Vaccine Adjuvants: Methods and Protocols (Methods in Molecular Biology, 626)

✍ Scribed by Gwyn Davies (editor)

• Publisher: Humana
• Year: 2010
• Tongue: English
• Leaves: 314
• Category: Library

✦ Synopsis

Spanning from discoveries in fundamental immunology to industrial and commercial concerns, the study of vaccine adjuvants has developed into an exciting area of work with great, vital potential in innovating techniques in which adjuvants may steer the immune system towards the responses required by unmet vaccination needs. In Vaccine Adjuvants: Methods and Protocols, expert researchers in the field provide clear and concise guidance on how to go about assessing the activity of adjuvant products. Rather than describing individual adjuvants, the volume strives to include detailed, practical information on measuring the responses produced by adjuvants in order to be relevant to the widest array of experiments. Written in the highly successful Methods in Molecular Biology™ series format, chapters include introductions to their respective topics, lists of the necessary materials and reagents, step-by-step, readily reproducible laboratory protocols, and notes on troubleshooting and avoiding known pitfalls.

Authoritative and versatile, Vaccine Adjuvants: Methods and Protocols will enable those already pursuing vaccine adjuvant research, while also serving to stimulate discussion on how to best standardize adjuvant testing in order to facilitate meaningful comparisons, and above all, to aid in the prediction of which new products will most effectively and safely help to solve the current challenges in vaccination.

✦ Table of Contents

Preface
Contents
Contributors
1 Immunology of Vaccine Adjuvants
1 Introduction
2 Signal 1 and Signal 2 Facilitators
3 Signal 1 Facilitators
3.1 Oil-Based Emulsions
3.2 Aluminum-Based Adjuvants
4 Signal 2 Facilitators
4.1 Signal Zero, PAMPs, and Stranger Motifs
4.2 Danger Signals, Alarm Signals, or Damage-Associated Molecular Patterns (DAMPs)
4.3 Recombinant Cytokines or Co-stimulatory Molecules Mimicking Endogenous Immune Amplifiers
4.4 Release of the Brakes
5 Outlook
References
2 Preclinical Development of AS04
1 Introduction
2 The AS04 Adjuvant System
2.1 Aluminum Salts
2.2 Monophosphoryl Lipid A (MPL)
2.3 Formulation
3 The Preclinical Development of AS04
3.1 Immunological Evaluation of MPL
3.2 Safety Studies
3.3 Functional Properties
4 The Added Value of AS04 in the Clinic
5 Conclusions
Acknowledgments
References
3 Nonclinical Safety Assessment of Vaccines and Adjuvants
1 Introduction
2 Regulatory Considerations
3 Toxicology Studies Performed for Vaccines
3.1 Selection of Animal Models
3.2 Immunogenicity Evaluation
3.3 Toxicology Studies
3.3.1 Single-Dose Toxicity Studies
3.3.2 Repeat-Dose Toxicity Studies
3.3.3 Local Tolerance Studies
3.3.4 Safety Pharmacology Studies
3.3.5 Developmental and Reproductive Toxicity Studies
3.3.6 Genotoxicity Studies
3.3.7 Carcinogenicity Studies
3.3.8 Other Toxicity Studies
3.4 Toxicology Studies Required for Adjuvants Alone
4 Evaluation of Toxicology Data and Risk Assessment
5 Conclusion
Acknowledgments
References
4 Aluminum Adjuvants: Preparation, Application, Dosage, and Formulation with Antigen
