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Assessment of the biodistribution of aluminum-based vaccine adjuvants using ¹¹¹/¹¹⁵In-AlO(OH) Esposito, Tullio Vito Francesco 2016

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Assessment of the Biodistribution of Aluminum-Based Vaccine Adjuvants using 111/115In-AlO(OH) by Tullio Vito Francesco Esposito B.Sc (Pharmacy), The University of British Columbia, 2013A THESIS SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE In The Faculty of Graduate and Postdoctoral Studies (Pharmaceutical Sciences) THE UNIVERSITY OF BRITISH COLUMBIA (Vancouver) August 2016 ©Tullio Vito Francesco Esposito, 2016ii  Abstract Aluminum-based adjuvants are found in a wide range of non-living vaccines to enhances antibody-mediated immune responses. Alhydrogel is one of the most common aluminum-based adjuvants and consists of 1-12 μm clusters of loosely aggregated aluminum oxyhydroxide (AlO(OH)) nanoparticles. Despite widespread use since in 1930s, the biodistribution of this adjuvant is poorly understood. The current assumption within the field is that AlO(OH) particles remain at the site of injection and slowly dissolve over time. However, rare neurological and muscle adverse effects, such as macrophagic myofasciitis, are thought to be due to the bio-persistence of the adjuvant at the site of injection and the distribution of AlO(OH) nanoparticles throughout the body.  The objective of this thesis was to examine if component nanoparticles of Alhydrogel distribute following administration.  In order the test this hypothesis, traceable forms of AlO(OH) were synthesized by doping the adjuvant’s crystal structure with different indium isotopes. Various physical, morphological and functional assessments showed that the doped adjuvant closely matched commercial Alhydrogel. Using a radioactive version of the tracer, 111In-AlO(OH), the biodistribution of the adjuvant was assessed using serial SPECT/CT imaging. Approximately 20% of the tracer was released from the site of injection over 15 days and could be found in organs like the draining lymph nodes and liver. A novel application of mass cytometry was used to examine the kinetics and cellular distribution of 115In-AlO(OH) particles within the draining lymph nodes; the adjuvant appeared at this site within 1 day and reached peak levels 15 days post-injection. The adjuvant was sequestered into antigen presenting cells and induced the up-regulation of maturation markers in adjuvant-positive dendritic cells within the draining lymph node. Finally, electron microscopy coupled with atomic mapping confirmed the presence of Alhydrogel component nanoparticles in the draining lymph node and liver.   This thesis presents the most detailed assessment to date of the biodistribution of aluminum-based adjuvants. Understanding Alhydrogel’s biodistribution can help shed light on the adjuvant’s immunostimulatory mechanisms and adverse events. Lessons learnt from one of the world oldest adjuvants can be applied to the design of next generation of immunopotentiators.         iii  Preface  Chapter 2:  This material is from a manuscript in preparation. Rahel Schneider (UBC/ University of Basel) helped under my guidance to develop the methods to synthesize the doped adjuvant (Sections 2.2.1 and 2.2.2). Staff at the UBC Structural Chemistry Facility carried out the PXRD analysis (Figures 2-4 and 2-6). Xin Zhang at SFU 4D Labs provided assistance with STEM imaging and elemental mapping (Figures 2-10 to 2-12). Patrick Hopkins (UBC) provided assistance and guidance for the functional immune assays (Figures 2-15 to 2-16). All other experiments and the data analysis were performed by myself. I conceived the doping strategy for Alhydrogel and Urs Hafeli and myself developed the experimental plan. None of this text is taken from previously published articles. Chapter 3:  This material is from a manuscript in preparation. Cristina Rodriguez developed the reconstruction parameters for the dual-isotope 67Ga/111In SPECT at the UBC in vivo Imaging Centre (Figure 3-2). Katayoun Saatchi (UBC) assisted with the animal dissection for the biodistribution study (Figure 3-8). All other experiments and the data analysis were performed by myself. Urs Hafeli and I conceived the experiments. None of this text is taken from previously published articles. Chapter 4:  This material is from a manuscript in preparation. Patrick Hopkins and Jacqueline Lai (UBC/ University of Gothenburg) helped with sample staining (Section 4.2.2.) and aspects of the data analysis (Figures 4-5 to 4-7). Andy Johnson from the UBC Flow Cytometry Centre provided technical assistance and instruction in the operation of the CyTOF instrument (Section 4.4.4).  All other experiments and the data analysis were performed by myself. I devised the concept of using mass cytometry for in vivo nanoparticle tracking. Urs Hafeli, Jan Dutz, Jacqueline Lai, Patrick Hopkins and myself developed the experimental plan. None of this text is taken from previously published articles. Chapter 5:  This material is from a manuscript in preparation. Derrick Horne at the UBC Bioimaging Facility processed the fixed tissue samples for electron microscopy (Section 5.2.2). Matthew Bilton at SFU 4D Labs assisted with STEM imaging and elemental mapping (Section 5.2.3.). All other experiments and the data analysis were performed by myself. Urs Hafeli and I developed the experimental plan. None of this text is taken from previously published articles.  Appendix:  Staff at the UBC Structural Chemistry Facility carried out the PXRD analysis (Figure A-1). Andrew Yung and Barry Bohnet at the UBC MRI Research Centre carrying out the phantom scans (Figures A-2 and A-3). All other experimental work was carried out by myself.  Urs Hafeli and I developed the experimental plan. None of this text is taken from previously published articles.  iv  Table of Contents   Abstract ................................................................................................................................................................... ii Preface .................................................................................................................................................................... iii Table of Contents ................................................................................................................................................... iv List of Tables ......................................................................................................................................................... vii List of Figures....................................................................................................................................................... viii List of Abbreviations ................................................................................................................................................x Acknowledgements .............................................................................................................................................. xiii Chapter 1: Introduction .............................................................................................................................................1 1.1. History and Clinical Use of Aluminum-Based Adjuvants........................................................................1 1.2. Structure and Adsorptive Mechanisms of Aluminum-Based Adjuvants: .................................................1 1.3. Immunostimulatory Mechanisms of Aluminum-Based Adjuvants: .........................................................2 1.4. Safety of Aluminum-Based Adjuvants. ....................................................................................................4 1.5. Current State of Knowledge for the Biodistribution of Aluminum-Based Adjuvants..............................5 1.6. Hypothesis and Research Objectives ........................................................................................................6 Chapter 2: Preparation and Characterization of Indium-Doped AlO(OH) ...............................................................7 2.1. Introduction ..............................................................................................................................................7 2.2. Materials and Methods .............................................................................................................................8 2.2.1. Synthesis of AlO(OH) ......................................................................................................................8 2.2.2. Synthesis of 115In-Doped AlO(OH) ..................................................................................................9 2.2.3. Synthesis of 111In-Doped AlO(OH) ..................................................................................................9 2.2.4. Encapsulation Efficiency of 111In within the AlO(OH) Matrix ........................................................9 2.2.5. Concentration and Specific Activity .................................................................................................9 2.2.6. Powder X-Ray Diffraction (PXRD) .................................................................................................9 2.2.7. Dynamic Scanning Calorimetry (DSC) ..........................................................................................10 2.2.8. Fourier Transform Infrared Spectroscopy (FTIR) ..........................................................................10 2.2.9. Dynamic Light Scattering (DLS) ...................................................................................................10 2.2.10. Scanning Transmission Electron Microscopy (STEM) ..................................................................10 2.2.11. Stability of 111In-AlO(OH) in Different Media over Time .............................................................10 2.2.12. Protein Adsorptivity .......................................................................................................................11 2.2.13. Protein De-Adsorption in Biological Fluids ...................................................................................11 2.2.14. Cellular Toxicity Assays ................................................................................................................11 2.2.15. Humoral Immune Response Assessment........................................................................................12 2.2.16. Cellular Immune Response Assessment .........................................................................................13 2.2.17. Data Analysis ..................................................................................................................................13 v  2.2.18. Ethics Statement .............................................................................................................................13 2.3. Results ....................................................................................................................................................14 2.3.1. Encapsulation Efficiency, Concentration and Specific Activity of 111/115In-AlO(OH) ...................14 2.3.2. Stability of 111In-AlO(OH) in Different Media ...............................................................................14 2.3.3. Physical Characterization of Alhydrogel ........................................................................................15 2.3.4. Physical Characterization of 111/115In-AlO(OH) ..............................................................................16 2.3.5. Hydrodynamic Size and Zeta Potential of 111/115In-AlO(OH) and Alhydrogel ...............................17 2.3.6. Particle Morphology and Spatial Location of 115In Dopant within AlO(OH) Nanoparticles .........18 2.3.7. Protein Adsorptivity and De-Adsorptivity Assays to 1/115In-AlO(OH) and Alhydrogel .................21 2.3.8. Cellular Toxicity of 1/115In-AlO(OH) and Alhydrogel ....................................................................22 2.3.9. Humoral and Cellular Immune Responses to 111/115In-AlO(OH) and Alhydrogel ..........................23 2.4. Discussion ...............................................................................................................................................25 Chapter 3: Biodistribution of 111In-AlO(OH) .........................................................................................................27 1.1. Introduction ............................................................................................................................................27 3.2. Materials and Methods ...........................................................................................................................28 3.2.1. Synthesis and Radiolabeling of 67Ga-NOTA-OVA ........................................................................28 3.2.2. Labeling Efficiency and Radiopurity Assessment of 67Ga-NOTA-OVA .......................................28 3.2.3. SDS and Native PAGE Analysis of 67Ga-NOTA-OVA .................................................................28 3.2.4. SPECT/CT Assessment of 67Ga-NOTA-OVA Adsorbed to 111In-AlO(OH) or Alhydrogel ..........29 3.2.5. Sacrifice-Based Biodistribution of 111In-AlO(OH) .........................................................................30 3.2.6. Data Analysis ..................................................................................................................................30 3.2.7. Ethics Statement .............................................................................................................................30 3.3. Results ....................................................................................................................................................31 3.3.1. Preparation and Characterisation of 67Ga-NOTA-OVA .................................................................31 3.3.2. Serial  SPECT/CT Assessment of 111In-AlO(OH) and 67Ga-NOTA-OVA at the Injection Site ....32 3.3.3. Sacrificed-Based Biodistribution of 111In-AlO(OH) .......................................................................34 3.4. Discussion ...............................................................................................................................................35 Chapter 4: Distribution of 115In-AlO(OH) Particles in the Lymph Node ...............................................................37 4.1. Introduction ............................................................................................................................................37 4.2. Materials and Methods ...........................................................................................................................38 4.2.1. Animal Immunizations and Tissue Preparation ..............................................................................38 4.2.2. CyTOF Staining and Analysis ........................................................................................................38 4.2.3. Establishing a Gating Strategy for 2.5% 115In-AlO(OH) ................................................................39 4.2.4. Statistical Analysis .........................................................................................................................39 4.2.5. Ethics Statement .............................................................................................................................39 4.3. Results ....................................................................................................................................................40 vi  4.3.1. Gating Strategy for 2.5% 115In-AlO(OH) Particles .........................................................................40 4.3.2. Cells Treated with Free 115In or 2.5% 115In-AlO(OH) can be Distinguished ..................................40 4.3.3. Levels of 115In within the Inguinal Lymph Node of Naïve Mice....................................................41 4.3.4. Kinetics of 115In-AlO(OH) within the Draining Lymph Node .......................................................41 4.3.5. Cellular Distribution of 115In-AlO(OH) within the Draining Lymph Node ....................................42 4.4 Discussion.....................................................................................................................................................46 Chapter 5: STEM-Based Assessment of Alhydrogel’s Biodistribution .................................................................48 5.1. Introduction ............................................................................................................................................48 5.2. Materials and Methods ...........................................................................................................................49 5.2.1. Animal Immunizations and Tissue Fixation ...................................................................................49 5.2.2. Tissue Embedding and Preparation for STEM Imaging .................................................................49 5.2.3. STEM Imaging and Data Analysis .................................................................................................49 5.2.4. Ethics Statement .............................................................................................................................49 5.3. Results ....................................................................................................................................................50 5.3.1. Persistence, Morphology and Cellular Infiltration of Alhydrogel and 2.5% 115In-AlO(OH) at the Site of Injection ..............................................................................................................................................50 5.3.2. Alhydrogel Particles within the Draining Lymph Node 70 Days Post-Injection ...........................52 5.3.3. Alhydrogel Particles within the Liver After 70 Days Post-Injection ..............................................52 5.4. Discussion ...............................................................................................................................................54 Chapter 6: Conclusion and Future Work ................................................................................................................55 6.1. Conclusion ..............................................................................................................................................55 6.2. Future Work ............................................................................................................................................56 References ..............................................................................................................................................................58 Appendix 1: Development of a MR-Traceable Form of AlO(OH) ........................................................................68           vii   List of Tables  Chapter 2: Table 2-1: Secondary antibodies used to detect anti-OVA antibodies…..