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UBC Theses and Dissertations

Investigation of the immunostimulatory activity and vaccine potential of lipid encapsulated plasmid DNA… Wilson, Kaley 2007

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INVESTIGATION OF THE IMMUNOSTIMULATORY ACTIVITY AND VACCINE POTENTIAL OF LIPID ENCAPUSLATED PLASMID DNA AND OLIGODEOXYNUCLEOTIDES by Kaley Dianne Wilson B.Sc., University of Guelph, 2001  A THESIS SUBMITTED IN PARTIAL FULFILMENT OF THE REQUIREMENT FOR THE DEGREE OF DOCTOR OF PHILOSOPHY in THE FACULTY OF GRADUATE STUDIES (Biochemistry and Molecular Biology)  THE UNIVERSITY OF BRITISH COLUMBIA December 2007  © Kaley Dianne Wilson, 2007  ABSTRACT DNA vaccines offer unique promise as a means of generating immunity against infectious and malignant disease. Unfortunately a number of obstacles, including rapid degradation of naked plasmid DNA (pDNA), poor cellular uptake by antigen presenting cells (APCs) and subsequent low levels of gene expression have limited the ability of DNA vaccines to raise sufficient immune responses towards the target antigen. This thesis is focused on investigating the immunostimulatory potential of liposomal nanoparticulate (LN) formulations of pDNA (stabilized plasmid lipid particles; SPLP) and  cytosine-guanine oligodeoxynucleotides (CpG-ODN; LN CpG-ODN), and  examining their ability to act together as a non-viral DNA vaccine in attempt to address the shortcomings of current DNA vaccine approaches. One focus of this thesis concerns investigating the immunostimulatory activity of LN formulations of CpG-ODN and pDNA. It is shown that despite dramatic differences in pharmacokinetics and biodistribution of LN CpG-ODN following intravenous (i.v.) and subcutaneous (s.c.) administration the resultant immune response is very similar, which is concluded to be due to the intrinsic ability of APCs to sequester LN CpG- ODN. In addition, it is demonstrated that lipid encapsulation dramatically enhances the immunostimulatory potential of pDNA and it is observed that SPLP maintains immunostimulatory activity in Toll-like receptor 9 (TLR9) knock-out mice. Together theses findings highlight the need for DNA-based therapies to consider both TLR9dependent and -independent immunostimulatory activities of pDNA when constructing non-viral vectors.  ii  Furthermore, a new role for SPLP as a non-viral gene delivery vehicle for the generation of a systemically administered genetic vaccine in the presence of LN CpGODN is introduced. The ability of vaccination with SPLP to act prophylactically, to protect mice from tumour challenge, and therapeutically, in a novel vaccination strategy where the antigen is expressed at the tumour site as a result of SPLP-mediated transfection, is explored, demonstrating that in the presence of LN CpG-ODN SPLP possesses potential as a non-viral delivery system for DNA-based cancer vaccines. In summary, this work represents a substantial advance in the understanding of the immunostimulatory potential of both SPLP and LN CpG-ODN and provides insight into their ability to work together as a non-viral DNA vaccine.  iii  TABLE OF CONTENTS ABSTRACT...............................................................................................................................ii TABLE OF CONTENTS ..........................................................................................................iv LIST OF TABLES ....................................................................................................................ix LIST OF FIGURES.................................................................................................................... x ACKNOWLEDGEMENTS...................................................................................................... xv CO-AUTHORSHIP STATEMENT........................................................................................xvii INTRODUCTION......................................................................................................................1 1.1 Vaccines ...........................................................................................................................1 1.1.1 Overview of vaccine immunology..............................................................................2 1.2 DNA vaccines ..................................................................................................................4 1.2.1 Mechanism of antigen presentation by DNA Vaccines ...............................................6 1.2.2 Routes of administration of DNA vaccines.................................................................8 1.2.3 Carrier systems for DNA vaccines .............................................................................9 1.2.4 Safety of DNA vaccines ...........................................................................................10 1.3 Cancer immunotherapy and cancer vaccines ...................................................................10 1.3.1 DNA vaccines for cancer .........................................................................................13 1.4 Immunostimulatory properties of unmethylated CpG motifs ...........................................14 1.4.1 Role of CpG in DNA vaccines .................................................................................18 1.4.2 Therapeutic applications & safety of CpG-ODN as an adjuvant ...............................21 1.5 Liposomal delivery systems for conventional drugs ........................................................22 1.5.1 Liposomes as adjuvants and carriers of peptide and protein vaccines........................24 1.5.2 Cationic lipid containing delivery systems for pDNA...............................................25 1.5.3 Systemic administration of cationic lipid containing DNA complexes ......................27 1.5.4 Cationic lipid-based carrier systems for DNA vaccines ............................................29 1.6 Stabilized plasmid lipid particles (SPLP) ........................................................................30 1.7 Liposomal nanoparticle encapsulated CpG ODN (LN CpG-ODN)..................................33  iv  1.8 Thesis objectives ............................................................................................................34 1.9 References ......................................................................................................................35 CHAPTER TWO ..................................................................................................................... 49 EFFECTS OF INTRAVENOUS AND SUBCUTANEOUS ADMINISTRATION ON THE PHARMACOKINETICS, BIODISTRIBUTION, CELLULAR UPTAKE AND IMMUNOSTIMULATORY ACTIVITY OF CpG ODN ENCAPSULATED IN LIPOSOMAL NANOPARTICLES .......................................................................................... 49 2.1 Introduction ....................................................................................................................49 2.2 Materials & Methods ....................................................................................................51 2.2.1 Materials ..................................................................................................................51 2.2.2 Animals ...................................................................................................................52 2.2.3 Preparation of liposomal ODN .................................................................................52 2.2.4 Pharmacokinetic & biodistribution studies ...............................................................53 2.2.5 Immune cell isolation ...............................................................................................53 2.2.6 Uptake studies..........................................................................................................54 2.2.7 Activation & cytotoxicity assays ..............................................................................54 2.2.8 Statistical analyses ...................................................................................................55 2.3 Results............................................................................................................................55 2.3.1 Blood levels of LN CpG ODN differ following i.v. or s.c administration..................55 2.3.2 I.V. administration of LN-CpG ODN results in enhanced delivery to liver and spleen, whereas s.c. administration enhances accumulation in regional lymph nodes. ...................57 2.3.3 LN-CpG ODN are accumulated by immune cells in the blood, spleen and lymph nodes following i.v. or s.c. administration.........................................................................59 2.3.4 Similar levels of immune cell activation are observed following s.c. or i.v. administration of LN-CpG ODN.......................................................................................63 2.3.5 Similar levels of NK cytolytic activity and ADCC are observed following s.c. or i.v. administration of LN-CpG ODN.......................................................................................64 2.4 Discussion ......................................................................................................................67 2.4 References ......................................................................................................................71 CHAPTER THREE.................................................................................................................. 75 STABILIZED PLASMID LIPID PARTICLES ACTIVATE APCs THROUGH TLR9DEPENDENT AND –INDEPENDENT MECHANISMS IN VIVO .......................................... 75 3.1 Introduction ....................................................................................................................75 v  3.2 Materials and Methods....................................................................................................77 3.2.1 Materials ..................................................................................................................77 3.2.2 Animals ...................................................................................................................77 3.2.3 Preparation of SPLP and LN-CpG ODN ..................................................................78 3.2.4 Immune activation studies........................................................................................79 3.2.5 Chloroquine study....................................................................................................79 3.2.6 Cell activation analysis.............................................................................................79 3.2.7 Plasma cytokine analysis..........................................................................................80 3.2.8 Statistical analyses ...................................................................................................80 3.3 Results............................................................................................................................80 3.3.1 Encapsulation of pDNA within lipid nanoparticles dramatically enhances immune cell activation following systemic administration ....................................................................80 3.3.2 SPLP demonstrates immunostimulatory activity in TLR9 KO mice..........................82 3.3.3 SPLPs elicit enhanced immunostimulatory activity in the presence of the endosomolytic agent chloroquine......................................................................................83 3.4 Discussion ......................................................................................................................87 3.5 References ......................................................................................................................91 CHAPTER FOUR .................................................................................................................... 97 THE COMBINATION OF STABILIZED PLASMID LIPID PARTICLES AND LIPID NANOPARTICLE ENCAPSULATED CpG-ODN FOR THE DEVELOPMENT OF A GENETIC VACCINE .............................................................................................................. 97 4.1 Introduction ....................................................................................................................97 4.2 Materials and methods. ...................................................................................................99 4.2.1 Materials. .................................................................................................................99 4.2.2 Animals and cell lines ............................................................................................ 100 4.2.3 Preparation of SPLP and LN-CpG ODN ................................................................ 100 4.2.4 Uptake and Transfection of APCs by SPLP............................................................ 102 4.2.5 SPLP-mediated Induction of Adaptive Immune Responses .................................... 104 4.2.6 Statistical Analysis................................................................................................. 106 4.3 Results.......................................................................................................................... 106  vi  4.3.1 APCs take up SPLP ex vivo.................................................................................... 106 4.3.2 SPLP transfect the cultured macrophage cell line RAW264.7, whole splenocyte populations and CD11b+ cells ex vivo............................................................................. 107 4.3.3 SPLP are taken up by APCs in the spleen following intravenous administration..... 109 4.3.4 SPLP transfect CD11b+ and CD11c+ cells in the spleen following intravenous administration................................................................................................................. 109 4.3.5 Vaccination with SPLP followed 24 h later by LN CpG-ODN primes the generation of transgene-specific humoral and cellular immune responses............................................. 111 4.4 Discussion .................................................................................................................... 115 4.5 References .................................................................................................................... 120 CHAPTER FIVE.................................................................................................................... 125 CHARACTERIZATION OF SPLP AND LN CpG-ODN AS A NON-VIRAL GENETIC CANCER VACCINE ............................................................................................................. 125 5.1 Introduction .................................................................................................................. 125 5.2 Materials and Methods.................................................................................................. 127 5.2.1 Materials ................................................................................................................ 127 5.2.2 Animals and cell lines ............................................................................................ 127 5.2.3 Preparation of SPLP and LN-CpG ODN ................................................................ 128 5.2.4 Determination of the temporal effects of LN CpG-ODN addition on its adjuvant activity in an SPLP-based vaccine................................................................................... 130 5.2.5 Prophylactic vaccination ........................................................................................ 130 5.2.6 Therapeutic vaccination ......................................................................................... 131 5.2.7 ELISA.................................................................................................................... 131 5.2.8 In Vitro Restimulation............................................................................................ 132 5.2.9 Determination of CTL activity by 51Cr release assay .............................................. 132 5.2.10 IFN-γ cytokine secretion assay ............................................................................. 133 5.2.11 MHC pentamer assay ........................................................................................... 133 5.2.12 Statistical analyses ............................................................................................... 134 5.3 Results.......................................................................................................................... 134  vii  5.3.1 LN CpG-ODN demonstrates optimal adjuvant activity when delivered 24 h after SPLP .............................................................................................................................. 134 5.3.2Investigation of SPLP as a prophylactic cancer vaccine in the presence and absence of LN CpG-ODN ................................................................................................................ 137 5.3.3Investigation of the ability of SPLP-mediated vaccination and transfection of tumours to act as a novel therapeutic cancer vaccine..................................................................... 140 5.3.4β-gal and tumour specific immune responses in vaccinated mice receiving therapeutic treatment with SPLP (βgal) ............................................................................................. 143 5.4 Discussion .................................................................................................................... 145 5.5 References .................................................................................................................... 151 CHAPTER SIX...................................................................................................................... 155 SUMMARY AND FUTURE DIRECTIONS.......................................................................... 155 APPENDIX............................................................................................................................ 159  viii  LIST OF TABLES Table 5.1: Test results from whole blood and serum samples obtained from mice euthanized as a result of distress following vaccination and therapeutic treatment with SPLP……………………………………………………………………………………143  ix  LIST OF FIGURES Figure 1.1: Schematic representation of antigen processing and presentation in an APC following genetic vaccination ..................................................................................................... 3 Figure 1.2: Schematic representation of the CpG-DNA/TLR9-mediated cellular signaling pathway.. ................................................................................................................................. 17 Figure 1.3: Mechanism by which CpG-ODNs facilitate innate and adaptive immune responses.................................................................................................................................. 18 Figure 1.4: A schematic diagram depicting the passive accumulation of liposomal formulations at diseased site tissues through the EPR effect...................................................... 24 Figure 1.5: Luciferase gene expression following a single intravenous administration of SPLP in neuro-2A tumour-bearing A/J mice............................................................................. 31 Figure 1.6: Cryo-transmission electron microscopy of SPLP ................................................... 32 Figure 2.1: Blood levels of free and LN CpG ODN differ dramatically following i.v. or s.c administration ..................................................................................................................... 56 Figure 2.2: Biodistribution of i.v. and s.c. administered free and LN-CpG ODN to liver, spleen and lymph nodes............................................................................................................ 58 Figure 2.3: Preferential Accumulation of LN-CpG ODN in the lymph nodes following s.c. administration .................................................................................................................... 60 Figure 2.4: LN-CpG ODN are accumulated by CD11c positive, Mac3 positive, CD11b positive and B220/CD45R positive cells in the blood, spleen and lymph nodes compartments following i.v and s.c. administration .................................................................. 61 Figure 2.4: LN-CpG ODN are accumulated by CD11c positive, Mac3 positive, CD11b positive and B220/CD45R positive cells in the blood, spleen and lymph nodes compartments following i.v and s.c. administration .................................................................. 62 Figure 2.6: Similar levels of NK cytolytic activity of splenic and peripheral blood immune cells are observed following i.v. and s.c. administration of LN-CpG ODN .................. 65 Figure 2.7: Similar levels of ADCC of splenic and peripheral blood immune cells against the human B-cell lymphoma cell Daudi are observed following i.v. and s.c. administration of LN-CpG ODN. ............................................................................................. 66 Figure 3.1: Comparison of immune cell activation following systemic administration of free or encapsulated plasmid..................................................................................................... 81 Figure 3.2: Comparison of plasma cytokine induction following systemic administration of free or encapsulated plasmid ................................................................................................ 82  x  Figure 3.3: SPLP promotes the upregulation of activation markers CD69 and CD86 on APCs in WT and TLR9 KO mice ............................................................................................. 84 Figure 3.4: Systemic administration of SPLP promotes the induction of proinflammatory cytokines in WT and TLR9 KO mice.................................................................. 85 Figure 3.5: Chloroquine enhances immune cell activation following treatment with SPLP ...... 86 Figure 3.6: Chloroquine enhances plasma cytokine levels following treatment with SPLP....... 87 Figure 4.1: SPLP is preferentially taken up by CD11b+, CD11c+ and B220/CD45R+ cells ex vivo ............................................................................................................................ 107 Figure 4.2: SPLP transfects the cultured macrophage cell line RAW264.7, whole splenocyte populations and primary macrophage ex vivo both in the presence and absence of LN CpG-ODN.................................................................................................................... 108 Figure 4.3: Uptake of SPLP by CD11b+, CD11c+ and B220/CDR45+ cells in the spleen following intravenous administration...................................................................................... 110 Figure 4.4: SPLP-mediated transfection of CD11b+, CD11c+ and B220/CDR45+ cells in the spleen following the intravenous administration of SPLP (βgal) alone or followed 24 hours later by LN CpG-ODN.................................................................................................. 112 Figure 4.5: Vaccination with SPLP (βgal) followed 24 hours later by LN CpG-ODN primes the generation of a βgal specific humoral immune response ........................................ 113 Figure 4.6: Vaccination with SPLP (βgal) followed 24 hours later by LN CpG-ODN increases the frequency of antigen-specific IFN-γ secreting CD8+ T-cells .............................. 114 Figure 4.7: Vaccination with SPLP (βgal) followed 24 hours later by LN CpG-ODN primes the generation of βgal specific CTLs ........................................................................... 116 Figure 5.1: Temporal effects of LN CpG-ODN administration on the humoral immune response to an SPLP-based DNA vaccine ............................................................................... 135 Figure 5.2: Temporal effects of LN CpG-ODN administration on the frequency of βgalspecific CD8+ T-cells following SPLP-based vaccination ...................................................... 136 Figure 5.3: Temporal effects of LN CpG-ODN administration on the frequency of βgalspecific IFN-γ secreting CD8+ T-cells following SPLP-based vaccination ............................. 137 Figure 5.4: Vaccination with SPLP (βgal) in the presence of LN CpG-ODN enhances median survival following tumour challenge with CT26.CL25 ............................................... 139 Figure 5.6: Mice vaccinated with SPLP (βgal) and LN CpG-ODN exhibit tumour growth delay and increased survival following therapeutic treatment with SPLP (βgal) ...................... 142  xi  Figure 5.7: Mice vaccinated with SPLP (βgal) and LN CpG-ODN exhibit increased CTL activity against both the βgal expressing cell line CT26.CL25 and the parental non-βgal expressing cell line CT26 following therapeutic treatment with SPLP (βgal). ......................... 144  xii  ABBREVIATIONS ACT APC BCG βgal CpG CMFDG CMV cpm CTL DC Di-I perchlorate DNA DODAC DODMA DOPE DOTMA trimethylammoniumchloride dsDNA DSPC ELISA EPR FBS HIV i.d. IFN IL i.m. i.p. IRAK i.v. KO LPS luc MAGE-1 MFI MHC MyD88 NK ODN ONPG PAMP PBS pDNA  adoptive cell transfer antigen-presenting cell bacillus of calmette guerin beta-galactosidase unmethylated cytosine-guanosine dinucleotide motif 5-chloromethylfluorescein di-β-D-galactopyranoside cytomegalovirus counts per minute cytotoxic T-lymphocyte dendritic cell 1,1'-dioctadecyl-3,3,3',3'- tetramethylindocarbocyanine deoxyribonucleic acid N,N-Dioleyl-N,N-dimethylammonium chloride 1,2-dioleyloxy-N,N-dimethylaminopropane 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine N-[-1-(2,3-dioleyloxy)proply]-N,N,Ndouble stranded DNA 1,2-distearoyl-sn-glycero-3-phosphocholine enzyme linked immunosorbant assay enhanced permeability and retention fetal bovine serum human immunodeficiency virus intradermal interferon interleukin intramuscular intraperitoneal interleukin-1 receptor activated kinase intravenous knock out lipopolysaccharide luciferase melanoma antigen-1 mean fluorescence intensity major histocompatability complex myeloid differentiation primary response gene 88 natural killer oligodeoxynucleotide o-nitrophenyl β-D-galactopyranoside pathogen associated molecular pattern phosphate buffered saline plasmid DNA  xiii  PEG PO POPC PS RES RNA SEM SD SVF TAA TLR Th1 Th2 TNF TRAF6  polyethylene glycol phosphodiester 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine phosphorothioate reticuloendothelial system ribonucleic acid standard error of the mean standard deviation spontaneous vesicle formation tumour associated antigen toll-like receptor T-helper 1 T-helper 2 tumour necrosis factor tumour necrosis factor receptor associated factor 6  xiv  ACKNOWLEDGEMENTS There are so many people that I would like thank for helping me achieve this goal. First and foremost I would like to thank Pieter for providing me with the opportunity to study in his laboratory and for providing me with the resources and freedom to complete this project. I would especially like to thank Ying Tam for acting as both a mentor and as a co-supervisor to me. Ying, thank you for your constant support and encouragement, for always entertaining my point of view, and for always offering yours. Thank you for patience, your enthusiasm, and your attempts to re-introduce me to the run-on sentence. It has been a pleasure working with you. I could not have done it without you and I could not have asked for a better mentor. To everyone in the Cullis lab, thank you for your support, your encouragement and for the constant entertainment. Sue, I would especially like to thank you for your help and support, both experimental and emotional, your constant willingness and encouragement, and your promise to see things through to the end, even if the end was the next morning. May, I would like to thank you for your support along the long road to understanding the complexity of performing animal-based research. Most importantly, I would like to thank you both for your friendship. On a personal note, I would like to thank the ladies of the biochemistry department, Sonya Cressman, Barbara Lelj, Leanne McHardy and Nicole Quenneville. Thank you for your friendship and your support. I would like to thank my family, Mom, Dad, Kristin, Kaidre and Pale. You have all been so supportive and so encouraging and I could not have done it without you. Pale, I would like to thank you for encouraging me  xv  to never give up, for helping me see this through to the end and for standing beside me as I learned the valuable lesson of how to fail and become stronger as a result of it.  xvi  CO-AUTHORSHIP STATEMENT Research in Chapter Two of this thesis, entitled “Effects of intravenous and subcutaneous administration on the pharmacokinetics, biodistribution, cellular uptake and immunostimulatory activity of CpG ODN encapsulated in liposomal nanoparticles” was performed in collaboration with staff at INEX Pharmaceuticals. The research included in Figures 2.1, 2.2, 2.3 and 2.4 was performed by myself in collaboration with INEX Pharmaceuticals staff, while the research included in Figures 2.5, 2.6 and 2.7 was performed by INEX Pharmaceuticals staff. All data analysis, with the exclusion of statistical analysis, was performed by myself and I was completely responsible for preparing the manuscript. Research in Chapter Three of this thesis, entitled “Stabilized plasmid lipid particles activate APCs through TLR9-dependent and independent pathways in vivo” was designed and performed by myself in conjunction with Ms. Susan D. de Jong. All data analysis, with the exclusion of statistical analysis, was performed by myself and I was completely responsible for preparing the manuscript. For the remainder of this thesis I am solely responsible for the identification and design of the research program, performance of the research, analysis of the data and preparation of the manuscripts, with the exception of statistical analysis which was performed throughout the thesis with the assistance of Susan D. de Jong and Ewan Ramsay.  xvii  INTRODUCTION 1.1 Vaccines Over the last century, the development and widespread use of vaccines against a variety of infectious diseases has had a tremendous impact on human health worldwide. Vaccination, a concept first demonstrated over 200 years ago by Edward Jenner, has resulted in the global eradication of smallpox, the elimination of poliomyelitis from the majority of the world, and the control/prevention of millions of deaths each year from other infectious diseases such as measles and influenza (Hilleman 2000). Despite the overall success of vaccination, effective vaccines against many pathogens are currently unavailable. Furthermore, traditional vaccines composed of attenuated live organisms, although efficacious in producing diverse and persistent immune responses, are associated with rare, but serious risks including reversion or mutation to a more pathogenic state, and provoking disease in immune-compromised individuals.  While new  generation vaccines, such as those using inactivated virus or recombinant proteins and peptides are viewed as potentially safer than traditional vaccines, they are often less immunogenic and, although efficient at inducing antibody responses, demonstrate a reduced (or lack of) ability to induce cell mediated responses required for effective vaccination against intracellular pathogens and tumours. This has led to the generation of alternate vaccination strategies aimed at inducing more effective cellular immune responses, such as the delivery of DNA encoding the pertinent antigen. Based on the efficacy of DNA vaccines in small animal models, they have moved very rapidly from the laboratory into clinical trials, however clinical findings highlight that while DNA vaccines hold promise as means of vaccinating against intracellular pathogens and tumours, further development is required to increase the potency of DNA vaccines (Gurunathan, Klinman et al. 2000; Cui 2005).  1  1.1.1 Overview of vaccine immunology The nature of an immune response against an antigen or infectious agent is determined by how the antigen is processed and presented by antigen presenting cells (APCs) to T-cells (Figure 1.1). For vaccines in which the antigen is provided exogenously, such as for recombinant peptide or protein vaccines or killed/inactivated pathogens, the antigen is taken up by APCs via phagocytosis or endocytosis, is processed in the endosome by the major histocompatability complex (MHC) class II pathway and is presented on the cell surface as MHC class II – antigenderived peptide complexes. Recognition of these complexes by antigen-specific CD4+ T-cells, in the presence of appropriate co-stimulatory signals provided by interaction of CD28 and CD40L on the surface of the T-cell with CD80, CD86 and CD40 on the surface of activated APCs, leads to the stimulation of these CD4+ T cells, and promotes the priming of T-helper 2 (Th2)-biased antigen-specific humoral immune responses. For vaccines which allow expression of the antigen endogenously by the host APCs (DNA vaccines or live viral vaccines) the relevant antigen is degraded by the proteasome, processed by the MHC class I pathway and presented on the cell surface as MHC class I – antigen-derived peptide complexes. Recognition of these complexes by antigen-specific CD8+ T-cells, through a similar process described for CD4+ cells, leads to the stimulation of antigen-specific CD8+ T cells and promotes the priming of Thelper 1 (Th1)-biased antigen-specific cytotoxic T-lymphocyte (CTL) mediated cellular immune responses (Gurunathan, Klinman et al. 2000). The generally accepted paradigm that MHC class I molecules present peptides generated from endogenous antigens, whereas antigenic peptides derived from exogenous proteins are displayed by MHC class II molecules, does not always hold true.  Professional APCs, particularly dendritic cells (DCs), have demonstrated the ability to  generate MHC-I-associated peptides from exogenous materials and present them to CD8+ CTL precursors through processes termed cross-presentation and cross-priming, respectively (discussed in more detail in section 1.2.1). Although many unresolved questions concerning the 2  exact molecular pathways of the cross-priming process still exist this alternative pathway has been implicated to play a role in the priming of CD8+ T-cell responses to pathogens as well as tumour associated antigens (TAA) in vivo and has recently attracted a lot of research interest as a better understanding of its mechanism may prove valuable for vaccine development (Melief 2003; Basta and Alatery 2007).  Figure 1.1: Schematic representation of antigen processing and presentation in an APC following genetic vaccination. Upon cellular uptake of DNA from a genetic vaccine (A), the gene is transcribed and translated (B) intracellularly to create a full length protein. This protein which represents an antigen is subsequently degraded into smaller peptide fragments by the proteosome (C), which are transported into the endoplasmic reticulum for loading onto MHC Class I molecules (D). The MHC I-peptide complex is then transported to the surface for presentation to antigen-specific CD8+ CTLs (E). This complex, along with the appropriate co-stimulatory signals on the surface of the APC, would result in the activation and proliferation of the CD8+ CTL. Alternatively, in the case of somatic cell expression and presentation of the antigen fragment (thereby marking it as a target), this CD8+ CTL would mediate lysis of the target cell. In APCs only, a MHC Class II complex is transported from the endoplasmic reticulum (F) in vesicles destined to fuse with endosomes containing exogenously captured antigen released from other transfected cells (G). The antigen is degraded into short peptides in the late endosomes to allow MHC Class II – peptide loading, which is then transported for expression on the surface in tandem with co-stimulatory molecules for the activation of CD4+ helper T-cells (H). The exclusivity of these pathways for processing and presentation of an endogenous or exogenous antigen is not complete and cross-presentation, a situation in which endogenously captured antigen is processed via the MHC I pathway for presentation to CD8+ CTLs is not depicted here. Adapted from Little and Langer 2005.  