1 Introduction
1.1 Preparation of Aluminum Adjuvants
1.2 Application of Aluminum Adjuvants
1.3 Dosing Aluminum Adjuvants
1.4 Temperature Stability of Aluminum Adjuvants
1.5 Relevant Monographs
1.6 Formulation with Antigen
2 Materials
2.1 Production of Aluminum Adjuvants
2.1.1 Aluminum Hydroxide ad modus Gupta and Rost
2.1.2 Method for Preparation of Aluminum Phosphate Adjuvant ad modus WHO ( 40 )
2.2 Testing of Aluminum Adjuvants
2.2.1 Determination of Aluminum
2.2.2 Determination of the Protein Adsorption Capacity
3 Methods
3.1 Production of Aluminum Adjuvants
3.1.1 Aluminum Hydroxide ad modus Gupta and Rost
3.1.2 Method for Preparation of Aluminum Phosphate Adjuvant ad modus WHO ( 40 )
3.2 Testing of Aluminum Adjuvants
3.2.1 Determination of Aluminum
3.2.2 Determination of the Protein Adsorption Capacity ( see Notes 6--8)
4 Notes
References
5 Freund's Complete and Incomplete Adjuvants, Preparation, and Quality Control Standards for Experimental Laboratory Animals Use
1 Introduction
1.1 Freund's Complete Adjuvant
1.2 Freund's Incomplete Adjuvant (FIA)
1.3 The Mineral Oil
2 Materials
2.1 Preparation of Freund's Complete Adjuvanted Experimental Vaccines
2.2 Preparation of Freund's Incomplete Adjuvanted Experimental Vaccines
2.3 Immunization Procedures
2.4 Comparative Tests to Measure the Safety and Efficacy of Adjuvants
3 Methods
3.1 Preparation of Freund's Complete Adjuvanted Experimental Vaccines
3.2 Preparation of Freund's Incomplete Adjuvanted Experimental Vaccines
3.3 Immunization Procedures
3.4 Comparative Tests to Measure the Safety and Efficacy of Adjuvants
3.4.1 Haemolytic Activity of Adjuvants: Spectrophotometric Determination of Haemoglobin Release
3.4.2 Haemolytic Activity of Adjuvants: Creatine Kinase Assay
3.4.3 Rabbit Pyrogenicity Assay
3.4.4 Endotoxin Assays: Mouse Weight Gain Test
3.4.5 Practical Procedures in the Preparation and Application of Adjuvant Mixtures
3.4.6 Recommended Volumes of Injection Doses and Routes of Inoculation for Animals
4 Notes
References
6 Liposomal Adjuvants: Preparation and Formulation with Antigens
1 Introduction
2 Materials
2.1 Starting Raw Materials and Equipment
2.1.1 Liposomal Lipids, Lipoidal Immunostimulants, and Selected Buffers
2.1.2 Equipment for the Homogenization and Filtration of Liposomes
2.1.3 Ethanol Injection
2.1.4 Detergent Dialysis
2.2 Purification of Liposomal Antigen
2.2.1 Sucrose Gradient
2.2.2 Ultracentrifugation
2.2.3 Size Exclusion Chromatography
2.3 Extraction and Determination of Liposomal Antigen
2.4 Equipment for the Characterization of Liposomes
2.4.1 HPLC of Lipids
2.4.2 Particle Sizers
2.4.3 Zetameter
2.4.4 SDS-Polyacrylamide Gel Electrophoresis (SDS-PAGE)
3 Methods
3.1 Liposome Preparation and Association with Antigens
3.1.1 Mixing Starting Lipid Ingredients
3.1.2 Preparation of Small Unilamellar Vesicles (SUVs) from Multilamellar Vesicles (MLVs)
3.1.3 Preparation of SUVs Directly by Ethanol Injection
3.1.4 Association of Antigens with Preformed Liposomes
3.1.5 Encapsulation of Soluble Antigens into Small Liposomes
3.1.6 Reconstitution of Hydrophobic Antigens into Liposomes by the Detergent Removal Technique
3.2 Separation of Liposomal Antigen from Non-associated Antigen
