancer. Journal of translational medicine. 2005 Dec;3(1):9.

Mourao-Sa D, Roy S, Blander JM. Vita-PAMPs: signatures of microbial viability. InCrossroads Between Innate and Adaptive Immunity IV 2013 (pp. 1-8). Springer, New York, NY.

Mueller DL. Mechanisms maintaining peripheral tolerance. Nature immunology. 2010 Jan;11(1):21.

Mugnier MR, Stebbins CE, Papavasiliou FN. Masters of disguise: antigenic variation and the VSG coat in Trypanosoma brucei. PLoS pathogens. 2016 Sep 1;12(9):e1005784.

Munita JM, Arias CA. Mechanisms of antibiotic resistance. Microbiology spectrum. 2016 Apr;4(2).

Munn DH, Mellor AL. IDO in the tumor microenvironment: inflammation, counter- regulation, and tolerance. Trends in immunology. 2016 Mar 1;37(3):193-207.

Muñoz-Carrillo JL, Cordero JF, Gutiérrez-Coronado O, Villalobos-Gutiérrez PT, Ramos-Gracia LG, Hernández-Reyes VE. Cytokine Profiling Plays a Crucial MaulanaRole in Activating Azad Library,Immune System Aligarh to Clear Muslim Infectious University Pathogens. InImmune Response Activation 2018 Oct 4. IntechOpen.

Muñoz-Wolf N, Lavelle EC. Innate immune receptors. InNLR Proteins 2016 (pp. 1- 43). Humana Press, New York, NY.

Munz C. Antigen processing for MHC class II presentation via autophagy. Frontiers in immunology. 2012 Feb 2;3:9.

Nagasawa M, Spits H, Ros XR. Innate lymphoid cells (ILCs): cytokine hubs regulating immunity and tissue homeostasis. Cold Spring Harbor perspectives in biology. 2018 Dec 1;10(12):a030304.

190

Bibliography

Nardacci R, Ciccosanti F, Marsella C, Ippolito G, Piacentini M, Fimia GM. Role of autophagy in HIV infection and pathogenesis. Journal of internal medicine. 2017 May;281(5):422-32.

Newton HJ, Ang DK, Van Driel IR, Hartland EL. Molecular pathogenesis of infections caused by Legionella pneumophila. Clinical microbiology reviews. 2010 Apr 1;23(2):274-98.

Nichols DB, De Martini W, Cottrell J. Poxviruses utilize multiple strategies to inhibit apoptosis. Viruses. 2017 Aug;9(8):215.

Nicholson LB. The immune system. Essays in biochemistry. 2016 Oct 31;60(3):275- 301.

Nicoletti, P., 1990. Vaccination. In: Nielsen, K., Duncan, J.R. (Eds.), Animal Brucellosis. CRC Press, Boca Raton, pp. 284–299.

Niemann S, Köser CU, Gagneux S, Plinke C, Homolka S, Bignell H, Carter RJ, Cheetham RK, Cox A, Gormley NA, Kokko-Gonzales P. Genomic diversity among drug sensitive and multidrug resistant isolates of Mycobacterium tuberculosis with identical DNA fingerprints. PloS one. 2009 Oct 12;4(10):e7407.

Noack M, Miossec P. Th17 and regulatory T cell balance in autoimmune and inflammatory diseases. Autoimmunity reviews. 2014 Jun 1;13(6):668-77.

Nolte-'t Hoen E, Cremer T, Gallo RC, et al. Extracellular vesicles and viruses: Are they close relatives? Proc Natl Acad Sci U S A 2016;113:9155-61. 10.1073/pnas.1605146113

Nunes-Alves C, Booty MG, Carpenter SM, Jayaraman P, Rothchild AC, Behar SM. In search of a new paradigm for protective immunity to TB. Nature Reviews Microbiology. 2014 Apr;12(4):289-99.

Nunes-Alves C, Booty MG, Carpenter SM, Jayaraman P, Rothchild AC, Behar SM. In searchMaulana of a new Azad paradigm Library, for protective Aligarh immunity Muslim to TB. University Nature Reviews Microbiology. 2014 Apr;12(4):289-99.

O'Garra A, Redford PS, McNab FW, Bloom CI, Wilkinson RJ, Berry MP. The immune response in tuberculosis. Annual review of immunology. 2013 Mar 21;31:475-527.

O’Hagan, D. T., and N. M. Valiante. 2003.Recent advances in the discovery and delivery of vaccine adjuvants. Nat. Rev. Drug Discov. 2: 727–735.

O’Leary MF, Hood DA. Denervation-induced oxidative stress and autophagy signaling in muscle. Autophagy. 2009 Feb 16;5(2):230-1.

191

Bibliography

Oliveira SC, Splitter GA. Subcloning and expression of the Brucella abortus L7/L12 ribosomal gene and T-lymphocyte recognition of the recombinant protein. Infection and immunity. 1994 Nov 1;62(11):5201-4.

Oliveira SC, Zhu Y, Splitter G. Sequences of the rplJL operon containing the L10 and L7/L12 genes from Brucella abortus. Gene. 1994 Jan 1;140(1):137-8.

Oliveira, S.C. and Splitter, G.A. 1996. Immunization of mice recombinant L7/L12 ribosomal protein confers protection against Brucellaabortusinfection. Vaccine. 14:959–62.

Orzalli MH, Broekema NM, Diner BA, Hancks DC, Elde NC, Cristea IM, Knipe DM. cGAS-mediated stabilization of IFI16 promotes innate signaling during herpes simplex virus infection. Proceedings of the National Academy of Sciences. 2015 Apr 7;112(14):E1773-81.

Osborn JA, Jardine FH, Young JF, Wilkinson G. The preparation and properties of tris (triphenylphosphine) halogenorhodium (I) and some reactions thereof including catalytic homogeneous hydrogenation of olefins and acetylenes and their derivatives. Journal of the Chemical Society A: Inorganic, Physical, Theoretical. 1966:1711-32.

Oswald E, Nougayrède JP, Taieb F, Sugai M. Bacterial toxins that modulate host cell- cycle progression. Current opinion in microbiology. 2005 Feb 1;8(1):83-91.

Ouyang W, Rutz S, Crellin NK, Valdez PA, Hymowitz SG. Regulation and functions of the IL-10 family of cytokines in inflammation and disease. Annual review of immunology. 2011 Apr 23;29:71-109.

Owais, M. and Gupta, C.M. 2000.Liposome mediated cytosolic delivery of macromolecules and its possible use in vaccine development. Eur J Biochem. 267:3946–56.