………………………………………13 Table 2-2: Panel for OVA-tetramer staining…………………………………………………………………….13  Chapter 3: Table 3-1: SPECT scan times and durations………………………………………………………………....…...29  Chapter 4:  Table 4-1: Panel for CyTOF staining………………………………………………………………………..…...38 Table 4-2: Definition of cell populations for the CyTOF analysis………………..………………………...……42  Appendix: Table A-1: Relaxivity values for Gd-AlO(OH) in a 7T MRI…………………………………………………….58                viii   List of Figures  Chapter 1: Figure 1-1: Adsorptive mechanisms of Alhydrogel………………………………………………………………2  Chapter 2: Figure 2-1: Overview of the doping strategy for Alhydrogel using indium………………………………………8 Figure 2-2: Encapsulation efficiency of 111In-AlO(OH) ……………...…………………………………….……14 Figure 2-3: Stability of 111In-AlO(OH) over time in different diluents………...………………………...………15 Figure 2-4: PXRD analysis of Alhydrogel……………………………………………………………………..…15 Figure 2-5: DSC and FTIR analysis of Alhydrogel………………………………………………………………16 Figure 2-6: PXRD analysis of 2.5-10% 115In-AlO(OH) ……………………………………………………….…16 Figure 2-7: DSC and FTIR analysis of 2.5% 115In-AlO(OH) ……………………………………………………17 Figure 2-8: Hydrodynamic size and zeta potential of 111/115In-AlO(OH) versus Alhydrogel……...…………..…17 Figure 2-9: Alhydrogel and 2.5% 115In-AlO(OH) before and after sonication for STEM imaging………………18 Figure 2-10: STEM images of Alhydrogel and 2.5% 115In-AlO(OH) …………………………………...………19 Figure 2-11: Elemental mapping of Alhydrogel………………...…………………………………………..……20 Figure 2-12: Elemental mapping of 2.5% 115In-AlO(OH) …………………………………………………..……21 Figure 2-13: Protein adsorptivity and de-adsorptivity to Alhydrogel and 111/115In-AlO(OH) ………………...….21 Figure 2-14: YOYO-1 and MTT cell toxicity assays of Alhydrogel versus 2.5% 115In-AlO(OH) ………………22 Figure 2-15: Humoral immune response to OVA adsorbed to Alhydrogel versus 111/115In-AlO(OH) ………..…23 Figure 2-16: Gating strategy for OVA-tetramer positive CD8+ T-cells…………..……………………………...24 Figure 2-17: Cell-mediated immune response to OVA adsorbed to Alhydrogel versus 111/115In-AlO(OH) …….24  Chapter 3: Figure 3-1: Gamma spectra of 67Ga, 111In and mixed isotopes…………………………………………………..27  Figure 3-2: Reconstruction and triple energy windows for 67Ga/111In dual-isotope SPECT scans………………29 Figure 3-3: Labeling efficiency and radiopurity of 67Ga-NOTA-OVA…………………………………………..31 Figure 3-4: SDS and native PAGE analysis of 67Ga-NOTA-OVA……………………………………………….31 Figure 3-5: Separation of 111In-AlO(OH) and 67Ga-NOTA-OVA signals within a mouse……………………….32 ix  Figure 3-6: VOI placement and draining of 67Ga-NOTA-OVA from the sites of injection over time…………...33 Figure 3-7: VOI analysis of 111In-AlO(OH) from the site of injection over time………………………………...33 Figure 3-8: Sacrifice-based biodistribution results for 111In-AlO(OH) …………………………………….….…34  Chapter 4:  Figure 4-1: Gating strategy for cell unassociated 2.5% 115In-AlO(OH) nanoparticles..………………………….40 Figure 4-2: Differentiation of cells stained with 115In-AlO(OH) and 115In-citrate...…………………………...…41 Figure 4-3: Kinetics of total 115In-positive events and cell unassociated 115In-AlO(OH) nanoparticles within the draining lymph node………………………………….………………………………………………..…………42 Figure 4-4: Gating strategy to assess the phenotype of 115In-positive cellular events in the draining lymph node……………………………………………………………………………………………………….………43 Figure 4-5: Changes in the cell numbers with the draining lymph node over time in mice treated with 111In-AlO(OH)………………………………………………………………………………………………….……....44 Figure 4-6: Changes in the proportion of the cell population that are 115In-positive in the draining lymph node over time……………………………………………………………………………………………………….…44 Figure 4-7: Expression of maturation markers in 115In-positive dendritic cells in the lymph node over time ......45  Chapter 5:  Figure 5-1: Injection site nodule 70 days post-administration…………………………………………………..50 Figure 5-2: Morphology of Alhydrogel at the site of injection 70 days post-administration……………………50 Figure 5-3: Morphology and elemental mapping of 2.5% 115In-AlO(OH) at the site of injection 70days post-administration………………………………………………………………………………………………….…51 Figure 5-4: Cellular infiltrate within the Alhydrogel injection site at 70 days post-administration …..…………51 Figure 5-5: Alhydrogel particle within the draining lymph node 70 days post-administration………………….52 Figure 5-6: Unequivocal proof of Alhydrogel nanoparticles within the liver 70 days post-administration……...53  Appendix: Figure A-1: FTIR and PXRD of 0.5-10% Gd-AlO(OH) ………………………………………………………...57 Figure A-2: Phantom scan of Alhydrogel, Gadovist and 0.5-2.5% Gd-AlO(OH) in a 7T MRI……….…………58 Figure A-3: Phantom scan of a 1/10 serial dilution of 1% Gd-AlO(OH) in a 7T MRI…………………………..59    x   List of Abbreviations  7AAD  7-amino-actinomycin ACK  Ammonium-Chloride-Potassium AF555  AlexaFluor-555 ANOVA Analysis of Variance  APC  Antigen Presenter Cell APS  Ammonium Persulfate AS04  Adjuvant System 4 (GlaxoSmithKline) ASIA  Autoimmune/Inflammatory Syndrome Induced by Adjuvants AUC  Area Under the Curve BSA  Bovine Serum Albumin CCL  Chemokine (C-C motif) Ligand CD  Cluster of Differentiation  cpm  Counts Per Minute CT  X-Ray Computed Tomography  CTL  Cytotoxic T-Lymphocyte CXCL  Chemokine (C-X-C motif) Ligand CyTOF  Mass Cytometry by Time of Flight CyFACS CyTOF Staining Buffer DAMPs Damage Associated Molecular Patterns DC  Dendritic Cell DLS  Dynamic Light Scattering DSC  Dynamic Scanning Calorimetry  DNA  Deoxyribose Nucleic Acid DNAse1 Deoxyribonuclease 1 DMEM  Dulbecco’s Modified Eagle Medium DMSO  Dimethyl Sulfoxide ELISA  Enzyme-Linked Immunosorbent Assay EDTA  Ethylenediaminetetraacetic Acid xi  EDX  Energy-Dispersive X-Ray Spectroscopy  FACS  Fluorescence-Activated Cell Sorting FBS  Fetal Bovine Serum  Fc  Fragment Crystallizable FDA  Food and Drug Administration FTIR  Fourier Transform Infrared Spectroscopy  HAADF High-Angle Annular Dark-Field HBeAg  Hepatitis B e Antigen HRP  Horseradish Peroxidase ICP-MS Inductively Couple Plasma Mass Spectrometry ID  Injected Dose IL  Interleukin Ig  Immunoglobulin ITLC  Instant thin layer chromatography JCPDS  Joint Committee on Powder Diffraction Standards kDa  Kilodalton MHC  Major Histocompatibility Protein MMF  Macrophagic Myofasciitis MTT  3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium MR  Magnetic Resonance  MWCO Molecular Weight Cut-Off  NK  Natural Killer Cell NLRP3  Nucleotide-Binding Domain, Leucine-Rich-Containing Family, Pyrin Domain-Containing-3 NOTA  1,4,7-triazacyclononane-1,4,7-trisacetic acid  OVA  Ovalbumin  PAGE  Polyacrylamide Gel Electrophoresis PBS  Phosphate Buffered Saline PBS-T  Phosphate Buffered Saline with Tween-20 PE  Phycoerythrin PET  Positron Emission Tomography PI3  Phosphoinositide 3 Kinase PXRD  Powder X-Ray Diffraction xii  SDS  Sodium Dodecyl Sulfate SPECT  Single-Photon Emission Computed Tomography  STEM  Scanning Transmission Electron Microscopy Syk  Spleen Tyrosine kinase TEM  Transmission Electron Microscopy TEMED Tetramethylethylenediamine TLC  Thin-Layer Chromatography TLR  Toll-like Receptor Th  CD4+ T-helper Cell TMB  3,3’,5,5’-Tetramethylbenzidine VOI  Volume of Interest                     xiii   Acknowledgements  I would like to thank Dr. Urs O. Häfeli, my supervisor and scientific mentor. Dr. Häfeli is the one who sparked my interest in the fields of nanomedicine and drug delivery science. I would also like to thank Drs. Katayoun Saatchi and Cristina Rodriguez for teaching me how to work with radioisotopes and perform SPECT/CT studies, respectively. I’m grateful to Rahel Schneider for her assistance in the lab at the early stages of the project. I would like to thank Dr. Jan Dutz as well as past and previous members of his lab (Dr. Jacqueline Lai and Patrick Hopkins) for their assistance and guidance on the many immunological aspects of this project.  A special thanks are owed to my mother (Sheila Esposito), sister (Demitria Esposito) and uncle (Paul Esposito) for all their support during my studies. This thesis is dedicated to my father (Tullio F. Esposito), who passed away in November 2013.                    1  Chapter 1: Introduction 1.1. History and Clinical Use of Aluminum-Based Adjuvants Adjuvants have been used in vaccines for almost a century. In 1925, Gaston Ramon administered diphtheria toxoid to horses in combination with a variety of substances, such as plant extracts, starch and fish oil (1-3). He observed that their addition increased the antibody response to the toxoid significantly (1-3). Ramon coined the term adjuvant from the latin adjuvare, ‘to help’(1-3). A year later, in 1926, Alexander T. Glenny and colleagues dissolved diphtheria toxoid in a solution of alum (KAl(SO4)2∙12H2O) that when neutralized entrapped the antigen within an aluminum-precipitate (3, 4). This precipitate resulted in a significantly enhanced immune response compared to the soluble toxoid in guinea pigs (3, 4). Minor variations however in the anions present at the time of precipitation, largely from the buffers and growth media used to prepare the antigen component, lead to significant heterogeneity amongst aluminum-precipitated vaccines (5, 6). This problem was solved by Maschmann et al. in 1931 by adsorbing antigens onto the surface of preformed, standardized aluminum precipitates (7). Such preparations are known as aluminum-adsorbed vaccines (7).  Preformed aluminum precipitates have become the most common type of adjuvant in non-living vaccines (3, 8, 9). They are found, for example, in inoculations against tetanus, diphtheria, hepatitis A and B, pertussis, anthrax and the human papilloma virus (3, 8, 9). Since the 1930s, it has been estimated that hundreds of millions of people have been administered nearly a billion doses of aluminum-adsorbed vaccines (8, 9). Preformed aluminum precipitates were the only adjuvants approved for clinical use by the American FDA until 2009 with the approval of AS04 (10, 11).   1.2.  Structure and Adsorptive Mechanisms of Aluminum-Based Adjuvants: Currently, there are four different types of preformed aluminum precipitates used in vaccines: amorphous aluminum hydroxyphosphate, amorphous aluminum hydroxysulfate, amorphous aluminum hydroxyphosphate sulfate and poorly crystalline aluminum oxyhydroxide (12). The focus of this thesis will be aluminum oxyhydroxide, AlO(OH).  Alhydrogel, manufactured by Brenntag Biosector A/S, was designated in the late 1980s as the international standard of AlO(OH) for use in vaccines (13). Alhydrogel is a viscous white suspension of 1-12 µm clusters of weakly aggregated AlO(OH) nanoparticles in water (12). The component nanoparticles have average dimensions of 4.5 x 2.2 x 10 nm and provide a surface area of approximately 500 m2/g for the adsorption of antigens (12, 14).  2  Antigens are adsorbed to AlO(OH) particles via hydrophobic interactions, hydrogen bonding, van der Waals forces, electrostatic attraction or ligand exchange; the latter two are the most predominant mechanisms (12). Negatively charged antigens are electrostatically attracted to AlO(OH) given that the adjuvant has an isoelectric point of 11.4 and is positively charged at physiological pH (Figure 1-1A) (12). Ligand exchange is a process by which the hydroxyls coordinated to aluminum atoms on the surface of AlO(OH) are substituted by phosphate groups on the antigen (Figure 1-1B) (12). Ligand exchange is the strongest of the adsorptive mechanisms, as it can even overcome electrostatic repulsion to adsorb positively charged antigens to Alhydrogel (12).  Figure 1- 1: Electrostatic (A) and ligand exchange (B) mechanisms by which Alhydrogel adsorbs antigens to its surface.  1.3.  Immunostimulatory Mechanisms of Aluminum-Based Adjuvants: Preformed aluminum precipitates promote antibody-mediated immunity against adsorbed antigens, but are poor at stimulating cell-mediated responses. The mechanisms by which this adjuvant class works are complex and the details have only recently begun to be elucidated. The following theories exist in the literature: • Recruitment of innate immune cells to the site of injection: preformed aluminum precipitates induce the secretion of pro-inflammatory mediators, such as IL-1β, IL-5, CCL2, CCL4, CCL11, CCL24, CXCL1 and CXCL2 upon administration (15, 16). These factors recruit neutrophils, eosinophils, macrophages, inflammatory monocytes and dendritic cells to the site of injection (15, 16). The degree of inflammation is significantly higher when antigens are adsorbed to aluminum precipitates compared to the soluble state (15). The recruitment of the innate immune cells helps to support the sequence of events required to develop antigen-specific adaptive responses, like antibodies.  • Increased antigen internalization and duration of presentation: antigen presenter cells (APCs), such as dendritic cells, more efficiently internalize antigen in particulate form (17-19). Ghimire et al. found that antigen accumulation in cultured dendritic cells was 100-fold higher when bound to Alhydrogel compared to the soluble protein (17). The magnitude and duration of antigen presentation by dendritic cells in vitro was also enhanced when the antigen was adsorbed (17).  3  • Formation of an antigen depot at the site of injection: the rate of antigen draining from the site of injection is slowed when adsorbed to aluminum precipitates to allow enough time for APCs to arrive, internalize and process the antigen. Glenny was the first to observe the formation of an antigen depot at the site of injection with aluminum-based adjuvants (20). Harrison et al. further demonstrated antigen persistence by using inflammatory nodules that form at the site of injection to immunize naïve animals (21-23).  More recently, Noe et al. used a radiolabeled protein, 111In-α-casein, to confirm that adsorbed antigens are retained at the injection site longer than free antigen (24). Excision of the injection site 4 days post-administraiton was found to interfer with the development of humoral immunity, demonstrating the importance of the antigen depot (6). However, a similarly designed study found that removal of the depot only 2 hours post-injection had no immunological consequences (25). There is still considerable controvery in the field whether or not an antigen depot at the site of injection is required to potentiate an immune response with this adjuvant class.  • Release of endogenous DAMPs: aluminum-based adjuvants induce local necrosis at the site of injection, which leads to the release of endogenous damage associated molecular patterns (DAMPs) (15, 26, 27). These molecules, such as uric acid and genomic DNA, bind to pattern recognition receptors (PRRs) that activate APCs to potentiate adaptive immunity (15, 26, 27).  Mice pre-treated at the site of injection with DNAse1, an enzyme that degrades genomic DNA, had a reduced humoral response to an aluminum-adsorbed vaccine (26).  • Activation of the NLRP3 inflammasome: following internalization by APCs, aluminum precipitates destabilize the phagolysosome leading to the assembly and activation of the NLRP3 inflammasome (28). Activation of this inflammasome results in caspase-1 processing IL-1β and IL-18 into their biologically mature forms (28). As mentioned above, IL-1β helps to recruit cells of the innate immune system to the site of injection (15, 16, 29). IL-1β and IL-18 together have been shown to polarize naïve CD4+ T-helper cells toward a Th2 phenotype, which in turn promotes humoral immunity (IgG1 and IgE isotypes in mice) (28, 30). There is significant controversy surrounding this theory since NRLP3 knockout mice administered an aluminum-adsorbed vaccine have shown a marked suppression of antigen-specific IgE and IgG1 production (31, 32), suppression of IgE but not IgG1 (29) or no effect on antibody production at all (33, 34). • Promotion of PI3-Syk kinase signalling: the interaction of extracellular aluminum precipitates with lipid rafts in the cell membrane of APCs can activate the PI3 and Syk kinases (35, 36). Activation of the PI3 kinase inhibits the production of IL-12p70, a key cytokine in the polarization of naïve CD4+ cells toward a cellular immunity promoting Th1 phenotype (35, 37). Downstream effects of Syk kinase activation include the release of prostaglandin E2 by APCs, a molecule that promotes Th2 polarization (36). 