3.2.1 Sucrose Gradient Method
3.2.2 Ultracentrifugation Method
3.2.3 Gel Chromatography Method
3.3 Extraction of Antigen from Liposomal Formulation
3.4 Characterization and Quality Control of Liposomes and Liposomal Vaccines
3.4.1 Lipid Composition
3.4.2 Particle Sizing
3.4.3 Zeta Potential
3.4.4 Total Antigen Content and Amount of Antigen Associated to the Liposomes
3.5 Use in Preclinical Studies
4 Notes
References
7 Micro/Nanoparticle Adjuvants: Preparation and Formulation with Antigens
1 Introduction
2 Materials
2.1 Synthesis of Anionic Blank PLG Microparticles
2.2 Synthesis of Cationic Blank PLG Microparticles
2.3 Synthesis of Encapsulated PLG Microparticles
2.4 Synthesis of Blank PLG Nanoparticles
2.5 Adsorption of Protein Antigens
2.6 Adsorption of DNA Plasmid Antigens
2.7 Lyophilization of PLG/Antigen Formulations
2.8 Characterization of Formulations
3 Methods
3.1 Synthesis of Anionic PLG Microparticles
3.2 Synthesis of Cationic Blank PLG Microparticles
3.3 Synthesis of Encapsulated PLG Microparticles
3.4 Synthesis of Blank PLG Nanoparticles
3.5 Adsorption of Protein Antigens
3.6 Adsorption of DNA Plasmid Antigens
3.7 Lyophilization of PLG/Antigen Formulations
3.8 Characterization of Formulations ( see Notes 6 and 7)
3.8.1 Sizing
3.8.2 Osmolarity
3.8.3 Endotoxin
3.8.4 SDS-PAGE and Agarose Gel Electrophoresis
3.8.5 HPLC
4 Notes
Acknowledgments
References
8 Adjuvant Activity on Human Cells In Vitro
1 Introduction
2 Materials
2.1 Monocyte-Derived Dendritic Cells
2.1.1 Preparation of Monocyte-Derived DCs and Incubation with TLR Ligands
2.1.2 Analysis of Ligand Activities on Monocyte-Derived DC Maturation
2.1.3 Analysis of Ligand Activities on Cytokine Production by Monocyte-Derived DCs
2.2 Whole Blood Assays
2.2.1 Stimulation of Human Blood Cells
2.2.2 Analysis of the Phenotype of Circulating DCs and Monocytes
2.2.3 Cytokine Production in Whole Blood
2.2.4 Determination of Cell Types in Whole Blood Responsible for Cytokine Production
2.2.5 Stimulation of Freshly Isolated DCs
2.3 HEK 293 Cells
3 Methods
3.1 Monocyte-Derived DCs
3.1.1 Preparation of Monocyte-Derived DCs and Incubation with TLR Ligands
3.1.2 Analysis of Ligand Activities on Monocyte-Derived DC Maturation
3.1.3 Analysis of Ligand Activities on Cytokine Production by Monocyte-Derived DCs
3.2 Whole Blood Assays
3.2.1 Stimulation of Human Blood Cells
3.2.2 Analysis of the Phenotype of Circulating DCs and Monocytes
3.2.3 Cytokine Determination
3.2.4 Determination of Cell Types in Whole Blood Responsible for Cytokine Production
3.2.5 Stimulation of Freshly Isolated DCs
3.3 HEK 293 Cell System
4 Notes
References
9 Adjuvant Activity on Murine and Human Macrophages
1 Introduction
2 Materials
2.1 Material for Bone Marrow-Derived Macrophages (BMDM) in Mice
2.2 Activation of BMDM in Culture and Measurement of Cytokine Production
2.3 Assessment of Cell Activation by Flow Cytometric Analysis
2.4 Preparation of Resting and Elicited Peritoneal and BAL Macrophages from Mice
2.5 Material for Human Monocytes/ Macrophages Culture
3 Methods
3.1 Preparation of Murine Bone Marrow-Derived Macrophages (BMDM)
3.2 Activation of BMDM in Culture and Measurement of Cytokine Production
3.3 Methods to Assess Cell Activation by Flow Cytometry
