Vaccine Design: Methods and Protocols, Volume 1. Vaccines for Human Diseases (Methods in Molecular Biology, 2410)

Dedication
Preface
Contents
Contributors
Part I: Vaccines: Introduction
	Chapter 1: Challenges for Vaccinologists in the First Half of the Twenty-First Century
		1 Introduction
			1.1 Change and Emerging Infectious Diseases
			1.2 Development of Vaccines to Protect against COVID-19
			1.3 Development of Vaccines to Protect against HIV
		2 Development of Vaccines for Flaviviruses
		3 Development of Vaccines for Norovirus
		4 Development of Vaccines for Influenza
		5 Development of Vaccines for Sepsis
		6 Development of Vaccines for Tuberculosis
		7 Development of Vaccines for Tick-Borne Diseases
		8 Development of Vaccines for Flesh-Eating Bacteria
		9 Development of Vaccines for Parasites
		10 Development of Vaccines for Malaria
		11 Development of Vaccines for Cancer, Neurodegenerative Diseases, Substance Abuse, and Autoimmune Diseases
		12 Antibody-Dependent Enhancement
		13 Future Challenges
		References
	Chapter 2: Principles in Immunology for the Design and Development of Vaccines
		1 Introduction
			1.1 A Brief History of Vaccination
		2 Basic Concepts of Vaccine Immunology
		3 Innate Immunity
		4 Adaptive Immunity
		5 T Cells
		6 B Cells
		7 Immune Memory
		8 How Do Vaccines Mediate Protection?
		9 Immune Correlates of Protection
		10 Principles of Vaccine Development
		11 Selecting Vaccine Antigens
		12 Improving Vaccines
		13 Future Prospects
		References
	Chapter 3: Revisiting the Principles of Designing a Vaccine
		1 Introduction
		2 Disruption in Antigenic Priming Affects Vaccine Efficacy
			2.1 Stage-Specific Vaccine Candidate Imparts Specificity
			2.2 Relative Antigen Abundance During Processing Inside APCs
			2.3 Subcellular Localization and Availability of Antigens for Processing
		3 Leishmania-Associated Inhibitions in Antigenic Processing/Presentation
			3.1 Endocytic Mechanisms for Leishmania Uptake
			3.2 Inhibition of Phagolysosome Formation
			3.3 Dysregulation of Protease Activity by Leishmania
			3.4 Destruction of Antigen Presentation Machinery inside Macrophages
			3.5 Immunodominance and Epitope Crypticity Affects Vaccination
			3.6 Dysfunctional Epitope Loading to MHC Molecule
			3.7 MHC-II Affinity Determines the Immunodominant Nature of Generated Epitopes
		4 Impairment of Immune Synapse at the APC-T Cell Junction
		5 T Cell Associated Events Affecting Vaccination Outcome
			5.1 T-Cell Plasticity
			5.2 T-Cell Anergy
			5.3 T Cell Exhaustion
			5.4 Programmed Cell Death or Apoptosis
		6 Reverse Vaccinology: An Extension of Immunoinformatics
		7 Reverse Vaccinology in T Cell-Based Vaccine Design
			7.1 Epitope Prediction and Mapping
			7.2 Utility of Comparative Genomics and Pangenome Analysis
			7.3 Genome Annotation, Subcellular Localization, and Antigenicity Prediction
		8 Reverse Vaccinology Against Leishmaniasis: Redefining Vaccine Candidate Selection
		9 Proteomics-Based Approaches for High-Throughput Vaccine Discovery against Leishmaniasis
		References
	Chapter 4: Status of COVID-19 Pandemic Before the Administration of Vaccine
		1 Introduction
		2 Origin and Transmission
		3 Symptoms Caused by the SARS-CoV-2 Virus
		4 Structure of SARS-CoV-2
		5 Variants of SARS-CoV-2
		6 Immediate Scenario After Partial or Complete Lockdown Due to COVID-19
		7 Drugs Used in the Treatment of COVID-19
		8 Behavioral Pattern that Slowed the Virus
		9 Poor Leadership Impacted the Spread of the Virus Globally
		10 COVID-19 in India
		11 Distribution of Vaccines
		12 Wastage of Vaccines
		13 Impact of COVID-19 on Climate Change
		14 Preparing for a Pandemic in the Future
		References
Part II: Trends in Vaccinology
	Chapter 5: mRNA Vaccines to Protect Against Diseases
		1 Introduction
		2 Nucleic Acid Vaccines Protect Against Infection
		3 Development of mRNA as a Vaccine
		4 Types of mRNA Vaccines
		5 Formulation and Delivery of mRNA Vaccines
		6 mRNA Vaccines Against SARS-CoV-2
		7 mRNA Vaccines Targeting Influenza
		8 mRNA Vaccines Targeting Rabies
		9 mRNA Vaccines Against Zika Virus
		10 mRNA Vaccines Against Bacterial and Parasite Infections
			10.1 mRNA Vaccines Against Cancer
		11 Challenges in the Development of mRNA Vaccines
		12 Future of mRNA Vaccines
		13 Brief History of Katalin Kariko, the Pioneer of mRNA Vaccine Technology
		References
	Chapter 6: Artificial Intelligence in Vaccine and Drug Design
		1 Introduction
		2 Use of AI to Determine Protein Structure
		3 AI in Drug Design and Vaccine Development
		4 Use of AI in Immunological Applications
		5 AI in Vaccine Design and Development
		6 In Silico Approaches to SARS-CoV-2 Drug and Vaccine Design and Diagnosis
		7 Conclusions
		References
Part III: Vaccines for Human Viral Diseases
	Chapter 7: Vaccines Targeting Numerous Coronavirus Antigens, Ensuring Broader Global Population Coverage: Multi-epitope and Mu...