4   The above theories all assume that Alhydrogel nanoparticles remain and slowly dissolve from the site of injection. Prominent figures within the field have proposed that the distribution of AlO(OH) nanoparticles to distant sites may help to explain controversial aspects of the adjuvant’s mechanism (38). For example, the movement of antigen-coated nanoparticles to the draining lymph nodes may explain why the injection site can be removed after 2 hours without immunological consequences (25, 38). The distribution of AlO(OH) nanoparticles is not inconceivable, given that fact that APCs are known to internalize Alhydrogel in vitro and that fluorescently-tagged antigens adsorbed to aluminum precipitates can be found within inflammatory dendritic cells migrating from the site of injection (15, 39).   1.4.  Safety of Aluminum-Based Adjuvants.  At the population level, Alhydrogel has an excellent safety profile and is associated with only mild local reactions, such as transient erythema (40, 41). The general safety of aluminum precipitates helps to explain the widespread and continued use of this adjuvant class (40, 41).  However, a small subset of patients treated with Alhydrogel develop a range of serious local and systemic adverse events that are collectively described as autoimmune/inflammatory syndrome induced by adjuvants (ASIA) (42). One condition within this syndrome, macrophagic myofasciitis (MMF), is characterized by prolonged inflammation and pain at the site of injection along with arthralgia, muscle weakness, chronic fatigue and cognitive dysfunction (42, 43).  MMF is strongly associated with the persistence of Alhydrogel nanoparticles at the site of injection; electron micrographs of injection site biopsies from this population have found adjuvant particles sequestered within macrophages over 12-years from the time of administration (43). The persistence of Alhydrogel can be explained by the poor solubility of the adjuvant in biological fluids. For example, Seeber et al. found that 55% of amorphous aluminum hydroxyphosphate dissolved over 12 hours in a simulated interstitial fluid whereas no AlO(OH) dissolved over the same timeframe (44). Under supra-physiological conditions to promote dissolution (100 times the concentration of citric acid found in vivo), only ~8% of the AlO(OH) dissolved over a 5-day period compared to 100% for the amorphous aluminum adjuvant (44). These findings help to explain why the only aluminum-based adjuvant associated with MMF, and ASIA in general, is poorly crystalline Alhydrogel (42, 43).  The systemic adverse effects of ASIA are hypothesized to be due in part to the distribution of Alhydrogel nanoparticles from the injection site, which in turn facilitates an unwanted inflammatory response and damage in the distant tissue (42, 45, 46). Although the biodistribution of aluminum-based adjuvants is sparsely studied, there is emerging evidence to support this hypothesis.   5  1.5. Current State of Knowledge for the Biodistribution of Aluminum-Based Adjuvants.  As mentioned above, Alhydrogel consists of 1-12 µm clusters of aggregated nanoparticles (12, 14). The forces holding the AlO(OH) nanoparticles together are rather weak, as exemplified by the fact that the clusters de-aggregate and re-aggregate during the process of antigen adsorption (47). De-aggregation of the adjuvant upon injection and exposure to interstitial fluid may release particles that can drain from the site of injection (47, 48). Particles <200 nm, such as the component nanoparticles of Alhydrogel, efficiently enter the afferent lymphatics and drain to the lymph nodes within hours of injection (25, 48). When coated with antigen, these small particles are particularly effective at stimulating humoral immune responses since the unprocessed antigen can be recognized and efficiently phagocytosed by antigen-specific B cells in the lymph nodes (48). Particles >500 nm, like Alhydrogel nanoparticle aggregates, largely remain at the site of injection and tend to also potentiate humoral immunity (19, 49). On the other hand, particles between 200-500 nm are efficiently phagocytosed, processed and transported over a period of days to the draining lymph node by dendritic cells; adjuvants within this size range tend to stimulate cell-mediated immune responses (48). Flarend et al. used AlO(OH) containing a small amount of the positron emitter 26Al to elucidate details about the distribution and elimination of Alhydrogel following intramuscular administration into 2 rabbits (50). The distribution of 26Al was highest in the kidney followed by the spleen, liver, heart, non-draining lymph nodes and brain at 28-days post injection (50). From the blood AUC profile, it was estimated that 17% of the injected dose had been absorbed from the site of injection and 6% of the adjuvant had been excreted in the urine after 4 weeks (50). Although AlO(OH) is poorly soluble in vivo, the authors observed a large spike of 26Al in the blood within the first 48 hours followed by a low, steady level of the radiotracer over the next 26 days (50). Experts in the field simply assumed that AlO(OH) somehow dissolved more rapidly in vivo than observed in vitro by Seeber et al. (44, 50, 51). Although not assessed by Flarend et al., the distribution of 26Al-AlO(OH) nanoparticles is an alternative explanation for the above pharmacokinetic results. Only a handful of studies have directly assessed the biodistribution of the nanoparticles within aluminum-based adjuvants. In 1955, White et al. observed granular masses in the sinuses and macrophages of the draining lymph node 24 hours post-administration of an aluminum-precipitated vaccine in guinea pigs and rabbits (52). The authors assumed the granular substance was the adjuvant since it could not be found in the lymph node of control animals (52). Nearly 60 years later, researchers used both fluorescent latex beads and gadolinium oxide cores coated with aluminum oxide and rhodamine as surrogates of Alhydrogel to explore the adjuvant’s biodistribution (45). The authors found that the surrogate materials translocated over time to distant organs, such as the spleen and brain, in a CCL2-dependent manner within predominantly macrophages (45). Ablation of the draining lymph nodes was found to significantly reduced the migration of the surrogate nanomaterials (45). Next, Eidi et al. functionalized the surface of Alhydrogel with hyperbranched polyglycerol (HPG) dendrimers conjugated to nanodiamonds as a way to trace the actual adjuvant in vivo (53). Like with the Alhydrogel analogues, fluorescent signals from the nanodiamonds could be found within sites like the draining 6  lymph node and spleen in a time-dependent manner (53). In 2015, Giusti et al. reported structures that resemble the component nanoparticles of an aluminum-based adjuvant in the draining lymph node 16-18 hours post-injection using electron microscopy (54).  There are indications in the literature that aluminum precipitates distribute from the site of injection and rather quickly appear within the blood and draining lymph node. However, the topic has been sparsely studied and remains rather controversial despite the widespread use of this adjuvant class. Novel tracers and techniques are required to better understand the behavior of Alhydrogel nanoparticles in vivo.  1.6. Hypothesis and Research Objectives  The central hypothesis of this thesis is that the component nanoparticles of Alhydrogel are released from the site of injection and distribute to other regions of the body. The overall objective of this work is to improve the understanding of the biodistribution of aluminum-based adjuvants, particularly Alhydrogel. The specific research objectives are as follows: 1. To synthesize and characterize stable, traceable forms of Alhydrogel by doping the adjuvant’s AlO(OH) crystal structure with different isotopes of indium 2. To assess the biodistribution of 111In-AlO(OH) using nuclear imaging techniques 3. To assess the kinetics and cellular distribution of 115In-AlO(OH) particles within the draining lymph nodes using a novel application of mass cytometry  4. To assess the persistence of Alhydrogel at the site of injection along with the distribution of adjuvant particles to the draining lymph node and liver using electron microscopy.  Knowing the biodistribution and cellular distribution of Alhydrogel can help better understand how the adjuvant potentiate immune responses and its side effect profile. Fundamental knowledge about Alhydrogel is also important as this adjuvant will continue to be widely used given its safety profile, ease of preparation, stability, immunostimulatory effects and incorporation into next-generation mixed-adjuvant systems like AS04 (55, 56).      7  Chapter 2: Preparation and Characterization of Indium-Doped AlO(OH)   2.1. Introduction Assessing the biodistribution of Alhydrogel is challenging since the adjuvant lacks any optical, magnetic or other properties that make it detectable (12). Flarend et al. developed the first traceable form of Alhydrogel, which consisted of AlO(OH) doped with the positron emitter 26Al (50). Although this radiotracer shed some light on the adjuvant’s clearance, it could not differentiate between 26Al within an AlO(OH) nanoparticle or 26Al atoms released if the adjuvant dissolved (50).  The first attempt to assess the biodistribution of the component nanoparticles in Alhydrogel used surrogate materials, such as fluorescent 500 nm latex nanoparticles, together with confocal microscopy and flow cytometry (45). Making conclusions from surrogate materials is difficult however since their composition, size, zeta potential, geometry and so forth differs substantially from Alhydrogel (45). The next approach to assess the pharmacokinetics of AlO(OH) nanoparticles electrostatically adsorbed nanodiamonds coated with hyperbranched polyglycerols onto the surface of Alhydrogel; epifluorescent microscopy was used to detect the location of the nanodiamonds within tissue sections (53). The problem with this strategy is that substances adsorbed to AlO(OH) purely via electrostatic interactions rapidly dissociate from the adjuvant upon exposure to biological fluids (57). For example, Morefield et al. found that dephosphorylated ovalbumin, which can only be adsorbed electrostatically, completely elutes from Alhydrogel within 30 minutes in lymph (57). It is unclear if the nanodiamonds remained bound to Alhydrogel in vivo, especially over the 21 day timeframe of this particular study (57). The traceable forms of Alhydrogel used so far have been less than ideal. Reports have suggested that the biodistribution and cellular location of AlO(OH) nanoparticles can be assessed by staining tissues sections and cell isolates with morin or lumogallion, compounds that fluoresce in the presence of aluminum (39, 53, 58). Morin forms fluorescent complexes with various other cations, such as calcium and magnesium, resulting in a high false positive rate (53, 59). Although lumogallion is more specific for aluminum, its wide emission spectrum makes it difficult to combine with other fluorophores for immunophenotyping techniques like flow cytometry (53, 58). Using lumogallion pre-stained Alhydrogel in vivo is also problematic since the fluorophore decreases the zeta potential and increases the hydrodynamic size of the adjuvant (58).  Giusti et al. have recently used electron microscopy to directly observe preformed aluminum precipitates in the draining lymph node (54). X-ray spectroscopy coupled with electron microscopy can provide details about the composition and structure of the nanomaterials within tissue (60). However, this technique is low-throughput and presents a challenge when the phenotype of the adjuvant-positive cells has to be determined (60). Other techniques, such as ICP-MS, are required to determine the concentration of the aluminum-based adjuvant within a particular tissue/ organ system (60). However, dissolution of the samples in acid to conduct such an analysis results in the loss of critical information, such as whether the aluminum atoms were in a nanoparticle form (60).  8  Novel tracers and techniques are required to better study the biodistribution of Alhydrogel. The objective of this chapter was to synthesize a stable and traceable form of Alhydrogel by doping its AlO(OH) crystal structure with indium (Figure 2-1).   Figure 2- 1: Crystal structure of AlO(OH)/Alhydrogel (A) and the traceable indium-doped version of the adjuvant (B). Either 111In or 115In can be incorporated into the AlO(OH) matrix.  Two different isotopes of indium, gamma emitting 111In and non-radioactive 115In, were incorporated into the AlO(OH) matrix in order to trace of the adjuvant in vivo using novel techniques such as SPECT/CT (chapter 3) and mass cytometry (chapter 4), respectively. A number of physical, morphological and functional assessments were carried out in this chapter to first ensure that the indium-doped adjuvant closely matched Alhydrogel before proceeding with more tracking studies.    2.2. Materials and Methods 2.2.1. Synthesis of AlO(OH) A protocol was derived from papers by Xu et al. and Yau et al. to synthesis AlO(OH) nanoparticle aggregates that closely resemble commercial Alhydrogel (61, 62). Briefly, 50 µL of 0.1 N HCl (Sigma 258148) was added to 1 mL of 0.32 M AlCl3∙6H2O (Alfa Aesar 10622) in 18 MΩ water (pH 1.5). After heating the solution to 50˚C in an oil bath, 175 µL of NH4OH (Fisher A669-212) was added rapidly to precipitate the metal salts (pH 10). After 2 hours of curing (50˚C, 200 rpm), the AlO(OH) was washed thrice with 2 mL of 18 MΩ water; centrifugation at 1800 g was used to pellet the adjuvant between washes and light vortexing was used to re-suspend the aggregates. The AlO(OH) was re-suspended to a final volume of 1.2 mL with 18 MΩ water and autoclaved using liquid settings (121˚C, 15 psi, 30 minute hold, slow vent). The sterile adjuvant was stored at room temperature until used.  9  2.2.2. Synthesis of 115In-Doped AlO(OH) 115InCl3∙4H2O (Strem Chemicals 93-4931) in 50µL 0.1N HCl, was added to 1 mL of 0.32 M AlCl3∙6H2O in 18 MΩ water (pH 1.5); up to 10% of the aluminum atoms were substituted for indium. The mixture of metal salts was co-precipitated with base and processed as described in section 2.2.1. 2.2.3. Synthesis of 111In-Doped AlO(OH) Up to 340 MBq of carrier free 111InCl3 in 0.05 N HCl (Nordion IPG-In-111) was diluted to a volume of 50 µL with 0.1 N HCl and added to 1mL of 0.32 M AlCl3∙6H2O in 18 MΩ water (pH 1.5). The mixture of metal salts was co-precipitated with base and processed as described in section 2.2.1.  2.2.4. Encapsulation Efficiency of 111In within the AlO(OH) Matrix Before washing the adjuvant thrice with 18 MΩ water, 1 μL was spotted onto an instant thin layer chromatography (ITLC) strip (BioDex 150-771) and developed with 0.1 M EDTA at pH 4 (Sigma E4884). In this system, 111In-AlO(OH) remains at Rf=0 while unincorporated 111In forms a complex with EDTA and migrates to Rf=1 (63). The location of the radioactivity on the ITLCs was visualized using a Cyclone Phosphor Imager with a photostimulable phosphor screen (Perkin Elmer) and analyzed with OptiQuant software (Perkin Elmer). Encapsulation efficiency was also assessed by measuring the amount of 111In in the supernatant as the adjuvant was washed using a CRC-55tR dose calibrator (Capintec). Encapsulation efficiency was calculated using the following equation: 𝐸𝐸𝐸𝐸𝐸𝐸𝐸𝐸𝐸𝐸𝐸𝐸𝐸𝐸𝐸𝐸𝐸𝐸𝐸𝐸𝐸𝐸𝐸𝐸𝐸𝐸 𝐸𝐸𝐸𝐸𝐸𝐸𝐸𝐸𝐸𝐸𝐸𝐸𝐸𝐸𝐸𝐸𝐸𝐸𝐸𝐸 (%) = �𝑇𝑇𝐸𝐸𝐸𝐸𝐸𝐸𝐸𝐸 𝐼𝐼𝐸𝐸 − 𝑈𝑈𝐸𝐸𝐸𝐸𝐸𝐸𝐸𝐸𝐸𝐸𝑈𝑈𝐸𝐸𝐸𝐸𝑈𝑈𝐸𝐸𝐸𝐸𝐸𝐸𝑈𝑈 𝐼𝐼𝐸𝐸 111  111𝑇𝑇𝐸𝐸𝐸𝐸𝐸𝐸𝐸𝐸 𝐼𝐼𝐸𝐸111 � 𝑥𝑥 100 2.2.5. Concentration and Specific Activity The concentration of 111/115In-AlO(OH) was determined by lyophilizing a known volume overnight in a FreeZone 2.5 Freeze Dry System (Labconco) and measuring the weight of the solid fraction. The specific activity of the 111In-AlO(OH) was determined by measuring the amount of radioactivity in a known weight of lyophilized adjuvant with a dose calibrator. Concentration was reported as %w/v while specific activity was reported as μCi/μg.  2.2.6. Powder X-Ray Diffraction (PXRD) After centrifuging the adjuvant at 10,000 g for 5 minutes and aspirating the supernatant, the pellet was dried at 80˚C for 2.5 hours. Each sample was ground into a fine powder and loaded into a D8-Advance Diffractometer (Bruker) in Bragg-Brentano configuration; a copper source was used with a nickel filter leaving CuKα radiation (λ = 1.54 Å). The generator was set at 40 kV and 40 mA and the slit sizes were 1.0 mm (divergence), 8.0 mm (anti-scatter) and 2.5° (soller). PXRD data was collected from 5˚ to 90˚ 2θ and analyzed with TOPAS software (Bruker).  10  2.2.7. Dynamic Scanning Calorimetry (DSC) Samples were dried as described for PXRD, loaded into aluminum pans (DSC Consumables 84006) and placed into a DSC Q100 (TA Instruments). DSC measurements were taken from 40-500˚C at a ramp rate of 20˚C/ minute. The data was analyzed using Universal Analysis software (TA Instruments) and reported as heat flow (mW/g) versus temperature (˚C). 2.2.8. Fourier Transform Infrared Spectroscopy (FTIR) Samples for FTIR analysis were dried as described for PXRD and loaded into a Frontier spectrometer outfitted with a UATR sampling system (Perkin Elmer). Data was recorded from 550 to 4000 cm-1 and analyzed with Spectrum software (Perkin Elmer).  2.2.9.  Dynamic Light Scattering (DLS) Particle size and zeta potential analysis were conducted using a Zetasizer Nano ZS (Malvern) and folded capillary zeta cell (Malvern DTS1070). 111/115In-AlO(OH) or commercial Alhydrogel were diluted 10-fold in 18 MΩ water (viscosity: 0.8872 cP, refractive index: 1.330, dielectric constant: 78.5) and allowed to equilibrate to 25˚C before being analyzed. The refractive index for the adjuvant was set at 1.650 with no absorptivity at 𝝀𝝀=632 nm (k=0.00). The angle of detection was 173˚ backscatter with automatic attenuation. Micro-electrophoretic measurements used a Smoluchowski model (F(ka)=1.5). The average (number %) of 10 measurements of 30 seconds was reported for the hydrodynamic size of each sample, unless otherwise specified. The average of at least 15 measurements was reported for the zeta potential of each sample.  