Pahari S, Kaur G, Negi S, Aqdas M, Das DK, Bashir H, Singh S, Nagare M, Khan J, Agrewala JN. Reinforcing the functionality of mononuclear phagocyte system Maulanato control tuberculosis. Azad Library, Frontiers Aligarh in immunology. Muslim 2018 University Feb 9;9:193.

Pappas, G., Papadimitriou, P., Akritidis, N., Christou, L., Tsianos, E.V. 2006. The new global map of human brucellosis. Lancet Infect Dis. 6(2):91-9.

Parameswaran N, Patial S. Tumor necrosis factor-α signaling in macrophages. Critical Reviews™ in Eukaryotic Gene Expression. 2010;20(2).

Parihar SP, Guler R, Khutlang R, Lang DM, Hurdayal R, Mhlanga MM, Suzuki H, Marais AD, Brombacher F. Statin therapy reduces the mycobacterium tuberculosis burden in human macrophages and in mice by enhancing autophagy and phagosome maturation. The Journal of infectious diseases. 2013 Oct 16;209(5):754-63.

192

Bibliography

Pasman L, Kasper DL. Building conventions for unconventional lymphocytes. Immunological reviews. 2017 Sep;279(1):52-62.

Pasqualetti P, Bonomini C, Dal Forno G, Paulon L, Sinforiani E, Marra C, Zanetti O, Rossini PM. A randomized controlled study on effects of ibuprofen on cognitive progression of Alzheimer’s disease. Aging clinical and experimental research. 2009 Apr 1;21(2):102-10.

Pasquevich KA, Ibanez AE, Coria LM, GarcíaSamartino C, Estein SM, Zwerdling A, Barrionuevo P, Oliveira FS, Seither C, Warzecha H, Oliveira SC, Giambartolomei GH, Cassataro J. An oral vaccine based on U-Omp19 induces protection against B. abortus mucosal challenge by inducing an adaptive IL-17 immune response in mice. PLoS One. 2011;6(1):e16203.

Pasquevich, K.A.I., Estein, S.M., GarcíaSamartino, C., Zwerdling, A., Coria, L.M., Barrionuevo, P., Fossati, C.A., Giambartolomei, G.H., Cassataro, J. 2009. Immunization with recombinant Brucella species outer membrane protein Omp16 or Omp19 in adjuvant induces specific CD4+ and CD8+ T cells as well as systemic and oral protection against Brucellaabortus infection. Infect Immun. 77(1):436-45.

Patel, M., Xu, D., Kewin, P., et al. 2005. TLR2 agonist ameliorates established allergic airway inflammation by promoting Th1 response and not via regulatory T cells. J Immunol. 174:7558–63.

Paulsen V. Completely bounded maps and operator algebras. Cambridge University Press; 2002.

Peddireddy V, Doddam SN, Ahmed N. Mycobacterial dormancy systems and host responses in tuberculosis. Frontiers in immunology. 2017 Feb 15;8:84.

Pereira MI. Tuberculosis infection in captive sloth bears (Melursus ursinus): a pilot study on diagnostic strategies(Doctoral dissertation, Universidade de Lisboa, Faculdade de Medicina Veterinária).

Perera PY, LichyMaulana JH, Waldmann Azad Library, TA, Perera Aligarh LP. The Muslim role of University interleukin-15 in inflammation and immune responses to infection: implications for its therapeutic use. Microbes and infection. 2012 Mar 1;14(3):247-61.

Perrie, Y., Mohammed, A.R., Kirby, D.J., McNeil, S.E., Bramwell, V.W. 2008. Vaccine adjuvant systems: enhancing the efficacy of sub-unit protein antigens. Int J Pharm. 8;364(2):272-80.

Pizarro-Cerdá J, Méresse S, Parton RG, van der Goot G, Sola-Landa A, Lopez-Goñi I, Moreno E, Gorvel JP. Brucella abortus transits through the autophagic pathway and replicates in the endoplasmic reticulum of nonprofessional phagocytes. Infection and immunity. 1998 Dec 1;66(12):5711-24.

193

Bibliography

Pollard AJ, Frasch C. Development of natural immunity to Neisseria meningitidis. Vaccine. 2001 Dec 8;19(11-12):1327-46.

Prister LL. The Role of a Guanine Quadruplex and Associated sRNA in Pilin Antigenic Variation of Neisseria gonorrhoeae (Doctoral dissertation, Northwestern University).

Pulendran B, Ahmed R. Immunological mechanisms of vaccination. Nature immunology. 2011 Jun;12(6):509.

Pulendran, B. and Ahmed, R. 2006. Translating innate immunity it immunological memory: implications for vaccine development. Cell. 124: 849–863.

Qazi, K.R., Gehrmann, U., Domange Jordo, E., Karlsson, M.C., Gabrielsson, S., 2009. Antigen-loaded exosomes alone induce Th1-type memory through a B- cell-dependent mechanism. Blood 113, 2673–2683.

Ram S, Cullinane M, Blom AM, Gulati S, McQuillen DP, Boden R, Monks BG, O'Connell C, Elkins C, Pangburn MK, Dahlbäck B. C4bp binding to porin mediates stable serum resistance of Neisseria gonorrhoeae. International immunopharmacology. 2001 Mar 1;1(3):423-32.

Rao, M., Peachmana, K. K., Lic, Q., Matyasa, G.R., Shivachandrac, S.B. Borscheld, R., Mortholee,V.I., Fernandez-Pradad, C., Alvinga, C.R., Rao, V.B. 2011. Highly effective generic adjuvant systems for orphan or poverty-related vaccines. Vaccine. 29:873–877.

Raposo G, Nijman HW, Stoorvogel W, et al. B lymphocytes secrete antigen- presenting vesicles. J Exp Med1996;183:1161-72. 10.1084/jem.183.3.1161 ]

RastogiMaulana RP, Sinha RP.Azad 2010 Library, Apoptosis: Aligarh molecular Muslim mechanisms University and pathogenicity. Rawal N, Rajagopalan R, Salvi VP. Stringent regulation of complement lectin pathway C3/C5 convertase by C4b-binding protein (C4BP). Molecular immunology. 2009 Sep 1;46(15):2902-10.

Razzaq MS, AlSaadi MA, Al-Yassari AK. Molecular study of virulence genes of Brucella isolated from human clinical cases in Babylon Province. Journal of University of Babylon. 2014;22(5):1531-44.

Redente EF, Higgins DM, Dwyer‐Nield LD, Orme IM, Gonzalez‐Juarrero M, Malkinson AM. Differential polarization of alveolar macrophages and bone marrow‐derived monocytes following chemically and pathogen‐induced

194

Bibliography

chronic lung inflammation. Journal of leukocyte biology. 2010 Jul;88(1):159- 68.