		1 Introduction
			1.1 Vaccine Using Whole Coronaviruses as Antigen
			1.2 Vaccines Targeting Subunit/Full-Length Proteins of Coronaviruses as Antigen
			1.3 Vaccines Targeting Multiple Coronaviruses Proteins
				1.3.1 Multi-epitope Vaccine
				1.3.2 Multi-patch Vaccine
		2 Materials
			2.1 Coronavirus Proteomes
			2.2 Adjuvants
			2.3 Linkers
			2.4 Validation Assays
			2.5 Purification of Multi-epitope Vaccine and Multi-patch Vaccine Candidates
		3 Methods
			3.1 Screening of the Coronavirus Proteome for Epitopes and Ag-Patches
				3.1.1 Screening for Cytotoxic T Lymphocyte (CTL) Epitopes
				3.1.2 Immunogenicity
				3.1.3 Screening of Helper T lymphocyte (HTL) Epitopes
				3.1.4 Identification of Protein Sequence and Protein Structure-Based B Cell Epitopes
				3.1.5 In Vitro Microarray-Based Screening of Epitopes from the Coronavirus Proteome
				3.1.6 A Novel ``Reverse Epitomics´´ Approach for the Identification of Antigenic Patches (Ag-Patches)
				3.1.7 Estimation of Population Coverage by Screened CTL and HTL Epitopes and Identified Ag-Patches
			3.2 Epitope Characterization
				3.2.1 Conservation Analysis of Epitopes and Antigenic Patches (Ag-Patches)
				3.2.2 Epitope Toxicity Prediction
				3.2.3 Overlapping Residue Analysis of CTL, HTL, and B Cell Epitopes
				3.2.4 In Silico Validation of Shortlisted Epitopes
				3.2.5 Molecular Interaction Analysis of Selected CTL Epitopes with TAP Transporter
			3.3 In Vitro Epitope-Antibody/Ag-Patches Antibody (from Patient Serum) Complex Formation Tendency
				3.3.1 Dot Blot and ELISA-Based Validation of Epitopes, Ag-Patches, MEVs, and MPVs
			3.4 Multi-epitope Vaccine and Multi-patch Vaccine Design
			3.5 In Silico Characterization of CTL and HTL Multi-epitope Vaccines
				3.5.1 Interferon Gamma-Inducing Epitopes Prediction from Designed  MEVs
				3.5.2 MEVs and MPVs Allergenicity and Antigenicity Prediction and Physicochemical Analysis
				3.5.3 In Silico Tertiary Structure Modeling, Refinement, and Validation of MEVs and MPVs
				3.5.4 Discontinuous B Cell Epitope Prediction from MEVs and MPVs
				3.5.5 Molecular Interaction Analysis of MEV and MPV with Immunological Receptor
				3.5.6 Analysis of cDNA of MEV and MPV for Cloning and Expression in Human Cell Lines
			3.6 Preparation of the MEV and MPV Constructs
				3.6.1 Expression and Purification of the MEV and MPV Constructs
				3.6.2 Complex Formation Tendency of MEV/MPV with Serum Antibodies from Coronavirus Patient and from Experimental Animal Model
		4 Notes
		Declaration: Patents filed: IN202011037585, IN202011037939, PCT/IN2021/050841.References
	Chapter 8: Use of Micro-Computed Tomography to Visualize and Quantify COVID-19 Vaccine Efficiency in Free-Breathing Hamsters
		1 Introduction
		2 Materials
			2.1 SARS-CoV-2 Strain
			2.2 Cell Culture and Media
			2.3 Vaccine
			2.4 Animals
			2.5 Micro-Computed Tomography (μCT)
			2.6 Anesthesia
			2.7 Network Connection and Data Storage
			2.8 Software
			2.9 Experimental Endpoint
		3 Methods
			3.1 Immunization of Hamsters
			3.2 Experimental SARS-CoV-2 Infection
			3.3 μCT Acquisition
				3.3.1 Skyscan 1278
				3.3.2 X-Cube
			3.4 μCT Scan Reconstruction
				3.4.1 Skyscan 1278
				3.4.2 X-Cube
			3.5 Micro-CT Data Visualization
			3.6 Micro-CT Data Quantification
				3.6.1 Semiquantitative Scoring of Visual Observations in Micro-CT  Data
				3.6.2 Quantification of μCT-Derived Biomarkers
			3.7 Experiment Termination, Endpoint, and Validation
		4 Notes
		References
	Chapter 9: Design of Replication-Competent VSV- and Ervebo-Vectored Vaccines Against SARS-CoV-2
		1 Introduction
		2 Materials
			2.1 Cloning
			2.2 Virus Rescue
			2.3 TCID50 Assay to Determine Live Virus Titers
			2.4 Growth of Recombinant VSV and Purification
		3 Methods
			3.1 Cloning
			3.2 Transfection and Rescue
			3.3 TCID50 Assay to Determine Virus Titers
			3.4 Stock Virus Production and Purification
		4 Notes
		References
	Chapter 10: CRISPR Engineering of Bacteriophage T4 to Design Vaccines Against SARS-CoV-2 and Emerging Pathogens
		1 Introduction
		2 Materials
			2.1 Plasmid Construction
			2.2 Recombinant Phage Construction
			2.3 Phage Production and Purification
		3 Methods
			3.1 Construction of CRISPR-Cas12a Spacers Targeting Hoc or  Soc
			3.2 Construction of Ee-Hoc and Soc-RBD Donors
				3.2.1 Ee-Hoc Donor Construction
				3.2.2 Soc-RBD Donor Construction
			3.3 Transformation of Spacer and Donor into E. coli
				3.3.1 Making B40 Competent Cells
					Day 1
					Day 2
					Day 3
				3.3.2 Spacer Plasmid Transformation into B40
				3.3.3 Making B40-Spacer Competent Cells
				3.3.4 Donor Plasmid Transformation into B40-Spacer Cells
			3.4 Construction of T4-Soc-RBD and T4-Ee-Hoc Recombinant Phages by CRISPR Engineering
				3.4.1 Measuring Efficiency of Plating (EOP)
				3.4.2 Recombinant Phages Construction
			3.5 Recombinant Phage Purification for Immunization
				3.5.1 Preparation of Phage Working Stock from ``Zero Stock´´
				3.5.2 Phage Production
				3.5.3 Phage Purification
		4 Notes
		References
	Chapter 11: Techniques for Developing and Assessing Immune Responses Induced by Synthetic DNA Vaccines for Emerging Infectious...