3.4 Preparation of Resting and Elicited Peritoneal and BAL Macrophages from Mice
3.4.1 Resident Peritoneal Macrophages
3.4.2 Thioglycollate Activated Macrophages
3.4.3 BAL Lavage Macrophages
3.5 Preparation and Culture of Human Blood-Derived Monocytes/Macrophages
3.5.1 Preparation of Human PBMC and Monocytes
3.5.2 Culture and Differentiation
3.5.3 Stimulation of Cytokine Production
3.5.4 mRNA Determination
4 Notes
References
10 In Vitro Effects of Adjuvants on B Cells
1 Introduction
2 Materials
2.1 Isolation and Culture of Human Peripheral Blood Mononuclear Cells (PBMC)
2.2 Isolation and Culture of Human B Cells
2.3 Culture of B Cell Lines
2.4 Cytokine Secretion
2.5 Cell Surface Activation
2.6 Intracellular Cytokine Measurement
2.7 B Cell Proliferation
2.8 Ab Secretion
3 Methods
3.1 Isolation and Culture of Human PBMC
3.2 Isolation and Culture of B Cells
3.3 Culture of B Cell Lines
3.4 Cytokine Secretion
3.5 Cell Surface Activation
3.6 Intracellular Cytokine Measurement
3.7 B Cell Proliferation
3.8 Ab Secretion
4 Notes
Acknowledgements
References
11 NKT Cell Responses to Glycolipid Activation
1 Introduction
2 Materials
2.1 NKT Cell Detection In Vitro
2.2 iNKT Cell Isolation and Culture
2.3 Measurement of Antigenic Potency
2.4 Measurement of Cytokine Release In Vitro
2.5 Serum Cytokine Measurement
2.6 NKT Activation In Vivo
2.7 In Vivo Dendritic Cell Maturation Assays
3 Methods
3.1 NKT Cell Detection In Vitro
3.1.1 Tetramer Staining of Mouse Mononuclear Cells
3.1.2 Tetramer Staining of Human or Nonhuman Primate (NHP) PBMC
3.1.3 Antibody Staining of Murine iNKT Cells
3.1.4 Human iNKT Cell Detection Using Antibodies
3.2 iNKT Cell Isolation and Culture
3.2.1 Mononuclear Cell (MNC) Preparations from Spleen
3.2.2 Mononuclear Cell Preparation from the Liver
3.2.3 Peripheral Blood Mononuclear Cell Preparation from Human and NHP PBMC
3.2.4 iNKT Cell Enrichment from Mouse MNCs Using Magnetic Beads
3.2.5 iNKT Cell Enrichment from Mouse MNCs Using FACS Sorting
3.2.6 Culture of NKT Cells in Mouse MNC Preparation from the Spleen, Thymus, LN, or Liver
3.2.7 Culture of iNKT Cells in Human or NHP PBMC
3.3 Measurement of Antigenic Potency
3.3.1 Measurement of Antigenic Potency Using iNKT Hybridoma
3.3.2 Measurement of Antigenic Potency Using APC
3.4 Measurement of Cytokine Release In Vitro
3.5 Serum Cytokine Measurements
3.6 NKT Activation In Vivo
3.7 In Vivo Dendritic Cell Maturation Assay
4 Notes
References
12 Tracking Dendritic Cells In Vivo
1 Introduction
2 Materials
2.1 Preparation of Y-Ae Antibody
2.2 Preparation and Purification of His-Tagged E 0 -GFP
2.3 Detection of E-- Peptide--MHC Complexes in Frozen Lymph Node Sections
2.4 Detection of E0 Peptide0MHC Complexes and Enhanced Detection of E0 - GFP in Tissue Sections
2.5 Co-localisation of pMHC Complexes and Dendritic Cell Markers
2.6 Immunofluorescence Microscopy
2.7 Immunohistochemical Detection of pMHC Complexes
2.8 Detection of pMHC Complexes by Flow Cytometry
2.9 Labelling Bone Marrow-Derived Dendritic Cells for In Vivo Imaging
2.10 Two-Photon Imaging of Dendritic Cells in Lymph Nodes In Vivo
3 Methods
3.1 Preparation of Y-Ae Antibody
3.2 Preparation and Purification of His-Tagged E 0 -GFP
