DNA Vaccines: Methods and Protocols

Preface
Contents
Contributors
Part I: Vector Design for Vaccination
	Chapter 1: A Novel Cre Recombinase-Mediated In Vivo Minicircle (CRIM) DNA Vaccine Platform for Veterinary Application
		1 Introduction
		2 Materials
			2.1 Construction of CRIM Plasmid
			2.2 Determination of Synthesis of Cre Recombinase
			2.3 Determination of the mcDNA Production
			2.4 Preparation of Bacteria Harboring CRIM Plasmid
		3 Methods
			3.1 Construction of CRIM Plasmid
			3.2 Determination of Synthesis of Cre Recombinase by Western  Blot
			3.3 Confirmation of the Production of mcDNA
			3.4 Bacterial Delivery of mcDNA In  Vivo
		4 Notes
		References
	Chapter 2: Vaccination with Messenger RNA: A Promising Alternative to DNA Vaccination
		1 Introduction
		2 The Emergence of Genetic Vaccination
		3 Pros and Cons of DNA and mRNA Vaccines
		4 mRNA Vaccine Types and Delivery
		5 Current Standing of the Field of mRNA Vaccines
			5.1 mRNA-Based Cancer Vaccines
			5.2 Infectious Disease mRNA Vaccines
		6 Challenges and Future Directions
		References
	Chapter 3: Impact of a Plasmid DNA-Based Alphavirus Vaccine on Immunization Efficiency
		1 Introduction
		2 Materials
			2.1 Reagents and Equipment
			2.2 Cell Lines
			2.3 Cell Culture Media
			2.4 Alphavirus Plasmid Vectors
		3 Methods
			3.1 Subcloning into SFV Vectors
			3.2 Transfection of Alphavirus Plasmid  DNA
			3.3 Verification of Transfection Efficiency
				3.3.1 GFP Detection
				3.3.2 X-Gal Staining
				3.3.3 Immunofluorescence
			3.4 Evaluation of Gene Expression
				3.4.1 Western Blotting
				3.4.2 Metabolic Labeling
			3.5 Immunizations
				3.5.1 DNA Immunization of C57BL/6  Mice
				3.5.2 DNA Immunization of BALB/c  Mice
				3.5.3 DNA Immunization of  Pigs
			3.6 Future Directions and Potential for Other Applications
		4 Notes
		References
Part II: DNA Vaccine Adjuvants and Immunostimulatory Response
	Chapter 4: CpG Oligonucleotides as Vaccine Adjuvants
		1 Introduction
		2 Activation of TLR9
			2.1 The TLR9 Signaling Cascade
			2.2 CpG  ODN
			2.3 Cellular Immunology
		3 CpG ODN as Vaccine Adjuvants
			3.1 Results from Preclinical Studies
				3.1.1 Impact of Delivery Method
				3.1.2 Use of CpG ODN in Combination with Other Immune Modifiers
			3.2 Results from Clinical Studies
				3.2.1 Vaccines Targeting Infectious Agents
				3.2.2 Vaccines Targeting Allergens
				3.2.3 Vaccines Targeting Cancer
			3.3 CpG ODN as Adjuvants for DNA Vaccines
			3.4 Safety
		4 Conclusion
		References
	Chapter 5: Molecular Adjuvants for DNA Vaccines: Application, Design, Preparation, and Formulation
		1 Introduction
			1.1 Cytokine-Encoding Plasmids
			1.2 Chemokine-Encoding Plasmids
			1.3 Co-Stimulatory Molecule-Encoding Plasmids
			1.4 Plasmids Encoding Pathogen-Recognition Receptor (PRR) Ligands and Immune-Signaling Molecules
		2 Materials
			2.1 Target Gene Preparation to  Use
			2.2 Directional Cloning
				2.2.1 Double-Digestion
				2.2.2 DNA Extraction and Clean-Up
				2.2.3 Ligation Process
			2.3 Introduction of the Recombinant Vector into the Competent Cell
			2.4 Evaluation of the Desired Protein Expression
			2.5 Evaluation of Immune Responses
				2.5.1 Immunization
				2.5.2 ELISA Assay
				2.5.3 Lymphocyte Proliferation Assay (LPA)
				2.5.4 Cytokine ELISpot Assay
		3 Methods
			3.1 Insertion of the DNA Fragment of Interest into the Desired Cloning Vector
				3.1.1 Double-Digestion
					If Single Compatible Buffer Is Available
					If Single Compatible Buffer Is Not Available
				3.1.2 DNA Extraction and Clean-Up
				3.1.3 Ligation of Purified DNA Inserts and Vector Molecules by the Bacteriophage T4 DNA Ligase (Preferably)
			3.2 Introduction of the Recombinant Vector into the CaCl2 Treated-Cells
				3.2.1 Preparation of Component Cells
				3.2.2 Transformation of Competent Cells by Heat-Shock Method
				3.2.3 Selection of Bacteria Colonies Containing Recombinant Plasmids
			3.3 Evaluation of the Desired Protein Expression
			3.4 Evaluation of Immune Responses
				3.4.1 Immunization
				3.4.2 Evaluation of Humoral Immune Responses
				3.4.3  Cellular Mediated Immune Responses by lymphocyte proliferation assay (LPA)
				3.4.4 Evaluation of the Cellular Mediated Immune Responses by ELISpot Assay
		4 Notes
		References
	Chapter 6: Assessing Antigen-Specific Cellular Immune Responses upon HIV/SIV Plasmid DNA Vaccination in the Nonhuman Primate M...