Reece ST, Kaufmann SH. Host defenses to intracellular bacteria. InClinical Immunology 2019 Jan 1 (pp. 375-389).

Ribeiro KD, Kowalski LP, Saba LM, Camargo BD. Epithelial salivary glands neoplasms in children and adolescents: a forty‐four‐year experience. Medical and pediatric oncology. 2002 Dec;39(6):594-600.

Ribet D, Cossart P. How bacterial pathogens colonize their hosts and invade deeper tissues. Microbes and infection. 2015 Mar 1;17(3):173-83.

Ricklin D, Hajishengallis G, Yang K, Lambris JD. Complement: a key system for immune surveillance and homeostasis. Nature immunology. 2010 Sep;11(9):785.

Riechmann L, Clark M, Waldmann H, Winter G. Reshaping human antibodies for therapy. Nature. 1988 Mar;332(6162):323.

Rivera J, Tessarollo L. Genetic background and the dilemma of translating mouse studies to humans. Immunity. 2008 Jan 18;28(1):1-4.

Robbins PD, Morelli AE. Regulation of immune responses by extracellular vesicles. Nat Rev Immunol2014;14:195-208. 10.1038/nri3622

Roh JS, Sohn DH. Damage-associated molecular patterns in inflammatory diseases. Immune network. 2018 Aug 13;18(4).

Roop RM, Gaines JM, Anderson ES, Caswell CC, Martin DW. Survival of the fittest: how Brucella strains adapt to their intracellular niche in the host. Medical microbiology and immunology. 2009 Nov 1;198(4):221-38.

Rueckert, C. andGuzman, C.A., 2012. Vaccines: from empirical development to rational design. PLoSPathog. 8(11):e1003001.

Sadarangani M,Maulana Pollard AJ, Azad Gray -Library,Owen SD. OpaAligarh proteins Muslim and CEACAMs: University pathways of immune engagement for pathogenic Neisseria. FEMS microbiology reviews. 2011 May 1;35(3):498-514.

Salgame P. Host innate and Th1 responses and the bacterial factors that control Mycobacterium tuberculosis infection. Current opinion in immunology. 2005 Aug 1;17(4):374-80.

Sambrook, J. and Russel, D.W. 2001. Molecular cloning: a laboratory manual, 3rd ed. Cold Spring Harbor, N.Y.

Sánchez-Ramón S, Conejero L, Netea MG, Sancho D, Palomares O, Subiza JL. Trained immunity-based vaccines: a new paradigm for the development of

195

Bibliography

broad-spectrum anti-infectious formulations. Frontiers in immunology. 2018;9.

Sasindran SJ, Torrelles JB. Mycobacterium Tuberculosis Infection and Inflammation: what is Beneficial for the Host and for the Bacterium?. Frontiers in microbiology. 2011 Jan 26;2:2.

Schauber J, Dorschner RA, Coda AB, Büchau AS, Liu PT, Kiken D, Helfrich YR, Kang S, Elalieh HZ, Steinmeyer A, Zügel U. Injury enhances TLR2 function and antimicrobial peptide expression through a vitamin D–dependent mechanism. The Journal of clinical investigation. 2007 Mar 1;117(3):803-11.

Schorey JS, Cheng Y, Singh PP, Smith VL. Exosomes and other extracellular vesicles in host–pathogen interactions. EMBO reports. 2015 Jan 1;16(1):24-43.

Schroeder Jr HW, Cavacini L. Structure and function of immunoglobulins. Journal of Allergy and Clinical Immunology. 2010 Feb 1;125(2):S41-52.

Schurig GG, Roop II RM, Bagchi T, Boyle S, Buhrman D, Sriranganathan N. Biological properties of RB51; a stable rough strain of Brucella abortus. Veterinary microbiology. 1991 Jul 1;28(2):171-88.

Schurig GG, Sriranganathan N, Corbel MJ. Brucellosis vaccines: past, present and future. Vet Microbiol. 2002; 90(1-4):479-96.

Schurig GG, Sriranganathan N, Corbel MJ. Brucellosis vaccines: past, present and future. Veterinary microbiology. 2002 Dec 20;90(1-4):479-96.

Seveau S. Multifaceted activity of listeriolysin O, the cholesterol-dependent cytolysin of Listeria monocytogenes. InMACPF/CDC Proteins-Agents of Defence, Attack and Invasion 2014 (pp. 161-195). Springer, Dordrecht.

Shafiani S, Tucker-Heard GS, Kariyone A, Takatsu K, Urdahl KB. Pathogen-specific regulatory T cells delay the arrival of effector T cells in the lung during early tuberculosis. Journal of Experimental Medicine. 2010 Jul 5;207(7):1409-20.

Shao Y,Maulana Shen Y, Azad Chen T, Library, et al. The Aligarh functions Muslim and clinical University applications of tumor- derived exosomes. Oncotarget2016;7:60736-51.

Sharif HS, Aideyan OA, Clark DC, Madkour MM, Aabed MY, Mattsson TA, Al- Deeb SM, Moutaery KR. Brucellar and tuberculous spondylitis: comparative imaging features. Radiology. 1989 May;171(2):419-25.

Shen H, Chen ZW. The crucial roles of Th17-related cytokines/signal pathways in M. tuberculosis infection. Cellular & molecular immunology. 2018 Mar;15(3):216.

196

Bibliography

Shenoda BB, Ajit SK. Modulation of Immune Responses by Exosomes Derived from Antigen-Presenting Cells.Clin Med Insights Pathol 2016;9:1-8.

Shotland Y, Krämer H, Groisman EA. The Salmonella SpiC protein targets the mammalian Hook3 protein function to alter cellular trafficking. Molecular microbiology. 2003 Sep;49(6):1565-76.

Siadat, S.D., Salmani, A.S. and Aghasadeghi MR. 2012. Brucellosis Vaccines: An Overview, Zoonosis, Dr. Jacob Lorenzo-Morales (Ed.), ISBN: 978-953-51- 0479-7, InTech.

Singh SB, Davis AS, Taylor GA, Deretic V. Human IRGM induces autophagy to eliminate intracellular mycobacteria. Science. 2006 Sep 8;313(5792):1438-41.

Singh, V., Jain, S., Gowthaman, U., Parihar, P., Gupta, P., et al. 2011. Co- Administration of IL-1+IL-6+TNF-a with Mycobacterium tuberculosis Infected Macrophages Vaccine Induces Better Protective T Cell Memory than BCG. PLoS ONE 6(1): e16097.