3.3 Detection of E-- Peptide--MHC Complexes in Frozen Lymph Node Sections
3.4 Detection of E0 Peptide0MHC Complexes and Enhanced Detection of E0 - GFP in Tissue Sections
3.5 Co- localisation of pMHC Complexes and Dendritic Cell Markers
3.6 Immunofluorescence Microscopy
3.7 Immunohistochemical Detection of pMHC Complexes
3.8 Detection of pMHC Complexes by Flow Cytometry
3.9 Labelling Bone Marrow-Derived Dendritic Cells for In Vivo Imaging
3.10 Two-Photon Imaging of Dendritic Cells in Lymph Nodes In Vivo
4 Notes
References
13 Adjuvant Effects on Antibody Titre
1 Introduction
1.1 Enzyme-Linked Immunosorbent Assay
1.2 Developing Technologies
1.3 Specific Antigen ELISA
2 Materials
3 Methods
3.1 Plate Washing
3.2 ELISA
3.2.1 ELISA Plate Antigen Coating
3.2.2 Preparation of Sera for ELISA
3.2.3 Transfer to ELISA Plate for Assay
3.2.4 Developing ELISA Assay
3.3 Data Analysis
4 Notes
Acknowledgements
References
14 Functional Antibody Assays
1 Introduction
1.1 Toxin Neutralisation
1.2 Serum Bactericidal Antibody
1.3 Opsonophagocytic Killing
2 Materials
2.1 Toxin Neutralisation
2.2 Serum Bactericidal Antibody
2.3 Opsonophagocytic Killing
3 Methods
3.1 Toxin Neutralisation
3.2 Serum Bactericidal Antibody
3.3 Opsonophagocytic Killing
4 Notes
References
15 Determining Adjuvant Activity on T-Cell Function In Vivo: Th Cells
1 Introduction
2 Materials
2.1 Isolation of PBMCs
2.2 Isolation of Cells from Spleen and Lymph Nodes
2.3 Cell Culture and Antigen Recall
2.4 Conventional Capture-ELISAsA: IFN- ELISA; B: IL-17A ELISA
2.5 Cytometric Bead Analysis---Th1/Th2 Cytokine CBA
2.6 ELISPOTA: IFN- ELISPOT; B: IL-5 ELISPOT
2.7 Proliferation: CFSE Dilution Assay
2.8 Intracellular FACS Staining for Multifunctional T Cells
3 Methods
3.1 Isolation of PBMCs
3.2 Isolation of Cells from Spleen and Lymph Nodes
3.3 Cell Culture and Antigen Recall
3.4 Conventional Capture-ELISAsA: IFN-; B: IL-17A
3.5 Cytometric Bead Analysis---Th1/Th2
3.6 ELISPOTA: IFN-; B: IL-5
3.7 Proliferation: CFSE Dilution Assay
3.8 Intracellular FACS Staining for Multifunctional T Cells
4 Notes
Acknowledgements
References
16 Quantitative Multiparameter Assays to Measure the Effect of Adjuvants on Human Antigen-Specific CD8 T-Cell Responses
1 Introduction
2 Materials
2.1 Isolation of Peripheral Blood Mononuclear Cells (PBMCs) from Human Blood
2.2 Isolation of Human Lymphocytes from Tumor-Infiltrated Lymph Nodes (TILNs) or Primary Tumor Material (TILs)
2.3 Cryopreservation of Human Cells
2.4 Thawing of Human Cells
2.5 Ex Vivo Multimer Staining and Phenotyping of Human PBMCs or TILNs and Limiting Dilution Analysis (LDA)
2.6 ELISPOT Assay for Detection of IFN- -Producing Human CD8 T Cells
2.7 Intracellular Cytokine Staining
2.8 Carboxyfluorescein Succinimidyl Ester (CFSE) Proliferation Assay
3 Methods
3.1 Isolation of Peripheral Blood Mononuclear Cells (PBMCs) from Human Blood
3.2 Isolation of Human Lymphocytes from Tumor-Infiltrated Lymph Nodes (TILNs) or Primary Tumor Material (TILs)
3.3 Cryopreservation of Human Cells
3.4 Thawing of Human Cells
3.5 Ex Vivo Multimer Staining and Phenotyping of Human PBMCs and TILNs and Limiting Dilution Analysis (LDA)
3.5.1 Ex Vivo Multimer Staining and Phenotyping of Human PBMCs and TILNs