2.2.10. Scanning Transmission Electron Microscopy (STEM) To better visualize the morphology of the nanoparticles within the adjuvant’s aggregates, samples were first diluted 100-fold with 18 MΩ water and sonicated at 60 W for 5 minutes on ice with a Ultrasonic 3000 equipped with a stepped micro-tip (BioLogics) (14). Between 0.5-1 µL of sample was placed onto formvar and carbon backed 200 mesh copper grids (Ted Pella 01810) and allowed to dry for at least 24 hours in a laminar hood. The samples were examined in a Technai Osiris STEM with a Schottky field emission electron source operating at 200 kV (FEI). An Ultrascan 1000XP-P CCD camera (Gatan) along with high-angle annular dark field (HAADF) and bright field STEM detectors (FEI) were used to perform a high-resolution assessment of the adjuvant’s morphology on the Osiris platform. This microscope was also equipped with a Super-X windowless energy-dispersive X-ray (EDX) detection spectrometer (FEI) for elemental mapping. Esprit 1.9 software (Bruker) was used to correct for Bremsstrahlung radiation and analyze the EDX data.  2.2.11. Stability of 111In-AlO(OH) in Different Media over Time Twenty microliters of autoclaved 111In-AlO(OH) was incubated under sterile conditions with 480 μL of saline, 0.1 M EDTA at pH 7 or fetal bovine serum (Life Technologies 12483020) at 37˚C with end-over-end 11  mixing for 10 days. At set times during the incubation (0, 2 and 10 days), the adjuvant was pelleted at 10,000 g for 5 minutes and 200 μL of the supernatant was removed to measure the amount of free 111In using a Wizard2 2470 gamma counter (Perkin Elmer). Fresh diluent was added to replace the removed 200 μL before continuing the incubation. Stability was reported as the cumulative amount of 111In released over time.  2.2.12. Protein Adsorptivity Alhydrogel or 111/115In-AlO(OH) were mixed with an equal volume of 0-20 mg/mL ovalbumin (OVA; Sigma A5503) in normal saline and allowed to adsorb for 30 minutes at room temperature (57). The adjuvant was pelleted at 10,000 g for 5 minutes and 10 µL of the supernatant was removed to detect unbound OVA using a fluorescamine assay. Briefly, the supernatant was diluted in 80 μL of 18 MΩ water before the addition of 10 μL of 3 mg/mL fluorescamine (Sigma F9015) in acetone (57, 64). Data was acquired using a Synergy MX plate reader (BioTek) measuring fluorescence at 380/464 nm (64).   2.2.13. Protein De-Adsorption in Biological Fluids The model antigen OVA was dissolved in 0.1 M sodium bicarbonate buffer pH 8.5 (Fisher S233-10) and fluorescently tagged by the addition of 10 molar-excess of AlexaFluor-555 succinimidyl ester (AF555; Life Technologies A20009) (65). After 2 hours at room temperature, unreacted dye was removed and the protein was buffer exchanged into saline using a 30 kDa MWCO micro-concentrator (Merck UFC503096). Alhydrogel or 111/115In-AlO(OH) was mixed with an equal volume of 2 mg/mL AF555-OVA and allowed to adsorb for 30 minutes at room temperature (57). Twenty microliters of the protein adsorbed adjuvant was incubated with 230 μL of saline or FBS for 30 minutes. The adjuvant was pelleted down at 10,000 g for 5 minutes and 100 μL of supernatant was removed to measure fluorescence at 555/572 nm using a Synergy MX plate reader (BioTek) (57, 65). The quantity of protein in the supernatant was determined using a standard curves of AF555-OVA in saline or FBS.   2.2.14. Cellular Toxicity Assays HEK293 is a cell line commonly used to assess cellular toxicity (66). HEK293 cells were cultured in high-glucose DMEM supplemented with L-glutamine and sodium pyruvate (Life Technologies 11995065) along with 10% FBS and 1% penicillin/ streptomycin (Life Technologies 15140122) (67). The cells were maintained in an incubator at 37˚C with 5% CO2 and split using a solution of 0.25% trypsin/EDTA (Life Technologies 25200056) when they reached ~70% confluence (67). To perform the toxicity assays, HEK293 cells were seeded at 5000 cells/well in 96-well plates (Corning 353072) and allowed to adhere overnight before the addition of 0-500 μg/mL adjuvant. Two different assays were used to assess toxicity: 12  1. MTT Assay for Cellular Respiration: two days post-addition of the adjuvant, 20 μL of 5 mg/mL thiazolyl blue tetrazolium bromide (MTT reagent; Sigma M5655) in 1x PBS was added to each well and placed back into the cell incubator for 2 hours (68, 69). The MTT reagent is converted into insoluble formazan crystals within the mitochondria of living cells (68, 69). After aspirating the supernatant, the formazan and adjuvant particles were dissolved with the addition of 150 μL of DMSO (Fisher D128) (68, 69).  Data was acquired using a Synergy MX plate reader (BioTek) measuring absorbance at 540 nm (68, 69). A correction factor was applied to account for a red-shift in the absorption spectrum of formazan due to the complexation of the molecule with aluminum and indium ions (70). Data was reported as percent respiration of untreated HEK293 cells.   2. YOYO-1 Assay for Cell Death: each well was supplemented with 0.75 μM of YOYO-1 dye (Life Technologies Y3601) at the same time as the addition of the adjuvant. YOYO-1 is membrane impermeable DNA-binding dye that fluorescently labels the nucleus of dead cells with a compromised membrane (71, 72). The plate was serially scanned every 2 hours for 2 days in an IncuCyte  Zoom (Essen Bioscience) recording both green fluorescence and phase contrast data (72). Cell death was reported as green channel fluorescence confluence versus time (72).  2.2.15. Humoral Immune Response Assessment C57BL/6 mice (6-8 weeks old) were injected in the posterior half of the dorsal surface with 100 μg of OVA adsorbed to 0.7 mg of either 111/115In-AlO(OH) or Alhydrogel in a total volume of 100 μL. Two weeks post immunization, blood was collected via heart puncture and allowed to clot on ice.  After centrifuging the blood at 5000 g for 10 minutes, the serum was removed and stored at -20˚C until analyzed via ELISA for anti-OVA antibodies.  To perform the ELISA, Nunc-Immuno MaxiSorp 96-well plates (Fisher 439454) were first coated with antigen by incubating 100 μL of 20 μg/mL OVA overnight at 4˚C in 50 mM sodium bicarbonate buffer at pH 9.6 per well (73). After removing the coating solution, the plate was blocked with 150 μL of 2% bovine serum albumin (BSA; Sigma A7030) in 1xPBS for 1 hour at 37˚C. The plate was washed twice with 200 µL of 0.05% Tween-20 (Sigma P9416) in 1xPBS (PBS-T) before the addition of 100 μL of diluted serum in 0.1% BSA in 1xPBS per well; the serum was diluted 1/4000-1/32000 to assess total IgG and IgG1 and 1/400-1/3200 for IgG2c (73). After 2 hours at room temperature, the plate was washed twice with PBS-T and once with 1xPBS before adding 100 μL of the HRP-conjugated secondary antibodies listed in Table 2-1 (73). Target Dilution       Vendor Catalogue Number Total IgG (goat anti-mouse) 1/50,000   Jackson ImmunoResearch 115-035-003 IgG1 (rabbit anti-mouse) 1/32,000 Zymed 61-0120 IgG2c (goat anti-mouse) 1/10,000 Jackson ImmunoResearch 115-035-208 Table 2- 1: Secondary antibodies used to detect anti-OVA antibodies. 13  After 1 hour incubation with the secondary antibody, each well was washed twice with 200 µL of PBS-T and twice with 200 μL of 1xPBS (73). One hundred microliters of TMB substrate (eBioscience 00420156) was added to each well and developed for 1 hour at room temperature before stopping the reaction with 50 μL of 2 N H2SO4 (Fisher 351293) (73). Data was read using a Synergy MX plate reader (BioTek) measuring absorbance at 450 nm (73). Data was always compared to a naïve/non-immunized age-matched mouse.  2.2.16. Cellular Immune Response Assessment C57BL/6 mice were immunized as previously described in section 2.2.15. One week post-administration, peripheral blood was collected from the saphenous vein and treated with ACK buffer (0.15 M NH4Cl, 10 mM KHCO3, 0.1 mM EDTA at pH 7.2) to osmotically lyse the red blood cells (74). The cells were washed with FACS buffer (4% v/v FBS, 2 mM EDTA in 1xPBS) and incubated with 2.4G2 tissue culture supernatant for 15 minutes to block the Fc receptors (74). The cells were then stained with the markers listed in Table 2-2 for 45 minutes on ice (74): Antibody/ Probe Fluorochrome Clone Dilution Vendor Catalogue Number CD3 AF405 KT31.1            1/25 AbLab (UBC) 45-0034-05 Ovalbumin Tetramer  PE Residues 257-164 of ovalbumin on H2-Kb tetramer 1/50 Tan Laboratory (UBC CFRI)  CD8 PE-Cy7 53-6.7 1/3000 eBioscience 25-0081-81 Table 2- 2: Panel for OVA-tetramer staining. The cells were then incubated with 1 μg/mL of 7-amino-actinomycin (7AAD; Sigma A9400) for 5 minutes as the live/dead discriminator (74). The cells were washed twice with FACS buffer and analyzed using a CytoFlex 13-color flow cytometer (Beckman). A minimum of 100,000 events/sample was collected and the data was analyzed using FlowJo V10 software (TreeStar). Data was always compared to a naïve age-matched mouse.  2.2.17. Data Analysis Data are presented as mean ± standard deviation unless otherwise specified. GraphPad Prism 7 was used for statistical analysis using Student’s t-test or ANOVA followed by multiple comparisons testing (Dunnet’s test). P<0.05 was considered statistically significant and was indicated by an asterisks (*). Data was graphed using either GraphPad Prism 7 or OriginPro 9.   2.2.18. Ethics Statement All animal experiments were carried out in accordance with the guidelines of the University of British Columbia’s Animal Care Committee under the approved protocol A14-0159. 14  2.3. Results 2.3.1.  Encapsulation Efficiency, Concentration and Specific Activity of 111/115In-AlO(OH)  ITLC analysis (Figure 2-1) showed that the 111InCl3 did not precipitate when added to the AlCl3 solution, since the radionuclide migrated to Rf=1. After the addition of base, the vast majority of 111In remained at Rf=0 since it was entrapped within the AlO(OH) matrix. Integration of the ITLC data found the encapsulation efficiency of 111In to be 99.6 ± 0.1%.   Figure 2- 2: Encapsulation efficiency of 111In within AlO(OH). 111InCl3 in 0.05N HCl from Nordion (A) and when mixed with 0.32M AlCl3 before (B) and after (C) the addition of base to co-precipitate the metal salts.  Although the encapsulation efficiency was high, ~15% of the 111/115In-AlO(OH) nanoparticles adsorbed to the wall of the polypropylene vessel used to autoclave the adjuvant and could not be recovered. The final concentration of the 111/115In-AlO(OH) was determined to be 1.39 ± 0.04% w/v; each batch of adjuvant was diluted with a specific amount of 18 MΩ water to match the concentration of Alhydrogel at 1.3% w/v. The specific activity of the 111In-AlO(OH) was 12.58 MBq/mg, a sufficiently high value to preform the SPECT/CT study described in chapter 3.    2.3.2.  Stability of 111In-AlO(OH) in Different Media  111In-AlO(OH) was incubated in different diluents at 37˚C to assess its stability over time (Figure 2-2). Only 1.60 ± 0.04% of the tracer dissolved after 10 days in FBS, a diluent used to simulate the interstitial fluid in contact with the adjuvant upon injection. Only 7.77 ± 0.22% of the adjuvant dissolved after 10 days in 0.1M 15  EDTA, a strong chelator for aluminum and indium atoms that promotes that dissolution of 111In-AlO(OH) (44, 75, 76). The adjuvant was stable in saline.    Figure 2- 3: Stability of 111In-AlO(OH) in different diluents over time.  2.3.3.  Physical Characterization of Alhydrogel PXRD analysis of Alhydrogel (Figure 2-3) confirmed previous reports that the adjuvant has a structure consistent with AlO(OH) (12). The broad diffraction peaks highlight that the adjuvant is poorly crystalline in nature (12). Signals at 18.774 and 20.224˚ 2θ suggest that the batch of Alhydrogel tested (Batch 5012) contained a small amount of more crystalline Al(OH)3 (5).  Figure 2- 4: PXRD of Alhydrogel.                                                                                                                                     0102030405060708090100110  5 10 20 30 40 50 60 70 80 9d=4.72278, 18.774 °d=4.38731, 20.224 °JCPDS AlO(OH) JCPDS Al(OH)3  2θ Scale Lin (cps) 16  DSC analysis of Alhydrogel (Figure 2-4A) showed characteristics endothermic peaks at 110˚C and 420˚C for AlO(OH) (77). The endothermic peak at ~290˚C was characteristic for Al(OH)3 and confirms the above PXRD results (78). The FTIR analysis of Alhydrogel (Figure 2-4B) matched the spectrum for AlO(OH) with a broad peak at 3100-3700 cm-1, representative of the hydroxylated surface, and additional peaks at 750, 883, 1072 and 1639 cm-1 (61).    Figure 2- 5: DSC (A) and FTIR(B) analysis of Alhydrogel. 2.3.4.  Physical Characterization of 111/115In-AlO(OH)  Batches of 115In-AlO(OH) with increasing amounts of dopant were characterized with PXRD (Figure 2-5). Up to 5% of the aluminum atoms could be replaced by 115In without affecting the adjuvant’s crystal structure. Above that level of doping, a combination of 115In(OH)3 and 115In-AlO(OH) were produced. None of the 115In-AlO(OH) adjuvants assessed using PXRD contained any Al(OH)3. 2.5% 115In-AlO(OH) was selected for downstream assessments as this formulation contained a sufficient amount of dopant for detection with a mass cytometer (See section 4.3.1).111In-AlO(OH) was not analysed by PXRD.  Figure 2- 6: PXRD of 2.5-10% 115In-AlO(OH).                                                                                                                                                                          0102030405060708090100110120  5 10 20 30 40 50 60 70 80 92.5% In-AlO(OH)  5% In-AlO(OH)  10% In-AlO(OH)  JCPDS AlO(OH)  JCPDS In(OH)3 2θ Scale Lin  17   DSC and FTIR analysis of 111In-AlOOH and 2.5% 115In-AlO(OH) confirmed that both adjuvants were AlO(OH) (Figure 2-6) (61, 77). These assays further confirmed the absence of Al(OH)3 in the doped-adjuvants.   Figure 2- 7: DSC (A) and FTIR (B) analysis of 2.5% 115In-AlO(OH). 2.3.5.  Hydrodynamic Size and Zeta Potential of 111/115In-AlO(OH) and Alhydrogel  Dynamic light scattering analysis (Figure 2-7) showed that the hydrodynamic size and zeta potential of 111In-AlO(OH) and 2.5% 115In-AlO(OH) were not statistically different from Alhydrogel. The adjuvants consisted of ~1500 nm aggregates with a slight positive charge.  These observations are consistent with previous reports in the literature for Alhydrogel from Stanley Hem’s group (12).  Figure 2- 8: Hydrodynamic size (A) and zeta potential (B) of 111/115In-AlO(OH) and Alhydrogel. 18  2.3.6. Particle Morphology and Spatial Location of 115In Dopant within AlO(OH) Nanoparticles  The aggregate nature of Alhydrogel makes it challenging to assess the morphology of the adjuvant (14). Alhydrogel and 2.5% 115In-AlO(OH) were sonicated to disrupt the aggregates to better visualize the nanoparticles under STEM imaging (14). Figure 2.8 shows that the hydrodynamic size of the adjuvants were reduced from ~1500 nm to between 10-100 nm upon sonication.    Figure 2- 9: Alhydrogel and 2.5% 115In-AlO(OH) before and after sonication in preparation for STEM imaging. Each line represents a triplicate measurement of 30 seconds.     STEM imaging (Figure 2-9) showed that 2.5% 115In-AlO(OH) was composed of aggregated nanoparticles with a similar high-aspect ratio morphology to the commercial adjuvant. The average size of the nanoparticles within the doped adjuvant was 10.02 +/- 2.11 nm by 5.41 +/- 1.29 nm, comparable to the dimensions of the Alhydrogel nanoparticles (12).  19   Figure 2- 10: STEM images comparing the morphology of Alhydrogel (left) to 2.5% 115In-AlO(OH) (right) at increasing levels of magnification from the top to bottom panels. Note that the concentration of the 2.5% 115In-AlO(OH) loaded onto the TEM grids was ~1.5x more concentrated than Alhydrogel. 20  Elemental mapping of Alhydrogel (Figure 2-10) confirmed that the adjuvant is composed of aluminum and oxygen atoms; hydrogen cannot be atomically mapped with STEM (12). Elemental mapping also confirmed that the commercial adjuvant does not contain any indium.   Figure 2- 11: Elemental mapping of Alhydrogel. The top panel shows a reference HAADF image and the distribution of aluminum (red), indium (green) and oxygen (blue) within the adjuvant. The bottom panel shows the EDX spectrum (left) and results from the quantification of the EDX data (right).   Elemental mapping of 2.5% 115In-AlO(OH) (Figure 2-11) showed that the dopant was evenly dispersed through the AlO(OH) nanoparticles. Quantification of the EDX spectrum from 3 different regions found that the amount of indium was equivalent to 2.55 ± 0.21% of the aluminum atoms. This finding shows that most, if not all, of the 115In was incorporated into the AlO(OH) matrix and corroborates the results from section 2.3.1 with 111In-AlO(OH). Radioactive 111In-AlO(OH) was not assessed with STEM.       21   Figure 2- 12: Elemental mapping of 2.5% 115In-AlO(OH). The top panel shows a reference HAADF image and the distribution of aluminum (red), indium (green) and oxygen (blue) within the adjuvant. The bottom panel shows the EDX spectrum (left) and results from the quantification of the EDX data (right). 2.3.7. Protein Adsorptivity and De-Adsorptivity Assays to 1/115In-AlO(OH) and Alhydrogel  Alhydrogel, 111In-AlO(OH) and 2.5% 115In-AlO(OH) adsorbed the model antigen OVA to a similar extent, as illustrated in Figure 2-12A. The adsorptive ability of the adjuvants, between 10-20 mg/mL, was similar to the manufacturer’s specifications for 1.3% w/v Alhydrogel using human serum albumin (12-20 mg/mL) (79). Upon exposure to FBS (Figure 2-12B), which was used as a surrogate for interstitial fluid at the injection site, ~65% of the adsorbed AF555-OVA was released from the AlO(OH) particles. The degree of antigen de-adsorption was not statistically different between 111/115In-AlO(OH) and commercial Alhydrogel. A similar degree of de-adsorption was reported for OVA coated to Alhydrogel in the presence of lymph by Morefield et al. (57). The antigen was not eluted upon dilution in saline for all adjuvants tested.   