		1 Introduction
			1.1 Challenge
			1.2 Vaccine and Immunization Assessment
			1.3 Development of Antigen-Specific Synthetic DNA Vaccines Against Emerging Infectious Diseases
			1.4 Design of Synthetic DNA Vaccines
			1.5 Immune Focusing Using Domain Minimization and Glycan Resurfacing
			1.6 Design of Next-Generation DNA-Launched Nanoparticle Vaccines
		2 Methods
			2.1 Western Blot (or Immunoblot) Analysis
				2.1.1 Materials
				2.1.2 Preparation of Lysate
				2.1.3 Sample Preparation
				2.1.4 Preparation, Loading, and Running of the  Gel
				2.1.5 Transfer of Proteins to Membrane and Blocking
				2.1.6 Staining with Primary and Secondary Antibody
			2.2 Immunofluorescence Assay
				2.2.1 Materials
				2.2.2 Sample Preparation
				2.2.3 Immunostaining
			2.3 Biophysical and Antigenic Profile Characterization of Produced Antigens
		3 Animal and Ethics
		4 Ex Vivo Immune Assays for Measuring Vaccine-Specific Immune Responses
			4.1 Enzyme-Linked Immunosorbent Assay
				4.1.1 Reagent Preparation
				4.1.2 Antigen Coating
				4.1.3 Measurement of Antibody Binding
			4.2 Enzyme-Linked Immunospot (ELISpot) Assay for IFN-γ Measurements
				4.2.1 Buffers and Reagents
				4.2.2 Preparation and Blocking of Plate (Sterile Conditions): Day 1
				4.2.3 Incubation of Cells in Plate (Sterile Conditions): Day 2
				4.2.4 Detection of Spots: Day 3
			4.3 Measurements of Vaccines-Specific Cytokines Production for T Cell Immunity by FACS Analysis
				4.3.1 Material Preparations
				4.3.2 Cell Preparation for FACS Analysis
				4.3.3 Extracellular and Intracellular Staining
		5 Neutralization Techniques
			5.1 Live Virus Neutralization Assays
			5.2 Plaque Reduction Neutralization Test (PRNT) Assay
			5.3 Antiviral-Based Cytopathic Effect Assay (CPE assay)
			5.4 Neutralization Assay with Pseudotyped Virus
				5.4.1 Materials to Produce Pseudovirus
		6 Antibody Glycosylation to Measure Humoral Response
			6.1 IgG N-glycan Analysis by Capillary Gel Electrophoresis
				6.1.1 Materials
				6.1.2 Deglycosylation and Labeling of Free N-glycans
				6.1.3 Clean Up the Labeled N-glycans
				6.1.4 N-glycans Profiling
		7 Protective Efficacy Assessment for Vaccine
		8 Summary
		References
	Chapter 12: Towards Determining the Epitopes of the Structural Proteins of SARS-CoV-2
		1 Introduction
		2 Materials
			2.1 Bioinformatics
			2.2 Animals and Immunization
			2.3 Reagents and Plasticwares
			2.4 Software
		3 Methods
		4 Notes
		References
	Chapter 13: Development, Production, and Characterization of Hepatitis B Subviral Envelope Particles as a Third-Generation Vac...
		1 Introduction
		2 Materials
			2.1 Lentiviral Production
			2.2 Recombinant Mammalian Cell Line Development
			2.3 Cell Line Characterization
				2.3.1 Flow cytometry
				2.3.2 Fluorescence Microscopy
			2.4 HBV-SVPs Preparation and Concentration
			2.5 HBV-SVPs Characterization
				2.5.1 ELISA Analysis
				2.5.2 Western Blot Analysis
				2.5.3 Transmission Electron Microscopy and Immunogold Analysis
			2.6 HBV-SVPs Immunization Protocol
				2.6.1 Humoral Immune Response Analysis
				2.6.2 Functional Characterization of Antibodies
		3 Methods
			3.1 Lentivirus Production and Titration
			3.2 CHO-K1 Recombinant Cell Line Development
			3.3 Cell Line Characterization
				3.3.1 Flow Cytometry
				3.3.2 Fluorescence Microscopy
			3.4 Preparation and Concentration of HBV-SVPs
			3.5 Characterization of the HBV-SVPs
				3.5.1 ELISA for HBV-VSPs Characterization
				3.5.2 Western  Blot
				3.5.3 Transmission Electron Microscopy and Immunogold Analysis
			3.6 Analysis of the Immune Response Triggered by HBV-SVPs
				3.6.1 Immunization Protocol
				3.6.2 Determination of Total S-Specific Antibody Endpoint Titers
				3.6.3 Functional Characterization of Antibodies
		4 Notes
		References
	Chapter 14: Generation of CpG-Recoded Zika Virus Vaccine Candidates
		1 Introduction
		2 Materials
			2.1 Software
			2.2 Infectious Subgenomic Amplicons (ISA) Transfection and ZIKV Stock Generation
			2.3 Virus Stock Titration
		3 Methods
			3.1 Design and Generation of Overlapping DNA Fragments
			3.2 Infectious Subgenomic Amplicons to Recover Wild-Type and CpG-Recoded ZIKV Variants
			3.3 Generation of Wild-Type and CpG-Recoded ZIKV Stocks
			3.4 Titration of CpG-Recoded ZIKV Stocks
		4 Notes
		References
Part IV: Vaccines for Human Bacterial Diseases
	Chapter 15: Salmonella Uptake into Gut-Associated Lymphoid Tissues: Implications for Targeted Mucosal Vaccine Design and Deliv...