Singha, H., Mallick, A.I., Jana, C., Fatima, N., Owais, M. Chaudhuria, P. 2011. Co- immunization with interlukin-18 enhances the protective efficacy of liposomes encapsulated recombinant Cu–Zn superoxide dismutase protein against Brucellaabortus. Vaccine. 29:4720– 4727.

Skokos D, Le Panse S, Villa I, Rousselle JC, Peronet R, David B, Namane A, Mécheri S. Mast cell-dependent B and T lymphocyte activation is mediated by the secretion of immunologically active exosomes. The Journal of Immunology. 2001 Jan 15;166(2):868-76.

Smith I. Mycobacterium tuberculosis pathogenesis and molecular determinants of virulence. Clinical microbiology reviews. 2003 Jul 1;16(3):463-96.

Sparrer KM, Gableske S, Zurenski MA, Parker ZM, Full F, Baumgart GJ, Kato J, Pacheco-Rodriguez G, Liang C, Pornillos O, Moss J. TRIM23 mediates virus- induced autophagy via activation of TBK1. Nature microbiology. 2017 Nov;2(11):1543.Maulana Azad Library, Aligarh Muslim University

Starr T, Child R, Wehrly TD, Hansen B, Hwang S, López-Otin C, Virgin HW, Celli J. Selective subversion of autophagy complexes facilitates completion of the Brucella intracellular cycle. Cell host & microbe. 2012 Jan 19;11(1):33-45.

Steinhagen F, Kinjo T, Bode C, Klinman DM.. TLR-based immune adjuvants. Vaccine. 2011. 29:3341–3355.

Stoermer KA, Morrison TE. Complement and viral pathogenesis. Virology. 2011 Mar 15;411(2):362-73.

197

Bibliography

Stolte M, Wellens E, Bethke B, Ritter M, Eidt H. Helicobacter heilmannii (formerly Gastrospirillum hominis) gastritis: an infection transmitted by animals?. Scandinavian journal of gastroenterology. 1994 Jan 1;29(12):1061-4.

Storey M, Jordan S. An overview of the immune system. Nursing Standard. 2008 Dec 17;23.

Strugnell RA, Wijburg OL. The role of secretory antibodies in infection immunity. Nature Reviews Microbiology. 2010 Sep;8(9):656.

Stutz MD, Clark MP, Doerflinger M, Pellegrini M. Mycobacterium tuberculosis: rewiring host cell signaling to promote infection. Journal of leukocyte biology. 2018 Feb 1;103(2):259-68.

Su SA, Xie Y, Fu Z, Wang Y, Wang JA, Xiang M. Emerging role of exosome- mediated intercellular communication in vascular remodeling. Oncotarget. 2017 Apr 11;8(15):25700.

Subauste CS. CD40 and the immune response to parasitic infections. InSeminars in immunology 2009 Oct 1 (Vol. 21, No. 5, pp. 273-282). Academic Press.

Suematsu N, Tsutsui H, Wen J, Kang D, Ikeuchi M, Ide T, Hayashidani S, Shiomi T, Kubota T, Hamasaki N, Takeshita A. Oxidative stress mediates tumor necrosis factor-α–induced mitochondrial DNA damage and dysfunction in cardiac myocytes. Circulation. 2003 Mar 18;107(10):1418-23.

Sullivan EV, McCullough G, Stager M. A developmental study of the relationship between conceptual, ego, and moral development. Child Development. 1970 Jun 1:399-411.

Sun Y, Han M, Kim C, et al. Interplay between interferon-mediated innate immunity and porcine reproductive and respiratory syndrome virus. Viruses 2012;4:424- 46.

Suresh R, Mosser DM. Pattern recognition receptors in innate immunity, host defense, Maulanaand immunopathology. Azad Library, Advances Aligarh inMuslim physiology University education. 2013 Dec;37(4):284-91.

Sysoeva TA, Zepeda-Rivera MA, Huppert LA, Burton BM. Dimer recognition and secretion by the ESX secretion system in Bacillus subtilis. Proceedings of the National Academy of Sciences. 2014 May 27;111(21):7653-8.

Takeda K, Akira S. Toll-like receptors in innate immunity. International immunology. 2005 Jan 1;17(1):1-4.

Tatum B. Talking about race, learning about racism: The application of racial identity development theory in the classroom. Harvard Educational Review. 1992 Apr 1;62(1):1-25.

198

Bibliography

Thaiss CA, Levy M, Suez J, Elinav E. The interplay between the innate immune system and the microbiota. Current opinion in immunology. 2014 Feb 1;26:41-8.

Thaiss CA, Zmora N, Levy M, Elinav E. The microbiome and innate immunity. Nature. 2016 Jul;535(7610):65-74.

Thakur A, Mikkelsen H, Jungersen G. Intracellular Pathogens: Host Immunity and Microbial Persistence Strategies. Journal of immunology research. 2019;2019.

Théry C, Zitvogel L, Amigorena S. Exosomes: composition, biogenesis and function. Nature Reviews Immunology. 2002 Aug;2(8):569.

Thoma-Uszynski, S., Kiertscher, S.M., Ochoa, M.T., et al. 2000.Activation of Toll- like receptor 2 on human DCs triggers induction of IL-12, but not IL-10. J Immunol.165:3804–10.

Tibor, A., Decelle, B., Letesson, J.J. 1999. Outer membrane proteins Omp10, Omp16, and Omp19 of Brucella spp. are lipoproteins. Infect Immun. 67(9):4960-2.

Tiwari BM, Kannan N, Vemu L, Raghunand TR. The Mycobacterium tuberculosis PE proteins Rv0285 and Rv1386 modulate innate immunity and mediate bacillary survival in macrophages. PloS one. 2012 Dec 17;7(12):e51686.

Tosh C, Sanyal A, Hemadri D, Venkataramanan R. Phylogenetic analysis of serotype A foot-and-mouth disease virus isolated in India between 1977 and 2000. Archives of virology. 2002 Mar 1;147(3):493-513.

Towbin H, Staehelin T, Gordon J. Electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets: procedure and some applications. Proceedings of the National Academy of Sciences. 1979 Sep 1;76(9):4350-4.

Tufail S, Owais M, Kazmi S, Balyan R, Khalsa JK, Faisal SM, Sherwani MA, Gatoo MA, Umar MS, Zubair S. Amyloid Form of Ovalbumin Evokes Native AntigenMaulana-specific Immune Azad ResponseLibrary, in Aligarh the Host Muslim PROSPECTIVE University IMMUNO- PROPHYLACTIC POTENTIAL. Journal of Biological Chemistry. 2015 Feb 13;290(7):4131-48.

