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Immunization using the skin : using topical Toll-like receptor 9 agonists as a method to increase vaccine… Cheng, Wing Ki 2013

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IMMUNIZATION USING THE SKIN: USING TOPICAL TOLL-LIKE RECEPTOR 9 AGONISTS AS A METHOD TO INCREASE VACCINE EFFICACY  by Wing-Ki Cheng  B.Sc. (First Class Honors), Simon Fraser University, 2006  A THESIS SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF  DOCTOR OF PHILOSOPHY in THE FACULTY OF GRADUATE STUDIES (Experimental Medicine)  THE UNIVERSITY OF BRITISH COLUMBIA (Vancouver)  May 2013  © Wing-Ki Cheng, 2013  ABSTRACT  Vaccination is a cost-effective method to prevent diseases. Yet, vaccine effectiveness remains lower than anticipated and infectious disease is still a leading cause of illness and death. Immunostimulatory oligodeoxynucleotides containing CpG motifs (CpG ODNs) activate Tolllike receptor 9 (TLR9), bridging innate and adaptive immune responses. CpG ODNs have been studied as adjuvants, agents to improve vaccine efficacy. However, split administration of topical TLR9 agonists with parenteral vaccines has been less explored. I hypothesized that topical CpG ODN 1826 activates the skin immune system in a unique fashion, increasing the efficacy of locally administered protein-based vaccines. I found that an effective strategy to administer CpG ODN was single dose topically at time of local parenteral vaccine delivery. The generation of antigen-specific antibodies, CD4+ T helper 1 cells and CD8+ T cells were augmented. In murine Listeria monocytogenes and influenza A infection models, application of topical CpG ODN at time of parenteral protein immunization reduced bacterial and viral burden. The mechanisms whereby topical CpG adjuvant improves vaccine outcomes were investigated. Using bone marrow chimeric mice, I found that TLR9 expression in the hematopoietic compartment was necessary while expression in the stromal compartment also contributed to the enhancement of specific CD8+ T cell generation. Keratinocytes responded to topical CpG ODN partly by up-regulating TLR9 expression. Topical CpG ODNs were internalized by CD11c+ cells and were detected within the skin draining lymph nodes (SLNs). Topical compared to subcutaneous administration of CpG ODN differentially modulated cytokine and chemokine gene expressions in the SLNs and induced a higher proportion of  ii  specific CD4+ T cells to express tissue-homing molecules. In the influenza model, topical CpG adjuvant increased the proportion of specific CD8+ memory T cells at the site of infection. This work demonstrates the effectiveness of a novel immunization strategy that separates topical CpG adjuvant from a protein-based vaccine in enhancing rapid and long-lasting protective humoral and cellular vaccine responses. These improved responses are the consequences of the activation of stromal and hematopoietic cells in the skin, which subsequently modulate the microenvironment of the SLNs and alter the migratory ability of the T cells to tissues.  iii  PREFACE Chapter 3  The part of research working with the Listeria monocytogenes infection model was performed in collaboration with Kathleen Wee, a graduate student from the laboratory of Dr. Tobias R. Kollmann (Department of Pediatrics, Child & Family Research Institute, The University of British Columbia (UBC), Vancouver, BC, Canada). I performed all the experiments, analyzed the results, prepared the figures and wrote the manuscript. K. Wee helped with sample collection and processing leading to figure 3.13 as well as edited the manuscript submitted for publication. Dr. J.P. Dutz supervised this research and the preparation of the manuscript. Dr. T.R. Kollmann provided the Listeria strains and reviewed the manuscript.  The part of research working with the influenza A infection model was performed in collaboration with Adam W. Plumb, a graduate student from the laboratory of Dr. Ninan Abraham (Department of Microbiology & Immunology, Life Sciences Centre, UBC, Vancouver, BC, Canada). I performed most of the experiments, analyzed all the results, and prepared all the figures. A.W. Plumb helped with sample collection, flow cytometry, and performed enzymelinked immunosorbent assay (ELISA) leading to figures 3.2, 3.14 and 4.10.  This research was performed under the approval of UBC Animal Care and Use Committee. UBC Application Number A09-0467: Epicutaneous Priming and Modulation of T Lymphocytes (August 01, 2003 – June 12, 2013). Canadian Council on Animal Care (CCAC) National Institutional Animal User Training (NIAUT) program certificate # 1421 and #5064-11 Biology and Husbandry of the Laboratory Rodent Workshop certificate # RBH-357-08 Laboratory Biological Safety certificate (issued on October 16, 2007) iv  TABLE OF CONTENTS  ABSTRACT ................................................................................................................................... ii	
   PREFACE..................................................................................................................................... iv	
   TABLE OF CONTENTS ............................................................................................................ iv	
   LIST OF TABLES ....................................................................................................................... xi	
   LIST OF FIGURES .................................................................................................................... xii	
   LIST OF ABBREVIATIONS .................................................................................................... xv	
   ACKNOWLEDGEMENTS .................................................................................................... xviii	
   DEDICATION ........................................................................................................................... xix	
   CHAPTER 1: INTRODUCTION................................................................................................ 1	
   1.1	
  Infectious disease ............................................................................................................... 1	
   1.1.1	
   Impact of infectious disease on humans....................................................................... 1	
   1.1.2	
   Overview of the innate and adaptive immune system .................................................. 2	
   1.1.3	
   Listeria monocytogenes – Model of intracellular bacterial infections ........................ 4	
   1.1.4	
   Influenza – Model of viral infections ........................................................................... 8	
   1.1.5	
   Growing concerns in pandemics ................................................................................. 9	
   1.2	
  Vaccines and immunizations ........................................................................................... 11	
   1.2.1	
   Traditional vaccines and advances ............................................................................ 11	
   1.2.2	
   Limitations of existing vaccines ................................................................................. 12	
   1.2.3	
   Routes of vaccine administration ............................................................................... 13	
   1.2.4	
   The ideal vaccine and challenges in vaccine development ........................................ 14	
   1.3	
  Adjuvants ......................................................................................................................... 15	
   v  1.3.1	
   Approved vaccine adjuvants ...................................................................................... 15	
   1.3.2	
   Overview of TLR9 ...................................................................................................... 17	
   1.3.3	
   TLR9 expressions and cells stimulated by TLR9 agonists ......................................... 21	
   1.3.4	
   Synthetic CpG oligodeoxynucleotides as adjuvants................................................... 22	
   1.4	
  The skin as an immune organ .......................................................................................... 25	
   1.4.1	
   The structure of human and murine skin ................................................................... 25	
   1.4.2	
   Immune cells in the skin ............................................................................................. 28	
   1.4.3	
   Using the skin as a route of immunization ................................................................. 31	
   1.5	
  Thesis objectives .............................................................................................................. 33	
   CHAPTER 2: MATERIALS AND METHODS ...................................................................... 36	
   2.1	
  Materials .......................................................................................................................... 36	
   2.1.1	
   Antibodies .................................................................................................................. 36	
   2.1.2	
   Reagents ..................................................................................................................... 37	
   2.1.3	
   Mice............................................................................................................................ 38	
   2.1.4	
   Bacteria and virus strains .......................................................................................... 39	
   2.2	
  Methods............................................................................................................................ 40	
   2.2.1	
   Immunization.............................................................................................................. 40	
   2.2.2	
   Sample collection ....................................................................................................... 41	
   2.2.3	
   Detection of antigen-specific antibodies .................................................................... 41	
   2.2.4	
   Cell isolations and adoptive transfer ......................................................................... 42	
   2.2.5	
   Flow cytometry........................................................................................................... 43	
   2.2.6	
   Detection of antigen-specific T cells, intracellular cytokines and tissue-homing molecule expression ................................................................................................... 43	
   vi  2.2.7	
   Infection models ......................................................................................................... 44	
   2.2.8	
   Analysis of gene expressions by real-time PCR and PCR array ............................... 46	
   2.2.9	
   Generation of bone marrow chimeric mice ............................................................... 47	
   2.2.10	
  Detection of FITC-labeled CpG ODN in SLNs.......................................................... 47	
   2.2.11	
  Detection of the internalization of CpG ODN by dendritic cells ............................... 48	
   2.2.12	
  Protein contact hypersensitivity and lymphocyte egress inhibition ........................... 48	
   2.2.13	
  Tissue microscopy ...................................................................................................... 49	
   2.2.14	
  Statistical analyses ..................................................................................................... 49	
   CHAPTER 3: TOPICAL CPG ODN ENHANCES HUMORAL AND CELL-MEDIATED IMMUNE RESPONSES TO PROTEIN-BASED VACCINES .............................................. 50	
   3.1	
  Introduction ...................................................................................................................... 50	
   3.2	
  Results .............................................................................................................................. 52	
   3.2.1	
   Topical administration of CpG ODN as adjuvant augments Ab production ............. 52	
   3.2.2	
   Topical CpG adjuvant induces a bias towards CD4+ Th1 cells generation .............. 55	
   3.2.3	
   Topical route of CpG ODN delivery is superior to other routes in antigen-specific CTL generation .......................................................................................................... 55	
   3.2.4	
   Rapid prime-boost immunization regimen promotes optimal CTL response with topical CpG ODN ...................................................................................................... 61	
   3.2.5	
   Single application of topical CpG ODN at the time of vaccine administration is sufficient to enhance CTL response ........................................................................... 61	
   3.2.6	
   Topical CpG adjuvant increases memory CD8+ T cell population ........................... 65	
   3.2.7	
   Topical CpG ODN improves the efficacy of protein-based vaccines against Lm ..... 71	
    vii  3.2.8	
   Topical CpG ODN improves the efficacy of protein-based vaccines against influenza A virus ........................................................................................................................ 72	
   3.3	
  Discussion ........................................................................................................................ 76	
   3.3.1	
   Data summary ............................................................................................................ 76	
   3.3.2	
   Strategy to deliver CpG ODN as adjuvant ................................................................ 77	
   3.3.3	
   Topical CpG adjuvant enhances vaccine immune responses .................................... 79	
   3.3.4	
   Topical CpG ODN improves protection against intracellular bacterial and viral infections .................................................................................................................... 81	
   CHAPTER 4: THE MECHANISMS WHEREBY TOPICAL CPG ODN RESULTS IN IMPROVED VACCINE OUTCOMES .................................................................................... 84	
   4.1	
  Introduction ...................................................................................................................... 84	
   4.2	
  Results .............................................................................................................................. 85	
   4.2.1	
   TLR9 deficiency in the mouse abrogates the adjuvant effect of topical CpG ODN... 85	
   4.2.2	
   TLR9 deletion in hematopoietic cells abrogates the generation of CD8+ T cells ..... 87	
   4.2.3	
   Mice lacking TLR9 expression in stromal cells have decreased CD8+ T cell priming following topical CpG ODN administration .............................................................. 88	
   4.2.4	
   Activation of keratinocytes by topical CpG ODN 1826 ............................................. 90	
   4.2.5	
   Mast cells do not contribute to the topical CpG ODN-induced enhancement of CTL response ..................................................................................................................... 92	
   4.2.6	
   Internalization of CpG ODN by CD11c+ DCs detected in the SLNs ......................... 94	
   4.2.7	
   Topical CpG ODN modulates the microenvironment of the SLNs ............................ 96	
   4.2.8	
   Topical CpG adjuvant increases P-selectin ligand and E-selectin ligand expressing CD4+ T cells ............................................................................................................. 101	
   viii  4.2.9	
   T cell migration from the SLNs to the skin is required to induce protein contact hypersensitivity ........................................................................................................ 103	
   4.2.10	
  Topical administration of CpG ODN is required to generate a protein CHS response.. ................................................................................................................. 105	
   4.2.11	
  Topical CpG adjuvant increases memory CD8+ T cells at the site of infection ...... 107	
   4.3	
  Discussion ...................................................................................................................... 109	
   4.3.1	
   Data summary .......................................................................................................... 109	
   4.3.2	
   Topical CpG adjuvant effect on hematopoietic-derived cells .................................. 110	
   4.3.3	
   Topical CpG adjuvant effect on skin stromal cells .................................................. 112	
   4.3.4	
   Topical CpG adjuvant effect on SLNs and T cells ................................................... 113	
   4.3.5	
   Topical CpG ODN increases antigen-specific CD8+ T cells at the site of infection…….. .......................................................................................................... 115	
   CHAPTER 5: OVERALL SUMMARY AND FUTURE DIRECTIONS ............................ 116	
   5.1	
  A novel split immunization method of antigen and adjuvant to enhance vaccine response and its implications ....................................................................................................... 116	
   5.1.1	
   Topical route of CpG ODNs administration as vaccine adjuvants ......................... 116	
   5.1.2	
   Mechanisms of action of topical CpG ODNs ........................................................... 118	
   5.1.3	
   Implications of this work .......................................................................................... 119	
   5.1.4	
   Major drawback of this work using mouse models .................................................. 119	
   5.2	
  Future directions ............................................................................................................ 122	
   5.2.1	
   Cutaneous DC subsets that are necessary for the adjuvant effect of topical and subcutaneous CpG ODNs ........................................................................................ 123	
    ix  5.2.2	
   Ability of topical and subcutaneous CpG ODN to enhance seeding of antigen-specific T cell in tissues ......................................................................................................... 125	
   5.2.3	
   Clinical trials to test the efficacy of using topical CpG ODN as an adjuvant ......... 126	
   REFERENCES.......................................................................................................................... 127	
    x  LIST OF TABLES  Table 4.1. Over-expressed genes detected in the SLNs 24 hours post-immunization. ............... 100	
   Table 4.2. Under-expressed genes detected in the SLNs 24 hours post-immunization. ............. 100	
    xi  LIST OF FIGURES  Figure 1.1 Overview of the adaptive immune system. ................................................................... 7	
   Figure 1.2 TLR9 structure and signaling. ..................................................................................... 20	
   Figure 1.3 Structure of the mouse skin. ........................................................................................ 27	
   Figure 1.4 Immune cells in the skin. ............................................................................................. 35	
   Figure 3.1 Using CpG ODN as vaccine adjuvant with soluble protein antigen enhances the production of antigen-specific IgG Abs. .................................................................... 53	
   Figure 3.2 Whole heat-killed influenza virus with CpG ODN increases the production of influenza-specific IgG Abs. ....................................................................................... 54	
   Figure 3.3 CpG ODN induces proliferation of CD4+ Th1 T cells that secrete IFN-γ. ................. 57	
   Figure 3.4 Topical administration of CpG ODN is the optimal route to enhance CTL response. 58	
   Figure 3.5 Topical administration of CpG ODN does not cause splenomegaly. .......................... 59	
   Figure 3.6 The adjuvant effect of CpG ODN is CpG-motif specific. ........................................... 60	
   Figure 3.7 Optimal interval between doses of a vaccine series to induce antigen-specific CTLs.63	
   Figure 3.8 Multiple doses of CpG ODN do not further enhance antigen-specific CTL generation. .................................................................................................................................... 64	
   Figure 3.9 Using CpG ODN as adjuvant promotes generation of antigen-specific effector and memory CD8+ T cell. ................................................................................................. 68	
   Figure 3.10 Antigen-specific CD8+ memory T cells induced by immunization with CpG ODN proliferate upon infection. .......................................................................................... 69	
   Figure 3.11 Topical CpG ODN may promote effector memory T cells generation. .................... 70	
    xii  Figure 3.12 Topical and subcutaneous CpG ODN enhance protective immunity against systemic Lm infection. ............................................................................................................ 73	
   Figure 3.13 Using topical CpG ODN as adjuvant provides long-term protective immunity against systemic Lm infection. ............................................................................................. 74	
   Figure 3.14 Topical and subcutaneous CpG ODN enhance long-term protective immunity against intranasal influenza infection................................................................................... 75	
   Figure 4.1 Enhanced CTL response induced by topical CpG ODN is TLR9-dependent.. ........... 86	
   Figure 4.2 Necessity of TLR9 expression in hematopoietic and stromal cells for the enhancement of CD8+ T cell response induced by topical CpG ODN. ........................................... 89	
   Figure 4.3 Topical CpG ODN up-regulates TLR9 mRNA expression on the epidermis. ............ 91	
   Figure 4.4 Mast cells do not contribute to the adjuvant effect of topical CpG ODN in antigenspecific CTL generation. ............................................................................................ 93	
   Figure 4.5 Topical or subcutaneously administration of CpG ODN internalized by CD11c+ cells is detected in the SLNs 48 hrs post-treatment............................................................ 95	
   Figure 4.6 The route of CpG ODN administration differentially modulates the gene expression of cytokines and chemokines in the SLNs. .................................................................... 98	
   Figure 4.7 Topical route of CpG ODN administration is better at inducing the expression of tissue-homing molecules on activated T cells in the SLNs. .................................... 102	
   Figure 4.8 Inhibition of lymphocytes egression from the SLNs prevents ear swelling in a protein CHS model. .............................................................................................................. 104	
   Figure 4.9 Topical CpG ODN promotes migration of antigen-specific T cells to tissues. ......... 106	
   Figure 4.10 Topical CpG ODN increases antigen-specific T cells at the site of infection. ........ 108	
   Figure 5.1 Overview of events triggered by topical CpG ODN to improve vaccine outcomes. 121	