3  Once activated in an antigen-specific manner, T-cells proliferate into clones capable of either destroying target cells expressing the relevant antigen in the context of MHC class I molecules (CD8+ CTLs) or clones capable of directing the type of immune response elicited (CD4+ T helper cells). Activated CD4+ T helper cells are important and often required for initiating and amplifying CD8+ T-cell responses, by directly providing co-stimulatory cytokines and by indirectly up-regulating co-stimulatory molecules on APCs. However, in cases where the antigen provides both strong inflammatory or danger signals and a source of antigen, activation of naïve CD8+ T cells can occur in the absence of CD4+ T-cells (Castellino and Germain 2006; Emens 2006). The cytokine milieu present at the time of initial T-cell priming polarizes the immune response to become either a Th1-biased cytotoxic cell-mediated response, characterized by secretion of cytokines such as interferon-gamma (IFN-γ), interleukin (IL)-2 and IL-12 along with the production of IgG2a antibodies, or a Th2 humoral response characterized by secretion of IL-4 and IL-10 along with IgG1 antibodies. Typically cytokines that promote the generation of Th1 responses (i.e. IL-12, IFN-γ) tend to inhibit the generation of Th2 responses and vice versa (Gurunathan, Klinman et al. 2000). As Th2-biased antibody responses are required for the immunity of preventive vaccines and the control of extracellular pathogens, while Th1-biased cell-mediated immune responses are critical for antitumour immunity and immunity against chronic viral or intracellular infections, it is essential that the appropriate type of immune response is raised during vaccination (Gurunathan, Klinman et al. 2000; Emens 2006) and it is therefore crucial that new generation vaccines are designed with the specific application in mind.  1.2 DNA vaccines The concept of DNA-based vaccination rests on the findings of Wolff et al in the 1990’s that intramuscular inoculation of plasmid DNA (pDNA) into an animal could result in protein 4  expression within the muscle cells, introducing the concept that naked DNA could be delivered and direct protein expression in vivo (Wolff, Malone et al. 1990). Studies demonstrating the generation of transgene-specific humoral immune responses in the serum of mice following gene gun administration of plasmid encoding human growth hormone (Tang, DeVit et al. 1992) and the ability of DNA vaccines to protect mice against a lethal influenza challenge (Fynan, Webster et al. 1993; Ulmer, Donnelly et al. 1993) through the generation of both antibody and CD8+ CTL responses (Ulmer, Donnelly et al. 1993) provided the first examples of the capability of vaccination with pDNA to produce protective immune responses.  Over the last decade  numerous attempts have been made to use plasmid-based immunization to generate prophylactic or therapeutic immunity against a wide variety of diseases, including infectious diseases (Laddy and Weiner 2006), cancers (Prud'homme 2005), and allergic diseases (Weiss, Hammerl et al. 2005). DNA vaccines provide several important advantages over current vaccine strategies such as live or attenuated viral vaccines or recombinant peptide/protein-based vaccines in that they mimic the effects of natural viral infection in their ability to endogenously express foreign protein and have it processed in a manner that induces MHC class I-restricted CD8+ T-cell responses without the need for replication or infectious agents. Furthermore, because plasmids used for genetic immunization inherently contain immunostimulatory motifs, they function both as a source of antigen and immune stimulation to promote the generation of strong Th1 type responses (discussed in detail in section 1.6). In addition, DNA vaccines can be quickly and easy produced with high purity on a large scale and are stable compared to proteins or biological agent vaccines, removing the need for cold-chain storage (Schalk, Mooi et al. 2006). DNA vaccines first entered the clinic in 1994 (MacGregor, Boyer et al. 1998) for human immunodeficiency virus (HIV), and since then Phase I/II trails for HIV, malaria, tuberculosis, influenza, hepatitis B and C and various cancers including prostate cancer, colon cancer and 5  metastatic melanoma have demonstrated that DNA vaccines are well tolerated with no serious local or systemic adverse effects (Schalk, Mooi et al. 2006). However, only a handful of DNA vaccines have progressed to Phase III testing and to date clinical studies in humans have shown lower efficacy than expected based on animal studies. While a DNA-based vaccine has not been approved for use in humans, three veterinary DNA vaccines have been licensed; one for West Nile Virus (in horses), a second for infectious hematopoetic necrosis (in salmon) (Laddy and Weiner 2006) and a third for the therapeutic treatment of canine melanoma. Despite their limited success in the clinic DNA vaccines still hold unique promise as a strategy for vaccination against infectious disease and cancer. Based on our growing understanding of the mechanism of action of DNA vaccines, rational approaches to increase the potency of DNA vaccines, such as formulation to enhance their uptake, the design of better promoters and the co-administration of adjuvants, are being taken and offer promise in overcoming the shortcomings of the first generation DNA vaccines currently in the clinic.  1.2.1 Mechanism of antigen presentation by DNA Vaccines There are at least three proposed mechanisms by which the antigen encoded by pDNA can be processed and presented to the immune system to elicit an immune response: (a) direct priming of antigen-specific T-cells by transfected somatic cells (MHC class II negative cells) (b) priming of antigen-specific T-cells by transfected APCs and (c) cross-priming in which transfection of somatic cells or APC results in protein expression and proteins released are taken up by other professional APCs and presented to T-cells via the MHC class I or MHC class II pathway (Gurunathan, Klinman et al. 2000; Melief 2003; Cui 2005; Basta and Alatery 2007). The role of bone marrow-derived APCs in the induction of the immune response after DNA vaccination was first demonstrated by Iwasaki et al. (Iwasaki, Torres et al. 1997) and Corr et al. (Corr, Lee et al. 1996) in studies where bone marrow reconstitution demonstrated that the 6  immune response generated following DNA vaccination was restricted to the haplotype of the reconstituted bone marrow, ruling out the possibility that somatic cells directly present antigen to T-cells. Similarly, the finding that removing the muscle within 10 minutes of immunization does not alter the subsequent immune response provided additional evidence against the need for the transfection of somatic cells at the site of injection (Torres, Iwasaki et al. 1997). The direct transfection of professional APCs, specifically DCs, has been documented following several delivery routes including gene gun mediated intradermal (Condon, Watkins et al. 1996; Porgador, Irvine et al. 1998; Garg, Oran et al. 2003), scarification (Akbari, Panjwani et al. 1999) or intramuscular (Chattergoon, Robinson et al. 1998). While most reports detect the presence of only a very small number of directly transfected DCs in the lymph nodes draining the site of vaccination (50-100 antigen positive DCs per inguinal lymph node, representing approximately 0.5% of the DC population) (Porgador, Irvine et al. 1998) more recent studies performed by Garg et al indicate that transfection levels following gene gun mediated vaccination may be much higher than previously observed, with approximately 12% of the purified DCs from draining lymph nodes positive for antigen expression 60 hours after immunization (Garg, Oran et al. 2003). Reports that as few as 500 DCs transfected ex vivo are sufficient to elicit an immune response of similar magnitude to that observed following standard gene gun immunization (Takashima and Morita 1999) support the notion that the direct transfection of APCs plays a key role in the induction of immune responses following genetic vaccination. The concept of cross-priming, where CD8+ T-cell responses are triggered by APCs that have taken up the antigen generated by transfected somatic cells or other APCs, rather than endogenously synthesizing antigen themselves, provides an additional mechanism by which DNA immunization can promote the generation of antigen-specific immune response. Although peptides derived from exogenous sources are generally excluded from presentation on MHC 7  class I molecules there are now examples demonstrating that this can occur in vivo particularly in the presence of proper adjuvants or delivery systems (Harding and Song 1994; Falo, Kovacsovics-Bankowski et al. 1995).  Pivotal studies demonstrating cross-priming from  myocytes to APCs were performed by Ulmer et al, in which the transfer of myoblasts expressing an influenza nucleoprotein into mice induced nucleoprotein-specific antibody and CTL responses restricted by the MHC haplotype of the recipient, not the donor (Ulmer, Deck et al. 1996). Overall, evidence suggests that APCs, not somatic cells, directly induce immune responses after DNA vaccination, however, transfected somatic cells may serve as a reservoir for antigen and the relative importance of the two mechanisms of antigen uptake, direct transfection vs. cross-priming, is not totally clear.  As the route and method of administration naturally  influences the cell-types encountered and transfected by a DNA vaccine, and subsequently the outcome of the immune response generated, the influence of direct transfection vs. cross-priming on the resultant immune response primed may depend heavily on the route of administration and this should be considered during the optimization and development of novel DNA vaccine approaches (Fynan, Webster et al. 1993; Yokoyama, Zhang et al. 1996; Alpar and Bramwell 2002; Cui 2005).  1.2.2 Routes of administration of DNA vaccines A variety of routes of pDNA administration, including intramuscular (i.m.), intradermal (i.d.), intravenous (i.v.), intraperitoneal (i.p.), oral, intranasal, vaginal, and direct application onto the skin or via scarification have been investigated for vaccination purposes (Gurunathan, Klinman et al. 2000; Alpar and Bramwell 2002). Traditionally, i.m. injection of naked DNA and genegun mediated i.d. delivery of DNA-coated microprojectiles have been the most popular routes of administration employed.  In terms of the amount of DNA used, gene-gun mediated 8  immunization is considered to be the most effective mode of delivery (Fynan, Webster et al. 1993), however a serious problem associated with gene-gun vaccination is that the immune responses elicited are usually Th2-polarized (Feltquate, Heaney et al. 1997). It is now clearly evident that the nature of the adjuvant, the route of administration and the choice of delivery vehicle all profoundly affect the outcome of DNA immunization (Fynan, Webster et al. 1993; Yokoyama, Zhang et al. 1996; Alpar and Bramwell 2002; Cui 2005). Although rarely investigated, due to rapid degradation of DNA by serum nucleases, i.v. administration of pDNA vaccines have demonstrated the ability to induce antigen specific immune responses, when delivered in the free form (Fynan, Webster et al. 1993; McCluskie, Brazolot Millan et al. 1999; Cui, Asada et al. 2003), by hydrodynamic limb vein delivery (Neal, Bates et al. 2007), or when complexed with cationic lipid carriers (Yamamoto, Yamamoto et al. 1992; Yokoyama, Zhang et al. 1996). Based on the advantage of direct access to the relatively large number of APCs in the spleen, systemic administration of genetic vaccines and/or associated adjuvants represents an attractive alternate approach to traditional routes of administration if the barriers of poor stability and delivery to target cells can be addressed.  1.2.3 Carrier systems for DNA vaccines Naked DNA is highly susceptible to serum and extracellular nucleases and naked pDNA is rapidly degraded following administration by the i.v. (Kawabata, Takakura et al. 1995; Lew, Parker et al. 1995) and i.m. routes (Davis, Michel et al. 1994).  It naturally follows that  protecting plasmid DNA from extracellular degradation thus enhancing its lifetime in vivo and promoting uptake by immunologically relevant cells should enhance the efficacy of DNA vaccines. In attempt to address this, several methods of carrier-mediated DNA transfection have been developed including gene-gun mediated delivery, encapsulation in cationic liposomal or polymer based carrier systems, targeted carrier systems and attenuated organisms (Gurunathan, 9  Klinman et al. 2000; Alpar and Bramwell 2002). Overall, strategies which protect the plasmid from the extracellular environment, and enhance its uptake or targeting to APCs increase the efficacy of the DNA vaccine (Chen and Huang 2005; Little and Langer 2005), however due to their cationic nature most non-viral DNA delivery systems are associated with significant toxicity which limits their use in vivo and restricts the routes by which they can be administered (discussed in section 1.5.2) (Filion and Phillips 1997; Tousignant, Gates et al. 2000; Loisel, Le Gall et al. 2001; Lv, Zhang et al. 2006). It is now understood that the development of safe and efficient gene carriers is one of the prerequisites for the success of DNA based vaccines.  1.2.4 Safety of DNA vaccines Although DNA vaccines are thought to be safer than traditional live viral vaccines, several safety concerns exist. These include the risk of integration of pDNA into the recipient’s genome which could result in malignant transformation, genomic instability or cell growth dysfunction; the induction of autoimmune responses; and the induction of tolerance rather than immunity. Although each of these safety concerns have, to some extent, been demonstrated to occur in murine and/or non-human primate models to date, DNA vaccines which have entered the clinic for initial safety and immunogenicity testing in humans for various infectious diseases and cancers have been well tolerated with no serious local or systemic adverse effects (Schalk, Mooi et al. 2006).  1.3 Cancer immunotherapy and cancer vaccines The majority of cancer patients are treated by a combination of surgery, radiation and/or chemotherapy. Whereas the primary tumor can, in many cases, be efficiently treated by a combination of these standard therapies, preventing the metastatic spread of the disease through disseminated tumor cells  is often not  possible.  Furthermore, conventional cancer 10  chemotherapeutic approaches which target general abnormal cancerous processes, such as rapid cell division, are accompanied by varying degrees of toxicity.  In contrast, cancer  immunotherapy approaches are based on the exceptional specificity of the immune system and offer a nontoxic approach with the potential to eradication disseminated tumor cells and generate long-term tumour-specific protective memory (Schuster, Nechansky et al. 2006; Ryan, Gantt et al. 2007). The concept of using the immune system to target tumours dates back to as early as the 1890’s when William B. Coley successfully treated patients with sarcoma using bacterial toxins (Coley 1896) and 1909, when Paul Ehrlich vaccinated animals with tumour cells suggesting, for the first time, that the immune system can be directed to recognize and eliminate developing tumours. Currently, one of the most active agents for superficial bladder cancer therapy is the nonspecific immune stimulation accompanying the administration of Bacillus of Calmette Guerin (BCG) (Bassi 2002). As a consequence of genomic instability, tumour cells differ from their normal counterparts in antigenic composition and biological behaviour. Since the identification of the first TAA, melanoma antigen-1 (MAGE-1), (van der Bruggen, Traversari et al. 1991; Traversari, van der Bruggen et al. 1992) it has been well established that autologous tumour-specific T-cells can be isolated from tumour-bearing mice and cancer patients (Rosenberg 1999). Although tumours express TAAs, which have the potential to be recognized by the immune system, tumours are usually poorly immunogenic. This is in part because TAAs are typically mutated, inappropriately expressed or overexpressed self-antigens, and are therefore generally weakly immunogenic as a consequence of the intra-thymic deletion of autoreactive T-cells as a mechanism of self-tolerance (Sioud 2007). In addition, due to their lack of expression of costimulatory molecules, tumors behave similar to normal nonlymphoid peripheral tissues and fail to activate immune responses even in the existence of self-specific T-cells, promoting peripheral 11  T-cell ignorance or tolerance (Speiser, Miranda et al. 1997). Furthermore, it is currently believed that in addition to being poorly immunogenic, tumours employ a number of active mechanisms to suppress host immunity, thus avoiding eradication (Zou 2005). Although beyond the scope the discussion here, it is now widely accepted that numerous innate and adaptive immune effector cells participate in the recognition, destruction and ultimately the progression of cancer cells, a process which is known as cancer immunosurveillence or cancer immunoediting (Pardoll 2003; Dunn, Old et al. 2004; Zitvogel, Tesniere et al. 2006; Bui and Schreiber 2007). The observations that tumours have the potential to naturally elicit immune responses both in animals and humans (Boon, Cerottini et al. 1994; Houghton 1994; Boon and van der Bruggen 1996) and that the infusion of ex vivo expanded autologous tumor-reactive CD8+ T cells to patients with cancer can mediate objective cancer regression (Dudley, Wunderlich et al. 2002; Dudley, Wunderlich et al. 2005) has promoted the development of numerous cancer immunotherapy approaches. Cancer vaccines, a form of active cancer immunotherapy, are focused on activating the immune system against TAAs expressed by the tumour, inducing de novo antitumour immune responses or potentiating existing antitumour immunity in the host. Vaccination approaches taken to date have included injection of irradiated whole tumor cell vaccines, tumour cell lysates, TAA-derived proteins or peptides, delivery of TAA encoding genes, as well as APC-based approaches employing the use of autologous DCs transfected ex vivo with TAA expressing DNA or RNA (Tabi and Man 2006).  Currently there are no  therapeutic cancer vaccines licensed for clinical use in Canada, Europe or the United States. Therapeutic cancer vaccines that are being tested or are about to be tested in Phase III trials are based on approaches which include TAA-pulsed DCs (Sipuleucil-T by Dendron), whole tumour cell vaccines (GVAX by Cell Genesys), personalized protein-based idiotype vaccines (MyVax by Genitope) and pDNA-based delivery of immunostimulatory molecules to tumours and APCs (Allovectin-& by Vical) (Tabi and Man 2006). 12  Alternatively, several non-specific or passive immunotherapies have been approved for the treatment of cancer and include cytokine (IL-2, IFN-α2a, IFN-α2b) and monoclonal antibody (Rituxan, Herceptin) based therapies (Reang, Gupta et al. 2006).  1.3.1 DNA vaccines for cancer Following initial studies which demonstrated that vaccination with pDNA could protect against infectious disease, it was shown that injection of pDNA encoding TAAs could result in the induction of immune responses which were protective against tumour challenge in animal models (Conry, LoBuglio et al. 1995; Bright, Beames et al. 1996). DNA vaccines for cancer immunotherapy possess a number of advantages over other cancer vaccine approaches. The ability of DNA vaccines to prime the generation Th1-biased cellular immune responses is of particular importance as animal models have demonstrated that T-cell mediated immunity is critical in the rejection of transplanted tumours (Melief 1992) and CD8+ T cells have been identified as potent effectors of the adaptive immune response against tumours in humans (Rosenberg 2001; Boon, Coulie et al. 2006). In addition DNA vaccines possess immunostimulatory motifs as an internal adjuvant (discussed in section 1.4.1) and are thus able to provide both a potent immunogenic stimulus and a source of antigen, which can remove the requirement of CD4+ T-cells for the activation of naïve CD8+ T cells (Castellino and Germain 2006; Emens 2006). Furthermore, vaccination with pDNA has demonstrated the potential to break natural immune tolerance to self-antigens (Davis, Brazolot Millan et al. 1997; Weber, Bowne et al. 1998; Amici, Smorlesi et al. 2000; Hawkins, Gold et al. 2000), a required characteristic of cancer vaccines due to the fact that TAAs are most often self-molecules and that the presence of established tumours indicates T-cell ignorance or tolerance towards TAAs in the tumour environment.  13  In the case of tumor vaccines, plasmid-based delivery using naked pDNA or synthetic non-viral vectors also has the advantage of focusing the immune response against the relevant tumour antigens in the absence of competing immunogenic molecules which can be carried by viral vectors, which is advantageous since TAAs are almost all poorly immunogenic selfmolecules (Tuettenberg, Jonuleit et al. 2003). Furthermore, the delivery of genes encoding TAAs can easily be coupled to the delivery of additional genes such as cytokines or costimulatory molecules to enhance and/or modify the immune response. In addition, unlike peptide-based vaccination strategies, vaccination with plasmid encoding the entire TAA circumvents limiting the prospective patient population on the basis of HLA restriction as well as providing additional T-helper epitopes to enhance immune responsiveness. DNA cancer vaccines are currently being tested in Phase I and II and to a lesser extent phase III clinical trials for a number of cancers including melanoma, leukemia, prostate cancer and colorectal cancer. TAAs targeted include gp100, tyrosinase, patient specific idiotypes, the prostate-specific membrane antigen, carcinoembryonic antigen and T54 (Prud'homme 2005). Although the full potential of DNA-based therapeutics has yet to be demonstrated in the clinic, it is well recognized that DNA vaccines offer unique promise, specifically for the generation of a cancer vaccine, and steps taken to enhance their potency should validate their capability as cancer immunotherapeutics.  1.4 Immunostimulatory properties of unmethylated CpG motifs Bacteria and bacterial extracts were originally used as adjuvants over a century ago by William Coley for the treatment of cancer (Coley 1896) and Freund, who used the whole mycobacterial extract as a major constituent in his adjuvant formula (Freund 1951). Their immunostimulatory activity is based on the fact that the innate immune system of vertebrates contains a number of highly conserved receptors that are activated by exposure to pathogen-associated molecular 14  patterns (PAMPs), expressed by a diverse group of infectious microorganisms. This provides a mechanism through which to rapidly respond to viral or bacterial infection, limiting early spread and priming the generation of adaptive immune responses directed against pathogen-specific antigens (Medzhitov and Janeway 1997). The immunostimulatory properties of bacterial DNA were first reported by Tokunaga and  Yamamoto  (Tokunaga,  Yamamoto  et al.  1984)  who  showed that  synthetic  oligodeoxynucleotides (ODN) with sequences patterned after those found in bacterial DNA could activate natural killer cells to secrete IFN-γ (Tokunaga, Yamamoto et al. 1984; Yamamoto, Yamamoto et al. 1992), but it was experiments performed by Kreig et al which identified unmethylated cytosine-guanine (CpG) dinucleotide motifs in the particular base context 5’purine-purine-CpG-pyrimidine-pyrimidine’3 as the cause of the observed immune stimulation (Krieg, Yi et al. 1995; Rankin, Pontarollo et al. 2001). Unmethylated CpG motifs are recognized by the innate immune system as PAMPs through interaction with the pattern recognition receptor Toll Like Receptor 9 (TLR9) which is located inside the endocytic compartment of professional APCs (Hemmi, Takeuchi et al. 2000; Takeshita, Leifer et al. 2001). Differences in the methylation status and utilization patterns of CpG dinucleotides in the genomes of prokaryotes and eukaryotes results in unmethylated CpG motifs being present at much higher frequency in the genomes of prokaryotes (Razin and Friedman 1981; Cardon, Burge et al. 1994). In humans, plasmacytoid DCs and B-cells are the primary cell types that express TLR9 and thus are directly activated by exposure to unmethylated CpG motifs, while in mice monocytes, macrophage and myeloid DCs also express this receptor (Hochrein, O'Keeffe et al. 2002). It has been proposed that evolutionary divergence among the TLR9 molecules has resulted in the optimal stimulatory sequence motif (CpG motif and flanking bases) differing between species (Rankin, Pontarollo et al. 2001), however recent findings indicate that 15  sequence-specific specificity may be an artifact of the use of ODN containing the phosphorothioate (PS) backbone (for stability reasons) as this is not observed for the natural phosphodiester (PO) ODN (Roberts, Sweet et al. 2005). Engagement of TLR9 with CpG-DNA in the endosome triggers a cell signaling cascade involving, sequentially, myeloid differentiation primary response gene 88 (MyD88), interleukin1 receptor activated kinase (IRAK), tumour necrosis factor receptor (TNFR)-associated factor 6 (TRAF6), and activation of NFkB (Takeshita, Gursel et al. 2004) (Figure 1.2). Activation by CpG DNA initiates an immunostimulatory cascade that culminates in the maturation, differentiation, and proliferation of DCs, natural killer (NK) cells, T cells, and monocytes/macrophages and the release of pro-inflammatory [IL-1, IL-6, IL-18, and tumour necrosis factor alpha (TNF-α)] and Th1-promoting cytokines (IFN-γ and IL-12) and chemokines (Krieg 2002) (Figure 1.3). The overall outcome of CpG-DNA mediated TLR9 stimulation is the promotion of a strong Th1-biased immune response (thus inhibiting the generation of Th2 responses as the two typically compete with one another) which is ideal for the generation of antigen specific CD8+ CTLs, making CpG-DNA a particularly useful adjuvant for vaccines against cancer or intracellular pathogens (Krieg 2006). One mechanism proposed to contribute to the strong adjuvant activity of CpG ODN is their ability to stimulate DC maturation and differentiation, which results in enhanced activation of Th1 cells and can promote strong CTL responses, even in the absence of CD4+ T-cell help (Sparwasser, Vabulas et al. 2000). Indeed, CpG ODN mediated stimulation results in the up-regulation of a variety of co-stimulatory molecules in professional APCs, including cell surface expression of MHCs, CD40 and CD86 (Jakob, Walker et al. 1998; Sparwasser, Koch et al. 1998) increasing their antigen-presenting capacity and ability to stimulate T cells (Jakob, Walker et al. 1998).  16  Cytoplasm  Endosome  Figure 1.2: Schematic representation of the CpG-DNA/TLR9-mediated cellular signaling pathway. Binding of CpG-DNA to TLR9 in the endosome promotes TLR9 dimerization which recruits the adaptor protein MyD88 to the cytoplasmic portion of TLR9. Interaction of IRAK4 with MyD88 promotes the activation of IRAK4 and the phosphorylation of IRAK1, which recruits TRAF6. Phosphorylated IRAK1, together with TRAF6, dissociates from the receptor where TRAF6 activates TAK1 which further activates the MAPK signaling cascades, JNK1 and p38. TRAF6 also activates the IKKs which phosphorylates IκB. Phosphorylated IκB then dissociates from NF-κB and becomes degraded. NF-κB translocates to the nucleus and can, alone or in conjunction with JNK1 and p38, induce expression of proinflammatory cytokines. Expression of these cytokines promotes several immunomodular effects such as cell-mediated immunity, apoptosis, and direct antimicrobial activity.  17  APC Mφ or DC  Activate NK cells  Figure 1.3: Mechanism by which CpG-ODNs facilitate innate and adaptive immune responses. CpG-ODN directly activate APCs (macrophage, DCs, B cells), creating an immune milieu that is rich in pro-inflammatory Th1-type cytokines. This innate immune response forms a foundation on which antigen-specific adaptive immunity is based. In particular, by improving the function of professional APCs, CpG ODNs facilitate the generation of humoral and cellular vaccine-specific immunity. Adapted from Klinman 2004 (Klinman 2004).  1.4.1 Role of CpG in DNA vaccines The unmethylated CpG motifs naturally present in pDNA activate APCs of the innate immune system, inducing the up-regulation of co-stimulatory molecules and the production of proinflammatory and Th1-biased cytokines, in a manner similar to that observed for CpG-ODN (Leclerc, Deriaud et al. 1997; Roman, Martin-Orozco et al. 1997; Zelenay, Elias et al. 2003). Thus, CpG motifs are believed to act as an internal adjuvant and contribute to the immunogenicity of DNA vaccines, promoting the generation of cellular immune responses towards encoded antigens (Sato, Roman et al. 1996; Klinman, Yamshchikov et al. 1997; Leclerc, Deriaud et al. 1997; Schneeberger, Wagner et al. 2004).  In support of this, it has been 18  demonstrated that reducing the number of unmethylated CpG motifs present (by methylase treatment) significantly reduced the immunogenicity of DNA vaccines (Klinman, Yamshchikov et al. 1997) while increasing the number of motifs enhances the induction of vaccine-specific humoral and cellular immune responses (Sato, Roman et al. 1996) (Schneeberger, Wagner et al. 2004). Similarly, it has been demonstrated that the humoral response, antigen-specific cellular response and in vivo antitumour efficacy elicited by a DNA vaccine can be augmented by coadministering free CpG ODN or non-coding pDNA (Klinman, Yamshchikov et al. 1997; Kojima, Xin et al. 2002; Ren, Zheng et al. 2004). However, observations made by numerous groups strongly suggest that the timing between the administration of free CpG-containing DNA, as an adjuvant, and a naked DNA vaccine is critical as it has been demonstrated that while delivering CpG-ODN after the vaccine typically results in an immunogenic boost (Kojima, Xin et al. 2002; Ren, Zheng et al. 2004) co-administration often inhibits the activity of the vaccine (Weeratna, Brazolot Millan et al. 1998). Reduction in the resultant immune response following co-administration is thought to stem from reduced gene expression due to competitive interference for uptake by APCs (Weeratna, Brazolot Millan et al. 1998) and/or the downregulation of viral promoters used by pDNA by CpG-induced cytokines (IFN-γ and TNF-α) (Harms and Splitter 1995; Tan, Li et al. 1999), both of which are proposed to be alleviated when CpG-ODN is administered after the DNA vaccine at a time when antigen is already being expressed (Kojima, Xin et al. 2002; Ren, Zheng et al. 2004). The observation that DNA vaccines remain immunogenic when administered to TLR9 knock-out (KO) mice has recently brought into question the impact of CpG motifs on DNA vaccine immunogenicity. Original results from Spies et al. demonstrating that DNA vaccines remain immunogenic when administered to TLR9 KO mice, and that the priming of antigenspecific CTLs and clonal expansion of CD8 T cells were equal in TLR9-positive and TLR9- or MyD88- negative mice (Spies, Hochrein et al. 2003) were later confirmed by Babiuk et al 19  (Babiuk, Mookherjee et al. 2004). However, more recent studies performed by Tudor et al demonstrate that although TLR9-deficient mice are able to mount Th1-biased antigen-specific antibody and IFN-γ responses following DNA vaccination, levels were lower than in wildtype mice. The observations made in these studies that DCs lose their ability to secrete IL-12 and IFN-γ in response to both CpG and naked pDNA ex vivo in the absence of TLR9, suggests that although TLR9 signaling is not needed for eliciting T and B-cell responses to DNA encoded antigens it plays a role in enhancing plasmid-adjuvant effects on antigen-specific immune responses (Tudor, Dubuquoy et al. 2005). Interestingly, when the immunostimulatory activity of pDNA is assessed ex vivo in TLR9 -/- macrophage and DCs it appears that naked pDNA acts in a completely TLR9-dependent manner (Spies, Hochrein et al. 2003; Tudor, Dubuquoy et al. 2005), however, complexing pDNA with cationic lipids confers the ability of pDNA to signal via TLR9-dependent and independent pathways, albeit responses are reduced in the absence of TLR9 (Tudor, Dubuquoy et al. 2005; Yasuda, Ogawa et al. 2005; Yasuda, Yu et al. 2005). Together these findings question the impact of CpG motifs on the immunogenicity of pDNA and raise the question of the possible involvement of a recently discovered TLR9-independent cytosolic pathway of DNA detection (Okabe, Kawane et al. 2005; Ishii, Coban et al. 2006) to the immunogenicity of pDNA. A better understanding of the undefined cellular and molecular mechanisms behind pDNA mediated activation of innate immune cells is clearly required. Although immunotherapies using DNA, such as DNA vaccines, benefit from concurrent immune cell activation the immunostimulatory activity of pDNA remains a major obstacle for therapeutic gene delivery. Thus, an understanding the mechanisms and contribution of TLR9-dependent and -independent recognition of pDNA will allow for manipulation and enhancement of DNA based therapeutics.  20  1.4.2 Therapeutic applications & safety of CpG-ODN as an adjuvant Adjuvants are functionally defined as components added to vaccine formulations that enhance the immunogenicity of antigens in vivo.  A great variety of experimental immunological  adjuvants are being investigated, however, the only vaccine adjuvants currently licensed for use in humans (aluminum hydroxide, salts, MF59, and virosomes) augment immune responses largely through enhancing antigen delivery and stimulate primarily humoral responses but fail to elicit CTL responses (Lindblad 2004; Petrovsky and Aguilar 2004). In contrast to currently licensed adjuvants, CpG ODN have demonstrated the potential to elicit both strong Th1-biased humoral and cellular immune responses to a wide variety of antigens including live or killed viruses and peptide or protein antigens in numerous animal models (Krieg 2006). Stemming from the natural function of TLR9 to stimulate protective immunity against intracellular pathogens, prophylactic treatment with CpG-containing DNA has the ability to protect against a wide range of viral, bacterial and some parasitic pathogens in mice (Elkins, Rhinehart-Jones et al. 1999; Gramzinski, Doolan et al. 2001; Rees, Gates et al. 2005; Krieg 2006). Furthermore, the strong Th1 bias induced by TLR9 stimulation has been applied to the development of allergy vaccines, which in mice are able to redirect the allergic Th2 response and prevent inflammatory disease manifestations, even in established allergic disease (Krieg 2006). In addition, the activity of CpG DNA in preventing or treating tumour development or metastasis has been demonstrated as a stand-alone therapy in several experimental murine models, however the effects of CpG monotherapy vary dramatically depending on tumour characteristics (Krieg 2004). While CpG monotherapy has demonstrated success in inducing Tcell mediated rejection of small tumours in mice, rejection of larger tumours typically requires combination with other antitumour strategies such as monoclonal antibodies, radiation, therapy, surgery or chemotherapy.  