Figure 2- 13: OVA adsorptivity (A) and de-adsorptivity (B) to Alhydrogel and 111/115In-AlO(OH). 22  2.3.8.  Cellular Toxicity of 1/115In-AlO(OH) and Alhydrogel  Figure 2-13A shows representative micrographs from the YOYO-1 assay for cells treated with up to 500 µg/mL over time. Dead cells with disrupted membranes become fluorescent due to the presence of the YOYO-1 indicator dye (72). Due to the presence of the adjuvant particles on the bottom of the microplate wells, the IncuCyte Zoom could track cell confluence. However, visual inspection of the micrographs suggests that the rate of cell proliferation was largely undisturbed, even in the presence of a large quantity of adjuvant. Alhydrogel and 115In-AlO(OH) at >125 µg/mL induced a level of cell death above untreated cell controls (Figure 2-13B). 115In-AlO(OH) at 500 ug/mL resulted in more cell death compared to Alhydrogel after 6-10 hours of exposure, suggesting that the doped adjuvant may be more acutely toxic.  The MTT assay for cellular respiration showed a similar toxicity pattern between Alhydrogel and 2.5% 115In-AlO(OH) (Figure 2-13C). 115In-AlO(OH) was statistically more toxic than untreated cells above 15.6 µg/mL while Alhydrogel showed reduced respiration only above 62.5 µg/mL.   Figure 2- 14: Representative micrographs (A) and analysis of cell death over time (B) from the YOYO-1 toxicity assay. Both adjuvants caused statistically more cell death compared to untreated cells above 125 µg/mL. Asterisks in panel B represent statistical differences between Alhydrogel and 2.5% 115In-AlO(OH) at a particular adjuvant concentration. MTT assay results for cellular respiration (C) after 48 hours of exposure to the adjuvants; an asterisks represent a statistical difference between the adjuvants and untreated cells.  23  2.3.9.  Humoral and Cellular Immune Responses to 111/115In-AlO(OH) and Alhydrogel Aluminum-based adjuvants promote humoral immunity and polarize CD4+ T-helper cells towards a Th2 phenotype (80). In mice, IgG1 and IgE are the antibody isotypes associated with a Th2-skewed immune response whereas IgG2c is associated with a Th1-polarized response (80). Figure 2-14 shows that 111In-AlO(OH) and 2.5% 115In-AlO(OH) increased total IgG and specifically promoted IgG1 over IgG2c against adsorbed OVA. These results suggest that the indium-doped adjuvants promotes a similar humoral response to published findings with Alhydrogel (80).  Figure 2- 15: Serum levels of anti-OVA total IgG, IgG1 and IgG2c in mice vaccinated with OVA adsorbed to either 2.5% 115In-AlO(OH) (left) or 111In-AlO(OH) (right). Each indium-doped adjuvant was compared to Alhydrogel and naïve control animals.  0 5000 10000 15000 20000 25000 30000 350000.00.51.01.5*/**/**/*ns Alhydrogel 2.5% 115In-AlO(OH) Naive ControlTotal IgG0 5000 10000 15000 20000 25000 30000 350000.00.51.01.52.0***nsTotal IgG Alhydrogel 111In-AlO(OH) Naive Control*/*0 500 1000 1500 2000 2500 3000 35000.00.20.4nsIgG2c0 5000 10000 15000 20000 25000 30000 350000.00.51.01.52.02.53.0*/**/**/**/*nsIgG10 5000 10000 15000 20000 25000 30000 350000.00.51.01.52.02.5*/**nsIgG10 500 1000 1500 2000 2500 3000 35000.00.20.4nsIgG2cDilution (1/x) Absorbance (450nm) 111In-AlO(OH) 2.5% 115In-AlO(OH) 24   OVA-tetramer staining was used to assess the formation of cell-mediated immunity to OVA adsorbed to the indium-doped adjuvants and Alhydrogel. The top panels of Figure 2-15 illustrates the gating strategy used to determine the percentage of the CD8+ T cells in the peripheral blood that are specific to the OVA antigen (74).   Figure 2- 16: Gating strategy for OVA-tetramer positive CD8+ T-cells. After drawing a gate around the lymphocyte population (A), live CD3+ T cells were selected as CD3hi 7AADlow events (B). CD8+ T-cells were identified as CD3hi CD8hi events (C) and OVA-tetramer high events were selected from this population (D).  Alhydrogel, 111In-AlO(OH) and 2.5% 115In-AlO(OH) promoted a slight, but statistically significant increase in the number of OVA-tetramer+ CD8+ T cells compared to naïve animals (Figure 2-15E). There was no statistical difference between the doped and commercial adjuvants tested though.  Figure 2- 17: Percentage of CD8+ T-cells in peripheral blood that are positive for OVA-tetramer. 25  2.4. Discussion ITLC and elemental mapping showed that the indium dopant was efficiently trapped and evenly dispersed within the AlO(OH) nanoparticles. Up to 5% of the aluminum atoms within the AlO(OH) matrix could be replaced by indium atoms without changing the adjuvant. An extensive array of physical and morphological assessments demonstrated that 111/115In-AlO(OH) closely match commercial Alhydrogel. The only physical difference observed between Alhydrogel and 111/115In-AlO(OH) was that the commercial adjuvant contained a small amount of Al(OH)3. The transition from AlO(OH) to more crystalline Al(OH)3 is an energetically favorable process and would likely be observed if the doped-adjuvant was stored for a sufficient period of time. The batch of Alhydrogel analyzed was 18 months from the time of precipitation whereas most batches of 111/115In-AlO(OH) were analyzed and used within a few days of preparation.   Stability testing of 111In-AlO(OH) corroborates previous work by Seeber et al., which showed that AlO(OH) dissolved poorly in physiological fluids (44). When Seeber and colleagues incubated Alhydrogel in a solution containing 100 times the physiological concentration of citric acid, a chelator that promotes adjuvant dissolution, only ~8% of the adjuvant dissolved after 5 days (44). A similar amount of 111In-AlO(OH) dissolved after 10-days in a high concentration of EDTA, another chelator that promotes dissolution (75, 76). The results of the stability test suggest that if the indium dopant is detected away from the site of injection, there is a high likelihood that it is in the form of a component nanoparticle.  The doped adjuvant had a similar toxicity profile to Alhydrogel, although 2.5% 115In-AlO(OH) may have been more acutely toxic. Aluminum-based adjuvants have been shown to induce cell death at the site of injection, leading to the release of genomic DNA and other DAMPs that in turn help to potentiate an immune response (26, 27). Eidi et al. previously used an MTT assay to assess the toxicity of Alhydrogel on NSC-34 neuron-like cells (53). Toxicity was observed at only 1 µg/mL of Alhydrogel for the NSC-34 line compared to 62.5ug/mL with HEK293 cells used in this thesis (53). Future toxicity assessments should use more representative cell lines for the injection site (i.e., C2C12 myocytes or 3T3 fibroblasts) and cells of the innate immune system that are drawn to this location (i.e., RAW264.7 monocytes). When used to vaccinate mice against the model antigen OVA, 111/115In-AlO(OH) promoted the formation of IgG1 over IgG2c antibodies suggesting that the adjuvant polarized naïve CD4+ Th cells towards a Th2 phenotype that is characteristic for Alhydrogel (80). The indium-doped adjuvants and Alhydrogel resulted in the clonal expansion of a small population of OVA-specific CD8+ T cells. Previous reports in the literature have shown that OVA adsorbed to Alhydrogel can be cross-presented by CD8+ dendritic cells to induce the activation and proliferation of OVA-specific CD8+ T cells (34, 81-83). However, these cells fail to differentiate into cytotoxic T cells (CTL), resulting in a weak cell-mediated response (34, 56, 81, 83). Only when Alhydrogel is incorporated into a mixed adjuvant system, like AS04 where the AlO(OH) surface is functionalized with a TLR4 ligand, is a robust cell-mediated immune response developed (56).  26  Overall, 111In and 115In were successfully incorporated into the AlO(OH) crystal structure while maintaining the physical and functional properties of Alhydrogel.  The doped-adjuvant can be used to better understand the commercial adjuvant’s biodistribution using techniques like SPECT/CT (chapter 3) and mass cytometry (chapter 4).                      27  Chapter 3: Biodistribution of 111In-AlO(OH)   1.1. Introduction The seminal study by Flarend et al. using AlO(OH) doped with the positron emitter 26Al helped to establish the safety profile of aluminum-based adjuvants by showing that serum aluminum levels are only  marginally elevated post-injection and below toxic levels (50). Although this study was the first to explore Alhydrogel’s pharmacokinetics, it did not assess the amount of adjuvant at key immunological sites like the draining lymph node (50). This chapter uses 111In-AlO(OH) to confirm certain aspects and expand upon Flarend’s work.  111In is a gamma-emitting isotope that is commercially produced for nuclear medicine applications (84). With a half-life of 2.8 days, 111In-AlO(OH) can be tracked over a period of ~2 weeks (84). Compared to 26Al-AlO(OH) with a half-life of 7.17x105 years, 111In-AlO(OH) is associated with fewer radiation safety issues (50, 84). To track 111In-AlO(OH) in vivo, single-photon emission computed tomography (SPECT) was used together with x-ray based computed tomography (CT) as an anatomical framework in this chapter (85). Compared to other imaging modalities (i.e., optical tomography, MR, etc…), SPECT/CT has amongst the highest sensitivity and is a fully quantitative technique; such a highly sensitive technique is required as only a small percentage of the administered adjuvant is expected to distribute from the site of injection (50, 84-88).  To further validate 111In-AlO(OH) as a useful tracer for Alhydrogel, the release rate of OVA antigen adsorbed to the adjuvant was assessed using SPECT/CT. For this purpose, OVA was modified with NOTA and radiolabeled with the gamma emitter 67Ga. The antigen was modified in this fashion due to the high in vivo stability of this chelation system and the similarity of this radioisotope’s half-life (3.2 days) to 111In (75, 84, 89). Since 67Ga and 111In have never been imaged in a quantitative fashion together with SPECT, we developed new physics protocols to compensate for cross-talk between the isotypes. Cross-talk is the detection of photons originating from one radionuclide within the acquisition window of the other radioisotope (90). Figure 3-1 shows the spectrums of 111In and 67Ga for which the new reconstruction parameters were developed.  Figure 3- 1: Gamma spectra of 67Ga (red), 111In (blue) and mixed isotopes (green). 28  3.2. Materials and Methods 3.2.1. Synthesis and Radiolabeling of 67Ga-NOTA-OVA OVA protein at 20 mg/mL in sodium bicarbonate buffer (pH 8.5) was reacted overnight at 10˚C with 2-S-(4-isothiocyanatobenzyl)-1,4,7-triazacyclononane-1,4,7-triacetic acid (p-SCN-Bn-NOTA; Macrocyclics B-605) at a stoichiometric ratio of 1:5 (91). The modified protein was isolated and buffer exchanged into 1xPBS using a    30 kDa MWCO micro-concentrator. To radiolabel the protein, 130 MBq of 67GaCl3 in 0.1N HCl (Nordion IPG-Ga-67) was added to 7.5 mg of NOTA-OVA at room temperature for 4 hours under light agitation (92). Excess activity was removed and the protein was buffer exchanged into saline using a 30 kDa MWCO micro-concentrator. The concentration of the radiolabeled antigen was determined using a fluorescamine assay, described previously, against a standard curve of NOTA-OVA.  3.2.2.  Labeling Efficiency and Radiopurity Assessment of 67Ga-NOTA-OVA  Thin layer chromatography (TLC) using a silica gel 60F254 (Merck 1055540001) stationary phase and 0.1 M citric acid solution at pH 4 (Sigma 251275) mobile phase was used to assess the radiolabeling of NOTA-OVA (93). In this system, 67Ga-NOTA-OVA remains at Rf=0 while free 67Ga forms a complex with the citric acid and migrates to Rf=1 (93). TLCs were spotted with 1 µL of 67Ga-NOTA-OVA before and after the removal of excess 67Ga to calculate the labeling efficiency and radiopurity, respectively. A photostimulable phosphor screen, Cyclone Phosphor Imager and OptiQuant software (Perkin Elmer) were used to analyze the TLC data, as described in chapter 1.  3.2.3.  SDS and Native PAGE Analysis of 67Ga-NOTA-OVA A Mini-PROTEAN electrophoresis system (Bio-Rad) was used to cast 0.75mm thick native and SDS polyacrylamide gels. The resolving portion consisted of 10% 1:29 bis:acrylamide, 375 mM Tris at pH 8.8, 0.1% APS and 0.05% TEMED  (± 0.1% SDS) and was polymerize under a layer of isopropanol (94, 95). The stacking region consisted of 5% 1:29 bis:acrylamide, 125 mM Tris at pH 6.8, 0.1% APS and 0.05% TEMED (± 0.1% SDS) (94, 95). All reagents for gel casting were obtained from Bio-Rad.  For SDS-PAGE, 10 µL of modified or unmodified OVA solution (0.5-2mg/mL) was mixed with 2 µL of 6x Laemelli sample buffer and heated at 95˚C before being loaded onto the gel (95). For native PAGE analysis, 5 µL of protein solution was mixed with an equal volume of 2x native sample buffer (Bio-Rad 1610738) and loaded directly onto the gel (94). The gels were run with constant current (15 mA) in a buffer contained 25 mM Tris and 192 mM glycine at pH 8.3 (± 0.1% SDS) (Bio-Rad 1610771/2) until the indicator dye reached the bottom of the gel.   The gels were stained using a solution of 85 mM Coomassie brilliant blue dye (Sigma 27816) in 35mM HCl with the assistance of microwave irradiation (96). The gels were de-stained with 18 MΩ with the aid of heat (96). The gels were documented using an Odessy CLx Imager (LiCor) measuring fluorescence in the     700 nm 29  channel. A photosimulable phosphor plate and Cyclone Phosphor Imager (Perkin Elmer) were used to assess the location of the radioactivity within the gel. 3.2.4. SPECT/CT Assessment of 67Ga-NOTA-OVA Adsorbed to 111In-AlO(OH) or Alhydrogel C57BL/6 mice (n=4) between 6-8 weeks old were injected into the left posterior region on the dorsal surface with 200 µg (3.7 MBq) of 67Ga-NOTA-OVA adsorbed to 0.7 mg (7.4 MBq) of 111In-AlO(OH) in a total volume of 100 μL. The same group of animals received a contralateral injection containing 200 μg (3.7 MBq) of 67Ga-NOTA-OVA adsorbed to 0.7 mg of Alhydrogel. A second group of mice (n=3) was administered on the dorsal surface 200 µg (3.7 MBq) of 67Ga-NOTA-OVA as a free antigen control. The 2 groups of animals were serially scanned (whole body minus tail) in list-mode under isoflurane in a VECTor PET/SPECT/CT system (MILabs) outfitted with an extra-ultra-high sensitivity mouse collimator (MILabs) (97, 98). BioVet software (m2m Imaging) was used to monitor the vital signs of the animals during the scans and make appropriate adjustments to the anesthesia when necessary. The time and duration of each SPECT scan post-injection of the tracers are shown in Table 3-1. The duration of the SPECT scan was increased over time to generate better quality data/obtain more counts as the 111In decayed. The CT scan was always 5 minutes in duration and followed the SPECT acquisition.  Time Post-Injection (hours) SPECT Acquisition Duration (minutes) 0  30 5  30 24 30 120 60 240 90 360 (Terminal Scan) 120 Table 3- 1: SPECT scan times and durations. CT data was reconstructed with a cone-beam filtered back-projection algorithm using NRecon software (Skyscan). The SPECT data was reconstructed from list-mode projection data using a pixel-based ordered subset expectation maximization (POSEM) algorithm with 6 iterations and 16 subsets with the voxel size defined as 0.4 mm3 using U-SPECT Rec2.5li software (MILabs) (99). The photopeak widow to reconstruct 67Ga was set at    96 ± 20 keV while 111In was reconstructed at 243 ± 15 keV (unpublished data, Hafeli Lab). Scatter correction was performed using triple energy window methods with two 20% wide scatter widows adjacent to the reconstruction windows (100).   Figure 3- 2: Location of reconstruction and triple energy windows used to separate 67Ga and 111In signals in dual-isotope SPECT. 67Ga 111In Reconstruction Windows Triple Energy Windows 30  U-SPECT Rec2.5li software was also used to apply CT-based attenuation correction and isotope decay correction to all SPECT reconstructions and to automatically register the SPECT and CT images. AMIDE software (UCLA) was used to generate renderings of the data; a 3D-Gaussian filter was applied (0.5 mm FWHM) to reduce noise unless otherwise specified (101). AMIDE software was also used to perform a volume of interest (VOI) analysis to assess the amount of 111In-AlO(OH) and 67Ga-NOTA-OVA remaining at the site of injection over time (101). Calibration factors (67Ga: 1522 MBq/mL, 111In: 1244 MBq/mL) were applied to convert the arbitrary units from the VOI analysis into units of radioactivity (unpublished data, Hafeli Lab). The data from the VOI analysis was standardized to the time of administration and reported as a percentage of the injected activity versus time.  3.2.5. Sacrifice-Based Biodistribution of 111In-AlO(OH) C57BL/6 mice (n=3) between 6-8 weeks old were injected into the posterior half of the dorsal surface with 100 µg (~3.7 MBq) of OVA adsorbed to 0.7mg (7.4 MBq) of 111In-AlO(OH) in a total volume of 100 μL. The animals were sacrificed 10-days post-administration to conduct a biodistribution study. Briefly, various organs and the site of injection were harvested, weighted and their activity determined using a Packard Cobra II gamma counter (92). Results were expressed as activity concentration (cpm/g), the percentage of the injected dose per organ (%ID/organ) and per gram of tissue (%ID/g) and as an organ-to-blood ratio (92). 3.2.6. Data Analysis Data are presented as mean ± standard deviation unless otherwise specified. GraphPad Prism 7 was used for statistical analysis using Student’s t-test, linear regression or ANOVA followed by multiple comparisons testing (Dunnet’s test). P<0.05 was considered statistically significant and was indicated by an asterisks (*). Data was graphed using either GraphPad Prism 7 or OriginPro 9.  