   xiii  LIST OF SYMBOLS  α  Alpha  β  Beta  °  Degree  δ  Delta  γ  Gamma  κ  Kapper  mφ  Macrophage  Δ  Mutant  %  Percent  ±  Plus - Minus  xiv  LIST OF ABBREVIATIONS  7AAD  7-amino-actinomycin D  Abs  Antibodies  ActA  Actin assembly-inducing protein  Ag  Antigen  APC  Antigen-presenting cell  BSA  Bovine serum albumin  CFU  Colony forming unit  CHS  Contact hypersensitivity  CTL  Cytotoxic T lymphocyte  DC  Dendritic cell  DMSO  Dimethyl sulfoxide  DTR  Diphtheria toxin receptor  ec  Epicutaneous or topical  EpCAM  Epithelial cell adhesion molecule  EDTA  Ethylenediaminetetraacetic acid  ELISA  Enzyme-linked immunosorbent assay  FBS  Fetal bovine serum  HA  Hemagglutinin  HAU  Hemagglutination unit  HBSS  Hank’s balance salt solution  xv  HRP  Horseradish peroxidase  IFN  Interferon  Ig  Immunoglobulin  IL  Interleukin  ip  Intraperitoneal  IRF7  Interferon regulatory factor 7  KC  Keratinocyte  KO  Knockout  LC  Langerhans cell  Lm  Listeria monocytogenes  MHC  Major histocompatibility complex  NFκB  Nuclear factor kappa B  NK cell  Natural killer cell  NP  Nucleoprotein  OCT  Optimal cutting temperature  O.D.  Optical density  ODN  Oligodeoxynucleotide  OVA  Ovalbumin  PA  Acid polymerase  PBS  Phosphate buffered saline  pDC  Plasmacytoid dendritic cell  PMA  Phorbol 12-myristate 13 acetate  xvi  RT-PCR  Reverse transcription polymerase chain reaction  sc  Subcutaneous  SEM  Standard error of the mean  SLN  Skin draining lymph node  STAT  Signal transducer and activator of transcription  Th  T helper  TIR  Toll-IL-1 receptor  TLR  Toll-like receptor  Tm  Memory T cell  TMB  Tetramethylbenzidine  TNF  Tumor necrosis factor  xvii  ACKNOWLEDGEMENTS  First and foremost, my utmost gratitude to my supervisor, Dr. Jan Dutz, for supporting my decision in transferring from the Experimental Medicine Master program into the PhD program. Without his excellent guidance, insightful advice, and his influential enthusiasm in translational research, I would not have come to my position right now in completing this degree. I am also indebted to my supervisory committee and collaborating principal investigators, Dr. Kirk Schultz, Dr. Tobias Kollmann and Dr. Ninan Abraham for their intellectual contributions to this work. I sincerely thank all of the members of the Dutz laboratory for their discussions, input, as well as encouragement throughout the duration of pursuing this degree. I thank Jackie Lai for her mentoring and the laughter she brought to me in those long experiment days. Thank you for the wonderful friendship that will last for years to come.  Financial supports from the Canadian Institute of Health Research (CIHR) through the CIHR Master’s award and the CIHR SRTC training scholarship were greatly appreciated.  Last but not least, my gratitude to my family and friends who have supported me throughout the years of my education extends beyond words.  xviii  DEDICATION  This work is dedicated to my parents, for their unique way of bringing me up. Without this experience, I would not have grown to who I am today and achieve my goal of being a scientist.  To my special love, Andy, thank you for your wonderful love. Without you, I would not be eating regularly during the countless days in the lab. Thank you for reminding me that I can make the impossible possible when I put in the effort and do my very best.  xix  CHAPTER 1: INTRODUCTION  1.1  1.1.1  Infectious disease  Impact of infectious disease on humans  Infectious disease is one of the leading causes of disability and death in humans. Although smallpox has been eradicated and poliomyelitis is on the way to extinction, many other infectious diseases still plague humanity. Acute lower respiratory infections, tuberculosis, and malaria, are among the dominating causes of death globally. Emerging and re-emerging infections such as influenza, human immunodeficiency virus infection, syphilis and yellow fever continue to be serious threats to human health (1),(2),(3),(4). In addition, infections caused by Helicobacter pylori, human papillomavirus and hepatitis B virus can lead to chronic diseases such as gastritis and gastric ulcers, cervical cancers, cirrhosis and liver cancer, respectively (5). Not only do infectious diseases increase the health care burden, they also decrease work productivity leading to economic decay (1). The issue of global warming may change the epidemiology of infectious diseases due to human migration and environmental alterations that can influence the infectious agents as well as their hosts (6). These changes make it difficult to predict disease patterns. Pandemic disease remains a public health concern. The outbreak of severe acute respiratory syndrome in Asia in 2007 and the H1N1 swine flu pandemic in 2009 highlighted the need to prepare for future pandemics (7). Learning from history as in the Black Death and the Spanish flu in 1918 that killed millions of people (8),(9), pandemic prevention remains one of the global focuses of human public health. 1  1.1.2  Overview of the innate and adaptive immune system  The immune system protects the host against infections. There are two major arms of the immune system: the innate immune system and the adaptive immune system. Innate immune system is the first line of defense but does not generate a memory response for subsequent pathogen encounters. The innate immune system includes antimicrobial proteins and peptides, complement, natural killer (NK) cells, NKT cells, gamma delta (γδ) T cells, macrophages, mast cells, dendritic cells (DCs), neutrophils and basophils (reviewed in (10),(11),(12)). Pathogens are recognized by germline-encoded receptors such as Toll-like receptors (TLRs). TLRs have broad specificity binding to molecules that commonly present on many pathogens called pathogenassociated molecular patterns (more details are discussed in section 1.3.2). The innate immune molecules and cells limit pathogen spread by pathogen clearance through phagocytosis and direct pathogen lysis, producing cytokines and chemokines, and inducing inflammation to recruit more phagocytic cells and effector molecules to the site of infection. Innate immune cells do not undergo clonal expansion so they act immediately to control pathogens while allowing time for specific adaptive immunity to develop. Professional antigen-presenting cells (APCs) such as DCs play important roles in linking innate immune responses to adaptive immune responses by processing pathogens and presenting the peptides generated to the adaptive immune cells such as T lymphocytes (T cells). A key characteristic of adaptive immune system is the unique ability to generate immunological memory, which means specific immune cells are generated to respond to subsequent exposures of the same pathogen. B cell receptors and T cell receptors on naive B and 2  T cells, respectively, bind specific antigens. Activated B cells differentiate into plasma cells and secrete free forms of the receptors called antibodies (Abs). Abs can mediate protection against pathogens by neutralization, opsonization and complement activation. These processes prevent pathogen adherence to host cells, promote phagocytosis of the pathogen into cells for destruction or lyse the pathogen directly. Cell-mediated immunity involves CD8+ cytotoxic T cells (CTL) and CD4+ T cells. APCs such as DCs process and present the peptides on major histocompatibility complex class I and class II (MHC I and MHC II) molecules to CD8+ and CD4+ T cells, respectively. CTLs release cytotoxins such as perforin and granzymes and utilize the Fas system to kill infected cells (13),(14). There are four major subtypes of CD4+ T cells based on cytokine production profiles and functions: T helper (Th) 1, Th2, Th17 and T regulatory (Treg) cells (reviewed in (15)). Th1 cells predominantly produce interferon gamma (IFN-γ). Th1 cells also produce tumor necrosis factor-alpha (TNF-α) that increase macrophage killing and promote CD8+ T cell proliferation. Th1 cells promote cell-mediated immunity against intracellular bacterial and viral infections. Th2 cells produce TNF-α, interleukin (IL-)4, IL-5, IL9, IL-13 and IL-25 that stimulate B cell proliferation and Ab class-switching (16),(17) against extracellular bacteria and parasites. Th2 cells may also play a role in the maintenance and function of memory CD8+ T cells (18),(19),(20). Th17 cells form a group of T cells, distinct from Th1 and Th2 subsets. They produce IL-17, IFN-γ, IL-21 and IL-22, which are critical in protection against extracellular bacteria and fungi. Studies showed that Th17 may promote autoimmune diseases such as psoriasis (21). The role of Th17 cells in infection is unclear because opposite functions have been found. For example, one study showed Th17 cells enhance viral persistence by inhibiting T cell cytotoxicity and apoptosis of infected cells in a Theiler's murine encephalomyelitis virus infection model (22). In contrast, others demonstrated that Th17 3  cells play protective roles in immunity against infectious pathogens such as Mycobacterium tuberculosis and Streptococcus pneumoniae (21). Another subset of CD4+ T cells is the CD4+ CD25+ Foxp3+ regulatory T cell subset that regulates lymphocyte homeostasis at steady state, activation and function. Cells from this subset suppress T cell responses by inhibitory cytokines such as IL-10, by cytolysis, metabolic disruption and modulation of DC maturation and function (23, 24). An overview of the T and B cell adaptive immune system is summarized in a schematic diagram (Figure 1.1). The types of CD4+ T cells are determined by the nature of the antigens, affinity to T cell receptors, cytokine milieu at the time of antigen encounter, and specific transcription factors. The master regulators and the signal transducer and activator of transcription (STAT) proteins are required for CD4+ T cell subset differentiation: Th1, Th2, Th17 and Treg cells require Tbet/STAT4, GATA-3/STAT6, RORγt/STAT3 and Foxp3/STAT5, respectively, for differentiation (15). A hallmark of adaptive immunity is immunologic memory. In particular, memory T cells respond to subsequent pathogen challenge in a more rapid and effective manner (25),(26).  1.1.3  Listeria monocytogenes – Model of intracellular bacterial infections  The immune system has evolved to protect the host from pathogen assault. Microorganisms such as Listeria monocytogenes (Lm) can be used to study the response of immune system to assault by infectious microorganisms. Lm is a gram-positive rod-shape facultative anaerobic bacterium, a food-borne pathogen that causes listeriosis. It can invade the intestinal epithelium, cross the feto-placental barrier and the blood-brain barrier leading to 4  gastroenteritis, septic abortion, meningitis and encephalitis (27),(28),(29). Lm can infect both phagocytic and non-phagocytic cells and reside in the cytosol (hence they are called intracellular bacteria) (30). Lm use listeriolysin O to form pores and escape from hydrolytic phagosomes (31),(32),(33). They spread from cell to cell with the actin assembly-inducing protein (ActA), which allows polymerization of F-actin (34). Lm has been used as a model pathogen (murine listeriosis) to study innate and adaptive immunity since 1960s (31). Lm infects humans by using the internalin A protein to enter into host epithelial by binding to host E-cadherin receptor cells in the intestine. However, a mutation in murine E-cadherin prevents this oral route of infection. Due to a single amino acid difference at position 16 of the surface receptor E-cadherin in mice, Lm internalin A cannot bind efficiently (35), (36). Thus, intravenous injection of Lm is commonly used to infect mice and assess their immune responses to the infection. Intravenous injection of Lm into mice leads to colonization of the spleen and the livers (35). Bacterial burden can easily be enumerated in these organs by plating. The reasons why Lm is an attractive mouse infection model include: Sub-lethal doses of Lm are able to induce strong immune responses and Lm can be genetically altered by inserting genes to express different antigens or by deleting various virulent factors. For example, Lm can be modified to express the CD8+ CTL epitope of ovalbumin (OVA257-264) (36) for antigen-specific CD8+ T cell studies, while the ActA gene can be deleted to generate a less virulent strain of Lm for vaccine research and development (36). Macrophages, neutrophils, and NK cells play important roles in the innate immune response to Lm involving TLRs and NOD-like receptors (31),(37). DCs link the innate and adaptive immune responses by presenting antigens to T cells (38). Since Lm can spread from cell to cell without being exposed to the extracellular milieu to avoid Abs and complement (30),(39), humoral immunity by B cells plays a minor role in Lm resistance. Instead, CD8+ T cells 5  dominate the adaptive immunity to Lm and T cell immunity is necessary for bacterial clearance (29),(32) although other cells also influence adaptive immunity against Lm (31),(40),(41),(42). Intracellular antigens are presented to CD8+ T cells by direct-presentation on MHC class I molecules. Extracellular antigens that normally presented by MHC class II molecules to CD4+ T cells can also be presented to CD8+ T cells by cross-presentation. In cross-presentation, antigens in endosomes are released into the cytoplasm of the APCs, and are then processed and presented to CD8+ T cells (43). Activated CD8+ T cells lyse infected cells using perforin and granzymes (31),(38). IFN-γ produced by T cells also activates macrophages to kill infected cells using reactive oxygen and nitrogen intermediates as well as other mechanisms (31). There are studies that demonstrate that B cells play a minor role in the protections against Lm through the production of specific antibodies (Abs). Yet, B cells support T cell responses against Lm (40),(42). Lm is a useful model to study cell-mediated adaptive immunity especially for vaccine studies in the generation, maintenance and challenge responses of memory T cells.  6  Figure 1.1 Overview of the adaptive immune system. Antigens are internalized by APCs such as DCs, which then process the antigens and present them as peptides on MHC molecules. Peptide-MHC class I and peptide-MHC class II complexes present antigens to CD8+ T cells and CD4+ T cells, respectively. Activated CD8+ T cells will differentiate into CTLs that secrete IFN-γ and cytotoxins that can lyse infected cells. Activated CD4+ T cells will differentiate into different subsets of Th cells including Th1, Th2 and Th17 cells. Th1 cells secrete cytokines that have antiviral or antibacterial effects while Th2 cells secrete cytokines that can assist B cell activation and Ab production. Th17 cells may either promote pathogenesis or protective immunity against infections. Regulatory T cells regulate the function of other T cells to prevent aberrant T cell effects. B cells are activated by cross-linking B cell receptors by antigen. They can differentiate into plasma cells that secrete Abs.  7  1.1.4  Influenza – Model of viral infections  Influenza viruses are common human viral pathogens that cause pandemic diseases. Influenza is an acute and contagious infectious respiratory disease. According to the World Health Organization (http://www.who.int/mediacentre/factsheets/fs211/en/index.html), it can affect all age groups including high-risk populations such as children of ages 2 and younger, the elderly of ages 65 and over, pregnant women, and individuals who are immunocompromised or have chronic medical conditions leading to complications and deaths. It is transmitted through aerosol or contact with infectious respiratory secretions (44). In humans, infected individuals may show symptoms of fever, chills, myalgia, headache, lethargy and anorexia (45). In addition, dry cough and nasal congestion will develop when disease progresses (45). Influenza is caused by negative RNA (antisense RNA strand that does not encode messenger RNA) viruses of the orthomyxoviridae family (44) with eight RNA strands encoding eleven viral genes in the influenza virus genome (46). There are three types of influenza viruses: Type A, B and C. While type C influenza viruses only cause mild illness, types A and B are responsible for epidemics of respiratory illness annually as well as serious complications and death (44). Influenza can be studied in mice and the murine influenza model is well-established (47). The A/Puerto Rico/8/1934 (H1N1) (PR8) virus and the A/HK/1/1968 (H3N2) (X31) virus are two influenza A viruses commonly used for murine infection (45). Influenza A virus replicates in epithelial cells and leukocytes (48). The virus life cycle begins with entry into the host cell with viral surface protein hemagglutinin (HA) attaching to sialic acid on the host cell and nuclear entry of viral ribonucleoproteins (49),(46). Next, transcription and replication of viral genome take place. The viral genome is then exported from the nucleus to be assembled and released 8  from the cell when neuraminidase cleaves the sialic acid residue on glycoproteins and glycolipids of the host (49),(46). Infection results in the production of chemotactic, proinflammatory and antiviral cytokines by epithelial cells, macrophages, DCs, NK cells and T cells (48). The T cell immune response to influenza is well-characterized in mice. It is known that viruses are cleared 10 days after infection without persistent antigen or viral RNA (49),(50), which allows the study of memory immune responses. CD8+ T cells recognizing two epitopes of the virus, the nucleoprotein NP366-374 and the acid polymerase PA224-233, dominating the T cell response (51). DCs carry antigens from the infected lungs to mediastinal lymph nodes to activate naive T cells that subsequently proliferate and differentiate into antigen-specific T cells. Murine immune responses to influenza viral infection, including Ab formation and T cell responses can be followed. Genetically engineered PR8 and X31 viruses, altered to express the CTL epitope of OVA257-264 (PR8-OVA and X31-OVA), can be used to study antigen-specific CD8+ T cell response (52). In addition, the pulmonary viral burden of infected lungs can be measured to evaluate vaccine efficacy using a murine influenza model. Although infection of mice by influenza viruses does not completely mimic the humans and the avian infections, it provides a clinically relevant model for the pre-clinical testing of potential vaccines and adjuvants that boost vaccine responses.  1.1.5  Growing concerns in pandemics  There are increasing concerns regarding the clinical burden of Lm infection. Lm is an opportunistic pathogen, with infants, elderly, pregnant women and the immunocompromised 9  individuals being more prone to infections (53). Listeriosis can present as septicemia, meningitis, miscarriage and death (54). Listeriosis has become increasingly common in the past two decades with growing concerns of antibiotic resistance. Recent outbreaks resulted in a high rate of hospitalization and deaths (53),(55),(54). Currently, no vaccines are licensed for Lm infection. There are also increasing concerns about the dangers of an imminent influenza pandemic. Influenza is an infectious disease and infected individuals have symptoms of the common cold but can lead to severe complications such as pneumonia. For example, the 1918 Spanish flu killed millions of individuals (9). The most recent pandemic was the H1N1 influenza outbreak in 2009 that killed tens of thousands in over 200 countries (56). Since influenza viruses are fastevolving, they pose a challenge to the immune system and make vaccine design difficult. Antigenic drift and antigenic shift are phenomena that produce new subtypes of influenza virus by genetic re-assortment and allow the viruses to evade the immune system. There is no crossreactive Abs for protection because Abs target antigens that are variable from year to year. Particularly, infants, pregnant women, the elderly, and the immunocompromised individuals, such as those with solid organ and hematopoietic cell transplantation, individuals with asthma or chronic lung disease, are highly vulnerable (57),(58),(59),(60),(61),(62). These individuals have a higher risk of developing complications because of an impaired memory response to previous infections and to vaccination. There is a growing global concern of an imminent and catastrophic influenza pandemic. Therefore, a novel strategy to improve influenza vaccine efficacy is needed.  10  1.2  1.2.1  Vaccines and immunizations  Traditional vaccines and advances  Edward Jenner is well-known for his contribution to immunization and smallpox eradication (63). His discovery of cowpox inoculation in 1796 to protect against smallpox was the first attempt to control infectious disease using the concept of live vaccines and immunization. Although live vaccines can induce both humoral and cell-mediated immune responses, live vaccines have the risk of virulence reversion as reported for the live-attenuated poliovirus, which can revert to virulence and cause paralytic poliomyelitis (64). Louis Pasteur next introduced the concept of killed or inactivated vaccines in his discovery of the rabies and the anthrax vaccines (65). Whole killed or attenuated microorganism vaccines are effective but there are concerns of high reactogenicity and side effects such as seizures and brain damage as described for the whole cell pertussis vaccine (66). This has led to the development of subunit vaccines by identifying specific antigens that best stimulate the immune system for desirable immune responses. The recombinant DNA hepatitis B vaccine (Engerix-B) and the acellular pertussis vaccine containing specific proteins from Bordetella pertussis in the diphtheria, tetanus and pertussis (DTaP) vaccine (Tripedia) are two examples of licensed subunit vaccines. The subunit strategy of vaccine design can avoid adverse events and thus increase vaccine safety. However, the drawback of subunit vaccines is reduced immunogenicity. An adjuvant is an agent often added to increase the efficacy of subunit vaccines; adjuvants can be categorized into three types: Particulate, non-particulates and others (67). For example, adjuvants that have been studied include aluminum salts, Freund’s complete adjuvant, liposomes, microspheres, peptides 11  of microbial origins, cholera toxin and heat labile enterotoxin produced by the bacterium Vibrio cholera and Escherichia coli (68). However, some of these adjuvants are unstable or cause side effects (see section 1.3 for more details on adjuvants).  1.2.2  Limitations of existing vaccines  Since the introduction of immunization by Jenner and Pasteur, immunization has had an immense impact on public health. It is a powerful tool to prevent diseases as exemplified by the eradication of smallpox and the elimination of poliovirus from the Western hemisphere (69). While there is a plethora of vaccines, they remain only partially effective. For example, annual influenza immunization is required due to antigenic drift and shift of the viruses. Most licensed vaccines have been designed to induce a humoral immune response to pathogens. However, humoral immunity is not sufficient to prevent infection from all pathogens. In addition, cellmediated immunity should also be induced, as it is important in the immune response against many pathogens. For example, CD4+ T follicular helper cells are crucial to generate high affinity Abs and immune memory (70). In addition, intracellular pathogens such as Lm are not exposed to the extracellular milieu and thus cell-mediated immune response is critical for infection control. Induction of cell-mediated immunity of CD8+ T cells that target less variable proteins, such as nucleoproteins and matrix proteins, might be a way to generate a “universal vaccine” against different strains of influenza viruses.  12  1.2.3  Routes of vaccine administration  In Canada and the United States, there are five different routes of administration for licensed vaccines according to the Public Health Agency of Canada and the Centers for Disease Control and Prevention of the United States. The most common route of immunization is intramuscular injection into the vastus lateralis muscle or the deltoid muscle. All inactivated vaccines except one formulation of a meningococcal vaccine are injected intramuscularly. Subcutaneous route is another vaccine administration method. Vaccines, such as the herpes zoster vaccine (Zostavax), are injected into the fat tissue below the dermis layer of the skin at the thigh or upper outer triceps of the arm. Recently, new vaccine administration routes have been utilized such as the intradermal route and the less invasive routes of oral and intranasal delivery of vaccines. Intradermal injection delivers vaccines into the dermis layer of the skin such as the influenza vaccine (Fluzone) and the Bacille Calmette-Guerin vaccine (BCG). Oral and intranasal immunizations deliver vaccines to the mucosal lining of the gut and the respiratory tract, respectively. The cholera vaccines (DUKORAL) and the live attenuated influenza vaccine (FluMist) are examples of oral and intranasal vaccines. The route of vaccine administration is one of the most important aspects in vaccine design because optimal immune response at the appropriate anatomic location is vital for protection against different pathogens. Vaccines should mimic the effect of natural infections and many human pathogens infect through mucosal surface. Studies have shown that local mucosal immune responses are crucial to combat pathogens as demonstrated by the importance of local memory T cells in the lungs for the protection against influenza virus challenge (71),(72). There is evidence that the route of immunization induces differential immune responses. For instance, 13  oral immunization mainly induces immune responses in the upper intestines while intranasal immunization induces immune responses in the upper respiratory tract (73). Studies have demonstrated the concept that an optimal immune response requires the appropriate route of vaccine administration for the pathogen. A single immunization via the intranasal route but not the intramuscular route protected against airway Mycobacterium turberculosis challenge in a murine model of pulmonary tuberculosis (74). Another study showed that intranasal administration of an influenza vaccine elicited higher Ab titers and stronger protective effect compared to oral, peritoneal and subcutaneous administration of the vaccine against intranasal influenza virus challenge (75).  1.2.4  The ideal vaccine and challenges in vaccine development  Ideal vaccines should be safe, effective and inexpensive. A universal vaccine, being able to rapidly induce life-long immunity to various pathogens to protect everyone, is desirable. However, this ideal vaccine does not exist because there are many challenges in vaccine development. Current problems include lack of understanding in what immune responses correlate with protection, unresponsiveness to vaccines in immunocompromised individuals, requirements in constant updates of vaccine formulation due to fast-evolving pathogens, high cost of vaccine development and inadequate access to populations in developing countries (76). Unlike therapeutic drugs that are used to treat patients who have already contracted the diseases, vaccines are administered into healthy individuals to prevent diseases. Therefore, the realistic goal is that benefits of the vaccines must outweigh the risks associated with immunization. Otherwise, it would be very difficult to have high compliance, especially in healthy children. 14  Vaccine development is multifaceted. Identifying specific antigens, determining route of vaccine delivery, targeting specific immune cells and cell compartments, tailoring to specific mode of immune responses, and minimizing side effects are all equally important factors to be considered. Understanding the pathogenesis of infectious disease may assist in determining relevant immune responses that correlate with protection and in identifying appropriate readouts to measure the efficacy of new vaccines. The process of translating bench discoveries into a human vaccine to be routinely administered is a long journey. Practical hurdles, including how to scale up the production, how to lower the cost of manufacturing and logistics, have to be overcome (77). Pre-clinical studies and human clinical trials are necessary to ensure safety and effectiveness of potential vaccines. Not all vaccines can induce sterilizing immunity but at least they can decrease severe disease and complications.  