Challenges and Opportunities of mRNA Vaccines Against SARS-CoV-2: A Multidisciplinary Perspective

Foreword
Preface
Acknowledgments
About the Author
What This Book Is All About
	References
Contents
Abbreviations
1 Introduction
	References
Part I Scientific Underpinnings, Early Expectations, and Emerging Challenges from the Global Inoculation Experience
	2 Appraisal of Some of the Key Postulates Underlying mRNA Vaccines
		2.1 Potential Uncertainties of the Model
			2.1.1 The Synthesized RNA Molecules May Not Play the Role of Messenger RNAs
			2.1.2 Maximizing the Expression of the Immunogenic Gene from a Network Perspective
			2.1.3 The Role of Metabolism
			2.1.4 The Spike Is a Functional Protein Whose Immunogenicity Is Determined and Targeted by Various Non-orthodox Modification Strategies
		2.2 The Assumption ``The IVT mRNA\'s self-adjuvant property is beneficial\'\'
			2.2.1 Harmful dsRNAs, as First Known from the in Vitro Synthesis of the mRNAs
			2.2.2 DsRNAs Play a Key Role in the Dichotomous Self-Adjuvant Effect of mRNA
			2.2.3 The mRNA Vaccine Effect of Type I IFNs Modulation on CD8+ T Cell Immunity Is a Double-Edged Sword
		2.3 The Assumption ``Vaccine mRNAs do not integrate into the human genome\'\'
			2.3.1 Implicit Assumptions Regarding Their Non-integrative and Non-mutagenic Nature
			2.3.2 Reverse-Transcriptase Activity in Human Cells
		2.4 The Assumption ``IVT mRNA is not mutagenic\'\'
			2.4.1 DsRNAs Are Mediators of RNAi Pathways and Potential Mutagens
		2.5 The Assumption That ``vaccine mRNAs immediately get degraded\'\'
		2.6 The Assumption ``Any excessive immune-stimulatory activity can be eliminated from IVT processing\'\'
			2.6.1 The Concern That Undesirable Immune Processes Get Triggered in Vivo
			2.6.2 dsRNA Production in Vivo from Synthetic mRNAs
			2.6.3 Vaccines with Self- and Trans-Amplifying mRNAs
		References
	3 Relevance for mRNA Vaccine Safety
		3.1 Detrimental Consequences of Type I IFN Stimulation and New Disease Patterns
			3.1.1 Dichotomous Immunogenicity and Cytotoxicity of Vaccine RNAs and Byproducts
			3.1.2 Autoimmune Conditions and New Pathologies
				3.1.2.1 Bell\'s Palsy and Guillain-Barre Syndrome
				3.1.2.2 Transverse Myelitis and Myelitis
				3.1.2.3 Blood Clots and Bleeding Disorders
		3.2 Cross-Reactivities, Vaccine Self-Adjuvancy, and Adverse Immune Responses
			3.2.1 mRNA Vaccines as Potential Agents in the Initiation of AI, or as Triggers of AI Flares
			3.2.2 Immune-Mediated Hepatitis Following mRNA Inoculation and Type III Hypersensitivity Reactions
			3.2.3 The Potential of Immunopathologic Th2 Responses
		3.3 Shooting the Messenger of Critical Human Proteins
			3.3.1 Impairment of the Adaptive Immune System
			3.3.2 Shooting the Messenger of Tumor-SuppressingProteins
		3.4 Contaminants of IVT Processing
		3.5 Dichotomous Immune Response and Attribution of Adverse Events
		3.6 The Real Potential of Creating Genetically Modified Humans
			3.6.1 DsRNAs and Their Role in Mutagenesis
				3.6.1.1 From Synthetic mRNAs to dsRNAs to Genetic Modifications
				3.6.1.2 DsRNAs, Even in Small Amounts Can Regulate Gene Expression
				3.6.1.3 Repeated Vaccination May Increase the Risk of Unintended Heritable Changes
			3.6.2 Pathogenic Endogenous RT Activity in Human Cells via Transposable Elements
			3.6.3 Retro-Integration of IVT RNAs May Be Triggered by Several Mechanisms
			3.6.4 Genomic Integration and False PCR Tests Following Vaccination
			3.6.5 Relevance for Clinical Trials and Antiviral Therapies
			3.6.6 Ribonucleotides as Harmful DNA Lesions
		3.7 Impact of Truncated IVT mRNA Species and Other Short RNAs Derived from RNA Vaccines
			3.7.1 Interfering with Endogenous RNAi Processes and miRNA-Regulated Gene Expression
				3.7.1.1 Vaccine Byproducts May Become Precursors of Regulatory RNAs
				3.7.1.2 The Initiation of RNAi Processing Does Not Hinge Upon Specific Genetic Features of the Instigating dsRNAs
				3.7.1.3 Overlapping Activities of si/miRNA Precursors Can Be Induced by a Range of Different dsRNAs
			3.7.2 Interfering with the Balance Between IFN and RNAi-Based Antiviral Defense Mechanisms in Mammalian Cells
				3.7.2.1 Unexpected Silencing Effects of RNA Vaccines
				3.7.2.2 Vaccine RNAs Interfering with miRNA Regulations
			3.7.3 Disrupting Other Activities of Human miRNAs at the Cellular Level
			3.7.4 Disrupting Circulating/Extracellular miRNAs
		3.8 External RNAs as miRNA Activity Modulators
		References
	4 From Challenges to Opportunities and Open Questions
		4.1 Differentiating Whether Adverse Reactions Are Geared Against the Lipid Nanoparticles or Against Vaccine RNAs and Their Byproducts