21  Based on their potential to elicit strong Th1-biased cellular immune responses, CpGODN represent a new class of adjuvants which show great promise in improving the efficacy of vaccines against cancer, infectious disease and allergy/asthma (Krieg 2006). In humans CpG ODN have shown potential as adjuvants for vaccination against hepatitis B surface antigen, anthrax, influenza and TAA-derived proteins and peptides in melanoma patients (Halperin, Van Nest et al. 2003; Cooper, Davis et al. 2004; Cooper, Davis et al. 2005). The safety of unmethylated CpG-ODN in humans has been observed in clinical trials over more than a 1000fold dose range. The primary adverse events are dose-dependent local injection reactions or systemic flu-like reactions, and a maximum tolerated dose in humans has not been reported (Krieg 2006). Despite the observations that CpG ODN treatment can exacerbate autoimmunity in mouse models of lupus (Hasegawa and Hayashi 2003), multiple sclerosis (Ichikawa, Williams et al. 2002), colitis (Obermeier, Dunger et al. 2002) and arthritis (Ronaghy, Prakken et al. 2002), clinical experience to date shows no indication that CpG ODN treatment of normal humans, cancer patients or individual infected with HIV induce autoimmune disease (Krieg 2006).  1.5 Liposomal delivery systems for conventional drugs Liposomes have been used extensively as a valuable tool in basic research, as model membrane systems for investigating the physical properties of biological cell membranes and lipids (Cullis and de Kruijff 1979), and also as effective carriers for the delivery of both conventional and genetic drugs in vivo. Encapsulation of conventional or genetic drugs in liposomal nanoparticles (LN) can dramatically improve their pharmacological activity by altering the pharmacokinetics and biodistribution of the encapsulated drugs and/or by acting as drug reservoirs allowing sustained release of the therapeutic (Allen and Cullis 2004). Alterations in the biodistribution of liposomal encapsulated drugs occurs through a mechanism known as the enhanced permeability and retention (EPR) effect or passive targeting (Figure 1.4) (Maeda, Wu et al. 2000). Certain 22  pathological conditions, such as inflammation or solid tumour growth, are associated with an increased permeability of the tissue vasculature which allows small particulate delivery systems, normally excluded from the tissues, to extravasate and localize in the intrastital space (Hashizume, Baluk et al. 2000). In particular, angiogenic blood vessels found in solid tumours can have gaps as large as 600 to 800 nm between adjacent endothelial cells, allowing liposomal drug delivery systems to extravasate in a size-dependent manner. In addition, because tumors have impaired lymphatic drainage, the carriers concentrate in the tumor, resulting in large increases in tumor drug concentrations (Jain 1987). In order for liposomes to effectively target tumour sites they must be small, long circulating and either have a net neutral surface charge, or contain a polyethylene glycol (PEG) coating which acts as a shield, preventing plasma proteins from binding to the liposome and promoting rapid clearance (Allen, Hansen et al. 1991). Currently, the most widely used clinical application of liposomes is for the systemic delivery of anticancer drugs to distal solid tumour sites.  The promise of liposomes as  conventional drug delivery systems has been demonstrated in the clinical use of various liposomal drug formulations such as Myocet (liposomal doxorubicin for use in metastatic breast cancer in combination with cyclophosphamide) and Doxil/Calyx (PEG-stabilized liposomal doxorubicin for use in metastatic ovarian cancer and advanced Kaposi’s sarcoma). Other indications for clinically approved liposomally encapsulated drugs include liposomal cytosine arabinoside for lymphomatous meningitis and liposomal amphotericin B for fungal infections (Allen and Cullis 2004).  23  Figure 1.4: A schematic diagram depicting the passive accumulation of liposomal formulations at diseased site tissues through the EPR effect. (A) Liposomes containing a genetic or conventional anticancer drug extravasate from the blood through gaps in vascular endothelial cells and accumulate in tumor solid tissue (dark green), but not in normal tissue (light green). (B) Drug is released from the liposomes in the vicinity of the tumor cells and taken up into the cells. (C) Liposomes containing anticancer nucleic acid-based therapeutics such as plasmid DNA or antisense oligonucleotides, bind to the cell surface through electrostatic charge interactions or ligand-mediated receptor processes triggering internalization of the liposomal carrier system into the endosomes. A portion of the encapsulated material escapes the endosomes and traffics to its intracellular site of action.  1.5.1 Liposomes as adjuvants and carriers of peptide and protein vaccines The adjuvant potential of liposomes was first recognized when strong humoral immune responses where obtained to liposomally encapsulated diphtheria toxoid in mice (Allison and Gregoriadis 1974) and it was demonstrated that repeat injections into patients could occur without serious side-effects (Gregoriadis, Weereratne et al. 1982). Adjuvants can be categorized based on their mechanism of action as immune potentiators, which activate innate immunity directly, such as cytokines or bacterial components (i.e. CpG-DNA), or as delivery systems, which better concentrate, target or display antigens to APCs.  The mechanism of action of  liposomal delivery systems as adjuvants for peptide and protein vaccines is thought to arise from factors including the ability of the liposomal delivery system to concentrate and enhance the  24  delivery of the antigen to APCs, as liposomal delivery systems are known to naturally target this cell population (Juliano 1986; Alving 1991), and their ability to enhance cytoplasmic delivery of the peptide or protein antigen thus allowing access to the MHC class I-restricted processing pathway and the generation of cell-mediated cytotoxic reactions (Rao and Alving 2000). Similarly, the inflammatory response induced by systemically administered CpG-ODN (Tan, Li et al. 1999; Mui, Raney et al. 2001; de Jong, Chikh et al. 2007) or pDNA (Scheule, St George et al. 1997; Freimark, Blezinger et al. 1998; Li, Wu et al. 1999; Yew, Wang et al. 1999; Loisel, Le Gall et al. 2001) is significantly enhanced when complexed within a cationic lipid-containing liposome, presumably due to protection of the nucleic acid payload and increased cellular uptake by target APCs (Juliano 1986; Oussoren, Zuidema et al. 1997; Oussoren, Velinova et al. 1998). Liposomal delivery of peptide and protein antigens has demonstrated the potential to enhance both cellular and humoral responses to peptide or protein antigens in animal models. Furthermore, liposomal carrier systems containing peptides and proteins have demonstrated success as vaccines for a variety of cancers and infectious diseases in Phase I/II clinical trials (Chen and Huang 2005). A liposomal formulation of the MUC1 derived peptide combined with monophosphoryl lipid A is currently in phase III clinical trials for non-small cell lung cancer.  1.5.2 Cationic lipid containing delivery systems for pDNA The efficient transfection of eukaryotic cells using DNA-cationic lipid complex systems composed  of  the  synthetic  cationic  lipid  N-[-1-(2,3-dioleyloxy)proply]-N,N,N-  trimethylammonium chloride (DOTMA) and the natural neutral phospholipid 1,2-dioleoyl-snglycero-3-phosphoethanolamine (DOPE) was first identified by Felgner et al in 1987 (Felgner, Gadek et al. 1987). Since then the development of a large variety of cationic lipids for the complexation of DNA has firmly established cationic lipid-based systems as useful agents for the delivery of genes into mammalian cells and revolutionized the use of nucleic acids as drugs 25  for gene and immunotherapy. The advantages of cationic lipids as gene delivery vehicles include their spontaneous condensation with negatively charged DNA via electrostatic interactions, their interaction with negatively charged biological surfaces (e.g. glycolipids and glycoproteins containing negatively charged sialic acid residues on cell surface membranes), and their ability to aid in membrane disruption which promotes the endosomal escape of large molecules such as pDNA (Felgner and Ringold 1989; Hafez, Maurer et al. 2001). While cationic lipid-containing DNA complexes demonstrate high levels of transfection in vitro, a number of obstacles limit their use in vivo. Cationic complexes often carry a strong net positive charge, which can lead to non-specific interactions with cells, proteins and other macromolecules in the circulation following systemic administration, resulting in the formation of large aggregates (> 300nm in diameter), which become trapped in the capillary beds of the lung (Mahato, Anwer et al. 1998). Furthermore, their overall positive charge promotes rapid clearance from the circulation by the reticuloendothelial system (RES) following systemic administration (Chen and Huang 2005). As a result, the majority of the accumulation and transfection is often restricted to first pass organs such as the liver and the lung.  It should be  noted that the charge ratio of cationic lipid to DNA has been demonstrated to markedly alter the biodistribution as preferential targeting of transgene expression to the lung has not been observed with lower lipid to DNA charge ratios (Ishiwata, Suzuki et al. 2000). In addition, although charge interactions have been demonstrated to offer partial protection to the complexed DNA from serum nucleases (Chiou, Tangco et al. 1994), a large amount of the pDNA in cationic complexes is degraded in the blood due to cleavage by serum nucleases, compromising the bioavailablity of the plasmid (Wheeler, Palmer et al. 1999). The half-life of free plasmid is typically less than 5 minutes in vivo (Lew, Parker et al. 1995). Furthermore, systemic administration of DNA-cationic lipid containing complexes in mice is often accompanied by an acute inflammatory response and dose-dependent toxicity. 26  Symptoms include the induction of high levels of proinflammatory cytokines (IFN-γ, IL-12, IL6, TNF-α), piloerection and lethargy in the days following administration and in more severe cases, liver damage (Tousignant, Gates et al. 2000). These toxic effects appear to be a result of the combination of the cationic lipid and DNA, rather than the toxicity of the individual components which do not display significant toxicity alone, even when much higher doses are injected (Yew and Scheule 2005). Immunostimulatory motifs present in the pDNA (discussed in section 1.4) have been demonstrated to be responsible for a significant portion of the acute inflammatory response to cationic/DNA complexes (Li, Wu et al. 1999; Yew, Wang et al. 1999; Loisel, Le Gall et al. 2001; Zhao, Hemmi et al. 2004). The immunostimulatory activity of pDNA, although beneficial for DNA vaccination, remains a major obstacle for therapeutic gene delivery as CpG-induced proinflammatory cytokines (IFN-γ and TNF-α) are known to downregulate the activity of viral promoters used by eukaryotic expression vectors thereby decreasing and limiting the duration of transgene expression (Harms and Splitter 1995; Tan, Li et al. 1999). It should be noted, that cationic lipids themselves, which biologically are very rare, are also associated with toxicity which is dependent on the structure of their hydrophilic head group and their biodegradability (Lv, Zhang et al. 2006).  1.5.3 Systemic administration of cationic lipid containing DNA complexes As discussed previously, systemic administration of cationic lipid containing DNA complexes is associated with a dose-dependent toxicity (Tousignant, Gates et al. 2000).  The associated  toxicity, which is thought to be CpG-dependent, stems from the production of a large amount of cytokines including IFN-γ, IL-12 and TNF-α, and is significantly enhanced when the pDNA or ODN is complexed with cationic lipid (Scheule, St George et al. 1997; Freimark, Blezinger et al. 1998; Li, Wu et al. 1999; Yew, Wang et al. 1999; Loisel, Le Gall et al. 2001; Mui, Raney et al. 2001). This is presumably due to protection of the nucleic acid payload and increased cellular 27  uptake by APCs, as these cell-types are known to avidly accumulate lipid particulate systems (Juliano 1986; Oussoren, Zuidema et al. 1997; Oussoren, Velinova et al. 1998). In line with this, toxicity/proinflammatory cytokine responses are typically not observed following the administration of free pDNA, most likely due to rapid degradation by nucleases in the blood following systemic administration (Li, Wu et al. 1999; Yew, Wang et al. 1999). In several instances antitumour activity and the development of tumour specific adaptive responses have been demonstrated following systemic administration of DNA-cationic lipidcontaining complexes (containing either CpG containing ODN or non-coding plasmid DNA) in murine models of pulmonary metastasis and cervical cancer (Dow, Fradkin et al. 1999; Whitmore, Li et al. 1999; Whitmore, Li et al. 2001) and an intraperitoneal tumour model (Lanuti, Rudginsky et al. 2000).  The antitumour response observed in these studies was  concluded to be a result of CpG-mediated stimulation as methylation resulted in a significant decrease in antitumour activity. Furthermore, depletion studies demonstrate the requirement for both NK and CD8+ cells in tumor eradication (Whitmore, Li et al. 2001). A model developed by Whitmore et al. proposes that the cytokine-mediated activation of the innate immune system by cationic lipid/DNA complexes could stimulate NK-dependent lysis of tumours, which would promote the clearing of lytic debris by stimulated APCs and subsequently the presentation of TAAs to CD8+ cells under the influence of Th1 cytokine support (Whitmore, Li et al. 2001). In addition, it has recently been indicated that some types of cationic lipids may themselves have immunostimulatory properties, as demonstrated by the ability of cationic lipids to stimulate DC in the absence of CpG-ODN, leading to the up-regulation of co-stimulatory molecules, CD80 and CD86 (Vangasseri, Cui et al. 2006; Yan, Chen et al. 2007).  28  1.5.4 Cationic lipid-based carrier systems for DNA vaccines Cationic lipid-based delivery systems represent an attractive means to deliver pDNA to APCs, the preferred target for DNA vaccines, as they not only have the potential to partially protect their DNA content from nuclease degradation, but it has also been long-established that APCs such as macrophages and DCs avidly accumulate lipid particulate systems following injection. This process occurs at the site of injection following s.c. administration (Oussoren, Zuidema et al. 1997; Oussoren, Velinova et al. 1998), or in the liver and spleen, following systemic administration (Juliano 1986) and is an important characteristic that has been implicated in the adjuvant activity of liposomal DNA delivery systems (Gregoriadis 1990). The accumulation of liposomal DNA into APCs can also result in enhanced transfection as suggested by experiments in which i.m. or s.c. injection of mice with cationic lipid/DNA complexes containing plasmid encoding the enhanced fluorescent green protein resulted in much more green fluorescence in the draining lymph nodes as compared to naked pDNA (Perrie, Frederik et al. 2001). Early experiments comparing the immune response following repeat (i.m.) vaccination with cationic lipid/DNA complexes containing pDNA encoding the hepatitis B surface antigen vs. naked pDNA, showed that plasmid associated with cationic complexes (DOTAP, DC-Chol or SA) elicited much greater IgG1 (up to 100-fold) and IgG2a (up to 10-fold) antibody responses (Gregoriadis, Saffie et al. 1997). Subsequently, association of pDNA with cationic lipid delivery systems has been demonstrated to enhance the CTL component of the immune response following s.c. administration as determined by specific killing of syngeneic target cells using ovalbumin as a model antigen, as compared to free pDNA for which no response was detected (Bacon, Caparros-Wanderley et al. 2002). More recently, approaches taken to enhance the uptake and/or transfection efficiency of cationic lipid/DNA delivery systems, such as the incorporation of targeting ligands or fusogenic peptides, have demonstrated the ability to enhance responses against the encoded antigen. 29  Specifically, incorporation of the targeting ligand mannose has demonstrated enhanced gene transfection in APCs resulting in enhanced CTL activity against melanoma, inhibition of tumour growth and prolonged survival after tumor challenge compared with unmodified liposomes and naked pDNA (Hattori, Kawakami et al. 2006; Lu, Kawakami et al. 2007). Although current cationic lipid/DNA delivery systems demonstrate potential as carrier systems for the delivery of DNA-based vaccines, due to their net positive charge they are often associated with one or more undesirable characteristics (discussed in section 1.5.2) which can inhibit their use in vivo.  1.6 Stabilized plasmid lipid particles (SPLP) Stabilized plasmid lipid particles (SPLP) are non-viral gene delivery systems composed of pDNA fully encapsulated within a lipid bilayer. Due to their small size (approximately 100 nm in diameter), long circulation lifetimes (t1/2 of 1 - 6 h depending on lipid composition) and net neutral charge, SPLP naturally and passively accumulate at distal tumor sites following i.v. administration through the enhanced permeability and retention (EPR) effect, resulting in reporter gene expression at the tumour site which is two to three orders of magnitude higher than that observed in all other organs (Figure 1.5). Unlike DNA/cationic lipid complexes formed by mixing preformed cationic lipid–containing vesicles with pDNA, SPLP fully protect DNA following incubation with Escherichia coli DNase I (Wheeler, Palmer et al. 1999; Tam, Monck et al. 2000; Ambegia, Ansell et al. 2005; Judge, McClintock et al. 2006). Traditionally, SPLP have been formulated using a detergent dialysis approach and most commonly consisted of the lipid composition N,N-Dioleyl-N,N-dimethylammonium chloride (DODAC):DOPE:PEG-lipid in the molar ratio 8:82:10 (Fenske, MacLachlan et al. 2002). More recently SPLP have also been prepared by destabilizing preformed empty vesicles in ethanol at 40% v/v and incubating in the presence of pDNA (Maurer, Wong et al. 2001) or by the drop 30  wise addition of lipid dissolved in ethanol to a rapidly mixing aqueous buffer containing DNA, referred to as spontaneous vesicle formation (SVF) (Jeffs, Palmer et al. 2005). Both methods overcome a number of limitations of the detergent dialysis procedure, making the formulation process simpler and more scalable to support preclinical and clinical development.  pg Luciferase / g Tissue  2000 1600 1200 800  Liver Spleen Lung Kidney Tumour  400  0  24  48  72  Time (hours)  Figure 1.5: Luciferase gene expression following a single intravenous administration of SPLP in neuro-2A tumour-bearing A/J mice. On day 0, 1.5 x 106 cells were injected subcutaneously in the hind flank of each mouse, when tumours were approximately 200mm3 (about day 9) SPLP (100 µg DNA) was administered i.v. in a total volume of 100uL. Twenty-four, 48 and 72 hours later mice were euthanized and organs were assayed for luciferase expression. Each time-point represents the average from 4 mice ± SD.  SPLP produced by the SVF procedure consist of cholesterol, 1,2-distearoyl-sn-glycero-3phosphocholine (DSPC), 1,2-dioleyloxy-N,N-dimethylaminopropane (DODMA) and a PEGdiacylglycerol lipid at molar ratios of 55:20:15:10 and possess the same desirable properties as those prepared by detergent dialysis (small size, protection from nuclease degradation, net neutral charge).  Unlike SPLP formed by detergent dialysis, which are predominantly  unilamellar, SPLP prepared by SVF consist of unilamellar, bilamellar and oligolamellar vesicles (Figure 1.6) however, this altered morphology appears to have no effect on the stability or activity of SPLP in vivo (Jeffs, Palmer et al. 2005).  31  A)  B)  Figure 1.6: Cryo-transmission electron microscopy of SPLP. (A) SPLP prepared by detergent dialysis. (B) SPLP prepared by spontaneous vesicle formation. Scale bars represent 200 nm. Adapted from Jeffs et al 2005 (Jeffs, Palmer et al. 2005).  Additional advantages of SPLP formulated by SVF include that they contain the ionisable cationic lipid DODMA, which has an apparent pKa of 6.8 in a lipid bilayer allowing it to associate with negatively charged DNA under acidic conditions but it become predominantly uncharged at physiologic pH, thus reducing the unfavourable pharmacokinetic and toxicity properties usually observed with cationic gene carrier systems in vivo (Jeffs, Palmer et al. 2005). Furthermore, incorporation of shorter chain PEGs [i.e. PEG-dimyristoylglycerol (DMG)], which dissociate more rapidly from the lipid bilayer following in vivo administration, results in decreased immunogenicity of the PEG component of the carrier system, allowing for repeat dosing of SPLP without the generation of anti-PEG antibody responses and subsequent hypersensitivity reactions (Judge, McClintock et al. 2006). The function of the PEG-lipid is to prevent aggregation during SPLP formulation, as well as to improve the circulation lifetime of the particle following intravenous administration by preventing uptake and removal of the particle from the circulation by the reticuloendothelial system (RES) (Allen, Hansen et al. 1991). The incorporation of shorter chain PEGs to allow for repeat administration does alter the pharmacokinetics (t1/2 reduce to approximately 1 hour) and the biodistribution profile of the particle, thus resulting in increased levels of intact plasmid DNA and transgene expression in organs such as the liver and the spleen following i.v. administration. Despite this, disease site 32  targeting is maintained when multi-dosing and gene expression at the tumor site is consistently two to three orders of magnitude higher than in any other tissues (Ambegia, Ansell et al. 2005; Jeffs, Palmer et al. 2005; Judge, McClintock et al. 2006). Due to their demonstrated potential for systemic gene transfer and the absence of undesirable properties which hamper the use of DNA-cationic lipid complexes, SPLP have been proposed as potential carrier systems for the delivery of DNA vaccines. The utility of SPLP for vaccine applications will be investigated in this thesis.  1.7 Liposomal nanoparticle encapsulated CpG ODN (LN CpG-ODN) Work performed in collaboration with our laboratory has demonstrated that the immune response to CpG-containing ODN are greatly enhanced when encapsulated in liposomal nanoparticles (LN) as determined by plasma cytokine levels (IL-12, IFN-γ, IL-6, and MCP-1) (Mui, Raney et al. 2001) and immune cell activation. In addition, lipid encapsulation results in a dramatic increase in the ability of CpG ODN to promote the development of antigen-specific humoral and adaptive responses following co-administration with protein antigen, which are protective against subsequent tumour challenge in both xenogeneic and syngeneic tumour models (de Jong, Chikh et al. 2007). LN CpG-ODN are small (~100nm in diameter) multilamellar vesicles, which can contain on the order of 1000 to 3000 ODN molecules per particle (Maurer, Wong et al. 2001). Similar to SPLP, they are formulated with the ionizable cationic aminolipid DODMA and a PEGconjugated lipid, typically consisting of the lipids 1-palmitoyl-2-oleoyl-sn-glycero-3phosphocholine (POPC) /cholesterol/DODMA/PEG-DMG at a molar ratio of 25/45/20/10, resulting in the generation of particles possessing optimal characteristics (i.e. neutral surface charge, small particle size) for i.v. delivery of ODN to target effector cells.  33  The enhanced immunopotency of lipid-encapsulated CpG-ODN stems from their ability, like other particulate delivery systems (Juliano 1986), to naturally target macrophage and other professional APCs in vivo. We have observed that LN CpG-ODN containing fluorescently labeled ODN are rapidly taken up by APCs in the blood, spleen and lymph node compartments following administration by the i.v. or s.c. route, and it has been demonstrated that liposomal encapsulation enhances intracellular delivery of ODN (compared to equivalent doses of free CpG ODN) through endocytosis-mediated cellular uptake (Semple, Klimuk et al. 2000). Furthermore, lipid encapsulation serves to enhance the circulation lifetime of the ODN, protecting the payload from nuclease degradation, resulting in the delivery of more intact sequences to tumours and sites of inflammation (Klimuk, Semple et al. 2000; Semple, Klimuk et al. 2000) and enabling the phosphodiester backbone to be employed if desired. Together, these findings strongly suggest that liposomal encapsulation is an effective strategy to optimize the activity of CpG-ODN and that encapsulation of CpG ODN within LN offers an attractive strategy for significantly enhancing the activity of free CpG ODN and improving its ability to act as a vaccine adjuvant for the treatment and prevention of cancer and other diseases.  1.8 Thesis objectives As described, current non-viral DNA cancer vaccine approaches are limited by a number of factors including the instability and poor uptake of pDNA by target APCs; limitations on the routes of administration which can be employed; toxicities associated with cationic carrier systems; identification of appropriate immunogenic TAAs towards which to direct the immune response; and the inability of DNA vaccines to raise sufficient immune responses towards the target TAA. It was the objective of this thesis to investigate the immunostimulatory potential of LN CpG-ODN and SPLP and the ability of SPLP and LN CpG-ODN to act together to produce a 34  potent non-viral DNA cancer vaccine that overcomes these obstacles. In Chapter Two, the pharmacokinetics, biodistribution and cellular uptake of LN-CpG ODN following i.v. and s.c. administration is characterized and correlated with immunostimulatory activity, demonstrating that, despite dramatic differences in tissue distribution profiles the resultant immune response is very similar, presumably due the intrinsic ability of APCs to sequester LN-CpG ODN.  In  Chapter Three, the immunostimulatory potential of SPLP is investigated following systemic administration as compared to free pDNA, demonstrating not only that lipid encapsulation dramatically enhances the immunostimulatory potential of pDNA but also that SPLP maintains immunostimulatory activity in TLR9 KO mice and that agents which promote the endosomal release of SPLP enhance its immunostimulatory potential suggesting the involvement of a cytoplasmic detection pathway for pDNA. In Chapter Four, we investigate the uptake and transfection potential of SPLP with respect to APCs and demonstrate a new role for SPLP as a non-viral gene delivery vehicle for the generation of a systemically administered genetic vaccine in the presence of LN CpG-ODN. 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"Immunosuppressive networks in the tumour environment and their therapeutic relevance." Nat Rev Cancer 5(4): 263-74.  48  CHAPTER TWO EFFECTS OF INTRAVENOUS AND SUBCUTANEOUS ADMINISTRATION ON THE PHARMACOKINETICS, BIODISTRIBUTION, CELLULAR UPTAKE AND IMMUNOSTIMULATORY ACTIVITY OF CpG ODN ENCAPSULATED IN LIPOSOMAL NANOPARTICLES1 2.1 Introduction Bacterial DNA and synthetic ODN that contain unmethylated CpG motifs activate a wide array of immune effector cells, stimulating potent, T-helper 1 biased immune responses.  Upon  exposure to APCs, CpG ODNs are rapidly internalized into the endosomal compartment where they interact with the Toll-like receptor 9 pattern recognition receptor (TLR9) (Hemmi, Takeuchi et al. 2000; Takeshita, Leifer et al. 2001). This leads to activation of cell signaling pathways that result in the up-regulation of co-stimulatory molecules and the secretion of proinflammatory and T-helper 1 biased cytokines. Stimulation of these primary immune effects promotes the activation of immune cells not directly responsive to CpG ODN, enhancing NK cell cytotoxicity and differentiation of naive CD4- and CD8- T cells into T-helper 1 cells and cytotoxic lymphocytes, respectively (Heeg and Zimmermann 2000; Kawarada, Ganss et al. 2001). As a result of their potent ability to stimulate and enhance innate and acquired immune responses, synthetic CpG ODN are currently undergoing Phase I-III clinical trials in the treatment and prophylaxis of cancer, allergy and asthma, and infectious disease (Cooper, Davis et al. 2004; Cooper, Davis et al. 2005; Friedberg, Kim et al. 2005; Speiser, Lienard et al. 2005). While the use of chemical modification has effectively reduced the sensitivity of ODN to nuclease degradation, such as with the phosphorothioated CpG ODN currently in clinical trials, and prolonged circulation lifetime, the use of free CpG-ODN still faces several significant challenges including unfavorable pharmacokinetics, a lack of specificity for target cells after 1  A version of this chapter has been published. Wilson KD, Raney SG, Sekirov L, Chikh G, deJong SD, Cullis PR, Tam YK. (2007) Effects of intravenous and subcutaneous administration on the pharmacokinetic, biodistribution, cellular uptake and immunostimulatory activity of CpG ODN encapsulated in liposomal nanoparticles. International Immunopharmacology 7(8):1064-75.  49  systemic administration and poor cellular uptake (Zhao, Matson et al. 1993; Sands, Gorey-Feret et al. 1994; Agrawal, Temsamani et al. 1995). Encapsulation of biologically active agents in liposomal nanoparticles can dramatically enhance their activity by increasing drug delivery to disease sites and by acting as drug reservoirs allowing sustained release of the therapeutic (Allen and Cullis 2004). It is well recognized that liposomal nanoparticles are removed from the circulation by phagocytic cells [i.e. macrophages and dendritic cells (DC)] of the reticular endothelial system, largely in the liver and spleen, or by extravasation in areas where the vascular membrane is more permeable, such as at sites of infection, inflammation and tumors (Oussoren, Velinova et al. 1998; Maeda, Wu et al. 2000; Allen and Cullis 2004). Liposomes have also been widely used in immunotherapeutic applications ranging from adjuvants to carriers of peptide, protein and DNA vaccines (Alving, Koulchin et al. 1995; Gluck 1995; Gregoriadis, Bacon et al. 2002; Chen and Huang 2005).  We have previously  demonstrated that immune responses to a CpG-containing ODN are greatly enhanced when encapsulated in liposomal nanoparticles as determined by plasma cytokine levels (interleukin12, interferon-gamma, interleukin-6, and macrophage chemoattractant protein-1) (Mui, Raney et al. 2001), immune cell activation and anti-tumor efficacy in animal models (de Jong, Chikh et al. 2007). The rationale for encapsulation of CpG ODN is distinctly different than for other therapeutic agents. In particular, we are now taking advantage of the natural ability of APCs to accumulate LN-CpG ODN to provide enhanced delivery, resulting in enhanced immune cell activation and induction of more potent, T-helper 1 biased immune responses. In the same vein, although i.v. delivery is the most common route of administration for conventional liposomal drugs, it may not represent the optimal route for immunostimulatory agents such as LN-CpG ODN. While i.v. administration would allow accumulation of LN-CpG ODN in circulating and tissue resident macrophages, s.c. administration would result in passage 50  of the particle through the lymphatic capillaries to the regional lymph nodes prior to reaching the general circulation (Allen, Hansen et al. 1993; Oussoren, Zuidema et al. 1997; Oussoren, Velinova et al. 1998). Thus the s.c. route of administration would be expected to provide enhanced access to immune cells found in high density in these regions, including dendritic cells, macrophages, as well as T- and B-lymphocytes. Clearly then, the route of administration influences the specific environment and immune cell populations that are exposed to LN-CpG ODN and therefore, has the capacity to exert a potentially significant influence on the nature, degree and duration of the resultant immune response. Here we evaluate the influence of the route of administration on the pharmacokinetics, biodistribution and uptake of lipid encapsulated CpG ODN and correlate these characteristics with immunopotency.  2.2 Materials & Methods 2.2.1 Materials. Palmitoyloleoylphosphatidylcholine (POPC) was purchased from Avanti Polar Lipids (Alabaster, AL), cholesterol from Sigma (St. Louis, MO). 1,2-dioleyloxy-3-N,Ndimethylaminopropane (DODMA) and polyethylene glycol-dimyristol glycerol (PEG-DMG) were provided by Inex Pharmaceuticals Corporation (Burnaby, BC, Canada). INEX-6295 and 6303,  16-mer  phosphorothioate  ODN  (5’TAACGTTGAGGGGCAT-3’)  containing  unmethylated and methylated cytosine residues in the CpG motifs respectively, were used in pharmacokinetic, biodistribution, immune cell activation and cytotoxicity studies. To assess pharmacokinetics and biodistribution, ODN was spiked with phosphorothioated INX-6295 containing two internal tritiated thymidine residues while for cell uptake studies, an irrelevant 5’-fluoroscene  isothiocyanate-bearing  15-mer  phosphorothioate  ODN  (5’-  CCGTGGTCATGCTCC-3’) was used. All ODN were synthesized by Trilink Biotechnologies (San Diego, CA).  51  2.2.2 Animals. Female, 6 to 8-week-old ICR and C3H mice were obtained from Charles River Laboratories (Wilmington, MA) and were quarantined for 3 weeks prior to use. All procedures involving animals were performed in accordance with the guidelines established by the Canadian Council on Animal Care.  2.2.3 Preparation of liposomal ODN.  ODN were encapsulated in lipid nanoparticles  containing an ionisable aminolipid using an ethanol dialysis procedure, as previously described (Maurer,  Wong  et  al.  2001).  Briefly,  lipid  mixtures  consisting  of  POPC/cholesterol/DODMA/PEG-DMG (molar ratio 25/45/20/10) were solubilized in ethanol, passed twice through stacked 200nm + 100nm polycarbonate membranes (Whatman Nuclepore, Clifton, NJ) using a thermobarrel extruder (Lipex Biomembranes, Vancouver, B.C., Canada) and mixed with 50 mM citrate buffer containing 3.33 mg/ml of ODN, to give a final ethanol concentration of 36%. The vesicles were dialyzed against citrate followed by HEPES-buffered saline  and  unencapsulated  ODN  removed  on  DEAE-Sepharose  CL-6B  columns.  Oligonucleotide and lipid concentrations were determined by UV spectroscopy (260nm) and an inorganic phosphorus assay after separation of the lipids from the ODN by a Bligh and Dyer extraction (Bligh and Dyer 1959), respectively. Studies similar to those previously described (Maurer, Wong et al. 2001; Semple, Klimuk et al. 2001) were performed to determine the optimal final ODN-to-lipid ratio of 0.1 (w/w) for this liposome formulation in terms of initial ratio and encapsulation efficiency (data not shown). Particle size was approximately 100nm in diameter as determined by quasi-elastic light scattering using a NICOMP submicron particle sizer (model 370, Santa Barbara, CA) was 100 ± 25 nm. All liposomal formulations used in these studies, including those containing radio- and fluorescently- labeled ODN, were assumed to have similar circulation/distribution properties based on identical lipid composition and biophysical characteristics (i.e. size, ODN:lipid ratio, etc). 52  2.2.4 Pharmacokinetic & biodistribution studies. ICR mice were injected i.v. or s.c. at ODN doses of 20mg/kg or 360-440µg/animal (140-160 µl administered via the lateral tail vein) and 4.5-5.5 mg/kg or 100µg/animal (100 µl administered into the hind flank) respectively, with free or LN CpG-ODN. Doses for these initial experiments were derived from studies evaluating the effect of CpG dose on immunological response (unpublished data) and selected from the linear portion of the response curve inducing maximal pharmacodynamic responses.  