Innate and adaptative immune mechanisms of COVID-19 vaccines. Serious adverse events associated with SARS-CoV-2 vaccination: A systematic review

Abstract

Introduction

The present review focuses on innate–adaptative immune stimulation by COVID-19 vaccines, especially by mRNA-iLNP vaccines. It describes iLNP and nucleoside-modified mRNA technologies, reverse transcription, inflammatory signals linked to reactogenicity, including vascular endothelial growth factor-mediated vascular cross-talk, induced by systemic and spike protein, which mimic COVID-persistent. Finally, the connection between the manifestation of severe forms of adverse reactions to vaccination and molecular mimicry, the production of particular autoantibodies and the role of certain vaccine adjuvants are discussed in detail.

Objectives

To identify articles that publish information on the adverse effects produced after the administration of COVID-19 vaccines in order to demonstrate their therapeutic potential in the treatment–prevention of disease; as well as to demonstrate the association of causality and temporal ocurrence.

Methodology

Systematic review of the scientific literature published between July 2021 and July 2023, which analyses all reports of inflammatory signatures of serious adverse effects caused by COVID-19 vaccines.

Results

The systematic review identified 2033 records which, after a screening process according to the inclusion criteria and the elimination of duplicated papers, work with methodological problems and work without open access, were reduced to 58 articles, of which 50 articles are human models and 2 are cellular models.

Conclusion

The results of this systematic review reveal the causal and temporal association of the various serious adverse events following administration of COVID-19 vaccines and the “peak effect” of COVID-19 vaccines is recognised.

Resumen

Introducción

La presente revisión se centra en la estimulación inmunitaria innata-adaptativa por las vacunas COVID-19, especialmente por las vacunas ARNm-iLNP. Se describen las tecnologías iLNP y ARNm modificado con nucleósidos, la transcripción inversa, las señales inflamatorias vinculadas a la reactogenicidad, incluye la diafonía vascular mediada por el factor de crecimiento endotelial vascular (VEGF), inducida por la proteína pico con efecto sistémico y, que imitan el COVID-persistente. Por último, se discuten en detalle la conexión entre la manifestación de las formas graves de las reacciones adversas a la vacunación y el mimetismo molecular, la producción de autoanticuerpos particulares y el papel de ciertos adyuvantes de las vacunas.

Objetivos

Identificar los artículos que publican información sobre los efectos adversos producidos después de la administración de las vacunas COVID-19 para demostrar su potencial terapéutico en el tratamiento y/o prevención de la enfermedad; así como evidenciar la asociación de causalidad y ocurrencia temporal.

Metodología

Revisión sistemática de la literatura científica publicada entre julio de 2021 y julio de 2023, que analiza todos los reportes sobre firmas inflamatorias de efectos adversos graves causados por las vacunas contra la COVID-19.

Resultados

La revisión sistemática ha permitido identificar 2033 registros que, tras un proceso de cribado de acuerdo con los criterios de inclusión y la eliminación de trabajos duplicados, de trabajos con problemas metodológicos y de trabajos sin acceso libre se redujeron a 58 artículos, de ellos, 50 artículos son modelos humanos y 2 corresponden a modelos celulares.

Conclusión

Los resultados de esta revisión revelan la asociación causal y temporal de los distintos efectos adversos graves posteriores a la administración de las vacunas COVID-19 y se reconoce el «efecto de pico» de las vacunas COVID-19.