3.2.7. Ethics Statement  All animal experiments were carried out in accordance with the guidelines of the University of British Columbia’s Animal Care Committee under the approved protocol A12-0172. 31  3.3.Results 3.3.1.   Preparation and Characterisation of 67Ga-NOTA-OVA TLC analysis showed that the labeling efficiency for 67Ga-NOTA-OVA, which remains at Rf=0, was 84.5 ± 4.3% (Figure 3-3B). After purifying the radiolabeled antigen and removing free 67Ga, the radiopurity was calculated to be 99.4 ± 0.9% (Figure 3-3C). The tracer was sufficiently pure for in vivo assessments.   Figure 3- 3: Labeling efficiency (B) and radiopurity of 67Ga-NOTA-OVA (C) in relation to free GaCl3 (A). SDS-PAGE analysis of NOTA-OVA and 67Ga-NOTA-OVA (Figure 3-4A) showed that the antigen had a similar molecular weight to the unmodified protein (44.3kDa) (102). No appreciable amount of degradation products were observed additionally. Native-PAGE analysis (Figure 3-4B) found that both NOTA-OVA and 67Ga-NOTA-OVA migrated further down the gel toward the cathode. This observation helps to confirm the conjugation of NOTA, as the added negative charge from the chelator reduced the isoelectric point of the antigen and improved its electrophoretic mobility. Native PAGE analysis showed no overt signs of aggregation in relation to the unmodified antigen. The location of the radioactivity matches the protein staining on the gel, confirming that the radiolabeling procedure was successful.    Figure 3- 4: 10% SDS PAGE (A) and native-PAGE (B) analysis of 67Ga-NOTA-OVA. The lanes are a molecular weight ladder (L), 2-0.25mg/mL calibration curve of unmodified OVA (1-4), OVA-NOTA (5-6) and 67Ga-NOTA-OVA (7-8). The Coomassie stain (gray scale) has been overlaid with a phosphorimage of the radioactivity within the gels (heat map). L 1 2 3 4 5 6 7 8 1 2 3 4 5 6 7 8 A 50 37 75 kDa B 32  3.3.2. Serial  SPECT/CT Assessment of 111In-AlO(OH) and 67Ga-NOTA-OVA at the Injection Site Using the SPECT reconstruction parameters described in section 3.2.4, 111In and 67Ga signals could be properly separated and independently quantified. Figure 3-5 helps to illustrate this point, since 111In-AlO(OH) can be distinguished from 67Ga-NOTA-OVA when injected into the same animal.    Figure 3- 5: Coronal view of a SPECT/CT scan taken immediately post-administration of the radiotracers. The leftmost panel illustrates that the mouse received an injection of 67Ga-NOTA-OVA adsorbed to 111In-AlO(OH) on the left flank and an injection of 67Ga-NOTA-OVA adsorbed to Alhydrogel on the right flank. The next 2 panels show the location of the 111In (green) and 67Ga (red) signal within the mouse. The rightmost panel is merged rendering of the 111In and 67Ga signals.    To help validate the 111In-AlO(OH) tracer, the kinetics of 67Ga-NOTA-OVA released from the doped adjuvant was compared to Alhydrogel in vivo. Figure 3-6A shows the location of the injection site VOIs used to perform the analysis. Both adjuvants retained 67Ga-NOTA-OVA for a longer duration at the site of injection when compared to the free antigen (Figure 3-7B). For example, at day 5 post-administration essentially no free antigen remained at the injection site compared to ~40% when adsorbed. This finding is in agreement with work by Noe et al., who found that aluminum-based adjuvants delayed the drainage of 111In-alpha-casein from the region of administration (24).  The release of 67Ga-NOTA-OVA from 111In-AlO(OH) was not statistically 33  different from Alhydrogel. Approximately 60% of the adsorbed 67Ga-NOTA-OVA was released from both adjuvants within the first 24 hours followed by a slower phase of release.   Figure 3- 6: Placement of the VOIs (blue regions) over the injection sites containing 111In (green) and or 67Ga (red) (A). Drainage of 67Ga- tagged antigen from the site of injection over time as free protein or when adsorbed to an aluminum-based adjuvant (B).  The VOI analysis of 111In-AlO(OH) showed that a small burst of 111In (~10%) was released from the site of injection within the first 24 hours post-administration followed by a slower phase (Figure 3-7). Approximately 20% less 111In was detected at the site of injection after 15 days. A linear regression through the data found that the slope of the line was significantly non-zero, adding confidence to the claim that the amount of radiotracer is decreasing from this location (Figure 3-7 Insert).   Figure 3- 7: VOI analysis of 111In-AlO(OH) from the site of injection. Asterisks designate a significant change at each time point in relation to the activity detected within the injection site VOI from the 0 hour post-injection scans. The graph insert shows a linear-regression through the data. 34  3.3.3.  Sacrificed-Based Biodistribution of 111In-AlO(OH)  Since the amount of 111In that distributed from the site of injection was rather low, quantification of the activity in the various organs was challenging using the SPECT/CT data. A group of animals was subsequently administered 111In-AlO(OH) and sacrificed 10 days post-administration to determine the amount of activity in the organs with a gamma counter.  There was no statistical difference between the amount of activity measured at the injection site using SPECT/CT (88.1 ± 3.3%) or a sacrifice-based biodistribution (87.0 ± 2.1%) at day 10. This finding helps to validate the VOI analysis and the dual-isotope SPECT reconstruction parameters from section 3.3.2. The distribution of activity beyond the injection site is displayed in Figure 3.8. 111In was found predominantly in the inguinal draining lymph nodes, kidneys, liver, spleen, bone, bladder and muscle. Minimal amounts of activity were found within the blood, heart, lung, small intestines, pancreas, brain and excreted into the feces on day 10 post-injection. The distribution of 111In was similar to that observed by Hem’s group using 26Al-AlO(OH) (50)  Figure 3- 8: Results of the biodistribution study presented as cpm/g (A), organ/blood ratio (B), %ID/g (C) and %ID/organ (D). 35  3.4. Discussion This chapter presented the first dual-isotope SPECT scan using 67Ga and 111In. Reconstruction parameters were developed that effectively prevented isotope cross-talk, as illustrated in Figure 3-1. This technique could be applied to other imaging experiments or diagnostic procedures that require 2 gamma emitters with relatively long yet similar half-lives.  Compared to the free antigen, 111In-AlO(OH) and Alhydrogel formed a long-lasting depot of 67Ga-NOTA-OVA at the site of injection. Other investigations have also shown that aluminum-based adjuvants slow the drainage of adsorbed antigens from the injection site, which may be mechanistically important in the generation of an immune response (6, 20, 22, 24). There was no statistically significant difference between the release of 67Ga-NOTA-OVA adsorbed to 111In-AlO(OH) or Alhydrogel, which helps to further validate the indium-doped AlO(OH) strategy. 67Ga-NOTA-OVA was released from the doped and commercial adjuvant in a 2-phase manner: a burst phase where ~60% of the antigen drained from the site of injection over the first 24 hours followed by a slower phase of distribution. Noe et al. observed a similar release profile at the site of injection for 111In-alpha-casein adsorbed to Alhydrogel in mice (24). This dual-phase release is likely the result of partial antigen de-adsorption upon exposure to constituents in interstitial fluid (24, 57, 103). Results from the de-adsorption assay in section 2.3.7. of this thesis found that ~65% of the adsorbed OVA was released from the adjuvant’s surface within 30 minutes of exposure to a biological fluid. Work out of Staneley Hem’s group observed a similar degree of de-adsorption with OVA from Alhydrogel in vitro (57).  The SPECT/CT VOI analysis of 111In-AlO(OH) showed that ~ 20% of the administered 111In distributes from the site of injection by day 15. Flarend et al. estimated that ~17% of their 26Al-doped adjuvant had dissolved from the site of injection based on blood AUC data (50). 111In-AlO(OH) appeared to also distribute as a small initial burst (~10%) followed by a slower secondary phase. Flarend observed a similar phenomena, reporting that the amount of 26Al in the blood spiked over the first 48 hours post-administration followed by a low, steady level of  26Al in the circulation over the next 26 days (50). As shown in section 2.3.2. of this thesis and in work by Seeber et al, AlO(OH) is poorly soluble in biological fluids with only ~1.5% of 111In-AlO(OH) dissolving after 10 days in FBS (44). Even highly favorable, non-physiological conditions (i.e., 0.1M EDTA) could only dissolve ~8% of 111In-AlO(OH) after 10 days (75, 76). These findings together question the assumption within the field that Alhydrogel particles remain at the site of injection and slowly dissolve over time (50, 51). The distribution of adjuvant nanoparticles may help to explain the pharmacokinetics of 111In-AlO(OH) and 26Al-AlO(OH), particularly the early burst phase. It is already known that Alhydrogel aggregates temporarily de-aggregate upon exposure to biological fluid as the adjuvant surface equilibrates with factors, such as phosphate ions and proteins, present in the new environment (47). De-aggregated AlO(OH) nanoparticles are an ideal size (~10 x 5.5 nm) to enter into afferent lymphatic vessels and distribute to distant tissues (48).  36  The biodistribution of 26Al-AlO(OH) and 111In-AlO(OH) both showed a high concentration of the radiotracer within the kidney, liver and spleen as well as a negligible amount of activity in the brain (50). The 111In tracer was also found at higher levels within sites not assessed by Flarend et al., like the draining inguinal lymph nodes and bone. Unfortunately, the biodistribution profiles observed with 26Al-AlO(OH) and 111In-AlO(OH) can both be explained from the context of nanoparticle associated or soluble radionuclide. For example, free indium forms a complex with transferrin in vivo that traffics the radiometal to the liver (75). As part of the reticuloendothelial system, the liver also is highly efficient at clearing nanomaterials, such as 111In-AlO(OH), from the blood (104). Kidney uptake may be due to the clearance of the solubilized radiometals or the filtration of adjuvant particles <5nm in diameter, which are below the size cut-off for the glomerulus (104). Bone marrow has been shown to take up both 111In-tranferrin and nanomaterials efficiently (104, 105). The results obtained with 111In-AlO(OH) helped to identify which tissues to assess with techniques that can determine if the adjuvant is in nanoparticle form, such as mass cytometry (chapter 4) and STEM imaging (chapter 5). One of the limitations with 111In-AlO(OH) is that the adjuvant could only be tracked for ~2 weeks before the radionuclide decayed to too great of an extent to be accurately quantified. Appendix 1 discusses options for the development of a longer-lasting traceable forms of Alhydrogel, such as MR-responsive Gd-AlO(OH).                 37  Chapter 4: Distribution of 115In-AlO(OH) Particles in the Lymph Node   4.1. Introduction White et al. and Giusti et al. have both reported structures that resemble preformed aluminum particles in the draining lymph node within 24 hours of administration (52, 54). Khan et al. found surrogate nanomaterials within the draining lymph node by day 1 post-injection that peaked by day 4 (45). Using nanodiamond-functionalized Alhydrogel, Eidi et al. observed 2552 ± 22 fluorescent structures within thin sections of the draining lymph node on day 7 and 308 ± 22 events on day 21 post-administration (53). Although there are limitations with the above studies, as outlined in chapter 2, they provide some preliminary evidence that Alhydrogel particles can distribute to the draining lymph node. Data from this thesis, namely the initial burst phase of 111In from the site of injection despite the poor solubility of 111In-AlO(OH), supports the hypothesis that a portion of the adjuvant distributes in particulate form. The high concentration of 111In observed within the draining lymph node on day 10 post-administration of 111In-AlO(OH) further suggests that this site is appropriate to investigate for adjuvant in particle form.  A technique using mass cytometry by time of flight (CyTOF) and indium-doped AlO(OH) was developed in this chapter to identify aluminum-based adjuvants within the draining lymph node. CyTOF is a rather new technique that combines the cellular analysis principle of fluorescent-based flow cytometry with the selectivity and quantitative power of ICP-MS (106). CyTOF replaces the fluorescent markers used in flow cytometry with metal isotopes having a molecular weight of >80 g/mol (106). For this project, the lumogallion stain for Alhydrogel was replaced with 2.5% 115In-AlO(OH). Due to minimal spill over between isotopes, mass cytometry can analyze >50 cellular parameters with little to no compensation (106). The indium doped adjuvant when coupled with mass cytometry overcomes one of the primary drawbacks with lumogallion, significant spectral overlap with other fluorophores that limits multi-parameter assessments (53, 58).  Researchers to date have only used the CyTOF technique for its original intended purpose of high dimensional immunophenotyping (107). This chapter uses mass cytometry for the first time for a different application, to evaluate the kinetics and cellular distribution of a heavy-metal tagged nanomaterials in vivo.     38  4.2. Materials and Methods 4.2.1. Animal Immunizations and Tissue Preparation C57BL/6 mice between 6-8 weeks of age were injected into the posterior region of the dorsal surface with 100 µg of OVA adsorbed to 0.7 mg of 2.5% 115In-AlO(OH) in a total volume of 100 µL. At set time points (1, 5, 10, 15, 40 and 70 days), 3 immunized mice and a naïve control were sacrificed by CO2 asphyxiation under anesthesia with isoflurane. The inguinal lymph nodes were excised and pressed through a 70 µm cell strainer (Fisher 352350) to form a single-cell suspension and rinsed with CyFACS buffer (1% BSA in 1x PBS) (108). 1x107 cells from each sample were transferred to a 96-well plate for staining.   4.2.2. CyTOF Staining and Analysis The collected cells were Fc blocked using tissue culture supernatant from a 2.4G2 hybridoma for 15 minutes on ice (74).  The cells were then stained with the antibody cocktail described in Table 4-1 for 30 minutes at room temperature:   Antibody/ Probe Clone Metal Tag Dilution Vendor Catalogue Number CD45 30-F11 147Sm      1/100      Fluidigm 3147003B CD3e 145-2C11 152Sm 3152004B CD19 6D5 166Er 3166015B CD11c N418 162Dy 3162017B CD11b M1/70 148Nd 3148003B Gr-1 RB6-8C5 141Pr 3141005B NK1.1 PK136 170Er 3170002B F4/80 BM8 159Tb 3159009B CD86 GL1 172Yb 3172016B MHCII M5/114.15.2 174Yb 3174003B Table 4- 1: CyTOF antibody panel. After washing the cells with 150 µL of CyFACS buffer, they were re-suspended in 100 µL of 1xPBS and 100 µL of 5 µM cisplatin (Fluidigm 201064) was then added to the cells for 5 minutes at room temperature (109). The reaction was quenched with the addition of 100 µL of CyFACS buffer (109). After washing the cells with 200 µL of CyFACS buffer, they were re-suspended in 250 µL of 0.125 µM 191/193Ir intercalator (Fluidigm 201192B) in fixation/permeation buffer (Fluidigm 201067) for 1 hour at room temperature (110). The cells were washed once with 300 µL of CyFACS buffer and re-suspended in 500 µL of CyFACS for transport to the CyTOF instrument on ice.  Within 1 hour of running the samples through the CyTOF, the cells were pelleted and re-suspended in 500 µL of 18 MΩ water to minimize cell loss due to osmotic lysis. Approximately 0.75x106 cells were then diluted to a volume of 600 µL with 18 MΩ water and 60 µL of 1x 4-metal calibration beads (Fluidigm 201078) was added to each sample. The cells were passed through a 40 µm strainer (Fisher 352340) before being injected 39  into a 500 µL sample loop of a CyTOF 2 mass cytometer (Fluidigm) at a flow rate of 45 µL/min. A minimum of 250,000 events were collected for each sample.  To account for minor changes in the sensitivity of the instrument between days, the data was normalized against the 151/153Eu calibration beads using software developed by the Nolan Lab at Stanford (111). The normalized data was analyzed using FlowJo V10 software (TreeStar).  4.2.3. Establishing a Gating Strategy for 2.5% 115In-AlO(OH)  To determine how to properly gate for 2.5% 115In-AlO(OH) particles, 0.1 µL of the adjuvant was stained as described above and injected alone into the mass cytometer. Although AlO(OH) is rather resistant to dissolution in biological fluids, controls were carried out to assess how 2.5% 115In-AlO(OH) and free 115In are taken up or bind to cells. This was assessed by incubating cells isolated from the inguinal lymph node of a naïve animal with 0.1 µL of 2.5% 115In-AlO(OH) or an equivalent amount of free 115In (as 115In-citrate) before staining and injection into the CyTOF. Data was normalized and analysed as described above.   4.2.4. Statistical Analysis Data are presented as mean ± standard deviation unless otherwise specified. GraphPad Prism 7 was used for statistical analysis using Student’s t-test. P<0.05 was considered statistically significant and was indicated by an asterisks (*). Data was graphed using either GraphPad Prism 7 or OriginPro 9.   4.2.5. Ethics Statement All animal experiments were carried out in accordance with the guidelines of the University of British Columbia’s Animal Care Committee under the approved protocol A14-0159.     40  4.3.  Results 4.3.1.  Gating Strategy for 2.5% 115In-AlO(OH) Particles  When 2.5% 115In-AlO(OH) was injected into the mass cytometer, the majority of the 115In events were within the 4-log dynamic range of the instrument (Figure 4-1A). 