		1 Introduction
		2 Materials
			2.1 Common Reagents, Materials, and Equipment
			2.2 Reagents and Materials for the Processing and Storage of NHP Blood Samples
			2.3 Reagents for the Processing of NHP Tissues
			2.4 Reagents List for ELISpot Assay
			2.5 Reagents, Materials, and Equipment for Flow Cytometry-Based Assay (Peptide Stimulation to Evaluate the Antigen-Specific T-...
			2.6 Reagents for Tetramer Staining
		3 Methods
			3.1 Processing of Blood Samples and Freezing and Thawing of Peripheral Blood Mononuclear Cells (PBMC)
				3.1.1 Plasma Separation
				3.1.2 PBMC Isolation Using Ficoll-Paque PLUS and Freezing Viable  PBMC
				3.1.3 Thawing Procedure of  PBMC
			3.2 Isolation of Lymphocytes from Tissues
			3.3 ELISpot Assay to Enumerate the Antigen-Specific Cells Secreting IFN-γ or Perforin upon Peptide Stimulation
			3.4 Peptide Stimulation to Evaluate the Antigen-Specific T-Cell Responses by ICS/Flow Cytometry
			3.5 Tetramer Staining to Assess Mucosal Dissemination of Cellular Responses
				3.5.1 Tetramer Staining
		4 Notes
		References
Part III: Biotechnological Processes to Obtain DNA Vaccines
	Chapter 7: Enhanced Biosynthesis of Plasmid DNA from Escherichia coli Applying Experimental Design
		1 Introduction
		2 Materials
			2.1 Plasmid and Strain
			2.2 Cell Banking
			2.3 Pre-culture and Batch Fermentations
			2.4 Plasmid DNA Quantification
		3 Methods
			3.1 Preparation and Transformation of Competent Cells
			3.2 Master and Working Cell  Bank
			3.3 Plasmid DNA Biosynthesis - Standard Culture Conditions
			3.4 Alkaline Cell Lysis and pDNA Quantification
			3.5 Plasmid DNA Quality Analysis
			3.6 Construction of a Box-Behnken Design
		4 Notes
		References
	Chapter 8: Primary Purification of Plasmid DNA Using Differential Isopropanol Precipitation
		1 Introduction
		2 Materials
			2.1 Agarose  Gel
			2.2 Urea Denaturing Polyacrylamide  Gel
			2.3 Plasmid Quantification
			2.4 Culture Media
			2.5 Alkaline Lysis
			2.6 Isopropanol Precipitation
		3 Methods
			3.1 Agarose Gel Electrophoresis
			3.2 Urea Denaturing Polyacrylamide Gel Electrophoresis
			3.3 Plasmid Quantification and Determination of pDNA Purity
			3.4 Bacterial Cell Growth
			3.5 Alkaline Lysis
			3.6 Differential Precipitation
		4 Notes
		References
	Chapter 9: Scale-Up of Plasmid DNA Downstream Process Based on Chromatographic Monoliths
		1 Introduction
			1.1 Downstream Processing of Plasmid DNA
			1.2 Process Analytical Technology for pDNA
		2 Materials
			2.1 pDNA Lysis
			2.2 pDNA Capture Using Anion-Exchange Chromatography
			2.3 pDNA Polishing Using Hydrophobic Interaction Chromatography (HIC)
			2.4 pDNA Analytics Using CIMac pDNA Column
		3 Methods
			3.1 Optimization of Cell Lysis and Clarification Step
				3.1.1 Optimization of Cell Lysis Time (See Note 6)
				3.1.2 Optimization of Clarification Step (See Note 9)
				3.1.3 pDNA Analytics (See Note 10)
			3.2 Chromatographic Capture Optimization
			3.3 Polishing Step Optimization
			3.4 pDNA Quality Control (See Note 34)
			3.5 Scaling-up the DSP-Intermediate  Step
			3.6 Scaling-up the DSP-Large-Scale Purification
		4 Notes
		5 Future Aspects
		References
	Chapter 10: Purification of Plasmid DNA by Multimodal Chromatography
		1 Introduction
		2 Materials
			2.1 Culture Media
			2.2 Primary Purification and Intermediate Recovery