Introduction

The leading vaccine technologies against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) include recombinant glycoprotein, weakened/inactivated adenovirus, and lipid-nanoparticle (LNP)-encapsulated mRNA (Pfizer-BioNTech Comirnaty, Moderna Spikevax).^1,2 Nucleoside-modified mRNA vaccines against coronavirus disease 2019 (COVID-19) are the first mRNA products to be approved by the Food and Drug Administration (FDA) and the European Medicines Agency (EMA). These vaccines' main components are: nucleoside-modified mRNA, which has the capacity to encode antigenic protein, in this case the SARS-CoV-2 spike (S) protein, and lipid nanoparticles containing ionizable lipid (iLNP), which function as a delivery vehicle for intact mRNA to the cytoplasm of cells that will translate the encoded protein.^3–5 These nucleoside-modified mRNA-iLNP vaccines are highly effective in inducing spike-specific adaptive immune responses in humans, especially neutralising antibodies to create protective immunity against SARS-CoV-2 infection, as well as promoting humoral immunity via T cells and preventing severe forms of COVID-19.^1,3,6–8 However, we know very little about the dynamics and structure of the spike protein of vaccine platforms, how innate immune pathways regulate adaptive immunity, or which immune responses are protective and which are dispensable.

The present review focuses on innate/adaptive immune stimulation by COVID-19 vaccines, especially by mRNA-iLNP vaccines. In the first part, we define the concept of iLNP and nucleoside-modified mRNA technologies, and provide a detailed study on the dynamics and structure of the S protein of COVID-19 vaccines, reverse transcription, exposure and subsequent dissemination to the cell line and integration into the host genome. Then, the inflammatory signals linked to reactogenicity, including vascular endothelial growth factor (VEGF)-mediated cross-talk, induced by the spike protein with systemic effect and mimicking long COVID. Finally, we discuss in detail the connection between the manifestation of severe forms of adverse reactions to vaccination and molecular mimicry, the production of particular autoantibodies, and the role of certain vaccine adjuvants.

Nucleoside-modified mRNA and ionisable lipid nanoparticle technologies

Safe and effective vaccines must stimulate the innate immune system in such a way that they achieve a balance between immunogenicity and reactogenicity. They must deliver the signals necessary to maintain and prime adaptive immune responses. This refers to the ability of vaccines to act in a specific situation without causing excessive local and systemic inflammatory effects.³

A number of factors must be considered that stand in the way of using synthetic mRNA for biomedical (vaccine or therapeutic) applications, such as the highly inflammatory mechanical and biocompatible properties for recognising mRNA molecules by innate sensors in subcellular compartments and the inefficient cytosolic delivery of mRNA in vivo.^3,9–11

First, it is important to highlight the need to select a manufacturing method adapted to innate immune recognition of mRNA. Karikó et al. (2005) identified the expression of certain modified ribonucleosides working as immune sensors discriminating self-RNA from foreign RNA versus unmodified ribonucleosides. In particular, the replacement of uridine with natural uridine derivatives (pseudouridine [ψ] and its derivatives) is used to attenuate inflammation and facilitate translation. The aforementioned mechanical properties are essential for mRNA to mitigate or escape detection by most immune sensors and mimic their microstructural features. This technological advance was essential to create the approved COVID-19 mRNA vaccines, uridines are replaced with N1-methylpseudouridine (m1ψ).^3,4,12,13

Finally, a key requirement in the manufacture of COVID-19 vaccines is that it is biocompatible. It must be chosen based on mRNA delivery efficiency, biodegradability, and tolerability to withstand cell culture manipulation and the biological activities of the host. The approved COVID-19 mRNA-iLNP vaccines use ionisable lipids (ALC-0315 in the Pfizer-BioNTech vaccine and SM-102 in the Moderna vaccine). iLNPs comprise a polyethylene glycol (PEG)-conjugated lipid that confers platform stability, cholesterol, a cationic “ionisable” amino lipid, and 1,2-diestearoyl-sn-glycero-3-phosphocholine.^3,14,15

The iLNP technology not only enables the delivery of mRNA into innate immune cells after vaccination, but also plays a special role with strong adjuvant activity in this type of vaccine platform. The main characteristic of nucleoside-modified mRNA is its ability to not produce inflammation, mRNA-iLNP vaccines are not immunosilent, as verified by the strong innate immune activation and local and systemic adverse event reports post-COVID-19 vaccination in humans.^{1,3,12,16–53} This would change our understanding of the current mRNA vaccine paradigm, of how it activates the innate immune system, and would raise the prospect for refining the design for more effective and safer mRNA vaccines and treatments in the near future. The advantage of mRNA-iLNP platforms is that they do not require the addition of adjuvants to induce robust protective immune responses against various pathogens.^1,3