		4.2 The Need for Clear Attribution
		4.3 Guarding Against Cross-Reactivities and Aberrant Immune Responses
		4.4 Guarding Against Other Medium and Longer-Term Side Effects
		4.5 The Opposing Role of mRNA Vaccine-Induced Type I IFN Signaling in the Regulation of T Cell Immunity
		4.6 Discerning the Function and Impact of Vaccine-Derived Regulatory RNAs
			4.6.1 The Interplay Between IFN Responses and RNAi Mechanisms in Self- and Non-self Recognition
			4.6.2 Mechanisms and Effects of Externally Derived Regulatory RNAs
			4.6.3 Off-Target Effects, Effects on the Human Microbiota, and the Larger Environment
			4.6.4 Small Vaccine-Derived RNAs in the Extracellular Space
		4.7 Can Vaccine RNAs Act as Micro RNA Activity Modulators?
		4.8 The Spike During Infection, as Opposed to the Spike Expressed via a Vaccine
			4.8.1 The Spike Itself as a Driver of Severe Disease
			4.8.2 Fate of the Vaccine Induced Spike Unclear Even at the Beginning of the Global Vaccination Campaign
			4.8.3 The Spike and Potential Analogs of Virally Mediated AI
		4.9 When the Vaccine Just ``Does Not Work\'\'
		4.10 The Need for a Clear Understanding of How mRNA Vaccines Affect Reproductive Health
			4.10.1 Pregnancy Safety Studies of mRNA Covid-19 Vaccines
				4.10.1.1 Rigorous Exclusion During the Trials
				4.10.1.2 How Has the Safety of mRNA Injections During Pregnancy Been Established?
			4.10.2 Incompletely Understood Mechanisms and Impact of Antibody Transfer to Infants
			4.10.3 Sperm Parameters Before and After mRNA Inoculation
		4.11 Mutagenic Risks Impacting Future Generations
		4.12 RNA Vaccines Need to Be Classified as Gene Therapies, with Corresponding Testing, Surveillance, and Long-Term Follow-up Practices
			4.12.1 RNA Vaccines Do Satisfy the Criteria of the FDA to Be Classified as GT Products
			4.12.2 Persistence, Resistance, and Viral Escape
			4.12.3 All GT Products, Including mRNA Vaccines, Require Long-Term Follow-up Studies
		References
	5 The Challenge of Evaluating Vaccine Safety and Effectiveness
		5.1 The Problem of Adequate and Unified Testing and Surveillance, and the Utilization of Statistics in the Biological Sciences
		5.2 The Challenge of Unified and Transparent Reporting of Adverse Events
		5.3 The Need for Unbiased Scientific Reporting and Interpretation of Vaccine Safety and Effectiveness
			5.3.1 Variations Among the Reported Case Numbers
			5.3.2 Global Patterns, as Opposed to Those at a Smaller Scale
		5.4 The Problem of Computing Vaccine Effectiveness
			5.4.1 Dying from or with
			5.4.2 Lack of a Single Cause in Population-Wide Infection Dynamics
			5.4.3 The Problem of Assessing Covid-19 Vaccine Effectiveness, During the Delta Time and Beyond
			5.4.4 Statistical Models and their Interpretations
			5.4.5 Other Statistical Measures and Complications
				5.4.5.1 The Number Needed to Treat (Vaccinate)
				5.4.5.2 When Vaccine Efficacy Parameters Drop, NNVs Quickly Increase