		1 Introduction
		2 Materials
			2.1 Quantifying Peyer´s Patch Invasion
			2.2 Quantification of Colonization by Fecal Shedding
			2.3 Isolation and Immunohistochemistry of Intestinal Samples Containing S. Typhimurium
		3 Methods
			3.1 Quantifying Peyer´s Patch Invasion
				3.1.1 Preparation of Bacteria
				3.1.2 Gavage
				3.1.3 Collection and Processing of Peyer´s Patches
				3.1.4 Count Colonies and Compute Competitive Indices (CIs)
			3.2 Quantification of Colonization by Fecal Shedding
				3.2.1 Preparation of Bacteria
				3.2.2 Collection of Fecal Pellets
				3.2.3 Processing of Fecal Pellets
				3.2.4 Count Colonies and Compute Competitive Indices (CIs)
			3.3 Isolation and Immunohistochemistry of Intestinal Samples Containing S. Typhimurium
				3.3.1 Preparation of Bacteria and Gavage
				3.3.2 Collection of Digestive Tract (Stomach, Small Intestine, Cecum, Large Intestine)
				3.3.3 IHC: Deparaffinization and Tissue Rehydration
				3.3.4 IHC: Antigen Retrieval (Proteinase K Method)
				3.3.5 IHC: Blocking (Rodent Block M and BLOXALL)
				3.3.6 IHC: Primary Antibody Application
				3.3.7 IHC: AP-Polymer and Chromogen Application
				3.3.8 IHC: Counterstain
				3.3.9 IHC: Dehydrate Tissue and Apply Coverslips
		4 Notes
		References
	Chapter 16: Development of Human Recombinant Leptospirosis Vaccines
		1 Introduction
		2 Materials
			2.1 Antigen Selection
			2.2 Design of Recombinant Constructions
			2.3 Cloning of Leptospira Coding Sequences
			2.4 Expression of Recombinant Proteins and Solubility Testing
			2.5 Solubilization, Purification, and Concentration of Recombinant Proteins
			2.6 Immunoblotting Components
			2.7 Vaccine Formulation
			2.8 Adsorption  Test
			2.9 Animals
			2.10 Blood Collections
			2.11 Immunization
			2.12 Humoral Immune Response (Indirect ELISA)
			2.13 Leptospira sp. Culture and Challenge
			2.14 Tissue Collection
		3 Methods
			3.1 Antigen Selection
			3.2 Design and Recombinant Constructions
			3.3 Cloning of Native Coding Sequences of Leptospira
			3.4 Recombinant Protein Production
			3.5 Vaccine Preparation
			3.6 Hamster Manipulation
			3.7 Blood Collections
			3.8 Immunization
			3.9 Humoral Immune Response Analyses
			3.10 Leptospira sp. Culture and Challenge
			3.11 Tissue Collection
				3.11.1 Evaluation of Kidney Colonization by Culture
				3.11.2 Evaluation of Kidney Colonization by  qPCR
		4 Notes
		References
	Chapter 17: Induction of T Cell Responses by Vaccination of a Streptococcus pneumoniae Whole-Cell Vaccine
		1 Introduction
			1.1 Subtypes and Functions of T Cells
			1.2 T Cell Induction by Infection of Bacteria and Virus
			1.3 T Cell Induction by Vaccination
			1.4 T Cells Induced by Whole-Cell Vaccine
		2 Materials
			2.1 Strain Selection
			2.2 Culture Medium Preparation
			2.3 Prepare Bacteria Culture
			2.4 Inactivation of Pneumococcal Whole  Cell
			2.5 Immunize Animals by Intranasal Route
			2.6 Immunize Animals by Subcutaneous Route
			2.7 Detection of T Cell Responses
			2.8 In Vitro Stimulation of Splenocytes
		3 Methods
			3.1 Prepare Bacteria Culture
			3.2 Inactivation of Pneumococcal Whole  Cell
			3.3 Immunize Animals by Intranasal Route
			3.4 Immunize Animals by Subcutaneous Route
			3.5 Detection of T Cell Responses
			3.6 In Vitro Stimulation of Splenocytes
		4 Notes
		References
	Chapter 18: Development of a Bacterial Nanoparticle Vaccine Against Escherichia coli
		1 Introduction
		2 Materials
			2.1 Bacterial Growth and Antigen Extraction
			2.2 Protein and Lipopolysaccharide Content Determination
			2.3 SDS Polyacrylamide Gel Components
			2.4 Immunoblotting Components
			2.5 Nanoparticle Formulation Preparation
			2.6 Nanoparticle Characterization
			2.7 Nanoparticle Loading Capacity
			2.8 Determination of Antigen Integrity
		3 Methods
			3.1 Bacterial Strain and Growth Conditions
			3.2 Antigenic Complex (HT Membrane Vesicles)
			3.3 Characterization of the Antigenic Extracts
			3.4 Preparation of HT-Loaded Nanoparticles
			3.5 Nanoparticle Characterization
			3.6 Nanoparticle Loading Capacity
			3.7 Determination of Antigen Integrity
		4 Notes
		References
	Chapter 19: Construction of Novel Live Genetically Modified BCG Vaccine Candidates Using Recombineering Tools
		1 Introduction
		2 Materials
			2.1 Chromosomal DNA Preparation
			2.2 Plasmid Construction for Recombination Substrate
			2.3 Preparation of Recombineering Substrates
			2.4 Preparation of Recombinogenic/Electrocompetent  BCG
			2.5 Electroporation of Recombineering Substrates
			2.6 Growth, Verification of Allelic Replacement Mutants, and Recombineering Plasmid Curation
		3 Methods
			3.1 Chromosomal DNA Preparation
			3.2 Plasmid Construction to Produce the Recombination Substrate
			3.3 Preparation of Recombinogenic/Electrocompetent Slow Growing Mycobacteria
			3.4 Electroporation of the Substrates for Homologous Recombination
			3.5 Growth, Verification of Double Homologous Recombination Events, and Curation of the Recombineering Plasmid
		4 Notes
		References
	Chapter 20: An Update on Tuberculosis Vaccines
		1 Introduction
		2 BCG: A Classical Vaccine
		3 Strategic Goal for a Candidate TB Vaccine
		4 Types of TB Vaccines
		5 Candidate TB Vaccines in Clinical Trials
			5.1 Live Attenuated Whole-Cell Vaccine
				5.1.1 VPM1002
				5.1.2 MTBVAC
				5.1.3 AERAS 422
			5.2 Killed Mycobacterial Vaccines
				5.2.1 RUT-1
				5.2.2 Dar-901
				5.2.3 M. Vaccae
				5.2.4 MIP
			5.3 Adjuvant Protein Subunit Vaccines
