Gabor Tamas Szabo, MD, PhD, is an Associate Director at BioNTech SE. His work is focuses on mRNA technology optimization and applications.

Norbert Pardi, PhD, is an Assistant Professor at the University of Pennsylvania, USA. His research is focused on the development of mRNA-based therapeutics with a focus on vaccines.

Preface xiii Preface from the Volume Editors xv Part I How mRNA Vaccines Work 1 1 A Historical Overview on mRNA Vaccine Development 3Rein Verbeke, Miffy H.Y. Cheng, and Pieter R. Cullis 1.1 Introduction 3 1.2 The Path of mRNA as an Unstable and Toxic Product to a New Class of Medicine 5 1.2.1 The Discovery and In Vitro Production of mRNA 5 1.2.2 The Inflammatory Nature of mRNA 7 1.3 How Studying Lipid Bilayer Structures in Cell Membranes Gave Rise to the Eventual Development of Lipid Nanoparticles for RNA Delivery 8 1.3.1 From Biological Cell Membranes to Liposomal Drugs 8 1.3.2 Ionizable Lipid Nanoparticles for Systemic Delivery of Nucleic Acids 10 1.4 The Journey of Developing Clinical mRNA Vaccines 12 1.5 Concluding Remarks 14 References 15 2 Immune Responses to mRNA Vaccine 29Jean-Yves Exposito, Claire Monge, Danielle C. Arruda, and Bernard Verrier 2.1 Introduction 29 2.2 Innate Sensing of RNA Molecules 30 2.3 Innate Immune Response to mRNA Vaccines 32 2.3.1 Innate Immune Response in Humans 33 2.3.2 Tissue Innate Immune Response in Mice 34 2.4 mRNA Design and Innate Immunity 35 2.4.1 Cap 35 2.4.2 Untranslated Regions 37 2.4.3 Poly(A) 39 2.4.4 Coding Sequence 41 2.5 Optimization and Production of mRNA for an Adequate Innate Immune Response 42 2.5.1 IVT Production 42 2.5.2 Posttranscriptional Modification 44 2.5.3 Purification 45 2.6 mRNA Delivery Systems and Immune Response: The Role of Formulation Composition 45 2.7 Concluding Remarks and Perspectives 51 Acknowledgments 54 References 54 3 Modified or Unmodified mRNA Vaccines? - The Biochemistry of Pseudouridine and mRNA Pseudouridylation 69Pedro Morais and Yi-Tao Yu 3.1 Pseudouridine (¿): The Fifth Nucleoside 69 3.2 RNA Pseudouridylation Mechanism 70 3.2.1 Naturally Occurring RNA Pseudouridylation 71 3.2.1.1 RNA-independent Pseudouridylation Catalyzed by PUS Enzymes 71 3.2.1.2 RNA-dependent Pseudouridylation Catalyzed by Box H/ACA snoRNP 71 3.2.2 Artificially Introduced RNA Pseudouridylation 73 3.2.2.1 Targeted Pseudouridylation of RNA Using Artificial Guide RNAs 73 3.2.2.2 Incorporation of ¿ During In Vitro Transcription of RNA 74 3.3 ¿ detectioninRNA 75 3.3.1 Indirect ¿ Sequencing Methods 76 3.3.2 Direct ¿ Sequencing Methods 76 3.4 Impact of ¿ in Pre-mRNA Splicing and Protein Translation 77 3.4.1 Effect of ¿ in snRNA and Pre-mRNA on Pre-mRNA Splicing 77 3.4.2 Effect of ¿ in rRNA and tRNA on Protein Translation 77 3.4.3 Effect of mRNA Pseudouridylation on Nonsense Suppression 78 3.4.4 Effect of mRNA Pseudouridylation on the Coding Specificity of Sense Codons 80 3.5 ¿ and the Immune System 80 3.6 Pseudouridylated Versus Unmodified mRNA Vaccines 82 3.6.1 ¿ Successor: N1-methyl-¿ 82 3.6.2 Nucleoside-modified COVID-19 mRNA Vaccines 84 3.6.3 Unmodified mRNA COVID-19 vaccines 85 3.6.4 Cancer mRNA Vaccines 89 3.7 Conclusions 90 Acknowledgments 92 Conflict of Interest 92 References 92 4 Self-Replicating RNA Viruses for Vaccine Development 109Kenneth Lundstrom 4.1 Introduction 109 4.2 Expression Systems For Self-Replicating RNA Viruses 109 4.3 Vaccines Against Infectious Diseases 113 4.4 Vaccines Against Cancers 130 