Turner OC, Basaraba RJ, Orme IM. Immunopathogenesis of pulmonary granulomas in the guinea pig after infection with Mycobacterium tuberculosis. Infection and immunity. 2003 Feb 1;71(2):864-71.

Umilta C, Nicoletti R. Spatial stimulus-response compatibility. InAdvances in psychology 1990 Jan 1 (Vol. 65, pp. 89-116). North-Holland.

199

Bibliography

Unsworth DJ, Kieffer MA, Holborow EJ, Coombs RR, Walker-Smith JA. IgA anti- gliadin antibodies in coeliac disease. Clinical and experimental immunology. 1981 Nov;46(2):286.

Uribe-Querol E, Rosales C. Control of phagocytosis by microbial pathogens. Frontiers in immunology. 2017 Oct 24;8:1368.

Vaerman JP, Heremans JF, Vaerman C. Studies of the Immune Globulins of Human Serum: I. A Method for the Simultaneous Isolation of the Three Immune Globulins (γss, γ1M and γ1A) from Individual Small Serum Samples. The Journal of Immunology. 1963 Jul 1;91(1):7-10.

Van Avondt K, van Sorge NM, Meyaard L. Bacterial immune evasion through manipulation of host inhibitory immune signaling. PLoS pathogens. 2015 Mar 5;11(3):e1004644. van Wilgenburg B, Loh L, Chen Z, Pediongco TJ, Wang H, Shi M, Zhao Z, Koutsakos M, Nüssing S, Sant S, Wang Z. MAIT cells contribute to protection against lethal influenza infection in vivo. Nature communications. 2018 Nov 9;9(1):4706.

Velikovsky CA, Cassataro J, Giambartolomei GH, Goldbaum FA, Estein S, Bowden RA, Bruno L, Fossati CA, Spitz M. A DNA vaccine encoding lumazine synthase from Brucella abortus induces protective immunity in BALB/c mice. Infection and immunity. 2002 May 1;70(5):2507-11.

Verma, J.N., Rao, M., Amselem, S., Krzych, U., Alving, C.R., Green, S.J., Wassef, N.M. 1992. Adjuvant effects of liposomes containing lipid A: enhancement of liposomal antigen presentation and recruitment of macrophages. Infect Immun. 60(6):2438-44.

Vieyra-Lobato MR, Vela-Ojeda J, Montiel-Cervantes L, López-Santiago R, Moreno- Lafont MC. Description of CD8+ Regulatory T Lymphocytes and Their Specific Intervention in Graft-versus-Host and Infectious Diseases, Autoimmunity, and Cancer. Journal of immunology research. 2018;2018. Maulana Azad Library, Aligarh Muslim University Vivier E, Raulet DH, Moretta A, Caligiuri MA, Zitvogel L, Lanier LL, Yokoyama WM, Ugolini S. Innate or adaptive immunity? The example of natural killer cells. science. 2011 Jan 7;331(6013):44-9.

Voth DE, Broederdorf LJ, Graham JG. Bacterial Type IV secretion systems: versatile virulence machines. Future microbiology. 2012 Feb;7(2):241-57.

Walker JD, Maier CL, Pober JS. Cytomegalovirus-infected human endothelial cells can stimulate allogeneic CD4+ memory T cells by releasing antigenic exosomes. J Immunol 2009;182:1548-59. 13. Montaner-Tarbes S, Borrás FE, Montoya M, et al. Serum-derived exosomes from non-viremic animals

200

Bibliography

previously exposed to the porcine respiratory and reproductive virus contain antigenic viral proteins. Vet Res2016;47:59.

Wallden K, Rivera‐Calzada A, Waksman G. Microreview: Type IV secretion systems: versatility and diversity in function. Cellular microbiology. 2010 Sep;12(9):1203-12.

Walsh CT, Wencewicz TA. Prospects for new antibiotics: a molecule-centered perspective. The Journal of antibiotics. 2014 Jan;67(1):7.

Warne B, Harkins CP, Harris SR, Vatsiou A, Stanley-Wall N, Parkhill J, Peacock SJ, Palmer T, Holden MT. The Ess/Type VII secretion system of Staphylococcus aureus shows unexpected genetic diversity. BMC genomics. 2016 Dec;17(1):222.

Waters JP, Pober JS, Bradley JR. Tumour necrosis factor in infectious disease. The Journal of pathology. 2013 Jun;230(2):132-47.

Weiss G, Schaible UE. Macrophage defense mechanisms against intracellular bacteria. Immunological reviews. 2015 Mar;264(1):182-203.

Witter AR, Okunnu BM, Berg RE. The essential role of neutrophils during infection with the intracellular bacterial pathogen Listeria monocytogenes. The Journal of Immunology. 2016 Sep 1;197(5):1557-65.

Wong KW. The role of ESX-1 in Mycobacterium tuberculosis pathogenesis. Microbiology spectrum. 2017 May;5(3)

Wu YL, Ding YP, Tanaka Y, Shen LW, Wei CH, Minato N, Zhang W. γδ T cells and their potential for immunotherapy. International journal of biological sciences. 2014;10(2):119.

Xu, S., F., Shi, and H. Wu., 1991. Schistosomajaponicum: some parameters affecting the development of protective immunity induced by a cryopreserved, irradiated schistosomula vaccine in guinea-pigs. Parasitology 102: 45–47. Maulana Azad Library, Aligarh Muslim University Yamagami H, Matsumoto T, Fujiwara N, Arakawa T, Kaneda K, Yano I, Kobayashi K. Trehalose 6, 6′-dimycolate (cord factor) ofmycobacterium tuberculosis induces foreign-body-and hypersensitivity-type granulomas in mice. Infection and immunity. 2001 Feb 1;69(2):810-5.

Yang G, Chen J, Wen Z, Ge K, Zhu L, Chen X, Chen X. The role of selenium in Keshan disease. InAdvances in nutritional research 1984 (pp. 203-231). Springer, Boston, MA.

Yang T, Zhang F, Zhai L, He W, Tan Z, Sun Y, Wang Y, Liu L, Ning C, Zhou W, Ao H. Transcriptome of porcine PBMCs over two generations reveals key genes

201

Bibliography

and pathways associated with variable antibody responses post PRRSV vaccination. Scientific reports. 2018 Feb 6;8(1):2460.

Yoneyama H, Matsuno K, Zhang Y, Murai M, Itakura M, Ishikawa S, Hasegawa G, Naito M, Asakura H, Matsushima K. Regulation by chemokines of circulating dendritic cell precursors, and the formation of portal tract–associated lymphoid tissue, in a granulomatous liver disease. Journal of Experimental Medicine. 2001 Jan 1;193(1):35-50.