ODN were  labeled by spiking with 3H-labeled phosphorothioate INX-6295 (Trilink Biotechnologies) while lipids were labeled with 14C-cholesterol-hexadecylether (CHE) to allow a per mouse dosing of approximately 3µCi of 3H-ODN and 1µCi of  14  C-lipid. Mice were euthanized by a terminal  dose of 3.2% (v/v) ketamine/0.8% (v/v) xylazine 0.5, 1, 2, 4, 6, 8, 16 or 24 hours following administration (3 mice per time-point). Blood was collected in Vacutainer (BD Biosciences, Canada) tubes containing EDTA while lymph nodes (bilateral, axial & inguinal), liver and spleen were harvested and chemically digested at room temperature using Solvable (Perkin Elmer, Wellesley, MA) followed by de-colorization with hydrogen peroxide (30% w/w). Blood and tissue digests were analyzed by liquid scintillation counting in Picofluor-40 (Perkin Elmer).  2.2.5 Immune cell isolation. For immunological assays, mice were euthanized as described previously. Blood was collected via cardiac puncture into Vacutainer (BD Biosciences) tubes containing EDTA and viable peripheral blood mononuclear cells (PBMCs) were isolated using Lympholyte (Cedarlane Laboratories, Canada). Spleens and lymph nodes were harvested and dissociated to single cells by passage through a sterile 100 um nylon mesh (BD Biosciences) and red blood cells were lysed (0.1M ammonium acetate, 10mM potassium bicarbonate, 70uM EDTA).  Cells were stained with fluorescently labeled antibodies (BD Biosciences) and  analyzed on 2 laser, 4-colour FACSort or FACScaliber flow cytometers (BD Biosciences, San 53  Jose, CA). Dead cells were excluded with propidium iodide and viable cells were gated based on forward and side scatter characteristics. Data was acquired and analyzed using CELLQuest Pro software V 4.0.1 (BD Biosciences).  2.2.6 Uptake studies. 6 to 8-week old ICR mice were injected i.v. or s.c. with 10 mg/kg fluoresceine isothiocyanate-labeled ODN encapsulated within lipid particles (140-160 µl delivered into the lateral tail vein and hindflank, respectively). Mice were euthanized 1.5, 4, 7.5 and 24.5 hours after administration and blood, lymph nodes and spleen were isolated. Cell suspensions were prepared, stained with antibodies against cell phenotype markers (CD11c for DCs, CD11b and Mac3 for macrophages, B220/CD45R for B-cells, CD4 for CD4 T-cells; BD Biosciences) and analyzed by flow cytometry.  2.2.7 Activation & cytotoxicity assays. C3H mice were injected i.v. or s.c. with free or encapsulated ODN at a 20mg/kg dose (140-160 µl delivered into the lateral tail vein and hindflank, respectively). Twenty-four, 48 and 72 hours following administration mice were euthanized as described.  In activation studies, splenocytes and PBMCs were stained with  fluorescently labeled antibodies against cell phenotype (CD11c for DCs, DX5 for NK cells, CD11b and Mac-3 for macrophages, CD8 and CD4 for T-cells, CD45R/B220 for B-cells) and cell activation (CD69 and CD86) markers, and analyzed using by flow cytometry. NK killing was assessed in a standard 4 hour  51  Chromium release assay using YAC-1 cells as targets.  Splenocytes or PBMC were co-cultured with 51Chromium labeled target cells at E:T cell ratios of 1:1, 5:1, 25:1, 100:1 and 1:1, 5:1, 20:1 and 50:1, respectively for 4 h at 37°C, 5% CO2 and the amount of 51Chromium released to the supernatants was quantitated as a measure of cell killing. Percentage of cytotoxicity was calculated using the equation: (Sample counts per minute spontaneous counts per minute)/(maximum counts per minute) x 100. Maximum counts were 54  determined using 20% Triton-X 100 while spontaneous counts were measured in culture medium. Activation of ADCC was assessed in a similar manner assessing lysis of Daudi cells in the presence/absence of Rituxan (100 µg/106 cells at a concentration of 10 µg/ml), a mAb directed against the CD20 Ag present on the target cells.  2.2.8 Statistical analyses.  A one-way analysis of variance (ANOVA) was used to evaluate  the differences between treatment groups. In the case of statistically significant results, the differences between treatment groups were assessed using Bonferroni adjusted t-tests. Probability values less than 0.05 were considered significant.  2.3 Results INX-6295 and INX-6303 are 16-mer ODNs with identical nucleotide sequences, the latter containing a methylated cytosine in the CpG motif.  When encapsulated  within a lipid nanoparticle, the immune activity of the methylated sequence is equivalent to or greater than the unmethylated sequence as determined through plasma cytokine profiling, ex vivo immune cell activation and anti-tumor efficacy in animal models (de Jong, Chikh et al. 2007). As a result, INX-6303 was used for all immunological studies.  2.3.1 Blood levels of LN CpG ODN differ following i.v. or s.c administration. Pharmacokinetic and biodistribution characteristics were assessed using [3H]-ODN and [14C]CHE to indicate the fate of the ODN and the lipid delivery system, respectively. Cholesterolhexadecylether has been used extensively as a marker and has been shown to be nonexchangeable and non-metabolizable (Stein, Halperin et al. 1980). Similarly, internally labeled 55  phosphorothioate ODN have been extensively documented as having good in vivo stability accompanied by slow metabolism (Sands, Gorey-Feret et al. 1994). Blood clearance profiles were compared over a 24 hour time course following i.v. or s.c. administration of encapsulated and free ODN (Figure 2.1; 8 hour comparison). As expected (Klimuk, Semple et al. 2000; Semple, Klimuk et al. 2000), ODN- and lipid- equivalents (3H-ODN- and  14  C-lipid- derived  radioactivity, representing both intact and metabolite forms) show similar clearance profiles following both i.v. and s.c. administration, suggesting that ODN remains encapsulated within the carrier system (data not shown). Following i.v. administration of LN-CpG ODN, approximately half of the injected ODN-equivalent dose is cleared from the circulation within 30 minutes, with 0.5% of the injected ODN-equivalent dose remaining after 24 hours.  100%  % Injected ODN Equivalents  100.000%  % Injected ODN Equivalents  90% 80% 70% 60% 50% 40%  10.000% 1.000% 0.100% 0.010% 0.001% 0.000%  30%  0  2  20%  4 Tim e (hours)  6  8  10% 0% 0  1  2  3  4  Tim e (hours)  Figure 2.1: Blood levels of free and LN CpG ODN differ dramatically following i.v. or s.c administration. Blood clearance profiles following intravenous or subcutaneous administration of encapsulated (∆ – i.v., □ – s.c.) and free (▲ – i.v., ■ – s.c.) CpG ODN, tracking 3H-labeled ODN, expressed as the percent of the injected dose detected ± SD (3 mice/group). LN-CpG ODN and free ODN were administered intravenously at an ODN dose of 20mg/kg or 360-440µg/animal, or subcutaneously at a dose of 4.5-5.5mg/kg or 100µg/animal. Data is shown on both a linear (main) and log (insert) scale.  Conversely, only low levels of ODN-equivalents enter the circulation after s.c. administration, reaching maximum levels representing only 0.2 ± 0.03% of injected ODNequivalent dose 24 hours after administration.  Similar trends are observed following 56  administration of free ODN although, based on ODN equivalents, clearance from the circulation following i.v. administration is 2-3 times quicker than with LN-CpG ODN (20.5 % of the injected ODN-equivalent dose present 30 minutes after administration) while s.c. administration of free ODN results in at least 3-fold higher levels of ODN-equivalent dose reaching the circulation, compared to LN-CpG ODN-equivalent dose, at all time-points investigated.  2.3.2 I.V. administration of LN-CpG ODN results in enhanced delivery to liver and spleen, whereas s.c. administration enhances accumulation in regional lymph nodes. Following i.v. administration of LN-CpG ODN, the majority of the ODN-equivalents accumulate in the liver, with peak levels (71.1 ± 4.7 % of injected dose) occurring 4 hours post-injection (Figure 2.2). Accumulation in the spleen follows a similar pattern with maximal ODN-equivalent accumulation of 1.57 ± 0.36 % of the injected dose occurring 4 hours post-injection. Encapsulation enhances hepatic and splenic accumulation following i.v. administration compared to free ODN due to clearance by the reticular endothelial system. ODN-equivalents levels in both the liver and spleen remain relatively constant after 4 hours with no significant decline over 24 hours, likely due to uptake of the stable phosphorothioated ODN by tissueresident phagocytic cells in the liver and spleen. Low, but detectable levels of ODN-equivalents are observed in the lymph nodes over the entire time-course with both encapsulated and free forms.  When LN-CpG ODN is administered s.c., relatively little ODN-equivalents  accumulation is observed in the liver and spleen with reductions of approximately 18- and 14fold respectively compared to i.v. administration. Maximum ODN-equivalents accumulation (3.9 ± 1.84 % and 0.11 ± 0.10 % respectively) is not reached until 8 hours post-administration. However, although ODN-equivalents levels are considerably lower, relative biodistribution patterns in the blood, liver and spleen are similar to those observed following i.v. administration,  57  indicating that when the particles reach the peripheral circulation they are subject to similar clearance mechanisms as those administered i.v.  A) % Injected ODN Equivalents  100% 80% 60% 40% 20% 0% 0  B)  5  10 15 Time (hours)  20  25  10 15 Time (hours)  20  25  % Injected ODN Equivalents  2.0%  1.5%  1.0%  0.5%  0.0% 0  C)  5  % Injected Dose  2.0%  1.5%  1.0%  0.5%  0.0% 0  5  10  15  20  25  Time (hours)  Figure 2.2: Biodistribution of i.v. and s.c. administered free and LN-CpG ODN to liver, spleen and lymph nodes. Tissue accumulation of CpG ODN following intravenous or subcutaneous administration of encapsulated (∆ – i.v., □ – s.c.) and free (▲ – i.v., ■ – s.c.) CpG ODN, tracking 3H-labelled ODN, in the liver (A), spleen (B) and lymph nodes (C), expressed as the percent of the injected dose detected ± SD (3 mice/group). LN-CpG ODN and free ODN were administered intravenously at an ODN dose of 20mg/kg or 360-440µg/animal, or subcutaneously at a dose of 4.5-5.5mg/kg or 100µg/animal.  58  In contrast, s.c. delivery of LN-CpG ODN results in levels of accumulation in lymph nodes (1.54 ± 0.28%) that are 50-fold greater than following i.v. administration, with maximal ODN-equivalents accumulation occurring by 8 hours post-injection. Levels declined thereafter to approximately 0.6% by 24 hours, likely due to ODN uptake by highly mobile phagocytic cells in the injection site and migration to and from the draining lymph nodes. Also, in contrast to i.v. delivery, liposomal delivery results in reduced CpG ODN-equivalents accumulation in the liver and spleen, with free ODN demonstrating enhanced hepatic and splenic ODN-equivalent localization following s.c. administration. Interestingly, enhanced accumulation in the lymph nodes is not observed for s.c. administered free ODN. Comparison of organ accumulation based on tissue weight demonstrates that when administered s.c., LN-CpG ODN-equivalents accumulate preferentially in the lymph nodes, with very low levels of ODN-equivalents being observed in the spleen and liver (Figure 2.3). Conversely, s.c. administration of free ODN or i.v. administration of free or encapsulated ODN resulted in low levels of ODN-equivalents in the lymph nodes.  2.3.3 LN-CpG ODN are accumulated by immune cells in the blood, spleen and lymph nodes following i.v. or s.c. administration. In the spleen, maximum levels of uptake by CD11c positive, Mac-3 positive, CD11b positive and B220/CD45R positive cells are observed 7.5 hours following both i.v. and s.c. administration (Figure 2.4).  Not surprisingly, based on  pharmacokinetic and biodistribution data, enhanced uptake is seen in the spleen and peripheral blood compartments following i.v. administration, both in terms of the total number of cells and on a per cell basis. In the spleen, i.v. administration results on average in 10 to 30% more APCs taking up LN-CpG ODN compared to s.c. administration. Furthermore, mean fluorescence intensity is 4 to 13-fold higher following i.v. administration, indicating enhanced uptake on a per cell basis. Similarly, 10 – 50% more peripheral blood CD11c positive, Mac-3 positive, CD11b 59  positive or B220/CD45R positive cells take up LN-CpG ODN following i.v. administration  B)  % Injected ODN Equivalents /g Tissue  A)  % Injected ODN Equivalents / g Tissue  (Figure 2.4), concomitant with 3 to 18-fold higher uptake on a per cell basis.  70% 60% 50% 40% 30% 20% 10% 0% 0  5  10 15 Time (hours)  0  5  10  20  25  120% 100% 80% 60% 40% 20% 0% 15  20  25  Time (hours)  Figure 2.3: Preferential Accumulation of LN-CpG ODN in the lymph nodes following s.c. administration. Tissue accumulation of encapsulated and free CpG ODN in the liver (LN-CpG ODN ∆; free ODN ▲), spleen (LN-CpG ODN □; free ODN ■) and lymph nodes (LN-CpG ODN ◊ ; free ODN ♦) tracking 3H-labelled ODN following intravenous (A) and subcutaneous (B) administration, expressed as the percent of the injected dose per gram of tissue ± SD, each data point represents three mice. Encapsulated and free CpG ODN was administered intravenously at an ODN dose of 20mg/kg or 360440µg/animal, or subcutaneously at a dose of 4.5-5.5mg/kg or 100µg/animal.  60  A)  Spleen  % Uptake  CD11c+  Blood 100%  100%  80%  80%  80%  60%  60%  60%  40%  40%  40%  20%  20%  20%  0%  % Uptake  5  10  15  20  25  % Uptake  10  15  20  25  100%  80%  80%  80%  60%  60%  60%  40%  40%  40%  20%  20%  20%  10  15  20  25  0  5  10  15  20  25  100%  100%  100%  80%  80%  80%  60%  60%  60%  40%  40%  40%  20%  20%  20%  5  10  15  20  0  25  5  10  15  20  25  100%  100%  100%  80%  80%  80%  60%  60%  60%  40%  40%  40%  20%  20%  20%  0%  0% 0  5  10 15 Tim e (h)  20  25  5  10  15  20  25  0  5  10  15  20  25  0  5  10  15  20  25  0  5  10 15 Time (h)  20  25  0%  0% 0  0  0%  0% 5  0%  % Uptake  5  100%  0  B220/ CD45R+  0  100%  0%  CD11b+  0%  0% 0  Mac3+  Lymph Nodes  100%  0% 0  5  10 15 Tim e (h)  20  25  Figure 2.4: LN-CpG ODN are accumulated by CD11c positive, Mac3 positive, CD11b positive and B220/CD45R positive cells in the blood, spleen and lymph nodes compartments following i.v and s.c. administration. Uptake of LN-CpG ODN containing FITC-labeled ODN by CD11c-, Mac3-, CD11b- and B220/CD45R- positive cells in the spleen, blood and lymph node compartments following intravenous (▲) or subcutaneous (□) administration at an ODN dose of 10mg/kg. Results are expressed as (A) the percent of cells positive for uptake and (B) MFI ± SD, as determined by flow cytometry. Each data point represents four mice, blood results represent 4 mice pooled.  61  B)  Spleen  30  120  25  25  20  20  15  15  40  10  10  20  5  5  MFI  100 80 60  0 0  5  10  15  20  25  1200  Mac3+ MFI  800 600  200 0 0  MFI  5  10  15  20  25  5  10  15  20  25  5  10  15  20  25  50 40  3  30  2  20  1  10  0 25  120 100 80  0  5  10  15  20  25  25  20  20  15  15  10  20  5  0  0 0  5  10  15  Time (h)  20  25  15  20  25  0  5  10  15  20  25  0  5  10  15  20  25  0  25  60 40  10  0 0  4  20  5  10  60  15  0  20  5  10  25  30  40 20 0  200  5  20  40  70  0  15  50  6  0  10  60  7  50  5  70  250  150  0  80  300  100  MFI  0 0  120 100 80 60  400  B220/ CD45R+  0  180 160 140  1000  CD11b+  Lymph Nodes  30  MFI  CD11c+  Blood  140  10 5 0  0  5  10  15  Tim e (h)  20  25  Tim e (h)  Figure 2.4: LN-CpG ODN are accumulated by CD11c positive, Mac3 positive, CD11b positive and B220/CD45R positive cells in the blood, spleen and lymph nodes compartments following i.v and s.c. administration. Uptake of LN-CpG ODN containing FITC-labeled ODN by CD11c-, Mac3-, CD11b- and B220/CD45R- positive cells in the spleen, blood and lymph node compartments following intravenous (▲) or subcutaneous (□) administration at an ODN dose of 10mg/kg. Results are expressed as (A) the percent of cells positive for uptake and (B) MFI ± SD, as determined by flow cytometry. Each data point represents four mice, blood results represent 4 mice pooled.  Greater accumulation in the lymph nodes is seen following s.c. administration which is also consistent with pharmacokinetic and biodistribution data. Two to 5-fold greater numbers of APCs positive for uptake and 5 to 10-fold higher uptake per cell is observed compared to i.v. administration. However, unlike the peripheral blood and splenic compartments that exhibit similar biodistribution and uptake patterns, APCs in the lymph nodes demonstrate significantly 62  divergent LN-CpG ODN uptake patterns based on route of administration. Uptake in lymph node APCs, both on a total cell number and a per cell basis, increase throughout the first 24.5 hours after s.c. administration for all cells with the exception of Mac-3 cells which reach maximal levels by 7.5 h and plateau thereafter. In contrast, after i.v. administration, maximum lymph node accumulation is observed for all cell-types by 7.5 hours, after which uptake levels either plateau or decline. 2.3.4 Similar levels of immune cell activation are observed following s.c. or i.v. administration of LN-CpG ODN. Administration of LN-CpG ODN by either route results in significant up-regulation of the activation markers CD69 or CD86 on NK cells, monocyte/macrophages, B- and T-lymphocytes and DCs (Figure 2.5). For all cells types, i.v. and s.c. treatment with LN-CpG ODN results in at least a 3-fold enhancement of activation marker expression compared to levels 48 hours after treatment with free ODN, which, in turn, is slightly elevated compared to control animals. We have previously found that maximal cell activation occurs 24-48h after administration of free CpG ODN (unpublished data). Similar levels of activation marker expression are observed following i.v. and s.c. administration for almost all cell types investigated, with the exception of splenic CD11b positive cells, where a notable increase in CD69 expression is observed following s.c. administration of LN-CpG ODN at the 24 hour time-point. Similar trends are observed in regards to the level of activation marker expression on a per cell basis (data not shown). Of note is that s.c. administration appears to result in a more prolonged activation of splenic APCs (Mac-3-, CD11b-, CD45R/B220- and CD11c- positive cells) and T-lymphocytes compared to i.v. as judged by CD69 and CD86 activation marker expression at the 48 and 72 hour time-points.  This same phenomenon,  however, is not observed in PBMCs.  63  A) 100% 90% % of Cell Population  80% 70% 60% 50% 40% 30% 20% 10% 0% 24h 48h 72h CD69/DX5 CD69/DX5  24h 48h 72h CD69/Mac-3 CD69/Mac-3  24h 48h 72h CD69/CD11b CD69/CD11b  24h 48h 72h  24h 48h 72h  24h 48h 72h  CD69/Mac-3  CD69/CD11b  24h 48h 72h CD69/CD45RB220 CD69/CD45RB220  24h 48h 72h CD69/CD4 CD69/CD4  24h 48h 72h CD69/CD8 CD69/CD8  24h 48h 72h CD86/CD11c CD86/CD11c  24h 48h 72h  24h 48h 72h  24h 48h 72h  CD69/CD8  CD86/CD11c  B) 100% 90%  % of Cell Population  80% 70% 60% 50% 40% 30% 20% 10% 0% CD69/DX5  24h 48h 72h CD69/CD45RB220  CD69/CD4  Figure 2.5: Similar levels of splenic and peripheral blood immune cell activation following i.v. and s.c. administration of LN-CpG ODN. Expression of cell activation markers CD69 or CD86 on splenocytes (A) and PBMCs (B) following intravenous (LN-CpG ODN ; free ODN ) (5 mice/group) or subcutaneous (LN-CpG ODN ; free ODN ) (4 mice/group) administration of LN-CpG ODN at an ODN dose of 20mg/kg. Data is expressed as the percent of CD69 or CD86 positive cells in each specific cell-type population, background fluorescence levels (1-20% depending on cell-type) were subtracted.  2.3.5 Similar levels of NK cytolytic activity and ADCC are observed following s.c. or i.v. administration of LN-CpG ODN. Levels of NK cytolytic activity, against the cell line Yac-1, are at least two-fold greater in splenocytes and PBMCs from mice treated with LN-CpG ODN than in animals 48 hours after treatment with free ODN administered by either route at all E:T ratios investigated (Figure 2.6). We have previously found that maximal NK activity occurs 2448h after administration of free CpG ODN (unpublished data). Overall, similar levels of NK activity are detected in splenocyte and PBMC populations following i.v. and s.c. administration 64  of LN-CpG ODN resulting in elevated cytotoxicity against Yac-1 cells throughout the entire 72 hour period following administration. Although levels of NK activity are similar, the kinetics do vary, with i.v. delivery resulting in peak cytolytic activity in both PBMC and splenocyte populations 24 h after i.v. administration and declining thereafter. Following s.c. administration, the activity remains relatively constant over the 72 hour time-course in PBMCs but peaks at 48 hours and declines thereafter, in splenocyte populations. A) % Chromium Release  25% 20% 15% 10% 5% 0%  B)  24 hours  48 hours  72 hours  24 hours  48 hours  72 hours  % Chromium Release  80%  60%  40%  20%  0%  Figure 2.6: Similar levels of NK cytolytic activity of splenic and peripheral blood immune cells are observed following i.v. and s.c. administration of LN-CpG ODN. Activation of NK-specific cytolytic activity in splenocyte (A) and PBMC (B) populations was assessed in a standard 4 hour Cr51 release assay using YAC-1 cells as targets following intravenous (LN-CpG ODN ■; free ODN ▲) (5 mice/group) and subcutaneous (LN-CpG ODN □; free ODN ∆) (4 mice/group) administration of LNODN at a dose of 20mg/kg or intravenous administration of an HBS control (♦). Splenocyte data is reported at an E:T ratio of 25:1, PBMC 20:1.  Furthermore, for splenocyte populations, enhanced levels of ADCC are observed ex vivo using the anti-CD20 monoclonal antibody Rituxan and the B-cell lymphoma line Daudi, following both i.v. and s.c. administration of LN-CpG ODN, with similar levels seen following delivery via both routes (Figure 2.7). In the case of PBMCs, although the NK activity is similar, analysis 65  reveals that i.v. administration of LN-CpG ODN induces a statistically significant 10-15 percent increase in ADCC levels compared to s.c. administration at all E:T ratios investigated at the 24 and 48 hour time-points, with a consistent but non-significant difference by 72 h (Figure 2.7) (Bonferroni adjusted t-tests; 24 hrs: p<0.05; and 48 hrs: p<0.05). At all time-points and E:T ratios, Daudi cell killing in the absence of Rituxan is equal to or less than 5.0% for PBMCs and 1.0% for splenocytes and Daudi cell killing in the presence of Rituxan following treatment with free ODN by either route of administration was only slightly greater than control at the 48 hour time-point.  Figure 2.7: Similar levels of ADCC of splenic and peripheral blood immune cells against the human B-cell lymphoma cell Daudi are observed following i.v. and s.c. administration of LN-CpG ODN. Activation of ADCC in splenocyte (A) and PBMC (B) populations was assessed in a  standard 4 hour Cr51 release assay using Daudi cells as targets in the presence of Rituxan following intravenous (LN-CpG ODN ■; free ODN ▲) (5 mice/group) and subcutaneous (LNCpG ODN □; free ODN ∆) (4 mice/group) administration of LN-CpG ODN at a dose of 20mg/kg or intravenous administration of an HBS control (♦). Splenocyte data is reported at an E:T ratio of 25:1, PBMC 20:1.  66  Of relevance to both the immune cell activation and NK/ADCC activity studies is the fact that dose titrations of LN-CpG ODN, including doses exceeding those administered in these studies, demonstrate a strong response between dose and immune cell activation and activity confirming that the similar immunostimulatory effects following i.v. and s.c. administration are not due delivery of excessive doses of CpG ODN (data not shown).  2.4 Discussion The results of this investigation show that despite large differences in LN-CpG ODN pharmacokinetics  and  biodistribution  following  i.v.  or  s.c.  administration,  the  immunostimulatory effects are largely similar. Four aspects of this work of particular interest concern first, the relation between the pharmacokinetics and biodistribution observed for LNCpG ODN as they relate to the mode of administration, second, the mechanism whereby similar immune responses are observed despite large differences in biodistribution, third, the areas in which the mode of administration does cause differences in immune response and, finally, the ways in which the immune response to LN-CpG ODN administered i.v. or s.c. differ from those observed by others for free ODN administered by either route. The pharmacokinetics and biodistribution characteristics of liposomally encapsulated drugs following i.v. (Klimuk, Semple et al. 2000; Semple, Klimuk et al. 2000; Gregoriadis, Bacon et al. 2002; Kamps and Scherphof 2004), and to a lesser extent, s.c. administration (Allen, Hansen et al. 1993; Oussoren, Velinova et al. 1998; Oussoren and Storm 2001), have been well characterized and are consistent with the results presented here for LN-CpG ODN. In particular, half of the injected LN-CpG ODN dose is cleared from the circulation approximately 30 minutes following i.v. injection, much longer than observed for free ODN. While the circulation lifetime of LN-CpG ODN is relatively short compared to liposomal formulations of drugs such as vincristine, where values of 10 hours or more are commonly observed (Boman, Masin et al. 67  1994), this can be attributed to the different lipid composition of the LN-CpG ODN system. The shorter circulation lifetime for the LN-CpG ODN reflect the fact that they are optimized for maximum immunostimulatory activity and are intended to be taken up by the APCs of the reticular endothelial system. For s.c. delivery, pharmacokinetic studies demonstrate that LN-CpG ODN appears in the circulation within one hour of administration and blood concentrations remain near constant below 0.1% of the injected dose over 24 hours. This behavior is consistent with that previously observed for liposomes (Allen, Hansen et al. 1993), where a continual supply of particles drain from the injection site through the regional lymph nodes into the bloodstream. The effective delivery of LN-CpG ODN to APCs in the lymph nodes following s.c. administration is dramatically illustrated by the increasing levels of uptake over the 24 hour period following injection. This is in contrast to uptake by splenic and peripheral blood APCs following i.v. and s.c. administration, as well as lymph node APCs following i.v. administration, where maximal levels were reached at 7.5 h after administration and declined thereafter. It is interesting to note that levels associated with the blood, liver and spleen were proportionately reduced relative to the i.v. route, indicating that when particles reach the blood they are removed in the liver and spleen by a mechanism similar to that following i.v. administration. The pharmacokinetic and biodistribution patterns of LN-CpG ODN indicate that particles reach APCs in the spleen, peripheral blood and lymph following both i.v.. and s.c. injection, albeit at very different levels. The fact that similar levels of immunostimulatory activity are observed is consistent with two features of APCs and their interactions with immunostimulatory particles. First, it has been long-established that APCs such as macrophages and DCs avidly accumulate exogenous particles such as liposomes (Juliano 1986; Oussoren, Zuidema et al. 1997; Oussoren, Velinova et al. 1998) which is consistent with results presented here. For example, although only a very small portion (approximately 0.1-0.2%) of the injected LN-CpG 68  ODN dose reaches the blood and spleen compartments following s.c. administration, the percentage of APCs positive for uptake is similar (50-95%) to that observed following i.v. administration. Similarly, although levels of LN-CpG ODN reaching the lymph nodes are some 50-fold lower for i.v. injection versus s.c., the percentage of APCs positive for uptake are still more than 20% of s.c. levels. The second feature concerns the fact that stimulation of immune activity requires only threshold levels of CpG ODN, and higher levels of uptake do not to lead to more activity. The fact that different effector cells from various target organs exhibit very similar levels of immune activation in spite of significant differences in LN-CpG ODN uptake most likely stems from the fact that the uptake of even a single LN-CpG-ODN results in the effective delivery of greater than 2000 CpG ODNs (Semple, Klimuk et al. 2001). The final area of discussion concerns differences between pharmacokinetics and tissue distribution of LN-CpG and free phosphorothioate ODN following s.c. and i.v. administration, and whether this results in significant differences in immune response. Stimulation of the immune system has been investigated following the i.v. & s.c., epicutaneous, intragastric, intramuscular and intradermal administration of free or lipid complexed CpG ODN, alone or combined with peptide, protein or DNA vaccination (Li, Bally et al. 2001; Whitmore, Li et al. 2001; Klimuk, Najar et al. 2004; Tengvall, Josefsson et al. 2005).  Few studies, however,  directly compare immune stimulation following different routes of CpG ODN administration. Comparative studies that have been performed investigated effects of administering free ODN by i.v. and s.c. routes in both mice (Wingender, Garbi et al. 2006) and humans (Krieg, Efler et al. 2004) and indicate a lack of immune stimulation following i.v. administration, as determined by cytotoxic T-lymphocyte activation and cytokine/chemokine profiling, respectively. This is consistent with results reported here, where relatively low levels of immune stimulation are observed for i.v. and s.c. administered free ODN. In contrast, comparison of expression levels of the activation markers CD69 and CD86 on splenocyte and PBMCs populations, 24, 48 and 72 69  hours following administration of LN-CpG ODN by either route indicates that there is no observable difference in the levels of cellular activation (DX5, Mac3, CD11b, CD45/B220, CD4, CD8 and CD11c). Treatment by either route resulted in at least a 3-fold enhancement of activation marker expression over peak levels observed after administration of free ODN, indicating that lipid encapsulation of the ODN enhances its immunostimulatory potential over that of free for all cell-types investigated, with the exception of those expressing Mac-3. On a functional level, it has been previously demonstrated that CpG-containing ODN are effective at inducing NK cell lytic activity (Ballas, Rasmussen et al. 1996). In our investigation, similar levels of NK-specific cytolytic activity were observed in splenocyte and PBMC populations of mice treated by both routes at all time-points investigated, although some differences in response kinetics was observed. Therefore, based on this measure, the data again indicates that both i.v. and s.c. administration of LN-CpG ODN results in potent, largely similar NK responses, despite disparate pharmacokinetic, biodistribution and uptake behavior. Further, levels of NK-specific cytolytic activity observed were at least two-fold greater than with free ODN. Given that CpG ODNs activate two effector cell populations that are effective mediators of ADCC (NK cells and monocytes/macrophages), activation of ADCC resulting from administration of LN-CpG ODN was studied in detail. Stimulation of ADCC is important as tumor-specific monoclonal antibodies can have significant clinical activity (Maloney, GrilloLopez et al. 1997) and most employ ADCC as part of their mechanism of action (Villamor, Montserrat et al. 2003). While similar levels of ADCC were observed for splenocytes isolated from mice treated with LN-CpG ODN by both routes, PBMCs exhibited a statistically significant increase in ADCC following i.v. administration, as compared to s.c. at all E:T ratios investigated at the 24, 48 and, to a lesser extent, 72 hour time-points. This suggests that i.v. administration of LN-CpG ODN could offer improved therapeutic benefit, compared with s.c., when used to improve the potency of monoclonal antibodies via ADCC. 70  In conclusion, results presented here indicate that LN-CpG ODN elicit similar immunostimulatory responses following i.v. and s.c. administration in spite of dramatic differences in biodistribution and cellular uptake. It is concluded that the inherent ability of APCs to accumulate liposomal nanoparticles results in very efficient uptake of LN-CpG ODN, even when present at very low concentrations, resulting in enhanced immune responses as compared to free ODN. Any differences that are observed are more of degree than of kind, indicating, for example, that i.v. administration may be preferred to s.c. administration if LNCpG ODN are used to increase the potency of monoclonal antibodies via ADCC. 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Klinman (2001). "Cutting edge: Role of Toll-like receptor 9 in CpG DNA-induced activation of human cells." J Immunol 167(7): 3555-8. Tengvall, S., A. Josefsson, J. Holmgren and A. M. Harandi (2005). "CpG oligodeoxynucleotide augments HSV-2 glycoprotein D DNA vaccine efficacy to generate T helper 1 response and subsequent protection against primary genital herpes infection in mice." J Reprod Immunol 68(1-2): 53-69. Villamor, N., E. Montserrat and D. Colomer (2003). "Mechanism of action and resistance to monoclonal antibody therapy." Semin Oncol 30(4): 424-33. Whitmore, M. M., S. Li, L. Falo, Jr. and L. Huang (2001). "Systemic administration of LPD prepared with CpG oligonucleotides inhibits the growth of established pulmonary metastases by stimulating innate and acquired antitumor immune responses." Cancer Immunol Immunother 50(10): 503-14. Wingender, G., N. Garbi, B. Schumak, F. Jungerkes, E. Endl, D. von Bubnoff, J. Steitz, J. Striegler, G. Moldenhauer, T. Tuting, A. Heit, K. M. 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Antisense Res Dev 3(1): 53-66.  74  CHAPTER THREE STABILIZED PLASMID LIPID PARTICLES ACTIVATE APCs THROUGH TLR9DEPENDENT AND –INDEPENDENT MECHANISMS IN VIVO2 3.1 Introduction It is widely appreciated that plasmid DNA (pDNA) activates antigen presenting cells (APCs) of the innate immune system, inducing the upregulation of co-stimulatory molecules and the production of pro-inflammatory and T-helper 1 (Th1) biased cytokines (Leclerc, Deriaud et al. 1997; Roman, Martin-Orozco et al. 1997; Zelenay, Elias et al. 2003), thus promoting the generation of Th1-biased immune responses towards encoded or co-delivered peptide or protein antigens (Sato, Roman et al. 1996; Klinman, Yamshchikov et al. 1997; Leclerc, Deriaud et al. 1997; Schneeberger, Wagner et al. 2004). The discovery that the immunostimulatory effects of bacterial DNA (Tokunaga, Yamamoto et al. 1984) could be mimicked by synthetic oligodeoxynucleotides (ODN) containing unmethylated cytosine guanosine (CpG) motifs (Krieg, Yi et al. 1995) has led to the hypothesis that the adjuvant properties of bacterial DNA are dependent on internal CpG motifs (Krieg 2002) which act via a Toll-like receptor 9 (TLR9) dependent mechanism, similar to that observed for synthetic CpG-ODN (Hemmi, Takeuchi et al. 2000; Bauer, Kirschning et al. 2001). In support of this hypothesis, the direct interaction of pDNA with TLR9 has been demonstrated to result in the upregulation of co-stimulatory molecules on APCs, and the secretion of Th1-biased pro-inflammatory cytokines by various cells of the innate immune system (Spies, Hochrein et al. 2003; Zelenay, Elias et al. 2003). Furthermore, cultured murine DCs deficient in TLR9 are unresponsive to both synthetic CpGODN and naked pDNA (Spies, Hochrein et al. 2003; Tudor, Dubuquoy et al. 2005). These and other findings clearly demonstrate that the immunopotency of pDNA vaccines is dependent on, 2  A version of this chapter has been prepared for submission as a short communication. Kaley D Wilson, Susan D. deJong, Mikameh Kazem, Jan P. Dutz, Pieter R. Cullis and Ying K. Tam. Stabilized plasmid lipid particles activated APCs through TLR9-dependent and –independent mechanisms in vivo.  75  (Sato, Roman et al. 1996) and can be influenced by, their CpG content (Klinman, Yamshchikov et al. 1997; Schneeberger, Wagner et al. 2004). However, the relative contribution of CpG motifs to the immunogenicity of pDNA are not understood. Currently evidence for a TLR9independent cytosolic pathway of DNA detection (Okabe, Kawane et al. 2005; Ishii, Coban et al. 2006), evidence that DNA–cationic liposome complexes can induce inflammatory responses in a TLR9-dependent and –independent manner (Zhao, Hemmi et al. 2004; Yasuda, Ogawa et al. 2005; Yasuda, Yu et al. 2005) and evidence that DNA vaccines remain immunogenic when administered to TLR9 knockout (KO) mice exists (Spies, Hochrein et al. 2003; Babiuk, Mookherjee et al. 2004; Tudor, Dubuquoy et al. 2005; Pavlenko, Leder et al. 2007). This therefore suggests a need to better understand the cellular and molecular mechanisms behind pDNA mediated activation of innate immune cells. Further, although immunotherapies using DNA, such as DNA vaccines, benefit from concurrent immune cell activation, the immunostimulatory activity of pDNA remains a major obstacle for therapeutic gene delivery. Understanding the mechanisms and contribution of TLR9-dependent and -independent recognition of pDNA will allow for rational design and manipulation of DNA based drugs to enhance their therapeutic activity. Here, using TLR9 KO mice, we demonstrate that the immunostimulatory activity of pDNA encapsulated in a lipid particle (stabilized plasmid lipid particles; SPLP) supports the proposal that TLR9-dependent and independent pathways exist for the recognition and immunostimulatory activity of pDNA in vivo. Furthermore we demonstrate that, in contrast to observations made by others with regards to CpG-ODN (Hacker, Mischak et al. 1998; Macfarlane and Manzel 1998; Ahmad-Nejad, Hacker et al. 2002; Rutz, Metzger et al. 2004), treatment with the endosomolytic agent chloroquine increases the immunostimulatory potential of SPLP. From these data, we propose that pDNA, delivered by non-viral vectors, has the potential to act through both a TLR9 –dependent (endosomal) and TLR9–independent 76  (cytoplasmic) pDNA detection system (Suzuki, Mori et al. 1999; Ishii, Suzuki et al. 2001; Okabe, Kawane et al. 2005; Ishii, Coban et al. 2006) to elicit immune responsiveness.  