The Novelty of mRNA Viral Vaccines and Potential Harms: A Scoping Review

: Pharmacovigilance databases are showing evidence of injury in the context of the modiﬁed COVID-19 mRNA products. According to recent publications, adverse event reports linked to the mRNA COVID-19 injections largely point to the spike protein as an aetiological agent of adverse events, but we propose that the platform itself may be culpable. To assess the safety of current and future mRNA vaccines, further analysis is needed on the risks due to the platform itself, and not speciﬁcally the expressed antigen. If harm can be exclusively and conclusively attributed to the spike protein, then it is possible that future mRNA vaccines expressing other antigens will be safe. If harms are attributable to the platform itself, then regardless of the toxicity, or lack thereof, of the antigen to be expressed, the platform may be inherently unsafe, pending modiﬁcation. In this work, we examine previous studies of RNA-based delivery by a lipid nanoparticle (LNP) and break down the possible aetiological elements of harm.


Introduction
Pharmaceutical drug and medical device approvals are predicated on the completion of a structured approval process through various regulatory agencies. Historically, the approval process has contributed to patient safety by subjecting all new approvals to a rigorous safety assessment. However, there are many examples of over-turnings of approvals of pharmaceuticals post facto, due to the emergence of oversights of particular safety factors that occurred during the approval process [1]. These failures of regulatory bodies to sufficiently assess safety during the approval process is costly in terms of health and economic harms [2]. To put this issue into perspective, of 309 novel cardiovascular, orthopaedic, and neurologic devices approved in the EU between 2005 and 2010, 73 (24%) were subjected to either a safety alert or product recall [3], consistent with reported rates for other medical devices [4]. Importantly, as the complexity of novel products increases, approval success rates decrease [5]; for example, new drug approvals are marred by low phase III trial success rates (~10%) [6].
Given the low success rates of novel and unprecedented drugs [6][7][8], and the potential risks to the population, it is important to adopt the precautionary principle [9] when approving any pharmacological products, especially those given to large populations. COVID-19 mRNA vaccine products have a novel delivery system, being the first mRNA vaccines approved for use in humans, as well as the first approved coronavirus vaccines in humans. The speed at which they were designed, developed, approved, and administered is also unprecedented in pharmaceutical history [10], and defies traditional timelines for testing of biological products for use in humans.
With the approval of the mRNA platform by health regulators across the globe, the industry is poised to develop new vaccines using mRNA, as it is a versatile platform that In this review, we summarise what is known about the distinct components of mRNA vaccines, by reviewing the literature on past therapeutics. Additionally, we review the known safety impacts of mRNA vaccines prior to COVID-19, as well as other coronavirus vaccines, which, while using a non-mRNA platform, inform us of safety risks when vaccinating against coronaviruses. There is a prior history of drug delivery by lipid nanoparticles (LNP), beginning with LNP-encapsulated small molecules (Ambisome, approved in the EU in 1990) [ [63], and laboratory science [64], there are open safety concerns to be addressed by future research.

mRNA Vaccine Elements and Potential for Harm
This review summarises known mechanisms of harm to mRNA vaccine recipients, where we examine historical data on mRNA vaccines to determine if safety signals were apparent during production or testing. Prior to the trials on COVID-19 vaccines involving tens of thousands of people, public data exist on only 285 patients administered various mRNA vaccines, with the earliest trials finishing in 2018 and exhibiting high rates (>10%) of severe adverse events (Supplementary Table S1). The novelty of mRNA/LNP products must be stressed in guiding their safety assessment, as current approvals still leave many questions unanswered, and serious risks cannot be definitively ruled out based on current evidence.
In this review, we summarise what is known about the distinct components of mRNA vaccines, by reviewing the literature on past therapeutics. Additionally, we review the known safety impacts of mRNA vaccines prior to COVID-19, as well as other coronavirus vaccines, which, while using a non-mRNA platform, inform us of safety risks when vaccinating against coronaviruses.

