Vaccine Technologies for Veterinary Viral Diseases: Methods and Protocols

Preface
Contents
Contributors
Chapter 1: An Overview of Veterinary Viral Diseases and Vaccine Technologies
	1 Viral Diseases of Animals and the Need for Vaccination
	2 Immunology Matters
	3 Vaccine Technologies
	4 Type I Vaccine Technologies
	5 Type II Vaccine Technologies
	6 Type III Vaccine Technologies
	7 Type IV Vaccine Technologies
	8 New Approaches for Vaccine Design
	9 Concluding Remarks
	References
Chapter 2: Production and Purification of Candidate Subunit Vaccines by IC-Tagging Protein Encapsulation
	1 Introduction
	2 Materials
		2.1 Baculovirus Expression System (MS)
			2.1.1 Cells and Tissue Culture
			2.1.2 Fluorescence Microscopy
			2.1.3 Microsphere (MS) Purification and Analysis
		2.2 Bacterial Expression System (NS)
			2.2.1 Bacterial Cell Cultures
			2.2.2 Nanosphere (NS) Purification and Analysis
	3 Methods
		3.1 Baculovirus Expression System (MS)
			3.1.1 Growing Sf9 Cells and Baculoviruses
			3.1.2 Titration of Baculovirus Stocks
			3.1.3 Validation of the IC-Tagging Method for Your Particular Epitope
			3.1.4 MS Production and Purification
		3.2 Bacterial Expression System (NS). Nanosphere Production and Purification
	4 Notes
	References
Chapter 3: Elastin-like Polymers as Nanovaccines: Protein Engineering of Self-Assembled, Epitope-Exposing Nanoparticles
	1 Introduction
	2 Materials
		2.1 Reagents
			2.1.1 Materials for Genes Construction and Expression
			2.1.2 Materials for Analysis of Gn Glycoprotein In Vitro Expression
			2.1.3 Materials for Analysis of In Vivo Biodistribution in  Mice
		2.2 Buffers and Media
		2.3 Equipment Required
	3 Methods
		3.1 ELR-Vaccine Device Synthesis
			3.1.1 Molecular Design Guidelines
			3.1.2 Chimeric Gene Construction
			3.1.3 ELR Expression and Purification: Isolation of a Highly Productive Colony
			3.1.4 ELR Production
			3.1.5 ELR Purification
			3.1.6 Endotoxin Removal from  ELRs
		3.2 ELR-Based DNA Vaccines
			3.2.1 Plasmid Mutagenesis and Cloning
			3.2.2 Synthesis of the DNA Vaccine Library
			3.2.3 Analysis Gn Glycoprotein Expression In Vitro
				Lipofectamine Transfection
				Turbofect Transfection
				Immunocytochemistry Analysis
				Confocal Microscopy
			3.2.4 Analysis of Biodistribution in Mice In  Vivo
				Fluorescent Labeling of DNA Constructs
				Intravenous Administration of Fluorescently Labeled DNA in Mice
	4 Notes
	References
Chapter 4: Display of Heterologous Proteins in Bacillus Subtilis Biofilms for Enteric Immunization
	1 Introduction
	2 Materials
		2.1 Transformation of B. subtilis
		2.2 Transduction of B. subtilis NCIB3610
		2.3 In Vitro B. Subtilis Biofilm
		2.4 Preparation of Biofilm Samples for Tas-AOI Fusion
		2.5 Preparation of Spores
			2.5.1 Growing Culture
			2.5.2 Spore Purification
			2.5.3 Spore Quantification
			2.5.4 Electron Microscopy of the Spores
	3 Methods
		3.1 Transformation of B. subtilis
			3.1.1 Competent Cells Preparation
			3.1.2 Competent Cells Transformation
			3.1.3 SPP1 Transduction of B. subtilis NCIB3610
			3.1.4 Genetic Background for the Expression of TasA-AOI Fusion Proteins
			3.1.5 Plasmids Construction
		3.2 Characterization of Biofilms
			3.2.1 Growing Biofilms of B. subtilis in MSgg Semi-Solid Media
			3.2.2 Growing Biofilms of B. subtilis in MSgg Liquid Medium
