Trends in mRNA Vaccine Research

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 xiiiPreface from the Volume Editors xvPart I How mRNA Vaccines Work 11 A Historical Overview on mRNA Vaccine Development 3Rein Verbeke, Miffy H.Y. Cheng, and Pieter R. Cullis1.1 Introduction 31.2 The Path of mRNA as an Unstable and Toxic Product to a New Class of Medicine 51.2.1 The Discovery and In Vitro Production of mRNA 51.2.2 The Inflammatory Nature of mRNA 71.3 How Studying Lipid Bilayer Structures in Cell Membranes Gave Rise to the Eventual Development of Lipid Nanoparticles for RNA Delivery 81.3.1 From Biological Cell Membranes to Liposomal Drugs 81.3.2 Ionizable Lipid Nanoparticles for Systemic Delivery of Nucleic Acids 101.4 The Journey of Developing Clinical mRNA Vaccines 121.5 Concluding Remarks 14References 152 Immune Responses to mRNA Vaccine 29Jean-Yves Exposito, Claire Monge, Danielle C. Arruda, and Bernard Verrier2.1 Introduction 292.2 Innate Sensing of RNA Molecules 302.3 Innate Immune Response to mRNA Vaccines 322.3.1 Innate Immune Response in Humans 332.3.2 Tissue Innate Immune Response in Mice 342.4 mRNA Design and Innate Immunity 352.4.1 Cap 352.4.2 Untranslated Regions 372.4.3 Poly(A) 392.4.4 Coding Sequence 412.5 Optimization and Production of mRNA for an Adequate Innate Immune Response 422.5.1 IVT Production 422.5.2 Posttranscriptional Modification 442.5.3 Purification 452.6 mRNA Delivery Systems and Immune Response: The Role of Formulation Composition 452.7 Concluding Remarks and Perspectives 51Acknowledgments 54References 543 Modified or Unmodified mRNA Vaccines? – The Biochemistry of Pseudouridine and mRNA Pseudouridylation 69Pedro Morais and Yi-Tao Yu3.1 Pseudouridine (Ψ): The Fifth Nucleoside 693.2 RNA Pseudouridylation Mechanism 703.2.1 Naturally Occurring RNA Pseudouridylation 713.2.1.1 RNA-independent Pseudouridylation Catalyzed by PUS Enzymes 713.2.1.2 RNA-dependent Pseudouridylation Catalyzed by Box H/ACA snoRNP 713.2.2 Artificially Introduced RNA Pseudouridylation 733.2.2.1 Targeted Pseudouridylation of RNA Using Artificial Guide RNAs 733.2.2.2 Incorporation of Ψ During In Vitro Transcription of RNA 743.3 Ψ detectioninRNA 753.3.1 Indirect Ψ Sequencing Methods 763.3.2 Direct Ψ Sequencing Methods 763.4 Impact of Ψ in Pre-mRNA Splicing and Protein Translation 773.4.1 Effect of Ψ in snRNA and Pre-mRNA on Pre-mRNA Splicing 773.4.2 Effect of Ψ in rRNA and tRNA on Protein Translation 773.4.3 Effect of mRNA Pseudouridylation on Nonsense Suppression 783.4.4 Effect of mRNA Pseudouridylation on the Coding Specificity of Sense Codons 803.5 Ψ and the Immune System 803.6 Pseudouridylated Versus Unmodified mRNA Vaccines 823.6.1 Ψ Successor: N1-methyl-Ψ 823.6.2 Nucleoside-modified COVID-19 mRNA Vaccines 843.6.3 Unmodified mRNA COVID-19 vaccines 853.6.4 Cancer mRNA Vaccines 893.7 Conclusions 90Acknowledgments 92Conflict of Interest 92References 924 Self-Replicating RNA Viruses for Vaccine Development 109Kenneth Lundstrom4.1 Introduction 1094.2 Expression Systems For Self-Replicating RNA Viruses 1094.3 Vaccines Against Infectious Diseases 1134.4 Vaccines Against Cancers 1304.4.1 Reporter Gene Expression 1314.4.2 Tumor-associated Antigens 1314.4.3 Cytotoxic and Anti-tumor Genes 1394.4.4 Immunostimulatory Genes 1394.4.5 Oncolytic Viruses 1404.5 Conclusions and Future Aspects 143References 1445 Circular RNA Therapeutics and Vaccines 161Xiang Liu and Guizhi Zhu5.1 Introduction 1615.2 The Biogenesis and Physiological Functions of Natural circRNA 1625.2.1 The Biogenesis of Natural circRNA 1625.2.2 The Physiological Functions of Natural circRNA 1625.3 The Design and Synthesis of Synthetic circRNA 1635.3.1 Design Considerations of Synthetic circRNAs for Vaccines 1635.3.2 Approaches to circRNA Synthesis 1645.4 The Applications of Synthetic circRNA as Novel Therapeutics and Vaccines 1685.5 The Delivery