			2.3 Multimodal Chromatography
				2.3.1 Capto Adhere MM Chromatography
				2.3.2 PPA HyperCell MM Chromatography
			2.4 Gel Electrophoresis and Densitometry Analysis
			2.5 Micro-Dialysis
			2.6 Plasmid Quantification
		3 Methods
			3.1 Bacterial Cell Growth
			3.2 Primary Purification and Intermediate Recovery
			3.3 Multimodal Chromatography
				3.3.1 Capto Adhere Multimodal Chromatography
				3.3.2 PPA HyperCell Multimodal Chromatography
			3.4 Micro-Dialysis
			3.5 Agarose Gel Electrophoresis
			3.6 Plasmid Quantification
		4 Notes
		References
	Chapter 11: Minicircle DNA Vaccine Purification and E7 Antigen Expression Assessment
		1 Introduction
		2 Materials
			2.1 Bacterial Growth for PP Amplification and Recombination for mcDNA Production
			2.2 mcDNA Extraction by Alkaline Lysis
			2.3 mcDNA Purification
			2.4 Agarose Gel Electrophoresis
			2.5 Cell Culture and Transfection
			2.6 Immunocytochemistry
		3 Methods
			3.1 PP Amplification by Bacterial Growth and PP Recombination
			3.2 Alkaline Lysis for mcDNA Extraction
			3.3 mcDNA Purification
			3.4 Horizontal Electrophoresis
			3.5 Cell Culture and Transfection
			3.6 Immunocytochemistry for E7 Antigen Expression Detection
		4 Notes
		References
Part IV: DNA Vaccine Delivery
	Chapter 12: Tumor-Specific CD8+ T-Cell Responses Induced by DNA Vaccination
		1 Introduction
		2 Materials
			2.1 DNA Vaccine
				2.1.1 Obtaining the  DNA
				2.1.2 CD8+ T-Cell Transfer
				2.1.3 Immunization
			2.2 Test CD8+ T-Cell Response
				2.2.1 Effector Phase
				2.2.2 Memory Phase
			2.3 Evaluate Antitumor Immune Response
		3 Methods
			3.1 DNA Vaccine
				3.1.1 Obtaining the  DNA
				3.1.2 CD8+ T-Cell Transfer
				3.1.3 Immunization
			3.2 Test CD8+ T-Cell Response
				3.2.1 Effector Phase
				3.2.2 Memory Phase
			3.3 Evaluate Antitumor Immune Response
				3.3.1 Adherent Cells
				3.3.2 Non-adherent Cells
		4 Notes
		References
	Chapter 13: Therapeutic DNA Vaccine Against HPV16-Associated Cancer
		1 Introduction
		2 Materials
			2.1 Animals
			2.2 Intradermal Immunization
			2.3 Skin Grafting Surgery and Monitoring
		3 Methods
			3.1 Intradermal Immunization
			3.2 Preparation of Ear Skin Grafts
			3.3 Recipient Surgery Preparations
			3.4 Grafting Procedure
			3.5 Bandaging Procedure
			3.6 Post-Surgery Care
			3.7 Monitoring and Measurement of Graft Rejection
		4 Notes
		References
	Chapter 14: Bulk and Microfluidic Synthesis of Stealth and Cationic Liposomes for Gene Delivery Applications
		1 Introduction
		2 Materials
			2.1 Bulk and Microfluidic Production of Cationic Liposomes (CLs)
			2.2 Stealth Cationic Liposomes Synthesis
			2.3 Synthesis of Lipoplexes for DNA Delivery
			2.4 Microfabrication Technique
		3 Methods
			3.1 Bulk and Microfluidic Production of Cationic Liposomes (CL)
				3.1.1 Lipid Solution (Stock Solution)
				3.1.2 Bulk Synthesis (Ethanol Injection)
				3.1.3 Microfluidic Synthesis
			3.2 Stealth Cationic Liposomes Synthesis
			3.3 Synthesis of Lipoplexes for DNA Delivery
				3.3.1 Molar Charge Ratio Between pDNA and Phospholipids for Lipoplex Synthesis
				3.3.2 Conventional Bulk Mixing
				3.3.3 Microfluidic Synthesis
			3.4 Microfabrication Technique
				3.4.1 Chip Design
				3.4.2 Plasma Treatment
				3.4.3 Surface Treatments: Hydrophobic Conditions
				3.4.4 Glass Slide Treatment
				3.4.5 Microfluidic Device Treatment