1.3  1.3.1  Adjuvants  Approved vaccine adjuvants  Subunit vaccines are safer than whole killed or attenuated vaccines. However, the drawback of subunit vaccines is that they have low immunogenicity and they confer partial protective immunity due to genetic variability of heterogeneous human population (68). Thus, adjuvants are substances often added to vaccines to boost immune responses against the vaccine antigens. In Latin, the word adjuvare means to help or to enhance. It was Alexander Glenny and his colleagues who first demonstrated the adjuvanticity of aluminum salts in the 1920s. They discovered that an antigen absorbed to aluminum potassium sulphate increases Ab response 15  compared to soluble antigen alone (78). In the past 90 years, many adjuvants have been developed to a pre-clinical stage. Yet, the number of licensed adjuvants to be used in human vaccines remains low. Currently, only a few adjuvants are licensed with human vaccines. These include the commonly used aluminum hydroxide (alum, mineral salts), MF59 (oil-in-water emulsion), AS04 (aluminum hydroxide and monophosphoryl lipid A (MPL), a TLR4 agonist), virosomes (liposomes), and AS03 (oil-in-water emulsion) (79). No adjuvant is licensed by itself. Instead, all adjuvants are licensed in the form of a particular antigen-adjuvant formulation. The slow advances in licensing novel adjuvants may be due to the lack of understanding in their mechanisms of action. Although alum has been used in human vaccine for over 90 years, its modes of action still remains unclear. Not until recent years that its modes of action and that of other adjuvants have been slowly revealed but controversies remain to be resolved. The effect induced by alum is TLR-independent and the modes of action include the following (80),(81),(82),(83),(84): (i) formation of depot and slow release of antigen, (ii) induction of inflammation by increasing chemokine and receptor expression to recruit neutrophils, eosinophils and NK cells as well as by activating inflammasomes to increase IL-1β and IL-18 secretion by DCs, (iii) induction of danger-associated molecular patterns such as uric acid causing cell necrosis, (iv) activation of monocytes to become mature DC and up-regulation of MHC class II molecule expression to increase antigen presentation, and (v) induction of Th2 cell generation and B cell class-switching. Adjuvants can enhance vaccine responses through five possible ways: immunomodulation, antigen presentation, CTL response promotion, antigen targeting to specific immune cells and compartments, and depot effect (85). Adjuvants often modulate the cytokine profile at the injection site or draining lymph nodes, which can polarize to a specific arm of immunity such as 16  induction of particular Ab isotypes or T cell subsets. For example, alum skews the immune response to Th2 response (86),(87), while MPL skews to Th1 response (88),(89). Adjuvants may conserve antigen conformation allowing recognition by certain immune cells through specific epitopes and consequently lead to increased relevant immune responses and signaling (85). Adjuvants may enhance CTL response through promoting IFN-γ production and presentation of antigen by MHC class I molecules. Adjuvants can also lower the amount of vaccine antigens required to induce immunity as demonstrated by intramuscular injection of CpG ODN as adjuvant with a hepatitis B vaccine (90). This effect may be due to targeting antigens to specific immune cells through aggregate forming or receptor binding (85). Depot effect of biodegradable microspheres may prolong stimulation of the immune system by continuous or pulse release of antigens as well as by avoiding degradation by serum proteases or removal by the liver (85). An ideal adjuvant should possess the characteristics of stability, biodegradability, low manufacturing cost, and ability to promote and enhance appropriate immune responses with the vaccines for protection needed against various pathogens without toxicity (91). Different adjuvants augment differential immune responses so it is important to make the right decision of which type of adjuvant should be used in a particular vaccine in order to induce relevant immune responses.  1.3.2  Overview of TLR9  Organisms have evolved pathogen recognition systems to protect against infections and that these systems might be exploited in adjuvant design. The discovery of the innate immune responses of Drosophila melanogaster to fungal infection through the Toll pathway paved the 17  way for studies of TLR immunity (92). TLRs are a family of phylogenetically conserved mediators of innate and adaptive immunity that are vital for host defense. They are type I transmembrane proteins with ectodomains containing fifteen leucine-rich repeats (93), which recognize pathogen associated molecular patterns derived from a plethora of microbes including bacteria, viruses, fungi and parasites (94). The intracellular Toll-IL-1 receptor (TIR) domains are required for downstream signaling. To date, a total of 13 TLRs have been identified with TLR19 being conserved between humans and mice. TLR11-13 are lost in the human genome while TLR10 is non functional in mice (95). TLRs can be classified into two groups. One group of TLRs consists of TLR1, 2, 4, 5, 6 and 11 that are expressed on cell surfaces and predominantly recognize microbial membrane components. Another group of TLRs consists of TLR3, 7, 8 and 9 that are expressed in intracellular vesicles including endoplasmic reticulum, endosomes, lysosomes and endolysosomes, which recognize microbial nucleic acids. Human and mouse TLR9 share 75.5% identity (96). TLR9 recognizes bacterial DNA with CpG motifs, synthetic CpG ODNs that mimic bacterial DNA, and insoluble crystal hemozoin (byproduct of hemoglobin digestion) (96),(97). Unmethylated 2’-deoxyribonucleic acid with CpG motifs are found in high frequency in bacterial or viral DNA but are rare in mammalian DNA. In addition, the intracellular localization of TLR9 prevents binding of extracellular self nucleic acids and protects hosts from mounting immune responses against self. Cell uptake of CpG ODN is CpG motif-independent (93). TLR9 ligand is localized within the endolysosome. In the steady state, TLR9 is sequestered in the endoplasmic reticulum. When the cell is stimulated, TLR9 is transported into the endolysosome to bind its ligand (95),(93). In the endolysosome, whether TLR9 cleavage by intracellular proteases is required to become functional remains ambiguous (98). Some studies suggest multimerization of TLR9 (cross-linking TLR9) is required 18  for optimal response (99). TLR9 recruits myeloid differentiation primary response gene 88 protein (MyD88), subsequently allowing the translocation of transcription factors nuclear factor kappa B (NF-κB) and interferon regulatory factor 7 (IRF7) to the nucleus for the production of inflammatory cytokines and type I IFNs to mount appropriate innate and adaptive immune responses (95). The structure of TLR9 and signaling are summarized in a schematic diagram (Figure 1.2).  19  Figure 1.2 TLR9 structure and signaling. Unmethylated single-stranded CpG ODNs are endocytosed into the cell inside endosomes, which fuse with other endosomes that have TLR9 recruited to them. CpG ODNs bind TLR9 and acidification of the endosomes allows TLR9 signaling through the adaptor MyD88. Downstream signaling leads to the activation and translocation of the transcription factors NK-κB and IRF7 into the nucleus and initiate the production of pro-inflammatory cytokines and type I IFNs.  20  1.3.3  TLR9 expression and cells stimulated by TLR9 agonists  TLR9 is expressed by both innate and adaptive immune cells in mice and in humans. Using reverse transcription polymerase chain reaction (RT-PCR), Western blot and flow cytometry, many cells have been shown to express TLR9. TLR9 is detected in un-stimulated murine cell lines including DCs, macrophages, neutrophils and B cells but is not detected in mast cells and T cells (100),(101),(102),(103). However, activated mast cells can up-regulate TLR9 expression, such as in the case of infection (104),(105). The role of mast cells in augmenting adaptive immunity is unclear but some studies suggest that they play an important role in early inflammation as well as DC and T cell recruitment to infected tissues and draining lymph nodes (106),(104),(107), (108). Other studies have detected TLR9 expression in murine activated invariant NK T cells, adrenal glands, liver endothelial cells, intestinal epithelial cell, and keratinocytes (109),(110),(102),(111),(112),(113). In humans, TLR9 is detected in plasmacytoid DCs (pDCs), B cells, plasma cells, NK cells, neutrophils as well as adrenal glands, intestinal epithelial cells and keratinocytes (112),(114),(115),(116),(117),(118),(119),(120),(121),(122). The greatest difference in the TLR9 expression between mice and humans is the restricted expression of TLR9 on pDCs in humans while it is also expressed on other subsets of DCs (myeloid DCs) in mice. As described, certain murine and human hematopoietic cells and stromal cells either express TLR9 constitutively or can be stimulated to up-regulate TLR9 expression. Many studies set to determine the consequences of these innate and adaptive immune cells upon TLR9 stimulation. Immunostimulatory effect of CpG ODNs must signal through TLR9. TLR9 stimulation may induce production of cytokines and chemokines that subsequently lead to the regulation of cell survival, activation and proliferation, antigen presentation, inflammation, and 21  other immune responses that are essential for host defense. A focus on using TLR9 agonists as adjuvants is due to their ability to induce Th1 and CTL responses that are critical to protection against tumors, intracellular bacterial and viral infections, which humoral immunity alone will not be effective. In addition, clinical trials demonstrated that TLR9 agonists are relatively safe to use in humans compared to the other TLR ligands.  1.3.4  Synthetic CpG oligodeoxynucleotides as adjuvants  Adjuvants often provide the “danger signal” to activate the immune system (67). In 1995, it was demonstrated that unmethylated CpG motifs in bacterial DNA flanked by two 5’ purines and 3’ pyrimidines activate B cells to proliferate and to secrete immunoglobulins (Ig) (123). The concept of using immunostimulatory CpG ODNs as vaccine adjuvants was introduced. It was not until the year of 2000 that TLR9 was identified as the mediator of the stimulatory effect of CpG ODNs (96). Bacterial DNA with CpG motifs can be mimicked by synthetic CpG ODNs that are classified into three families: Class A, B, and C. The phosphodiester backbone of ODNs can be modified to a phosphorothioate (PS) backbone, with one of the non-bridging oxygens replaced by sulfur. CpG ODNs with a PS backbone are nuclease resistant, have high cellular intake and high affinity for TLR9, which confers an increased ability to activate TLR9 (124). Class A CpG ODNs have phosphodiester backbones with 5’ and 3’ PS ends and poly G motifs that may form higher-ordered structures (125),(126). They contain one or more CpG motifs, are potent activators of NK cells, induce high level of type I IFNs but activate B cells weakly (125),(127),(126). Class B CpG ODNs have PS backbones with 1 or more CpG motifs and do not form higher-ordered structure (125). They induce low levels of type I IFNs but are potent 22  activators of B cells (125),(127),(126). Class C CpG ODNs have PS backbones and palindromic sequence at the 3’ end that may form higher-ordered structures. They possess characteristics intermediate between class A and B CpG ODNs (125),(127),(126). All classes of CpG ODNs signal through TLR9 as the adjuvant effects are abrogated in TLR9 deficient mice (128). CpG ODNs have been explored as adjuvant for both prophylactic and therapeutic vaccines due to their ability to stimulate immune cells to enhance both innate and adaptive immune responses. In particular, CpG ODNs promote Th1-biased and CTL adaptive immune responses (129),(130),(131). There are many pre-clinical studies that explore CpG ODNs as vaccine adjuvants for the prevention and treatment of cancers, allergies and infectious diseases while many have advanced to human clinical trials. The first human clinical trial using CpG ODNs as adjuvants was designed with the prophylactic Engerix BTM vaccine against hepatitis B started in 1999 (132). This trial demonstrated that a class B CpG ODNs administered intramuscularly with the vaccine enhanced Ab and CTL production without severe adverse events (133). Since then, many clinical trials have been performed to evaluate the potential of CpG ODNs as vaccine adjuvants for allergies, cancers and infectious diseases. A phase I/IIa clinical trial showed that 10 weeks of subcutaneous therapy with class A CpG ODNs associated with virus-like particle and house dust mite allergen extract freed patients from allergy for 38 weeks (134). Many clinical trials using CpG ODNs as adjuvants are for cancer therapeutic vaccines such as recurrent glioblastoma (135),(136), nonHodgkin lymphoma (137), melanoma (138),(139),(140), cutaneous T cell lymphoma (141) and prostate cancer (142). Various routes and technologies were tested in these studies, which include intratumoral, subcutaneous, intradermal and intranasal injection, intravenous infusion and virus-like particle administration. Not only have CpG ODNs been investigated as potential 23  therapeutic vaccine adjuvants, they have also been explored as prophylactic vaccine adjuvants for infectious diseases such as influenza (143), hepatitis B (144),(133),(145), pneumococcal disease (146), and malaria (147),(148),(149),(150). Immunocompromised individuals, such as patients infected with human immunodeficiency virus (HIV), were also responsive to the adjuvant effect of CpG ODNs and long-term sero-protection was achieved. Clinical trials showed increased humoral immune responses using CpG ODNs as adjuvant with hepatitis B or pneumococcal conjugate vaccine. Most of the human clinical trials evaluated class B CpG ODNs as vaccine adjuvants due to their nuclease resistant PS backbone. Recently, new technologies such as using virus-like particles to stabilize class A CpG ODNs (140) expand the possibilities of using different classes of CpG ODNs as adjuvants for discrete biological effects. CpG ODNs suppress Th2-driven allergic responses by skewing to Th1 immune responses as seen with therapeutic vaccines for allergies. CpG ODNs induce the secretion of IL-1β, IL-6, IL-8, and IFN-γ. In therapeutic cancer vaccines and prophylactic vaccines for infectious diseases, CpG ODNs may act by decreasing the proportion of CD4+ CD25+ Foxp3+ regulatory T cells and tumor growth factor-β (151),(138), augmenting antigen presentation, accelerating and enhancing Th1-type antigen-specific Ab production and cell-mediated immune responses (NK cells, DC, CD4+ and CD8+ T cells). The exact mechanisms of the various immune stimulatory effects induced by the three classes of CpG ODNs when used as vaccine adjuvants still remain unclear (126). Together, these studies have demonstrated the great potential of using CpG ODNs as adjuvants. The advantages of using CpG ODNs as adjuvants include the ability to induce Th1 and CTL cell-mediated immune responses as well as increase vaccine responses in immunocompromised individuals.  24  Although these studies have demonstrated some efficacies of using CpG ODNs as adjuvants, concerns remain about the local and systemic side effects observed. In mice, CpG ODNs can induce a toxic amount of TNF-α by macrophages and cause septic shock (152),(153). In humans, CpG ODNs seem to be well-tolerated since no severe adverse events were observed except in rare cases. Side effects are usually mild to moderate. The most frequently observed local reactions include pain, erythema, warmth and bruising at the injection site as well as limited arm motion. Systemic reactions most often observed include flu-like symptoms such as headache, nausea and pyrexia. Other systemic reactions were also observed such as neutropenia, leucopenia, thrombocytopenia and myalgia. When CpG ODNs were administered intracerebrally, seizures and transient neurological worsening were observed. Reassuringly, no clinical event related to autoimmunity or autoimmune disease has been reported due to CpG ODNs when they are used as vaccine adjuvants.  1.4  1.4.1  The skin as an immune organ  The structure of human and murine skin  Vaccines are generally administered parenterally or subcutaneously. Nevertheless, new appreciation of the skin being immunocompetent suggests that it might be a site for further vaccine and adjuvant development. The human and the murine skin both have three layers: epidermis, dermis and hypodermis (Figure 1.3). The hypodermis is less complex than the other layers and mainly contains adipocytes for insulation and connective tissues that attach the skin to bones and muscles. The epidermis and dermis are more complex. In the epidermis, keratinocytes 25  compose over 90% of the cellular component of the layer. Keratinocytes proliferate and progressively differentiate from the deeper layer to the top layer forming five layers termed the stratum basale, spinosum, granulosum, lucidum and the stratum corneum. Stratum corneum at the top layer of the epidermis consists of dead and fully differentiated keratinocytes and is regularly sloughed off and replaced (154). Between these keratinocytes are lipids such as cholesterol, fatty acids and ceramides. The dermis consists of connective tissues with blood vessels, lymphatics, nerves, hair follicles, and sebaceous and apocrine glands. Nonetheless, there are differences between human and murine skin. The major differences being mouse skin lacks apocrine glands. Yet, it has a thin muscle layer called the panniculosus carnosus and dendritic epidermal T cells in the epidermis, which are not found in human skin (155),(156).  26  Figure 1.3 Structure of the mouse skin. This is a cross section of mouse dorsal back skin with hematoxylin and eosin stain. The skin has five main layers. The epidermis is only a few layers thick and over 90% of cells are keratinocytes. The outermost layers of epidermis consist of dead and differentiated keratinocytes called stratum corneum. Under the epidermis are the dermis, the smooth muscle layer and the hypodermis (subcutaneous) layer.  27  1.4.2  Immune cells in the skin  The skin not only has a unique structure but it consists of a network of different types of cells. Some of these cells play important roles in immune responses. Studies have shown that keratinocytes, dendritic epidermal γδ T cells, Langerhans cells (LCs) and melanocytes in the epidermis play vital roles in regulating immune responses (157),(158),(159),(160). It is more complex in the dermis with many cells involved in immune responses, which include γδ T cells, different subtypes of dermal DCs, macrophages, NK cells, mast cells, NKT cells and T cells. Under homeostasis, it is critical to maintain a balance between immunity against pathogens and tolerance to self to prevent autoimmunity and autoimmune disease. The different subtypes of DCs and other main types of immune cells found in the skin are summarized in a schematic diagram (Figure 1.4). Keratinocytes were once thought of as a mere physical barrier, separating the external environment from the internal environment of the host to prevent damage from pathogens, ultraviolet radiation, heat and water loss. However, recent studies put keratinocytes in the spotlight for their ability to directly or indirectly influence immune responses through secretion of immune mediators. Keratinocytes express receptors such as TLRs (158),(121) to sense pathogens and they are involved in innate and adaptive immunity through various actions. They act as APCs, secrete antimicrobial peptides, cytokines and chemokines, and express cellular adhesion molecules (161),(158) ,(111) ,(162). These actions may prevent pathogen invasion, lead to inflammation, recruitment and activation of other immune cells such as T cells and DCs, which ultimately promote humoral and cell-mediated immune responses.  28  There are skin resident T cells located in the epidermis and dermis. Surprisingly, there are twice as many T cells in the skin than in the blood (158). One subtype of T cell that is common in the skin is the γδ T cell. In murine epidermis, 90% of T cells are special γδ T cells called dendritic epidermal T cells (DETC) while γδ T cells in the dermis do not have the dendritic structure (161). In humans, they are a small subset of T cells in the skin. γδ T cells are CD4CD8- with γδ chains in the T cell receptor with limited diversity. They recognize ligands such as heat shock proteins induced by stress in a non-classical MHC restricted manner but using MHCrelated molecules such as non polymorphic MHC class Ib (161),(163) ,(164). γδ T cells as well as other subsets of T cells expressing cutaneous lymphocyte–associated antigen reside in the epidermis and dermis or can be recruited in humans and in mice (165). For example, CD4+ and CD8+ T cells play important roles in the immediate control of local infections by producing perforin and granzymes that are cytolytic, or cytokines and chemokines that recruit and stimulate other immune cells (160),(163). It has been demonstrated that memory T cells reside in the skin and they can enhance local immunity against herpes simplex virus (166). Another type of immune cell in the skin are DCs, and they also express TLRs (114), (167), (168), (169), (170). There are various subsets of DCs that localize to particular regions of the skin. In the epidermis, DCs called Langerhan cells (LCs) express Langerin (CD207), a type II Ctype lectin receptor that binds mannose and related sugars in a calcium-dependent manner (171),(172), on the cell surface and constitutively associated with Birbeck granules present in the cytoplasm (173). LCs comprise 2-8% of epidermal cells and reside predominantly at the suprabasal layer of the epidermis (163). Different subsets of DCs including Langerin+ DCs, Langerin- DCs and pDCs reside in the dermis. LCs can be distinguished from the Langerin+ 29  dermal DCs by the expression of epithelial cell adhesion molecule (EpCAM) that is absent in Langerin+ dermal DCs (174). DCs are professional APCs that are crucial to the initiation of adaptive immune responses. There are active debates on the roles of the different subtypes of cutaneous DCs in the skin immune system. The recent generation of transgenic mice in which investigators can selectively deplete subtypes of DCs allows the design of studies to decipher the functions of these DCs. For example, transgenic mice with the gene for a primate diphtheria toxin receptor (DTR) linked to the Langerin gene can be used to deplete both LCs and Langerin+ dermal DCs conditionally with diphtheria toxin (175),(176). LCs reside in the epidermis, an ideal location as the first line of defense. LCs can efficiently acquire antigens, migrate to the skin draining lymph nodes (SLNs) and present antigens to naive and memory T cells (177). LCs have been thought to play a role in contact hypersensitivity (CHS) and in the cross-priming of CD8+ T cells that are important in fighting viruses and intracellular bacteria. However, when the Langerin+ dermal DCs were identified, it has become clearer that the old LC paradigm, which stated that LCs are the primary APCs in the skin (178),(179), should be revised: Studies showed that LCs and dermal DCs migrate to the SLNs under different kinetics and populate differential anatomic locations (180),(174). Studies demonstrated that LCs are not required for the induction of CHS immune responses, while dermal DCs are necessary for CHS (181). In addition, Langerin+ dermal DCs instead of LCs were shown to cross-present antigens to CD8+ T cells in the SLNs (182),(183). Langerin+ CD103+ dermal DCs are the only DCs able to cross-present the glycoprotein B antigen to CD8+ T cells in a herpes simplex virus skin infection model (182). LCs, Langerin+ dermal DCs and Langerin- dermal DCs all are capable of presenting antigens to CD4+ T cells. LCs present antigens to induce antigen-specific CD4+ T cells but not CD8+ T cells in a Candida skin 30  infection model (176). Instead, Langerin+ dermal DCs are required for Th1 and CD8+ T cell induction. Some studies have also suggested that antigens can be transferred from skin DCs to the SLN resident DCs under certain circumstances such as when the pathogens are cytolytic, killing the skin DCs (184), (185). pDCs are normally absent in the skin under steady state (186). However, they will migrate from the blood into the skin during infection of the skin (187). pDCs express TLR7 and TLR9 and secrete type I IFNs. They have antiviral and antitumor effects (187),(188) ,(189) ,(190). DCs can also directly activate T cells in the skin leading to the production of cytokines that recruit other immune cells to the infection site to fight invading pathogens (191),(192). These studies demonstrate that DCs have specialized functions in the induction of distinct immune responses.  