It is necessary to understand how nucleoside-modified mRNA components versus iLNP excipients function in the overall immune response elicited by the new generation of COVID-19 mRNA vaccines. As we shall discuss below, the main driver of adjuvanticity and reactogenicity of the mRNA-iLNP vaccines is the iLNP carrier. It activates a variety of specific signals including pro-inflammatory cytokines and chemokines. For example, granulocyte-macrophage colony-stimulating factor (GM-CSF); tumour necrosis factor (TNF); interferon-gamma (IFN-γ); interleukins (IL): IL-1β, IL-6; CC chemokines, e.g., CC motif chemokine ligand 2, 3, and 4 (CCL2, CCL3, CCL4); CXC motif chemokine ligand: 2 and 10 (CXCL2, CXCL10).^{3,15,16,54–57}

Pharmacokinetics of nucleoside-modified mRNA-iLNP vaccines. Biodistribution and dynamics of innate immune cells after administration of mRNA-iLNP vaccines

mRNA-iLNP vaccines travel in the body according to the route of administration and iLNP formulation. Intramuscular administration of COVID-19 mRNA vaccines and other similarly designed vaccines results in the uptake and production of the encoded antigen at the site of inoculation and subsequent drainage into the lymph nodes (LNs). In addition, limited mRNA and/or lipid spread was detected in other non-draining tissues such as lungs, liver, spleen, and LNs.^3,14,15,54 Verbeke et al. (2022) identify a similar biodistribution in adjuvant protein subunit vaccines.^3,58 Other studies showing lower seropositivity of the SARS-CoV-2 spike protein in plasma from humans and mice receiving Pfizer's BNT162b2 vaccine show how the spike protein, or its mRNA template (mRNA-iLNP or cell-associated mRNA) can spread systemically after intramuscular inoculation.^3,15,58–60Fig. 1 summarises the mechanisms of innate immune cells after administration of mRNA-iLNP vaccines (biodistribution and dynamics).

Fig. 1.

Dynamics and biodistribution of innate immune cells following mRNA-iLNP vaccination. (A) Intramuscular administration of nucleoside-modified mRNA-iLNP vaccines leads to local inflammation, recruitment of neutrophils, dendritic cell (DC) subsets, and monocytes through the production of chemokines and other inflammatory mediators involved in immune cell extravasation. (B) Antigen expression. mRNA-iLNPs spread to lymph nodes and drain. Biodistribution, cellular uptake, and protein uptake (opsonisation), limited by surface characteristics and size of innate immune cells. (C) Monocytes/macrophages and DCs are involved in antigen expression and T-cell priming. (D) Induction of affinity maturation. Follicular helper T cells (Tfh) drive B cells in germinal centre (GC) reactions in the presence of follicular DCs. Dysfunction in inflammatory signalling, e.g., iLNP-induced IL-6 in stimulating B cell Tfh and GC responses, IFN type 1 induces CTL (cytotoxic T lymphocyte) responses. Verbeke et al (2022).^2,3,15,17,57

Proteins produced after mRNA-iLNP inoculation appear rapidly. In pre-clinical studies, they peak 4–24 h after administering the vaccine and decline progressively, ranging from several days to weeks or months. This process will depend on the encoded protein, the mRNA dose, the route of mRNA-iLNP administration, and the type of iLNP.^{3,13–15,25,35,43,59,61,62} These features not only offer high and sustained antigen availability, but also favour more robust antibody responses, making them support for adherent cells, and improve survival of cell infiltration and differentiation, favouring protein production from mRNA vaccination compared to other vaccine platforms. For example, both spike-encoding mRNA and spike protein are detectable 60 days after the second dose of BNT162b2 and mRNA-1273 in vaccinated humans, located in axillary germinal centres (GCs).^{1,3,16,25,27,31,38,40,59,63,64}