				5.3.1 H1: IC31
				5.3.2 H56:IC31
				5.3.3 H4: IC31
				5.3.4 ID93: GLA/SE
				5.3.5 M72/AS01E
				5.3.6 GamTBVac
			5.4 Viral-Vectored Vaccines
				5.4.1 MVA85A
				5.4.2 ChAdOx1.85A/MVA85A
				5.4.3 Ad5Ag85A
				5.4.4 Crucell Ad35
				5.4.5 TB/FLU-04L
		6 Conclusion
		References
	Chapter 21: Structure-Based Design of Diagnostics and Vaccines for Lyme Disease
		1 Introduction
			1.1 History of Lyme Disease
			1.2 B. burgdorferi Structure
		2 Materials
			2.1 Bioinformatics
			2.2 Peptides and Reagents
			2.3 ELISA
			2.4 Use of Peptides as a Vaccine
			2.5 Software
		3 Methods
			3.1 Identification of Antigenic Epitopes and Peptide Synthesis
			3.2 Isolation of Serum from Patients
			3.3 Peptides for Diagnostic Applications
			3.4 Use of Peptides as a Vaccine to Protect Against B. burgdorferi
		4 Notes
		References
	Chapter 22: Development of a SONIX Vaccine to Protect Against Ehrlichiosis
		1 Introduction
		2 Materials
			2.1 Cell Culture
			2.2 Animals and Consumables
			2.3 Quantitative PCR
			2.4 ELISA
			2.5 Software
		3 Methods
		4 Notes
		References
Part V: Vaccines for Human Parasitic Diseases
	Chapter 23: Development of the Antileishmanial Vaccine
		1 Introduction
			1.1 Timeline of Antileishmanial Vaccine Development
				1.1.1 First-Generation Vaccine
				1.1.2 Second Generation Vaccine
				1.1.3 Third Generation Vaccine
			1.2 Importance of Vaccine Candidate and Strategy Selection
			1.3 Determinants of an Effective Antileishmanial Vaccine
			1.4 Major Techniques Used in Antileishmanial Vaccine Development
				1.4.1 Electroporation
				1.4.2 Ni-NTA Affinity Chromatography
				1.4.3 Peritoneum-Derived Primary Macrophages
				1.4.4 Mass Spectrometry
				1.4.5 Indirect and Sandwich ELISA
				1.4.6 Macrophage T-Cell Co-Culture System
				1.4.7 In Vivo Parasitic Load
				1.4.8 Flow Cytometry
		2 Materials
			2.1 Maintenance and In Vitro Passaging of L. major and L. donovani Strain
			2.2 Generation of Dominant-Negative Mutant Parasites
			2.3 PCR-Based Molecular Cloning and Sequence Confirmation
			2.4 Recombinant Protein Purification
			2.5 SELDI
			2.6 Mass Spectrometry
			2.7 Peritoneal Macrophages (PM) Collection and In Vitro Infection of Macrophages by Leishmania
			2.8 Giemsa Staining for Amastigote Count
			2.9 Cytokine/Indirect ELISA
			2.10 NO Release Assay
			2.11 Macrophage T-Cell Co-Culture Assay
			2.12 Antibody/Sandwich ELISA
			2.13 Western Blotting
			2.14 In Vivo Challenge Infection or Priming with Leishmania
			2.15 Footpad Measurement for CL Progression
			2.16 In Vivo Parasite Load Assay
			2.17 Ldu for VL Severity
			2.18 qPCR
			2.19 Flow Cytometry
		3 Methods
			3.1 Preparation of Vaccine Formulation
				3.1.1 Generation of In Vitro Culture-Based Avirulent Strain
				3.1.2 Generation of Overexpression Based Avirulent Strain (See Note 3)
				3.1.3 Preparation of E. coli DH5α and BL21 Competent Cells
				3.1.4 Clone Development
				3.1.5 Ni-NTA Affinity Chromatography for Protein Purification (See Note 9)
			3.2 Characterization of Whole-Parasite Based Vaccine
				3.2.1 In Vitro Growth Kinetics of Avirulent Strain
				3.2.2 In Vivo Infectivity of Avirulent Strain
				3.2.3 Isolation of Peritoneum-Derived Primary Macrophages
				3.2.4 In Vitro Infection of Macrophages by Leishmania sp. (See Note 16)
				3.2.5 In Vitro Intra-Macrophage Survival of Leishmania
				3.2.6 Characterization of Immune Response Elicited by Avirulent Strain
					Cytokine/Indirect ELISA from the Culture Supernatant
					Cytokine Profiling of Lymphocytes
					Macrophage T-Cell Co-Culture for Assessing the Antileishmanial Function of T-Cells
				3.2.7 Proteome Characterization
					SELDI Analyses of LP and HP Strain of L. major and L. donovani
					Mass Spectrometry
			3.3 Immunization of Mice: (See Note 21)
			3.4 Immunogenicity Assessment Post-Immunization
				3.4.1 Antibody/Sandwich ELISA for Antileishmanial IgG Determination
				3.4.2 Probing Native Protein with Mouse Sera
			3.5 Protection Studies
				3.5.1 In Vivo Challenge Infection
				3.5.2 Footpad Thickness Measurement for Assessing CL
				3.5.3 Ldu in Affected Organs for Assessing VL
				3.5.4 In Vivo Parasite Load in Draining Lymph Node
				3.5.5 In Vitro Processing of Splenic Tissue
				3.5.6 Antigen-Specific Immune Response Profiling by Indirect ELISA
				3.5.7 Cytokine Profiling by qPCR
				3.5.8 Antibody ELISA for Post-Challenge Antileishmanial IgG Determination
				3.5.9 The Leishmanicidal Activity of Ag-Primed T-Cells by the Co-Culture Assay System
				3.5.10 The Subset Analysis by Multi-Chromatic Flow Cytometry
		4 Notes
		References
	Chapter 24: In Silico Design of Recombinant Chimera T Cell Peptide Epitope Vaccines for Visceral Leishmaniasis
		1 Introduction
		2 Materials
			2.1 Construction of the Gene Encoding the Chimera Protein
				2.1.1 FASTA Sequence of Target Proteins
				2.1.2 Prediction of T Cell Epitopes
				2.1.3 Prediction of B Cell Epitopes
			2.2 Analysis of Peptide Identity
			2.3 Peptide Characterization
			2.4 Chimera Assembly
			2.5 Chimera Gene Synthesis
		3 Methods
			3.1 Construction of the Gene Encoding the Chimera Protein
				3.1.1 FASTA Sequence of Target Proteins
				3.1.2 Prediction of T Cell Epitopes
				3.1.3 Prediction of B Cell Epitopes
			3.2 Analysis of Peptide Identity
			3.3 Peptide Characterization
			3.4 Chimera Assembly
			3.5 Chimera Gene Synthesis
		4 Notes
		References
	Chapter 25: Preclinical Assessment of the Immunogenicity of Experimental Leishmania Vaccines