3.2 Materials and Methods 3.2.1 Materials.  Distearoylphosphocholine (DSPC) and palmitoyloleoylphosphatidycholine  (POPC) was purchased from Avanti Polar Lipids (Alabaster, AL) and cholesterol was obtained from Sigma (St. Louis, MO). 1,2-dioleyloxy-3-N,N-dimethylaminopropane (DODMA) and polyethylene  glycol-dimyristol  glycerol  (PEG-DMG)  were  provided  by  Tekmira  Pharmaceuticals Corporation (Burnaby, BC, Canada). Lipopolysaccharide (LPS) from E. coli was purchased from Sigma (Oakville, Canada) and pharmaceutical grade hydroxychloroquine sulphate USP was purchased from Wiler Fine Chemicals (London, Canada). The pCMVβgal plasmid encoding the reporter gene beta-galactosidase (βgal), under the control of the cytomegalovirus promoter, was propagated in E. coli strain DH5α and purified by standard alkaline lysis with two rounds of cesium chloride density gradient centrifugation. Endotoxin levels in pDNA were less than 10 EU/mg as determined by the limulus amoebocyte lysate (LAL) chromogenic endpoint assay as per the manufactures instructions (Charles River Laboratories; Wilmington, MA).  INX-6295, a 16-mer phosphorothioate (PS) ODN  (5’TAACGTTGAGGGGCAT-3’) containing unmethylated cytosine residues in the CpG motif, was synthesized by Trilink Biotechnologies (San Diego, CA).  3.2.2 Animals.  Eight to 12-week-old C57BL/6 mice were obtained from Charles River  Laboratories (Saint-Constant, PQ, Canada) and quarantined for 2 weeks prior to use. TLR9 KO mice (Kaisho et al 2001) were obtained from Oriental Biosciences Inc and backcrossed to C57BL/6 mice over 8 generations. Mice were held in a pathogen-free environment and all  77  procedures involving animals were performed in accordance with the guidelines established by the Canadian Council on Animal Care.  3.2.3 Preparation of SPLP and LN-CpG ODN.  Plasmid was encapsulated in lipid  nanoparticles containing an ionisable aminolipid using an ethanol dialysis procedure, as previously described (Maurer, Wong et al. 2001; Jeffs, Palmer et al. 2005). Briefly, a lipid mixture consisting of DSPC/cholesterol/DODMA/PEG-DMG at a molar ratio of 20/55/15/10 was solubilized in absolute, anhydrous ethanol and then diluted by adding distilled water to achieve an ethanol concentration of 90%. The total concentration of lipid in solution was 20 mM.  A plasmid solution was prepared by combining pDNA in 10 mM Tris EDTA (TE)  buffer with 100 mM citrate (pH 5.0) and distilled deionized water to achieve a pDNA concentration of 0.9 mg/ml in 20 mM citrate. Equal volumes of both lipid and plasmid solutions were heated to 37°C prior to vesicle formation.  Vesicles were prepared by the  dropwise addition of equal volumes of lipid dissolved in ethanol to a rapidly mixing aqueous buffer containing DNA, resulting in an ethanol concentration of 45%, and were then diluted dropwise to 36% ethanol with 10 mM citrate, pH 5.0, 750 mM NaCl resulting in a final concentration of 10mM citrate, 150 mM NaCl. The diluted vesicles were then incubated at 37°C for 1 h prior to dialysis first against 10 mM citrate (pH 6.0) containing 150 mM NaCl for 2 h, followed by PBS overnight at pH 7.5. Unencapsulated pDNA was subsequently removed by anion exchange chromatography on DEAE-Sepharose CL-6B columns equilibrated in PBS. SPLP were characterized with respect to plasmid entrapment using a previously described PicoGreen (Invitrogen Molecular Probes, Burlington, Canada) assay (Jeffs, Palmer et al. 2005), and mean particle diameter was determined using a submicron quasi-elastic light scattering particle sizer (Nicomp, Santa Barbara, CA). SPLP formulations used in this study demonstrated  78  a plasmid-to-lipid ratio of 0.04 - 0.05 (w/w), a maximum of 5–8 % unentrapped plasmid following purification and particle sizes of 145 ± 50 nm (χ2 = 0.3) in diameter. ODN  were  encapsulated  in  lipid  nanoparticles  consisting  of  POPC/cholesterol/DODMA/PEG-DMG at a molar ratio of 25/45/20/10 containing an ionisable aminolipid using an ethanol dialysis procedure, as previously described (Maurer, Wong et al. 2001; Wilson, Raney et al. 2007). The ODN-to-lipid ratio was typically 0.1 (w/w) and particle size was 100 ± 25 nm.  3.2.4 Immune activation studies. Eight to 12-week-old C57BL/6 mice or TLR9 KO mice were injected intravenously with HBS, free pDNA (5 mg/kg), SPLP (5 mg/kg), free CpG-ODN (20mg/kg), LN CpG-ODN (20 mg/kg) or LPS (2mg/kg). Mice were terminally anaesthetized with ketamine/xylazine (3.2%/0.8%, v/v) at the indicated time-points.  3.2.5 Chloroquine study. Six to 8-week old C57BL/6 mice were injected subcutaneously with 15 mg/kg of pharmaceutical grade hydroxychloroquine sulphate or HBS once per day for 3 days. Five hours following the final administration, mice were injected subcutaneously with HBS, free CpG-ODN, free pDNA (5 mg/kg) or SPLP (5 mg/kg) at the same site, and were euthanized 24 h later.  3.2.6 Cell activation analysis.  For cellular activation assays, spleens were collected and  processed to single cell suspensions by passage through a sterile 100 µm nylon mesh filter (Becton Dickenson, Franklin Lakes, NJ). Splenocytes were depleted of red blood cells by ammonium chloride lysis and analyzed for immune stimulation as judged by cell activation marker/phenotype expression. Cell suspensions were stained with fluorescently labeled phenotype antibodies (anti-CD11b, anti-CD11c, anti-B220/CD45R) in combination with labeled 79  antibodies directed against the activation markers CD69 or CD86. Cell activation was analyzed using a LSRII flow cytometer with FACSDiva v4.1 software (BD Biosciences, San Jose, CA) and data was analyzed with FlowJo flow cytometry analysis software v7.2.2 (Ashland, OR).  3.2.7 Plasma cytokine analysis. For plasma cytokines, mice were terminally anaesthetized as previously described and blood was collected via cardiac puncture. Plasma was isolated by centrifugation and frozen at –20oC. Plasma concentrations of IL-6, IFN-γ and MCP-1 were determined using a cytometric bead array kit (BD Biosciences), as per manufacturer’s instructions.  3.2.8 Statistical analyses.  All statistical analyses were performed using SPSS Version 14.0.  For TLR9 KO studies, an ANOVA was used to statistically evaluate the differences between treatment groups. In the case of statistically significant results, the differences between treatment groups were assessed through the use of a Tukey HSD adjusted t-test. A Student’s t-test was used to examine differences in immune activations between animals treated with and without chloroquine, equal variances were assumed based on the Levene’s test for equality of variances. Probability (p) values less than .05 were considered significant.  3.3 Results 3.3.1 Encapsulation of pDNA within lipid nanoparticles dramatically enhances immune cell activation following systemic administration. The effect of lipid encapsulation on the immunostimulatory potential of pDNA following systemic administration was assessed by monitoring the expression of the early activation marker CD69 and the co-stimulatory molecule CD86, both of which are known to be up-regulated by activated APCs. Over the 24 h timecourse investigated, a 15 to 100-fold increase in CD69 and CD86 expression was observed on 80  CD11c+ (DCs), CD11b+ (monocytes) and B220/CD45R+ (B-lymphocytes) splenocytes isolated from mice treated with SPLP, as compared to naked pDNA, for which activation marker expression was similar to that observed for the HBS control for all cell-types investigated (Figure 3.1). Similarly, measures of the production of the Th1 cytokine, IFN-γ, the proinflammatory cytokine, IL-6, and the macrophage chemokine, MCP-1, following the systemic administration of SPLP demonstrated a significant increase in plasma cytokine levels, which peaked between 4 and 7 h, while little or no cytokine induction was observed for naked pDNA (Figure 3.2). No appreciable immunostimulatory activity, based on immune cell activation or plasma cytokines was detectable in animals treated with empty liposomes (unpublished data).  MFI  A)  900  CD11c - SPLP  800  CD11c - Free Plasmid  700  CD11b - SPLP  600  CD11b - Free Plasmid B220/CD45R - SPLP  500  B220/CD45R - Free Plasmid  400 300 200 100 0 0  5  10  15  20  25  Time (hours)  B)  140 120  MFI  100 80 60 40 20 0 0  5  10  15  20  25  Time (hours)  Figure 3.1: Comparison of immune cell activation following systemic administration of free or encapsulated plasmid. Free or encapsulated plasmid (SPLP) was administered i.v. to C57BL/6 mice (n = 3) at a dose of 5 mg/kg. Splenocytes were harvested 4, 7 and 24 h after administration and were analyzed for the expression of the cell surface activation markers CD86 (A) and CD69 (B) in conjunction with phenotype markers by flow cytometry. Data represents MFI ± SEM, background fluorescence levels (MFI of 2-50 depending on cell-type) were subtracted.  81  A)  B) 2000  2400  SPLP Free Plasmid  1600 IL-6 (pg/mL)  IFN-γ (pg/mL)  2000 1600 1200 800  1200 800 400  400  0  0 0  5  10  15  20  25  0  5  C)  10  15  20  25  Tim e (hours)  Tim e (hours)  12000  MCP-1 (pg/mL)  10000 8000 6000 4000 2000 0 0  5  10 15 Tim e (hours)  20  25  Figure 3.2: Comparison of plasma cytokine induction following systemic administration of free or encapsulated plasmid. Free or encapsulated plasmid (SPLP) was administered i.v. to C57BL/6 mice (three animals per group) at a dose of 5 mg/kg. Blood was collected from animals by cardiac puncture, processed to collect plasma and cytokine levels (pg/ml ± SEM) were determined by cytometric bead analysis in conjunction with flow cytometry. (A) IFN-γ, (B) IL-6 and (C) MCP-1. Baseline cytokine levels observed in controls have been subtracted.  3.3.2 SPLP demonstrates immunostimulatory activity in TLR9 KO mice. The contribution of TLR9 to the immunostimulatory activity of SPLP in vivo was assessed by comparing immune cell activation and cytokine secretion in WT and TLR9 KO mice following treatment with SPLP. While SPLP elicited significant immunostimulatory activity in both WT and TLR9 KO mice, as assessed by the upregulation of the activation markers CD69 and CD86 on CD11b+, CD11c+ and B220/CD45R+ cells (p < .001), the peak levels of expression of the activation marker CD69 was significantly decreased (p < .001) in TLR9 KO mice for all cell-types assessed at 7 h. A similar, but less dramatic decrease, which approached statistical significance, was observed for peak levels of CD86 expression at 24 h (Figure 3.3). Concomitantly, TLR9  82  KO mice also demonstrated a statistically significant induction of pro-inflammatory cytokines IFN-γ, MCP-1 and IL-6 in response to SPLP (p< .05, p< .001, p< .05 respectively), which were significantly lower (p< .05, p< .001, p< .01 respectively) than levels observed following SPLP administration to WT mice (Figure 3.4). As expected, the immunostimulatory activity of LN CpG-ODN observed in WT mice was completely abolished in TLR9 KO mice, confirming that PS CpG-ODN signaling is mediated exclusively through TLR9 (Figure 3.3 & 3.4). Both WT and TLR9 KO mice were responsive to LPS (data not shown), confirming that immune cells from TLR9-deficient mice are responsive to signaling through other TLRs. Of note, LPS-mediated immune stimulation was slightly enhanced in TLR9 KO mice for all parameters investigated, an observation which has not been previously reported but is assumed to reflect compensation by the innate immune system of TLR9 KO mice. Animals treated with naked pDNA or empty liposomes (data not shown) demonstrated little or no immunostimulatory activity in either strain, indicating that the immunostimulatory activity of SPLP in TLR9 KO mice was not an artifact of residual endotoxin contamination of pDNA (≤ 10 EU/mg).  3.3.3 SPLPs elicit enhanced immunostimulatory activity in the presence of the endosomolytic agent chloroquine. Endosomal maturation/acidification is required for CpG DNA-mediated activation via TLR9 and inhibitors of endosomal acidification such as chloroquine abolish the immune responses by CpG motifs in vitro (Hacker, Mischak et al. 1998; Macfarlane and Manzel 1998; Ahmad-Nejad, Hacker et al. 2002; Rutz, Metzger et al. 2004). To examine whether endosomal acidification is required for the immunostimulatory activity of SPLP in vivo, we investigated the ability of SPLP to promote the upregulation of the activation markers CD69 and CD86 on APCs, and induce the secretion of the pro-inflammatory cytokines IFN-γ, IL-6 and MCP-1 in the presence of chloroquine in a murine model. 83  A)  D) SPLP - WT SPLP - TLR9 KO Free Plasmid - WT Free Plasmid - TLR9 KO LN CpG-ODN - WT LN CpG-ODN - TLR9 KO Free CpG-ODN - WT Free CpG-ODN - TLR9 KO  500  MFI  400 300  600 500 400 MFI  600  200  100  100  0  0  0  10  20  30  40  50  0  10  20  E)  Time (hours )  600  600  500  500  400  400 MFI  MFI  B)  300  30  40  50  30  40  50  30  40  50  Tim e (hours)  300  200  200  100  100  0  0 0  10  20  C)  30  40  50  0  10  20  F)  Tim e (hours )  600  600  500  500  400  400 MFI  MFI  300  200  300  300  200  200  100  100  0  Tim e (hours )  0 0  10  20  30 Tim e (hours )  40  50  0  10  20 Tim e (hours )  Figure 3.3: SPLP promotes the upregulation of activation markers CD69 and CD86 on APCs in WT and TLR9 KO mice. Five mg/kg of free or encapsulated plasmid (SPLP), 20 mg/kg of free or encapsulated CpG ODN (LN CpG-ODN) or 2 mg/kg LPS was administered i.v. to C57BL/6 WT or C57BL/6 TLR9 KO mice (three animals per group). Mice were euthanized at 7, 24, 48 h and spleens harvested. Splenocytes were analyzed for expression of CD69 (Panels A-C) and CD86 (Panels D-F) cell surface activation markers (MFI ± SEM) on CD11b+ve monocytes/macrophages (Panels A and D), CD11c+ve DCs (Panels B and E) and CD45R/B220 B-lymphocytes (Panels C and F); background fluorescence levels (MFI of 2-50 depending on cell-type) were subtracted.  84  A)  10000  TRL9 +/+ TLR9 -/-  1725 615  IFN-γ (pg/mL)  1000  100 25 13  10  0  0  1 Free CpG-ODN  B)  LN CpG-ODN  0  0  Free Plasmid  813  1000  IL-6 (pg/mL)  SPLP  359  82  100 35  10 2  0  1  Free CpG-ODN  C)  0  LN CpG-ODN  0  Free Plasmid  100000 10116  10000  4106  2755  2284  MCP-1 (pg/mL)  SPLP  1000  100 9  10  1  0  Free CpG-ODN  0  LN CpG-ODN  0  Free Plasmid  SPLP  Figure 3.4: Systemic administration of SPLP promotes the induction of pro-inflammatory cytokines in WT and TLR9 KO mice. Five mg/kg of free or encapsulated plasmid (SPLP), 20 mg/kg free or encapsulated CpG-ODN (LN CpG-ODN) or 2 mg/kg LPS was administered i.v. to C57BL/6 WT or C57BL/6 TLR9 KO mice (three animals per group). Mice were euthanized at 7, 24, 48 h blood was collected by cardiac puncture, processed to collect plasma and cytokine levels (pg/ml ± SEM) were determined by cytometric bead array in conjunction with flow cytometry. (A) IFN-γ, (B) IL-6 and (C) MCP-1. Baseline cytokine levels observed in WT and KO controls were similar and have been subtracted.  85  Surprisingly, consistent and statistically significant increases in immune cell activation were observed, as assessed by the upregulation of activation markers CD69 and CD86 in each cell type examined (p< .05; CD11b+, CD11c+ and B220/CD45R+) (Figure 3.5). Statistically significant increases in cytokine secretion (p< .05; IFN-γ & IL-6 and MCP-1) in mice which had been pre-treated with chloroquine were also observed (Figure 3.6).  Similar to the results  obtained in previous studies, free pDNA demonstrated little or no immunostimulatory activity in the presence or absence of chloroquine and mice treated with chloroquine alone revealed no immune cell activation above that observed for the PBS control (data not shown). A)  2000 1600  Free plasmid w ith chloroquine SPLP w /out chloroquine SPLP w ith chloroquine  MFI  1200  Free plasmid w /out chloroquine  800 400 0  B)  CD11b+  CD11c+  CD11b+  CD11c+  B220/CD45R +  300 250  MFI  200 150 100 50 0 B220/CD45R +  Figure 3.5: Chloroquine enhances immune cell activation following treatment with SPLP. C57BL/6 mice (three animals per group) were treated s.c. with HBS or chloroquine (15 mg/kg) once a day for 3 days. Five h following the final injection mice were administered free or encapsulated plasmid (SPLP) s.c. at a dose of 5 mg/kg and 24 h later the expression of the cell surface activation markers CD69 (A) and CD86 (B) by splenocytes (CD11b+, CD11c+, B220/CD45R+) were analyzed by flow cytometry. Data represents MFI ± SEM, background fluorescence levels (MFI of 2-50 depending on cell-type) were subtracted.  86  B)  A)  IFN-γ (pg/mL)  400  600  w /out chloroquine  500  w ith chloroquine  IL-6 (pg/mL)  500  300 200 100  400 300 200 100 0  0 Free Plasmid  Free Plasmid  SPLP  C)  SPLP  10000  MCP-1 (pg/mL)  8000 6000 4000 2000 0 Free Plasmid  Figure 3.6:  SPLP  Chloroquine enhances plasma cytokine levels following treatment with SPLP.  C57BL/6 mice (three animals per group) were treated s.c. with HBS or chloroquine (15 mg/kg) once a day for 3 days. Five h following the final injection mice were administered free or encapsulated plasmid (SPLP) s.c. at a dose of 5 mg/kg. Mice were euthanized 24 h later and blood was collected by cardiac puncture, processed to collect plasma and cytokine levels (pg/ml ± SEM) were determined by cytometric bead array in conjunction with flow cytometry. (A) IFNγ, (B) IL-6 and (C) MCP-1. Baseline cytokine levels observed in WT and KO controls were similar and have been subtracted. 3.4 Discussion Activation of the innate immune system by DNA has been studied extensively by numerous groups. While the immunostimulatory activity of CpG-ODN phosphorothioates are clearly TLR9-dependent (Hemmi, Takeuchi et al. 2000; Bauer, Kirschning et al. 2001; Yasuda, Yu et al. 2005) the mechanisms of recognition of double-stranded bacterial or vertebrate DNA have yet to be completely elucidated. It has been proposed that unmethylated CpG motifs are responsible for the induction of innate immunity by bacterial DNA via a TLR9 dependent mechanism, and findings from 87  numerous groups support this hypothesis (Yamamoto, Yamamoto et al. 1992; Hemmi, Takeuchi et al. 2000; Krieg 2002; Akira and Takeda 2004; Beutler 2004). However, the discovery of a TLR9-independent cytosolic pathway of dsDNA detection (Suzuki, Mori et al. 1999; Ishii, Suzuki et al. 2001; Okabe, Kawane et al. 2005; Ishii, Coban et al. 2006) and evidence that DNA–cationic liposome complexes containing bacterial or mammalian DNA can induce inflammatory responses by macrophage (Yasuda, Ogawa et al. 2005) in a TLR9-dependent and independent manner (Yasuda, Yu et al. 2005) has brought into question both the impact of CpG motifs on pDNA immunogenicity and the mechanisms behind the immunostimulatory activity of pDNA. Here we provide in vivo evidence that, when encapsulated in a lipid nanoparticle, the immunostimulatory activity of pDNA is mediated through both TLR9-dependent and independent processes. Furthermore we demonstrate that the immunostimulatory potential of SPLP is enhanced in the presence of the endosomolytic agent chloroquine, supporting the ability of pDNA delivered in a non-viral vector to act through a cytoplasmic dsDNA detection system. We, and others, have previously demonstrated that the inflammatory response induced by systemically administered CpG-ODN (Tan, Li et al. 1999; Mui, Raney et al. 2001; de Jong, Chikh et al. 2007) or pDNA (Scheule, St George et al. 1997; Freimark, Blezinger et al. 1998; Li, Wu et al. 1999; Yew, Wang et al. 1999; Loisel, Le Gall et al. 2001) is significantly enhanced when complexed within a cationic lipid-containing liposome. This is presumably due to protection of the nucleic acid payload and increased cellular uptake by APCs, as these cell-types are known to avidly accumulate liposomes (Juliano 1986; Oussoren, Zuidema et al. 1997; Oussoren, Velinova et al. 1998; Wilson, Raney et al. 2007). In agreement with these observations, we observe here that liposomal encapsulation dramatically increases the immunogenicity of pDNA following systemic administration when compared to free pDNA, promoting the induction of pro-inflammatory cytokines (IFN-γ, MCP-1, IL-6) and the upregulation of the activation markers CD86 or CD69 on CD11b+, CD11c+ and B220/CD45R+ 88  APCs. Failure of naked pDNA to mount a detectable immune response following systemic administration is not surprising as the phosphodiester backbone is known to be highly susceptible to rapid degradation by serum nucleases (Kawabata, Takakura et al. 1995; Lew, Parker et al. 1995). It has been demonstrated that unmethylated CpG residues present in pDNA are a major contributor to the induction of proinflammatory cytokines following the systemic administration of cationic lipid complexed pDNA (Li, Wu et al. 1999; Loisel, Le Gall et al. 2001; Zhao, Hemmi et al. 2004) as methylation of these motifs significantly suppress the inflammatory response (Yew, Wang et al. 1999) and TLR9 KO mice demonstrate dramatically reduced toxic responses (adverse hematological changes and liver damage) following treatment with cationic-lipid pDNA complexes (Zhao, Hemmi et al. 2004). Interestingly, when the immunostimulatory activity of pDNA is assessed ex vivo in TLR9 -/- macrophage and DCs it appears that naked pDNA acts in a completely TLR9-dependent manner (Spies, Hochrein et al. 2003; Tudor, Dubuquoy et al. 2005). However, complexing pDNA with cationic lipids confers the ability of pDNA to signal via TLR9-dependent and independent pathways, although the responses are reduced in the absence of TLR9 (Tudor, Dubuquoy et al. 2005; Yasuda, Ogawa et al. 2005; Yasuda, Yu et al. 2005).  In contrast to PS CpG-ODN, whose immunostimulatory activity is  entirely dependent on TLR9 both in the free and lipid encapsulated form, we demonstrate in vivo, that when encapsulated within a stabilized lipid particle, the immunostimulatory activity of pDNA is mediated through both TLR9-dependent and independent pathways. Although SPLPmediated immune stimulation was approximately 2-fold lower in TLR9 KO mice as compared to WT for all parameters investigated, SPLP demonstrates significant immunostimulatory activity in both WT and TLR9 KO mice as assessed by the upregulation of activation markers on APCs and the induction of pro-inflammatory cytokines. Therefore, our data strongly supports the existence of a TLR9-independent pathway of pDNA recognition and that this pathway plays a 89  major role in immune stimulation when pDNA is delivered as SPLP in vivo. This is somewhat contrary to the findings of Zhao et al where a very significant decrease in plasma cytokine levels (1000 to 5000-fold for IFN-γ and IL-6, respectively) were observed following the administration of cationic-lipid pDNA complexes to TLR9 KO mice. A possible reason for this difference is the enhanced ability of SPLP to protect pDNA from cleavage by serum nucleases, as compared to cationic-lipid pDNA complexes (Wheeler, Palmer et al. 1999; Tam, Monck et al. 2000), thus promoting improved delivery and uptake of intact plasmid by target APCs. In addition, it has been well documented that endocytosis of CpG-ODN and endosomal acidification (maturation) are essential processes for TLR9 recognition of CpG-DNA as compounds such as chloroquine, which interfere with endosomal acidification, inhibit CpGDNA-driven signaling (Hacker, Mischak et al. 1998; Macfarlane and Manzel 1998; AhmadNejad, Hacker et al. 2002). Interestingly, chloroquine is also widely used in conjunction with non-viral plasmid delivery systems to improve the efficacy of gene delivery (Luthman and Magnusson 1983; Ciftci and Levy 2001). The enhanced efficacy has been attributed to the chloroquine-mediated increase in lysosomal pH, which reduces enzymatic degradation of pDNA (Cotten, Wagner et al. 1992; Wagner, Zatloukal et al. 1992) and results in osmotic swelling. Such swelling promotes disruption of the endosome (de Duve, de Barsy et al. 1974), enhancing the fraction of internalized pDNA released into the cytoplasm intact (Luthman and Magnusson 1983). Here we report the novel observation that the endosomolytic agent chloroquine also enhances pDNA mediated immune stimulation in vivo, when pDNA is delivered as SPLP. This discovery, when combined with observations made in TLR9 KO mice, indicates that lipid nanoparticulate pDNA has the potential to act through a TLR9-independent pathway, particularly when access to the cytoplasm is promoted and suggests that pDNA delivered in lipid nanoparticles has the potential act through a cytoplasmic dsDNA detection system such as those previously proposed (Suzuki, Mori et al. 1999; Ishii, Suzuki et al. 2001; Okabe, Kawane et al. 90  2005; Ishii, Coban et al. 2006). Furthermore, we hypothesize that based on the inherent ability of cationic lipids to aid in endosomal membrane destabilization (Zhou and Huang 1994; El Ouahabi, Thiry et al. 1997; Wattiaux, Jadot et al. 1997; Hafez, Maurer et al. 2001), cationic lipid containing non-viral pDNA delivery systems may naturally promote access to cytoplasmic TLR9-independent pathways of dsDNA recognition. Thus providing a possible explanation as to why naked pDNA appears to acts in a completely TLR9-dependent manner (Spies, Hochrein et al. 2003; Tudor, Dubuquoy et al. 2005), while complexing pDNA with cationic lipids confers the ability of pDNA to signal via TLR9-dependent and independent pathways (Tudor, Dubuquoy et al. 2005; Yasuda, Ogawa et al. 2005; Yasuda, Yu et al. 2005). The data presented here suggests that TLR9-dependent and –independent pathways of pDNA play a cumulative role in pDNA-mediate immune stimulation when pDNA is delivered as SPLP. Furthermore, in addition to protecting the nucleic acid payload and increasing its uptake by APCs, liposomal encapsulation may provide a mechanism through which to enhance the immunostimulatory potential of pDNA by promoting access to a cytoplasmic, TLR9independent pathway of dsDNA recognition. Our findings highlight the need for DNA vaccines and DNA-based gene therapies to consider both TLR9-dependent and independent immunostimulatory activities of DNA when constructing non-viral vectors.  3.5 References Ahmad-Nejad, P., H. Hacker, M. Rutz, S. Bauer, R. M. Vabulas and H. Wagner (2002). "Bacterial CpG-DNA and lipopolysaccharides activate Toll-like receptors at distinct cellular compartments." Eur J Immunol 32(7): 1958-68. Akira, S. and K. Takeda (2004). "Toll-like receptor signalling." Nat Rev Immunol 4(7): 499511. Babiuk, S., N. Mookherjee, R. Pontarollo, P. Griebel, S. van Drunen Littel-van den Hurk, R. Hecker and L. Babiuk (2004). 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Taniguchi and S. Nagata (2005). "Toll-like receptorindependent gene induction program activated by mammalian DNA escaped from apoptotic DNA degradation." J Exp Med 202(10): 1333-9. Oussoren, C., M. Velinova, G. Scherphof, J. J. van der Want, N. van Rooijen and G. Storm (1998). "Lymphatic uptake and biodistribution of liposomes after subcutaneous injection . IV. Fate of liposomes in regional lymph nodes." Biochim Biophys Acta 1370(2): 259-72. Oussoren, C., J. Zuidema, D. J. Crommelin and G. Storm (1997). "Lymphatic uptake and biodistribution of liposomes after subcutaneous injection. II. Influence of liposomal size, lipid compostion and lipid dose." Biochim Biophys Acta 1328(2): 261-72. Pavlenko, M., C. Leder, S. Moreno, V. Levitsky and P. Pisa (2007). "Priming of CD8+ T-cell responses after DNA immunization is impaired in TLR9- and MyD88-deficient mice." Vaccine. Roman, M., E. Martin-Orozco, J. S. Goodman, M. D. Nguyen, Y. Sato, A. Ronaghy, R. S. Kornbluth, D. D. Richman, D. A. Carson and E. Raz (1997). "Immunostimulatory DNA sequences function as T helper-1-promoting adjuvants." Nat Med 3(8): 849-54. Rutz, M., J. Metzger, T. Gellert, P. Luppa, G. B. Lipford, H. Wagner and S. Bauer (2004). "Tolllike receptor 9 binds single-stranded CpG-DNA in a sequence- and pH-dependent manner." Eur J Immunol 34(9): 2541-50. Sato, Y., M. Roman, H. Tighe, D. Lee, M. Corr, M. D. Nguyen, G. J. Silverman, M. Lotz, D. A. Carson and E. Raz (1996). "Immunostimulatory DNA sequences necessary for effective intradermal gene immunization." Science 273(5273): 352-4. Scheule, R. K., J. A. St George, R. G. Bagley, J. Marshall, J. M. Kaplan, G. Y. Akita, K. X. Wang, E. R. Lee, D. J. Harris, C. Jiang, N. S. Yew, A. E. Smith and S. H. Cheng (1997). "Basis of pulmonary toxicity associated with cationic lipid-mediated gene transfer to the mammalian lung." Hum Gene Ther 8(6): 689-707. Schneeberger, A., C. Wagner, A. Zemann, P. Luhrs, R. Kutil, M. Goos, G. Stingl and S. N. Wagner (2004). "CpG motifs are efficient adjuvants for DNA cancer vaccines." J Invest Dermatol 123(2): 371-9. Spies, B., H. Hochrein, M. Vabulas, K. Huster, D. H. Busch, F. Schmitz, A. Heit and H. Wagner (2003). "Vaccination with plasmid DNA activates dendritic cells via Toll-like receptor 9 (TLR9) but functions in TLR9-deficient mice." J Immunol 171(11): 5908-12. Suzuki, K., A. Mori, K. J. Ishii, J. Saito, D. S. Singer, D. M. Klinman, P. R. Krause and L. D. Kohn (1999). "Activation of target-tissue immune-recognition molecules by double-stranded polynucleotides." Proc Natl Acad Sci U S A 96(5): 2285-90. Tam, P., M. Monck, D. Lee, O. Ludkovski, E. C. Leng, K. Clow, H. Stark, P. Scherrer, R. W. Graham and P. R. Cullis (2000). "Stabilized plasmid-lipid particles for systemic gene therapy." Gene Ther 7(21): 1867-74. 94  Tan, Y., S. Li, B. R. Pitt and L. Huang (1999). "The inhibitory role of CpG immunostimulatory motifs in cationic lipid vector-mediated transgene expression in vivo." Hum Gene Ther 10(13): 2153-61. Tokunaga, T., H. Yamamoto, S. Shimada, H. Abe, T. Fukuda, Y. Fujisawa, Y. Furutani, O. Yano, T. Kataoka, T. Sudo and et al. (1984). "Antitumor activity of deoxyribonucleic acid fraction from Mycobacterium bovis BCG. I. Isolation, physicochemical characterization, and antitumor activity." J Natl Cancer Inst 72(4): 955-62. Tudor, D., C. Dubuquoy, V. Gaboriau, F. Lefevre, B. Charley and S. Riffault (2005). "TLR9 pathway is involved in adjuvant effects of plasmid DNA-based vaccines." Vaccine 23(10): 125864. Wagner, E., K. Zatloukal, M. Cotten, H. Kirlappos, K. Mechtler, D. T. Curiel and M. L. Birnstiel (1992). "Coupling of adenovirus to transferrin-polylysine/DNA complexes greatly enhances receptor-mediated gene delivery and expression of transfected genes." Proc Natl Acad Sci U S A 89(13): 6099-103. Wattiaux, R., M. Jadot, M. T. Warnier-Pirotte and S. Wattiaux-De Coninck (1997). "Cationic lipids destabilize lysosomal membrane in vitro." FEBS Lett 417(2): 199-202. Wheeler, J. J., L. Palmer, M. Ossanlou, I. MacLachlan, R. W. Graham, Y. P. Zhang, M. J. Hope, P. Scherrer and P. R. Cullis (1999). "Stabilized plasmid-lipid particles: construction and characterization." Gene Ther 6(2): 271-81. Wilson, K. D., S. G. Raney, L. Sekirov, G. Chikh, S. D. Dejong, P. R. Cullis and Y. K. Tam (2007). "Effects of intravenous and subcutaneous administration on the pharmacokinetics, biodistribution, cellular uptake and immunostimulatory activity of CpG ODN encapsulated in liposomal nanoparticles." Int Immunopharmacol 7(8): 1064-75. Yamamoto, S., T. Yamamoto, S. Shimada, E. Kuramoto, O. Yano, T. Kataoka and T. Tokunaga (1992). "DNA from bacteria, but not from vertebrates, induces interferons, activates natural killer cells and inhibits tumor growth." Microbiol Immunol 36(9): 983-97. Yasuda, K., Y. Ogawa, I. Yamane, M. Nishikawa and Y. Takakura (2005). "Macrophage activation by a DNA/cationic liposome complex requires endosomal acidification and TLR9dependent and -independent pathways." J Leukoc Biol 77(1): 71-9. Yasuda, K., P. Yu, C. J. Kirschning, B. Schlatter, F. Schmitz, A. Heit, S. Bauer, H. Hochrein and H. Wagner (2005). "Endosomal translocation of vertebrate DNA activates dendritic cells via TLR9-dependent and -independent pathways." J Immunol 174(10): 6129-36. Yew, N. S., K. X. Wang, M. Przybylska, R. G. Bagley, M. Stedman, J. Marshall, R. K. Scheule and S. H. Cheng (1999). "Contribution of plasmid DNA to inflammation in the lung after administration of cationic lipid:pDNA complexes." Hum Gene Ther 10(2): 223-34. Zelenay, S., F. Elias and J. Flo (2003). "Immunostimulatory effects of plasmid DNA and synthetic oligodeoxynucleotides." Eur J Immunol 33(5): 1382-92.  95  Zhao, H., H. Hemmi, S. Akira, S. H. Cheng, R. K. Scheule and N. S. Yew (2004). "Contribution of Toll-like receptor 9 signaling to the acute inflammatory response to nonviral vectors." Mol Ther 9(2): 241-8. Zhou, X. and L. Huang (1994). "DNA transfection mediated by cationic liposomes containing lipopolylysine: characterization and mechanism of action." Biochim Biophys Acta 1189(2): 195203.  96  CHAPTER FOUR THE COMBINATION OF STABILIZED PLASMID LIPID PARTICLES AND LIPID NANOPARTICLE ENCAPSULATED CpG-ODN FOR THE DEVELOPMENT OF A GENETIC VACCINE3 4.1 Introduction Currently, a wide variety of approaches are being investigated for the generation of vaccines against infectious and malignant disease. DNA vaccines provide an important advantage over other vaccine strategies in that they mimic natural viral infection in their expression and processing of foreign proteins in a manner that induces T helper 1 (Th1)-biased major histocompatibility complex (MHC) class I-restricted CD8+ T-cell responses as well as humoral responses. Early work demonstrated the ability of naked plasmid DNA (pDNA) to lead to gene expression (Wolff, Malone et al. 1990) following i.m. injection, and the subsequent elicitation of both cellular and humoral immune responses, leading to protection from a targeted disease in animal models (Tang, DeVit et al. 1992; Fynan, Webster et al. 1993; Ulmer, Donnelly et al. 1993). In later studies attempts have been made to use plasmid-based immunization to generate protective immunity against a wide variety of infectious, malignant, autoimmune and allergic diseases (Cui 2005; Prud'homme 2005; Laddy and Weiner 2006).  Although naked DNA  vaccines have proven effective in small animal models, in human clinical studies they have often been found to prime sub-optimal immune responses. This can be attributed in part, to low in vivo transfection efficiencies stemming from the rapid degradation of the phosphodiester (PO) backbone (Kawabata, Takakura et al. 1995; Lew, Parker et al. 1995) as well as inefficient uptake of free DNA by antigen-presenting cells (APCs).  3  A version of this chapter has been prepared for submission. Kaley D Wilson, Susan D. deJong, Mikameh Kazem, Michael J. Hope, Pieter R. Cullis and Ying K. Tam. The combination of stabilized plasmid lipid particles and lipid nanoparticle encapsulated CpG-ODN for the development of a genetic vaccine.  