Young EJ. Serologic diagnosis of human brucellosis: analysis of 214 cases by agglutination tests and review of the literature. Reviews of infectious diseases. 1991 May 1;13(3):359-72.

Zaks, K., Jordan, M., Guth, A., Sellins, K., Kedl, R., Izzo, A., Bosio, C., and Dow. S. 2006. Efficient Immunization and Cross-Priming by Vaccine Adjuvants Containing TLR3 or TLR9 Agonists Complexed to Cationic Liposomes. J Immunol. 176:7335-7345.

Zeng W, Eriksson E, Chua B, Grollo L, Jackson DC. Structural requirement for the agonist activity of the TLR2 ligand Pam2Cys. Amino Acids. 2010 Jul 1;39(2):471-80.

Zeng, W., Ghosh, S., Lau. Y.F., Brown, L.E., Jackson, D.C. 2002. Highly immunogenic and totally synthetic lipopeptides as selfadjuvanting imm unocontraceptivevaccines. J Immunol. 169(9):4905-12.

Zhao X, Drlica K. Restricting the selection of antibiotic-resistant mutants: a general strategy derived from fluoroquinolone studies. Clinical Infectious Diseases. 2001 Sep 15;33(Supplement_3):S147-56.

Zhou LF, Zhu Y, Cui XF, Xie WP, Hu AH, Yin KS. Arsenic trioxide, a potent inhibitor of NF-κB, abrogates allergen-induced airway hyperresponsiveness and inflammation. Respiratory research. 2006 Dec;7(1):146.

Zhu J, Paul WE. Peripheral CD4+ T‐cell differentiation regulated by networks of Maulanacytokines Azad and transcriptionLibrary, Aligarh factors. Muslim Immunological University reviews. 2010 Nov;238(1):247-62.

Zhu Y, Yao S, Chen L. Cell surface signaling molecules in the control of immune responses: a tide model. Immunity. 2011 Apr 22;34(4):466-78.

Zipfel C. Early molecular events in PAMP-triggered immunity. Current Opinion in plant biology. 2009 Aug 1;12(4):414-20.

Zitvogel L, Regnault A, Lozier A, et al. Eradication of established murine tumors using a novel cell-free vaccine: dendritic cell-derived exosomes. Nat Med 1998;4:594-600. 10.1038/nm0598-594

202

LIST OF PUBLICATIONS

 Mujeeb AA, Alam KF, Alshameri AW, Jamal F, Farheen S, Kashif M, Ahmed A, Ghazi IA, Owais M. Chaperone Like Attributes of Biogenic Fluorescent Gold Nanoparticles: Potential to Alleviate Toxicity Induced by Intermediate State Fibrils Against Neuroblastoma Cells. Frontiers in Chemistry. 2019;7.

 Kazmi S, Mujeeb AA, Owais M. Cyclic undecapeptide Cyclosporin A mediated inhibition of amyloid synthesis: Implications in alleviation of amyloid induced neurotoxicity. Scientific reports. 2018 Nov 23;8(1):17283.

 Mohammad O, Faisal SM, Ahmad N, Rauf MA, Umar MS, Mujeeb AA, Pachauri P, Ahmed A, Kashif M, Ajmal M, Zubair S. Bio-mediated synthesis of 5-FU based nanoparticles employing orange fruit juice: a novel drug delivery system to treat skin fibrosarcoma in model animals. Scientific reports. 2019 Aug 23;9(1):1-4.

 Farhi A, Firdaus F, Saeed H, Mujeeb AA, Shakir M, Owais M. A quinoline- based fluorescent probe for selective detection and real-time monitoring of copper ions–a differential colorimetric approach. Photochemical & Photobiological Sciences. 2019.

Upcoming publications

 Olax scandens mediated biogenic synthesis of Ag-Cu nanocomposites: potential against inhibition of drug-resistant microbes Anzar Abdul MaulanaMujeeb, Nuha Azad Abeer Library, Khan, FauziaAligarh Jamal, Muslim Khan FarheenUniversity Badrealam, Haris Saeed, Shadab Kazmi,Ansam Wadia Faid Alshameri, Mohammad Kashif, Irfan Ahmad Ghazi and Mohammad Owais*, Front. Chem. - Nanoscience Manuscript ID: 516421

 A Leishmania specific promiscuous membrane protein Tubulin Folding Cofactor D divulges Th1/Th2 polarization in the host via ERK-1/2 and p38 MAPK signaling cascade. Fauzia Jamal, M Owais* , Manish Kumar Singh, Jagadish Hansa, Pushp Anjali, Ghufran Ahmad, Manas R Dikhit,Anzar Abdul Mujeeb, Sanjiva Bimal, Pradeep Das, Shubhankar K Singh* , Swaleha Zubair* and MOHD SAAD UMAR, Front. Immunol. - Vaccines and Molecular Therapeutics, Manuscript ID: 506961

 Aptamer mediated targeted delivery of Docetaxel to androgen independent prostate cancer cells: possible implication in cancer theranostics Shadab Kazmi , Anzar Abdul Mujeeb ,Syed Mohd. Faisal , Mohammad Owais Journal of cellular physiology Manuscript ID JCP-19-5786

Maulana Azad Library, Aligarh Muslim University ORIGINAL RESEARCH published: 19 November 2019 doi: 10.3389/fchem.2019.00787

Chaperone Like Attributes of Biogenic Fluorescent Gold Nanoparticles: Potential to Alleviate Toxicity Induced by Intermediate State Fibrils Against Neuroblastoma Cells

Anzar Abdul Mujeeb 1, Khan Farheen Badre Alam 1, Ansam Wadia Faid Alshameri 1, Fauzia Jamal 1, Saba Farheen 1, Mohd Kashif 2, Anees Ahmed 1, Irfan Ahmad Ghazi 3* and Mohammad Owais 1*

1 Interdisciplinary Biotechnology Unit, Aligarh Muslim University, Aligarh, India, 2 CSIR-National Botanical Research Institute, Lucknow, India, 3 Department of Plant Sciences, School of Life Sciences, University of Hyderabad, Hyderabad, India