Harms Due to Lipid Nanoparticle (LNP)
Lipid nanoparticles have been used in the delivery of drugs for decades, beginning with the 1990 EU approval of the drug AmBisome (LNP-encapsulated amphotericin B) for fungal infections [52]. In the US environment, the first LNP-administered drugs were Doxil (LNP-encapsulated doxorubicin) for Kaposi's sarcoma and Abelcet (LNP-encapsulated amphotericin B) for aspergillosis [52].
The simplest form of LNP is a liposome, which is produced endogenously [65]. This consists merely of a lipid bilayer that separates the contents from the outside environment [66]. While simple liposomes are detected and destroyed by the body's immune system [67,68], the addition of polyethylene glycol (PEG) enables the liposome to evade the host's immune response and last longer in the body to deliver the encapsulated product [69]. While PEG is often inert in the body, the injection of PEG does elicit anti-IgM antibodies, and subsequent injections containing PEG are cleared faster due to this immune response [70]. Additionally, a small proportion of the population has an allergy to PEG, and injection can trigger anaphylaxis, as did happen for several people receiving COVID-19 vaccines [71][72][73][74].
The safety of 1,2-Distearoyl-Sn-Glycero-3-Phosphocholine (DSPC), a component of the LNP used in both the Pfizer and Moderna COVID-19 vaccines, has been studied [75]. Studies in mice ruled that it was likely not toxic to humans, as no clinical manifestations were present [75]. LNPs have been claimed as safe for the delivery of therapeutic agents, according to a review [76]. However, pro-inflammatory concerns remain over LNPs, even in isolation [77,78].

Harms Due to Exogenous RNA
Foreign RNA triggers an inflammatory response, as toll-like receptors (TLR) [79] and retinoic acid-inducible gene I (RIG-I) [80] are activated. Extracellular RNA exists as a pro-coagulant [81], and increases the permeability of the endothelial cells in brain microvasculature [82]. The initial reason for modification of the RNA by pseudouridylation was to bypass activation of TLR [83]. As pseudouridylated RNA was translated at lower fidelity than RNA [84], the nucleosides were modified to N1-methyl-pseudouridine, which brought translation fidelity to near that of RNA [85].
The properties of both ΨRNA and N1-mΨRNA have been studied in some depth, though questions still remain. For example, through some application of the central dogma of molecular biology, it is assumed that RNA vaccines cannot be incorporated into the genome. This statement is not supported by experiments [86], and is, in fact, contradicted by experiments showing reverse transcription of the Pfizer BioNTech COVID-19 mRNA vaccine into a human liver cell line [64].
ΨRNA exists in nature and comprises between 0.2% and 0.6% of uridine content in human cell lines, and has biologically significant differences from RNA [87]. While N1-mΨRNA exists in nature, found within archaea [88], studies on its properties go back only as recently as 2015 [44]. Additionally, important biological differences exist between unmodified and modified RNA.

Harms Due to In-Vitro-Transcribed (IVT) RNA
The next step in complexity is moving onto RNA therapeutics that are actively transcribed by host ribosomes. These applications typically replace a damaged protein of interest by supplying it exogenously [89]. Using an LNP-mRNA platform here is better than supplying the protein itself, as a protein expressed from IVT RNA is more likely to have the correct post-transcriptional modifications (and subsequent conformation) for its target cell type than an exogenously supplied protein [90]. For these applications, it is typically necessary for the drug to be administered repetitively over long time periods [90,91]. With repetitive dosing, safety is very important, as even a low per-dose AE rate can compound over the many doses of the treatment.
Most studies of this therapeutic modality so far focus on drug efficacy, and limited safety data exist. In a 2021 review of non-immunologic application of mRNA, all studies using LNP-mRNA as protein replacement therapy demonstrated liver toxicity or lacked safety data [90]. Several studies also demonstrate the development of anti-drug antibodies (ADAs) [92][93][94], which can deactivate the drug and prevent treatment [95][96][97][98]. Immunemediated toxicity is also a cause for concern [99,100].
Another concern is the potential development of cross-reactivity to endogenous proteins, which can occur if the endogenous protein possesses similar structural motifs to the protein expressed from the administered mRNA [101]. Thromboembolic events have been observed in ADA reactions [96]. Typically, ADA reactions are decreased in cases where the encoded protein is a 'self' protein, as opposed to an exogenous protein [102].
Recent work demonstrated a class switch towards an IgG4 antibody response, observed after three doses of Pfizer BNT162b2 (COVID-19 vaccine) and not adenoviral vector COVID-19 vaccines [103], which raises concerns over possible immune tolerance, which is linked to an IgG4-dominated response [104,105].