1.4.3  Using the skin as a route of immunization  In 1983, Streilein coined the term “skin-associated lymphoid tissues” to describe keratinocytes, LCs and lymphocytes that together provide immune surveillance against cutaneous neoplasms and infections (193). Most of the current licensed vaccines are administered into the muscles or the subcutaneous fat with a few administered into the dermis and the nasal passage. The skin harbors many types of immune cells and thus it has the potential to be an accessible and effective route of immunization. The first consideration of using the skin as a site of immunization is the skin barrier, formed by the layers of dead keratinocytes called stratum corneum, that limits the penetration of large molecules over 500 kilo-dalton (194). Tapestripping, degreasing with acetone and hydration are methods to minimize this barrier. New  31  technologies have also been developed to overcome this barrier such as microneedles, liquid jet immunization, thermal ablation, ultrasound and electroporation (195),(196),(197),(198). There is strong evidence to support the concept that the skin is immunologically active and vaccines can be delivered through the skin to mount specific immune responses. In the past, cholera toxin and heat labile enterotoxin produced by the bacterium Vibrio cholera and Escherichia coli, respectively, were used as transcutaneous adjuvants to enhance immune responses. In combination with whole protein antigens such as diphtheria toxoid and tentaus toxoid or antigenic peptides, these toxins, when given topically, enhance antigen-specific humoral and T cell responses even in the elderly who are in general poorly responsive to vaccines (199),(200),(195), (201),(202). TLR7 agonists have been explored as topical adjuvants because no pre-treatment of the skin is required for their small molecular mass (203). They have been found to have a Th1-type immune response stimulating effect when given topically (204),(205),(206). However, using CpG ODNs as topical adjuvants is less studied. Transcutaneous antigen administration with topical CpG ODN augments the production of Th1type CD4+ T cells and Abs as well as CTL responses (207),(208),(209). Split administration of topical CpG ODNs and antigen promote cross-presentation of antigens and increase effector and memory CTLs compared to co-administered subcutaneous or intramuscular antigen (210). Split administration of topically administered CpG ODNs also enhanced the antitumor effect of dacarbazine in a mouse melanoma model (211). Although CpG ODNs have been studied as vaccine adjuvants administered via the same route as the antigens, the split delivery of CpG ODNs and antigens is rarely studied. To date, there are no clinical trials, and few pre-clinical studies that have explored the use of topical CpG ODNs as adjuvants, when administered separately from the route of vaccine administration. 32  1.5  Thesis objectives  Vaccine antigens, such as in subunit vaccines, are often not highly immunogenic and adjuvants can be added to increase immunogenicity to boost relevant immune responses. TLR9 is a pattern recognition receptor that links the innate immunity to adaptive immunity. TLR9 agonists include bacterial DNA and synthetic ODNs with CpG motifs (123),(96). In humans, TLR9 is constitutively expressed on B cells and pDCs, and TLR9 expression can also be induced in other cell types that are of hematopoietic and stromal origin. In the murine model, TLR9 is more broadly expressed not only on B cells and pDCs but on monocytes, myeloid DCs and other immune cells as well. Although TLR9 agonists have been explored as vaccine adjuvants administered via the parenteral routes, safety concerns remain for undesired systemic reactions or toxicity observed in human clinical trials. Administration of TLR9 agonists as adjuvants separated from the vaccine antigen has been less commonly studied but the administration mode introduces the advantage of flexibility, allowing tailoring of adjuvant administration to fit the clinical situation. Since the skin harbors many immune cells that constitutively express or can be induced to express TLR9, I hypothesized that topical administration of TLR9 agonists as adjuvants could be used to harness the skin immune system, in a uniquely effective way to increase the efficacy of locally administered protein-based vaccines. The first objective of this thesis was to compare the adjuvant effect of CpG ODN 1826 administered topically or parenterally in enhancing Ab production as well as CD4+ and CD8+ T cell responses. Also, an effective schedule to administer CpG ODN 1826 as topical adjuvant was determined. In order to determine whether topical CpG 33  ODN delivery (the proposed optimal route) provides advantages over standard immunization strategies, the ability to enhance protection against intracellular bacterial and viral infections was determined. Previous investigators have studied the cellular mechanisms of action of TLR9 agonists and how these ligands are being recognized and lead to signaling. The cellular immune responses to topical CpG ODNs and the mechanisms underlying these responses have been less commonly studied. I hypothesized that TLR9 agonists improve vaccine responses by enhancing the rapid induction of long-lasting humoral and cell-mediated immunity, through effects of both hematopoietic cells and skin stromal cells. The second objective of this thesis was to determine the mechanisms whereby topical CpG adjuvant results in improved vaccine outcomes. Immune cells including keratinocytes, mast cells and DCs were studied for their contribution to the adjuvant effect of topical CpG ODN 1826. The influence of topical CpG ODN to the microenvironment of the SLNs was examined. Our long-term goal is to enhance vaccine efficacy without the need to re-formulate existing vaccines using a split delivery method of topical CpG ODN 1826 as vaccine adjuvant.  34  Figure 1.4 Immune cells in the skin. The skin harbors many immune cells. Keratinocytes, T cells and LCs reside in the epidermis. The dermis harbors more types of immune cells compared to the epidermis, which include the different subsets of DCs, macrophages (mφ), NK cells, NKT cells, γδ T cells and other T cells. The dermis also contains blood vessels and lymphatics that allow recruitment of cells into the skin under certain circumstances such as infection and inflammation.  35  CHAPTER 2: MATERIALS AND METHODS  2.1  2.1.1  Materials  Antibodies  The following anti-mouse antibodies were used for flow cytometry: B220-peridinin chlorophyll protein (PerCP) or -isothiocyanate (FITC) (RA3-6B2), CD8α-allophycocyanin (APC) or -APC-Cy7 (53-6.7), IFNg-APC (XMG1.2), CD62L-FITC (MEL-14), CD127-APC (A7R34), CD8a-A700 (53.67), CD4-pacific blue (RM4-5), Thy1.1-phycoerythrin (PE) (OX-7), IL-4-APC (11B11), IL-17A-PECy7 (eBio17B7), IFN-g-FITC (XMG1.2), CD11c-PE (HL3), streptavidin-APC, CD45.1-PE (A20), CD45.2-PerCP-Cy5.5 (104), and anti-human IgG-Biotin (polyclonal) were purchased from BD Biosciences Inc. (Mississauga, ON), eBioscience Inc. (San Diego, CA), BioLegend (San Diego, CA), or the AbLab of The University of British Columbia (Vancouver, BC). PE conjugated OVA MHC class I tetramer specific for the Kb-restricted OVA8 peptide (OVA257-264, amino acid sequence SIINFEKL) was generated by conjugation of biotinylated monomers to streptavidin-PE in Dr. R. Tan's laboratory (Child & Family Research Institute, Vancouver, BC). MHC class I tetramer specific for the Db-restricted nucleoprotein protein (NP366-374, ASNENMETM) and polymerase acidic protein (PA224-233, SSLENFRAYV), conjugated to PE and APC, respectively, were provided by the National Institute of Health Tetramer Core Facility (Atlanta, GA). The anti-Fc receptor monoclonal Ab (2.4G2) was obtained from American Type Culture Collection (Rockville, MD). These antibodies were used for enzyme-linked immunosorbent assay (ELISA) to detect OVA-specific immunoglobulins (Igs): 36  horseradish peroxidase (HRP)-labeled goat anti-mouse IgG (H+L) or IgG2c Ab from Jackson ImmunoResearch (West Grove, PA), or rabbit anti-mouse IgG1 Ab from Zymed Laboratories (San Francisco, CA). For ELISA to detect influenza-specific Igs, HRP-labeled goat anti-mouse IgG1 and IgG2b Abs in the SBA ClonotypingTM System - B6C57J-HRP kit from SouthernBiotech (Birmingham, AL) were used.  2.1.2  Reagents  Ketalean (ketamine hydrochloride) and Rompum (xylazine) for mouse anesthesia were from Bimeda-MTC Animal Health Inc. (Cambridge, ON) and Bayer Inc. (Toronto, ON), respectively. Acetone for skin treatment was from Fisher Scientific (Edmonton, AB). Chicken ovalbumin protein grade V (OVA), ionomycin, phorbol 12-myristate 13-acetate (PMA), trypsin, 7-amino-actionmycin D (7AAD), dimethyl sulfoxide (DMSO), ethylenediaminetetraacetic acid (EDTA), and isopentane were from Sigma-Aldrich Inc. (Saint Louis, MO). Synthetic high performance liquid chromatography-purified, single-stranded, phosphothioated CpG ODN 1826 (5'-TCCATGACGTTCCTGACGTT-3') and bovine serum albumin (BSA) were purchased from Sigma-Aldrich Inc. (Saint Louis, MO). Control ODN without CpG motifs (5’TCCAGGACTTCTCTCAGGTT-3’) was from Integrated DNA Technologies, Inc. (San Diego, CA). The immunodominant Kb-restricted OVA8 peptide (OVA257-264, amino acid sequence SIINFEKL) was synthesized by Kinexus (Vancouver, BC). Purified mouse P-selectin-Ig fusion protein was purchased from BD Biosciences Inc. (Mississauga, ON). The tetramethylbenzidine (TMB) substrate, intracellular fixation and permeabilization buffers were from eBioscience Inc. (San Diego, CA). The recombinant mouse E-selectin Fc chimera was purchased from R & D 37  Systems Inc. (Minneapolis, MN). GolgiStopTM Protein transport inhibitor containing monensin was from BD Biosciences Inc. (Mississauga, ON). CpG ODN 1826 conjugated to FITC (CpGFITC) was from InvivoGen (San Diego, CA). FTY720 was from Cayman Chemical (Ann Arbor, MI). The EasySepTM Mouse CD4+ T cell Enrichment kit was from STEMCELL Technologies Inc. (Vancouver, BC). The Tissue-Tek optimal cutting temperature (OCT) compound was from Sakura Finetek USA Inc. (Torrance CA). The RNeasy Fibrous Tissue Mini Kit for RNA extraction was from QIAGEN Inc.(Toronto, ON). For reverse transcription, the QuantiTect Reverse Transcription Kit and the RT2 First strand kit were from QIAGEN Inc. (Toronto, ON). The SABiosciences RT2 qPCR Primer Assay for mouse beta actin, Tlr9 and Il1b and the 384wells Mouse Cytokines & Chemokines RT2 Profiler PCR Array were from QIAGEN Inc. (Toronto, ON). Neomycin 325 was from Vetoquinol NA. Inc. (Lavaltrie, QC). Fetal bovine serum, Hank's Balanced Salt Solution (HBSS) and the ProLong® Gold Antifade Reagent with DAPI were from Life Technologies Inc. (Burlington, ON).  2.1.3  Mice  C57BL/6 mice were purchased from Charles River Laboratories (Wilmington, MA). TLR9 deficient mice on C57BL/6 background (TLR9 KO mice) were obtained from Oriental Bioservice (Tokyo, Japan), bred and maintained in CFRI animal care facility (Vancouver, Canada). The B6.Cg-Tg(TcraTcrb)425cbn/J mice (stock #004194) that express a T cell receptor specific to OVA323-339 (amino acid sequence ISQAVHAAHAEINEAGR) in the context of I-Ab (OT-II transgenic mice), the B6.PL-Thy1a mice that carry the Thy1.1 allele (Thy1.1 mice), the WBB6F1/J-KitW/KitW-v mice (stock #100410) and the corresponding congenic +/+ mice, and the 38  B6.SJL-PtprcaPepcb/BoyJ (stock #002014), which were C57BL/6 mice that carried the CD45.1 allele were from The Jackson Laboratory (Bar Harbor, ME). The OT-II and the Thy1.1 mice were crossed to generate OT-II+ Thy1.1+ mice. All mice were housed in a specific pathogen-free animal care facility at Child & Family Research Institute (CFRI) (Vancouver, Canada) or at Centre for Disease Modeling (Vancouver, Canada). Animal experiments were conducted in accordance with protocols approved by the University Animal Care Committee and Canadian Council of Animal Care guidelines. Age-matched female mice were used between 6 to 12 weeks of age.  2.1.4  Bacteria and virus strains  Wild-type Lm strain 10403s and mutant strain Lm-OVA, which has been modified to express the CTL epitope of OVA257-264 (Lm-OVA) was provided by Dr. H Shen (University of Pennsylvania, Philadelphia, PA). The construction of the less virulent form of ΔactA-Lm-OVA with the targeted deletion in the virulence determinant ActA has been described elsewhere (36). For immunization and infection experiments, frozen infection aliquots of Lm strains were prepared as described previously (212). Briefly, Lm strains were grown in brain-heart infusion broth to mid-logarithmic phase (OD600, 1.0) at 37°C, washed twice with endotoxin-free, isotonic saline (0.9% NaCl), resuspended in 20% (v/v) glycerol in 0.9% NaCl, and stored at -80°C until use. Purified human influenza A virus A/PR/8/34 (H1N1 serotype) (PR8 virus) and the less virulent form influenza A virus X-31, A/Aichi/68 (H3N2 serotype) (X31 virus) were purchased from Charles River Laboratories (Wilmington, MA). The PR8 and the X31 viruses that  39  expressed the CTL epitope of OVA257-264 (PR8-OVA and X31-OVA) were made as previously described (52). Influenza viruses were stored at -80°C until use.  2.2  2.2.1  Methods  Immunization  Mice 6-12 weeks of age were anesthetized by intraperitoneal (ip) injection of ketamine and xylazine. Mice were shaved on the dorsal back, tape-stripped fifteen times using cellophane tape purchased from Staples (Vancouver, BC), and the skin was treated with acetone using a cotton swab. 100 µg of OVA protein, was injected either subcutaneously or intramuscularly. 250 µg or 50 µg of CpG ODN 1826, was administered either epicutaneously, subcutaneously, or intramuscularly in an epifocal manner overlaying antigen injection site. PBS/DMSO (1:1 v/v) or control ODN was applied when no adjuvant was administered as indicated. The area was then covered with waterproof tape to prevent oral ingestion of topically applied reagent on the dorsal skin. For a prime-boost regimen, mice were immunized twice, 7 days or 3 weeks apart, at the same site on the dorsal back. For a prime-only regimen, mice were immunized once. For multiple applications of adjuvant, CpG ODN 1826 was administered at the time of antigen administration and again at 1 and 2 days post initial immunization: Day 0, 1 and 2 for a primeonly regimen and day 0, 1, 2, 7, 8 and 9 for a prime-boost regimen. Mice were euthanized for analysis on days indicated accordingly.  40  2.2.2  Sample collection  Blood, serum, spleen, SLNs (axillary and branchial lymph nodes), lungs, livers and bronchoalveolar lavage (BAL) fluids were collected for experiments as indicated. Blood was collected by cardiac puncture and 200 µl of 20 mM EDTA per sample was added to prevent clotting. Sera were obtained by allowing the collected blood to clot and then collected the supernatant after centrifugation and stored at - 80°C. To obtain BAL fluids, a catheter was inserted into the trachea of the mouse and secured in place with suture thread and lungs were flushed four times with 1 mL PBS. Spleens and SLNs were filtered through 70 µm cell strainers while lungs were first digested with collagenase D at 37°C for 1 hr and then filtered through 70 µm cell strainers to obtain single cell suspension for flow cytometry. For analysis using the ImageStreamX Mark II, SLNs were digested with collagenase D at 37°C for 20 min and then filtered through 100 µm cell strainers to obtain single cell suspension. Erythrocytes were lysed for the blood and spleen samples before staining for flow cytometry. Spleens and lungs were homogenized and stored at -80°C for bacterial and viral count by plating and plaque assay. RNA from the skin and SLNs were extracted using the RNeasy Fibrous Tissue Mini Kit.  2.2.3  Detection of antigen-specific antibodies  ELISA was used to measure isotype-specific Abs in serial dilutions of sera. For OVAspecific Ig isotypes, 96-well flat-bottomed ELISA plates were coated with 2 µg of OVA protein in carbonate buffer (pH 9.6) overnight at 4°C. Plates were blocked with PBS supplemented with 2% BSA for 1 hr at 37°C and then washed with PBS-0.05% Tween and PBS. Serially diluted 41  sera were incubated for 2 hrs at room temperature and then washed. OVA-specific IgG isotypes were detected by HRP-labeled goat anti-mouse IgG (H+L), IgG2c, or rabbit anti-mouse IgG1 Abs. The TMB substrate was used for signal detection with a color change and the reaction was stopped with sulfuric acid. Color absorbance was measured at 450 nm with the Dynex MRX Revelation Microplate reader from Dynex Technologies (Chantilly, VA). For the detection of influenza-specific Ig titers, 96-well flat-bottomed ELISA plates were coated with 500 hemagglutination units (HAU) of PR8 virus overnight at 4°C. Plates were blocked with PBS supplemented with 1% BSA for 1 hr at room temperature and washed with PBS-0.05% Tween. Serially diluted sera were incubated for 1 hr at room temperature and washed. Plates were next incubated with HRP-labeled secondary Abs for 1 hr at room temperature before washing and then detected by addition of 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulphonic acid) and absorbance was measured at 405 nm.  2.2.4  Cell isolations and adoptive transfer  OT-II cells were isolated from the pooled lymph nodes and spleens of naïve OT-II female mice. CD4+ T cells were enriched to over 85% purity by negative selection using the EasySepTM Mouse CD4+ T Cell Enrichment Kit. 2 x 106 cells were injected into the mice’s lateral tail vein 1 day prior to immunization.  42  2.2.5  Flow cytometry  For staining of cells from blood, spleens, SLNs, lungs, and BAL fluids, 2 x 106 to 4 x 106 cells were first blocked with 50 µl of 2.4G2 tissue culture supernatant on ice for 15 min before labeling with Abs. Cells were then washed once with FACS buffer (PBS supplemented with 4% v/v fetal bovine serum (FBS) and with 2mM EDTA if stained for DCs) and then labeled with 50 µl of Abs diluted with FACS buffer at 4°C for 30 min. OVA-tetramer staining performed at 4°C for 45 min and influenza-tetramer staining was performed at room temperature for 10 min. Cells were washed twice with FACS buffer before resuspended in 200 µl of FACS buffer. Samples were analyzed on either the FACSCalibur or the LSRII Flow Cytometer from BD Biosciences Inc. (San Jose, CA) with the FACSDiva software version 6.0 (Mississauga, ON). Unlabeled and single fluorochrome labeled samples were used for compensation settings. Flow cytometry data were analyzed by the FlowJo flow cytometry analysis software for Macintosh version 8.8.2 (Tree Star, Inc., Ashland, OR).  2.2.6 Detection of antigen-specific T cells, intracellular cytokines and tissue-homing molecule expression  Single cell suspension of tissues were prepared either 5 or 30 days post-immunization for the detection of effector and memory T cells with corresponding surface markers using flow cytometry as described above. For detecting intracellular cytokines produced by T cells, single cell suspensions of tissues were prepared either 5 or 30 days post-immunization. Cells were restimulated with PMA and ionomycin, or OVA8 peptide, with GolgistopTM in vitro at 37°C for 4 43  hrs when only staining for IFN-γ alone or 6 hrs when staining for IFN-g, IL-4 and IL-17 at the same time. Cells were blocked with 50 µl of 2.4G2 tissue culture supernatant before surface marker staining as described above. Cells were then fixed and permeabilized with intracellular fixation and permeabilization buffer, respectively. Intracellular cytokine staining was performed subsequently at room temperature for 30 min and analyzed by flow cytometry. For detecting expression of tissue-homing molecules, OT-II CD4+ cells were adoptively transferred into mice via lateral tail vein and the mice were immunized 1 day after as described above. Single cell suspensions were prepared from the SLNs 3 days post-immunization. Cells were labeled with recombinant E-selectin Fc chimera, or P-selectin-IgG fusion protein containing the Fc region of human IgG at 20 µg/mL in HBSS supplemented with 0.2% BSA. The cells were then labeled with biotin-conjugated anti-human IgG Ab followed by streptavidin-APC Ab. L-selectin expression was detected by labeling cells with the CD62L Ab. Labeling and staining were performed at 4°C for 30 min. The adoptively transferred CD4+ T cells were tracked with the CD4 and Thy1.1 surface markers and analyzed by flow cytometry.  2.2.7  Infection models  For the Lm infection model, mice were ip injected with 2 x 106 colony forming units (CFUs) of ΔactA-Lm-OVA, an attenuated strain of Lm for immunizing infection. To challenge the mice, they were infected with 5 x 105 CFUs of the Lm-OVA in 100 µl 0.9% NaCl systematically via the tail vein. 3 days after infection, naive or immunized mice were euthanized, spleens and livers were collected and homogenized mechanically in 5 mL 0.9% NaCl containing 0.05% Triton-X100. To enumerate the number of viable bacteria colonizing the spleens and the 44  livers post-infection, serial dilutions of the mechanically lysed cell suspensions were plated on brain heart infusion agar. For the influenza infection model, mice were intranasally infected with 10 HAU of X31 or 20 HAU of X31-OVA virus on day 0 and day 7 for immunizing infection. To challenge mice, naive or immunized mice were intranasally infected with 5 HAU of PR8 virus or 10 HAU of PR8-OVA virus on 12 days or 37 days post-initial immunization to assess acute and long-term immune responses. To enumerate the number of viable virus colonizing the lung post influenza virus challenge, the lung tissues were homogenized in 10 mL RPMI 1640 medium per gram of tissues. Homogenates were centrifuged at 1200 xg for 20 minutes at 4°C and supernatants were collected. Madin Darby canine kidney (MDCK) cells (8.0 x 105 cells per well) obtained from American Type Culture Collection were cultured in Eagle’s Minimal Essential Medium supplemented with 10% FBS and 1% penicillin and streptomycin in 6-well plates at 37°C, 5% CO2, for 2 days until 80% confluent. Medium was removed and plates were washed twice with PBS. 200 ul of 1:5 diluted viral samples were added per well per sample to MDCK cells and incubated at room temperature for 1 h with gently rocking on a shaker. Viral samples were then removed and each well was overlaid with 1.5 ml agarose in Minimal Essential Medium supplement with 0.25% trypsin. Plates wells incubated at 37°C, 5% CO2, for 4 days. To count the number of plaques formed, virus was first inactivated by 1 ml Carnoy's Reagent at room temperature for 1 h. Agarose was discarded and plates were washed with distilled water. Cells were then fixed with 4% paraformaldehyde in PBS at room temperature for 20 min. Cells were washed once and stained with 1% crystal violet in 20% methanol at room temperature for 30 min, washed and air-dried. Number of plaques formed were counted and averaged between triplicates. Numbers of plaques were calculated per gram of lung tissues.  45  2.2.8  Analysis of gene expressions by real-time PCR and PCR array  Mice were euthanized and RNA was extracted using the QIAGEN Fibrous Tissue Mini Kit according to the company’s provided protocol to measure gene expressions on keratinocytes. Briefly, epidermis was homogenized and proteins were digested with proteinase K. RNA was extracted using the mini column and DNase was added to ensure no residual DNA was retained in the sample. RNA was quantified using Thermo Scientific Nanodrop 1000 Spectrophotometer. Then, 0.1 µg of RNA was reverse transcribed into cDNA using the QuantiTect Reverse Transcription Kit. The genomic DNA elimination reaction was performed at 42°C for 2 min and the reverse transcription reaction was performed for 30 min and stopped by inactivating the Quantiscript reverse transcriptase at 95°C for 3 min using the PTC-100TM Programmable Thermal Controller from MJ Research, Inc. (St. Bruno, QC). Real-time RT-PCR was performed using the Applied Biosystems 7500 Fast Real-Time PCR System from Life Technologies Inc. (Burlington, ON). The house-keeping b-actin gene was used as the internal control of gene expression and fold changes of gene expressions post-treatments were calculated by normalizing to a reference sample (untreated epidermis). To measure the gene expression of the chemokines and cytokines in the SLNs after immunization, the 384-wells Mouse Cytokines & Chemokines RT2 Profiler PCR Array was used. RNA was extracted as above. 400 ng of RNA was reverse transcribed into cDNA using the RT2 First strand kit and real-time RT-PCR was performed using the Applied Biosystems ViiATM 7 Real-Time PCR System from Life Technologies Inc. (Burlingto, ON). PCR array data were analyzed using the QIAGEN web-based RT2 Profiler PCR Array Data Analysis software version 3.5. 46  2.2.9  Generation of bone marrow chimeric mice  Mice to be irradiated were given neomycin (2 mg/ml) in drinking water 1 week prior to irradiation. To generate bone marrow chimeric mice, mice were irradiated with 1100 rad of gamma ray at The Biomedical Research Centre in University of British Columbia (Vancouver, BC). Donor mice were euthanized and bone marrow was taken from the femurs and tibias, erythrocytes were lysed and resuspended to 5.0 x 107 cells/ml in PBS. 1 x 107 cells were injected into irradiated recipient mice via the lateral tail vein. Hair on the dorsal back was shaved and the mice were then subjected to 2125 J/m2 of ultraviolet light treatment for 4 consecutive days. Following bone marrow transplantation, mice were set aside for 8 weeks for the bone marrow to be reconstituted before using them for experiments. These mice were housed in a pathogen-free environment and were given acidified water (0.008% hydrochloric acid) until the end of experiments. Reconstitution with donor cells in blood and the skin of recipient mice were monitored by staining for the pan-hematopoietic markers CD45.1 and CD45.2 using flow cytometry.  2.2.10 Detection of FITC-labeled CpG ODN in SLNs  To detect CpG ODN in vivo, FITC-labeled CpG ODN 1826 was administered topically on dorsal back skin of the mice. 2 hrs after treatment, mice were euthanized and SLNs were collected. The tissues were rinsed in ice-cold PBS, embedded in optimal cutting temperature (OCT) compound, frozen with isopentane and liquid nitrogen. Serial sections were cut at a 47  thickness of 6 µm and observed under a Zeiss AxioImager microscope. Images were taken using an AxioCam HRm camera operating through the AxioVision software version 4.4.  