		1 Introduction
		2 Materials
			2.1 Leishmania spp. Culture
			2.2 Soluble Leishmania Antigen (SLA)
			2.3 Evaluation of Antigen Concentration
			2.4 Preparation of Injections
			2.5 Immunization
			2.6 Mouse Challenge with Leishmania spp.
			2.7 Preparation of Mouse Serum After Immunization or After Immunization and Challenge
			2.8 Isolation of Mouse Splenocytes for Cell Culture
			2.9 Stimuli for Splenocytes Culture
			2.10 Isolation of Spleen, Liver, and Lymph Nodes for Parasite Culture
			2.11 Isolation of Bone Marrow for Parasite Culture
			2.12 Evaluation of the Parasite Load in Mouse Organs
			2.13 Flow Cytometry for Analysis of Cytokines
			2.14 Enzyme-Linked Immunosorbent Assay for Cytokines
			2.15 Enzyme-Linked Immunosorbent Assay for Antibodies
			2.16 Measurement of Nitrite Using Griess Reaction
				2.16.1 Preparation of Nitrite Standard Reference Curve
				2.16.2 Nitrite Measurement (Griess Reaction)
				2.16.3 Determination of Nitrite Concentrations in Experimental Samples
		3 Methods
			3.1 Leishmania spp. Culture
			3.2 Soluble Leishmania Antigen (SLA)
			3.3 Evaluation of Antigen Concentration
			3.4 Preparation of Injections
			3.5 Immunization
			3.6 Mouse Challenge with Leishmania spp.
			3.7 Preparation of Mouse Serum After Immunization or After Immunization and Challenge
			3.8 Isolation of Mouse Splenocytes for Cell Culture
			3.9 Stimuli for Splenocytes Culture
			3.10 Isolation of Spleen, Liver, and Lymph Nodes for Parasite Culture
			3.11 Isolation of Bone Marrow for Parasite Culture
			3.12 Evaluation of the Parasite Load in Mouse Organs
			3.13 Flow Cytometry for Analysis of Cytokines
			3.14 Enzyme-Linked Immunosorbent Assay for Cytokines
			3.15 Enzyme-Linked Immunosorbent Assay for Antibodies
			3.16 Measurement of Nitrite Using Griess Reaction
				3.16.1 Preparation of a Nitrite Standard Reference Curve
				3.16.2 Nitrite Measurement (Griess Reaction)
				3.16.3 Determination of Nitrite Concentrations in Experimental Samples
		4 Notes
		References
	Chapter 26: Production of Oral Vaccines Based on Virus-Like Particles Pseudotyped with Protozoan-Surface Proteins
		1 Introduction
		2 Materials
			2.1 VLP Production
				2.1.1 Plasmids
				2.1.2 Generation of a Cell Line That Stably Expresses VSP-G (HEK293-1267)
				2.1.3 Immunofluorescence Microscopy to Validate the Correct Expression of the Antigens
				2.1.4 Cell Transfection to Produce VLPs
				2.1.5 VLP Purification
			2.2 VLP Validation
				2.2.1 Western Blotting
				2.2.2 Hemagglutination Assay (HA)
				2.2.3 Nanoparticle Tracking Analysis (NTA)
				2.2.4 Immunoelectron Microscopy (Immuno-EM)
			2.3 VLP Immune Response
				2.3.1 VLP Immunization
				2.3.2 Ag-Specific Humoral Immune Response Analysis
					Fluid Collection
					Enzyme-Linked Immunosorbent Assay (ELISA) Tests
					Antibody Microneutralization Assays
				2.3.3 Ag-Specific Cellular Immune Response Analysis
					Splenocytes Isolation
					Cytokines Determination by  CBA
					IFN-γ ELISPOT Assay
					Flow Cytometry-Based Cytotoxic Assay
				2.3.4 Challenge with Live Virus
		3 Methods
			3.1 VLP Production
				3.1.1 Plasmids (Described in Subheading 2.2.1)
				3.1.2 Generation of a Cell Line That Stably Expresses VSP-G (HEK293-1267)
				3.1.3 Immunofluorescence Microscopy to Validate the Correct Expression of the Antigens
				3.1.4 Cell Transfection to Produce VLPs
				3.1.5 VLP Purification
			3.2 VLP Validation
				3.2.1 Western Blotting
				3.2.2 Hemagglutination Assay
				3.2.3 Nanoparticle Tracking Analysis (NTA)
				3.2.4 Immunoelectron Microscopy (Immuno-EM)
			3.3 VLP Immune Response
				3.3.1 VLP Immunization
				3.3.2 Antigen-Specific Humoral Immune Response Analysis
					Fluid Collection
					Enzyme-Linked Immunosorbent Assay (ELISA)
					Antibody Microneutralization Assays
				3.3.3 Ag-Specific Cellular Immune Response Analysis
					Isolation of Splenocytes
					Cytokine Determination by Cytokine Bead Array (CBA)
					IFN-γ Enzyme-Linked Immunospot (ELISPOT) Assay
					Flow Cytometry-Based Cytotoxic Assay
			3.4 Challenge with Live Virus
		4 Notes
		References
	Chapter 27: A Fast-Track Phenotypic Characterization of Plasmodium falciparum Vaccine Antigens through Lyse-Reseal Erythrocyte...