2.5% 115In is therefore an appropriate concentration of dopant for downstream in vivo assessments. The threshold for an 115In-positive event for all future analyses was set at >101.1. The indium-doped particles stained in a unique way such that they can be easily distinguished from cellular events. First, 2.5% 115In-AlO(OH) was poorly stained by the 191/193Ir intercalator, a compound used to label DNA to identify cellular events and distinguish singlets from doublets (Figure 4-1B) (110). Cisplatin (195Pt) is used as a viability marker in mass cytometry since this chemotherapeutic preferentially enters cells with a compromised membrane and reacts with protein nucleophiles (109). Since 2.5% 115In-AlO(OH) readily adsorbs proteins, it is not surprising that the adjuvant stained high for 195Pt (Figure 4-1C). The BSA within the CyFACS buffer effectively blocked the adsorption of the metalated antibodies to the surface of the adjuvant. These antibodies are used to bind cell makers, such as CD45 and MHCII, to identify cell populations and assess the functional status of the cells (Figure 4-1D). Extracellular 2.5% 115In-AlO(OH) nanoparticles are therefore defined as 115Inhi, 191/193Irlo, 195Pthi, phenotypic markerlo events. Live cells on the other hand stain quite differently as 191/193Irhi, 195Ptlo, phenotypic markerhi events.   Figure 4- 1: CyTOF staining and gating strategy for non-cell associated 2.5% 115In-AlO(OH) adjuvant. With 2.5% of the aluminum atoms replaced with 115In, the adjuvant particles fell within the dynamic range of the mass cytometer (A). The adjuvant bound poorly to 191Ir nuclear stain (B), strongly to cisplatin (C) and poorly to metalated antibodies (D)  4.3.2. Cells Treated with Free 115In or 2.5% 115In-AlO(OH) can be Distinguished Cells isolated from the inguinal lymph node of a naïve mouse were incubated briefly with 2.5% 115In-AlO(OH) or free 115In, in the form of 115In-citrate, to assess how to gate for the metal dopant within a nanoparticle or solubilized state. As discussed in the above section, non-cellular 2.5% 115In-AlO(OH) can be identified as a distinct population (Figure 4-2A). Exposure of cells to soluble 115In found few events within the 41  2.5% 115In-AlO(OH) nanoparticle gate (Figure 4-2B). This observation adds confidence in the specificity of the gating strategy outlined in section 4.3.2 for cell unassociated nanoparticles.  Figure 4- 2: 191Ir nuclear stain versus 115In in living cells incubated with 2.5% 115In-AlO(OH) (A) or 115In-citrate (B).  Cellular events (191/193Irhi) positive for 115In were observed in the both the samples treated with 2.5% 115In-AlO(OH) and 115In-citrate. Without an equivalent to side scatter in flow cytometers, CyTOF cannot distinguish whether the adjuvant particles are extracellularly associated or internalized into a cell (112).  Free 115In has been shown to form complexes with various proteins, such as transferrin and albumin, which helps to explain how the free metal labels cells (113). Subsequently, 191/193Irhi 115Inhi events represent cells with either extracellularly bound indium-doped AlO(OH) nanoparticles, internalized nanoparticles or protein-bound 115In released from adjuvant that has dissolved.    4.3.3. Levels of 115In within the Inguinal Lymph Node of Naïve Mice Since 115In is a rather rare element, it is not surprising that naïve mice do not have many 115In-positive events within the draining lymph node. On average, control mice had 0.0056 ± 0.0008 % total indium events in the draining lymph node and no 115In-positive cellular events were observed. 115In-positive events found in the lymph node of mice treated with 2.5% 115In-AlO(OH) can therefore be attributed to the adjuvant itself.    4.3.4. Kinetics of 115In-AlO(OH) within the Draining Lymph Node Figure 4-4A illustrates the kinetics of total 115In-postiive events and non-cell associated 115In-AlO(OH) nanoparticles within the draining lymph node;115In was elevated significantly elevated when naïve animals at all time points, with the exception of day 5. Representative plots for day 15 post-injection showed that 4.2% of the total events in the lymph node are 115In-positive (Figure 4-4B). Nearly 2% of the events within the lymph node cell unassociated 2.5% 115In-AlO(OH) nanoparticles (Figure 4-4C). Examination of the nanoparticle population 42  confirmed that these events were stained with cisplatin and poorly labeled with phenotypic markers (Figure 4-4D and E).  Figure 4-3: Kinetics of total 115In-postitive events and non-cell associated 115In-AlO(OH) nanoparticles within the draining lymph node (A). Representative plots showing the gating for total 115In-postitive events (B) and cell-free 115In-AlO(OH) nanoparticles (C). The cell-free particles were cisplatin positive (D) and phenotypic marker negative (E). 115In-positive events appeared within the draining lymph node within 1 day, peak at 15 days and slowly clear from that point onward. Cell unassociated 2.5% 115In-AlO(OH) nanoparticles represent on average 33.9 ± 10.1 % of the total 111In events within the lymph node. The fact that the AlO(OH) dissolves poorly in vivo and the presence of a significant population within the cell-free nanoparticle gate suggests that a portion of the 115In-positive cellular events are likely adjuvant nanoparticles.  4.3.5. Cellular Distribution of 115In-AlO(OH) within the Draining Lymph Node  Table 4-2 lists the phenotypic markers used to define various cellular populations within the draining lymph node (15, 114, 115). Figure 4-5 outlines the gating strategy used to assess the fraction of each cell population that were positive for the 115In tracer.  Cell Population Phenotypic Markers T cells CD45+, CD3+, CD19- B cells CD45+, CD19+, CD3- Natural Killer (NK) CD45+, CD3-, CD19-, NK1.1+ Dendritic cells (DC) CD45+, CD3-, CD19-, NK1.1-, CD11c+, CD11b+/- Macrophages  CD45+, CD3-, CD19-, NK1.1-, F480+, CD11b+/- Granulocytes CD45+, CD3-, CD19-, NK1.1-, CD11b+, Gr1+ Table 4- 2: Definition of cell populations for the CyTOF analysis.  43    Figure 4- 4: Gating strategy to assess the phenotype of 115In-positive cellular events. Non-calibration beads were initially selected (A) followed by live singlets (B and C). CD45+ events representing pan-leukocytes (D) were divided into CD3+, CD19+ and CD3-/CD19- populations (E). 115In-positive B cells (F) and T cells (G) were then selected. NK cells were identified from the CD3-/CD19- population (H) its 115In-positive subset (I). Non-NK cells were divided based on their expression of CD11b and Gr-1 (J) and 115In- positive granulocytes were identified (K). Non-granulocytes with were divided into macrophages (L) and dendritic cells (M); 115In-high regions were selected from these populations (N and O).   44   Figures 4-6A and B show that there was a statistically significant increase in the number of B cells, dendritic cells, macrophages and total cell count within the draining lymph nodes 10 days post-administration of 2.5% 115In-AlO(OH). Previous reports have observed a similar changes in animals administered an aluminum-based adjuvant (52).   Figure 4- 5: Changes in the number of total cells, T cells and B cells (A) along with NK cells, DCs, macrophages and granulocytes (B) in the draining lymph node over time. An asterisks indicates a significant change in the size of the cell population in relation to naïve mice not treated with 2.5% 115In-AlO(OH).   Furthermore, a statistically significant increase in the percentage of macrophages and dendritic cells for 115In was observed on days 5, 10 and 15 post-injection (Figure 4-6). A population of 115In-positive B cells was identified on days 1, 5, 10 and 15 post-injection. Although there were more 115In-positive B cells dendritic cells and macrophages, the proportion of B cells positive for the dopant was small due to the higher number of B cells within the lymph node. The increased proportion of 115In within the B cells, dendritic cells and macrophages suggests that these APCs may be actively internalizing or sequestering the adjuvant.   Figure 4- 6: Changes in the proportion of a cell population over time that are 115In-positive. The boxed insert helps to visualize the sequestration of the 115In dopant within the B cell population. An asterisks indicates a significant increase compared to control mice not exposed to 2.5% 115In-AlO(OH). 45  Dendritic cells within the lymph node that stained 115In-positive on day 1 post-injection showed upregulation of maturation markers (MHCII and CD86) compared to 115In-negative dendritic cells (Figure 4-7). There was a non-significant trend towards higher expression of MHCII/ CD86 in 115In-postive dendritic cells on the other assessed days.    Figure 4- 7: Upregulation of maturation markers in 115In-postiive dendritic cells.               1 5 10 15 40020406080100120% of Cells MHCIIhi CD86hiTime (days) All DCs 115In-Positive DCs*46  4.4 Discussion Although nanomaterials have been used in mass cytometry before (i.e., NaHoF4 nanoparticles), they were only used to increase the amount of metal per antibody to more efficiently detect surface markers that are expressed at low levels (116). These nanoparticle-labeled antibodies are akin to using a brighter fluorophore in conventional flow cytometry. CyTOF has not been used for any other purpose beyond immunophenotying to date (107). A novel application of mass cytometry was developed in this chapter to assess the kinetics and cellular distribution of 2.5% 115In-AlO(OH). This technique has widespread applications within the nanomedicine field since any nanomaterials containing heavy metals, such as gold or silver nanoparticles, can be directly tracked in vivo in a high-dimensional single-cell manner.   Mass cytometry was used in this chapter to show that nanoparticles of indium-doped AlO(OH) distribute from the site of injection to the draining lymph node. 115In-positive events were detected as early as 1 day post-administration and peaked at day 15. Using 500 nm fluorescent latex beads as a surrogate for Alhydrogel, Khan et al. found that the beads were observed in the draining lymph node within 1 day, but reached peak levels only at day 4 post-injection (45). Differences in the size, zeta potential, morphology and so forth between 115In-AlO(OH) and the surrogate material provide an explanation for the above discrepancy. As discussed in chapter 2, indium-doped AlO(OH) very closely matches the properties of Alhydrogel and is a more representative tracer for the commercial adjuvant than latex beads.  Approximately 30% of the observed 115In events within the lymph node at each time point were identified as 115In-AlO(OH) nanoparticles not associated with a cell (115Inhi, 191/193Irlo, 195Pthi, phenotypic markerlo events). The presence of these particles corroborates the 1955 report by White et al. that observed extracellular granular masses within the sinus of the draining lymph node 1 day post-injection (52). Khan et al. however, only observed the surrogate tracer within cells in the draining lymph node (45). At 500 nm in diameter, the surrogate material used in Khan’s study were too large to enter the afferent lymphatic vessels and must be transported by an APC into the lymphatic system (48). The component nanoparticles of aluminum-based adjuvants are smaller enough (~10 x 5.5 nm) to efficiently enter the lymphatic vessels on their own from the site of injection (48).  There are currently 2 theories in the literature to explain how antigen adsorbed to an aluminum-based adjuvant enters the draining lymph node. First, de-adsorbed antigen can freely enter the lymph and be captured by resident dendritic cells and follicular B cells in the lymph node (117-119). Alternatively, adsorbed antigens can be phagocytosed at the site of injection and transported by APCs to the lymph node (15, 120). Since the adjuvant is coated with antigen, the cell unassociated particles may help to carry antigen to the lymph node and establish an additional depot at this site (38). A secondary antigen depot may help to clarify some of the controversy around the requirement of an injection site depot to generate an immune response to an aluminum-adsorbed vaccine (6, 25). Additional studies are required to assess this new theory. 47  The remainder of the 115In events observed in the lymph node with CyTOF were associated with cellular events. Unfortunately, mass cytometry cannot differentiate between extracellularly bound indium-doped AlO(OH) nanoparticles, internalized nanoparticles or cell-bound 115In released from dissolved adjuvant particles. However, the presence of a large population of cell unassociated adjuvant particles and the poor solubility of AlO(OH) suggests that a proportion of the 115In-positive cellular events are nanoparticles (44).  A statistically significant increase in the proportion of dendritic cells and macrophages positive for 115In was observed on days 5, 10 and 15 post-injection. Previous work in vitro has shown that APCs can internalize aluminum-based adjuvants (15, 39). Although the depletion of macrophages had a minimal impact on the immune response to an aluminum-adsorbed vaccine, depletion of monocytes and dendritic cells abrogated the response (29, 34). 115In-positive dendritic cells had increased expression of maturation markers (MHCII and CD86) on day 1 post injection. Work by Sokolovska et al. confirms that murine dendritic cells activate/mature in the presence of aluminum-based adjuvants (30). Monocyte-derived migratory dendritic cells from the injection site have been shown to orchestrate the adaptive immune response of aluminum-adsorbed vaccines, not residence dendritic cells in the lymph node (16, 117). It is currently unknown from the CyTOF analysis if the 115In-positive dendritic cells migrated to the lymph node or are resident cells in the lymph node. Khan et al. found that the surrogate materials in the lymph node were sequestered within macrophages and monocyte-derived dendritic cells 4 days post-administration (45). White et al. observed aluminum adjuvant-like granular masses in macrophages at day 1 post-injection (52). The immunological effects of aluminum precipitates within these populations in the draining lymph node remains to be explored.  A population of 115In-positive B cells was identified on days 1, 5, 10 and 15 post-injection. Nanoparticles <200 nm coated with adjuvant in the lymph node have been previously shown to be particularly effective at stimulating humoral immune responses since the unprocessed antigen can be recognized and efficiently phagocytosed by antigen-specific B cells (48). When administered intraperitoneally, aluminum-based adjuvants were found to be taken up by resident B-cells (15). It is unknown if only antigen specific B cells phagocytose aluminum-based adjuvants and the general immunological consequences of the adjuvant within this cell type.       48  Chapter 5: STEM-Based Assessment of Alhydrogel’s Biodistribution 5.1. Introduction Alhydrogel is poorly soluble in physiological fluids and has been shown to persist at the site of injection for upwards of 12 years (43, 44). Electron micrographs of the injection site of Alhydrogel-adsorbed vaccines in patients with MMF have shown that the adjuvant particles are phagocytosed by macrophages and maintain their needle-like morphology within these cells (43). To further validate 2.5% 115In-AlO(OH) as a tool to study Alhydrogel, STEM imaging was used to assess the morphology of the adjuvant within the injection site nodule after 70 days. Atomic mapping was also used to assess the long-term persistence and concentration of the indium dopant in vivo.  Data from the previous CyTOF analysis suggested that nanoparticles of 2.5% 115In-AlO(OH) reached peak levels in the draining lymph node by day 15 post-injection before slowly clearing from the tissue. STEM imaging was used in this chapter to confirm the presence of Alhydrogel particles within the inguinal lymph nodes. A later time point, 70 days, was assessed seeing how other investigators have already observed structures that resemble aluminum adjuvant particles at this site within a day of administration (52, 54). The translocation of AlO(OH) particles to the liver is supported by the biodistribution data from surrogate nanomaterials, nanodiamonds functionalized Alhydrogel, 111In-AlO(OH) and 26Al-AlO(OH) (45, 50, 53). The distribution of Alhydrogel to the liver is further supported by the results of a recent phase IIa clinical trial of a therapeutic hepatitis B vaccine (121). Patients in the control group of this trial received 6 intramuscular injection of an aluminum-based adjuvant with no adsorbed antigen (121). Surprisingly, this group had a reduced viral load and developed antibodies against the hepatitis B e antigen (HBeAg) to a similar extent as the treatment group (121). Wang et al. replicated the trial in mice and observed that repeated injections of Alhydrogel alone resulted in a high number of spotty necrotic foci in the liver, the site of infection for the hepatitis B virus (122). Although the cause of these observations is currently unknown, the presence of Alhydrogel particles in the liver may provide some clarity since the adjuvant is well known to cause necrosis at the site of injection (15, 26, 27). The accumulation of AlO(OH) nanoparticles in the liver would not be surprising, given that this organ is part of the reticuloendothelial system and is efficient at sequestering nanomaterials (104). STEM imaging combined with atomic mapping was used to search for Alhydrogel particles in the liver after 70 days post-administration in this chapter.   49  5.2. Materials and Methods 5.2.1. Animal Immunizations and Tissue Fixation C57BL/6 mice (n=2/ group) between 6-8 weeks old were injected into the posterior region of the dorsal surface with 100 µg of OVA adsorbed to 0.7 mg of 2.5% 115In-AlO(OH) or Alhydrogel in a total volume of    100 µL. After 70 days, the animals were sacrificed via CO2 asphyxiation while under anesthesia with isoflurane. The site of injection/ nodule was removed in all animals as well as the draining inguinal lymph nodes and liver in those administered Alhydrogel. A 2-3 mm3 block of each tissue was fixed for 1 hour in a solution of 2.5% glutaraldehyde (Polyscience BLI1909) and 2.5% paraformaldehyde (Polysciences 18814) in 0.1M sodium cacodylate buffer at pH 7.4 (Ted Pella 18851) (123, 124). The samples were then placed into fresh fixative for an additional 24 hours at 4˚C (123, 124).   