2.2.11 Detection of the internalization of CpG ODN by dendritic cells  To detect internalization of CpG ODNs into DCs in SLNs, mice were administered with FITC-labeled CpG ODNs topically or subcutaneously to allow the tracing in vivo and ex vivo. Mice were euthanized 2 days after treatment, SLNs were collected, and single cell suspensions were prepared as described above. Cells were blocked with 50 µl of 2.4G2 tissue culture supernatant and stained with CD11c surface marker as described above. Cells were also stained with 1 µg/ml of 7AAD for 5 min to exclude non-viable cells. Data were acquired using the ImageStreamX imaging flow cytometer from Amnis Corporation (Seattle, WA) using the INSPIRE software. The acquired digital imagery was analyzed using the IDEAS statistical image analysis program version 4.0.  2.2.12 Protein contact hypersensitivity and lymphocyte egress inhibition  For the initiation phase of contact hypersensitivity, mice were immunized on the dorsal back with either subcutaneous OVA with or without CpG ODN administered topically or subcutaneously as described above for 2 consecutive days on days 0 and 1. For the elicitation phase, mice were challenged on day 6 as follows: the right ear was tape-stripped twice, rolled with acetone using a cotton swap twice and 100 µg of OVA and CpG ODN were administered topically. The left ear was the negative control, which was treated the same except DMSO was 48  applied instead of OVA and CpG ODN. To inhibit lymphocyte egression from the SLNs, FTY720 was injected ip at 1 mg/kg of mice 1 day post-CHS elicitation. Ear swelling calculated as follow: (Ttreated-Tinitial)treated ear - (Ttreated-Tinitial)untreated ear, where T is the thickness of the ear.  2.2.13 Tissue microscopy  Skin tissues were viewed using a Zeiss AxioImager microscope. Hematoxylin and eosin stained skin sections were viewed using the brightfield setting. Images were obtained using an AxioCam HRm camera operating through AxioVision software version 4.4.  2.2.14 Statistical analyses  All quantitative data were presented as mean ± standard error of the mean (SEM). Statistical analyses were performed using Prism 4 for Macintosh version 4.0b or version 5.0c. The Kolmogorov-Smirnov test was used to test whether data followed normal distribution. Data that followed normal distribution were analyzed by unpaired two-tailed Student’s t-test (two groups comparison) or One-way ANOVA test followed by Tukey’s post-test (for three or more groups comparison) to determine the statistical significance. Data that did not follow normal distribution were analyzed by Mann-Whitney test or by Kruskal-Wallis test followed by Dunn’s post-test to determine the statistical significance. Repeated measures were analyzed by the Twoway ANOVA test followed by Bonferroni post-test. p < 0.05 was considered significant.  49  CHAPTER 3: TOPICAL CPG ODN ENHANCES HUMORAL AND CELL-MEDIATED IMMUNE RESPONSES TO PROTEIN-BASED VACCINES  3.1  Introduction  Infectious diseases are the leading cause of illness in humans. Although immunization is the most cost-effective method to improve population health against infectious diseases, current vaccines remain ineffective in part due to poor ability to elicit CTLs, delayed protection, and lack of long-lasting protective effect. An adjuvant is often added to increase the immunogenicity of vaccine antigens. Synthetic CpG ODNs mimic unmethylated bacterial and viral DNA with CpG motifs, which bind the endosomal pattern recognition receptor TLR9 (96). Many studies used synthetic CpG ODNs as adjuvants for both therapeutic and prophylactic vaccines. CpG ODNs uniquely enhance CD8+ and Th1-type CD4+ T cell responses, which are particularly valuable for combating tumors, intracellular bacteria and viruses. The enhanced vaccine responses induced by CpG ODNs administered parenterally or intranasally have been demonstrated in human clinical trials. However, TLR9 agonists have not been adopted for widespread clinical use in humans as concerns regarding systemic toxicity and safety persist (152),(153),(213),(149). Strategies to limit the systemic toxicity of CpG ODNs include conjugation of the CpG ODN to antigen (214) and co-encapsulation of CpG ODN and antigen (215). These strategies are cumbersome and require further validation in humans. The skin is the most accessible organ of our bodies harboring many immune cells that can be harnessed to induce immune responses. Our group and others have explored the skin as a route of immunization by co-administration of an antigen and an adjuvant. Topical peptide with 50  cholera toxin induced robust cell-mediated immune responses in mice (216). However, using cholera toxin as an adjuvant may have significant toxicity and makes it difficult to license for human use. Studies from my laboratory showed that topical CpG ODNs co-administrated with an antigen promoted antigen-specific CTL production (217). Adjuvants are routinely combined with antigens for efficacy and ease of formulation. Split administration, where vaccine antigens and adjuvants are delivered separately, has been less commonly studied but has the advantages of flexibility and simplicity (1),(218). Our group started to explore split administration of CpG ODNs as adjuvants to enhance vaccine efficacy and to limit toxicity. Topical application compared to subcutaneous administration of CpG ODN 1826 as adjuvant promoted cross-presentation of injected soluble protein antigens with less systemic cytokine production (210). Topical CpG ODN 1826 applied overlaying the antigen administration site augmented the CTL response against melanoma in a mouse model (211), demonstrating the success of split administration of vaccines and adjuvants. The aim of the studies in this chapter was to determine an effective strategy to administer the type B CpG ODN, CpG ODN 1826, as vaccine adjuvant as well as the effect of topical CpG ODN 1826 in humoral and cell-mediated immune responses. CpG ODN 1826 was used because it is a class B CpG ODN, a strong B cell activator (127),(128). The phosphorothioate backbone is gives it a longer half-life and increases cellular intake and affinity for TLR9 binding. Importantly, CpG 1826 can skew immune responses to Th1-type Ab and CD4+ T cell production as well as promote CTL generation.  51  3.2  3.2.1  Results  Topical administration of CpG ODN as adjuvant augments Ab production  A standard method for assessing vaccine protection is still Ab production (219). I first assessed the ability of CpG ODN 1826 (thereafter refer as CpG ODN) administered topically on the skin to augment antigen-specific Ab production. Mice were immunized twice, 3 weeks apart, to mimic prime-boost immunization regimen in humans (Figure 3.1A). Ab production was tested with whole OVA protein or whole heat-killed PR8 influenza virus as antigens. Whole OVA protein was used because it is a whole protein antigen. OVA-specific IgG titers can be measured by HRP-labeled Abs against OVA-specific IgG2c and IgG1. The heat-killed influenza virus was used as antigen because it mimics whole influenza vaccines. Mice subcutaneously injected with OVA alone slightly increased the amount of total OVA-specific IgG Abs, while those immunized with OVA in combination with CpG ODN further increased these Abs in sera (Figure 1B). No difference was detected between the overall level of antigen-specific Abs between topical and subcutaneous administration of CpG ODN. However, the relative serum levels of IgG2c and IgG1 Abs were significantly higher when CpG ODN was delivered topically compared to subcutaneously (Figure 3.1C, D). In a similar fashion, increased level of the influenza-specific IgG2b Abs was detected when using whole heat-killed PR8 influenza as the antigen although no difference was detected with or without adjuvant (Figure 3.2A-C). Low level of influenza-specific IgG1 Abs was detected post-immunization. Thus, topical application compared to subcutaneous administration of CpG ODN promotes IgG2 more than IgG1 antibody production, indicative of a Th1-biased immune response. 52  Figure 3.1 Using CpG ODN as vaccine adjuvant with soluble protein antigen enhances the production of antigen-specific IgG Abs. (A) Schematic diagram of immunization and analysis schedule. C57BL/6 mice were immunized twice, 3 weeks apart. OVA protein (100 µg) was injected sc while CpG ODN was administered sc (50 µg) or ec (250 µg). Sera were collected 4 weeks post-immunization. Ab titers in serial diluted sera were measured by indirect ELISA. (B, C, D) Graphical representation of the average ± SEM absorbance of total IgG, IgG2c and IgG1 at optical density (O.D.) 405 nm. Data summarize three independent experiments. Naïve (black, n = 6), sc OVA (blue, n = 6) and sc OVA + sc CpG (red, n = 6), and sc OVA + ec CpG (green, n = 5). * p < 0.05; ** p < 0.01; *** p < 0.001. 53  Figure 3.2 Whole heat-killed influenza virus with CpG ODN increases the production of influenza-specific IgG Abs. (A) Schematic diagram of immunization and analysis schedule. C57BL/6 mice were immunized twice, 3 weeks apart. Whole heat-killed PR8 influenza virus was administered sc (20 µg) with CpG ODN sc (50 µg) or ec (250 µg). Sera were collected 4 weeks post-immunization. Abs titers in serially diluted sera were measured by indirect ELISA. (B, C) Graphical representation of the average ± SEM absorbance of IgG2b and IgG1 at O.D. 405 nm. Data summarize two independent experiments. Naïve (black, n = 4), sc PR8 (blue, n = 7) and sc PR8 + sc CpG (red, n = 7), and sc PR8 + ec CpG (green, n = 10). * p < 0.05.  54  Topical CpG adjuvant induces a bias towards CD4+ Th1 cells generation  3.2.2  Previous work has shown that CpG ODNs administered through the parenteral and the intranasal routes skew the immune response from Th2- to Th1-type T cell response. I further investigated whether this is also true when CpG ODN is administered topically on the skin as an adjuvant. Since the generation of Th1-type antibody IgG2 is promoted by the cytokine IFN-γ, I expected that CD4+ T cells would skew to Th1 cell differentiation. Mice were adoptively transferred with OT-II CD4+ T cells via the tail vein, immunized on the next day, and the spleen and the SLNs were analyzed 3 days post-immunization (Figure 3.3A). Cells were re-stimulated with PMA and ionomycin ex vivo to detect IFN-γ-producing cells. Increased frequencies of adoptively transferred OT-II cells that secrete IFN-γ were detected in the SLNs with a similar trend observed in the spleen (Figure 3.3B, C). Interestingly, topical compared to subcutaneous administration of CpG ODN further increased the frequencies of CD4+ Th1 cells by 2-fold and 3-fold in the spleen and the SLNs (Figure 3.3B, C), respectively, suggesting topical CpG ODN further enhances the production of functional Th1 CD4+ T cell compared to subcutaneous CpG ODN.  3.2.3  Topical route of CpG ODN delivery is superior to other routes in antigen-specific CTL generation  Our laboratory previously showed that topical CpG ODNs augment CTL priming (210). However, it is unclear whether the topical route indeed is the better route to enhance antigen55  specific CTL response. Hence, the effects of changing the route of CpG ODN delivery as an adjuvant on antigen-specific CTL induction were investigated. The route of CpG ODN administration varied while the route of antigen administration stayed the same, which is either subcutaneously or intramuscularly. Three routes of CpG ODN administration were tested with a prime-boost immunization regimen (Figure 3.4A): topical, subcutaneous, and intramuscular. Topical application compared to subcutaneous or intramuscular administration of CpG ODN increased the frequencies of OVA-specific CTLs amongst CD8+ T cells by 9-fold and 2.5-fold, respectively, as detected in the spleens (Figure 3.4B, C). The same trend was observed in the peripheral blood (Figure 3C), supporting the speculation that topical route is the more effective route to administer CpG ODN as an adjuvant to enhance CTL response. Toxicities caused by CpG ODNs include follicle microarchitecture disturbance as well as splenomegaly due to erythroid and myeloid expansion in red pulp (213). Thus, the spleens of immunized and untreated mice (naive mice) were weighted as an indirect method to evaluate systemic toxicity. In contrast to parenteral injection of CpG ODN, significant increase of spleen weight was not detected in mice with topical administration (Figure 3.5). Focusing on immunization using topical CpG ODN as a vaccine adjuvant with a subcutaneous protein-based antigen to enhance CTL response, I next confirmed that the CpG-motif specificity in the adjuvant effect of CpG ODN. Immunization with the control ODN without CpG motifs abrogated OVA-specific CTL generation to the level detected in mice immunized with OVA protein alone 5 days postimmunization in all the compartments tested, which include peripheral blood, the spleen and the SLNs (Figure 3.6).  56  Figure 3.3 CpG ODN induces proliferation of CD4+ Th1 T cells that secrete IFN-γ. (A) Schematic diagram of mice treatment and analysis schedule. OT-II CD4+ T cells were adoptively transferred into C57BL/6 mice on day 0 and then the mice were immunized on day 1. Mice were immunized with sc OVA protein with ec or sc CpG ODN. Spleens were collected 3 days post-immunization and re-stimulated ex vivo with PMA and ionomycin at 37°C for 4 hrs. IFN-γ production by the OT-II CD4+ T cells was detected intracellularly by flow cytometry. (B) Representative flow cytometric dot plots showing the gating of CD4+ cells that secrete IFN-γ amongst the OT-II cells. Cells were gated on OT-II cells with the Thy1.1 cell surface marker and then CD4 and IFN-γ double positive population. (C) The percentage of CD4+ cells that secrete IFN-γ amongst the OT-II cells are summarized in the scatter plots. Data summarize three independent experiments (n = 7-10). * p < 0.05; ** p < 0.01; *** p < 0.001.  57  Figure 3.4 Topical administration of CpG ODN is the optimal route to enhance CTL response. (A) Schematic diagram of immunization and analysis schedule. C57BL/6 mice were immunized twice, 7 days apart. Mice were immunized with sc OVA protein with or without CpG ODN administered sc, ec, or intramuscularly (im). Blood and spleen were collected 5 days postimmunization to enumerate the frequencies of OVA-specific CTLs in an acute immune response by flow cytometry. (B) Representative flow cytometric dot plots showing the gating of OVAspecific CTL amongst CD8+ T cells. Cells were gated on the B220- population and then CD8 and OVA-tetramer double positive population (C) The percentage of OVA-specific CTLs amongst CD8+ T cells are summarized in the scatter plots. Data summarize two to three independent experiments (n = 5-9). * p < 0.05; ** p < 0.01; *** p < 0.001. 58  Figure 3.5 Topical administration of CpG ODN does not cause splenomegaly. C57BL/6 mice were immunized twice, 7 days apart. OVA protein was injected sc or im with or without CpG ODN administered sc, ec or im. Spleens were collected 5 days post-immunization and weighed on a scale. Spleen weights are summarized in the scatter plots. Data summarize two independent experiments (n = 5-9). *** p < 0.001.  59  Figure 3.6 The adjuvant effect of CpG ODN is CpG-motif specific. (A) Schematic diagram of immunization and analysis schedule. C57BL/6 mice were immunized twice, 7 days apart. Mice were immunized with sc OVA protein with ec CpG ODN or control ODN. Peripheral blood, spleen and SLNs were collected 5 days post-immunization for flow cytometric analysis. (B) Representative flow cytometric dot plots showing the gating of OVAspecific CTLs. Cells were gated on the B220- population and then CD8 and OVA-tetramer double positive population. (C, D) The percentage of OVA-specific CTLs amongst CD8+ T cells and total number of OVA-specific CTLs in the spleens and SLNs are summarized in the scatter plots. Data summarize two independent experiments (n = 3-8). * p < 0.05; ** p < 0.01; *** p < 0.001. 60  3.2.4  Rapid prime-boost immunization regimen promotes optimal CTL response with topical CpG ODN  Proper spacing between vaccine doses is essential to elicit optimal immune responses (220). Therefore, a 0, 7 day and a 0, 21 day prime-boost immunization schedule (Figure 3.7A) were assessed for the optimal induction of antigen-specific CTLs. In a prime-boost immunization schedule 7 days apart, topical compared to subcutaneous delivery of CpG ODN increased OVAspecific CTL generation detected in the spleens 5 days post-immunization by over 3-fold (Figure 3.7B, C). In addition, the frequency of OVA-specific CTLs dramatically increased with a primeboost immunization schedule 7 days apart and was also seen with a prime-boost schedule 21 days apart (Figure 3.7B, C). The rapid prime-boost immunization regimen (0, 7 day schedule) led to stronger acute CTL response.  3.2.5  Single application of topical CpG ODN at the time of vaccine administration is sufficient to enhance CTL response  Although I have demonstrated that a prime-boost immunization strategy with topical CpG ODN as an adjuvant enhanced both humoral and cell-mediated immune responses, it would be ideal if a single dose of vaccine with the adjuvant was sufficient to induce protective immunity. However, since multiple topical applications of a TLR7 agonist further enhanced CTL response (206), I hypothesized that a single application of adjuvant may be insufficient to induce CTL response but multiple applications of topical CpG ODN may enable sufficient CTL response. Thus, I compared the effects of multiple applications of topical CpG ODN in a one-time 61  immunization regimen and the prime-boost immunization regimen. In the one-time immunization regimen, OVA protein was injected subcutaneously on day 0 with one time application of topical CpG ODN or three times in 3 consecutive days on day 0, 1 and 2 (Figure 3.8A). The same regimen was applied to the prime-boost regimen but a booster dose of antigen was also injected on day 7 with one time CpG ODN application or three times in 3 consecutive days on days 7, 8 and 9 (Figure 3.8A). Topical CpG ODN increased IFN-γ-producing CD8+ T cells 5 days post-immunization in both the one-time and the prime-boost immunization regimen (Figure 3.8B, C). However, 3 consecutive days of CpG ODN application did not further increase the frequency of CTLs than a single topical adjuvant application (Figure 3.8B, C). In addition, immunization with the prime-boost compared to a one-time regimen induced 2- to 3-fold higher fraction of OVA-specific CTLs that secrete IFN-γ independent of single dose or multiple doses CpG ODN topical administration (Figure 3B, C). Thus, single dose topical CpG ODN at time of antigen injection in a prime-boost immunization strategy is optimal to enhance CTL response.  62  Figure 3.7 Optimal interval between doses of a vaccine series to induce antigen-specific CTLs. (A) Schematic diagram of immunization and analysis schedule. C57BL/6 mice were immunized twice, 7 days (7d) or 21 days (7d) apart. Mice were immunized with sc OVA protein with ec or sc CpG ODN. Spleens were collected for flow cytometric analysis. (B) Representative flow cytometric dot plots showing the gating of OVA-specific CTLs. Cells were gated on B220population and then and CD8 and OVA-tetramer double positive population. (C) The percentage of OVA-specific CTLs amongst CD8+ T cells are summarized in the scatter plot. Data summarize three (7d) and two (21d) independent experiments (n = 4-11). * p < 0.05.  63  Figure 3.8 Multiple doses of CpG ODN do not further enhance antigen-specific CTL generation. (A) Schematic diagram of immunization and analysis schedule. C57BL/6 mice were immunized with sc OVA protein once in a prime-only regimen or twice, 7 days apart. CpG ODN was either administered ec once at time of OVA protein administration or three times in 3 consecutive days. Spleens were collected 7 days or 12 days post-initial immunization and re-stimulated ex vivo with OVA8 peptide at 37°C for 4 hrs. IFN-γ production by cells was detected intracellularly by flow cytometry. (B) Representative flow cytometric dot plots showing the gating of CD8+ T cells that secrete IFN-γ amongst B220- population. Cells were gated on B220- population and then CD8 and IFN-γ double positive population. (C) The percentage of IFN-γ-producing cells amongst CD8+ T cells are summarized in the scatter plot. Data summarize two independent experiments (n = 4). * p < 0.05; ns = not statistically significant.  64  3.2.6  Topical CpG adjuvant increases memory CD8+ T cell population  Memory T cells are T cells that have experienced antigen. Memory T cells respond to pathogen encountered in a faster and stronger manner in subsequent exposures due to their primed state (221). It is important for vaccines to generate memory T cells to provide long-term protection. There are two major subsets of memory T cells: central-memory and effectormemory T cells (222),(223). Central-memory T cells are CD127+CD62L+, have a higher proliferative potential than naive cells, and preferentially reside in lymphoid organs. Effectormemory T cells are CD127+CD62L-, have a faster effector function, and preferentially reside in non-lymphoid (stromal) tissues. Since higher frequencies of antigen-specific memory T cells correlate with enhanced protection (166),(224),(71), I investigated whether CpG ODN augments antigen-specific CD8+ memory T cell generation. Mice were immunized twice, 7 days apart, with OVA protein administered subcutaneously in combination with CpG ODN as an adjuvant administered either topically or subcutaneously. Peripheral blood samples were collected at 5 days and 4 weeks post-immunization to enumerate OVA-specific CD8+ effector and memory T cells, respectively (Figure 3.9A). As shown above, CpG ODN robustly increased OVA-specific CD8+ effector T cells 5 days post-immunization (Figure 3.9B). Topical compared to subcutaneous administration of CpG ODN increased the frequency of OVA-specific CD8+ effector T cells by 6-fold (Figure 3.9B). At 30 days post-immunization, levels of OVA-specific CD8+ memory T cells were 10-fold and 6-fold higher for mice immunized with topical and subcutaneous CpG ODN, respectively, compared to immunization without adjuvant (Figure 3.9B). However, no significant difference in the percentage of OVA-specific memory CD8+ T cells was detected between topical and subcutaneous administration of CpG ODN. Thus, topical 65  compared to subcutaneous administration of CpG ODN does not enhance the proportion of circulating memory T cells. To further assess the function of the antigen-specific memory T cells, I next assessed the proliferative ability of the induced OVA-specific CD8+ memory T cells after infection. The same immunization strategy was utilized using OVA protein as the antigen. The levels of OVAspecific CD8+ memory T cells in peripheral blood were determined 4 weeks post-immunization before and after systemic infection (pre- and post-infection) by Lm-OVA. As a positive control for OVA-specific CD8+ memory T cell generation, mice were immunized with ΔactA Lm-OVA. Strikingly, mice immunized with topical CpG ODN generated even higher level of OVA-specific CD8+ memory T cells compared to those immunized with ΔactA Lm-OVA detected in peripheral blood pre-infection (Figure 3.10A, B). All groups of immunized mice had an increased percentage of OVA-specific memory T cells in the peripheral blood post-infection, indicating proliferation of these memory T cells upon Lm challenge (Figure 3.10A, B). There was a trend of higher frequency of OVA-specific memory CD8+ T cells post-infection in mice immunized with topical compared to subcutaneous CpG ODN. In addition, I noticed that the memory T cells induced by immunization with ΔactA Lm-OVA proliferated more than with topical CpG ODN (Figure 3.10A, B). To further characterize these memory T cells into central-memory and effector-memory T cells, cells were stained with the cell surface markers CD62L and CD127. Effector-memory T cells were identified as CD127+CD62L- while central-memory T cells were CD127+CD62L+. Mice immunized with subcutaneous CpG ODN induced equivalent levels of effector-memory T cells and central-memory T cells as mice immunized with ΔactA Lm-OVA. Interestingly, there was a trend with topical CpG to promote the generation of higher proportion of effector-memory T 66  cells, which was not observed with subcutaneous administration of the adjuvant (Figure 3.11A, B).  67  Figure 3.9 Using CpG ODN as adjuvant promotes generation of antigen-specific effector and memory CD8+ T cell. (A) Schematic diagram of immunization and analysis schedule. C57BL/6 mice were immunized twice with sc OVA protein with or without CpG ODN administered sc or ec, 7 days apart. Peripheral blood samples were collected 5 days and 4 weeks post-immunization for flow cytometric analysis. (B) Graphical representation of the average ± SEM percent OVA-specific CTLs amongst CD8+ T cells on day 5 and day 30 post-immunization. Data summarize two independent experiments (n = 3-6). *** p < 0.001; ns = not statistically significant.  68  Figure 3.10 Antigen-specific CD8+ memory T cells induced by immunization with CpG ODN proliferate upon infection. C57BL/6 mice were immunized twice, 7 days apart, with sc OVA protein with or without CpG ODN administered sc or ec. Mice were then infected with Lm-OVA intravenously 4 weeks postimmunization. Peripheral blood samples were collected 1 day pre-infection and 3 days postinfection for flow cytometric analysis. (A) Representative flow cytometric dot plots showing the gating of OVA-specific memory CD8+ T cells. Cells were gated on B220- population and then OVA tetramer and CD8 double positive population. (B) The percentage of OVA-specific cells amongst CD8+ memory T cells in peripheral blood pre- and post-infection are summarized in the scatter plots. Data summarize two independent experiments (n = 3-6). * p < 0.05; ** p < 0.01; *** p < 0.001.  