		1 Introduction
		2 Materials
			2.1 Production and Validation of Loaded Erythrocytes
			2.2 Assessment of miRNA Enriched Erythrocytes for Parasite Invasion
			2.3 Monitoring Translocation of micro-RNA into Parasites and Repression of the Target Protein
		3 Methods
			3.1 Production and Validation of Loaded Erythrocytes
				3.1.1 Preparation of Erythrocyte Ghosts and Loading of Candidate miRNA Mimics Using LyRED Method
				3.1.2 The Measurement of Resealing Efficiency
			3.2 Assessment of miRNA Enriched Erythrocytes for Parasite Invasion
				3.2.1 Parasite Culture of P. falciparum in miRNA Enriched Erythrocytes
				3.2.2 Monitoring Percent Merozoite Invasion
			3.3 Monitoring Translocation of micro-RNA to Parasites and Repression of the Target Protein
				3.3.1 Monitoring Translocation of miR-150-3p to the Parasite
				3.3.2 Confirmation of the Translocation of miRNA Mimic in Parasites from the Cargo Loaded Erythrocytes Through Fluorescence Im...
				3.3.3 Monitoring the Repression of the Target Protein
		4 Notes
		References
	Chapter 28: Plasmodium falciparum Antigen Expression in Leishmania Parasite: A Way Forward for Live Attenuated Vaccine Develop...
		1 Introduction
		2 Materials
			2.1 Medium and Reagents (See Note 1)
			2.2 Equipment
		3 Methods
			3.1 Cloning of P. falciparum Gene Encoding the Antigen
			3.2 Transient Transfection and Selection of L. donovani Promastigotes to Express PfAARP
			3.3 Confirmation of Antigen Expression in L. donovani Promastigotes
			3.4 Immunization of BALB/c Mice with L. donovani Promastigotes Expressing P. falciparum Antigen
			3.5 Evaluation of Malaria Infection Protection in Mice Immunized with L. donovani Expressing P. falciparum Antigen
		4 Notes
		References
	Chapter 29: Molecular Characterization of a Vector-Based Candidate Antigen Using the 3′-RACE and Genome Walking Methods and In...
		1 Introduction
		2 Materials
			2.1 RT-PCR
			2.2 PCR
		3 Methods
			3.1 Determining the Middle Region of the Target  Gene
				3.1.1 RNA Extraction
				3.1.2 Reverse Transcription Reaction
				3.1.3 Middle-Part Sequence Determination
			3.2 Determining the 3′ and 5′  Ends
				3.2.1 Primer Design for 3′- and 5′-RACE
				3.2.2 3′-RACE
				3.2.3 5′-RACE
				3.2.4 In Silico Analysis
					Sequence Assemble
					Structural Analysis
		4 Notes
		References
	Chapter 30: In Vitro Culture of Plasmodium falciparum for the Production of Mature Gametocytes for Performing Standard Membran...
		1 Introduction
		2 Materials
			2.1 Required Equipment
		3 Methods
			3.1 Preparing the Pooled AB+ Serum
			3.2 Preparing the Basic Culture Medium (BCM)
			3.3 Preparing the Complete Culture Medium (CCM)
			3.4 Preparing the Washed Red Blood Cells
			3.5 Mixed Atmosphere for Culture
			3.6 In Vitro Culture of P. falciparum and Gametocyte Production
			3.7 Parasite Synchronization
			3.8 In Vitro Culture of Gametocytes
		4 Notes
		References
	Chapter 31: Plasmodium berghei Infection in BALB/c Mice Model as an Animal Model for Malaria Disease Research
		1 Introduction
		2 Materials
			2.1 Required Equipment
		3 Methods
			3.1 Infecting Mouse with Cryopreserved P. Berghei
				3.1.1 Parasite Preparation
				3.1.2 Mouse Specifications
				3.1.3 Injection of Parasites
			3.2 Evaluating Mouse Infection
				3.2.1 Preparing the Blood Smear
			3.3 Counting the Parasites Number
			3.4 Parasite Passage
			3.5 Third Passage into the Phenylhydrazine-Injected Mice
		4 Notes
		References
	Chapter 32: Standard Membrane Feeding Assay for Malaria Transmission Studies
		1 Introduction
		2 Materials
			2.1 Required Equipment
		3 Methods
			3.1 Preparation of Mosquitoes
				3.1.1 Mosquito Selection and Rearing
				3.1.2 Female Mosquito Selection and Grouping
			3.2 Preparing the Artificial Feeder Instrument
			3.3 Exflagellation and Preparing the Gametocyte Culture
			3.4 Mosquito Feeding
			3.5 Mosquito Midgut Dissection and Oocyte Evaluation
			3.6 Transmission-Blocking Activity Calculation
		4 Notes
		References
Part VI: Development of Cancer Vaccines
	Chapter 33: Generation of Tumor Targeted Dendritic Cell Vaccines with Improved Immunogenic and Migratory Phenotype
		1 Introduction
			1.1 DC Vaccines for Cancer Immunotherapy
			1.2 CCL3-Mediated Enhancement of Intradermal DC Vaccine Migration
			1.3 Serum-Free DC Culture for Superior Tumor Antigen-Specific Immunogenicity
		2 Materials
			2.1 Bone Marrow-Derived DC Vaccine Preparation and Electroporation
				2.1.1 Preparation of Serum-Free Mouse Dendritic Cells
				2.1.2 In Vitro-Transcribed (IVT) mRNA Electroporation
				2.1.3 CCL3 Incubation
			2.2 Flow Cytometric Immunophenotyping of DC Vaccine
			2.3 Intradermal DC Vaccination and Vaccine Migration Quantification