Edited by: Sadiq Umar, In general, neurodegenerative disorders have a great deal ofcorrelationwiththe University of Illinois at Chicago, United States misfolded as well as aggregated forms of protein-based macromolecules. Among various Reviewed by: species formed during the aggregation process, protein oligomers have been classified Imran Ahmad, as most toxic entities against several types of living cells.Aseriesofchemicalshavebeen University of Illinois at Chicago, developed to inhibit protein aggregation as a measure to regulate neurodegenerative United States Abdul Malik, diseases. Recently, various classes of nanoparticles have also been reported to inhibit Emory University, United States protein aggregation. In the present study, we synthesized fluorescent gold nanoparticles *Correspondence: (B-AuNPs) employing Olax scandens leaf extract. Next, an in vitro study was performed Irfan Ahmad Ghazi [email protected] to assess the effect of as-synthesized B-AuNPs on the aggregation behavior of the Mohammad Owais ovalbumin (OVA) and other related model proteins. We performed an extensive study [email protected] to elucidate anti-amyloidogenic properties of nano-sized entities and established that small-sized B-AuNPs manifest chaperone potential against protein aggregation. Further, Specialty section: This article was submitted to we exploited as-synthesized B-AuNPs as a mean to prevent protein aggregation Nanoscience, mediated toxicity in neuroblastoma cells. asectionofthejournal Frontiers in Chemistry Keywords: gold nanoparticles, biogenic nanoparticles, chaperone, amyloid, fibril Received: 24 AugustMaulana 2019 Azad Library, Aligarh Muslim University Accepted: 01 November 2019 Published: 19 November 2019 INTRODUCTION Citation: Mujeeb AA, Alam KFB, Amyloidal protein toxicity has a strong implication in several neurodegenerative disorders such Alshameri AWF, Jamal F, Farheen S, as Alzheimer’s, Parkinson’s (PD), Huntington’s, and Prion disease, etc. (Chung et al., 2018). Kashif M, Ahmed A, Ghazi IA and Fibrillation has been considered as a centric aggregation process that involves loss of native Owais M (2019) Chaperone Like conformation, unfolding and inappropriate refolding of protein-based macromolecules (Chiti and Attributes of Biogenic Fluorescent Dobson, 2017). In general, the protein aggregation encompasses formation ofoligomericand Gold Nanoparticles: Potential to other intermediate supramolecular entities, which subsequently associate to form insoluble fibrils Alleviate Toxicity Induced by β Intermediate State Fibrils Against comprising of unbranched -sheet rich fibrillar structure (Rahimi et al., 2008). Under normal Neuroblastoma Cells. physiological conditions, the aggregation-prone residues of a native protein remain buried deep Front. Chem. 7:787. in the hydrophobic core (Beerten et al., 2012). Surface exposure of β-strand forming short amino doi: 10.3389/fchem.2019.00787 acid residues ensues in the formation of protein aggregates (Wei et al., 2017). Various subspecies

Frontiers in Chemistry | www.frontiersin.org 1 November 2019 | Volume 7 | Article 787

OPEN Cyclic undecapeptide Cyclosporin A mediated inhibition of amyloid synthesis: Implications in Received: 23 November 2017 Accepted: 12 October 2018 alleviation of amyloid induced Published: xx xx xxxx neurotoxicity Shadab Kazmi, Anzar Abdul Mujeeb & Mohammad Owais

or polypeptide precursors under both physiological as well as simulated ambience. Amyloid synthesis is a multistep process that involves formation of several metastable intermediates. Among various intermediate species, the as-formed soluble oligomers are extremely toxic to the neuronal cells. In the present study, we evaluated cyclosporine A (CsA), an undecapeptide, for its potential to prevent aggregation of model protein ovalbumin (OVA). In an attempt to elucidate involved operative concentration dependent manner. The present study further suggests that exposure to CsA during impervious to the CsA treatment. The cyclic undecapeptide CsA was also found to successfully alleviate amyloid induced toxicity in neuroblastoma cells.

Aggregated forms of protein amyloids have a great deal of correlation with a growing list of diseases, including diabetes, Alzheimer’s, Parkinson’s, Huntington’s and Prion disease etc1. During !brillogenesis, misfolded proteins transform into a long, unbranched, β–sheet rich !brillar structure2. Protein aggregation generally ensues in generation of amyloid species in both in vitro as well as in vivo conditions3. In general, native proteins keep aggregation-prone residues buried in the hydrophobic core to avoid aggregation4. "e self-assemblage of β-strand forming short, aggregation-prone amino acid residues, is supposed to result in the formation of protein aggre- gates5 that may o#en acquire toxic properties6. MaulanaAmyloid-beta Azad (A βLibrary,) peptides (comprised Aligarh of 39–43 Muslim amino acid residues) University are main components of amyloid plaques7. "ey have been widely implicated in both familial as well as sporadic Alzheimer’s disease. "e amyloid cascade hypothesis presumes that amyloid aggregates, self-assembled from misfolded Aβ peptides, a$ect the structure and function of neuronal cells and stimulate apoptosis8,9. "is eventually results into synaptic dysfunction and neurodegeneration10. By stabilizing native state, small molecules/peptides can hinder the !brillation process and also reverse the misfolding of the protein thus can serve as promising clinical agent against many associated debilitating dis- eases11,12. "e observation is substantiated from the fact that there is a remarkable increase in the number of research articles, during recent past, reporting short peptides mediated inhibition of amyloid aggregation12–14. Recent advent in molecular biology and peptide synthesis technology has made it possible to fabricate speci!c peptides that have potential to inhibit aggregation. While it remains to be seen whether short peptides can be exploited as therapeutic agents to prevent the amyloid related diseases, nonetheless, such inhibitors can help us to comprehend intricacies associated with protein aggregation process14. "e short peptide based inhibitors can

India. Correspondence and requests for materials should be addressed to M.O. (email: [email protected])

SCIENTIFIC REPORTS | (2018) 8:17283 1 www.nature.com/scientificreports

OPEN based nanoparticles employing orange fruit juice: a novel drug Received: 20 May 2016 Accepted: 19 February 2019 delivery system to treat skin Published: xx xx xxxx Owais Mohammad, Syed Mohd. Faisal, Nadeem Ahmad, Mohd. Ahmar Rauf, Mohd Saad mar, Anzar Abdul Mujeeb, Piyush Pachauri, Anees Ahmed, Mohammad Kashif, Mohammad Ajmal & Swaleha Zubair

Nano-sized drug delivery systems (NDDS) have been widely exploited to achieve targeted delivery of pharmaco-materials. Traditional pharmaceutical approaches, implied in the synthesis of nano- formulations, are obscure owing to the incompatible physico-chemical properties of the core drug as well as some other factors crucial in development of NDDS. Infact, most of the existing methods used in development of NDDS rely on usage of additives or excipients, a special class of chemicals. Barring few exceptions, the usage of synthetic excipients ought to be curtailed because of several associated undesirable features. Such issues necessitate strategies that lead to development of the synthetic excipient free drug delivery system. Plant based extracts have great potential to induce synthesis of nano-sized particles. Considering this fact, here we propose a prototype employing orange fruit juice