97  Considerable effort is now being focused on developing ways to improve the efficacy of DNA vaccines, and it is well recognized that the route and method of DNA vaccine administration strongly influences both the strength and nature of the resultant immune response (Alpar and Bramwell 2002). While systemic administration of genetic vaccines possesses the advantage of direct access to the relatively large number of APCs in the spleen and has, in a number of instances, demonstrated the potential to induce antigen-specific humoral and cellular responses (McCluskie, Brazolot Millan et al. 1999; Fang, Liu et al. 2002; Cui, Asada et al. 2003; Neal, Bates et al. 2007), the instability of naked pDNA in the blood makes this approach very inefficient. It is clear that nucleic acid-based vaccines for the immunotherapy of infectious disease and cancer would benefit from the development of vector systems that can be safely and effectively administered systemically. Cationic lipid-based delivery systems, which partially protect the nucleic acid payload from nuclease degradation and improve uptake by APCs, (Juliano 1986; Oussoren and Storm 1997; Oussoren, Velinova et al. 1998) can dramatically enhance the potency of DNA vaccines following a number of routes of administration (Chen and Huang 2005; Little and Langer 2005). However, cationic lipid-based delivery systems have a limited ability to protect plasmid DNA from degradation by serum nucleases and the systemic administration of these complexes is often associated with significant toxicity (Filion and Phillips 1997; Lv, Zhang et al. 2006). We have previously described stabilized plasmid lipid particles (SPLP) as non-viral gene delivery vehicles for the delivery of pDNA to distal tumour sites following systemic administration (Monck, Mori et al. 2000; Tam, Monck et al. 2000; Cullis 2002; Fenske, MacLachlan et al. 2002; Ambegia, Ansell et al. 2005; MacLachlan and Cullis 2005; Judge, McClintock et al. 2006). However, as a result of their ability to protect pDNA from nuclease degradation, their small size (140 nm ± 50 nm), net neutral charge and low toxicity following systemic administration, combined with the fact that liposomal nanoparticles are naturally 98  cleared from the circulation by APCs (Juliano 1986; de Jong, Chikh et al. 2007; Wilson, Raney et al. 2007), SPLP represent a prime candidate for the delivery of systemic genetic vaccines. Here we investigate the ability of SPLP to be taken up by and transfect APCs following systemic administration, and the ability of SPLP-mediated delivery of a transgene to APCs to induce transgene-specific humoral and cellular immune responses.  Furthermore, we characterize the  resultant immune response following vaccination with SPLP alone or in the presence of liposomal nanoparticle encapsulated CpG containing oligodeoxynucleotides (LN CpG-ODN), a potent vaccine adjuvant previously demonstrated to promote immune cell activation and the induction of adaptive cellular responses against co-administered peptide or protein antigen (Mui, Raney et al. 2001; de Jong, Chikh et al. 2007; Wilson, Raney et al. 2007).  4.2 Materials and methods. 4.2.1 Materials. Distearoylphosphocholine (DSPC) was purchased from Avanti Polar Lipids (Alabaster, AL) while cholesterol was obtained from Sigma (St. Louis, MO). 1,2-dioleyloxy-3N,N-dimethylaminopropane (DODMA) and polyethylene glycol-dimyristol glycerol (PEGDMG) were provided by Tekmira Pharmaceuticals Corporation (Burnaby, BC, Canada). The fluorescently labeled lipid 1,1'-dioctadecyl-3,3,3',3'- tetramethylindocarbocyanine perchlorate (Di-I) was purchased from Invitrogen Molecular Probes (Burlington, ON). The pCMVluc and pCMVβgal plasmids, encoding luciferase (luc) and beta-galactosidase (βgal) reporter genes, respectively, under the control of the cytomegalovirus (CMV) promoter, were propagated in E. coli strain DH5α and purified by standard alkaline lysis with two rounds of cesium chloride density gradient centrifugation. Endotoxin levels in pDNA were less than 10 EU/mg as determined by the limulus amoebocyte lysate chromogenic endpoint assay as per the manufacturer’s instructions (Charles River Laboratories). INX-6295, a 16-mer phosphorothioate  99  (PS) ODN (5’TAACGTTGAGGGGCAT-3’) containing unmethylated cytosine residues in the CpG motif, was synthesized by Trilink Biotechnologies (San Diego, CA).  4.2.2 Animals and cell lines. Female, 6 to 8-week-old BALB/c mice were obtained from Jackson Laboratories (Bar Harbor, Maine) and were quarantined for at least 2 weeks prior to use. All procedures involving animals were performed in accordance with the guidelines established by the Canadian Council on Animal Care. The macrophage cell line, RAW264.7, the parental colon carcinoma cell line, CT26, and the colon carcinoma cell line stably expressing βgal, CT26.CL25, were obtained from the American Type Culture Collection (Manassas, VA). RAW264.7 cells were cultured in Dubelco’s Minimum Essential Medium (DMEM) supplemented with 10% fetal bovine serum (FBS), 4 mM L-glutamine, 100 U/ml penicillin-G and 100 µg/ml streptomycin sulphate at 37◦C with 5% CO2. CT26 and CT26.CL25 cells were cultured in RPMI 1640 medium supplemented with 10% FBS, 2 mM L-glutamine, 1.0 mM sodium pyruvate and adjusted to contain 4.5 g/L glucose. CT26.CL25 media also contained 0.4 mg/mL of the selection antibiotic G418. Splenocytes, isolated from BALB/c mice, were cultured in RPMI-1640 medium supplemented with 10% FBS, 2 mM L-glutamine, 100 U/ml penicillin G, 100 µg/ml streptomycin sulphate, 1 mM sodium pyruvate, 0.1 mM non-essential amino acids, 10 mM HEPES and 5x10-5 M β-mercaptoethanol. All cell culture reagents were obtained from Invitrogen (Burlington, Canada).  4.2.3 Preparation of SPLP and LN-CpG ODN.  Plasmid was encapsulated in lipid  nanoparticles containing an ionisable aminolipid using an ethanol dialysis procedure, as previously described (Maurer, Wong et al. 2001; Jeffs, Palmer et al. 2005). Briefly, a lipid mixture consisting of DSPC/cholesterol/DODMA/PEG-DMG at a molar ratio of 20/55/15/10 was solubilized in absolute, anhydrous ethanol and then diluted by adding distilled water to 100  achieve an ethanol concentration of 90%. The total concentration of lipid in solution was 20 mM.  A plasmid solution was prepared by combining pDNA in 10 mM Tris EDTA (TE)  buffer with 100 mM citrate (pH 5.0) and distilled deionized water to achieve a pDNA concentration of 0.9 mg/ml in 20 mM citrate. Equal volumes of both lipid and plasmid solutions were heated to 37°C prior to vesicle formation.  Vesicles were prepared by the  dropwise addition of equal volumes of lipid dissolved in ethanol to a rapidly mixing aqueous buffer containing DNA, resulting in an ethanol concentration of 45%, and were then diluted dropwise to 36% ethanol with 10 mM citrate, pH 5.0, 750 mM NaCl resulting in a final concentration of 10mM citrate, 150 mM NaCl. The diluted vesicles were then incubated at 37°C for 1 h prior to dialysis first against 10 mM citrate (pH 6.0) containing 150 mM NaCl for 2 h, followed by PBS overnight at pH 7.5. Unencapsulated pDNA was subsequently removed by anion exchange chromatography on DEAE-Sepharose CL-6B columns equilibrated in PBS. SPLP were characterized with respect to plasmid entrapment using a previously described PicoGreen (Invitrogen Molecular Probes, Burlington, Canada) assay (Jeffs, Palmer et al. 2005), and mean particle diameter was determined using a submicron quasi-elastic light scattering particle sizer (Nicomp, Santa Barbara, CA). SPLP formulations used in this study demonstrated a plasmid-to-lipid ratio of 0.04 - 0.05 (w/w), a maximum of 5–8 % unentrapped plasmid following purification and particle sizes of 145 ± 50 nm (χ2 = 0.3) in diameter. ODN  were  encapsulated  in  lipid  nanoparticles  consisting  of  POPC/cholesterol/DODMA/PEG-DMG at a molar ratio of 25/45/20/10 containing an ionisable aminolipid using an ethanol dialysis procedure, as previously described (Maurer, Wong et al. 2001; Wilson, Raney et al. 2007). The ODN-to-lipid ratio was typically 0.1 (w/w) with a particle size of 100 ± 25 nm.  101  4.2.4 Uptake and Transfection of APCs by SPLP 4.2.4.1 In vitro and in vivo uptake of SPLP by APCs. For in vitro studies, splenocytes isolated from BALB/c mice were prepared as single cell suspensions as described previously. Cells were plated at a density of 5 x 106 cells/mL and were incubated at 37°C + 5% CO2 overnight. Cells received 4 µg/mL of SPLP (luc) formulated to contain the fluorescently labeled lipid Di-I (Invitrogen Molecular Probes) (Di-I-SPLP) at a final lipid concentration of 0.5 mole percent and were incubated at 37°C or 4°C for the indicated time-points. Splenocytes were harvested and washed with PBS prior to staining with phenotype antibodies. For in vivo studies, 6 to 8-week old BALB/c mice were injected intravenously (i.v.) with 5 mg/kg Di-I-SPLP. Mice were euthanized by a terminal dose of 3.2% (v/v) ketamine/0.8% (v/v) xylazine 1, 4, 12 and 24 h after administration and lymph nodes and spleens were harvested. Single cell suspensions were prepared and stained for phenotype markers (CD11c for DCs, CD11b and Mac3 for macrophages, B220/CD45R for B-lymphocytes) with fluorescently labeled antibodies (eBioscience, San Diego, CA) according to the manufacturer's instructions and analyzed on a LSRII flow cytometer (BD Biosciences; Mississauga, Canada) for SPLP uptake by specific immune cell populations. Dead cells were excluded with propidium iodide (Invitrogen Molecular Probes) and data was acquired using FACSdiva software and analyzed using FloJo software V (BD Biosciences).  4.2.4.2  SPLP-mediated transfection of APCs in vitro. RAW264.7 cells were plated at a  concentration of 4 x 104 cells/mL. Splenocytes isolated from BALB/c mice were prepared as single cell suspensions by passage through a sterile 100 µm nylon mesh filter (BD Biosciences, ON, Canada) and red blood cells were lysed in ACK buffer (0.1 M ammonium acetate, 10 mM potassium bicarbonate, 70 µM EDTA) on ice. Splenocytes were washed and plated at a density of 5 x 106 cells/mL. Cells were treated with either PBS (control) or some combination of LN102  CpG ODN (3 µg/mL, added 4 h after plating for 12 h at 37°C + 5% CO2) and SPLP (luc) containing the luciferase encoding plasmid pCMVluc (4 µg/mL for the indicated time points). RAW264.7 cells and splenocytes were harvested manually and washed with PBS.  For  appropriate studies, CD11b+ cells were isolated from whole splenocyte populations by immunomagnetic sorting with CD11b microbeads (Miltenyi Biotech, Auburn, CA). For each treatment, 5.0 x 107 splenocytes were magnetically labeled and sorted. All cell populations were lysed with 0.25 mL of Reporter Lysis Buffer (Promega, Madison WI) followed by a freeze-thaw cycle.  Luciferase assays were performed using the Promega Luciferase Assay reagent kit  (Promega), according to the manufacturer’s instructions, in a Centro LB 960 microplate luminometer (Berthold Technologies, Oak Ridge, TN). A standard curve was generated from purified firefly luciferase (Roche, QC, Canada) and used to calibrate luminescence readings and luciferase activity was normalized against cell protein concentration determined using the Micro BCA protein assay reagent kit (Pierce, Rockford, IL).  4.2.4.3 SPLP-mediated transfection of APCs in vivo. BALB/c mice were seeded with 2 x 105 CT26 tumour cells in 100 µL PBS by subcutaneous hind flank injection. Tumours were allowed to reach an approximate volume of 200 mm3 [volume = (length * width2)/2] prior to i.v. administration of 5 mg/kg of SPLP (βgal) containing the βgal encoding plasmid pCMVβgal alone or followed 24 h later by LN CpG-ODN (10 mg/kg) via the lateral tail vein. For the 24-h time-point, LN CpG-ODN was administered 12 h after the addition of SPLP. Animals were sacrificed 24, 48, 72 and 96 hrs after SPLP administration, spleens were harvested and single cell suspensions were prepared as previously described. βgal activity was detected using the fluorogenic substrate 5-chloromethylfluorescein di-β-D-galactopyranoside (CMFDG) in the DetectaGene Green lacZ Detection Kit as per manufacturer’s instructions (Invitrogen Molecular  103  Probes). Briefly, 2 x 106 splenocytes were incubated in media containing 200 µM chloroquine for 30 min at 37°C, 5% CO2. Cells were loaded with CMFDG using the Influx Reagent and incubated for 3 h at 37°C, 5% CO2, following which the reaction was terminated by the addition of the competitive inhibitor phenylethyl β-D-thiogalactopyranoside at 1 mM. Verapamil was included in all solutions at a final concentration of 100 µM to block the efflux of the fluorescent product. Cells were analyzed immediately on a flow cytometer as previously described using propidium iodide to exclude dead cells.  4.2.5 SPLP-mediated Induction of Adaptive Immune Responses 4.2.5.1 Vaccination. 6 to 8-week old BALB/c mice were injected i.v. by lateral tail vein injection with SPLP (βgal) or SPLP (luc) corresponding to a dose of 5 mg/kg once a week for three weeks. Mice receiving both SPLP and LN CpG-ODN received LN-CpG ODN at a dose of 10 mg/kg 24 h following the administration of SPLP. One week following final vaccination, mice were euthanized as described above and blood was collected via cardiac puncture in Vacutainer tubes containing EDTA (BD Biosciences). Serum was isolated and stored at -70ºC until use in ELISAs. Spleens were harvested and processed as described above.  4.2.5.2 Serum Immunoglobulin Analysis.  Pooled serum samples were analyzed in  quadruplicate for anti-βgal antibodies by ELISA. In brief, plates (Nunc-Immuno MaxiSorp Nalge Nunc, Denmark) were coated overnight at 4ºC with 10 µg/ml βgal protein (Calbiochem) in a solution containing 0.1 M sodium carbonate, pH 9.5. After blocking with 10% FBS in PBS (pH 7.4) for 1 h at room temperature (RT), plates where incubated for a further 2 h at RT with the serially diluted experimental mouse serum and then washed with PBS + 0.05% Tween 20. Mouse IgG1 and IgG2a antibodies were detected by biotin-conjugated rabbit anti-mouse  104  isotype-specific  antibodies  (Rockland,  Gilbertsville,  PA)  and  avidin-HRP  (BD  Biosciences)/TMB substrate (BD Biosciences) at OD 492 nm corrected for absorbance at 520 nm. Antibody titre was defined as the highest serum dilution giving an absorbance twice that of serum from non-immune control animals.  4.2.5.3 Determination of CTL activity by chromium-51 release assays. One week following the final vaccination, cytotoxic T-lymphocyte (CTL) responses were assessed ex vivo or following 5 days of in vitro restimulation using a standard chromium-51 (51Cr) release assay. For in vitro restimulation, splenocytes from immunized animals were co-cultured with control splenocytes pulsed with 0.2 µM of the βgal-immunodominant peptide TPHPARIGL (SigmaGenosys, ON, Canada) and treated with mitomycin C (50 µg/mL), at a ratio of 10:1 for 5 days with the addition of human recombinant interleukin (IL)-2 (100 IU/ml; BD Biosciences). βgalspecific cytotoxicity was assessed using a standard 5 h  51  Cr release assay where splenocytes  were mixed in effector to target (E:T) ratios of 5:1, 25:1, 50:1, 100:1 with 51Cr-loaded parental CT26 or βgal-expressing CT26.CL25 cells.  The percentage of cellular cytotoxicity was  calculated on the basis of 51Cr released to the supernatant using the formula: [Sample counts per min (cpm) - spontaneous cpm)]/(maximum cpm) x 100, where maximal cpm was achieved by complete lysis of 51Cr labeled targets in 10% TritonX-100, spontaneous cpm was determined by incubating labeled targets in media and antigen-specific killing was determined by comparison of cytotoxicity of 51Cr-labeled βgal expressing and non-expressing CT26.CL25 and CT26 cells, respectively.  4.2.5.4 Interferon (IFN)-γ cytokine secretion assay.  CD8+ T lymphocytes capable of  responding to antigen-specific stimulation by secretion of IFN-γ were detected using the IFN-γ secretion assay (Miltenyi Biotec Inc., Auburn, CA) according to the manufacturer’s instructions. 105  Briefly, splenocytes from immunized mice were re-stimulated with 10 µg/mL of the peptide TPHPARIGL for 5 h prior to incubation with a bi-specific antibody designed to bind to activated T-cells via the CD25 activation marker and capture secreted IFN-γ. The frequency and phenotype of cells responding to βgal-stimulation were determined by flow cytometry as described previously using a fluorescently labeled anti-IFN-γ antibody in combination with the previously described fluorescently labeled phenotype antibodies.  4.2.6 Statistical Analysis. All statistical analyses were performed using SPSS Version 14.0. A one-way analysis of variance (ANOVA) was used to evaluate the differences between treatment groups. In the case of statistically significant results, the differences between treatment groups were assessed using the Tukey HSD modified t-test where probability (p) values less than 0.05 were considered significant.  4.3 Results 4.3.1 APCs take up SPLP ex vivo. Studies employing Di-I-labeled SPLP demonstrate that ex vivo, SPLP is taken up by splenic CD11b+, CD11c+ and B220/CD45R+ APCs when administered at concentrations typically used in transfection experiments (Figure 4.1). The maximum percentage of cells positive for uptake was reached between 4 and 8 h for all three cell-types and was greatest for CD11b+ cells (37%), followed by CD11c+ cells (30%) and B220/CD45R+ cells (21%).  On a per cell basis as judged by mean fluorescence intensity  (MFI), CD11b+ and CD11c+ cells showed a similar level of uptake, while uptake was approximately 2-fold lower in B220/CD45R+ cells at 8 h. Uptake by CD8+ and CD4+ cells was only visible at 8 h where 3.0% and 1.3% of cells were positive for uptake, respectively. Nonspecific binding was less than 2% in all instances as determined by incubation at 4°C (data not shown). 106  A)  45%  CD11b+ CD11c+ B220+ CD8+ CD4+  % Positive for Uptake  40% 35% 30% 25% 20% 15% 10% 5% 0% 0  B)  2  4 Tim e (hours)  2  4 e (hours) Tim  6  8  25  20  MFI  15  10  5  0 0  6  8  Figure 4.1: SPLP is preferentially taken up by CD11b+, CD11c+ and B220/CD45R+ cells ex vivo. Splenocytes (5 x 106 cells/mL) isolated from BALB/c mice were treated with Di-I labeled SPLP (4.0 µg/mL) ex vivo. Percent uptake (A) and MFI (B), as determined by flow cytometry, was calculated by subtracting non-specific binding for each cell-type at 4°C from uptake at 37°C. Each data point represents an n = 3 ± standard deviation.  4.3.2 SPLP transfect the cultured macrophage cell line RAW264.7, whole splenocyte populations and CD11b+ cells ex vivo. Investigation of the ability of SPLP (luc) to transfect the cultured macrophage cell line RAW264.7 and cultured splenocytes ex vivo, demonstrates that SPLP (luc) is capable of transfecting both, albeit at low levels, and that similar levels of gene expression are obtained when cells are pretreated with LN CpG-ODN. Detectable levels of luciferase expression were observed in RAW264.7 cells 48 and 72 h after incubation with SPLP in both the presence and absence of LN CpG-ODN (Figure 4.2A) and similarly in cultured 107  splenocytes 36 and 48 h after incubation with SPLP in both the presence and absence of LN CpG-ODN (Figure 4.2B). Similar expression levels were also observed when cells were treated with LN CpG-ODN 12 h after the addition of SPLP (data not shown). A)  B)  1600  1000  PBS Control SPLP  fg Luciferase/mg protein  fg Luciferase/mg protein  2000  SPLP & LN CpG-ODN 1200 800 400 0 24 hours  48 hours  C) fg Luciferase/mg protein  800 700  72 hours  800 600 400 200 0 24 hours  36 hours  48 hours  Initial CD11b Depleted CD11b Enriched  600 500 400 300 200 100 0 SPLP  SPLP & LN CpG-ODN  Figure 4.2: SPLP transfects the cultured macrophage cell line RAW264.7, whole splenocyte populations and primary macrophage ex vivo both in the presence and absence of LN CpG-ODN. Raw264.7 cells (A) (2 x 104 cells/mL) or splenocytes (B & C) (5 x 106 cells/mL) were plated 16 hours prior to treatment with SPLP (luc) (4.0 µg/mL), SPLP (luc) (4.0 µg/mL) and LN CpG-ODN (3.0 µg/mL) or (♦) PBS. Wells receiving LN CpG-ODN were treated 12 hours prior to SPLP addition. Luciferase expression was normalized to protein concentration, error bars represent n = 4 ± standard deviation (A & B). (C) Forty-eight hours after treatment with SPLP splenocyte populations were immunomagnetically sorted to obtain CD11b+ enriched and CD11b+ depleted populations prior to the quantitation of luciferase expression, data represents the findings from 2 independent experiments.  Immunomagenetic sorting was used to specifically demonstrate SPLP-mediated transfection of CD11b+ cells in whole splenocyte populations isolated from BALB/c mice. A 23 fold increase in luciferase expression per mg of protein was observed at 48 h in CD11b+ enriched populations over that observed for unseparated or CD11b depleted populations 108  indicating that CD11b+ cells are transfected by SPLP ex vivo (Figure 4.2C).  On average  immunomagnetic sorting resulted in a 12 - 20 fold increase in the percentage of CD11b+ cells over that of the depleted population or unsorted population.  4.3.3 SPLP are taken up by APCs in the spleen following intravenous administration. Using Di-I labeled SPLP, we were able to demonstrate the uptake of SPLP by CD11b+, CD11c+ and B220/CD45R+ cells in the spleen within 1 h of i.v. administration. The percentage of cells positive for uptake peaked one hour following administration for CD11b+ cells (66%) and CD11c+ cells (51%), and while B220/CD45R+ cells showed a similar level of cells positive for uptake (58%) this cell-type displayed a different uptake profile, exhibiting maximum uptake between 4 and 12 h following administration (Figure 4.3A). Although B220/CD45R+ cells demonstrated a similar level of uptake based on the percentage of cells positive for uptake as compared to CD11b+ and CD11c+ cells, based on MFI, they were found to have a 8 to 10-fold lower level of uptake on a per cell basis compared to CD11b+ and CD11c+ cells (Figure 4.3B). 4.3.4 SPLP transfect CD11b+ and CD11c+ cells in the spleen following intravenous administration. The ability of SPLP (βgal) to transfect APCs in the spleen 24, 48, 72 and 96 h following i.v. administration was demonstrated by flow cytometry using the fluorogenic substrate CMFDG in conjunction with fluorescently labeled phenotype markers. βgal activity was observed in both CD11b+ and CD11c+ cell populations 48 to 96 h and 72 to 96 h, respectively, after SPLP administration both in mice receiving SPLP (βgal) alone, or SPLP (βgal) followed 24 h later by LN CpG-ODN.  A distinct population of B220/CD45R+ cells  demonstrating high levels of βgal expression was not observed however it appears that a low level of transfection may occur in a small subset of cells at 48 and 72 h (Figure 4.4).  109  % Positive for Uptake  A) 80%  CD11b+  70%  CD11c+  60%  B220/CD45R+  50% 40% 30% 20% 10% 0% 0  5  10  15  20  25  10 15 Tim e (hours)  20  25  Time (hours)  B)  1000 800  MFI  600 400 200 0 0  5  Figure 4.3: Uptake of SPLP by CD11b+, CD11c+ and B220/CDR45+ cells in the spleen following intravenous administration. Uptake of Di-I labeled SPLP by CD11b (■), CD11c (▲) and B220/CD45R (◊) positive cells in the spleen following i.v. administration to BALB/c mice at a plasmid dose of 5 mg/kg. Results are expressed as the percent of cells positive for uptake (A) and mean fluorescence intensity (B), as determined by flow cytometry. Each data point represents four mice ± SEM.  In CD11b+ cell populations, peak levels of cells positive for gene expression were observed 72 h following transfection with SPLP (βgal) alone, or in combination with LN CpGODN. While the percentage of cells positive for gene expression at 72 h in the presence of LN CpG-ODN were on a whole similar to that observed when mice where treated with SPLP (βgal) alone (9.4% and 12.6%, respectively) treatment with LN CpG-ODN appeared to alter expression levels and kinetics as the level of expression on a per cell basis peaked 72 h after administration of SPLP (βgal) in the presence of LN CpG-ODN and 96 h after the administration of SPLP alone (MFI of 166 and 168, respectively). 110  Detectable levels of βgal expression where not observed in CD11c+ cells until 72 h following either treatment. Treatment with SPLP alone resulted in observable transfection levels (6.5%) at the 72 h time-point only, while combination treatment with LN CpG-ODN resulted in observable levels of transfection at 72 and 96 h (peak level of 7.9% at 96 h). Mock transfection with SPLP (luc) containing a plasmid encoding luciferase under the control CMV promoter alone or in combination with LN CpG-ODN resulted in background levels of fluorescence at 48 h which were similar to that observed for the PBS control (data not shown).  4.3.5 Vaccination with SPLP followed 24 h later by LN CpG-ODN primes the generation of transgene-specific humoral and cellular immune responses. To evaluate the ability of SPLP to act as a vaccine and prime the generation of adaptive antigen-specific humoral and cellular immune responses animals were immunized with SPLP (βgal) or the mock vaccine SPLP (luc) alone or followed 24 h later by LN CpG-ODN once a week for three weeks.  βgal-specific  immune responses were assessed one week following the final vaccination. With respect to the generation of antigen-specific humoral immune responses, immunization with SPLP (βgal) followed 24 h later by LN CpG-ODN resulted in the generation of βgal specific IgG1 and IgG2a responses, with a titre of 100, while minimal responses (titre ≤ 1) were observed following vaccination with SPLP (βgal) alone or the mock vaccine (Figure 4.5).  111  A) 24 hours  48 hours  72 hours  96 hours  CD11b+  CD11c+  B220/ CD45R+  CMFDG - MFI  B)  SPLP (βgal)  PBS Control 0.06  91.7  SPLP (βgal) & LN CpG-ODN 0.61  92.9  0.44  CMFDG - MFI  90.1  0.72  2.39  2.14  CD11b+  Figure 4.4: SPLP-mediated transfection of CD11b+, CD11c+ and B220/CDR45+ cells in the spleen following the intravenous administration of SPLP (βgal) alone or followed 24 hours later by LN CpG-ODN. Transfection of CD11b+, CD11c+ and B220/CD45R+ cells in the spleen of BALB/c mice bearing CT26 tumours as determined 24, 48, 72 and 96 hours following the i.v. administration of PBS (shaded curve), SPLP (5mg/kg) encoding βgal alone (solid line - black) or followed 24 hours later by LN CpG-ODN (dashed line - grey) (10mg/kg). Mice receiving LN CpG-ODN were treated 24 hours after the administration of SPLP, except for the 24 hour time-point where mice received LN CpG-ODN 12 hours after SPLP. βgal expression was detected using the fluorogenic substrate CMFDG in conjunction with fluorescently labeled phenotype markers and flow cytometry. Control mice receiving PBS showed similar background as mice receiving SPLP (luc) encoding luciferase in the presence or absence of LN CpG-ODN. (A) Histogram representation of data obtained from 3 mice pooled, (B) dot plot representation for CD11b+ cells at the 72 hour time-point.  112  β gal-specific Antibody Titre  100  80  IgG1 IgG2a  60  40  20  0 SPLP (luc)  SPLP (luc) & LN CpG-ODN  SPLP (bgal)  SPLP (bgal) & LN CpG-ODN  Figure 4.5: Vaccination with SPLP (βgal) followed 24 hours later by LN CpG-ODN primes the generation of a βgal specific humoral immune response. βgal specific IgG1 and IgG2a serum antibody responses in BALB/c mice vaccinated with SPLP (βgal) or SPLP (luc) (5mg/kg) alone or followed 24 hours later by LN CpG-ODN (10mg/kg) once a week for three weeks. Each bar represents the antibody titre of plasma isolated from 5 mice pooled as assessed by ELISA one week following the final vaccination. Antibody titre was defined as the highest serum dilution giving an absorbance twice  that of serum from non-immune control animals at 492 nm. Data represents findings from two independent experiments. The ability of vaccination with SPLP (βgal) to promote the generation of βgal-specific CTLs was assessed functionally by quantitating the number of CD8+ T-cells capable of responding to antigen-specific stimulation by secreting IFN-γ (a Th1 response indicator) in a standard cytokine secretion assay. Vaccination with SPLP (βgal) followed 24 h later by LN CpG-ODN resulted in a 6-fold increase in the percentage of CD8+ cells producing IFN-γ following antigenic stimulation with a βgal-derived peptide (Figure 4.6) while no such increase in the frequency of CD8+/IFN-γ+ T-cells was observed following antigen exposure in splenocytes isolated from control or mock vaccinated mice.  113  A)  Unstimulated 22.6  Stimulated  0.01  0.01  23.8  SPLP (luc) & LN CpG-ODN 0.11  CD8  0.09  19.3  0.02  19.9  0.11  SPLP (βgal) & LN CpG-ODN 0.12  0.84  IFN -γ  B) 0.6%  0.55%  % of CD8+ IFNγ +  0.5% 0.4% 0.3% 0.2% 0.1%  0.03%  0.01%  PBS Control  SPLP (luc)  0.03%  0.03%  0.0% SPLP (luc) & LN SPLP (bgal) CpG-ODN  SPLP (bgal) & LN CpG-ODN  Figure 4.6: Vaccination with SPLP (βgal) followed 24 hours later by LN CpG-ODN increases the frequency of antigen-specific IFN-γ secreting CD8+ T-cells. Frequency of IFN-γ+ CD8+ T-cells in BALB/c mice vaccinated with SPLP (βgal) or SPLP (luc) (5mg/kg) alone or followed 24 hours later by LN CpG-ODN (10mg/kg) once a week for three weeks. One week following the final vaccination the frequency of antigen-specific, IFN-γ secreting CD8+ was assessed by an IFN-γ secretion assay following stimulation for 5 hours with the immunodominant peptide of βgal (TPHPARIGL). (A) Dot plot representation of splenocytes isolated from mock vaccinated mice [SPLP (luc) & LN CpG-ODN] or mice vaccinated with SPLP (βgal) & LN CpG-ODN, before and after peptide stimulation. (B) Bar graph representation of the percentage of CD8+ cells positive for IFN-γ secretion following peptide stimulation for all vaccination strategies. Data represents splenocytes isolate from 5 mice pooled and is representative of the findings from two independent experiments.  114  Additional functional assessment of the induction of antigen-specific CTL responses following vaccination was determined in a standard  51  Cr release cytotoxicity assay where the  relative ability of splenocytes, isolated from immunized animals, to lyse βgal-expressing CT26.CL25 target cells in an antigen-specific manner was assessed immediately after isolation or following 5 days of in vitro restimulation with peptide-pulsed APCs. In both primary and secondary assays, performed following in vitro restimulation, an increase in antigen-specific CTL responses were observed in mice vaccinated with SPLP (βgal) followed 24 h later by LN CpG-ODN. Specifically, at an effector to target ratio of 100:1 significantly higher CTL activity was observed in splenocytes isolated from mice vaccinated with SPLP (βgal) and LN CpG-ODN (16.8%), over that observed for control animals vaccinated with PBS (2.5%) or with the mock vaccine (1.3%) (p < .05) for which only minimal levels of target cell lysis (less than 4%) were observed at all effector to target ratios (Figure 4.7).  4.4 Discussion The concept of using pDNA for immunization purposes is a promising approach for immunotherapy, however the relatively low levels of gene expression and weak immune responses attained following immunization with naked pDNA constitute significant hurdles to clinical application. A number of approaches have been taken to enhance pDNA delivery to APCs in the hope of improving immunogenicity and enhancing vaccine activity, but sub-optimal responses and/or serious safety and toxicity issues remain concerns (Gurunathan, Klinman et al. 2000; Chen and Huang 2005; Lv, Zhang et al. 2006). Here we show that SPLP, which have previously been used as a non-toxic systemic delivery system for gene therapy, can function as a systemically administered genetic vaccine. Specifically, we demonstrate first that SPLP are taken up by and effectively transfect APCs of the spleen compartment both ex vivo and in vivo following i.v. administration, and second that the SPLP-mediated transfection of APCs when 115  combined with a strong immunostimulatory signal such as that provided by the adjuvant LN CpG-ODN, can promote the priming of transgene-specific humoral and cellular immune responses. 20%  Control SPLP (L055)  16%  SPLP (L055) & LN CpG-ODN SPLP (Bgal)  % Specific Lysis  SPLP (Bgal) & LN CpG-ODN 12%  8%  4%  0% 5:1  25:1  50:1  100:1  Effector : Target Ratio  Figure 4.7: Vaccination with SPLP (βgal) followed 24 hours later by LN CpG-ODN primes the generation of βgal specific CTLs. βgal specific cytolytic activity in BALB/c mice vaccinated with SPLP (βgal) or SPLP (luc) (5mg/kg) alone or followed 24 hours later by LN CpG-ODN (10mg/kg) once a week for three weeks. One week following the final vaccination splenocytes were restimulated for 5 days in vitro with βgal-peptide pulsed splenocytes and cytotoxicity was assessed in a standard Cr51 release assay. Results are expressed as the percent specific lysis, calculated by subtracting the percentage of nonspecific lysis (CT26) from the percentage of specific lysis (CT26.CL25) after 5 hours of coincubation at various effector to target ratios. Each data point represents the specific lysis from 5 mice pooled ± SD. Data represents findings from two independent experiments.  It has been conclusively demonstrated that bone marrow-derived APCs play a key role in the induction of the immune response following vaccination with pDNA (Corr, Lee et al. 1996; Doe, Selby et al. 1996; Iwasaki, Torres et al. 1997), making the efficient delivery of plasmid to APCs a primary goal for DNA vaccines. As it has been long-established that APCs avidly accumulate particulate lipid delivery systems ex vivo and in vivo (Juliano 1986; Oussoren and Storm 1997; Oussoren, Velinova et al. 1998), it was of considerable interest to investigate the  116  ability of SPLP to passively target to, be taken up by, and transfect APCs.  Ex vivo, we were  able to demonstrate that SPLP is taken up by approximately 21-38% of B220/CD45R+, CD11c+ and CD11b+ cells when present at the same concentrations as those typically used for in vitro transfection.  Furthermore, SPLP demonstrates the ability to transfect both the cultured  macrophage cell line RAW264.7 and primary splenocytes ex vivo, albeit at low levels, and enhanced luciferase expression per mg of protein is observed in populations enriched for CD11b+ cells, indicating that SPLP is able to transfect this cell-type ex vivo. Although numerous attempts have been made to design vectors which both protect pDNA from degradation and target its delivery to APCs, few studies have directly characterized the uptake of nonviral vectors by APCs and their subsequent transfection in vivo.  