Harms of RNA Vaccination
In addition to the other harms present in IVT RNAs, RNA vaccines also have the additional safety challenges of expressing an exogenous protein for the express purpose of generating an immune response and immune memory [106]. Of the RNA therapeutic systems introduced so far in this review, the mRNA vaccines are the most complex drug-like therapeutic biologic.
Given their novelty, mRNA vaccines have limited long-term safety data. While the type of vaccination (i.e., attenuated live virus, inactivated virus, mRNA) should not have a significant impact on the IgG antibodies produced, an important consideration must be mentioned: mRNA vaccines encode for a single antigen in most cases, which better enables immune escape rather than a broader antibody response including other proteins. Recent evidence revealed a subclass switch from IgG1 to IgG4 in the context of the Comirnaty mRNA product, which may have consequences with regard to cancer [108], pregnancy [109], and IgG4-related diseases [103,110]. COVID-19 mRNA vaccines are commonly used in Europe and North America; these encode specifically and exclusively for the spike (S) protein [111,112]. Since the introduction of vaccines, mutations have occurred, lessening the neutralizing capacity of these vaccines [113,114].

Harms of Coronavirus Vaccination
In addition to the considerations on the novelty of mRNA vaccines, the C19 mRNA vaccines are also unprecedented in terms of another quality, namely, they are the first coronavirus vaccines approved in humans. Following the 2002/2003 outbreak of SARS-CoV [115] and the 2012 outbreak of MERS-CoV [116], vaccines against coronaviruses infecting humans gained more attention, and were subsequently tested on both animal models as well as on human subjects [117].
A SARS-CoV candidate vaccine given to ferrets elicited enhanced hepatitis [118]. Animal trials on four SARS vaccine candidates in ferrets demonstrated an initial protective period against infection, followed by hypersensitivity to rechallenge with SARS-CoV. The ferrets developed histopathological changes in the lungs induced from virus challenge after all four vaccine candidates, suggesting immune-mediated damage [119]. However, a study of MERS-CoV vaccines on mice and rhesus macaques [120] demonstrated protection without visible histopathology.
Mice given an inactivated virus later developed a pro-inflammatory pulmonary response upon challenge [121]. Anti-spike IgG antibodies are produced by all mRNA COVID-19 vaccines [122], and at significantly lower levels by other COVID-19 vaccines [123]. Anti-spike IgG antibodies are demonstrated to cause severe acute lung injury in rhesus macaques on re-exposure to the virus, suggesting a negative impact of a narrow immune response [124].
Immune-mediated danger from vaccines has been widely acknowledged to be an extant issue in the development of coronavirus vaccines [125][126][127][128][129][130][131], and is supported by current evidence [132]. During the rapid development of COVID-19 vaccines, it was an issue of concern that sufficient long-term monitoring for antibody-dependent enhancement (ADE) be established [133,134]. Unfortunately, as of the time of writing, there are no data available on the long-term impacts of COVID-19 vaccines, including effects resulting from rechallenge with the virus.
Veterinary vaccines for other coronaviruses are available, and are summarised in a recent review [135]. Evidence of immune-dependent enhancement was present for cell culture experiments on vaccination against feline coronaviruses [136][137][138]. ADE is also a concern for avian infectious bronchitis virus (IBV), a coronavirus [139,140]. In IBV, suboptimal vaccination alters the evolutionary dynamics of the viruses and can contribute to the production of escape mutants [141][142][143]. Finding broadly neutralizing IBV vaccines remains a significant challenge for the poultry industry [144][145][146][147][148].
Early canine coronavirus vaccines were withdrawn due to neurological symptoms [149,150], though current vaccines do not carry the same safety issues [151,152]. Bovine coronavirus vaccinations often fail to provide immunity against subsequent reinfections [153][154][155]. Immunizations against transmissible gastroenteritis virus (TGEV) in swine have historically had issues in inducing immune protection [156,157], but are widely used now. Too-frequent exposure to vaccine antigens can lower the immune response against TGEV [158]. Another swine coronavirus vaccine, porcine epidemic diarrhoea virus (PEDV), is widely used [159]. Extant safety concerns for the PEDV vaccine are minor, and mostly deal with lack of efficacy; these are summarised in a review [159].
There were several human trials of coronavirus vaccines prior to the approval of COVID-19 vaccines (Table 1). In addition to the endemic coronaviruses that infect humans, several epidemic strains of coronaviruses have occurred in the past two decades, namely, the coronaviruses associated with severe acute respiratory syndrome (SARS-CoV) in 2003 [115] and Middle East respiratory syndrome (MERS-CoV) in 2012 [160]. These outbreaks impelled the production of coronavirus vaccine candidates, summarised in a recent review [117] (Table 1). In total, before the development of the COVID-19 vaccines, data existed on a total of 179 human participants given a SARS or MERS vaccine candidate, of which, 7 (4 ± 2%) experienced a serious adverse event (Table 1). A human trial of 63 adults for a MERS vaccine candidate showed no severe adverse events, but infections in 36% of participants [161,162]. Studies of coronavirus vaccines have a limited number of human participants and still represent a novel technique, though the recent implementation of large-scale vaccination programs for COVID-19 increases the data available to assess the safety of human coronavirus vaccines.