				2.3.1 Intradermal DC Vaccination
				2.3.2 DC Vaccine Migration Quantification
		3 Methods
			3.1 Preparation of Serum-Free Mouse Dendritic Cells
				3.1.1 In Vitro-Transcribed (IVT) mRNA Electroporation
				3.1.2 CCL3 Incubation
			3.2 Flow Cytometric Immunophenotyping of DC Vaccine
			3.3 Intradermal DC Vaccination and Vaccine Migration Quantification
				3.3.1 Intradermal DC Vaccination
				3.3.2 DC Vaccine Migration Quantification
					Vaccine Site-Draining Lymph Node Harvest
					Vaccine Site-Draining Lymph Node Processing
					Vaccine Site-Draining Lymph Node Staining
		4 Notes
		References
	Chapter 34: Monocytes as a Cellular Vaccine Platform to Induce Antitumor Immunity
		1 Introduction
		2 Materials
			2.1 Purification of BM-Derived Ly-6Chi Monocytes
			2.2 Immunophenotyping of Ly-6Chi Monocytes
			2.3 Monocyte Antigen Loading
			2.4 Monocyte Vaccine Administration
			2.5 Tetramer Staining of CD8+ T Cells to Examine Vaccine Efficacy
			2.6 Tumor Cell Culture and Inoculation
		3 Methods
			3.1 Purification of BM-Derived Ly-6Chi Monocytes
			3.2 Immunophenotyping Ly-6Chi Monocytes
			3.3 Monocyte Antigen Loading
			3.4 Monocyte Vaccine Administration
			3.5 Tetramer Staining of CD8+ T Cells to Examine Vaccine Efficacy
			3.6 Tumor Cell Culture and Inoculation
		4 Notes
		References
	Chapter 35: Beyond Sequencing: Prioritizing and Delivering Neoantigens for Cancer Vaccines
		1 Introduction
		2 Development of Neoantigen-Based Cancer Vaccines
		3 The Prediction and Prioritization of Immunogenic Tumor Neoantigens
			3.1 Alternative Sources of Neoantigens
			3.2 Neoantigen Presentation and the Impact of HLA Binding
			3.3 Clonal Heterogeneity: Selecting Driver vs. Passenger Mutations
		4 Formulation and Delivery of Neoantigen-Based Vaccines
			4.1 Peptide Vaccines
			4.2 Nucleic Acid Vaccines
			4.3 Viral Vector Vaccines
		5 Timing of Vaccination and Synergy with Other Therapies
		6 Neoantigen Vaccine Toxicity Considerations
		7 Conclusion
		References
Part VII: Vaccines for Allergy
	Chapter 36: Proteomics for Development of Food Allergy Vaccines
		1 Introduction
		2 Materials
			2.1 Reference Food/Fish Samples
			2.2 Protein Extraction
			2.3 Allergen Purification
			2.4 Protein Concentration
			2.5 Protein Digestion with Trypsin
			2.6 Shotgun Liquid Chromatography Tandem Mass Spectrometry (LC-MS/MS) Analysis
			2.7 MS Data Processing
			2.8 Bioinformatics Analysis of Allergen Sequences and B Cell Epitopes
			2.9 Synthetic Peptide Epitopes
			2.10 Sera from Different Fish Allergic Patients and Healthy Donors
			2.11 Enzyme-Linked Immunosorbent Assay (ELISA)
			2.12 Statistical Analysis
			2.13 3D Structural Modeling
		3 Methods
			3.1 Shotgun Proteomics Analysis of β-PRVBs
				3.1.1 Reference Fish Species
				3.1.2 Allergen Protein Extraction and Purification
				3.1.3 Trypsin Protein Digestion
				3.1.4 LC-MS/MS Analysis
				3.1.5 MS Data Processing
			3.2 Bioinformatics Analysis of β-PRVB Sequences
			3.3 Bioinformatics Analysis of B Cell Epitopes
			3.4 Synthesis of Selected B Cell Peptide Epitopes
			3.5 Sera from Different Fish Allergic Patients and Healthy Donors
			3.6 Immunoassay Using Sera from Healthy and Allergic Patients by ELISA
			3.7 Statistical Analysis
			3.8 3D Structural Modeling
		4 Notes
		References
Part VIII: Vaccines for Toxins
	Chapter 37: Estimating Vaccine Potency Using Antibody-Based Competition Assays
		1 Introduction
		2 Materials and Equipment
			2.1 Reagents
			2.2 Instrumentation
			2.3 Materials
		3 Procedure
			3.1 RiCoE
				3.1.1 Coating ELISA Plates
				3.1.2 Blocking Microtiter Plates
				3.1.3 Prepare Serial Dilutions of  BDP
				3.1.4 Prepare PB10 and SyH7
				3.1.5 Sample Application
				3.1.6 Addition of Secondary Antibody
				3.1.7 ELISA Development
				3.1.8 Measurement of Optical Density
				3.1.9 Analysis of Results
			3.2 Force Degradation (FD) Studies
				3.2.1 Forced Degradation of  BDP
				3.2.2 RiCoE Analysis of FD Samples
				3.2.3 RiCoE Analysis of FD Samples
			3.3 In Vivo Potency Determinations
				3.3.1 Study Design
				3.3.2 Assemble Mouse Groups
				3.3.3 Vaccination Preparation
				3.3.4 Vaccination: Prime
				3.3.5 Vaccination: Boost
				3.3.6 Serum Collection
				3.3.7 Antibody Titer Determinations by ELISA
					Coat ELISA Plates
					Blocking Microtiter Plates
					Serum Preparation
					Sample Application
					Secondary Antibody and TMB Detection
					ELISA Development
					Data Analysis
				3.3.8 Ricin Toxin (RT) Challenge
					RT Preparation
					Mouse Weight and Blood Glucose Determinations
					RT Challenge
					Monitoring Mice Post-RT Challenge
					Euthanasia
					Data Analysis
		4 Notes
		References
Index