				5.4.5.3 The Dilemma of the Study Population in a Dynamic Context
				5.4.5.4 The Number Needed to Harm
				5.4.5.5 The Need to Observe the Bigger Picture
				5.4.5.6 Pros and Cons of Vaccinating Trial Participants
		References
	6 Safeguarding Against the Analog of Antimicrobial-Resistance Development
		6.1 Resistance Development to SARS-CoV-2 Vaccines
			6.1.1 Breakthroughs Involving the SARS-CoV-2 Variants B.1.1.7 and B.1.351
			6.1.2 Breakthroughs Involving the SARS-CoV-2 Variant B.1.617
			6.1.3 Further Lab Experiments
			6.1.4 Breakthroughs After First and Second Doses
		6.2 Risks of Common Public Antibody Responses
			6.2.1 Non-traditional Antibodies
			6.2.2 Selection by Neutralizing Antibodies May be a Key Driver to Induce a Viral Escape Mutant
		References
	7 Scales, Pseudoscales, the Human Factor, and a Way Forward
		7.1 Scale Relationships and Human Activities
		7.2 Pseudoscales as Problems
			7.2.1 The Pseudoscale PCR Positiveness
			7.2.2 ``Naturalness\'\' of Vaccine-Induced Immune Response
				7.2.2.1 Basic Issues Regarding the pseudoscale ``Naturalness of Antibody Response\'\'
				7.2.2.2 Amount and Biodistribution of the Spike Produced
				7.2.2.3 The Spike in Circulation: A Critical Scaling Issue
			7.2.3 The Pseudoscale Preventiveness of Disease and Death
				7.2.3.1 Vaccine Effectiveness Decreases with Time
				7.2.3.2 Estimates and Trends of Weaning Immunity (Up to the Delta Variant)
			7.2.4 The Pseudoscale ``Predictiveness of Sequence Differences and Determinants\'\'
			7.2.5 The Pseudoscale ``Antibody-Titer\'\'
			7.2.6 Natural Immunity Versus Vaccine-Induced Immunity
				7.2.6.1 Early Signals of Natural Immunity During the Delta Waves
				7.2.6.2 Protective Immunity After Recovery from SARS-CoV-2 Infection
			7.2.7 Similar Viral Load Among the Vaccinated and Unvaccinated
		7.3 Critical Control Points via Genuine Scaling Features
			7.3.1 Contamination and Quality Control Are Distinctive Scaling Concerns
			7.3.2 Negative Scaling of Antibody Diversity and Flexibility Exerted by Vaccination
			7.3.3 ``Sky-High\'\' Antibody Levels: A Vaccine Induced Scaling Feature with Potentials for Adverse Effects
			7.3.4 Scaling of Antibody-Driven Selection Pressure Leading to the Emergence of Viral Variants in Immune-Compromised Individuals
			7.3.5 Scaling of Antibody-Driven Selection Pressure Leading to the Emergence of New VOCs In Vitro
			7.3.6 Epidemiological Findings: Increased Dominance of VOCs Scales with the Increased Rate of Mass Vaccination Across Nations
			7.3.7 Boosters and Scaling of Disease Severity and Adverse Events
				7.3.7.1 Increased Disease Severity Upon SARS-CoV-2 Infection
				7.3.7.2 ADE as a Scaling Feature
				7.3.7.3 Scaling of Unselective Tissue Tropism of the LNPs, from Individual Patients to the Human Race
				7.3.7.4 Scaling of mRNA Vaccine Integration into, and Expression from, the Human Genome
			7.3.8 ``Vaccines\'\' Themselves, as Newly Defined by the CDC, as the Main Scale Driver