		3.3 Characterization of TasA-Heterologous Protein Fusion by Western Blotting
			3.3.1 Sample Preparation
			3.3.2 Gel Migration and Immunoblotting
			3.3.3 Data Analysis
		3.4 Preparation of Spores
			3.4.1 Growing Culture
			3.4.2 Spore Purification
			3.4.3 Spores Quantification
			3.4.4 Electron Microscopy of the Spores
			3.4.5 Spores Storage
		3.5 Animal immunization
			3.5.1 Pretreatment with an Antibiotic Mixture
			3.5.2 Oral Gavage
			3.5.3 Collection of Blood and Feces Samples
				Blood Sample Collection
				Feces Sample Collection
		3.6 Data Analysis
			3.6.1 Determination of Recombinant B. subtilis in Mouse Feces
			3.6.2 ELISA for Detection of the Humoral Response
	4 Notes
	References
Chapter 5: Production of Influenza H5 Vaccine Oligomers in Plants
	1 Introduction
	2 Materials
		2.1 Plant Transient Expression
		2.2 SDS-PAGE and Immunoblotting
		2.3 ELISA
		2.4 Hemagglutination Assay
		2.5 Hemagglutination Inhibition Assay
		2.6 Mouse Immunization
	3 Methods
		3.1 Transient Expression of Recombinant Vaccine Antigen in Plants
		3.2 Western Blot Analyses
		3.3 Hemagglutination Assay
			3.3.1 Total Soluble Protein Extraction
			3.3.2 Hemagglutination Assay
		3.4 Mouse Vaccination
			3.4.1 Total Protein Extraction
			3.4.2 Mouse Vaccination
			3.4.3 Hemagglutination Inhibition Assay
		3.5 ELISA
	4 Notes
	References
Chapter 6: DNA Vaccines in Pigs: From Immunization to Antigen Identification
	1 Introduction
	2 Design and Deliver a DNA Vaccine
		2.1 The Use of Liposomes
		2.2 The Gene  Gun
		2.3 Electroporation
		2.4 Microneedle Array Patch
	3 Enhancing the Immunogenicity of DNA Vaccines
		3.1 The Use of Cytokines as Plasmid-Encoded Adjuvants
		3.2 Targeting the Encoded  Ag
	4 Searching for Potential Antigens in Complex Pathogens: The Cocktail Option and the ELI Strategy
	5 A prototypic Approach to Obtain and Test a DNA Vaccine Specifically Designed for Swine
		5.1 Construction of a DNA Vaccine
		5.2 Assessing Ag Expression In Vitro
		5.3 Swine Immunization
	6 Concluding Remarks
	References
Chapter 7: Use of Foot-and-Mouth Disease Virus Non-coding Synthetic RNAs as Vaccine Adjuvants
	1 Introduction
	2 Materials
		2.1 RNA Synthesis
		2.2 Removal of DNA Template
		2.3 Purification of RNA Transcripts
		2.4 RNA Formulation
		2.5 Vaccine Formulation
	3 Methods
		3.1 RNA Synthesis
		3.2 Removal of DNA Template
		3.3 Purification of RNA Transcripts
		3.4 RNA Folding and Emulsification
		3.5 Vaccine Formulation
		3.6 RNA Delivery and Vaccination in Pigs
		3.7 RNA Delivery and Vaccination in Mice
	4 Notes
	References
Chapter 8: Evaluation of Innate Lymphoid Cells and Dendritic Cells Following Viral Vector Vaccination
	1 Introduction
	2 Materials
		2.1 Immunization
		2.2 Sample Collection and Preparation
		2.3 Flow Cytometry Analysis (FACS)
		2.4 Immunofluorescence
	3 Methods
		3.1 Immunization
		3.2 Sample Collection and Preparation
			3.2.1 Lung Tissue Collection and Preparation Following Intranasal Immunization
			3.2.2 Muscle Tissue Collection and Preparation Following Intramuscular Immunization
		3.3 Flow Cytometry Analysis
			3.3.1 ILC Staining
			3.3.2 DC Surface Staining
			3.3.3 DC Intracellular Staining
		3.4 Immunofluorescence to Detect Intracellular and Extracellular Receptor Expression (IL-13 and IFN-γ) Using Confocal Microsco...