		4 Notes
		References
	Chapter 15: Conception of Plasmid DNA and Polyethylenimine Delivery Systems with Potential Application in DNA Vaccines Field
		1 Introduction
		2 Materials
			2.1 PEI/pDNA Complexes
			2.2 Agarose Gel Electrophoresis
			2.3 Fourier-Transform Infrared Spectroscopy
			2.4 Confocal Microscopy
		3 Methods
			3.1 Formation of PEI/pDNA Vectors
			3.2 Characterization of Developed Polyplexes
				3.2.1 Nanoparticles Morphology
				3.2.2 pDNA Complexation
				3.2.3 FTIR
				3.2.4 Size and Surface Charges
			3.3 Cellular Uptake and Internalization
				3.3.1 FITC pDNA Labeling
				3.3.2 Confocal Fluorescence Microscopy
		4 Notes
		References
	Chapter 16: Main Features of DNA-Based Vectors for Use in Lactic Acid Bacteria and Update Protocols
		1 Introduction
		2 Materials
			2.1 Confection and Test of Escherichia coli (E. coli) Electrocompetent Cells (See Note 1)
				2.1.1 Efficiency Test of Electrocompetent E. coli Cells (See Note 1)
			2.2 Transformation of E. coli Electrocompetent Cells (See Note 1)
			2.3 Plasmid Extraction (Alkaline Lysis) (See Note 1)
			2.4 Clone Confirmation
				2.4.1 Polymerase Chain Reaction (PCR)
				2.4.2 Enzymatic Digestion
				2.4.3 Sequencing
			2.5 Plasmid Expression Confirmation
				2.5.1 Culture of Caco-2 Cells (See Note 1)
				2.5.2 Lipofection of Eukaryotic Cells with the Plasmid (See Note 1)
				2.5.3 Confocal Microscopy
				2.5.4 Flow Cytometry (See Note 1)
			2.6 Confection of Electrocompetent Lactococcus sp. Cells (See Note 1)
			2.7 Transformation in L. lactis Electrocompetent Cells (See Note 1)
			2.8 Plasmid Extraction (See Note 1)
			2.9 Plasmid Verification
			2.10 Dose Preparation for Gavage (See Note 1)
		3 Methods
			3.1 Confection of Electrocompetent E. coli Cells (See Notes 1 and 8)
			3.2 Efficiency Test of E. coli Electrocompetent Cells (See Note 1)
			3.3 Transformation in Electrocompetent E. coli Cells (See Note 1)
			3.4 Plasmid Extraction (See Note 1)
			3.5 Clone Confirmation
				3.5.1 Polymerase Chain Reaction
				3.5.2 Enzymatic Digestion
				3.5.3 Sequencing
			3.6 Plasmid Expression Confirmation
				3.6.1 Culture of Caco-2 Cells (See Notes 1 and 16)
				3.6.2 Lipofection of Eukaryotic Cells with the Plasmid (See Note 1)
				3.6.3 Confocal Microscopy (See Note 1)
				3.6.4 Flow Cytometry (See Note 1)
			3.7 Confection of Electrocompetent Lactococcus sp. Cells (See Note 1)
			3.8 Transformation of Electrocompetent Lactococcus sp. Cells (See Note 1)
			3.9 Plasmid Extraction
			3.10 Plasmid Verification
			3.11 Doses Preparation for Gavage (See Note 1)
		4 Notes
		References
Part V: DNA Vaccine Transfer to Clinical Trials
	Chapter 17: Ethics of DNA Vaccine Transfer for Clinical Research
		1 Introduction
		2 Ethical Issues for the Development of DNA Vaccines: Oversight and Regulatory Framework
		3 Population and Location Selection
		4 Definition of Benefits and Risks in Clinical Trials
		5 Informed Consent
		6 Independent Review and Conflict of Interests
		References
	Chapter 18: Preparation of an Academic Clinical Trial
		1 Introduction
		2 Clinical Trials of Investigational Medicinal Products (IMPs) and Advanced Therapy Medicinal Products (ATMPs)
		3 Designing the Clinical Trial Protocol
		4 Stakeholders´ Responsibilities
		5 Regulatory Submission
		6 Feasibility Assessment
		References
Index