There is little literature on the dynamics of protein/iLNP interactions for lymphatic and cellular dissemination, although it is acknowledged that the opsonisation of mRNA-iLNP induces detection and uptake by innate immune cell receptors. Nucleoside-modified mRNA-iLNP vaccines elicit T follicular helper cell (Tfh) responses and the creation of GCs, essential for the generation of particular autoantibodies, with high affinity and persistence.^3,65 Verbeke et al. (2022) report a persistence of more than 6 months after the second dose of 30 μg mRNA in draining LNs in humans, the resulting GC B and Tfh cells leading to the generation of affinity mature B cells and long-lived bone marrow plasma cells. The versatility of COVID-19 mRNA vaccines in humans, COVID-19 mRNA vaccines induce antigen-specific circulating Tfh cells, as well as CD4⁺ T cells with T helper 1 (Th1) polarisation and IFN-γ-producing CD8⁺ T cells which remain detectable up to 6 months post-vaccination.^{1,3,15,66–71} Another point to consider is the strong immunogenicity associated with the surface decoration of nanoparticles with PEG that modifies the topological structure to prevent aggregation of nanomaterials and facilitate distribution in the lymphatic system, slowing opsonization processes, and their phagocytosis.^3,15,57 Accordingly, PEG lipids desorb from the iLNPs, to support binding between endogenous proteins and lipids in the extracellular space, forming a biomolecular “corona” around the iLNP, including various lipoproteins, immunoglobulins, and complement fragments abundant in blood.^3,72–74 It is also possible that neutrophils could compete with other immune cells for the uptake of MRNA-iLNP vaccines by efficiently internalising at the injection site, but they show weak reporter protein encoding. In contrast, monocyte, and dendritic cell (DC) subsets take up and translate mRNA-iLNP better.^3,15,57,75

In these studies, neutrophils are dispensable for B-cell Tfh and GC responses compared to frequencies of monocytes and/or myeloid DC subsets and macrophages that are increased in draining LNs and express a greater number of co-stimulatory markers such as CD80 and CD86 compared with cells in the contralateral non-draining LNs.^3,60,76 The mechanisms of iLNP-mRNA uptake by the innate immune system and their physicochemical parameters such as surface composition, morphology, and size of the iLNPs are not yet well understood, which may condition the pharmacokinetics of these vaccines.

Immunological identification of nucleoside-modified and unmodified mRNA

There is little public information on the exact methods of RNA preparation (transcription, protection, and purification) used by Pfizer-BioNTech, Moderna, and other RNA vaccines, which makes research in this field difficult. Three categories are investigated of innate immune sensing of synthetic mRNA, which are: (1) uridine-dependent recognition of various RNA species,^68,77 (2) recognition of double-stranded RNA (dsRNA),^78–80 and (3) recognition of the 5′ end of mRNA if not properly capped.^3,81–83 Identification of uridine-containing RNAis associated with increased expression of pro-inflammatory cytokines, particularly type 1 interferon (IFN-1), which further promotes the expression of RNA sensors, causing inhibition of antigen expression from the mRNA via protein kinase R (PKR) and 2′,5′-oligoadenylate synthetase (OAS).^3,4 The modification of nucleosides is required for clinical success and the widespread deployment of these vaccines today. However, it is not clear how nucleoside modification per se induces reactions in GCs compared to unmodified RNA. Verbeke et al. (2022) propose several hypotheses that are not mutually exclusive: (1) The modified mRNA protein is presented with higher and longer parameters over time than unmodified mRNA. This would drive GC reactions, which are promoted by prolonged antigen availability, giving the platform better kinetics,^3,64 (2) the main difference between modified and unmodified mRNA is not (only) the overall protein expression, but also differential expression in key antigen-presenting cell types (monocytes, macrophages, and DC). These cell types may be especially sensitive to the translation-inhibiting effects of unmodified mRNA due to higher expression of RNA sensors, for example, TLR7/8 genes, hindering a key pathway of T-cell priming: direct presentation of translated antigens to CD4 + and CD8 + T cells,^{3,13,54,75,84} (3) the cytokine dysfunction generated by modified mRNA-iLNP would produce increased reactogenicity, conditioning GC responses compared to responses induced by unmodified mRNA-iLNP.³