Recent progression in the !eld of nanotechnology has made remarkable impact on human health and safety. Several recent reports have described a direct correlation between nanoparticles (NPs) size and higher e"cacy of the associated drug1,2. Nanoparticle based drug delivery vehicles have been reported to preferentially accumulate at the site of injury, infection and in#ammation, mostly because of endothelial dysfunction and blood vessel Maulanafenestration Azad at such locations. Library, $e nano-dimensions Aligarh of Muslim the NPs promote University their ability to traverse various parts of the body. In general, NPs have tendency to accumulate into liver or spleen (components of reticulo-endothelial system) as well as other vital organs including lungs and brain1–3. Surface modi!cation of NPs with speci!c ligands such as antibodies as well as aptamers etc. can facilitate their homing at desired site inside the host body. Upon gaining access to a speci!c biological niche, the nano-sized materials tend to interfere with a variety of cellular functions including cell proliferation, cell cycle regulation and various vital metabolic activities, etc. 5-Fluorouracil (5-FU), is an anti-metabolite showing broad spectrum anti-cancer activity against solid tum- ors4. Primarily, a pyrimidine analog, 5-FU is a purely ‘S-phase’ active chemotherapeutic agent (with no activity when cells are in G0 and G1 phase of cell cycle) that acts as a thymidylate synthase inhibitor and thereby inter- feres with DNA synthesis5. However, 5-FU drug has several limitations when used in clinical setting that include its short biological half-life, wide systemic distribution and marked toxic e%ects on bone marrow6. Numerous

Interdisciplinary Biotechnology Unit, AMU, Aligarh, India. University of Jeddah, Jeddah, Saudi Arabia. National Institute of Immunology, New Delhi, India. Jawaharlal Nehru Medical College, AMU, Aligarh, India. Present address: Department of Computer Science, AMU, Aligarh, India. Women’s College, AMU, Aligarh, India. Correspondence and requests for materials should be addressed to O.M. (email: [email protected]) or S.Z. (email: [email protected])

SCIENTIFIC REPORTS | (2019) 9:12288 | 1 1 Photochemical & 1 Photobiological Sciences

5 PAPER 5

A quinoline-based ﬂuorescent probe for selective 10 detection and real-time monitoring of copper ions 10 Cite this: DOI: 10.1039/c9pp00247b – adiﬀerential colorimetric approach†

Q1 Atika Farhi,a Farha Firdaus, *b Haris Saeed,c Anzar Mujeeb,c Mohammad Shakira 15 and Mohammad Owaisc 15

A quinoline moiety was used as a building block for designing a probe for the selective detection of copper ions in a partially aqueous medium. We have developed a molecular sensing system which gives insight into the complex physiological and redox aspects of labile copper. The probe provides a colori- 20 20 metric approach for distinguishing cuprous and cupric ions along with their simultaneous discrimination from other metal ions in the visible range of the spectrum. The chemosensor showed a remarkable fluorescence enhancement along with a significant bathochromic shift of about 35 nm. The detection limit of the probe was found to be 1.03 µM which is optimally favorable to be applied in real-time moni- 25 toring. Fabrication of paper strips with the probe was done to detect the presence of cuprous ions in the 25 real sample. The value of the binding constant (1.37 × 104 M−1) suggests stable complex formation Received 30th May 2019, between the metal ion and the sensing probe. The photoluminescence and structural aspects of the Accepted 26th September 2019 chemosensor were characterized by using fluorescence, absorption, ESI-MS, and 1H NMR spectroscopy. DOI: 10.1039/c9pp00247b Furthermore, the cytotoxic nature and bioimaging properties of the probe were interpreted in vitro on 30 rsc.li/pps RAW 264.7 macrophage cell lines and peripheral blood mononuclear cells (PBMCs) respectively. 30

Introduction electronic environment, etc.3 The presence of potentially toxic metal ions leads to the evolution of advance quantitative 35 The designing of an ideal sensing system lies in the concept of and qualitative analytical methods, to understand the nature 35 mutual cooperative operation of all the molecular components of the pollutant and to check their concentration for the present in it. Most synthetic receptors are based on the con- welfare of the environment.4,5 Due to the vast area of appli- struction of recognition sites that are capable of binding cation of chemosensors, there are practically an infinite actively to the host. Another keystone feature of the chemo- number of analytes of concern such as heavy metal ions, tran- 40 40 sensor is to apprehend the information regarding the analyte sition metal ions, phenols, volatile organic compounds, dyes – in a selective fashion that is encoded to allow distinction etc.6 10 In this work, we are narrowing it down to the sensing from other closely related chemical species.1 A typical chemo- of a transition metal ion, i.e., copper, which is of special inter- sensing system possesses a recognition site (receptor) and an est because of its versatility and ubiquity in different biological 2,11 45 active unit (fluorophore/chromophore)Maulana Azad that modifies Library, one or Aligarhsystems. MuslimDue to the University intrinsic paramagnetic behavior of 45 2,11 more of its properties upon analyte interaction. The Cu(II) ions, it is relatively easy to develop a fluorescence turn- mechanism of such systems involves an alteration in the mode off type or a colorimetric sensor for it. Also, colorimetric of operation (ON/OFF) in response to the external stimuli sensors with imine moieties offer the advantages of uncompli- like the presence of metal ions, magnetic environment, cated synthesis and low cost instrumentation.12 Therefore, 50 many sensing systems have been devised to date for cupric 50 ions, leaving the domain of cuprous ion sensors nearly 13–17,33 aDivision of Inorganic Chemistry, Department of Chemistry, scarce. Aligarh Muslim University, Aligarh 202002, India Fluorescence based sensing has dramatically changed the bChemistry Section, Women’s College, Aligarh Muslim University, Aligarh 202002, way of visualizing and interpreting the biological and cellular 55 India. E-mail: [email protected]; Tel: +91-9045320178 events as well as drug designing.18,19 Fluorescent chemo- 55 cMolecular Immunology Group Lab, Interdisciplinary Biotechnology Unit, sensors are gaining attention nowadays because of their versa- Aligarh Muslim University, Aligarh 202002, India †Electronic supplementary information (ESI) available. See DOI: 10.1039/ tility, precision, low cost, sensitivity and real-time 20–22 c9pp00247b detection. Recognition and assessment of bio-relevant

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Urkund Analysis Result

Analysed Document: 4151 Chapter B Anzar Mujeeb GB5982 Bio Tech AMU.docx (D60578406) Submitted: 12/9/2019 7:01:00 AM Submitted By: [email protected] Significance: 0 %

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