Harms of RNA Vaccination with SARS-CoV-2 Spike (S) Antigen
There is reason to believe that vaccines encoding the spike (S) protein of SARS-CoV-2 have additional mechanisms of harm, owing to the biological impacts of S protein specifically. There is some research in the literature [169][170][171][172], and it is beyond the scope of this review to cover this in significant depth. However, the addition of spike protein adds another factor in assessing the complexity of RNA vaccines. The complexity, as well as uncertainties about possible harms, are non-trivial and cannot be dismissed based on current data. This section briefly covers some of the hypothesised mechanisms of harm from spike-protein-encoding mRNA vaccines and the evidence for each from a clinical/epidemiological outlook, as well as any mechanistic data from laboratory work.
Several observations have been made that contradict fundamental claims of RNA vaccine safety. For example, it was assumed that the RNA was relatively labile and transient in the cell. However, several studies identified spike protein and vaccine mRNA months post-injection [61,62]. Spike protein has been shown in laboratory settings to cause inflammation [173,174], vascular damage [175], and to act as a seed for amyloid formation [176].

Discussion
There is limited information to make a safety assessment of mRNA vaccines. In the category of mRNA vaccines, there are patient data for 385 patients. For mRNA vaccines against an infection, there are data for 285 patients. The rate of serious adverse events was 64 out of 385 for the broad category of RNA vaccines (including cancer vaccines), or 17%; restricting the definition to vaccines against infection, the rate of SAEs is 41/285 or 14%. While high levels can be expected for trials of a novel technology where dosage levels must be determined (many of these trials are phase I) [177], these findings showcase the relative immaturity of mRNA vaccination as a strategy. Given the low efficacy and short duration of protection of SARS-CoV-2 mRNA products [178,179], and the low risk of many populations from COVID-19 complications [180], it may be advisable to suspend mRNA vaccines in certain risk cohorts.
The key to the reactivity of mRNA vaccines is the fact that they express a foreign antigen, for which the antigen-presenting cells are marked for destruction. While the lipid nanoparticle exhibits an acute inflammatory response by itself [77,78,181], the trials using LNPs, so far, have not found a large safety signal when using LNPs to deliver small molecules, non-expressing RNAs, or RNAs for endogenous proteins [77,78,181].
In addition to there being harms attributable to the general immune response from an LNP-RNA delivery system, there are also some harms specific to the spike protein.
Several of these mechanisms are supported by laboratory experiments and clinical findings, but need more investigation. Medicine is replete with cases for which safety was assumed without adequate evidence at the time, which later regretfully led to loss of health and life. mRNA vaccines are demonstrating great unintended harms, and these harms demand further investigation into the mechanisms, which is important for identifying treatment modalities.
Novel biomedical technologies can bring relief for a wide variety of conditions and diseases. However, their use must take into consideration their possible harms. Here, we argue that the mRNA technology is novel enough that safety concerns in current and future products cannot be definitively ruled out, and further research must be performed to ensure their safety for current and future users. Other vaccine platforms have longer term data on their mechanisms, and these have fewer unknown long-term impacts. Considering the lack of data on the platform itself, we recommend a robust, independent, and wide-ranging safety audit of mRNA-LNP formulations and call on regulators to hold manufacturers to high safety standards, especially for products used prophylactically in the general population.