			7.3.9 Scaling of Human Intervention: When Apparent Benefits Escalate Risks Instead
		7.4 Ethical Considerations
		7.5 Critical Control Points Allow for Optimal Risk Mitigation
		References
Part II A Deeper Dive into mRNA Vaccine Safety and Security, and Developments Until Delta
	8 mRNA Vaccine Safety and Efficacy—Official Criteria When AEs Are Caused by the Injection
		8.1 Death and Other Adverse Events Following Immunization: The Three Basic Levels of the WHO Manual
			8.1.1 Evaluation of AEFIs in the Context of mRNA Vaccines—The Population Level
				8.1.1.1 Lack of a Baseline—A Consequence of Vaccinating Almost All Trial Participants
				8.1.1.2 Concerns with Trials
				8.1.1.3 Trials Are Not Completed Yet
				8.1.1.4 Challenges in Regard to Database Reporting
			8.1.2 AEFIs in the Context of mRNA Vaccines—The Individual Level
				8.1.2.1 Established Causality at the Population Level Necessary to Judge Causality at the Individual Level
				8.1.2.2 A Lack of Knowledge to Fulfill the Official Criteria of ``individual causal relationship\'\'
		8.2 Individual Criteria in the WHO-AEFI Manual in Regard to mRNA Vaccines—A Closer Look
			8.2.1 The Concept of Biological Plausibility Does Not Extend to mRNA Vaccines
			8.2.2 Is the Event Classifiable?
			8.2.3 Causality in the WHO Manual Requires No Other Factors Involved
		8.3 The Traditional vs. the Modified Definition of a Vaccine: From the Perspective of Causation
			8.3.1 ``Covid-19 Vaccines Prevent Infection and Transmission\'\' as a Causal Relationship
				8.3.1.1 Causality from a Logical Perspective
				8.3.1.2 Vaccine Effectiveness from a Causal Perspective
			8.3.2 A Moving Endpoint—Goal of the Vaccine
			8.3.3 The Moving Target as to Who Counts as Vaccinated
		8.4 Lessons Learned from the Pandemic—The WHO-AEFI Criteria Revisited
			8.4.1 Covid-19 Vaccines as Single Causative Agents
			8.4.2 Impact on WHO-AEFI Criteria to Determine AEFIs
			8.4.3 How Signals Are Being Identified, Measured, and Interpreted
			8.4.4 Optimism, Bias, and Disparate Interpretations
			8.4.5 Comparing a Subtle Outcome and Computer Decisions
		8.5 The Investigation of Signals—Assessing Causation Without Data
			8.5.1 Data Not Getting Published
				8.5.1.1 ``The C.D.C. Isn\'t Publishing Large Portions of the Covid Data It Collects\'\'
				8.5.1.2 The Pfizer Documents
			8.5.2 Another Aspect of Underreporting
		8.6 Criteria for Assessing Causation—Too Ambiguous vs. Too Strict
			8.6.1 Notions and Criteria to Assess Vaccine Safety Are Too Ambiguous
			8.6.2 Criteria Are Too Strict—``No causation shown\'\' Does Not Mean There Is No Causation
		References
	9 mRNA Covid-19 Vaccines Best Reflect Effective Pharmaceuticals—Basic Considerations and LNPs
		9.1 Concerns About mRNA ``Vaccines,\'\' Regarding Their Potentials as Vaccines
			9.1.1 Do mRNA Injections Resemble Actual ``Vaccines\'\'?
			9.1.2 Do mRNA Inoculations Act Like Therapeutics?
				9.1.2.1 ``Therapeutic Vaccines\'\'