Using  fluorescently labeled SPLP it is demonstrated here that SPLP are rapidly taken up by CD11b+, CD11c+ and B220/CD45R+ cell populations in the spleen within one hour of i.v. administration. These results are in agreement with previous findings which demonstrate the efficient uptake of physically similar, stabilized lipid nanoparticles containing immunostimulatory ODN by APCs following systemic administration (Wilson et al, 2007). Furthermore, using a very sensitive flow cytometry-based assay for the detection of βgal activity (Fiering, Roederer et al. 1991), we are able to quantitatively characterize the transfection of CD11b+ and CD11c+ cells in the spleen following i.v. administration of SPLP encoding βgal. While detectable levels of βgal expression were evident in a high percentage of CD11b+ (12.6%) cells from 48 to 96 h following administration, the transfection of CD11c+ (6.5%) cells was considerably lower and more transient. Although transgene expression by macrophage and DC populations following genetic vaccination has been previously assessed using detection methods which include the expression of a fluorescent product (i.e. GFP) in combination with fluorescent microscopy, expression levels reported are very low (ie. 50-100 antigen positive DCs per inguinal lymph node) and it is difficult to directly compare these finding with our results as they are typically assessed 117  following intramuscular or gene-gun administration of naked pDNA (Condon, Watkins et al. 1996; Chattergoon, Robinson et al. 1998; Porgador, Irvine et al. 1998). Our observations are in agreement, however, with those observed by Garg et al (Garg, Oran et al. 2003) who, using a Cre/loxP recombination strategy which results in βgal expression by transfected cells, observed that approximately 12% of the purified CD11c+ DCs from draining lymph nodes of gene gun immunized mice were βgal positive 60 h after immunization. These authors did not detect transfected macrophage following vaccination, a discrepancy that is most likely due to differences in the method and route of delivery. It is proposed that the efficient uptake and transfection of APCs in the spleen observed here depends largely on our use of newly formulated SPLP particles which have been modified compared to their traditional counterparts to allow for repeat administration (Judge, McClintock et al. 2006). The shorter circulation lifetime (t1/2 = 1 h) of the SPLP formulated here with the shorter chain PEG-lipid PEG-DMG, as compared to the long circulation halftimes (t1/2 = 7 h) observed for traditional SPLP formulated with longer chain PEG-lipid PEG-DSG, results in increased accumulation of the particle in the spleen (Ambegia, Ansell et al. 2005), which is well suited to the role for SPLP as a genetic vaccine carrier. The efficient transfection of CD11b+ and CD11c+ by SPLP in vivo may also rely in part on the fact that SPLP and LN CpG-ODN have been demonstrated to promote the proliferation of these cell-types in vivo within 24 h of systemic administration, as assessed by bromodeoxyuridine (BrdU) incorporation (unpublished results), since transfection of cells by SPLP depends strongly on mitotic activity (Mortimer, Tam et al. 1999). Importantly, co-administration of the adjuvant LN CpG-ODN did not downregulate SPLP-mediated gene expression levels in either of the cell-types investigated. It is well documented that unmethylated CpG motifs present in pDNA and synthetic ODN promote the production of cytokines, including IFN-γ and TNF-α, which can act to down-regulate gene expression from viral promoters such as CMV (Harms and Splitter 1995; Lee, Yee et al. 1999; 118  Tan, Li et al. 1999), particularly when administered systemically as cationic lipidcontaining/DNA particles (Freimark, Blezinger et al. 1998; Li, Wu et al. 1999; Mui, Raney et al. 2001). The strength and nature of immune responses primed by DNA vaccines are strongly influenced by their route and method of administration (Yokoyama, Zhang et al. 1996; McCluskie, Brazolot Millan et al. 1999; Alpar and Bramwell 2002). Although the efficacy of systemically administering genetic vaccines has been evaluated in several instances for both free or cationic lipid-complexed forms of pDNA (Yokoyama, Zhang et al. 1996; McCluskie, Brazolot Millan et al. 1999; Cui, Asada et al. 2003; Hattori, Kawakami et al. 2004) the instability of naked DNA combined with the toxicity and rapid clearance often associated with cationic carriers has significantly reduced the viability of this approach. However, due to the advantage of direct access to the relatively large number of APCs in the spleen, systemic administration of genetic vaccines still represents an attractive alternate approach to traditional routes of administration if the obstacle of poor delivery can be overcome. Here we demonstrate the generation of functional antigen-specific cellular responses, as assessed by cytokine secretion and cytotoxicity, and humoral responses, as assessed by serum immunoglobulin titres, which directly support the ability of SPLP to act as a systemically administered genetic vaccine when combined with a strong immunostimulatory signal. While very low levels of antigen-specific CTLs are detected in splenocytes of control or mock vaccinated mice, a concomitant increase in βgal-specific IFN-γ secretion and cytolytic activity is detected in the splenocytes of animals immunized with SPLP in conjunction with LN CpG-ODN. Not surprisingly, it should be noted that the systemic administration of SPLP (βgal) alone can, on some occasions, prime antigenspecific humoral and cellular responses (data not shown), however, these responses are sporadic and consistently weaker than those observed in the presence of LN CpG-ODN.  Our  observations that LN CpG-ODN can act as an adjuvant for a DNA-based vaccine, enhancing the 119  generation of antigen-specific humoral and cellular immune responses are in agreement with several groups who have also demonstrated an enhancement in resultant immune responses from a DNA vaccine following administration of CpG containing DNA (Klinman, Yamshchikov et al. 1997; Kojima, Xin et al. 2002; Ren, Zheng et al. 2004). Consistent with observations made by other groups (Kojima, Xin et al. 2002; Ren, Zheng et al. 2004), we have observed (unpublished data) that the relative timing of administration of the DNA vaccine and CpG ODN adjuvant is critical, with delivery of the CpG-ODN after the vaccine resulting in an enhanced immunogenic boost. In conclusion, our studies demonstrate that SPLP represents an effective candidate for the non-viral delivery of a systemic genetic vaccine when combined with additional immune stimulation provided by LN CpG-ODN. It is evident that SPLP are taken up by and effectively transfect CD11b+ and CD11c+ cells in the spleen compartment following systemic administration, and, in the presence of additional immune stimulation provided by LN CpGODN, induces the generation of detectable antigen-specific humoral and cellular responses. These data highlight the benefit of additional immune stimulation, particularly CpG-mediated, in enhancing the generation of immune responses to genetic vaccines. While optimization of dosing and dosing schedules are required, our demonstration of the ability of SPLP combined with LN CpG-ODN to promote the priming of antigen-specific immune responses while at the same time being well-tolerated following systemic administration supports the potential of SPLP as a genetic vaccine.  4.5 References Alpar, H. O. and V. W. Bramwell (2002). "Current status of DNA vaccines and their route of administration." Crit Rev Ther Drug Carrier Syst 19(4-5): 307-83.  120  Ambegia, E., S. 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L. Whitton (1996). "DNA immunization: effects of vehicle and route of administration on the induction of protective antiviral immunity." FEMS Immunol Med Microbiol 14(4): 221-30.  124  CHAPTER FIVE CHARACTERIZATION OF SPLP AND LN CpG-ODN AS A NON-VIRAL GENETIC CANCER VACCINE4  5.1 Introduction The common goal of cancer vaccine strategies is the activation of the immune system against tumour-associated antigens (TAA) expressed by the tumour or supporting tissues, resulting in the induction of tumour-specific immune responses in the host and ultimately, anti-tumour activity. A wide variety of vaccination approaches, including the injection of whole tumour cells or tumour cell lysates, TAA-derived peptides or proteins, the delivery of genes encoding TAAs as well as modified antigen-presenting cell (APC)-based methods have been investigated in an attempt to induce potent TAA-specific immune responses in animal models and, particularly, in humans (Prud'homme 2005; Tabi and Man 2006). Although tumours differ from their normal counterparts in antigenic composition and it is well established that TAAs have the potential to be recognized by the host immune system (Kawakami, Eliyahu et al. 1994; Rosenberg 1999), TAAs are typically self-antigens and are poorly immunogenic. Difficulties in raising potent immune responses against TAAs in tumour-bearing hosts arise from complexities associated with central and peripheral immune tolerance and/or tumour-associated immune suppression (Mocellin, Rossi et al. 2004; Tabi and Man 2006; Curiel 2007). Therefore, novel approaches which aid the immune system in overcoming these hurdles and recognizing tumours are clearly required. DNA vaccines offer unique advantages as a means of generating immune responses against cancer because the administration of TAA encoding plasmid DNA (pDNA) can induce the generation of both TAA-specific antibody responses and potent cytotoxic T lymphocyte 4  A version of this chapter has been prepared for submission. Kaley D Wilson, Susan D. deJong, Mikameh Kazem, Pieter R. Cullis and Ying K. Tam. Characterization of SPLP and LN CpG-ODN as a non-viral genetic cancer vaccine.  125  (CTL) responses which have been shown to be protective against tumor challenge in animal models (Conry, LoBuglio et al. 1995; Bright, Beames et al. 1996). Furthermore, vaccination with pDNA has demonstrated the potential to break natural immune tolerance against selfantigens in animal models, an important property for cancer vaccines due to the fact that most TAAs are self-derived proteins (Davis, Brazolot Millan et al. 1997) (Amici, Smorlesi et al. 2000) (Hawkins, Gold et al. 2000) (Weber, Bowne et al. 1998). Despite their potential, DNA cancer vaccines have met with only limited success in the clinic, which is at least partly due to suboptimal priming of immune responses in patients. To overcome the barrier of low transfection efficiency associated with poor cellular uptake and rapid degradation of naked DNA vaccines by nucleases (Kawabata, Takakura et al. 1995; Lew, Parker et al. 1995) we have previously employed Stabilized Plasmid Lipid Particles (SPLP) as non-viral gene delivery vehicles for the delivery of a DNA vaccine to APCs in a murine model, demonstrating efficient uptake and transfection of APCs in the spleen compartment following systemic administration. Furthermore, using beta-galactosidase (βgal) as a model antigen, we have demonstrated that vaccination with SPLP in conjunction with an immunostimulatory signal provided by the adjuvant LN CpG-ODN (liposomal nanoparticulate encapsulated CpG oligodeoxynucleotides) results in the priming of βgal specific humoral and cellular immune responses (K. Wilson, unpublished data). Here, we confirm these observations and evaluate the efficacy of a SPLP/LN-CpG ODNbased vaccine in an animal model of cancer. Using βgal as an artificial TAA, we first define the impact of the relative timing of SPLP and LN CpG-ODN administration on vaccine induced immune responses. The ability of prophylactic vaccination with SPLP to protect against tumour challenge is then assessed to evaluate the feasibility of using SPLP as a conventional DNA cancer vaccine in a strategy where SPLP-mediated delivery of pDNA-encoded TAAs, in combination with the immune adjuvant LN-CpG ODN, are used to induce TAA-specific anti126  tumour immune responses. Furthermore, based on the documented ability of SPLP to deliver transgenes to both APCs and distal tumour sites, we also investigate the application of SPLP in a novel therapeutic cancer vaccine strategy in which pDNA encoded transgenes are delivered to both APCs and tumour cells to allow for the expression of an artificial TAA at the tumour site and the initiation of an immune response against this TAA.  5.2 Materials and Methods 5.2.1 Materials.  1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC) was purchased from  Avanti Polar Lipids (Alabaster, AL), cholesterol was obtained from Sigma (St. Louis, MO) and 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) was purchased from Northern Lipids (Vancouver,  Canada).  1,2-dioleyloxy-3-N,N-dimethylaminopropane  polyethylene  glycol-dimyristol  glycerol  (PEG-DMG)  were  (DODMA)  provided  by  and  Tekmira  Pharmaceuticals Corporation (Burnaby, BC, Canada). The pCMVluc and pCMVβgal plasmids, encoding the luciferase (luc) and βgal reporter genes, respectively, under the control of the cytomegalovirus promoter, were propagated in E. coli strain DH5α and purified by standard alkaline lysis with two rounds of cesium chloride density gradient centrifugation. Endotoxin levels in pDNA were less than 10 EU/mg as determined by the limulus amoebocyte lysate (LAL) chromogenic endpoint assay as per the manufacturer’s instructions (Charles River Laboratories).  INX-6295, a 16-mer phosphodiester (PO) or phosphorothioate (PS) ODN  (5’TAACGTTGAGGGGCAT-3’) containing unmethylated cytosine residues in the CpG motif, was synthesized by Trilink Biotechnologies (San Diego, CA).  5.2.2 Animals and cell lines. Female, 6 to 8-week-old A/J mice and BALB/c mice were obtained from Jackson Laboratories (Bar Harbor, Maine) and quarantined for at least 2 weeks prior to use in these studies. All procedures involving animals were performed in accordance 127  with the guidelines established by the Canadian Council on Animal Care. The cell line CT26 is an N-nitroso-N-methylurethane induced BALB/c (H-2d) undifferentiated colon carcinoma that grows progressively in BALB/c mice after subcutaneous (s.c.) or intravenous (i.v.) injection. CT26.CL25 is a variant of CT26 which was generated by stable transfection with the lacZ gene encoding the model TAA, βgal, and has been demonstrated to possess similar growth characteristics to the parental cell line in vivo (Wang, Bronte et al. 1995). CT26 and CT26.CL25 cells were cultured in RPMI 1640 medium supplemented with 10% heat inactivated fetal bovine serum (FBS), 2 mM L-glutamine, 1 mM sodium pyruvate (Invitrogen ON, Canada) and adjusted to contain 4.5 g/L glucose. CT26.CL25 media also contained 0.4 mg/mL of the selection antibiotic G418.  P815, a mastocytoma cell line originating from DBA/2 (H-2d) mice was used  as haplotype matched control in chromium-51 (Cr51) release assays, and was cultured in Dulbecco's modified Eagle's medium (DMEM) with 4 mM L-glutamine, 4.5 g/L glucose and 10% FBS. All cell lines were obtained from the American Type Culture Collection (Manassas, VA). Splenocytes, isolated from BALB/c or A/J mice, were cultured for in vitro restimulation in RPMI-1640 medium supplemented with 10% FBS, 2 mM L-glutamine, 100 U/ml penicillin G, 100 µg/ml streptomycin sulphate, 1 mM sodium pyruvate, 0.1 mM non-essential amino acids, 10 mM HEPES, 5x10-5 M β-mercaptoethanol and 100 IU/ml human recombinant interleukin (IL)-2 (eBioscience, San Diego, CA).  5.2.3 Preparation of SPLP and LN-CpG ODN.  Plasmid was encapsulated in lipid  nanoparticles containing an ionisable aminolipid using an ethanol dialysis procedure, as previously described (Maurer, Wong et al. 2001; Jeffs, Palmer et al. 2005). Briefly, a lipid mixture consisting of DSPC/cholesterol/DODMA/PEG-DMG at a molar ratio of 20/55/15/10 was solubilized in absolute, anhydrous ethanol and then diluted by adding distilled water to 128  achieve an ethanol concentration of 90%. The total concentration of lipid in solution was 20 mM.  A plasmid solution was prepared by combining pDNA in 10 mM Tris EDTA (TE)  buffer with 100 mM citrate (pH 5.0) and distilled deionized water to achieve a pDNA concentration of 0.9 mg/ml in 20 mM citrate. Equal volumes of both lipid and plasmid solutions were heated to 37°C prior to vesicle formation.  Vesicles were prepared by the  dropwise addition of equal volumes of lipid dissolved in ethanol to a rapidly mixing aqueous buffer containing DNA, resulting in an ethanol concentration of 45%, and were then diluted dropwise to 36% ethanol with 10 mM citrate, pH 5.0, 750 mM NaCl resulting in a final concentration of 10 mM citrate and 150 mM NaCl. The diluted vesicles were then incubated at 37°C for 1 hour prior to dialysis first against 10 mM citrate (pH 6.0) containing 150 mM NaCl for 2 hour, followed by phosphate-buffered saline (PBS) overnight at pH 7.5. Unencapsulated plasmid was subsequently removed by anion exchange chromatography on DEAE-Sepharose CL-6B columns equilibrated in PBS.  SPLP were characterized with respect to plasmid  entrapment using a previously described PicoGreen assay (Jeffs, Palmer et al. 2005) (Molecular Probes), and particle mean diameter was determined using a submicron quasi-elastic light scattering particle sizer (Nicomp, Santa Barbara, CA). SPLP formulations used in this study demonstrated a plasmid-to-lipid ratio of 0.04 - 0.05 (w/w), a maximum of 5–8 % unentrapped plasmid following purification and particle sizes of 145 ± 50 nm (χ2 = 0.3) in diameter. Oligodeoxynucleotides (ODN) were encapsulated in lipid nanoparticles consisting of POPC/cholesterol/DODMA/PEG-DMG at a molar ratio of 25/45/20/10 containing an ionisable aminolipid using an ethanol dialysis procedure, as previously described (Maurer, Wong et al. 2001; Wilson, Raney et al. 2007). The ODN-to-lipid ratio was typically 0.1 (w/w) and particle size was 100 ± 25 nm.  129  5.2.4 Determination of the temporal effects of LN CpG-ODN addition on its adjuvant activity in an SPLP-based vaccine. Eight week old female A/J mice were vaccinated i.v. via the lateral tail vein once a week for three weeks (q7 x 3 regimen) with 5 mg/kg SPLP (βgal). LN CpG-ODN (INX-6295 PO) was administered at 10 mg/kg by the same route immediately following SPLP or 24, 48 or 72 h after vaccination. One week following the final vaccination, mice were euthanized by a terminal dose of 3.2% (v/v) ketamine/0.8% (v/v) xylazine, blood was collected via cardiac puncture into Vacutainer tubes containing EDTA (BD Biosciences) and plasma was isolated and stored at -70ºC until analysis. Spleens were harvested and prepared as single cell suspensions by passage through a sterile 100 µm nylon mesh filter (BD Biosciences, ON, Canada). Red blood cells were lysed in ACK buffer (0.1 M ammonium acetate, 10 mM potassium bicarbonate, 70 µM EDTA) on ice, washed and used in various immunological assays.  5.2.5 Prophylactic vaccination. Eight week old BALB/c mice were vaccinated i.v. by lateral tail vein injection on a q7 x 3 regimen with SPLP (βgal) alone (5 mg/kg) or followed 24 h later by LN CpG-ODN (INX-6295 PS) at 10 mg/kg. Control mice were mock vaccinated with PBS, SPLP (luc) encoding the unrelated antigen luciferase (5 mg/kg) or LN CpG-ODN (INX-6295 PS) (10 mg/kg). Two weeks following the final vaccination mice were challenged with the βgal expressing tumour cell line CT26.CL25 (2 x 105 cells) by s.c. hind flank injection. Tumour volume was monitored every other day using calipers and calculated using the equation Volume = (Length x Width2)/2. Mice were euthanized when tumours reached a volume of 1000 mm3. Tumour βgal expression was quantitated using the β-galactosidase reporter gene activity detection kit, based on the colourimetric conversion of the substrate o-nitrophenyl β-Dgalactopyranoside (ONPG), as per manufacturer’s instructions (Sigma Aldrich) and was 130  normalized against cell protein concentration as determined by the Micro BCA protein assay reagent kit (Pierce, Rockford, IL). Mice able to completely clear CT26.CL25 tumours were rechallenged with CT26.CL25 (2 x 105 cells) 8 weeks after clearance (11 weeks after the initial challenge) on the contralateral flank and with the parental, non-βgal expressing tumour cell line CT26 (2 x 105 cells) on the flank that received the initial challenge.  5.2.6 Therapeutic vaccination. Eight week old BALB/c mice were vaccinated i.v via the lateral tail vein on a q7 x 3 regimen with SPLP (βgal) alone (5 mg/kg) or followed 24 h later by 10 mg/kg LN CpG-ODN (INX-6295 PS).  Two weeks following the final vaccination mice  were challenged with the parental non-βgal expressing tumour cell line CT26 (2 x 105 cells) by s.c. hind flank injection and tumour volume was monitored as described. Twelve days after seeding, when tumours had reached an average volume of 100 mm3, vaccinated mice were treated with 5 mg/kg SPLP (βgal) or SPLP (luc) encoding the unrelated antigen luciferase once a week for two weeks. Five days following the final vaccination, 4 mice were sacrificed from each treatment group to assess immune parameters and the remaining mice (6-9 per group) were maintained for efficacy studies. Mice were euthanized when tumours reached a volume of 1000 mm3 or if animals exhibited signs of distress (hunched posture, piloerection, lethargy, lack of grooming) or greater than 20% loss in body weight. Analyses were performed on blood samples isolated from mice euthanized for reasons other than tumour burden by the Central Laboratory for Veterinarians (Langley BC).  5.2.7 ELISA. Pooled plasma samples were analyzed for anti-βgal specific antibodies by ELISA. In brief, 96-well plates (Nunc-Immuno MaxiSorp - Nalge Nunc, Denmark) were coated overnight with 10 µg/ml βgal protein (Calbiochem) in a solution containing 0.1 M sodium 131  carbonate, pH 9.5. After blocking with 10% FBS in PBS (pH 7.4), plates where incubated for a further 2 h at RT with serially diluted experimental mouse plasma. All washes were performed with PBS + 0.05% Tween 20. Mouse IgG1 and IgG2a antibodies were then detected by biotinconjugated rabbit anti-mouse IgG1 and IgG2a antibodies (Rockland, Gilbertsville, PA) and visualized with avidin-HRP (BD Biosciences) and TMB substrate (BD Biosciences) at OD 492 nm corrected for absorbance at 520 nm. Antibody titre was defined as the highest serum dilution giving an absorbance twice that of serum from non-immune control animals.  5.2.8 In Vitro Restimulation. For cytokine secretion and MHC pentamer assays, splenocytes from immunized animals were co-cultured with control splenocytes pulsed with 0.2 µM of the βgal immunodominant peptide TPHPARIGL (Sigma-Genosys, ON, Canada) and treated with mitomycin C (50 µg/mL), at a ratio of 10:1 for 5 days in the presence of human recombinant IL2 (100 IU/ml; BD Biosciences). For 51Cr release assays, splenocytes from immunized mice were co-cultured with mitomycin C treated CT26.CL25 (βgal expressing) colon carcinoma cells at a ratio of 40:1.  5.2.9 Determination of CTL activity by  51  Cr release assay. βgal-specific CTL responses in  vaccinated animals were assessed immediately after euthanasia or following 5 days of in vitro restimulation, as described, using a standard 5-h  51  Cr release assay where splenocytes were  mixed in effector-to-target (E:T) ratios of 5:1, 25:1, 50:1, 75:1 or 100:1 with parental CT26 or βgal-expressing CT26.CL25 cells.  51  Cr-loaded  The percent cellular cytotoxicity was  calculated on the basis of 51Cr released to the supernatant using the formula: [sample counts per minute (cpm) - spontaneous cpm)/(maximum cpm) x 100, where maximal cpm was achieved by complete lysis of 51Cr labeled targets in 10% TritonX-100, spontaneous cpm was determined by  132  incubating labeled targets in media and βgal-specific killing was determined by comparison of cytotoxicity of  51  Cr-labeled βgal expressing and non-expressing CT26.CL25 and CT26 cells,  respectively. P815, a mastocytoma cell line originating from DBA/2 (H-2d) mice was used as a haplotype matched control for non-specific cell lysis. 5.2.10 IFN-γ cytokine secretion assay. Antigen-specific interferon (IFN)-γ secreting CD8 Tcells were detected using the IFN-γ secretion assay (Miltenyi Biotec Inc., Auburn, CA) according to the manufacturer’s instructions. Briefly, splenocytes from immunized mice were restimulated with the βgal peptide TPHPARIGL for 5 h prior to incubation with a bi-specific antibody designed to bind to activated T lymphocytes via the CD25 activation marker and capture secreted IFN-γ. The frequency and phenotype of cells that responded to βgal-stimulation by actively secreting cytokines were analyzed on a LSRII flow cytometer (BD Biosciences) using a fluorescently labeled anti-IFN-γ antibody in combination with a fluorescently labeled anti-CD8 antibody. Dead cells and B-cells were excluded using a combination of propidium iodide and anti-B220/CD45R antibodies (Invitrogen Molecular Probes) and data was acquired using FACSdiva software and analyzed using FloJo software V (BD Biosciences).  5.2.11 MHC pentamer assay. After immunization, the frequency of βgal-specific CD8+ Tcells was determined using an MHC-pentamer assay. Briefly, 2 x 106 spleen cells were incubated with PE-coupled H-2dL MHC pentamers containing the immunodominant peptide of βgal (TPHPARIGL; ProImmune, Springville, VA) and fluorescently labeled anti-CD8 and anti-TCRβ phenotype antibodies (eBioscience) prior to analysis on a flow cytometer as previously described. Dead cells were excluded using propidium iodide and 5 x 105 events were collected to analyze the frequency of βgal-specific CD8 T lymphocytes in immunized animals.  133  5.2.12 Statistical analyses. A one-way analysis of variance (ANOVA) was used to evaluate the differences between treatment groups. In the case of statistically significant results, the differences between treatment groups were assessed using the Tukey test.  Comparison of  survival times between treatment groups was made using the Mantel-Cox test. Probability values less than 0.05 were considered significant.  5.3 Results 5.3.1 LN CpG-ODN demonstrates optimal adjuvant activity when delivered 24 h after SPLP.  Antigen-specific humoral and cellular immune responses were evaluated following  vaccination with SPLP (βgal) alone or SPLP (βgal) with LN CpG-ODN, administered simultaneously 24, 48 or 72 h after SPLP. Immunization with SPLP followed 24 or 48 h later by LN CpG-ODN resulted in the generation of detectable βgal specific IgG2a antibody responses (titre of 100 and 10, respectively; Figure 5.1), while minimal responses (titre ≤ 1) were observed following vaccination by all other regimens. Furthermore, minimal levels of βgal specific IgG1 antibodies (titre = 1) were only detected in the plasma of mice vaccinated with SPLP followed 72 h later by LN CpG-ODN (Fig 5.1). The influence of LN CpG-ODN on the ability of vaccination with SPLP (βgal) to generate βgal specific CD8 T-cells was assessed quantitatively, using a standard MHC pentamer assay, and functionally using an IFN-γ secretion assay. Splenocytes were isolated one week following the final vaccination and assessed immediately or following 5 days of in vitro restimulation with βgal peptide pulsed APCs. Splenocytes analyzed immediately following isolation from vaccinated mice demonstrated that vaccination with SPLP followed 24 h later by LN CpG-ODN resulted in a 2-fold enhancement in the percentage of CD8/TCRβ/βgal-pentamer+ cells over that observed for control mice vaccinated with PBS (Figure 5.2) while all other 134  vaccination strategies demonstrated either no enhancement or enhancement which was less than 1.5-fold.  Consistent with data obtained for the pentamer assay, a 4-fold increase in the  percentage of CD8+ cells producing IFN-γ following antigenic stimulation was observed in splenocytes isolated from mice which had been vaccinated with SPLP followed 24 h later by LN CpG-ODN compared to that observed for the PBS-treated control (Figure 5.3).  100  IgG1  Anti- β gal Antibody Titre  IgG2a 80  60  40  20  0 SPLP alone  Same time  24 hours later  48 hours later  72 hours later  Figure 5.1: Temporal effects of LN CpG-ODN administration on the humoral immune response to an SPLP-based DNA vaccine. βgal specific IgG1 and IgG2a antibody responses in A/J mice following vaccination with SPLP (βgal) alone, or following administration of LN CpG-ODN at the same time or 24, 48 or 72 h after SPLP (βgal) vaccination. Each bar represents the antibody titre of plasma isolated from 5 mice pooled as assessed by ELISA one week following the final vaccination. Titre was calculated by endpoint dilution where the endpoint was defined as the highest serum dilution giving an absorbance at 492 nm that was twice that of the non-immune control.  135  % of TCRβ+ /CD8+ Pentamer +  A)  0.4% 0.32% 0.3% 0.2%  0.24% 0.16%  0.16%  0.13%  0.11% 0.1% 0.0% PBS Control  SPLP alone  Same time  24 hours later 48 hours later 72 hours later  B) % of TCRβ+ /CD8+ Pentamer +  1.2% 0.95% 0.80%  0.91% 0.74%  0.8%  0.53% 0.4% 0.20% 0.0% PBS Control  SPLP alone  Same time  24 hours later 48 hours later 72 hours later  Figure 5.2: Temporal effects of LN CpG-ODN administration on the frequency of βgal-specific CD8+ T-cells following SPLP-based vaccination. Frequency of TPHPARIGL-MHC pentamer+ CD8+ T-cells in splenocyte populations isolated from A/J mice following vaccination with SPLP (βgal) alone, or following administration of LN CpG-ODN at the same time or 24, 48 or 72 h after vaccination. (A) Splenocytes isolated from A/J mice (5 mice per group pooled) were incubated with TPHPARIGL-MHC pentamers in conjunction with anti-CD8 and TCRβ antibodies and analyzed by flow cytometry to quantitate the frequency of βgal-specific CD8+ T-cells following immunization. (B) Frequency of TPHPARIGL-MHC pentamer+ CD8+ T-cells following 5 days of in vitro restimulation with TPHPARIGL-pulsed splenocytes.  Antigen-specific responses detected following vaccination with SPLP and LN CpG-ODN were confirmed by the ability of in vitro restimulation with βgal peptide-pulsed splenocytes to amplify antigen-specific T-cells, resulting in a clear increase in the percentage of pentamer positive CD8+ T-cells and CD8+ T-cells secreting IFN-γ following antigen stimulation in vaccinated mice (Figure 5.2 & 5.3). In agreement with observations made in initial assays, splenocytes isolated from mice which had been vaccinated with SPLP followed 24 h later by LN CpG-ODN demonstrated a dramatic increase in the percentage of CD8+ secreting IFN-γ in response to antigen stimulation following in vitro restimulation. An increase in the percentage  136  of pentamer positive CD8+ T-cells was observed in all vaccinated groups following in vitro restimulation, which was not observed in splenocytes isolated from unvaccinated controls. A)  % of CD8+ IFN-γ +  0.15%  0.10%  0.10%  0.07% 0.05% 0.02% 0.01%  0.02% 0.00%  0.00% PBS Control  B)  SPLP alone  Same time  2.5%  24 hours later  48 hours later  72 hours later  2.21%  % of CD8+ IFN-γ+  2.0% 1.5% 1.0% 0.52% 0.5% 0.0%  0.25% 0.01%  0.01%  0.04%  PBS Control  SPLP alone  Same time  24 hours later  48 hours later  72 hours later  Figure 5.3: Temporal effects of LN CpG-ODN administration on the frequency of βgal-specific IFN-γ secreting CD8+ T-cells following SPLP-based vaccination. Frequency of TPHPARIGL-specific IFN-γ secreting CD8+ cells following immunization of A/J mice with SPLP (βgal) alone, or following administration of LN CpG-ODN at the same time or 24, 48 or 72 h after vaccination. (A) Splenocytes isolated from A/J mice (5 mice per group pooled) were incubated with the βgal immunodominant peptide TPHPARIGL for 5 h and the frequency of IFN-γ secreting CD8+ T-cells in response to stimulation were quantitated by flow cytometry. (B) Frequency of TPHPARIGL-specific IFN-γ secreting CD8+ T-cells following 5 days of in vitro restimulation with TPHPARIGL -pulsed splenocytes. Data is represented as the percentage of CD8+ cells positive for IFN-γ secretion following stimulation minus unstimulated splenocytes.  5.3.2 Investigation of SPLP as a prophylactic cancer vaccine in the presence and absence of LN CpG-ODN. The efficacy of SPLP as a prophylactic cancer vaccine, in the presence and absence of LN CpG-ODN, was investigated in studies where mice were vaccinated against βgal as an artificial TAA and the ability of vaccination to protect against challenge with the βgal expressing tumour cell line CT26.CL25, or delay growth of tumours stably expressing βgal, was monitored. To control for the contribution of non-specific activation of innate immunity to 137  tumour rejection or growth delay following vaccination with lipid/DNA, mice were also vaccinated with SPLP (luc) encoding the unrelated antigen luciferase, or LN CpG-ODN alone. SPLP (luc) is known to exhibit a similar level of immunostimulatory activity as SPLP (βgal) based on the upregulation of activation markers (CD69 & CD86) by APCs and the induction of proinflammatory cytokines (IFN-γ, IL-6, MCP-1) following systemic administration (unpublished data). All mice used in the study showed palpable tumours 12 days after challenge. Sixty percent of mice vaccinated with SPLP (βgal) in the presence of LN CpG-ODN and 40% of mice vaccinated with SPLP (βgal) alone were found to be tumour-free 5 weeks following challenge with the βgal expressing tumour cell line CT26.CL25, as compared to PBStreated control mice which all developed tumours reaching a volume of 1000 mm3 by the end of the fifth week. Twenty percent of mice that had been vaccinated with SPLP (luc) or LN CpGODN alone also remained tumour-free at this time. Mice immunized with SPLP (βgal) in the presence of LN CpG-ODN had an increase in median survival to 45 days compared with all other groups for which a median survival of 19 to 26 days was observed (Figure 5.4). Vaccinated mice that initially developed tumours did not exhibit an observable delay in tumour growth when compared to PBS treated controls (Figure 5.4).  138  A) Median Tumour Volume (mm3)  1400  PBS Control  1200  LN CpG-ODN  1000  SPLP (luc) SPLP (bgal)  800  SPLP (bgal) & LN CpG-ODN  600 400 200  B)  0 0  5  10  15  20  25  30  Days Post Tum our Challenge  B)  PBS Control; (26)  100%  LN CpG-ODN; (22) SPLP (luc); (19)  % Surviving  80%  SPLP (bgal); (19) SPLP (bgal) & LN CpG-ODN; (45)  60% 40% 20% 0% 0  5  10  15  20 25 30 35 40 45 Days Post Tumour Challenge  50  55  60  65  Figure 5.4: Vaccination with SPLP (βgal) in the presence of LN CpG-ODN enhances median survival following tumour challenge with CT26.CL25. BALB/c mice (n = 5) were vaccinated with SPLP (βgal) alone or followed 24 h later by LN CpG-ODN, SPLP (luc) alone, LN CpG-ODN alone or PBS on days 0, 7 and 14. Mice were challenged 14 days following the final vaccination subcutaneously with 2 x 105 CT26.CL25 cells. (A) Median tumour volume following tumour challenge (B) Kaplan-Meir survival curves, number in brackets in figure legend denotes median survival for that treatment group in days. Note: PBS control group has an n=4 as one mouse was euthanized prior to reaching a tumour volume = 1000 mm3 due to tumour-related paralysis.  To assess if tumour growth in vaccinated animals was a result of antigen loss, tumours were isolated from mice when they reached a terminal volume of 1000mm3 and assayed for βgal expression. Tumours isolated from control and vaccinated animals all showed detectable levels of βgal expression (data not shown) with the exception of tumours isolated from the two mice 139  which showed delayed tumour development (days 45 and 62).  Tumours isolated from these  mice, one mouse from the group vaccinated with SPLP (βgal) alone and one mouse from the group vaccinated with SPLP (βgal) and LN CpG-ODN, demonstrated no detectable levels of tumour βgal expression indicating the outgrowth of tumour cells which did not express βgal. Mice surviving initial tumour challenge were rechallenged with both the parental (CT26) and the βgal expressing (CT26.CL25) tumour cell lines 11 weeks after the initial challenge (approximately 8 weeks after tumours cleared). Mice originally vaccinated with SPLP (βgal) alone, SPLP (βgal) and LN CpG-ODN or LN CpG-ODN alone were protected against rechallenge with the βgal expressing cell line CT26.CL25 and the parental, non-βgal expressing cell line CT26, indicating the generation of protective tumour-specific immune responses.  In  contrast, the surviving mouse from the group initially vaccinated with SPLP (luc) encoding the unrelated antigen luciferase was protected against challenge with the βgal expressing cell line (CT26.CL25), but not the with the parental cell line (CT26) for which tumour growth was similar to that observed for age matched controls (data not shown).  