4.4.1 Reporter Gene Expression 131 4.4.2 Tumor-associated Antigens 131 4.4.3 Cytotoxic and Anti-tumor Genes 139 4.4.4 Immunostimulatory Genes 139 4.4.5 Oncolytic Viruses 140 4.5 Conclusions and Future Aspects 143 References 144 5 Circular RNA Therapeutics and Vaccines 161Xiang Liu and Guizhi Zhu 5.1 Introduction 161 5.2 The Biogenesis and Physiological Functions of Natural circRNA 162 5.2.1 The Biogenesis of Natural circRNA 162 5.2.2 The Physiological Functions of Natural circRNA 162 5.3 The Design and Synthesis of Synthetic circRNA 163 5.3.1 Design Considerations of Synthetic circRNAs for Vaccines 163 5.3.2 Approaches to circRNA Synthesis 164 5.4 The Applications of Synthetic circRNA as Novel Therapeutics and Vaccines 168 5.5 The Delivery Systems of Synthetic circRNA 170 5.6 Conclusion 170 References 171 6 Good Manufacturing Practices and Upscaling of mRNA Vaccine Production 177Eleni Stamoula, Theofanis Vavilis, Ioannis Dardalas, and Georgios Papazisis 6.1 Introduction 177 6.2 Plasmid Production 178 6.3 Considerations of In Vitro Transcription Stage 180 6.3.1 The In Vitro Transcription Reaction 180 6.3.2 Purification of the In Vitro Transcribed RNA 181 6.4 Considerations of Lipid Nanoparticles (LNPs) 185 6.4.1 Synthesis of LNP and mRNA Encapsulation 185 6.4.2 Scaling Up Production of LNPs to Industrial Standards 186 6.5 Considerations of Fill-to-Finish and Storage 186 6.6 mRNA and mRNA-LNP Critical Quality Attribute Analysis 187 6.7 General Remarks and Further Considerations 189 References 191 7 mRNA Vaccination for Induction of Immune Tolerance Against Autoimmune Disease 201Mark C. Gissler, Felix S.R. Picard, Timoteo Marchini, Holger Winkels, and Dennis Wolf 7.1 Role of Adaptive Immune Cells in Autoimmunity and Tolerance 201 7.1.1 Development of the Adaptive Immune System - Defining the Boundaries of Autoimmunity 201 7.1.2 Diversity of Adaptive Immunity 201 7.1.3 Antigen-Specific T Cells 202 7.1.4 T-cell Phenotypes and Functions 203 7.1.4.1 CD4 + T Cells 203 7.1.4.2 CD8 + T Cells 204 7.1.5 Role of B Cells and Autoantibodies 204 7.1.5.1 Development of B Cells 204 7.1.5.2 Somatic Hypermutations in B Cells - Refining High-Affinity Antibodies 205 7.1.5.3 Noncanonical Functions of B Cells 206 7.1.6 Autoimmune Diseases - Break of Tolerance Against Self-antigens 206 7.1.7 Immunomodulation of Autoimmune Diseases 207 7.1.7.1 Tolerogenic Vaccination to Dampen MHC-II-Dependent Autoimmunity 208 7.2 Atherosclerosis - An Unprecedented Autoimmune Disease 208 7.2.1 Autoimmune Component of Atherosclerosis 208 7.2.1.1 Role of Antigen-Specific T-Helper Cells in Atherosclerosis 209 7.2.1.2 B Cells and Autoantibodies in Atherosclerosis 209 7.2.2 Established Autoantigens in Atherosclerosis 210 7.2.2.1 LDL-C and ApoB 210 7.2.2.2 Heat-Shock Proteins 211 7.2.2.3 Beta-2-Glycoprotein I 211 7.2.2.4 Virus-Derived Antigens 211 7.2.3 Mechanism of Tolerogenic Peptide Vaccination in Atherosclerosis 212 7.2.4 Alternative Immunomodulation Against Cardiovascular Disease (CVD) Autoimmunity 212 7.2.4.1 DNA and mRNA Vaccination 212 7.2.4.2 Immunotherapy with Immunoglobulins 214 7.2.4.3 TCR/CAR T-cell Immunotherapy 215 7.3 The Autoimmune Component of MS 215 7.3.1 Pathophysiology of MS 215 7.3.2 Role of Antigen-Specific Immunity in MS 215 7.3.3 Mimicking MS by EAE Model 216 7.3.4 Vaccination Approaches to Prevent EAE 216 7.3.4.1 mRNA-Based Tolerogenic Vaccination Against EAE 217 7.4 Framework and Rationale for Future