				9.1.2.2 Is the Underlying Mechanism That of some Ill-defined Immune Activation?
			9.1.3 No Clearly Specified end of mRNA-LNP Activity, Amplifying Cell Damage and Adverse Reactions After Each Inoculation
		9.2 Tissue Tropism of the LNP ``Delivery System\'\'
			9.2.1 Selective Versus Unselective Tissue Tropism
			9.2.2 The Leaked Japanese Study
			9.2.3 Potential Consequences
		9.3 Findings from Related Vaccines
			9.3.1 Biodistribution of Moderna\'s 2017 Flu ``Vaccine\'\' Candidate
				9.3.1.1 Basic Findings of the 2017 Study
				9.3.1.2 The Results and Interpretations Lead to Some Open Questions
			9.3.2 Moderna\'s 2022 Flu Vaccine Candidate
			9.3.3 A Common Denominator
		9.4 Lipid NPs as Active Compounds
			9.4.1 LNPs Used for Preclinical Studies Are Highly Inflammatory and May Be Key Drivers of the Antibody Response
			9.4.2 Mechanism of the LNPs as Active Compounds Are Poorly Understood
			9.4.3 Polyethylene Glycol (PEG) and More
		References
	10 mRNA Covid-19 Pharmaceuticals and the Spike Antigen
		10.1 Design Criteria of the Spike
			10.1.1 Basic Assumptions and Open Questions
			10.1.2 Toxicity of the Spike in Covid-19 Disease and When Produced Upon Vaccination
				10.1.2.1 The Same Spike S1 Subunit Released in Both the Virus and the Product of the Genetic Inoculations, and Additional Concerns with the Vaccine-Spike
				10.1.2.2 Unique Toxic Features of the Spike
				10.1.2.3 The Vaccine-Induced Spike
				10.1.2.4 Unique Features of the Inoculations Lead to More Reasons for Concern
		10.2 Biodistribution and Persistence of the Vaccine-Induced Spike
			10.2.1 Spike Protein Fragments and Entire Spike Protein Detected in the Plasma of mRNA-1273 Vaccine Recipients
			10.2.2 Spike Protein Induced by BNT162b2 Found on Exosomes
			10.2.3 Vaccine Spike and mRNA Found to Persist in Lymph Node Germinal Centers and Confirmed in the Blood of Vaccinated Individuals
			10.2.4 More Evidence That the Vaccine mRNA Is Not Degrading But Continues to Produce Protein
		10.3 Vaccine-Derived Products Are Not Limited to the Cytosol But Found in the Nucleus
			10.3.1 Reverse-Transcriptase Activity Following SARS-CoV-2 Infection or Injection
			10.3.2 In vitro, Pfizer Vaccine mRNA Becomes DNA in Liver Cells
			10.3.3 Spike Protein Goes to the Nucleus and Impairs DNA Repair
				10.3.3.1 The Viral Spike and Other Viral Proteins Seem to Have the Potential to Block the Nucleus from Forming the Very Machinery to Repair Itself
				10.3.3.2 Concerns: With the Study or Rather with Premature Critique?
				10.3.3.3 Increased Concerns About the Injections
				10.3.3.4 Detection of Early Signals
				10.3.3.5 Covid Vaccines and Cancer
			10.3.4 Controversies or Incomplete Model?
		References
	11 Other Facets of SARS-CoV-2 Immunity, the Risk of Immune Tolerance and T Cell Exhaustion
		11.1 Sars-CoV-2 Immunity Beyond B Cell Protection
			11.1.1 Beyond the Notion of Antibodies as Necessary and Sufficient Agents
			11.1.2 Beyond Antibodies: The Impact of Vaccination on Innate and Adaptive Immunity
				11.1.2.1 The Potential of mRNA Vaccines to Impair Both Adaptive and Innate Immune Responses
				11.1.2.2 Pfizer\'s Vaccine Candidate BNT162b1 Was Shown to Lead to Reduces Lymphocyte Counts Following Vaccination
				11.1.2.3 Evidence of a Temporarily Blunted Innate Immune Response Immediately Following Injection
				11.1.2.4 Activation of Natural Killer Cells Through Virus-Specific Antibodies
		11.2 Covid-19 as a Mucosal Disease and the Risk of Vaccine-Induced Tolerance
			11.2.1 Mucosal Immune Tolerance, a Foundational Pillar of the Mucosal Immune System
			11.2.2 Covid-19 as a Two-Part Disease and mRNA Injections from a Mucosal Perspective
			11.2.3 mRNA Injections May Be Subject to Suppressor Functions of the Mucosal Immune System