Unmodified mRNA can also elicit protein expression and neutralising antibody reactions, thus promoting strong antigen expression and immunogenicity.^3,11 This theory verifies the decrease in inflammation caused by uridine following mRNA administration. However, the likelihood of yielding an immunosilent mRNA is low; not all uridines can be removed from the mRNA sequence. The alternative could be partial removal of uridine,^1,3,6,17 this would explain the differences in immunogenicity and reactogenicity between COVID-19 vaccines.

In addition, dsRNA (unwanted RNA) is involved in “deleterious” innate immune recognition. In vitro transcription (IVT) by T7 RNA polymerase yields the desired RNA, but also a set of unwanted RNA species, including short abortive transcripts. It also produces antisense RNAs transcribed from the promoter-less end of the DNA template.^3,85 This could drive the formation of dsRNA, inducing a potent inflammatory response and translational blockade via the recognition of various intracellular receptors: PKR, OAS, melanoma differentiation-associated protein 5 (MDA-5), endosomal Toll-like receptor 3 (TLR3), retinoic acid-inducible cytosolic receptor gene I (RIG-1), laboratory of genetics and physiology 2 (LGP-2), mitochondrial antiviral signalling protein (MAVS), DEAH-box helicase 33 (Homo sapiens, human [DHX33]), etc., initiate an interferon response to single-stranded RNA.^{3,12,13,15,78,86} DsRNA sensors play an essential role in translational blockade, these are the IFN-inducible cytosolic sensors OAS and PKR. Secondary and tertiary double-stranded structures also form based on the sequence of the mRNA.³ It can be argued that the preparation of the mRNA vaccine involves destroying any therapeutic mRNA. These deleterious innate immune responses can be reduced chemically by 2 methods; first, as discussed above, the inclusion of modified nucleosides such as ψ, m1ψ, and 5-methylcytidine to reduce the activation of PKR and OAS sensors, and second, the removal of dsRNA from IVT mRNA by another chemical treatment known as purification.^3,12,13 Finally, the 5′ co-transcriptional capping strategy impacts the translatability and immune activation of IVT mRNA.^3,83

The use of modified uridines is now recognised as a key aspect of the effectiveness of COVID-19 mRNA vaccines used by Pfizer-BioNTech and Moderna. Unfortunately, both the inflammatory capacity of the mRNA component and its translation are altered by other factors.

Activity of vaccine adjuvants. Inflammatory signature similar to long COVID. Innate immune sensing of ionizable lipid nanoparticles (iLNP)

iLNPs function as delivery agents, both empty iLNPs and mRNA-iLNP complexes act as adjuvants. The ionisable lipid component is necessary for the adjuvant effect, alone they do not induce robust antibody responses. Importantly, mRNA-iLNP and iLNP-adjuvanted protein vaccines induce similar, much stronger, humoral, and cellular immune responses in Tfh and GC B cells.^{3,13,15,16,65} An understanding is beginning to emerge of the potent adjuvant activity of iLNPs and the inflammatory milieu that they stimulate. After immunisation, in the draining LNs, both nucleoside-modified mRNA-iLNPs and empty iLNPs or iLNPs comprised of non-coding RNA stimulate the production of several CC- and CXC-motif chemokines (CXCL1, CXCL2, CXCL5, CXCL10, CCL3, CCL4) and cytokines IL-1β, IL-6, leukaemia inhibitory factor (LIF) and GM-CSF.^3,16,57 The rapid, efficient, and potent production of these inflammatory signals explains the induction of Tfh cells and GC B cells, as well as the infiltration of immune cells in the injected tissues.^3,56