3.5.2 Limiting Dilution Analysis (LDA)
3.6 ELISPOT Assay for Detection of IFN- -Producing Human CD8 T Cells
3.7 Intracellular Cytokine Staining
3.8 Carboxyfluorescein Succinimidyl Ester (CFSE) Proliferation Assay
4 Notes
References
17 Large-Animal Model for Establishing E/T Ratio of Adjuvants
1 Introduction
2 Materials
2.1 Immunization
3 Methods
3.1 Immunization (see Note 2 )
3.2 Determination of Efficacy
3.3 Determination of Toxicity
3.3.1 Quantification of Local Reaction
3.4 References and Controls
3.5 Data Processing
4 Notes
References
18 Determining the Activity of Mucosal Adjuvants
1 Introduction
2 Materials
2.1 Preparation of Immunogen/Adjuvant Vaccine Formulations
2.2 Immunization of Animals
2.3 Serum and Mucosal Sample Collection and Storage
2.4 Assessment of Adjuvant Activity on Antibody Responses by Enzyme-Linked Immunosorbent Assay (ELISA)
2.4.1 Antibody Response to Immunogen and LTK63 or Other LT Mutants
2.5 Assessment of Adjuvant Activity on T-Cell Responses
2.6 Assessment of Adjuvant Activity on B Cells ( ELISPOT)
3 Methods
3.1 Preparation of Immunogen/Adjuvant Mixture
3.1.1 Immunogen/Adjuvant Mixture for Intranasal Immunization
3.1.2 Immunogen/Adjuvant Mixture for Intragastric Immunization
3.1.3 Immunogen/Adjuvant Mixture for Vaginal and Rectal Immunizations
3.1.4 Immunogen/Adjuvant Mixture for Intramuscular Immunizations
3.2 Immunization of Animals
3.2.1 Intranasal Immunization of Mice
3.2.2 Intragastric Immunization of Mice
3.2.3 Vaginal Immunization of Mice
3.2.4 Rectal Immunization of Mice
3.2.5 Intramuscular Immunization of Mice
3.3 Mucosal Sample Collection and Storage
3.3.1 Nasal Washes
3.3.2 Intestinal Washes
3.3.3 Vaginal Washes
3.3.4 Blood Collection
3.3.5 Organ/Tissue Collection
3.4 Assessment of Adjuvant Activity on Antibody Responses by Enzyme-Linked Immunosorbent Assay (ELISA)
3.4.1 Antibody Response to LTK63 and to Other LT Mutants
3.4.2 Antibody Response to Vaccine Antigens
3.5 Assessment of Adjuvant Activity on T Cells
3.5.1 Preparation of Single-Cell Suspensions
3.5.2 T-Cell Proliferation Assay
3.5.3 Cytokine Production Assay
3.6 Assessment of Adjuvant Activity on B Cells
3.6.1 Assessment of LTK63 Mutant-Specific and Vaccine Antigen-Specific Antibody-Secreting Cells (ASCs) by ELISPOT
3.7 Measurement of Functionally Active Antibodies In Vitro and In Vivo Evaluation of Efficacy
4 Notes
Acknowledgment
19 Adjuvant Activity of Cytokines
1 Introduction
1.1 Pattern-Recognition Receptors, Cytokines, Innate Immunity, and Establishment of the Adaptive Immune Response
2 The Human Interferon Superfamily
2.1 Type I Interferons
2.2 Type II Interferons
2.3 Type III Interferons
2.4 Activation of the Type I Interferon Pathway
2.5 Effect of Type I Interferons on the Innate Immune Response and Induction of Adaptive Immunity
2.6 Effect of Type I Interferons on Apoptosis and Antigen Presentation
2.7 Type I Interferons and Mucosal Immunity
2.8 Type I Interferons: Clinical Studies
2.9 Type II Interferons: Interferon (IFN- )
3 Interleukin 2 (IL-2)
4 Interleukin 12 (IL-12)
5 Interleukin 15 (IL-15)
6 Interleukin 18 (IL-18)
7 Interleukin 21 (IL-21)
8 Granulocyte Macrophage Colony-Stimulating Factor (GM-CSF)
9 Flt-3 Ligand
10 Conclusions
References
Subject Index