Systems of Synthetic circRNA 1705.6 Conclusion 170References 1716 Good Manufacturing Practices and Upscaling of mRNA Vaccine Production 177Eleni Stamoula, Theofanis Vavilis, Ioannis Dardalas, and Georgios Papazisis6.1 Introduction 1776.2 Plasmid Production 1786.3 Considerations of In Vitro Transcription Stage 1806.3.1 The In Vitro Transcription Reaction 1806.3.2 Purification of the In Vitro Transcribed RNA 1816.4 Considerations of Lipid Nanoparticles (LNPs) 1856.4.1 Synthesis of LNP and mRNA Encapsulation 1856.4.2 Scaling Up Production of LNPs to Industrial Standards 1866.5 Considerations of Fill-to-Finish and Storage 1866.6 mRNA and mRNA–LNP Critical Quality Attribute Analysis 1876.7 General Remarks and Further Considerations 189References 1917 mRNA Vaccination for Induction of Immune Tolerance Against Autoimmune Disease 201Mark C. Gissler, Felix S.R. Picard, Timoteo Marchini, Holger Winkels, and Dennis Wolf7.1 Role of Adaptive Immune Cells in Autoimmunity and Tolerance 2017.1.1 Development of the Adaptive Immune System – Defining the Boundaries of Autoimmunity 2017.1.2 Diversity of Adaptive Immunity 2017.1.3 Antigen-Specific T Cells 2027.1.4 T-cell Phenotypes and Functions 2037.1.4.1 CD4 + T Cells 2037.1.4.2 CD8 + T Cells 2047.1.5 Role of B Cells and Autoantibodies 2047.1.5.1 Development of B Cells 2047.1.5.2 Somatic Hypermutations in B Cells – Refining High-Affinity Antibodies 2057.1.5.3 Noncanonical Functions of B Cells 2067.1.6 Autoimmune Diseases – Break of Tolerance Against Self-antigens 2067.1.7 Immunomodulation of Autoimmune Diseases 2077.1.7.1 Tolerogenic Vaccination to Dampen MHC-II-Dependent Autoimmunity 2087.2 Atherosclerosis – An Unprecedented Autoimmune Disease 2087.2.1 Autoimmune Component of Atherosclerosis 2087.2.1.1 Role of Antigen-Specific T-Helper Cells in Atherosclerosis 2097.2.1.2 B Cells and Autoantibodies in Atherosclerosis 2097.2.2 Established Autoantigens in Atherosclerosis 2107.2.2.1 LDL-C and ApoB 2107.2.2.2 Heat-Shock Proteins 2117.2.2.3 Beta-2-Glycoprotein I 2117.2.2.4 Virus-Derived Antigens 2117.2.3 Mechanism of Tolerogenic Peptide Vaccination in Atherosclerosis 2127.2.4 Alternative Immunomodulation Against Cardiovascular Disease (CVD) Autoimmunity 2127.2.4.1 DNA and mRNA Vaccination 2127.2.4.2 Immunotherapy with Immunoglobulins 2147.2.4.3 TCR/CAR T-cell Immunotherapy 2157.3 The Autoimmune Component of MS 2157.3.1 Pathophysiology of MS 2157.3.2 Role of Antigen-Specific Immunity in MS 2157.3.3 Mimicking MS by EAE Model 2167.3.4 Vaccination Approaches to Prevent EAE 2167.3.4.1 mRNA-Based Tolerogenic Vaccination Against EAE 2177.4 Framework and Rationale for Future mRNA-Based Peptide Vaccination Strategies in Autoimmune Diseases 2187.4.1 Evidence for mRNA Vaccination to Induce Tolerance in Animal Models 2197.4.2 Limitations of Traditional Peptide Vaccination 2207.4.3 Challenges of Future Vaccination Strategies 2217.4.3.1 Antigen Targets and MHC Variability 2217.4.3.2 Clinically Applicable Adjuvants and Routes of Administration 2227.4.3.3 Effectiveness and Safety of Peptide Vaccination 2237.4.3.4 Requirement of Clinical Biomarkers 2237.4.4 Outlook: Chances of mRNA-Based Approaches in Future Clinical Immunomodulation in Allergy 224List of Abbreviations 227Acknowledgements 228Conflict of Interest 228References 228Part II Recent Progress in Vaccine Research and Development 2418 Design and Development of mRNA Vaccines to Combat the COVID-19 Pandemic 243Istvan Tombacz8.1 Introduction 2438.2 SARS-CoV-2 Vaccine Design 2448.3 Development of SARS-CoV-2 mRNA Vaccines 2478.3.1 mRNA-1273 – Moderna 2478.3.2 BNT162b2 – Pfizer/BioNTech 2488.4 Other SARS-CoV-2 mRNA Vaccines Developments 2498.4.1 CVnCoV – CureVac 2498.4.2 Additional mRNA-based SARS-CoV-2 Vaccines Evaluated in Clinical Trials 2508.5 Booster Immunizations and Variants of Concern 2518.6 Future Directions 252References 2539 mRNA