69  Figure 3.11 Topical CpG ODN may promote effector memory T cells generation. C57BL/6 mice were immunized twice, 7 days apart, with sc OVA protein with sc or ec CpG ODN. Mice were infected with Lm-OVA intravenously 4 weeks post-immunization. Peripheral blood samples were collected 1 day pre-infection and 3 days post-infection for flow cytometric analysis. (A) Representative flow cytometric zebra plots showing CD127+ CD62L- (effectormemory) and CD127+CD62L+ (central-memory) population in the upper left and upper right quadrants, respectively. Cells were gated on B220- population and then OVA tetramer and CD8 double positive population. Effector-memory and central-memory cells were identified by gating on CD127+CD62L- and CD127+CD62L+ populations, respectively. (B) The percentage of effector memory and central memory T cells pre- and post-infection are summarized in the scatter plots. Data summarize two independent experiments (n = 3-6). * p < 0.05; ** p < 0.01; ns = not statistically significant.  70  3.2.7  Topical CpG ODN improves the efficacy of protein-based vaccines against Lm  Whether the increased levels of antigen-specific CD8+ T cells correlate stronger in vivo protective immune resposnes was next investigated. CD8+ T cells are crucial in the immune response against the intracellular bacteria Lm (29),(32). Thus, the systemic Lm infection model was utilized to address this question. Using CpG ODN as an adjuvant, I tested both the one-time and the prime-boost immunization regimen in providing acute protective immunity against Lm (Figure 3.12A). Mice were immunized and then infected with Lm-OVA intravenously as indicated. The CFUs of Lm in infected spleens were enumerated. Significant reductions of bacterial burden were detected in the spleens when mice were immunized with topical CpG ODN with a prime-only immunization strategy. Approximately 3-log lower of bacterial counts were detected in mice immunized with topical CpG ODN compared to without the adjuvant (Figure 3.12B). In addition, further reduction of bacterial burdens were detected in mice immunized with a prime-boost regimen, which lowered bacterial burdens by 4-log in the spleens compared to mice immunized without topical CpG ODN (Figure 3.12B). Similar to the results of equivalent levels of CD8+ effector T cell induced after immunization with topical and subcutaneous CpG ODN, no further reduction of bacterial burden was detected in the spleens with topical compared to subcutaneous delivery of CpG ODN (Figure 3.12C). Using this Lm infection model, I also investigated whether increased antigen-specific memory T cells induced by CpG ODN correlate with strong long-lasting in vivo protective immunity. Mice were immunized with a prime-boost regimen and infected with Lm-OVA intravenously 4 weeks after (Figure 3.13A). Bacterial burdens were enumerated in the spleens 3 days post-infection by plating the spleen homogenates (Figure 3.13A). Although it was less 71  dramatic than with the enhanced acute immune response provided by CpG ODN, bacterial burdens were also significantly reduced in mice immunized with CpG ODN as the adjuvant compared to mice immunized without CpG adjuvant (Figure 3.13B). Topical but not subcutaneous administration of CpG ODN reduced Lm CFU counts in the spleens by approximately 2-fold (Figure 3.13B). 3.2.8  Topical CpG ODN improves the efficacy of protein-based vaccines against influenza A virus  An intranasal influenza infection model was utilized to assess the in vivo long-term protective ability of using topical CpG ODN as adjuvant against viral infections. Unlike the Lm infection model, the influenza infection model does not limit the assessment of the adjuvant effect to antigen-specific CD8+ T cells but can also account the effects of CD4+ Th1 cells and humoral immunity. In addition, the protection provided by topical and subcutaneous CpG ODN at the site of infection can be addressed. Mice were immunized with a prime-boost regimen, 7 days apart, and then infected with PR8-OVA intranasally 4 weeks post-immunization. Viral burdens were measured in the lungs 3 days post-infection by plaque assay (Figure 3.14A). It is clear that using CpG ODN as the adjuvant decreased viral titers in the lungs. Immunization with topical CpG ODN reduced viral titers by 27-fold while subcutaneous CpG ODN reduced viral titers by 4-fold compared to immunization without CpG adjuvant (Figure 3.14B). Topical application of CpG ODN as the adjuvant compared to subcutaneous administration provided stronger long-term protective effect against intranasal influenza infection as indicated by further reduction of viral titers in the lungs. 72  Figure 3.12 Topical and subcutaneous CpG ODN enhance protective immunity against systemic Lm infection. (A) Schematic diagram of immunization, infection and analysis schedule. C57BL/6 mice were immunized with sc OVA protein with or without CpG ODN administered sc or ec in a primeonly or prime-boost regimen 7 days apart. Mice were then infected with Lm-OVA via the tail vein 7 days or 5 days post-immunization as indicated. Bacterial burdens in the spleens were enumerated 3 days post-infection. (B, C) CFU counts are summarized in the scatter plots with log10 scale y-axis. Data summarize three independent experiments (n = 3-10). * p < 0.05; ** p < 0.01; *** p < 0.001; ns = not statistically significant.  73  Figure 3.13 Using topical CpG ODN as adjuvant provides long-term protective immunity against systemic Lm infection. (A) Schematic diagram of immunization, infection and analysis schedule. C57BL/6 mice were immunized twice, 7 days apart, with sc OVA protein with or without CpG ODN administered sc or ec. Mice were then infected with Lm-OVA via the tail vein 4 weeks post-immunization. Bacterial burdens in the spleens were enumerated 3 days post-infection. (B) CFU counts are summarized in the scatter plot with log10 scale for the y-axis. Data summarize three independent experiments (n = 3-12). ** p < 0.01.  74  Figure 3.14 Topical and subcutaneous CpG ODN enhance long-term protective immunity against intranasal influenza infection. (A) Schematic diagram of immunization, infection and analysis schedule. C57BL/6 mice were immunized twice, 7 days apart, with sc OVA protein with or without CpG ODN administered sc or ec. Mice were then infected with PR8-OVA intranasally 4 weeks post-immunization. (B) Viral titers in the lungs were enumerated 3 days post-infection. Viral titers are summarized in the scatter plot. Data summarize two independent experiments (n = 9-10). * p < 0.05.  75  3.3  3.3.1  Discussion  Data summary  In this study, the potential of utilizing a TLR9 agonist, CpG ODN 1826, as vaccine adjuvant to improve both humoral and cell-mediated immune responses was explored. I focused on investigating the adjuvant effects of topical versus subcutaneous administration of CpG ODN. CpG ODN 1826 was selected for experiments because it is a class B CpG ODN, potent activator of B cells (127),(128),(129). The phosphorothioate backbone is nuclease-resistant gives it a longer half-life (48 hrs) in vivo (246). Also, class B CpG ODN has higher cellular intake property and greater affinity for TLR9 binding (246). The best route to administer CpG ODN as adjuvant for protein-based vaccines was found to be the topical route on the skin overlying the site of antigen administration either subcutaneously or intramuscularly. Topical CpG ODN in combination with subcutaneous OVA protein as antigen rapidly elicited robust acute OVAspecific T cell responses. Higher frequencies of OVA-specific CTLs and IFN-γ producing CD4+ T cells were detected. Long-term antigen-specific immune responses were achieved including increased level of antigen-specific (OVA or influenza) IgG Abs as well as CD8+ memory T cells. An increased proportion of effector-memory T cells was detected with immunization using topical CpG ODN compared to subcutaneous delivery of the adjuvant. In contrast to parenteral administration of CpG ODN that led to the enlargement of the spleen, splenomegaly was not detected in mice immunized with topical CpG ODN. Based on these results, the effective strategy to administer topical CpG ODN was then determined. Single dose of topical CpG ODN adjuvant at the time of antigen administration with a prime-boost regimen, 7 days apart, was the 76  better immunization strategy to administer CpG ODN 1826 as the adjuvant. Next, in vivo infection models were used to validate the ability of topical and subcutaneous CpG ODN to confer systemic and mucosal protective immunity. Topical and subcutaneous deliveries of CpG ODN as vaccine adjuvant provided equivalent level of acute protection against systemic Lm infection and the intranasal influenza infection model. Interestingly, topical administration of CpG ODN conferred stronger long-term protective immunity.  3.3.2  Strategy to deliver CpG ODN as adjuvant  Separate administration of vaccine and adjuvant at immunization has several advantages over standard co-administration. These advantages include: being more efficacious in terms of eliciting immune responses, increasing safety and tailoring adjuvant use to specific human populations. The structural and immune properties of the skin enable a split adjuvant and antigen vaccination strategy first demonstrated by Guebre-Xavier with the enterotoxin patch (225),(202), showing relevance of this approach in humans. TLR7 agonists were also studied as topical adjuvants (205),(226),(227). However, safety concerns remain with handling enterotoxins and even limited amounts of topical TLR7 agonists may induce undesirable systemic symptoms and side effects such as systemic cytokine storms (206),(228). CpG ODNs have been explored for immunotherapeutic uses, including the reduction of allergic responses and the enhancement of cancer therapy and innate immune responses (229). They have also been extensively studied for the ability to improve vaccine efficacy. These studies have always used co-administration of antigen and adjuvant. Previous studies showed CpG ODN injections led to systemic responses such as splenomegaly and disrupted follicle 77  architecture in the spleens of the mice (230),(213). CpG ODNs are larger molecules that have less penetration after topical application than TLR7 agonists such as imiquimod. Thus, topical administration of CpG ODNs perhaps can limit the toxicity to local injection reactions and avoid systemic side effects. Current protein vaccines require booster doses to achieve effective immunity. Additionally, current vaccines do not often induce a protective CD8+ T cells threshold with a single immunization (231). Thus, a prime-boost immunization regimens are required for long-lasting protection (232). Since proper spacing between doses of a given vaccine series is essential for optimal immune responses, I tested two prime-boost immunization regimens: one with 7 days apart and another one with 21 days apart to optimize the strategy to induce a better CTL response. These intervals were chosen because a 0, 7 day immunization schedule is common in murine cancer vaccine protocols while a 0, 21 day immunization schedule is the standard timing for antibody production. I found that a prime-boost immunization schedule 7 days apart is superior to 21 days apart as higher frequency of OVA-specific CTL was detected in the spleens 5 days post-immunization. The advantage of topical administration of CpG ODN as the adjuvant is that, at least in the mouse model, a rapid prime-boost immunization schedule of 7 days instead of the standard 3 weeks apart is sufficient to enhance cell-mediated immune responses. The ability of topical CpG adjuvant to enhance rapid induction of robust protective immunity is theoretically desirable to combat infection pandemics or bioterrorist attacks. Although multiple applications of topical adjuvants may further enhance CTL responses (206), this is not the case when using topical CpG ODN as the adjuvant. Further benefit in CTL generation was not detected with multiple applications of CpG ODN. A single dose of topical CpG ODN at time of protein antigen administration in a prime-boost regimen 7 days apart is 78  optimal for antigen-specific CTL generation. More importantly, CpG ODN administered topically does not induce splenomegaly verifying their safety, unlike that noted with TLR7 agonists.  3.3.3  Topical CpG adjuvant enhances vaccine immune responses  Topical administration of CpG ODNs as adjuvants with co-administered parenteral antigens enhances antigen-specific CTL response (210),(211). In addition, studies in non-human primates demonstrate that CpG ODNs are as effective as TLR7 adjuvants in their ability to induce long-lasting protective immunity but without the induction of associated skin inflammation (233). Here, I have expanded on previous observations from our laboratory and demonstrated that split skin administration of CpG ODN 1826 as a vaccine adjuvant with protein antigen is a safe and effective method to improve vaccine responses. Existing vaccine design has focused on the induction of humoral immune responses. Pathogen-specific Abs have important roles in preventing pathogen invasion and in providing long-term protection against infection. Using OVA protein and whole heat-killed influenza as model protein antigens, the effect of topical compared to subcutaneous delivery of CpG ODN 1826 on Ab production was compared. Surprisingly, topical CpG ODN was shown to be superior to subcutaneous administration of the adjuvant in generating antigen-specific Th1-type and Th2 type IgG antibodies detected in sera. Thus, CpG ODN can be used as adjuvant to enhance Ab production to subcutaneous protein or subunit antigens. Many studies showed the bias of Th1 CD4+ T cells with parenteral injection of CpG ODN (see section 1.3). Whether this is also the case with topical delivery of CpG ODN or does the topical route further promote CD4+ Th1 cell 79  generation was determined. The OT-II cell adoptive transfer system was used to monitor the differentiation of antigen-specific CD4+ T cells into IFN-γ producing CD4+ Th1 cells. In parallel with the effect on humoral response, topical compared to subcutaneous CpG ODN induced greater frequency of antigen-specific CD4+ Th1 cells that secrete IFN-γ. CTL response is crucial in intracellular bacterial and viral pathogen control and clearance. However, cell-mediated immunity has only recently been a focus for vaccine development. Memory is unique in adaptive immunity, allowing faster and higher response against reencountered pathogens. Memory T cells have a higher affinity for antigens and a decreased threshold for activation (234). Antigen-specific T cells pass through a contraction phase as memory T cells are generated. Memory T cells are crucial to combat reencountered pathogens while chronic infections are often due to suboptimal or exhausted T cell function (235). I demonstrated that topical CpG ODN not only improved antigen-specific humoral immune responses but also CTL responses. Topical was superior to subcutaneous administration of CpG ODN as the adjuvant at rapidly initiating robust antigen-specific CTL generation. I also demonstrated that topical or subcutaneous CpG ODN induced antigen-specific memory T cells forming a stable pool of circulating population detected at 4 weeks post-immunization. There is evidence that memory T cells rapidly proliferate on antigenic recall to generate a large pool of secondary effectors. Since the ability of antigen-specific memory T cells to proliferate upon infection is vital to protection, the proliferative abilities of these T cells induced by immunization with topical and subcutaneous CpG ODN were assessed. Increased frequencies of these T cells after systemic Lm infection indicated their ability to proliferate upon reencounter of pathogens. Interestingly, in contrast to pre-infection, equivalent frequencies of these T cells were detected post-infection in peripheral blood between topical and subcutaneous CpG ODN. This may be due 80  to the fact that a large population of these T cells had already migrated into the infected spleen and livers to confer protection and these cells were therefore less present in the peripheral blood (236),(237). Based on these data, I speculate that memory T cells induced by topical CpG ODN may be better at migrating into stromal tissues including the potential sites of infection. Two types of CD8+ memory T cells with differential proliferative and protective potential have been identified based on surface expression of CD62L: CD62L+ central-memory T cells with low proliferative potential and CD62L- effector-memory T cells with high proliferative potential (238). However, the relationship between proliferative and protective potential remains unclear. Both central- and effector-memory T cells may be crucial to combat pathogens, in which they act at different times following infection (238). Effector-memory T cells may act rapidly while central-memory T cells may expand and act in concert with effector-memory T cells at a later time during infection. I found that topical administration of CpG ODN resulted in a higher proportion of effector-memory cells compared to subcutaneous administration. I speculate that a bias towards effector-memory cell generation using topical CpG ODN as vaccine adjuvant may provide more rapid immunity to control and to clear the pathogens in the body.  3.3.4  Topical CpG ODN improves protection against intracellular bacterial and viral infections  Based on the findings summarized above, I hypothesized that enhancement of antigenspecific humoral and cell-mediated immune responses detected using topical CpG ODN as a vaccine adjuvant correlate with protective immunity against infection. The purpose of prophylactic vaccines for infectious diseases is to educate the immune system prior to actual 81  infections so that it can quickly respond when pathogens invade the body. Using two different murine infection models, the effectiveness of topical CpG ODN in conferring protection against infections caused by intracellular bacteria and viruses has been demonstrated. T cells are required for protection against Lm infection (239),(240). Topical route of CpG ODN administration conferred both acute and long-term protection against systemic Lm infections, with a stronger effect using the prime-boost immunization regimen compared to a one-time immunization regimen. Repeated administration of topical CpG ODN without antigen decreased bacterial load slightly, suggesting that topical application of the adjuvant can have a broad, antigen non-specific effect, as noted following intraperitoneal administration (241), which may be due to the effect of secreted IL-12 and IFN-γ. Since topical and subcutaneous administration of CpG ODN as adjuvant induced equivalent level of CD8+ memory T cells, I speculated that this correlated to equivalent level of long-term protective immunity in vivo. Interestingly, administration of CpG ODN subcutaneously only conferred acute but not longterm protection in this model, suggesting topical CpG ODN may be better at inducing memory T cell responses. The systemic Lm-OVA infection model is limited by the dominating CD8+ T cell response for bacterial control and clearance while Ab response only plays a minor role. On the other hand, influenza viruses cause respiratory infections, which require both humoral and cell-mediated immunity for viral control and clearance (242),(243). In mice, this route of infection can be mimicked by intranasal infection. In addition, protection against mucosal infection using topical CpG ODN in combination with protein-based antigen remains unknown. Therefore, an intranasal influenza model was utilized to assess the adjuvant effects of topical and subcutaneous CpG ODN. In parallel to the Lm model, topical administration of CpG ODN as an adjuvant provided 82  stronger long-term protection against influenza A infection. Data again suggest that topical CpG ODN-induced memory T cells either have greater migratory ability to the site of infection or the memory T cells are already resided at the site prior to infection to confer protection. In this chapter, I have demonstrated the promising potential of using topical CpG ODN as a prophylactic instead of a therapeutic vaccine adjuvant. These findings provide solid pre-clinical data that support the initiation of human clinical trials using topical CpG ODN as a vaccine adjuvant to improve the effectiveness of existing vaccines.  83  CHAPTER 4: THE MECHANISMS WHEREBY TOPICAL CPG ODN RESULTS IN IMPROVED VACCINE OUTCOMES  4.1  Introduction  In Chapter 3, an effective immunization strategy using CpG ODN as vaccine adjuvant was determined. In addition, I demonstrated that CpG ODN 1826 is able to enhance both acute and memory immune responses that are protective against Lm and influenza infections. Little is known about the topical route, also called transcutaneous, of immunization. It is believed that DCs in the skin take up the vaccine immunogen, process it and present it to T and B cells in the SLNs (244). It has been revealed that CpG ODNs can activate different immune cells including monocytes, DCs, B cells, macrophages, NK cells, NKT cells and keratinocytes (132),(245),(246),(247),(248),(249),(250),(251). It is thought that immune effects of CpG ODNs in humans result from activating DCs and B cells to increase the co-stimulatory molecule expressions, cytokine secretion, Ig secretion, Ig class-switching and B cell survival. CpG ODNs also promote antigen cross-presentation by DCs (252) and LC mobilization by decreasing the expression of E-cadherin and α6 integrin, molecules that retain DCs in the skin (251). In turn, activated DCs and B cells directly or indirectly stimulate other immune cells to induce adaptive immune responses such as T cell proliferation. CpG ODNs activate DCs and B cells, bypassing the help requirement from Th cells and the CD40L-CD40 interactions to induce CTL responses (250). Keratinocytes can secrete many cytokines (253) and some studies suggest that keratinocytes cross-talk with other immune cells to respond to CpG ODNs (251).  84  Although the molecular signaling pathways of CpG ODNs have been made clearer recently (247), the mechanisms of how CpG ODNs administered topically enhance adaptive immunity remain unclear. Hence, this thesis chapter explores the mechanisms of how topical CpG ODN 1826 confers adjuvant effects to enhance protective adaptive immunity. In particular, whether skin stromal cells and/or hematopoietic cells are necessary and what influences CpG ODN 1826 have on keratinocytes, SLNs and T cells were investigated.  4.2  4.2.1  Results  TLR9 deficiency in the mouse abrogates the adjuvant effect of topical CpG ODN  Previous studies show that uptake of CpG ODNs by cells are CpG motif-independent and some studies suggest that CpG ODNs induce TLR9-independent immune effects (254),(255). Thus, I first wished to confirm that the adjuvant effect of CpG ODN in the augmentation of an antigen-specific CTL responses signals through TLR9. Wild-type (WT) and TLR9 -/- (TLR9 KO) mice were immunized with subcutaneous OVA protein and topical CpG ODN with the prime-boost regimen. Mice were then infected with Lm-OVA intravenously 5 days postimmunization. Bacterial burdens were enumerated in the spleens 3 days post-infection. Bacterial loads dramatically reduced in WT mice but not in TLR9 KO mice, confirming the adjuvant effect of topical CpG ODN is TLR9-dependent.  85  Figure 4.1 Topical CpG adjuvant effect in CTL response is TLR9-dependent. (A) Schematic diagram of immunization, infection and analysis schedule. C57BL/6 or TLR9 KO mice were immunized twice, 7 days apart, with sc OVA protein with ec CpG ODN. Mice were then infected with Lm-OVA via the tail vein 5 days post-immunization. Bacterial burdens in the spleens after infection were enumerated 3 days post-infection. (B) CFU counts are summarized in the scatter plot with log10 scale on the y-axis. Data summarize two independent experiments (n = 5). ** p < 0.01.  