		3.5 Flow Cytometry Gating Strategy for ILC and DC Analysis
			3.5.1 ILC Gating Strategy (Figs. 1 and 2)
			3.5.2 DC Gating Strategy (Fig. 3)
	4 Notes
	References
Chapter 9: Generation of Replication Deficient Human Adenovirus 5 (Ad5) Vectored FMD Vaccines
	1 Introduction
	2 Materials
		2.1 Construction of Ad5-Vectored FMD
		2.2 Confirmation of Cloning
		2.3 Production and Amplification of Recombinant Ad5-O1M-FMD Virus
		2.4 Purification of Recombinant Ad5-FMD Virus by Density Gradient Centrifugation
		2.5 Measuring Titers for Ad5-O1M-FMD
		2.6 Western Blotting
	3 Methods
		3.1 Directional Cloning for the Construction of Ad5-O1M-FMD
		3.2 Confirmation of Cloning by Restriction Enzyme Diagnostics and Sequencing
		3.3 Production and Amplification of Recombinant Ad5-O1M-FMD
			3.3.1 Derivatization of Recombinant Ad5-O1M-FMD
			3.3.2 Amplification of Recombinant Ad5-O1M-FMD
		3.4 Purification of Ad5-O1M-FMD Recombinant Virus
		3.5 Measuring Titers of Recombinant Ad5-O1M-FMD
		3.6 Confirmation of the Expression of Capsid Proteins by Western Blot
	4 Notes
	References
Chapter 10: Recombinant Modified Vaccinia Virus Ankara Development to Express VP2, NS1, and VP7 Proteins of Bluetongue Virus
	1 Introduction
	2 Materials
		2.1 Cells and Viruses
		2.2 Cell Culture Reagents
		2.3 Construction of Recombinant MVAs
		2.4 Confocal Microscopy
		2.5 Immunoprecipitation Analysis
		2.6 Purification of rMVAs
	3 Methods
		3.1 Cloning of VP2, NS1, and VP7 BTV-4 Genes for Generation of Recombinant MVAs
		3.2 Construction of Recombinant MVAs
			3.2.1 Infection/Transfection of DF-1 Cells with MVA-wt and pSC11 Plasmid
			3.2.2 Plaque Purification of Recombinant MVA Viruses
		3.3 Amplification, Escalation, and Titration of Virus Working Stocks
		3.4 Protein Expression Analysis
			3.4.1 Immunofluorescence Assay
			3.4.2 Analysis of BTV Proteins Expression by Radiolabeling, Immunoprecipitation, and SDS-PAGE
		3.5 Purification of rMVAs by Using a Sucrose Gradient
	4 Notes
	References
Chapter 11: Cloning Strategies for the Generation of Recombinant Capripoxvirus Through the Use of Screening and Selection Mark...
	1 Introduction
	2 Materials
	3 Methods
		3.1 Infection of Transfected OA3.T Cells with  LSDV
		3.2 Transfection of OA3.T Cells
		3.3 Selection of Recombinant Viruses
		3.4 Isolation of Individual Recombinant Plaques
		3.5 Removal of gpt Gene Through Selective Pressure
	4 Notes
	References
Chapter 12: Using RVFV as a Vector Platform for the Expression of Ruminant Disease Antigens
	1 Introduction
	2 Materials
		2.1 Cell Culture
		2.2 Plasmid Generation
		2.3 Virus Rescue and Growth
		2.4 Expression Analysis
		2.5 In Vivo Analysis in Mice
	3 Methods
		3.1 Cloning of Heterologous Genes for Generation of Recombinant RVFVs
		3.2 Generation of Recombinant rRVFV
			3.2.1 Transfection of HEK-293 and BHK-21 Cells with pHH21 and pI.18 Plasmids
			3.2.2 Plaque Purification of Recombinant Viruses and Working Stock Preparation
		3.3 Protein Expression Analysis
			3.3.1 Immunofluorescence Assay
			3.3.2 Western Blot Analysis
		3.4 Phenotypic Characterization of the Recombinant Viruses
			3.4.1 Growth Curve
		3.5 Analysis of Stability in Cell Culture
			3.5.1 Phenotypic Stability
			3.5.2 Genetic Stability
		3.6 Analysis of Immunogenicity In Vivo
	4 Notes
	References
Chapter 13: Generation and Characterization of Single-Cycle Infectious Canine Influenza A Virus (sciCIV) and Its Use as Vaccin...