5.2.2. Tissue Embedding and Preparation for STEM Imaging The fixed tissue was rinsed thrice with 0.1 M sodium cacodylate buffer at pH 7.4 and post-fixed in buffer 1% OsO4 (Polysciences 0972B) for 2 hours at 4˚C (123, 124). The samples were rinsed thrice with 18 MΩ water and then taken through a graded dehydration with ethanol (123, 124). Post-dehydration, the samples were infiltrated with Spurr resin (Ted Pella 18300-4221) using a Pelco 3441 laboratory microwave under vacuum; the first infiltration was in 50% resin using acetone as the diluent followed by three 100% resin steps (125, 126). The resin was then polymerized over a period of 72 hours at 65˚C (125, 126). The blocks were sectioned 200 nm thick on a Leica Ultracut 7 ultramicrotome before mounting onto formvar and carbon backed 200 mesh copper grids (Ted Pella 01810).   5.2.3.  STEM Imaging and Data Analysis The mounted sections were systematically searched under HAADF TEM mode with the Technai Osiris microscope described in chapter 2. When a region of higher atomic number with a similar morphology to AlO(OH) nanoparticles was identified, it was atomically mapped under STEM mode. A minimum of 3 slices/ tissue type/ animal were assessed. The EDX data was analyzed and corrected for Bremstralung radiation using Esprit 1.9 software (Bruker). Representative pictures are shown below.  5.2.4. Ethics Statement All animal experiments were carried out in accordance with the guidelines of the University of British Columbia’s Animal Care Committee under the approved protocol A14-0159. 50  5.3.  Results 5.3.1. Persistence, Morphology and Cellular Infiltration of Alhydrogel and 2.5% 115In-AlO(OH) at the Site of Injection 70-days post-administration, a considerable amount of adjuvant could still be found at the site of injection within an inflammatory nodule (Figure 5-1). These nodules form within 4 hours of injection in a fibrinogen/fibrin-dependent manner (127). The long-term persistence of Alhydrogel nodules helps to explain why the adjuvant is administered into deep tissue layers, such as the muscle, to minimize adverse events like pruritus (128, 129).  Figure 5- 1: Injection site nodule from the surface of the skin (A) and after retracting the skin (B) 70 days post-administration. Both 2.5% 115In-AlO(OH) and Alhydrogel maintained their characteristic needle-like morphology after a prolonged period in vivo (Figures 5-1 and 5-2). Elemental mapping of the 2.5% 115In-AlO(OH) injection site showed that the heavy metal dopant was still associated with the adjuvant. The amount of indium detected was equivalent to 2.16 ± 0.28% of the aluminum atoms, a non-statistical difference from before administration (Figure 5-2).   Figure 5- 2: Alhydrogel at the site of injection (A). Atomic mapping for aluminum (B) in the region outlined by the green box in panel A. Higher magnification view of the region in the yellow box in panel A showing that the needle-like morphology of Alhydrogel was maintained after 70 days in vivo (C).  51   Figure 5- 3: Morphology and elemental mapping of 2.5% 115In-AlO(OH) at the site of injection 70 days post-administration. The top panel shows a reference HAADF image and the distribution of aluminum (red), indium (green) and oxygen (blue). The bottom panel shows the EDX spectrum (left) and results from the quantification of the EDX data (right).  Cells could be found dispersed throughout the aluminum-based adjuvant within the injection site nodule. The exact phenotype of the cell infiltrate could not be determined with the data collected. Figure 5-3 is a representative image from the Alhydrogel injection site 70-days post-administration. The presence of cells was confirmed via elemental mapping for osmium, which stains the DNA within nuclei (123, 124). Other investigators have previously shown that aluminum-based adjuvants attract various cell types, such as monocytes and B cells, to the site of injection (15, 16, 52).   Figure 5- 4: Cellular infiltrate within the Alhydrogel injection site. Aluminum is displayed as a heat map while the insert shows the distribution of osmium in purple. 52  5.3.2. Alhydrogel Particles within the Draining Lymph Node 70 Days Post-Injection Figure 5-4 shows an representative image of an aluminum aggregate within the dLN of a mouse administered Alhydrogel 70 days earlier into the subcutaneous fat. This finding helps to validate the observations from the CyTOF assessment in chapter 4.  Figure 5- 5: An representative aggregate of Alhydrogel within the draining lymph node. The distribution of aluminum is presented as a heat map.   5.3.3. Alhydrogel Particles within the Liver After 70 Days Post-Injection Nanoparticles of Alhydrogel with a distinct needle-like morphology were identified in the liver 70 days post-administration of the adjuvant. The adjuvant nanoparticles appear to be intracellular, although the type of cell and its subcellular location were difficult to determine. The liver is part of the reticuloendothelial system and contains a large number of Kupffer cells, a specialized type of macrophage, that are highly efficient at clearing nanomaterials from the systemic circulation (104).  53   Figure 5- 6: Alhydrogel nanoparticles within 3 different sites of the liver from 2 different animals. The distribution of aluminum as a heat map is shown in the rightmost panels     54  5.4. Discussion Results from this chapter help to further validate indium-doped AlO(OH) as a traceable form of Alhydrogel. Both 2.5% 115In-AlO(OH) and the commercial adjuvant were found to maintain their needle-like morphology after an extensive duration in vivo. This finding is corroborated by the persistence of Alhydrogel nanoparticles at the site of injection in MMF patients (43).  The concentration of 115In within the adjuvant at the injection site was not statistically different from before administration, demonstrating the long-term stability of the doped particles. 2.5% 115In-AlO(OH) overcomes the stability issues associated with other tracer designs, such as electrostatically adsorbed nanodiamonds to Alhydrogel’s surface (53). The stability of the doped tracer and the direct observation of Alhydrogel particles in the lymph node at day 70 support the notion that most of 115In-positive events observed with CyTOF analysis are 2.5% 115In-AlO(OH) nanoparticles (chapter 4).  This chapter presented the first observation of an aluminum-based adjuvant distributing from the site of injection to the liver. The adjuvant nanoparticles maintained their distinct morphology in the liver and appeared to be intracellular. Additional assessments are required to determine the exact subcellular location and phenotype of the adjuvant positive cells. Since the liver clears nanomaterials from the systemic circulation, the Alhydrogel particles must have entered the blood (104). Although the details have yet to be elucidated, one can hypothesize that the particles entered via the lymphatic system since extracellular 115In-AlO(OH) nanoparticles are found in the draining lymph node and ablation of the nodes reduced the distribution of surrogate materials into the blood (45). The presence of AlO(OH) particle in the liver combined with the poor solubility of AlO(OH) under physiological conditions suggests that the initial burst phase of 111In-AlO(OH) and 26Al-AlO(OH) is due at least in part to the distribution of the adjuvant in particulate form. Although the presence of Alhydrogel particles within the liver may help to explain the necrotic foci observed by Wang et al., more detailed assessments are required before making such a claim (122).         55  Chapter 6: Conclusion and Future Work 6.1. Conclusion  The primary objective of this thesis was to improve the understanding of the biodistribution of aluminum-based adjuvants like Alhydrogel. Although the adjuvant has been used for nearly a century clinically, its biodistribution has been sparsely studied. This is not surprising, considering that even regulatory agencies do not require pharmacokinetic data for adjuvants (130).  In order to study the biodistribution of Alhydrogel in detail, a novel tracing strategy was developed where indium isotopes were incorporated into the AlO(OH) matrix using a highly controlled co-precipitation technique. Both the gamma emitter 111In and non-radioactive 115In were efficiently encapsulated into the adjuvant. A number of physical and functional assays were used to confirm that the adjuvant closely matched Alhydrogel. Specifically, the indium-doped adjuvant matched Alhydrogel with regards to its FTIR and DSC spectrum, protein adsorptivity and de-adsorptivity, size and morphology of the component nanoparticles, stability in biological media, size and zeta potential. The 111/115In-AlO(OH) tracer generated a similar humoral and cellular immune response when compared to Alhydrogel (34, 80-83). The rate release for adsorbed antigens (i.e., 67Ga-NOTA-OVA) between the indium-doped adjuvant and Alhydrogel from the site of injection was identical. In-AlO(OH) was found to persistent at the site of injection and maintain it’s needle-like morphology as has been observed for Alhydrogel in animals models and humans (21, 23, 43). Only minor difference were observed between the doped and commercial adjuvant, namely the presence of a small amount of Al(OH)3 in Alhydrogel and a minor increase in the cytotoxicity of 115In-AlO(OH). Compared to the other tracers used to date, indium-doped AlO(OH) is the most stable and representative tracer of Alhydrogel (45, 52).   The biodistribution data of both 111In-AlO(OH) and 26Al-AlO(OH) found that a small burst of activity was released from the injection site of shortly after administration followed by a slower phase of distribution (50). This pharmacokinetic profile is poorly explained by the dissolution of the tracer from the injection site given the poor solubility of AlO(OH) in biological media (44, 50, 51). A novel application of CyTOF and STEM imaging was used to show that the radioactive signal in the liver and draining lymph node was at least partially due to the presence of adjuvant nanoparticles. Within the draining lymph node, the adjuvant particles reached peak levels at day 15 and were sequestered into populations of APCs; adjuvant-positive dendritic cells also showed signs of maturation. The distribution of Alhydrogel nanoparticles supports the hypothesis of antigen depots forming in tissues beyond the site of administration (38).   This study provided the most detailed assessment to date of the biodistribution of Alhydrogel. The observation of adjuvant particles beyond the site of injection departs from the long term belief in the vaccinology community that aluminum-based adjuvants remain at the site of injection and slowly dissolve over time. This 56  finding opens a Pandora’s box of questions as the immunological and toxicological effects of the adjuvant now have to be studied at these new sites.   6.2. Future Work The biodistribution data from 111In-AlO(OH) in chapter 3 found radioactivity in tissues such as the draining lymph node, spleen, liver, kidney and bone. Work in this thesis found that the 111In signal was at least in part attributed to the adjuvant in nanoparticle form within the draining lymph node and liver. The other organ systems with 111In uptake need to be assessed with techniques like CyTOF and STEM imaging to determine if signal is due to the radionuclide in soluble form or nanoparticles of the adjuvant. Techniques such as ImmunoGold can be used to obtain data about the subcellular location of the adjuvant nanoparticles with electron microscopy (131). To better understand Alhydrogel’s toxicity, MTT and related assays should be carried out on the predominant cell types found where the nanoparticles of the adjuvant distribute. Additional work is required to assess if rare side effects, such as ASIA, are due to the direct toxicity of the particles or the long-term pro-inflammatory effects of bio-persistent nanomaterials at distant sites (42, 45, 46).  To better understand the immunological consequences of the adjuvant, the CyTOF panel used in the thesis can be readily expanded (106). Additional phenotypic markers will allow the different cell populations to be more specifically defined and answer key questions. For example, are the 115In-positive dendritic cells with upregulated maturation markers in the lymph node resident cells or a migratory cell from the site of injection?  To assess if a secondary antigen depot is formed in the lymph node, as theorized in this thesis, a variety of techniques are required. ImmunoGold staining, electron microscopy and atomic mapping can be used together to determine if the extracellular Alhydrogel in the lymph node is coated with antigen (131). By labeling the antigen with a heavy metal, CyTOF can be used to assess the cellular distribution of the antigen in relation to the adjuvant. Finally, 67Ga-NOTA-OVA, described in chapter 3, can be used to quantify the amount of antigen within the draining lymph node when administered as a soluble antigen or adsorbed to an aluminum precipitate.   Long-lived inflammatory nodules at the site of injection form rapidly following administration to help entrap the adjuvant (127). Interestingly, when pre-formed aluminum precipitates are administered into fibrinogen-deficient mice, a nodule does not form and cell-mediated immunity is enhanced to the adsorbed antigen (127). Next-generation aluminum-based adjuvants that minimize aggregation of the component nanoparticles have also shown enhanced cellular immunity (132-135). The techniques established in this thesis can be used to assess the distribution of Alhydrogel particles in a fibrinogen knockout model and next-generation aluminum-based adjuvants to determine if the pharmacokinetics of the particles correlates with enhanced cellular immunity. It would also be interesting to assess if the application of ultrasound to the injection site, which is 57  known to disrupt Alhydrogel aggregates in vitro, can enhances the distribution of the adjuvant and alter the type of immune response generated (14).  This thesis focused on the distribution of Alhydrogel from the subcutaneous space. In clinical practice, the adjuvant is commonly also administered intramuscularly and it would be interesting to assess if the site of injection impacts the distribution of the adjuvant (3, 8, 9). Finally, this thesis concentrated solely on the biodistribution of crystalline AlO(OH); techniques developed here should be applied to study the movement of the amorphous aluminum-based adjuvants, such as aluminum hydroxyphosphate (12).                    58  References  1. Ramon G. Sur l'augmentation anormale de l'antitoxine chez les chevaux producteurs de sérum antidiphtérique. Bull Soc Centr Med Vet. 1925;101:227-34. 2. Ramon G. 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One option would be to dope the crystal structure with a longer lived radioisotope, like 51Cr (27.8 day half life), which allows the adjuvant to be quantitatively tracked for ~140 days. However, most labs do not have access to high-level radiation facilities and nuclear imaging equipment to use such tracers. A non-radioactive adjuvant that can be tracked for a long period of time might be useful to other groups that wish to examine the biodistribution of aluminum-based adjuvants.  Gadolinium (Gd) is a paramagnetic element that shortens both longitudinal (T1) and transverse (T2) relaxation times in magnetic resonance (MR) imaging (136). Given recent articles about Gd-doped nanoparticles and layered double hydroxides as contrast agents, experiments were undertaken to explore the potential of Gd-AlO(OH) as a MR-traceable form of Alhydrogel (137-139).  GdCl3∙6H2O was co-precipitated with AlCl3∙6H2O as described in chapter 2 of this thesis. PXRD and FTIR data show that Gd-AlO(OH) closely matches commercial Alhydrogel (Figure A-1). The crystal structure appears to become more amorphous as the [Gd] is increased.   Figure A- 1: FTIR (A) and PXRD (B) of 0.5-10% Gd-AlO(OH).     69  In order to evaluate the effectiveness of Gd-AlO(OH) as a MR contrast agent, the relaxation properties of the adjuvant were examined using a Biospec 7.0 T pre-clinical scanner (Bruker). Figure A-2 shows that 0.45 %w/v solution of 0.5-2.5% Gd-AlO(OH) (equivalent 380, 760 and 1900µM Gd) provided MR contrast. The relativity values for the MR responsive adjuvants are listed in Table A-1. . Gd-AlO(OH) maybe useful to assess the amount of adjuvant remaining at the site of injection over a longer timeframe that was achievable with 111In-AlO(OH).    Figure A- 2: MR phantom scan of Alhydrogel (1), 1000µM Gadovist (2), 0.5% Gd-DTPA (Gadovist; 2), 0.5% Gd-AlO(OH) (3), 1% Gd-AlO(OH) (4-5) and 2.5% Gd-AlO(OH) (6).  Adjuvant ([Gd]) T1 T2* R1 R2* 0.5% Gd-AlO(OH) (380μM) 127.49 8.08 0.0078 0.12 1% Gd-AlO(OH) (760μM) 62.91 2.05 0.015 0.48 2.5% Gd-AlO(OH) (1900μM) 49.89 0.78 0.020 1.3 Table A- 1: Relaxivity values for Gd-AlO(OH) in a 7T MRI.     1 2 3 4 5 6 70    Figure A-3 shows a phantom scan of 1% Gd-AlO(OH) serially diluted by a factor of 10 from 0.45 w/v% (760µM Gd). No appreciable contrast was observed after a 1/100 dilution of the adjuvant. Gd-AlO(OH) may not be a sensitive enough tracer to assess the distribution of Alhydrogel to sites beyond the injection site.   Figure A- 3: MR phantom of 1% Gd-AlO(OH) adjuvant at 0.45%w/v (760µM Gd) (1). The adjuvant was diluted 1/10 serially (3-6). 1000µM Gadovist control (2).   1 2 3 4 5 6 

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