mRNA-Based Peptide Vaccination Strategies in Autoimmune Diseases 218 7.4.1 Evidence for mRNA Vaccination to Induce Tolerance in Animal Models 219 7.4.2 Limitations of Traditional Peptide Vaccination 220 7.4.3 Challenges of Future Vaccination Strategies 221 7.4.3.1 Antigen Targets and MHC Variability 221 7.4.3.2 Clinically Applicable Adjuvants and Routes of Administration 222 7.4.3.3 Effectiveness and Safety of Peptide Vaccination 223 7.4.3.4 Requirement of Clinical Biomarkers 223 7.4.4 Outlook: Chances of mRNA-Based Approaches in Future Clinical Immunomodulation in Allergy 224 List of Abbreviations 227 Acknowledgements 228 Conflict of Interest 228 References 228 Part II Recent Progress in Vaccine Research and Development 241 8 Design and Development of mRNA Vaccines to Combat the COVID-19 Pandemic 243Istvan Tombacz 8.1 Introduction 243 8.2 SARS-CoV-2 Vaccine Design 244 8.3 Development of SARS-CoV-2 mRNA Vaccines 247 8.3.1 mRNA-1273 - Moderna 247 8.3.2 BNT162b2 - Pfizer/BioNTech 248 8.4 Other SARS-CoV-2 mRNA Vaccines Developments 249 8.4.1 CVnCoV - CureVac 249 8.4.2 Additional mRNA-based SARS-CoV-2 Vaccines Evaluated in Clinical Trials 250 8.5 Booster Immunizations and Variants of Concern 251 8.6 Future Directions 252 References 253 9 mRNA Vaccines for HIV- 1 259Paolo Lusso 9.1 Introduction 259 9.1.1 A Long and Winding Road: 40 Years and Counting 259 9.1.2 A Very High Bar: Failure of Traditional Approaches 260 9.1.3 A New Era: An HIV-1 Vaccine Is Feasible 260 9.2 Strategies for HIV-1 Vaccine Design 261 9.2.1 Main Strategies 261 9.2.1.1 Lineage-Based Vaccines 261 9.2.1.2 Mutation-Guided Vaccines 262 9.2.1.3 Structure-Based Vaccines 262 9.2.1.4 Epitope-Based Vaccines 262 9.2.1.5 Combination Strategies 263 9.3 mRNA-Based HIV-1 Vaccines 263 9.3.1 Why mRNA? 263 9.3.2 Key Technological Breakthroughs 265 9.3.3 Main Platforms for mRNA-Based HIV-1 Vaccines 265 9.3.3.1 mRNA-Transduced Dendritic Cells 267 9.3.3.2 Direct In Vivo mRNA Delivery 267 9.3.3.3 The Rise of the LNPs 269 9.3.3.4 Self-Amplifying mRNA 270 9.4 Recent Advances in HIV-1 mRNA Vaccine Design 271 9.4.1 The Medium Is Not the Message 271 9.4.2 Specific Approaches 271 9.4.2.1 A VLP-Forming env-gag mRNA Platform 272 9.4.2.2 Self-Assembling Nanoparticles 274 9.4.2.3 Engineered Germline-Engaging gp120 Cores 274 9.5 The Future 275 9.5.1 Room for Improvement 276 9.5.1.1 Mucosal Delivery and Other Alternative Routes 276 9.5.1.2 Slow Delivery 276 9.5.1.3 Env-Gag VLP Optimization 277 9.5.1.4 Multiple-Array Antigen Presentation 277 9.5.1.5 Supplemental Adjuvants 278 9.5.1.6 Combination of mRNA with Other Platforms 278 9.6 Concluding Remarks 279 Acknowledgment 279 References 279 10 mRNA Vaccines Against Tick-borne Diseases 285Gunjan Arora and Erol Fikrig 10.1 Introduction 285 10.2 Vector-borne Diseases 285 10.3 Tick-borne Diseases 286 10.4 Tick Saliva Antigens as Vaccine Candidates 286 10.5 Vaccines Targeting Pathogens That Cause Tick-borne Diseases 288 10.6 mRNA Vaccines 288 10.7 An mRNA Vaccine Against Ticks 289 10.8 Powassan Vaccine 291 10.9 RNA Vaccine Against Crimean-Congo Hemorrhagic Fever Virus 291 10.10 Conclusions 292 References 293 11 mRNA Vaccines for Malaria and Other Parasitic Pathogens 303Leroy Versteeg and Jeroen Pollet 11.1 The Global Burden of Parasitic Pathogens 303 11.2 Challenges of Vaccine Development Against Parasitic Pathogens 305 11.3 mRNA Technology to Accelerate the Development of Advanced Next-Generation...