				11.2.3.1 Intersection Between mRNA Injections and the Mucosal Immune System
			11.2.4 Is the Suppressive Effect of Mucosal Immunity Responsible for the Decline of mRNA Vaccine Efficacy?
			11.2.5 The Opposite: Boosters Evoking Hyperinflammatory Immune Responses
		11.3 T Cell Exhaustion
			11.3.1 The EMA Concern of Boosters Potentially Weakening the Immune System
			11.3.2 Plausibility of the Concern
		11.4 Signals of Adverse Immunological Reactions
			11.4.1 Large-Scale Study to Assess the Fourth Dose of BNT162b2
				11.4.1.1 Overall Study Design and Implications
				11.4.1.2 Study Outcome in Line with the Notion That mRNA Injections Resemble Drug-Therapeutics Rather Than Vaccines
			11.4.2 A Deeper Analysis of the Study Outcome: A Signal of an Adverse Immune Effect?
		11.5 The Impact of Vaccination During the Pandemic: Declining VE, No VE, or Negative Effects?
			11.5.1 Immune System Habituation and More
			11.5.2 No Effect or a Negative Effect?
			11.5.3 Possible Scenarios of Vaccine Protection and Decline
				11.5.3.1 Signals of Actual Negative Effects
			11.5.4 The Main Surrogates of Vaccine Protection Proved Inappropriate
		References
Part III The Omicron Variants
	12 Omicron
		12.1 Indicators of an Essentially New Virus
			12.1.1 High Degree of Escape from Previous Protection
			12.1.2 A Drastic Increase in Infectivity
			12.1.3 Overall, Omicron is Causing Less Severe Disease Compared to Previous SARS-CoV-2 Variants
		12.2 Why Is Omicron so Much More Infectious?
		12.3 Decline in Vaccine-Induced Immunity Intensifies During the Omicron Wave(s)
			12.3.1 Overall Declining VE Data
			12.3.2 For Children, VE Shows an Immediate and Radical Decline
			12.3.3 Recognition that Next-Generation Vaccines are Needed
			12.3.4 Accumulating Evidence of Negative VE Against Omicron
		12.4 Evidence of Increased Vaccine Mismatch
			12.4.1 Neutralization of Omicron Compared to Other VOCs, up to 3 Doses
			12.4.2 Neutralization of Omicron Compared to Other VOCs: 3 and 4 Doses
			12.4.3 A Synopsis of Factors that Seem to Drive Viral Neutralization Resistance
		12.5 ``Number of Doses Administered\'\' as a Scaling Feature
			12.5.1 Secondary Attack Rate (SAR) Differences Between Delta and Omicron in Denmark Point to the Number of Doses as an Important Scale Driver
				12.5.1.1 More Evidence of Omicron\'s Immune Evasiveness
				12.5.1.2 Differences in Transmission Between the Vaccinated and Unvaccinated Remains an Open Question
				12.5.1.3 The Odds Ratio (OR) is Significantly Greater than 1 for the Fully Vaccinated and Booster Vaccinated
				12.5.1.4 A Clear Dose–Response Relationship Between the OR and the Number of Injections
			12.5.2 A Vaccine-Dose-Dependent Rise in Omicron Infections Also Seen from a Large Study from California
			12.5.3 The Number of Shots: Likely the Main Scale Driver on Both the Viral and the Host Side
		12.6 Omicron, as an Escape Mutant, Can Use a New Way to Enter Cells
		12.7 Emerging Trends and Open Questions
			12.7.1 IgG Bias, High Ab Levels, and Immune Imprinting
			12.7.2 Omicron-Specific Boosters and Immune Priming
			12.7.3 The Origin of Omicron, and Why It Matters
				12.7.3.1 Several Theories About Omicron\'s Origin
				12.7.3.2 Could Omicron Have Arisen as a Vaccine-Escape Mutant?
				12.7.3.3 Beyond the Current Models
			12.7.4 Future Variants: A Guaranteed Trajectory of Common-Cold CVs?
		References
	13 Conclusion
		Modeling and Predictions Vs. Clinical and Human Reality
		With mRNA Vaccines, Risks and Benefits Do Scale Differently
		The Necessity of Mass Vaccination, Revisited
		Lessons (to Be) Learned from Omicron
		Looking Ahead
		References