5.3.3 Investigation of the ability of SPLP-mediated vaccination and transfection of tumours to act as a novel therapeutic cancer vaccine. The potential of SPLP to act as a novel therapeutic cancer vaccine, in a situation where SPLP acts both to promote the generation of an immune response against an artificial TAA and as a vehicle for the delivery of the artificial TAA to the tumour site, was investigated using βgal as an artificial TAA. Mice vaccinated with SPLP (βgal) alone or followed 24 h later by LN CpG-ODN were seeded with the syngeneic tumour cell line CT26 two weeks after the final vaccination. When tumours were approximately 100 mm3 mice were administered SPLP (βgal) i.v. once a week for two weeks to promote the expression of βgal at the tumour site. To control for the contribution of non-specific activation 140  of innate immunity following systemic vaccination with lipid/DNA and the expression of a foreign protein at the tumour site to tumour rejection or tumour growth delay, vaccinated mice were also treated with SPLP (luc). Tumours grew at a similar rate in all treatment groups up until day 22 (3 days following the final treatment with SPLP) when a visible tumour growth delay was observed in mice that had been originally vaccinated with SPLP (βgal) and LN CpG-ODN and were therapeutically receiving SPLP (βgal) in the second set of treatments (Figure 5.6). A reduced tumour growth delay was also observed for mice that had initially been vaccinated with SPLP (βgal) alone and were receiving SPLP (βgal) therapeutically. No such delay was observed in unvaccinated mice receiving SPLP (βgal) or vaccinated mice receiving SPLP (luc), indicating that tumour growth delay was not a consequence of non-specific immune stimulation following systemic delivery of SPLP or the expression of a foreign antigen at the tumour site. Unfortunately, by day 30 surviving mice began to show signs of distress (weight loss, hunched posture, lack of grooming and lack of mobility) and between days 34 and 40, the surviving mice were euthanized due to what appeared to be a result of cationic lipid-related toxicity unrelated to tumour burden.  Autopsy revealed severe splenomegaly and varying  degrees of liver toxicity in all 5 mice, but no visual signs of tumour metastasis were observed in any of the major organs. Testing of whole blood and serum samples from 4 of the 5 mice demonstrated elevated levels of alanine aminotransferase, gamma-glutamyltransferase, total protein and globulin levels all indicative of liver damage (Table 5.1). Although the study was terminated early, a significant increase (Mantal-Cox test p < .05) in median survival to 37.5 days was observed for mice vaccinated with SPLP (βgal) & LN CpG-ODN receiving SPLP (βgal) therapeutically, when compared to vaccinated mice receiving SPLP (luc) therapeutically (median survival of 25 days), and all other control treatments, again indicating that tumour  141  growth delay was not a non-specific consequence of the systemic delivery of SPLP or the expression of a foreign antigen at the tumour site (Figure 5.6). A)  PBS / PBS  Median Tumour Volume (mm3)  1200  PBS / SPLP (bgal) 1000  SPLP (bgal) / SPLP (bgal) SPLP (bgal) & LN CpG-ODN / SPLP (bgal)  800  SPLP (bgal) & LN CpG-ODN / SPLP (luc)  600 400 200 0 0  5  10  15  20  25  30  35  40  Days Post Tumour Challenge  B) 100%  % Surviving  80%  60%  PBS / PBS; (25) PBS / SPLP (bgal); (27)  40%  SPLP (bgal) / SPLP (bgal); (30) SPLP (bgal) & LN CpG ODN / SPLP (bgal); (37.5) SPLP (bgal) & LN CpG ODN / SPLP(luc); (25)  20%  0% 0  5  10  15  20  25  30  35  40  Days Post Tumour Challenge  Figure 5.6: Mice vaccinated with SPLP (βgal) and LN CpG-ODN exhibit tumour growth delay and increased survival following therapeutic treatment with SPLP (βgal). BALB/c mice vaccinated  with SPLP (βgal) alone or followed 24 h later by LN CpG-ODN were challenged 14 days following the final vaccination subcutaneously with 2 x 105 CT26 cells. Twelve days after seeding when tumours had reached an average volume of 100mm3 vaccinated mice were treated with 5mg/kg SPLP (βgal) or SPLP (luc) once a week for two weeks to promote transgene expression at the tumour site (arrows indicate therapeutic treatment). Groups are labeled as Vaccination Regimen / Therapeutic Treatment (A) Tumour growth expressed as median tumour volume (n = 6 to 8) (B) Kaplain-Meir survival plot (n = 6 to 8), number in brackets in figure legend denotes median survival for that treatment group in days. Note: dashed lines indicate mice which where euthanized as a result of distress, not tumour burden. 142  Table 5.1: Test results from whole blood and serum samples obtained from mice euthanized as a result of distress following vaccination and therapeutic treatment with SPLP. A,B and C represent mice which were originally vaccinated with SPLP (βgal) and LN CpG-ODN and were receiving SPLP (βgal) therapeutically, while D represents a mouse which was originally vaccinated with SPLP (βgal) alone and was receiving SPLP (βgal) therapeutically. Analysis was performed by the Central Laboratory for Veterinarians, values in red represent levels which fall outside of the normal range, N.D. = not determined. Hematology White Blood Cell Count Red Blood Cell Count Hemoglobin Hematocrit RDW Platelet Count Differentials Neutrophils Banded Neutrophils Lymphocytes Monocytes Chemistry Glucose Blood Urea Nitrogen Creatinine Total Protein Albumin Globulin Total Bilirubin Aspartate Aminotransferase (AST) Gamma-Glutamyltransferase Alanine Aminotransferase (ALT)  Normal Range 2.5 - 10.5 6.9 - 11.1 128 - 181 0.38 - 0.51 8.0 - 20.0 630 - 1430 Normal Range 0.4 - 2.8 0 - 0.1 2.1 - 6.5 0 - 0.4 Normal Range 5.5 - 10.4 6.0 - 17.1 30 - 56 35 - 48 17 - 26 12 - 24 0-7 70 - 900 0-1 0 - 50  A 562 7.7 101.2 0.31 24.5 2046 A 522.66 16.86 5.62 11.24 A 6 10.1 22.4 55 21.9 33 1 1138 3 793  B 189 8.07 110 0.334 29.7 2213 B 185.22 N.D. 1.89 1.89 B 0.9 14.5 1.1 55 17.9 37 3 379 7 97  C 173.2 8.6 127 0.359 26.2 2748 C 133.36 1.73 15.59 22.52 C 1.2 8 24.9 59 20.3 39 4 537 6 403  D 220.8 7.6 100 0.32 26.6 2775 D 189.9 6.62 6.62 17.66 D 0.9 11.3 32.4 58 19.5 39 2 1755 2 1294  5.3.4 β-gal and tumour specific immune responses in vaccinated mice receiving therapeutic treatment with SPLP (βgal). To evaluate the levels of βgal specific and tumourspecific CTL responses in vaccinated mice receiving SPLP (βgal) or SPLP (luc) therapeutically, standard  51  Cr release assays were performed on splenocytes isolated from vaccinated mice  immediately and following five days of in vitro restimulation. While both groups of mice originally vaccinated with SPLP (βgal) followed 24 h later by LN CpG-ODN demonstrated increased killing of βgal expressing target cells CT26.CL25 at an E:T ratio of 75:1 (p < .05) (Figure 5.7A), only vaccinated mice treated with SPLP (βgal), but not SPLP (luc), demonstrated a significant increase in tumour-specific CTL responses (p < .05) as indicated by an increase in 143  the lysis of the cell line CT26 at the highest E:T ratio, 75:1 (Figure 5.7B). No increase in killing of the nonsyngeneic haplotype matched tumour cell line P815 was observed for any treatment group (data not shown).  A) % Chromium Release  40% 35% 30% 25% 20% 15% 10% 5% 0%  B)  PB S Co ntro l  PB S / SP LP (bgal)  SP LP (bgal) / SPLP (bgal)  SPLP (bgal) & LN CpG-ODN / SPLP (luc)  SPLP (bgal) & LN CpG-ODN / SPLP (bgal)  P B S Co ntro l  P B S / SP LP (bgal)  SP LP (bgal) / SP LP (bgal)  SP LP (bgal) & LN CpG-ODN / SP LP (luc)  SP LP (bgal) & LN CpG-ODN / SP LP (bgal)  % Chromium Release  14% 12% 10% 8% 6% 4% 2% 0%  Figure 5.7: Mice vaccinated with SPLP (βgal) and LN CpG-ODN exhibit increased CTL activity against both the βgal expressing cell line CT26.CL25 and the parental non-βgal expressing cell line CT26 following therapeutic treatment with SPLP (βgal).. BALB/c mice vaccinated with SPLP (βgal) alone or followed 24 h later by LN CpG-ODN were challenged 14 days following the final vaccination s.c. with 2 x 105 CT26 cells. Twelve days after seeding when tumours had reached an average volume of 100 mm3 vaccinated mice were treated with 5mg/kg SPLP (βgal) or SPLP (luc) once a week for two weeks to promote transgene expression at the tumour site. Groups are labeled as Vaccination Regimen / Therapeutic Treatment. Spleens isolated five days following the final vaccination were analyzed for (A) CT26.CL25 and (B) tumour-specific (CT26) CTL activity using a standard 51Cr release assay following 5 days of in vitro restimulation with the βgal expressing cell line CT25.CL25. Effector to target ratio of 75:1 is shown for all cell lines, each bar represents splenocytes pooled from 5 mice performed in quadruplicate ± standard deviation.  144  5.4 Discussion Although DNA vaccines offer a convenient approach to elicit cellular immune responses against cancer, clinical outcomes to date have not been satisfactory, mainly because immune responses elicited by DNA vaccines are not sufficiently potent to suppress cancer progression. A number of strategies focused on breaking immunological tolerance to TAAs are currently being evaluated to enhance the potency of DNA vaccines, some of which involve broad stimulation of the immune system via co-expression or co-administration of immunomodulatory agents (Petrovsky and Aguilar 2004) and vaccination with xenoantigens. Here we demonstrate the feasibility of a SPLP/LN-CpG ODN-based cancer vaccine and introduce a novel approach for the generation of a non-viral DNA cancer vaccine where the immune response is raised against a foreign, immunogenic artificial TAA, which is not normally expressed at the tumour site but can be delivered there for expression by SPLP. It is proposed that the expression of a foreign immunogenic antigen at the tumour site, combined with a pre-existing immune response against the antigen primed by the SPLP/LN CpG-ODN-based vaccine, will promote the targeted destruction of transfected tumour cells in a situation where tolerance against the TAA does not exist. CpG-ODN based adjuvants prime the generation of strong Th1-biased immune responses and have demonstrated the potential to act as potent tumour vaccine adjuvants when antigen is delivered as a peptide, protein or autologous cellular vaccine (Krieg 2004; Klinman 2006). We have previously reported that LN CpG-ODN is an effective adjuvant for an SPLP-based DNA vaccine, enhancing antigen-specific humoral and cellular immune responses, when LN CpG-ODN is provided 24 h after the administration of SPLP (K. Wilson, unpublished results).  Observations made by others strongly suggest that the timing between the  administration of free CpG-containing DNA, as an adjuvant, and a naked DNA vaccine is critical. In line with this it has been demonstrated that delivering CpG-DNA after the vaccine 145  typically results in an immunogenic boost (Kojima, Xin et al. 2002; Ren, Zheng et al. 2004) while co-administration often inhibits the activity of the vaccine (Weeratna, Brazolot Millan et al. 1998). This effect is presumably due to competitive interference for uptake by APCs (Weeratna, Brazolot Millan et al. 1998) and/or the down-regulation of viral promoters used by the pDNA by CpG-induced cytokines (IFN-γ and TNF-α) (Harms and Splitter 1995; Tan, Li et al. 1999), both of which are proposed to be alleviated when CpG-DNA is administered after the DNA vaccine at a time when antigen is already being expressed (Kojima, Xin et al. 2002; Ren, Zheng et al. 2004). To examine the temporal effects of LN CpG-ODN as an adjuvant for our SPLP-based vaccine we administered LN CpG-ODN immediately following SPLP vaccination or 24, 48 or 72 h thereafter. The results demonstrate that administering LN CpG-ODN 24 h after DNA vaccination optimally increases the generation of antigen-specific IgG2a humoral immune responses, and the frequency of antigen-specific CD8+ T-cells as assessed using MHC-pentamer analysis and IFN-γ secretion assays. Antigen-specific cellular responses in vaccinated animals, particularly following in vitro restimulation, clearly demonstrates the presence of βgal specific cellular responses in mice vaccinated with SPLP and LN CpG-ODN; most noticeably in mice vaccinated with SPLP followed 24 h later by LN CpG-ODN. DNA vaccines encoding TAAs have demonstrated the potential to induce TAA-specific immune responses, which are protective against tumour challenge, in numerous animal models (Conry, LoBuglio et al. 1995; Bright, Beames et al. 1996; Prud'homme 2005). Here using the well-defined, syngeneic CT26.CL26/BALB/c colon carcinoma model in which immunized animals are challenged with syngeneic βgal-expressing CT26.CL25 tumor cells, we investigated the ability of vaccination with SPLP to act prophylactically to protect against tumour challenge. Prophylactic vaccination with SPLP (βgal) and LN CpG-ODN was demonstrated to increase the  146  median survival of mice to 45 days, as compared to 19-26 days for all other treatment groups, concomitant with an increase in the percentage of tumour-free mice in groups which had been vaccinated with SPLP (βgal) & LN CpG-ODN or SPLP (βgal) alone (60% and 40% tumour-free at 5 weeks, respectively). Furthermore, animals from these groups which remained tumour free were protected against rechallenge with both the βgal expressing cell line CT26.CL25, as well as with non-βgal-expressing CT26 cells, demonstrating the induction of effective secondary antitumour immune responses against other TAAs. Interestingly, 20% of mice vaccinated with SPLP encoding the non-related antigen luciferase or LN CpG-ODN also remained tumour-free implicating non-specific immune activation as being able to provide some level of protection against tumour challenge, even up to two weeks after treatment. Although somewhat surprising, it has been previously reported that administration of CpG-DNA can protect against challenge with viral or bacterial pathogens (Krieg 2006) and also increase Th1-biased responses to protein antigens delivered, in some cases, up to 2 weeks following CpG ODN administration (Kobayashi, Horner et al. 1999). As with vaccinated mice, the mouse originally treated with LN-CpG ODN alone, which survived the initial tumour challenge exhibited protection against re-challenge with both CT26.CL25 and CT26 βgal expressing and non-expressing tumour cell lines while mice treated with SPLP (luc) alone were protected against CT26.CL25 but not CT26 rechallenge. Although it is known that vaccination with SPLP (βgal) and followed 24 h later by LN CpG-ODN promotes βgal specific immune responses in mice, it is not possible using this model system to quantitate the specific contribution of vaccine primed antigen-specific immune responses to protection against tumour challenge. Results from these studies appear to support a contribution of non-specific immune responses to protection afforded by vaccines which are themselves potent stimulators of innate immunity.  147  The goal of priming Th1-biased TAA-specific T-cell responses in cancer vaccines is often hampered by the fact that TAAs are derived from self-proteins, and thus poorly immunogenic due central and peripheral tolerance mechanisms. In attempt to overcome this obstacle, we investigated the ability of SPLP-mediated delivery of an artificial TAA to established tumours to promote tumour growth delay or tumour rejection in mice that had been vaccinated against the same artificial TAA by SPLP. The systemic administration of cationic lipid/DNA complexes (containing either CpG containing ODN or non-coding plasmid DNA) has demonstrated the potential to promote non-specific anti-tumour activity and the subsequent development of tumour specific adaptive responses against established tumours in several murine models (Dow, Fradkin et al. 1999; Whitmore, Li et al. 1999; Lanuti, Rudginsky et al. 2000; Whitmore, Li et al. 2001). Vaccinated mice were therefore also treated therapeutically with SPLP (luc), which possesses similar immunostimulatory activity, to control for both the influence of non-specific immunostimulatory activity and the expression of a foreign protein at the tumour site on tumour rejection/tumour growth delay. Tumour growth delay and an increase in median survival was observed for mice therapeutically treated with SPLP (βgal) which had been vaccinated with SPLP (βgal) and LN CpG-ODN, and to a lesser degree in mice which had been vaccinated with SPLP (βgal) alone, as compared to vaccinated mice treated with SPLP (luc), indicating that tumour growth delay was a result of antigen-specific immune responses in combination with the expression of the relevant antigen at the tumour site. Only a slight tumour growth delay and increase in survival was observed in unvaccinated mice receiving SPLP (βgal) or vaccinated mice receiving SPLP (luc), as compared to the PBS control, indicating that the systemic administration of SPLP in the absence of a pre-existing antigen-specific immune response is not sufficient to promote therapeutic tumour growth delay in this model.  148  As SPLP-mediated delivery of a transgene to distal tumour sites is known to result in the transfection of only a small percentage of the tumour cells (approximately 3 – 6%; unpublished data), it is hypothesized that tumour growth delay observed in vaccinated mice treated therapeutically with SPLP (βgal) may be a result of the generation of secondary tumour specific responses through epitope spreading, which has been observed to occur in both humans and in animal models following immunization with cancer vaccines (Disis, Gooley et al. 2002; Butterfield, Ribas et al. 2003). This hypothesis is supported by the observation that splenocytes isolated from mice that demonstrated tumour-growth delay following therapeutic treatment with SPLP (βgal) also demonstrated a significant increase in CTL activity against the parental, nonβgal expressing tumour cell line CT26, which was not observed in any of the vaccinated controls. While SPLP and LN-CpG ODN are able to induce immune responsiveness and antitumour efficacy as standalone agents, administration in combination as a cancer vaccine consistently induces more potent adaptive immune responses concomitant with enhanced antitumour activity. This should not surprising in view of our recent observations that SPLPdelivered plasmind DNA immunostimulation is mediated, at least in part, through a TLR-9independent pathway (manuscript in preparation). This indicates that a SPLP/LN-CpG cancer vaccine will induce immune stimulation through multiple pathways and raises the possibility that coactivation of these two pathways will ultimately results in additive or syngeristic immunostimulatory activity. Unfortunately, despite observable tumour growth delay, surviving mice were euthanized between days 34 and 40 due to non-tumour related causes. While liver damage stemming from toxicity associated with repetitive administration of DODMA, a cationic lipid which has recently demonstrated toxicity in several murine models, or repetitive administration of cationic lipid containing DNA particles is the most probable cause of death (Tousignant, Gates et al. 2000; 149  Loisel, Le Gall et al. 2001), a role for targeted immune-mediated destruction of transfected liver cells cannot be excluded. Although SPLP are known to passively accumulate at tumour sites resulting in levels of transgene expression which are 2-3 orders of magnitude higher than those observed in any other tissue, up to 40% of the injected dose per gram of tissue can be found in the liver within 12 h of administration (Ambegia, Ansell et al. 2005) and low levels of transgene expression are known to occur (Monck, Mori et al. 2000; Ambegia, Ansell et al. 2005; Judge, McClintock et al. 2006). Alternatively, it should be noted that a third therapeutic injection of SPLP, attempted in mice that had been previously vaccinated with SPLP (βgal) and LN CpG-ODN, resulted in acute mortality with symptoms characteristic of antibody-mediated anaphylactic reactions including lethargy, facial puffing, and laboured respiration (Roy, Mao et al. 1999; Judge, McClintock et al. 2006). While generation of anti-PEG antibodies and the systemic release of platelet-activating factor following repeat administration of nucleic acid containing PEGylated liposomes has been previously demonstrated (Semple, Harasym et al. 2005; Judge, McClintock et al. 2006), formulation of SPLP with shorter chain PEG-lipids, as employed here, has been found to alleviate this problem by allowing rapid dissociation of the PEG-lipid from the immunostimulatory payload (Judge, McClintock et al. 2006). Our observations may indicate that despite shorter chain PEG-lipids, repeat dosing with these particles can, over an extended period of time, result in the generation of antibody titres which are sufficient to cause anaphylactic-mediated mortality. In conclusion, these studies demonstrate that LN CpG-ODN is most effective as an adjuvant for an SPLP-based DNA vaccine when administered 24 h after the vaccine. Furthermore, although the ability of antigen-specific responses generated by an SPLP vaccine to act prophylactically to protect against tumour challenge was confounded by the potent nonspecific immunostimulatory activity of SPLP and LN CpG-ODN, the potential of SPLP to act as 150  a novel therapeutic cancer vaccine, in a situation where SPLP acts both to promote the generation of an immune response against an artificial TAA and as a vehicle for the delivery of the artificial TAA to the tumour site, was demonstrated. In addition, these studies highlight the adverse side-affects associated with the systemic administration of SPLP and LN CpG-ODN which become more apparent following repeat dosing and which need to be addressed prior to further investigation of the vaccine potential of SPLP.  5.5 References Ambegia, E., S. Ansell, P. Cullis, J. Heyes, L. Palmer and I. MacLachlan (2005). "Stabilized plasmid-lipid particles containing PEG-diacylglycerols exhibit extended circulation lifetimes and tumor selective gene expression." Biochim Biophys Acta 1669(2): 155-63. Bright, R. K., B. Beames, M. H. Shearer and R. C. Kennedy (1996). "Protection against a lethal challenge with SV40-transformed cells by the direct injection of DNA-encoding SV40 large tumor antigen." Cancer Res 56(5): 1126-30. Butterfield, L. H., A. Ribas, V. B. Dissette, S. N. Amarnani, H. T. Vu, D. Oseguera, H. J. Wang, R. M. Elashoff, W. H. McBride, B. Mukherji, A. J. Cochran, J. A. Glaspy and J. S. Economou (2003). "Determinant spreading associated with clinical response in dendritic cell-based immunotherapy for malignant melanoma." Clin Cancer Res 9(3): 998-1008. Conry, R. M., A. F. LoBuglio, F. Loechel, S. E. Moore, L. A. Sumerel, D. L. Barlow, J. Pike and D. T. Curiel (1995). "A carcinoembryonic antigen polynucleotide vaccine for human clinical use." Cancer Gene Ther 2(1): 33-8. Curiel, T. J. (2007). "Tregs and rethinking cancer immunotherapy." J Clin Invest 117(5): 116774. Davis, H. L., C. L. Brazolot Millan, M. Mancini, M. J. McCluskie, M. Hadchouel, L. Comanita, P. Tiollais, R. G. Whalen and M. L. Michel (1997). "DNA-based immunization against hepatitis B surface antigen (HBsAg) in normal and HBsAg-transgenic mice." Vaccine 15(8): 849-52. Disis, M. L., T. A. Gooley, K. Rinn, D. Davis, M. Piepkorn, M. A. Cheever, K. L. Knutson and K. Schiffman (2002). "Generation of T-cell immunity to the HER-2/neu protein after active immunization with HER-2/neu peptide-based vaccines." J Clin Oncol 20(11): 2624-32. Dow, S. W., L. G. Fradkin, D. H. Liggitt, A. P. Willson, T. D. Heath and T. A. Potter (1999). "Lipid-DNA complexes induce potent activation of innate immune responses and antitumor activity when administered intravenously." J Immunol 163(3): 1552-61.  151  Harms, J. S. and G. A. Splitter (1995). "Interferon-gamma inhibits transgene expression driven by SV40 or CMV promoters but augments expression driven by the mammalian MHC I promoter." Hum Gene Ther 6(10): 1291-7. Jeffs, L. B., L. R. Palmer, E. G. Ambegia, C. Giesbrecht, S. Ewanick and I. MacLachlan (2005). "A scalable, extrusion-free method for efficient liposomal encapsulation of plasmid DNA." Pharm Res 22(3): 362-72. Judge, A., K. McClintock, J. R. Phelps and I. Maclachlan (2006). "Hypersensitivity and loss of disease site targeting caused by antibody responses to PEGylated liposomes." Mol Ther 13(2): 328-37. Kawabata, K., Y. Takakura and M. Hashida (1995). "The fate of plasmid DNA after intravenous injection in mice: involvement of scavenger receptors in its hepatic uptake." Pharm Res 12(6): 825-30. Kawakami, Y., S. Eliyahu, C. H. Delgado, P. F. Robbins, K. Sakaguchi, E. Appella, J. R. Yannelli, G. J. Adema, T. Miki and S. A. Rosenberg (1994). "Identification of a human melanoma antigen recognized by tumor-infiltrating lymphocytes associated with in vivo tumor rejection." Proc Natl Acad Sci U S A 91(14): 6458-62. Klinman, D. M. (2006). "Adjuvant activity of CpG oligodeoxynucleotides." Int Rev Immunol 25(3-4): 135-54. Kobayashi, H., A. A. Horner, K. Takabayashi, M. D. Nguyen, E. Huang, N. Cinman and E. Raz (1999). "Immunostimulatory DNA pre-priming: a novel approach for prolonged Th1-biased immunity." Cell Immunol 198(1): 69-75. Kojima, Y., K. Q. Xin, T. Ooki, K. Hamajima, T. Oikawa, K. Shinoda, T. Ozaki, Y. Hoshino, N. Jounai, M. Nakazawa, D. Klinman and K. Okuda (2002). "Adjuvant effect of multi-CpG motifs on an HIV-1 DNA vaccine." Vaccine 20(23-24): 2857-65. Krieg, A. M. (2004). "Antitumor applications of stimulating toll-like receptor 9 with CpG oligodeoxynucleotides." Curr Oncol Rep 6(2): 88-95. Krieg, A. M. (2006). "Therapeutic potential of Toll-like receptor 9 activation." Nat Rev Drug Discov 5(6): 471-84. Lanuti, M., S. Rudginsky, S. D. Force, E. S. Lambright, W. M. Siders, M. Y. Chang, K. M. Amin, L. R. Kaiser, R. K. Scheule and S. M. Albelda (2000). "Cationic lipid:bacterial DNA complexes elicit adaptive cellular immunity in murine intraperitoneal tumor models." Cancer Res 60(11): 2955-63. Lew, D., S. E. Parker, T. Latimer, A. M. Abai, A. Kuwahara-Rundell, S. G. Doh, Z. Y. Yang, D. Laface, S. H. Gromkowski, G. J. Nabel and et al. (1995). "Cancer gene therapy using plasmid DNA: pharmacokinetic study of DNA following injection in mice." Hum Gene Ther 6(5): 55364. Loisel, S., C. Le Gall, L. Doucet, C. Ferec and V. Floch (2001). "Contribution of plasmid DNA to hepatotoxicity after systemic administration of lipoplexes." Hum Gene Ther 12(6): 685-96. 152  Maurer, N., K. F. Wong, H. Stark, L. Louie, D. McIntosh, T. Wong, P. Scherrer, S. C. Semple and P. R. Cullis (2001). "Spontaneous entrapment of polynucleotides upon electrostatic interaction with ethanol-destabilized cationic liposomes." Biophys J 80(5): 2310-26. Mocellin, S., C. R. Rossi and D. Nitti (2004). "Cancer vaccine development: on the way to break immune tolerance to malignant cells." Exp Cell Res 299(2): 267-78. Monck, M. A., A. Mori, D. Lee, P. Tam, J. J. Wheeler, P. R. Cullis and P. Scherrer (2000). "Stabilized plasmid-lipid particles: pharmacokinetics and plasmid delivery to distal tumors following intravenous injection." J Drug Target 7(6): 439-52. Petrovsky, N. and J. C. Aguilar (2004). "Vaccine adjuvants: current state and future trends." Immunol Cell Biol 82(5): 488-96. Prud'homme, G. J. (2005). "DNA vaccination against tumors." J Gene Med 7(1): 3-17. Ren, J., L. Zheng, Q. Chen, H. Li, L. Zhang and H. Zhu (2004). "Co-administration of a DNA vaccine encoding the prostate specific membrane antigen and CpG oligodeoxynucleotides suppresses tumor growth." J Transl Med 2(1): 29. Rosenberg, S. A. (1999). "A new era for cancer immunotherapy based on the genes that encode cancer antigens." Immunity 10(3): 281-7. Roy, K., H. Q. Mao, S. K. Huang and K. W. Leong (1999). "Oral gene delivery with chitosan-DNA nanoparticles generates immunologic protection in a murine model of peanut allergy." Nat Med 5(4): 387-91. Semple, S. C., T. O. Harasym, K. A. Clow, S. M. Ansell, S. K. Klimuk and M. J. Hope (2005). "Immunogenicity and rapid blood clearance of liposomes containing polyethylene glycol-lipid conjugates and nucleic Acid." J Pharmacol Exp Ther 312(3): 1020-6. Tabi, Z. and S. Man (2006). "Challenges for cancer vaccine development." Adv Drug Deliv Rev 58(8): 902-15. Tan, Y., S. Li, B. R. Pitt and L. Huang (1999). "The inhibitory role of CpG immunostimulatory motifs in cationic lipid vector-mediated transgene expression in vivo." Hum Gene Ther 10(13): 2153-61. Tousignant, J. D., A. L. Gates, L. A. Ingram, C. L. Johnson, J. B. Nietupski, S. H. Cheng, S. J. Eastman and R. K. Scheule (2000). "Comprehensive analysis of the acute toxicities induced by systemic administration of cationic lipid:plasmid DNA complexes in mice." Hum Gene Ther 11(18): 2493-513. Wang, M., V. Bronte, P. W. Chen, L. Gritz, D. Panicali, S. A. Rosenberg and N. P. Restifo (1995). "Active immunotherapy of cancer with a nonreplicating recombinant fowlpox virus encoding a model tumor-associated antigen." J Immunol 154(9): 4685-92. Weeratna, R., C. L. Brazolot Millan, A. M. Krieg and H. L. Davis (1998). "Reduction of antigen expression from DNA vaccines by coadministered oligodeoxynucleotides." Antisense Nucleic Acid Drug Dev 8(4): 351-6. 153  Whitmore, M., S. Li and L. Huang (1999). "LPD lipopolyplex initiates a potent cytokine response and inhibits tumor growth." Gene Ther 6(11): 1867-75. Whitmore, M. M., S. Li, L. Falo, Jr. and L. Huang (2001). "Systemic administration of LPD prepared with CpG oligonucleotides inhibits the growth of established pulmonary metastases by stimulating innate and acquired antitumor immune responses." Cancer Immunol Immunother 50(10): 503-14. Wilson, K. D., S. G. Raney, L. Sekirov, G. Chikh, S. D. Dejong, P. R. Cullis and Y. K. Tam (2007). "Effects of intravenous and subcutaneous administration on the pharmacokinetics, biodistribution, cellular uptake and immunostimulatory activity of CpG ODN encapsulated in liposomal nanoparticles." Int Immunopharmacol 7(8): 1064-75.  154  CHAPTER SIX SUMMARY AND FUTURE DIRECTIONS In this work the immunostimulatory activity of SPLP and LN CpG-ODN, and their potential to act together as a non-viral DNA vaccine, is described.  In Chapter Two, the  pharmacokinetics, biodistribution and cellular uptake of LN-CpG ODN following i.v. and s.c. administration was characterized and correlated with immunostimulatory activity in a murine model. Results presented show that despite dramatic differences in tissue distribution profiles and considerable differences in uptake by APCs following i.v and s.c. administration, the resultant immune response is very similar with respect to levels of cellular activation and cytolytic activity of immune cells in the spleen and blood compartments. It is concluded that the inherent ability of APCs to accumulate liposomal nanoparticles results in very efficient uptake of LN-CpG ODN, even when present at very low concentrations, resulting in enhanced immune responses as compared to free ODN. In Chapter Three, the immunostimulatory potential of SPLP is examined and it is demonstrated that lipid encapsulation dramatically enhances the immunostimulatory potential of pDNA. Furthermore, it is demonstrated in vivo, that the immunostimulatory activity of SPLP is not abrogated in TLR9-knockout animals and that, in contrast to that previously documented for free CpG-ODN, the endosomolytic agent chloroquine enhances the immunostimulatory potential of SPLP. From these data it is concluded that the immunostimulatory activity of SPLP-delivered pDNA is mediated through both TLR9-dependent and independent pathways, and implicates a role for a cytoplasmic pDNA detection pathway in the immunostimulatory activity of SPLP. In Chapter Four, the ability of SPLP to function as a systemically administered genetic vaccine in the presence and absence of additional immune stimulation provided by LN CpGODN is described. It is demonstrated that SPLP have the potential to be taken up by and 155  transfect APCs (CD11b+ and CD11c+) in the spleen compartment following i.v. administration. Furthermore, using beta-galactosidase (βgal) as a model antigen it is demonstrated that in the presence of a strong immunostimulatory signal, provided by LN CpG-ODN, SPLP is able to promote the priming of transgene-specific humoral and cellular responses, neither of which can be consistently detected in the absence of LN CpG-ODN. In Chapter Five, the ability of SPLP & LN CpG-ODN to act together as a non-viral DNA cancer vaccine, both prophylactically and as a novel therapeutic cancer vaccine, is assessed in preliminary studies in a murine model. Using βgal as an artificial TAA the ability of antigenspecific immune responses generated by SPLP in the presence of LN CpG-ODN to act prophylactically to protect against tumour challenge is indicated.  Furthermore, the ability of  SPLP to act as a novel therapeutic cancer vaccine, in a model where SPLP acts both as a delivery vehicle for the generation of an immune response against an artificial TAA and for the delivery of plasmid DNA to tumour cells allowing for the expression of the artificial TAA at the tumour site is demonstrated. However, these studies also introduce the serious adverse sideaffects associated with repeat systemic administration of SPLP and LN CpG-ODN which were previously unrecognized. In summary, this work firstly improves our understanding of the immunostimulatory potential of both SPLP and LN CpG-ODN, providing a broader foundation on which their use as adjuvants can be based and highlights the role of TLR9-independent mechanisms in the immunostimulatory activity of non-viral gene delivery systems. Future work focused on increasing our understanding of the cellular mechanisms involved in the immunostimulatory activity of pDNA and the role that lipid encapsulation plays on access to pathways of pDNA detection is required for the design of optimized DNA-based therapeutics. Specifically, studies which investigate the immunostimulatory activity of pDNA delivered as SPLP in the presence and absence of agents that promote endosomal release are required in TLR9 KO mice to confirm 156  the hypothesis that enhanced immunostimulatory activity is a result of increased access to TLR9-independent cytoplasmic detection pathways. Comprehensive understanding of the cellular mechanism of pDNA detection will aid in the design of superior non-viral vectors for DNA vaccination and may provide insight into means by which to inhibit these pathways to allow for optimized gene-based therapy of disease. Secondly, this work introduces the concept that when combined with a strong immunostimulatory signal SPLP have the potential to act as a non-viral gene delivery vehicle for genetic vaccination. These studies not only provide proof of concept validation of an SPLPbased genetic vaccine but also identify the obstacles which need to be addressed to optimize this application.  Future work focused on optimizing SPLP delivery systems with respect to  reducing the toxicity observed following repeat administration is required prior to their use as genetic vaccines.  Improvements could be made by formulating SPLP with an alternate, less  toxic, cationic lipid than DODMA, which has been identified in these studies as a particularly toxic agent. Alternatively, strategies focused on improving the ability of SPLP to target and transfect APCs, through the incorporation of targeting ligands or components to enhance intracellular delivery properties, could increase the potency of SPLP as a genetic vaccine and reduce the dose required to generate an effective immune response. Furthermore, based on the observation that TLR9-dependent and –independent pathways play a cumulative role in pDNA-mediate immune stimulation when pDNA is delivered as SPLP, an investigation of the potential of using SPLP as an adjuvant, as compared to LN CpG-ODN, for an DNA-based vaccine should be conducted. These studies, when performed in TLR9 KO mice, could provide insight into whether TLR9-dependent and TLR9-independent pathways of immune stimulation play an additive or synergistic role to adjuvant SPLP-based vaccination. Although antigen-specific immune responses primed by SPLP following the vaccination regime employed in this thesis are weak, SPLP-mediated expression of the relevant antigen at 157  the tumour-site in the presence of an antigen-specific immune response demonstrated great potential as a therapeutic intervention in tumour-bearing mice. The observations made in this thesis therefore suggest that in addition to investigating optimized dosing regimes for improved immune responses, that future work should investigate combining SPLP-mediated expression of an artificial TAA at the tumour site with other vaccination strategies, in order to address the full potential of using SPLP-mediated antigen delivery to the tumour site in the presence of a strong immune response. In line with this, preliminary studies not discussed in this thesis demonstrate enhanced antigen-specific immune responses and anti-tumour efficacy in mice vaccinated with SPLP (βgal), LN CpG-ODN and βgal protein as compared to mice receiving SPLP (βgal) and LN-CpG-ODN or βgal protein and LN CpG-ODN, indicating that the investigation of prime/boost strategies using different vaccination methods may prove rewarding.  158  APPENDIX  159  160  

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