86  4.2.2  TLR9 deletion in hematopoietic cells abrogates the generation of CD8+ T cells  CpG ODNs activate many hematopoietic immune cells (101),(102),(109),(110),(111),(112),(113), which include the following: monocytes, DCs, B cells, macrophages, NK cells and NKT cells. The necessity of hematopoietic cells in the adjuvant effects of topical CpG ODN was determined by constructing bone marrow chimeric mice. WT mice were the recipients while the TLR9 KO mice were the bone marrow donors, creating mice with TLR9-deficient hematopoietic cells but normal stromal cells (Figure 4.2B). Mice were then treated with ultraviolent light 2 weeks after bone-marrow transplantation to deplete radioresistant cutaneous DCs to generate complete chimeras (256),(257). Chimerism of the grafted mice was confirmed 8 weeks post-transplantation by flow cytometry (Figure 4.2A). The percentage of recipient and donor cells in the peripheral blood and the SLNs as well as the skin were determined using the CD45.1 and CD45.2 cell surface markers, respectively. In peripheral blood and the SLNs, at least 80% of cells were of donor phenotype (data not shown). In the skin, at least 80% of cells were of donor phenotype except one mouse that was 40% of donor phenotype (data not shown). Chimeric mice were then immunized with subcutaneous OVA protein with topical CpG ODN and the fraction of OVA-specific CTLs amongst CD8+ T cells in the spleens were determined 5 days post-immunization (Figure 4.2A). OVA-specific CD8+ T cell priming was totally abrogated in mice with TLR9-deficient hematopoietic cells. The frequency of OVA-specific CTLs decreased by 19-fold in chimeric mice with TLR9-deficient hematopoietic cells compared to chimeric mice with WT hematopoietic and stromal cells, which was close to the baseline level of untreated mice (Figure 4.2C). Therefore, TLR9 expression in  87  hematopoietic cells is absolutely required for the enhanced CTL response induced by topical CpG ODN.  4.2.3  Mice lacking TLR9 expression in stromal cells have decreased CD8+ T cell priming following topical CpG ODN administration  The contribution of stromal cells such as keratinocytes in immune responses have only recently been recognized. The necessity of these cells in the adjuvant effect of topical CpG ODN with subcutaneous protein antigen remains unknown. Thus, the contribution of TLR9 signaling in skin stromal cells in the adjuvant effects of topical CpG ODN was determined also by constructing bone marrow chimeric mice. All the experimental procedures and mice treatments were the same as stated above. However, opposite to the chimeras generated above, TLR9 KO mice were the recipients while the WT mice were the bone marrow donors, creating mice with TLR9-deficient stromal cells but normal hematopoietic cells (Figure 4.2B). The percentage of OVA-specific CTLs amongst CD8+ T cells decreased by 3-fold in chimeric mice with TLR9deficient stromal cells compared to chimeric mice with WT hematopoietic and stromal cells (Figure 4.2C). OVA-specific CTL priming was diminished but not completely abolished in mice with TLR9-deficient stromal cells, suggesting TLR9 expression in stromal cells also contributes to the adjuvant effects of topical CpG ODN in enhancing CTL response.  88  Figure 4.2 Necessity of TLR9 expression in hematopoietic and stromal cells for the enhancement of CD8+ T cell response induced by topical CpG ODN. (A) Schematic diagram of bone marrow transplantation, immunization and analysis schedule. Recipient mice were lethally irradiated with gamma ray and injected with donor bone marrow intravenously on day 0. Mice were subjected to ultraviolet light treatment at 2 weeks posttransplantation and the chimeric mice were allowed to reconstitute for 8 weeks under a pathogenfree environment. Chimeras were checked for donor cell reconstitution and then immunized with OVA protein and ec CpG ODN respectively. Spleens were collected for flow cytometric analysis 5 days post-immunization. (B) The different combinations of chimeras, which were deficient in TLR9 expressions in hematopoietic, stromal cells or neither. (C) The percentage of OVAspecific CD8+ T cells are summarized in the scatter plots with log10 scale on the y-axis. Data summarize three (top panel) and four (lower panel) experiments ( n = 2-20). * p < 0.05; ** p < 0.01. 89  4.2.4  Activation of keratinocytes by topical CpG ODN  Keratinocytes consists of over 90% of the stromal cells in the skin. Recent studies demonstrated that keratinocytes are immuno-competent and can secrete a plethora of cytokines. IL-1, IL-6, IL-12 and TNFα can be induced by intradermal injection of CpG ODN (258). Both human and murine keratinocytes express TLR9 (121),(111). However, whether keratinocytes express TLR9 and respond to topical CpG ODN remains unclear. To address this question, epidermal sheets of mice treated with or without topical CpG ODN were analyzed 2 hrs after treatment by real-time PCR (Figure 4.3A). The relative TLR9 mRNA level in keratinocytes were compared between the different treatments. Since all the mice treated with topical CpG ODN were tape-stripped, mice subjected to tape-stripping or topical CpG ODN administration alone acted as controls to the effect of topical CpG ODN. A trend of relatively higher level of TLR9 mRNA was detected in the epidermis of mice that were tape-stripped and treated with topical CpG ODN compared to mice with single treatment (Figure 4.3B), suggesting keratinocytes are not inactive but are responsive to topical CpG ODN.  90  Figure 4.3 Topical CpG ODN up-regulates TLR9 mRNA expression on the epidermis. (A) Schematic diagram of skin treatment and analysis schedule. Dorsal back skins of C57BL/6 mice were shaved and either tape-stripped, applied topical CpG ODN, or both. Hair was removed from the treated areas of the skin using hair remover and RNAs were extracted for realtime PCR 2 hrs post-treatment. (B) Average ± SEM relative fold changes of TLR9 mRNA are summarized in the bar graph. Data summarize three independent experiments (n = 4).  91  4.2.5  Mast cells do not contribute to the topical CpG ODN-induced enhancement of CTL response  Mast cells are long-lived immune sentinel cells that are well-known for their innate effector cell function and their roles in allergies (104). They can express TLRs including TLR7 and TLR9 and reside at potential infection sites such as the skin and the airways. There are great similarities between human and murine mast cells in terms of anatomic distribution, morphology and mediators they secrete (259). Masts cells are crucial to DC migration to the SLNs and are required for peptide-specific CTL response in transcutaneous immunization (107),(260),(108). Nonetheless, the contribution of mast cells to the adjuvant effect of topical CpG ODN remains unknown. To determine the contribution of mast cells in antigen-specific CTL response enhanced by topical CpG ODN, antigen-specific CTL priming was compared in mast cellsdeficient mice KitWKitW-v and the congenic +/+ mice. Mice were primed and boosted 7 days apart with subcutaneous OVA with topical CpG ODN (Figure 4.4A). When mice were immunized with topical CpG ODN, the percentage of OVA-specific CTLs amongst CD8+ T cells were equivalent between mast cell-deficient and the congenic +/+ mice, indicating mast cells do not contribute to the CpG ODN-induced enhancement of antigen-specific CTL response (Figure 4.4B, C).  92  Figure 4.4 Mast cells do not contribute to the adjuvant effect of topical CpG ODN in antigen-specific CTL generation. (A) Schematic diagram of skin treatment and analysis schedule. C57BL/6 mice were immunized twice, 7 days apart, with sc OVA protein and CpG ODN sc or ec. Spleens and the SLNs were collected 5 days post-immunization for flow cytometric analysis. (B) Representative flow cytometric dot plots showing the percentage of OVA-specific CTLs amongst CD8+ T cells. Cells were gated on B220- cells and then OVA-tetramer and CD8 double positive population. (C) Percents OVA-specific CTLs amongst CD8+ T cells are summarized in the scatter plots. Data summarize two to three independent experiments (n = 1-9). ns = not statistically significant.  93  4.2.6  Internalization of CpG ODN by CD11c+ DCs detected in the SLNs  CpG ODNs promote LC mobilization by decreasing E-cadherin and α6 integrin that retain DCs in the skin (251). After barrier disruption and topical CpG ODN administration, CpG ODN had penetrated through the skin by 1 hr post-treatment (261). Hence, I hypothesized that CpG ODN reach the SLNs rapidly via DCs after topical administration. The SLNs from mice treated with FITC-labeled CpG ODN either topically or subcutaneously were collected 48 hrs after treatment (Figure 4.5A). Serial sections of the SLNs were cut at a thickness of 6 µm, mounted with DAPI as the nucleus marker and observed under a fluorescent microscope. No green fluorescence was observed in the SLNs of untreated mice while it is apparent that FITC-labeled CpG ODNs were observed as green fluorescence in the SLNs 48 hours after treatment despite the routes of administration (Figure 4.5B). In addition, imaging flow cytometry was performed to determine internalization of CpG ODNs by CD11c+ cells detected in the SLNs 48 hrs after topical or subcutaneous CpG ODN administration. Gated on 7AAD- CD11c+ cells, CD11c cell surface staining was observed at the membranes of cells in red while FITC-CpG ODNs were observed inside the CD11c+ cells in green (Figure 4.5C), suggesting CpG ODNs were internalized by CD11c+ cells and present in the SLNs after 48 hrs of treatment.  94  Figure 4.5 Topical or subcutaneously administration of CpG ODN internalized by CD11c+ cells is detected in the SLNs 48 hrs post-treatment. (A) Schematic diagram of skin treatment and analysis schedule. Dorsal back skins of C57BL/6 mice were tape-stripped and treated with FITC-CpG ODN sc or ec. SLNs were collected for analysis 48 hrs after treatment. (B) Representative cross-section images of the SLNs of two independent experiments: Blue is DAPI stain for nuclei and green is the FITC-CpG ODN. (C) Representative images of two independent experiments of the internalization of FITC-CpG ODNs (green) into CD11c+ cells (red) was measured by ImageStream and analyzed using the IDEAS software. 95  4.2.7  Topical CpG ODN modulates the microenvironment of the SLNs  Although it is well established that CpG ODNs affect DCs and other immune cells, the impact of CpG ODNs on the microenvironment of SLNs has not been studied. Based on the findings in chapter 3 that using topical and subcutaneous administration of CpG ODN as adjuvant induced different levels of antigen-specific effector and memory T cell generation and protective immunity, I hypothesized that the various routes of CpG ODN administration differentially modulate the cytokine and chemokine gene expression in the SLNs. In order to test this hypothesis, a PCR array was performed to profile the expression of a panel of cytokine and chemokine genes in the SLNs. The cytokine and chemokine gene profiles of the SLNs 24 hrs (Figure 4.6A) and 72 hrs after immunization were compared between mice immunized with OVA protein alone with or without CpG ODN administered topically or subcutaneously. As predicted, many genes are up-regulated while a few genes being down-regulated at least ± 2-fold as shown in the scatter plots (Figure 4.6B) and in the tables (Table 4.1, 4.2). To further compare significant changes in cytokine and chemokine gene expression between topical and subcutaneous administration of CpG ODN, a volcano plot was used to display the data (Figure 4.6C). Cxcl3, Ifnγ and Il11 were significantly over-expressed by 5- to 7-fold while Cxcll3 and Il7 were under-expressed by 4- to 6-fold in the SLNs when comparing topical to subcutaneous administration of CpG ODN (Figure 4.6C). At 72 hrs, there was no significant difference in cytokine and chemokine mRNA levels between topical and subcutaneous administration of CpG ODN (data not shown). Thus, topical and subcutaneous route of CpG ODN administration  96  differentially modulate the expression of cytokines and chemokines in the SLNs detected at 24 hrs post-immunization.  97  Figure 4.6 The route of CpG ODN administration differentially modulates the gene expression of cytokines and chemokines in the SLNs. (A) Schematic diagram of mouse immunization and analysis schedule. Mice were immunized with sc OVA protein alone with or without CpG ODN administered sc or ec. The SLNs were collected 24 hrs post-immunization for PCR array. (B) The cytokines and chemokines that showed gene expression changes greater or lower than at least ± 2-fold are shown above (green) or below (red) the boundary lines in the scatter plots, respectively. (C) A volcano plot displaying statistical significance (p < 0.05) on the y-axis versus fold-changes on the x-axis. Genes that 98  were significantly over- (red) or under-expressed (green) are shown left of and right of the boundary lines, respectively. The genes that had significant fold changes of at least ± 2-fold are highlighted in rectangular boxes. Data summarize three independent experiments (n = 3).  99  Table 4.1. Over-expressed genes detected in the SLNs 24 hrs post-immunization. sc CpG ODNa Vs No CpG  Gene Symbol Ccl2 Ccl3 Ccl4 Ccl7 Csf3 Cxcl11 Cxcl9 Ifng Il1rn  Fold Regulation 4 7 6 4 4 6 7 5 7  ec CpG ODNb Vs No CpG  Gene Symbol Ccl3 Ccl4 Ccl7 Csf3 Cxcl10 Cxcl11 Cxcl3 Cxcl9 Ifng Il10 Il11 Il1b Il1rn Il27 Il6 a Mice were immunized with sc OVA protein and sc CpG ODN. b Mice were immunized with sc OVA protein and ec CpG ODN. p-value 0.0001 0.0043 0.0020 0.0029 0.0369 0.0120 0.0000 0.0006 0.0006  Fold Regulation 19 23 6 15 6 8 7 11 32 5 6 5 18 5 49  p-value 0.0000 0.0298 0.0092 0.0033 0.0120 0.0009 0.0078 0.0188 0.0080 0.0057 0.0158 0.0198 0.0010 0.0004 0.0439  Table 4.2. Under-expressed genes detected in the SLNs 24 hrs post-immunization.  sc CpG ODNa Vs No CpG Gene Symbol  Fold Regulation  ec CpG ODNb Vs No CpG  Gene p-value Symbol Cxcl12 None Il4 Il7 a Mice were immunized with sc OVA protein and sc CpG ODN. b Mice were immunized with sc OVA protein and ec CpG ODN.  Fold Regulation -7 -4 -15  p-value 0.0003 0.0273 0.0064  100  4.2.8  Topical CpG adjuvant increases P-selectin ligand and E-selectin ligand expressing CD4+ T cells  Topical and subcutaneous deliveries of CpG ODN as adjuvant differentially modulate the microenvironment of SLNs as shown above. I also demonstrated that using CpG ODN as adjuvant increased antigen-specific Ab and CD8+ T cell generation. However, the specific effects of the altered microenvironment of SLNs have on the antigen-specific T cells remain unclear. Therefore, the impact of topical and subcutaneous CpG ODN on T cells was investigated. In particular, the expression of tissue-homing molecules on T cells in the SLNs were determined. The OT-II (OVA-specific CD4+ T cells) instead of the OT-I (OVA-specific CD8+ T cells) adoptive transfer system was used because tissue-homing molecules could not be analyzed when antigen-specific T cells are undetected without TLR9 stimulation as with the OT-I adoptive transfer system. OT-II CD4+ T cells were adoptively transferred into recipient mice via the tail vein (Figure 4.7A). These mice were immunized with OVA protein subcutaneously with or without CpG ODN administered topically or subcutaneously. The SLNs were collected 3 days post-immunization for flow cytometric analysis (Figure 4.7A). Prominently, topical CpG ODN compared to subcutaneous CpG ODN administration significantly increased the frequency of OT-II cells expressing P-selectin ligand and E-selectin ligand (Figure 4.7B, C). On the contrary, lower frequency of OT-II cells expressing L-selectin (CD62L) was detected in the SLNs of mice immunized with topical compared to subcutaneous CpG ODN (Figure 4.7B, C). These data indicate that topical compared to subcutaneous administration of CpG ODN is better at promoting tissue-homing molecule expression while decreasing lymph node-homing molecules expression on activated T cells in the SLNs. 101  Figure 4.7 Topical route of CpG ODN administration is better at inducing the expression of tissue-homing molecules on activated T cells in the SLNs. (A) Schematic diagram of OT-II T cell adoptive transfer, immunization and analysis schedule. C57BL/6 mice were adoptively transferred with 2 x 106 CD4+ T cell enriched OT-II cells intravenously. C57BL/6 Mice were then immunized twice, 7 days apart, with sc OVA protein with or without CpG ODN administered sc or ec. The SLNs were collected for flow cytometric analysis 3 days post-immunization. (B) Representative flow cytometric dot plots of cells that express L-selectin (CD62L), P-selectin ligand (PSL), or E-selectin ligand (ESL). Cells were gated on CD4 and Thy1.1 double positive population. (C) The percentage of single positive of CD62L+, PSL+, or ESL+ T cells amongst OT-II cells in the SLNs are summarized in the scatter plots. Data represent two independent experiments (n = 6). * p < 0.05; ** p < 0.01.  102  4.2.9  T cell migration from the SLNs to the skin is required to induce protein contact hypersensitivity  Topical CpG ODN differentially altered the microenvironment of the SLNs when compared to subcutaneous CpG ODN. Topical administration of CpG ODN also resulted in increased frequencies of activated antigen-specific CD4+ T cells that express tissue-homing molecules. With the ultimate aim being to assess the migratory ability of these T cells expressing tissue-homing molecules induced by topical CpG ODN, I first determined whether a mouse protein contact hypersensitivity (CHS) model could be used for this purpose. In the CHS initiation phase, mice were treated with OVA protein subcutaneously and CpG ODN topically on the dorsal back (Figure 4.8A). CHS was elicited on the ears with topical OVA protein and CpG ODN with or without injection of FTY720 (Figure 4.8A). I verified that total lymphocyte count was higher in the SLNs after 1 day of FTY720 injection but almost back to baseline numbers after 3 days (Figure 4.8B). Ear thickness was measured daily after CHS elicitation and ear swelling was calculated. Clearly, ear swellings were equivalent between mice before FTY720 injection (Figure 4.8C). However, the swelling slowed down over the 6 days in mice injected with FTY720 (Figure 4.8C), indicating that egress of lymphocytes from the lymph nodes is required for full expression of protein CHS.  103  Figure 4.8 Inhibition of lymphocytes egression from the SLNs prevents ear swelling in a protein CHS model. (A) Schematic diagram of CHS initiation and elicitation as well as FTY720 injection and ear thickness measurements schedule. Mice were immunized with sc OVA protein and ec CpG ODN on the dorsal back for 2 consecutive days. The right ear was sensitized with ec OVA protein and CpG ODN 5 days after with or without intraperitoneal injection of FTY720 on day 6. The left ear was treated the same with DMSO as control. Ear thickness was measured for 7 days from the day of CHS elicitation. (B) Total lymphocyte counts in the SLNs were determined by flow cytometry gated on the lymphocyte population based on size. Representative bar graphs showing total lymphocyte counts in mice with or without FTY720 injection. (C) Ear swellings were calculated and summarized in a graphical representation of average ± SEM mm of ear swelling daily postCHS elicitation. Ear swelling = (T-T0)treated – (T-T0)sham. Data summarize two independent experiments (n = 7-8).  104  4.2.10 Topical administration of CpG ODN is required to generate a protein CHS response  The migratory ability of antigen-specific T cells in the SLNs induced by immunization using CpG ODN as adjuvant delivered through the topical and the subcutaneous route is unknown. To investigate this, the mouse protein CHS model described above was used. In the CHS initiation phase, mice were immunized with OVA protein subcutaneously and CpG ODN topically or subcutaneously on the dorsal back (Figure 4.9A). CHS was elicited on day 6 and ear thickness was measured daily for 6 days (Figure 4.9A). Interestingly, mice immunized with CpG ODN delivered topically developed a higher degree of ear swelling compared to subcutaneous delivery started from day 3 and peaked on day 5 post-CHS elicitation (Figure 4.9B). Mice immunized with subcutaneous CpG ODN demonstrated a low degree of ear swelling that also peaked on day 5 post-CHS elicitation. This indicates that topical administration of CpG ODN may be required for optimal immune recall response in the skin. Thus, topical may be superior to subcutaneous administration of CpG ODN in promoting activated T cells to migrate out of the SLNs into the skin for their immune effects (see Chapter 5).  105  Figure 4.9 Topical CpG ODN promotes migration of antigen-specific T cells to tissues. (A) Schematic diagram of CHS initiation and elicitation and ear thickness measurements schedule. Mice were sensitized with OVA protein sc and CpG ODN ec on the dorsal back for 2 consecutive days. The right ear was challenged ec with OVA protein and CpG ODN on day 6. The left ear was treated the same with DMSO as control. Ear thickness was measured for 6 days from day 7. (B) Ear swellings were calculated and summarized in a graphical representation of average ± SEM mm of ear swelling days post CHS elicitation. Data summarize two independent experiments (n = 5).  106  4.2.11  Topical CpG adjuvant increases memory CD8+ T cells at the site of infection  The ability of the induced antigen-specific T cells to localize to the site of infection is vital to provide rapid T cell immunity at tissues where pathogens invade. Jiang et al. demonstrated that expression of E- and P-selectin ligands on CD8+ T cells are required to recruit the T cells to the skin after acute vaccinia virus skin infection (262). I investigated whether using topical CpG ODN as adjuvant can increase the level of antigen-specific memory CD8+ T cells at the site of infection. Mice were immunized with subcutaneous OVA protein with or without topical CpG ODN using the primte-boost immunization strategy (Figure 4.10A). An attenuated strain of influenza virus expressing the MHC class I epitope of OVA protein (X31-OVA) was used to immunize the mice intranasally as a positive control. Mice were challenged intranasally with PR8 virus, which also express the MHC class I epitope of OVA protein (PR8-OVA) 4 weeks post-immunization to assess memory immunity. Peripheral blood, spleens, BALs and lungs were collected to detect OVA-specific CD8+ T cells in these compartments after infection by flow cytometry. The proportion of OVA-specific CD8+ T cells were increased by over 15-fold as detected in peripheral blood, BAL, and lungs of mice immunized with topical CpG ODN compared to those immunized without the adjuvant (Figure 4.10B, C). Importantly, both the BALs and the lungs of mice that were immunized with topical CpG ODN as adjuvant contained the highest frequencies of OVA-specific CD8+ T cells amongst these four compartments. These results demonstrate the enhanced ability of topical CpG ODN to promote the presence of antigen-specific CD8+ T cells at the site of infection.  107  Figure 4.10 Topical CpG ODN increases antigen-specific T cells at the site of infection. (A) Schematic diagram of immunization, infection and analysis schedule. C57BL/6 mice were immunized twice, 7 days apart, with sc OVA protein with or without ec CpG ODN. Mice were then infected with PR8-OVA intranasally 4 weeks post-immunization. Peripheral blood, spleens, BALs and lungs were collected 3 days post-infection for flow cytometric analysis. (B) Representative flow cytometric dot plots showing OVA-specific T cells. Cells were gated on B220- population and then CD8+ and OVA tetramer double positive population. (C) The percentage of OVA-specific cells amongst CD8+ memory T cells in peripheral blood, spleens, BALs and lungs are summarized in the scatter plots. Data summarize three (PB, Spleen, BAL) and two (Lung) independent experiments (n = 6-9). *** p < 0.001.  108  4.3  4.3.1  Discussion  Data summary  In this study, the mechanisms of how CpG ODN 1826 improves vaccine outcomes with the focus on the topical route of administration were investigated. The hypothesis that both skin stromal cells and hematopoietic cells contribute to the adjuvant effects of topical CpG ODNs was supported by experimental data in this chapter. I first confirmed that the adjuvant effect of CpG ODN 1826 is indeed TLR9-dependent using TLR9 KO mice. Bone marrow chimeric mice with TLR9-deficient hematopoietic cells completely abrogated the enhancement of antigen-specific CTL generation induced by topical CpG ODN. The percentage of OVA-specific CTL decreased to the level detected in mice immunized with antigen alone. In addition, chimeric mice with TLR9-deficient skin stromal cells also diminished T cell generation when using topical CpG ODN as vaccine adjuvant. Further investigation demonstrated that mouse keratinocytes respond to TLR9 stimulation and up-regulate expression of TLR9 upon topical CpG ODN application. However, mast cell-deficient mice had no effect on antigen-specific CTL generation compared to the congenic +/+ control mice. CpG ODNs internalization into CD11c+ cells was detected in the SLNs 48 hrs after topical administration. CpG ODN induces changes in both hematopoietic and stromal cells. At the level of the SLNs, there was the up-regulation of genes including Cxcl3, Ifng and Il11 and down-regulation of genes such as Il7 when comparing the cytokine and chemokine profile between mice treated with or without CpG ODN 24 hrs post-treatment. When examined closely the cytokine and chemokine profile between topical and subcutaneous administration of CpG ODN, the level of 109  Cxcl3, Ifng and Il11 mRNA were significantly higher while Cxcl13 and Il7 genes were significantly lower with the topical route. Topical CpG ODN applied at time of immunization resulted in an increased fraction of antigen-specific CD4+ T cells expressing tissue-homing molecules P-selectin ligand and E-selectin ligand and were detected in the SLNs while less T cells express L-selectin. The necessity of lymphocyte egression out of the SLNs to confer immune effects was confirmed in a protein CHS model. Injection of FTY720, an inhibitor of lymphocyte egression, decreased ear swelling induced by topical CpG ODN. Focusing on topical administration of CpG ODN, equivalent frequencies of OVA-specific CTL were detected in the lungs of mice immunized with subcutaneous OVA protein and topical CpG ODN compared to those infected with an attenuated strain of influenza virus expressing the OVA MHC class I epitope. Thus, topical CpG ODN is required for skin immune responses but also allows the generation of lung immune responses that exceed or equal to those of viral infection. These effects of topical CpG ODN presumably required lymphocytes including the antigen-specific T cells to exit the SLNs.  4.3.2  Topical CpG adjuvant effect on hematopoietic-derived cells  The CpG-motif paradigm states that TLR9 activation strictly requires DNA sequences with CpG motifs, which restricts TLR9 activation to pathogen-derived DNA (124). CpG ODNs with phosphorothioate backbone absolutely required the CpG motifs to activate TLR9 for their stimulatory effects (124),(96),(263),(123). However, some studies suggested that CpG ODNs also have TLR9-independent effects on cells such as anti-apoptotic (254) and cytokine secretions (255). I demonstrated that the adjuvant effect of topical CpG ODN in enhancing CTL response is 110  completely TLR9-dependent. The abrogation of acute protection against Lm infection in TLR9 KO mice confirmed the TLR9 dependency. The contribution of hematopoietic cells in the adjuvant effect of topical CpG ODN 1826 on CTL response was investigated. Results revealed that TLR9 expression in hematopoietic cells is necessary. However, which population of hematopoietic cells are the ones responding? Many studies showed that when CpG ODNs are injected into the dermis, CpG ODNs activate cutaneous DCs including LCs and pDCs. Activation of cutaneous DCs increase expressions of MHC class II molecules costimulatory molecules as well as decrease expression of adhesion molecules such as E-cadherin and α6 integrins (264),(251),(265). Activated DCs then depolarize and migrate to the SLNs. I demonstrated that when CpG ODNs are administered topically, they also signal through TLR9 expressed in DCs as I showed that CpG ODNs are internalized into DCs detected in SLNs 48 hours after treatment. However, the subset of cutaneous DCs that topical CpG ODNs act upon remains to be determined (see Chapter 5). Interestingly, recent studies suggested that mast cells may also play important roles in initiating a cascade of events that subsequently regulate the development of adaptive immune responses (104). It has been shown that dermal mast cells can be activated through TLR7 to produce TNF-α and IL-1β (107). To determine whether mast cells are required for the adjuvant effect of topical CpG ODNs in enhancing antigen-specific CTL response, mast cell-deficient mice were utilized to address this question. KitWKitW-v mice have only approximately 0.3% mast cells in the skin compared to congenic +/+ mice, while absent in other organs. I showed that mast cell-deficient mice did not prevent antigen-specific CTL generation. Thus, mast cells in the skin do not play a major role in the adjuvant effect of topical CpG ODNs in CTL response or other  111  immune cells have redundant cellular and mediator pathways that masked the effect of mast cells in the immune response (259).  