Vaccines for HIV- 1 259Paolo Lusso9.1 Introduction 2599.1.1 A Long and Winding Road: 40 Years and Counting 2599.1.2 A Very High Bar: Failure of Traditional Approaches 2609.1.3 A New Era: An HIV-1 Vaccine Is Feasible 2609.2 Strategies for HIV-1 Vaccine Design 2619.2.1 Main Strategies 2619.2.1.1 Lineage-Based Vaccines 2619.2.1.2 Mutation-Guided Vaccines 2629.2.1.3 Structure-Based Vaccines 2629.2.1.4 Epitope-Based Vaccines 2629.2.1.5 Combination Strategies 2639.3 mRNA-Based HIV-1 Vaccines 2639.3.1 Why mRNA? 2639.3.2 Key Technological Breakthroughs 2659.3.3 Main Platforms for mRNA-Based HIV-1 Vaccines 2659.3.3.1 mRNA-Transduced Dendritic Cells 2679.3.3.2 Direct In Vivo mRNA Delivery 2679.3.3.3 The Rise of the LNPs 2699.3.3.4 Self-Amplifying mRNA 2709.4 Recent Advances in HIV-1 mRNA Vaccine Design 2719.4.1 The Medium Is Not the Message 2719.4.2 Specific Approaches 2719.4.2.1 A VLP-Forming env-gag mRNA Platform 2729.4.2.2 Self-Assembling Nanoparticles 2749.4.2.3 Engineered Germline-Engaging gp120 Cores 2749.5 The Future 2759.5.1 Room for Improvement 2769.5.1.1 Mucosal Delivery and Other Alternative Routes 2769.5.1.2 Slow Delivery 2769.5.1.3 Env-Gag VLP Optimization 2779.5.1.4 Multiple-Array Antigen Presentation 2779.5.1.5 Supplemental Adjuvants 2789.5.1.6 Combination of mRNA with Other Platforms 2789.6 Concluding Remarks 279Acknowledgment 279References 27910 mRNA Vaccines Against Tick-borne Diseases 285Gunjan Arora and Erol Fikrig10.1 Introduction 28510.2 Vector-borne Diseases 28510.3 Tick-borne Diseases 28610.4 Tick Saliva Antigens as Vaccine Candidates 28610.5 Vaccines Targeting Pathogens That Cause Tick-borne Diseases 28810.6 mRNA Vaccines 28810.7 An mRNA Vaccine Against Ticks 28910.8 Powassan Vaccine 29110.9 RNA Vaccine Against Crimean–Congo Hemorrhagic Fever Virus 29110.10 Conclusions 292References 29311 mRNA Vaccines for Malaria and Other Parasitic Pathogens 303Leroy Versteeg and Jeroen Pollet11.1 The Global Burden of Parasitic Pathogens 30311.2 Challenges of Vaccine Development Against Parasitic Pathogens 30511.3 mRNA Technology to Accelerate the Development of Advanced Next-Generation Vaccines 30711.4 Accessibility, Manufacturing Capacity, and Logistics of mRNA for Lowand Mid-Income Countries 30811.5 Published Data on mRNA Vaccines Against Parasitic Pathogens 31111.5.1 Malaria 31111.5.2 Toxoplasmosis 31411.5.3 Leishmaniasis 31511.5.4 Chagas Disease 31611.5.5 Helminths 31711.6 Conclusions and Prospects 317References 31812 Current State of mRNA Vaccine Development Against Mycobacterium tuberculosis 325Ilke Aernout, Rein Verbeke, Stefaan C. De Smedt, Francis Impens, and Ine Lentacker12.1 Introduction 32512.2 Immune Responses Responsible for Protective Immunity Against Mycobacterium tuberculosis 32612.3 Suitability and Advantages of an mRNA Vaccine Platform Against Mycobacterium tuberculosis 32812.4 mRNA TB Vaccines in (Pre-)clinical Development 330Acknowledgments 332References 33213 Cancer Vaccines Based on mRNA: Hype or Hope? 337Wout de Mey, Dorien Autaers, Giada Bertazzon, Arthur Esprit, Marta Marco Aragon, Lorenzo Franceschini, and Karine Breckpot13.1 Tumors: Setting the Scene for Cancer Immunotherapy 33713.2 Cancer Vaccination 33913.3 Vaccine Development Rules: A Brief Overview of Lessons Learned 34213.3.1 Use Multiple and Highly Immunogenic Tumor-Specific Antigens 34213.3.2 Use a Potent Adjuvant 34313.3.3 Use an Efficacious, Flexible, Safe, and Preferably Low-Cost Vaccine Vector 34513.3.4 Choose the Best Route of Delivery 34613.3.5 Incorporate Strategies to Subdue Tumor-Mediated Immunosuppression 34913.4 mRNA: From Discovery to Application in Vaccinology 35113.5 mRNA Manufacturing and Design 35313.6 mRNA Delivery and Formulation 35813.7 Controlling the Innate Immune Sensing of mRNA 36213.8 Adjuvants for mRNA-Based Vaccines 36613.9 Clinical Application 36813.10 Conclusion 373References 374Index 401