	1 Introduction
	2 Materials
		2.1 Viruses
		2.2 Tissue Culture Media and Reagents
		2.3 Mammalian Cell Lines
		2.4 Cloning A/canine/NY/dog23/2009 H3N8 HA
		2.5 Components for Immunofluorescence Assay
		2.6 SDS-PAGE and Immunoblotting Reagents
		2.7 Mouse Immunization and ELISA Components
	3 Methods
		3.1 Cloning of CIV H3N8 HA in pCAGGS
		3.2 Generation of CIV HA Protein-Expressing MDCK Cells
		3.3 Characterization of MDCK CIV H3N8 HA Cells
			3.3.1 Immunofluorescence Analysis (IFA)
			3.3.2 Western Blot Analysis
			3.3.3 sciIAV Complementing Assay
		3.4 Generation of sciCIV ΔHA/GFP
		3.5 Multicycle Growth Analysis of sciCIV ΔHA/GFP
		3.6 In Vivo Characterization of sciCIV ΔHA/GFP
			3.6.1 Intranasal (i.n.) Infection
			3.6.2 Evaluation of Viral Titers in Lungs
		3.7 Analysis of Immunogenicity Induced by sciCIV ΔHA/GFP
			3.7.1 Mouse Bleeding by Submandibular Puncture
			3.7.2 sciCIV UV Inactivation
			3.7.3 Preparation of Infected MDCK Cell Extracts
			3.7.4 ELISA
		3.8 Analysis of Protection Efficacy
	4 Notes
	References
Chapter 14: Reverse Genetics for Influenza A and B Viruses Driven by Swine Polymerase I Promoter
	1 Introduction
	2 Materials
		2.1 Influenza A and B Viral RNA (vRNA) Extraction
		2.2 Influenza A and B cDNA Synthesis
		2.3 Amplification of Influenza A and B Gene Segments from  cDNA
		2.4 Bidirectional Reverse Genetics pPIG2012 Plasmid Vector
		2.5 Cloning of Influenza A and B Virus Gene Segments into pPig2012
		2.6 Cell Culture Growth and Maintenance
		2.7 Generation of Influenza A and B Viruses by Reverse Genetics Using the pPig2012 Plasmid  Sets
		2.8 Propagation of Rescued Influenza A and B Viruses in MDCK Cells
		2.9 Propagation of Rescued Influenza A and B Viruses in Embryonated Chicken  Eggs
	3 Methods
		3.1 vRNA Extraction from Influenza A and B Viruses
		3.2 IAV cDNA Synthesis (See Note 5)
		3.3 IBV cDNA Synthesis (See Note 5)
		3.4 Amplification of IAV and IBV Gene Segments from  cDNA
		3.5 Cloning of IAV and IBV Gene Segments into pPIG2012 (See Fig. 2)
		3.6 Screening of Transformant Colonies by  cPCR
		3.7 Cell Culture Growth and Maintenance
		3.8 Rescue of IAV and IBV Using the Swine polI Reverse Genetics Plasmid  Sets
		3.9 Propagation of IAV and IBV in MDCK Cells
		3.10 Propagation of IAV and IBV in  ECEs
	4 Notes
	References
Chapter 15: Analysis of the Cellular Immune Responses to Vaccines
	1 Introduction
	2 Materials
		2.1 Flow Cytometry
		2.2 TCR Avidity
		2.3 ELISpot
		2.4 CTL Cytotoxicity Assay
		2.5 Immunoinformatics
	3 Methods
		3.1 Flow Cytometry
		3.2 TCR Avidity Assay
		3.3 ELISpot Assay for Detection of Bovine Interferon-Gamma (IFNγ)
			3.3.1 MACS Sorting for CD8, CD4, and Monocytes from Ex Vivo PBMC or Bulks
			3.3.2 ELISpot
		3.4 Cytotoxicity
			3.4.1 Generation of CTL Lines to Theileria parva from PBMC in Cattle
			3.4.2 Chromium-51 Release Assay Using Infected Cells or Peptide Pulsed PBMC as Targets
		3.5 Immunoinformatics
	4 Notes
	References
Index