4.3.3  Topical CpG adjuvant effect on skin stromal cells  Keratinocytes do not only act as a physical barrier and provide structural integrity but have also been shown to be immuno-competent, linking innate and adaptive immune responses. Recent studies suggested that they can produce a plethora of cytokines (159) and these cytokines lead to cross-talk between keratinocytes and other immune cells, which in turn initiate inflammation (253),(266),(267). TLR9 mRNA is undetected in normal mouse skin but TLR9 expression and the production of inflammatory cytokines including IL-1, IL-6, IL-12 and TNFα can be induced by intradermal injection of CpG ODNs (258). Keratinocytes indirectly augment functions of DC including antigen presentation and co-stimulatory molecule expressions (253). Cytokines produced by keratinocytes can also activate endothelial cells and subsequently increase adhesion molecules and chemotactic factors to recruit more immune cells into the skin. Based on these observations, I investigated the requirement of skin stromal cells for topical CpG ODN 1826 to enhance CTL response. Results revealed that TLR9 expression in skin stromal cells contribute but are not absolutely required. However, incomplete seeding of WT DCs in the skin could not be ruled out. CD11c+ cells in the skin of the chimeric mice were identified but not 100% of the cells were of donor phenotype. Furthermore, using the real-time PCR technique, I showed that there is a trend of increased TLR9 mRNA level after topical CpG ODN treatment. Thus, topical administration of CpG ODN as vaccine adjuvant induces activation of both keratinocytes and cutaneous DCs. Keratinocytes respond to topical CpG 112  ODNs in part by up-regulating TLR9 expressions on themselves creating a positive feedback loop. Keratinocytes may also cross-talk with cutaneous DCs and act in concert to relay the adjuvant effect of topical CpG ODN to the SLNs. Stromal cells are not necessary but are required for the optimal adjuvant effect of topical CpG ODN. The identity of the cytokines or factors secreted by keratinocytes under the effect of topical CpG ODN remains to be determined.  4.3.4  Topical CpG adjuvant effect on SLNs and T cells  Very rapidly, at 1 hr after barrier disruption and topical CpG ODN administration, CpG ODNs penetrates through the skin (261). It is well established that DCs are professional APCs that present antigens to T and B cells in lymphoid organs. Some studies suggested that after obtaining antigen, DCs in the skin migrate to the SLNs to activate T and B cells. The migratory kinetics of the different subsets of cutaneous DCs varies. After FITC painting on the skin, it was observed that dermal DCs reached the SLNs faster than LCs peaking 24 hours and 72 hours after treatment, respectively (180). I showed, at 48 hrs, after topical administration of CpG ODN, CpG ODNs were detected in SLNs inside CD11c+ cells. However, which subsets of cutaneous DCs are necessary for the adjuvant effect of topical and subcutaneous CpG ODN requires further study (see SUMMARY and FUTURE DIRECTIONS in Chapter 5). Using the PCR array technique, I compared the cytokines and chemokines gene profiles of the SLNs 24 hrs and 72 hrs post-treatment in mice treated with or without CpG ODN. Analyses of the SLNs at 24 hrs and 72 hrs dissect the involvement of dermal DCs and LCs in the modulation of SLN microenvironment because dermal DCs migrate to the SLNs faster than LCs (180). CpG ODN 1826 was administered either topically or subcutaneously. Despite the route of 113  administration, CpG ODN 1826 altered the microenvironment of SLNs as shown by the changes in gene profiles. Interestingly, the different routes of CpG ODN administration differentially modulated the microenvironment of SLNs at 24 hrs but not 72 hrs post-treatment. Thus, topical application may differ from subcutaneous administration of CpG ODN in the activation of dermal DCs, which subsequently migrate to the SLNs and modulate the microenvironment. The Il11, Ifng and Cxcl3 genes were significantly over-expressed while the Cxcl13 and Il7 genes were significantly under-expressed. These genes have different functions on immune and stromal cells. IL-11 induces of proliferation of hematopoietic stem cells and megakaryocytes plasma cell development. IFN-γ activates macrophage and direct killing of pathogens, inhibits proliferation of transformed cells and viral replication, induces Th1 cell differentiation, enhances immunogenicity of transformed cells as well as the antiviral and antitumor effects of type I IFNs (268). CXCL3 is chemotactic for neutrophils (269) and monocytes (270). A recent study found that neutrophils are early responders in inflammatory responses that may link the innate and the adaptive immunity. Neutrophils release chromatin fibers with antimicrobial proteins called extracellular traps. These traps activate pDCs and modulate inflammatory responses. In addition, they can directly prime T cells by decreasing T cell activation threshold (271). Cxcl13 is a B cell chemoattractant and IL-7 is important to B and T cell lymphopoiesis, which is also crucial to lymph node remodeling (272). Differential expressions of these cytokines and chemokines create a particular microenvironment in the SLNs that may explain the detected differences in T cell phenotypes and protective immunity subsequent to immunization with topical and subcutaneous CpG ODN as adjuvant. I only investigated the cytokines and chemokines gene profile because they can influence B and T cell development in SLNs as well as immune cell recruitment to SLNs. Many other genes may have also been differentially expressed in the SLNs between 114  topical and subcutaneous administration of CpG ODNs leading to the differences, which remains to be determined.  4.3.5  Topical CpG ODN increases antigen-specific CD8+ T cells at the site of infection  The mouse CHS model is a T cell mediated and antigen-specific skin inflammation induced by haptens of sensitized mice (273),(274). I demonstrated that using the novel immunomodulator FTY720 that blocks egress of lymphocytes from the SLNs (275), migration of antigen-specific T cells out of the SLNs is required to elicit protein CHS. Topical compared to subcutaneous administration of CpG ODN induced greater degree of ear swelling in the protein CHS model suggesting that topical CpG ODN may be required for effective skin homing, increasing seeding of antigen-specific memory T cells to the skin to generate skin-resident effector memory T cells. In addition, topical CpG ODN may also increase effector memory T cells seeding in other tissues such as the lungs. Increased expression of P- and E-selectin ligands detected on the OT-II cells in the SLNs in mice immunized with topical compared to subcutaneous CpG ODN also supports this hypothesis. Together, topical CpG ODN may enhance vaccine responses by increasing tissue antigen-specific T cells to rapidly control invading pathogens.  115  CHAPTER 5: OVERALL SUMMARY AND FUTURE DIRECTIONS  5.1  A novel split immunization method of antigen and adjuvant to enhance vaccine  response and its implications  5.1.1  Topical route of CpG ODN administration as vaccine adjuvants  Current vaccines remain inefficient in part due to their poor ability to induce cell-mediated immune responses, delayed responses, and lack of long-lasting protective effect. Class B CpG ODNs are potent B cell activators that do not strongly stimulate the production of type I IFNs (125),(127),(126). They signal through TLR9, linking innate and adaptive immune responses. What is interesting and attractive is that CpG ODNs induce non-specific innate immune responses but are capable of enhancing antigen-specific adaptive immunity. Their strong adjuvant effects in humoral immune responses are due to their ability to synergize with B cell receptor and preferentially activate antigen-specific B cells, improve B cell survival and enhance maturation of antibodies such as Ig class-switching. CpG ODNs skew CD4+ T cell to the Th1 type immune response and promote CTL responses (126). Many pre-clinical studies explored CpG ODNs as vaccine adjuvant for allergies, cancers and infectious diseases. Some of these studies have advanced to human clinical trials to boost immune responses for vaccines such as the hepatitis B (144),(133),(145), melanoma (138),(139),(140), influenza (143) and malaria (147),(148),(149),(150) vaccines . Nevertheless, all of the studies to date that explore CpG ODNs as vaccine adjuvants have not considered the topical route of administration, separating it from the route of antigen administration. 116  The work of this thesis advocates a novel immunization method where vaccine and adjuvant are administered separately using the skin as a site of immunization by administering a TLR9 agonist, CpG ODN 1826, as a topical adjuvant to enhance humoral and cell-mediated immune responses of protein-based vaccines. Low efficacy of vaccines in part is due to the poor ability to induce long-lasting protective cell-mediated immune responses. My work demonstrated that single dose of topical CpG ODN 1826 at the time of subcutaneous protein antigen administration in a rapid prime-boost immunization regimen, 7 days apart, is an effective strategy to enhance CTL responses. The efficacy of inducing rapid or long-lasting protection with this immunization strategy has been verified in two animal infection models. Pre-clinical animal studies with mice using the systemic Lm infection model and the intranasal influenza A infection model demonstrated the improved protection provided by topical CpG ODN 1826. With a 7-day prime-boost regimen, rapid protection is achieved within 5 days postimmunization. This immunization strategy induces protection against intracellular bacterial or viral infections. This split delivery of antigen and adjuvant immunization strategy has the added advantage of not requiring novel vaccine re-formulation of existing vaccines. Thus, this immunization strategy can be applied in the event of pandemics or bioterrorist attacks selectively to at risk populations such as the elderly and the very young to enhance protection. Most importantly, long-lasting protection, both humoral and cell-mediated immune responses, can be achieved using topical CpG ODNs as vaccine adjuvants.  117  5.1.2  Mechanisms of action of topical CpG ODNs  In this thesis, I unraveled in part the mechanisms of how topical CpG ODN improves antigen-specific CTL responses. Based on the results of this work, I propose that topical CpG ODN 1826 acts on both skin stromal cells and hematopoietic cells. It up-regulates TLR9 expressions on keratinocytes, cells known to secrete cytokines and chemokines to augment the functions of cutaneous DCs. Cutaneous DCs then migrate to the SLNs with CpG ODNs to present antigens to T cells and to alter the microenvironment of SLNs. In turn, these changes modulate tissue-homing molecule expressions on the activated T cells, providing them with a greater ability to migrate to tissues. I speculate that preferential induction of effector memory T cells allocates these T cells to tissues for rapid control of invading pathogens while awaiting central memory T cells to proliferate and act in a second wave to boost protective immunity. Jiang et al. showed that local vaccinia skin infection generated CD8+ skin-resident T cells but surprisingly provided global skin immunity (262). A summary of the proposed events induced by topical CpG ODN as vaccine adjuvant is shown in a schematic diagram (Figure 5.1). In summary, this work provides insights into the mechanisms of the action of topical CpG ODNs. Understanding how topical CpG ODNs confer its adjuvant effects can ensure appropriate immune cells are being targeted in order to optimize their adjuvant effects.  118  5.1.3  Implications of this work  Current vaccines lack the ability to induce long-term cell-mediated immune responses that are crucial to control and to eliminate invading intracellular bacteria and viruses. In addition, mucosal sites tend to induce tolerance to maintain homeostasis and thus mucosal immunity is difficult to achieve with parenteral routes of immunization. The oral and the respiratory tract routes of vaccine delivery are two methods to induce mucosal immunity. In the past few decades, new human infectious diseases have arisen and those that are thought to be extinct reemerged. Infectious diseases continue to be one of the major causes of morbidity and mortality in both developed and developing countries. An adjuvant that can boost the efficacy of existing vaccines would be beneficial and economical. My work demonstrates that topical CpG ODNs have great potential to be safe and effective vaccine adjuvants. This thesis provides a solid foundation as the pre-clinical study for the basis of future human clinical trials to assess the effective dose, safety and the efficacy of using topical CpG ODNs as adjuvants for prophylactic vaccines against intracellular and viral infections. Thus, the novel immunization strategy proposed in this thesis may revolutionize vaccination programs, which in turn have great impact in improving global health and alleviating the economic burdens of modern societies.  5.1.4  Major drawback of this work using mouse models  This thesis demonstrates a novel split immunization strategy of antigen and adjuvant to enhance protective immunity. It has laid down the foundation for others to continue in investigating the mechanisms of the adjuvant effect of topical CpG ODNs. The major drawback 119  inherent in this study is that TLR9 expression patterns differ between human and mice. TLR9 is expressed on murine myeloid DCs, pDCs, B cells, monocytes, and mast cells (112),(246). In normal mouse skin, TLR9 mRNA expression is induced following intradermal injection of CpG ODN (258). In humans, TLR9 expression is more restricted than in mice to plasmacytoid DCs and B cells but can also be induced in monocytes and keratinocytes (276),(265),(121) suggesting that topical application may have an additional adjuvant effect in humans as well. As others and as I have demonstrated in this thesis, the skin is not dormant but actively participates in immunological responses. Cholera toxin and TLR7 agonists are small molecules that easily penetrate through the skin. CpG ODNs are relatively large molecules and are more difficult to penetrate the skin. In mouse models of this work, the mice were shaved, tapestripping and treated with acetone prior to the application of CpG ODNs to increase penetration. Tape-stripping removes the dead keratinocytes and thus thinning the stratum corneum layer while acetone removes lipids such as cholesterol and fatty acids found between the dead keratinocytes allowing penetration of CpG ODNs. In humans, the skin thickness and lipid composition varies among individuals. It is not practical to perform these treatments but there are methods being developed to overcome this limit. Skin treatment with a hand-held abrader containing emery paper can be used to remove a few layers of stratum corneum allowing better penetration of CpG ODNs as adjuvants. Another method being developed is the use of microneedles where CpG ODNs can be delivered through the stratum corneum directly into the epidermis.  120  Figure 5.1 Overview of events triggered by topical CpG ODN to improve vaccine outcomes. Schematic diagram showing the proposed mechanisms of how topical CpG ODNs enhance antigen-specific CTL response. KC, keratinocytes; Ag, antigen; Tm, memory T cells.  121  5.2  Future directions  There are many questions that remain unanswered. I demonstrated that both skin stromal cells and hematopoietic cells are necessary for the adjuvant effect of topical CpG ODNs. However, specifically which type of stromal cells and which subsets of cutaneous DCs contribute to the adjuvant effect remain to be determined. CpG ODNs promote antigen crosspresentation by DCs (252) and the mobilization of DCs by decreasing E-cadherin and α6 integrins that retain DCs in the skin (251). Recent studies suggested that although LCs reside at the interface closest to the external environment, they are not required for immunity but for tolerance. In a herpes simplex virus skin infection model, DC subsets including LCs, Langerin+ dermal DCs and other dermal DCs all can present antigen to CD4+ T cells. However, only Langerin+ CD103+ DCs were able to cross-present antigen to CD8+ T cells (182). Based on the observations in Chapter 4 and studies by others, I hypothesize that both dermal DCs and LCs are involved in the adjuvant effect of topical CpG ODNs, with dermal DCs being the cutaneous DCs that shuttle CpG ODNs into the SLNs in the first wave followed by LCs at later time points. Subsets of cutaneous DCs with different functions cooperate with each other to induce optimal immune responses.  122  5.2.1  Cutaneous DC subsets that are necessary for the adjuvant effect of topical and subcutaneous CpG ODNs  It is important to resolve the contradicting results in published literatures of the role of LCs in the context of vaccine adjuvants to ensure the effectiveness of using topical CpG ODNs as vaccine adjuvants. It is ideal if LCs only play a role in immunity but perhaps we also have to consider their role in tolerance. Whether LCs and/or dermal DCs are responsible for the adjuvant effect of topical CpG ODNs should be further investigated. It has been shown that dermal DCs migrate from the skin to the SLNs faster than LCs after stimulation; dermal DCs can be detect at 12 hrs after FITC painting on the skin and peak at 24 hrs while LCs can be detected starting at 24 hrs and peak at 72 hrs (180). Whether LCs, dermal DCs, or both are responsible for CpG ODN uptake and transport to the SLNs can be determined by examining the presence of FTIC-CpG ODN in SLNs at earlier time points such as at 12 hrs and 24 hrs post-topical treatment. If FITCCpG ODNs were detected in SLNs internalized by DCs at 12 hrs time point, I can conclude that dermal DCs are the DCs responsible for CpG ODN uptake. Immunofluorescent staining with Langerin can be used to identify LCs. Presence of FITC-CpG ODN in Langerin- and/or Langerin+ cells can confirm which subset of DCs are responsible for CpG ODN uptake. To further address the importance of LCs, dermal Langerin+ DCs, and dermal DCs, three different mutant mice can be utilized. First, mutant mice containing a human diphtheria toxin receptor (DTR) and an enhanced green fluorescent protein downstream of the stop codon of the langerin gene (The Jackson Laboratory stock #016940, B6.129S2-Cd207<tm3Mal>/J) will be a useful tool to decipher the role of LCs and Langerin+ dermal DCs. Selective systemic in vivo depletion of LCs in the epidermis can be induced conditionally with ip injection of diphtheria 123  toxin (277). To confirm LC ablation in the epidermis, the dose of diphtheria toxin will be titrated. The epidermis of the experimental mice will be checked for the presence or absence of LCs by the green fluorescence detection using confocal laser scanning microscopy or flow cytometry to ensure complete LC depletion. Secondly, Batf3 -/- mutant mice that lack the Baft3 transcription factor constitutively deficient in Langerin+ CD103+ dermal DCs, CD103+ and CD8+ DCs in other tissues (278),(179). The mutant mice with human DTR and the Batf3 -/- mice together can determine whether LCs or Langerin+ dermal DCs are necessary for the adjuvant effect of topical and subcutaneous CpG ODN. Thirdly, all Langerin+ DCs can be ablated in the murine Langerin-DTR mice upon diphtheria toxin injection (179). Murine Langerin-DTR mice can be used to determine the necessity of LCs and Langerin+ dermal DCs or other subsets of dermal DCs in the adjuvant effect of topical and subcutaneous CpG ODN. The mutant mice injected with or without diphtheria toxin, will be immunized with OVA subcutaneously with or without CpG ODN topically or subcutaneous to determine the requirement of LCs, Langerin+ dermal DCs and other dermal DCs in the adjuvant effect in enhancing CTL response by CpG ODNs. If LCs are necessary for the adjuvant effect, antigen-specific CTL generation will be abrogated in both the human Langerin-DTR and the murine Langerin-DTR mice but not in the Batf3 -/- mice. If the Langerin+ dermal DCs are necessary, antigen-specific CTL generation will be abrogated in the Batf3 -/- and the murine Langerin-DTR mice but not in the human LangerinDTR mice. If other dermal DCs are necessary, antigen-specific CTL generation will not be abrogated in any of these mutant mice but only in CD11c-DTR mice with all the DCs ablated. However, if these subsets of DCs are not required or they only contribute in part to the adjuvant effect of topical and subcutaneous CpG ODN, antigen-specific CTL generation will not be affected or they will be diminished but not completely abolished, respectively. Antigen-specific 124  T cells can be induced in the mutant mice but they may become non-functional. To assess the functionality of the T cells, mice can be infected with influenza A virus intranasally and evaluate the protective immunity by enumerating viral burden in the lungs.  5.2.2  Ability of topical and subcutaneous CpG ODN to enhance traffic of antigen-specific T  cell to tissues  Recently, a novel population of memory CTL, termed tissue-resident memory cells, has been identified. Skin resident tissue memory T cells are particularly effective in protecting against cutaneous infection (262). In Chapter 4, delayed ear swelling with FTY720 injection and increased ear swellings with topical compared to subcutaneous CpG ODNs in the protein CHS model suggests that topical CpG ODN may promote skin resident memory T cells to rapidly confer protection upon infection. The protein CHS model can be used to assess the migratory ability of T cells to the skin induced by topical compared to subcutaneous administration of CpG ODNs. Skin infections provide global skin immunity by generating non-migratory resident memory T cells. I hypothesize that using topical CpG ODNs as adjuvants, more closely mimicking natural skin infection, will preferentially increase the tissue-resident memory CTL population. These T cells are akin to soldiers, already at the front line ready to fight pathogenic invasions without delay. To test this hypothesis, two models can be used: the protein CHS model and the parabiotic mouse model. In the protein CHS model, activated antigen-specific CTLs migrate out of the SLNs to the skin and can be inhibited with FTY720 immunomodulator. Elicitation of protein CHS can be performed 4 weeks post-initiation phase and ear swellings will be measured. The 125  presence of elevated frequency of tissue-resident memory T cells in the skin should promote rapid ear swellings, peaking on an earlier day compared to the presence of lower frequency of these T cells. Thus, if topical CpG ODN compared to subcutaneous administration of CpG ODNs increases the frequency of tissue-resident memory CTLs, ear swelling will peak on an earlier day post-CHS elicitation. In the parabiotic mice model, the blood circulation of a naive mouse and a mouse immunized with OVA subcutaneously with or without CpG ODN topically or subcutaneously are joined together by surgery 4 weeks post-immunization but the tissueresident memory T cells cannot not be shared as they remain sessile. The mice will then be separated and challenged with a cutaneous virus such as vaccinia virus on the skin or intranasally infected with influenza A virus in the lungs with FTY720 immunodulator injection to prevent lymphocytes exiting out of the lymphoid organs. The contribution of circulating antigen-specific memory T cells and tissue-resident memory T cells are still to be determined. 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