Available data on innate immune activation in humans following mRNA-iLNP administration are limited, however, the studies published to date are consistent with previous reports in animals. They indicate a similar inflammatory signature in the serum of mice after vaccination with BNT162b2: CXCL10, CCL4, IL-6, interferon alpha and gamma (IFN-α, IFN-γ).⁵⁴ Li et al. (2022) highlight how vaccine-stimulated IFN-I and IFN-stimulated gene (ISG) signatures in various cell types including monocytes, macrophages, DCs, natural killer (NK) cells, peak at day 1 and return to baseline levels by day 7, whereas NK cells and T cells exhibit a continuous increase in expression of cell-cycle- and transcription-associated genes (analysed by single-cell transcriptional profiling in draining LNs). Six hours after administration of a second vaccine dose, serum IFN-γ is 8.6-fold higher, coming largely from CD4 + and CD8 +, compared with 6 h after the first dose, when most of the IFN-γ is derived from NK cells.^3,54 It is not clear which component of the mRNA-iLNP vaccine can induce type 1 IFNs. They state that IFN-γ signalling activates antigen-presenting cells, but, in this condition, plasmacytoid DCs produce IFN-α only when exposed to mRNA-iLNP and not empty LNPs, supporting the theory that the mRNA component may be responsible for inducing type I IFN.⁷⁵

A higher concentration of IFN-γ is generally observed, i.e., a general immune dysfunction following vaccination, consistent with reports of adverse effects following approved mRNA COVID-19 vaccinations, mainly for systemic reactions, and it is theorised that enhanced T cell and myeloid cell activation results in cross-talk between lymphocytes and myeloid cells that increases their responsiveness to subsequent vaccine encounters and/or COVID-19 infection.^{2,3,31,54,74,86–91} It is suggested that mRNA-iLNP vaccines have persistent enhancing effects on trained immunity (myeloid cells).^3,60,91,92 There may be important species-dependent differences in inflammatory responses that need to be considered when testing vaccines in animal models, including reactogenicity, fever, and induction of other inflammatory cytokines stimulated by mRNA-iLNP. Human peripheral blood mononuclear cells (PBMCs) treated in vitro with m1ψ-modified mRNA-iLNPs formulated with SM-102 lipid release IL-β, IL-6, TNF, CCL5, vascular endothelial growth factor (VEGF-A), GM-CSF, and other molecules have been detected, and research is needed in this area.^3,15,55 Plant-Hately et al. (2022) note the stimulation of basophils in the generation and release of histamine within the vascular system.^50,57

The innate immune signalling pathways implicated in the iLNP adjuvant effect are: (1) oxidised phospholipids or metabolised and/or modified lipid products, e.g., oxidative impurities of ionisable lipid⁹³; (2) individual cholesterol and lipids, e.g., ionisable lipids^16,56,57; (3) the entire nanoparticle or other multimolecular structures; (4) endogenous molecules (apolipoprotein (ApoE) or complement proteins) that bind to iLNPs after inoculation with the mRNA vaccine are involved in both sensing mechanisms and receptor-mediated uptake by innate immune cells; (5) finally, the presence of a cellular receptor, such as a TLR, that specifically detects iLNPs.^3,13,15 Certain vaccine components such as LNPs and cationic liposomes are sensed by nucleotide binding domain, TLR2, TLR4, the stimulator of interferon genes (STING), and protein 3 (including the leucine-rich repeat pyrin domain-containing protein 3 [NLRP3]).^3,57,94–96 In contrast, Li et al. (2022) and Pichmair et al. (2009) suggest that certain nucleic acid and microbial lipid sensors, including inflammasome mediators, are not required for a strong immune response to this vaccine.^54,97 It is therefore possible that SARS-CoV-2 vaccines composed of viral vectors (AstraZeneca's Vaxzevria and Johnson & Johnson's Janssen) and protein subunits (Novavax's Nuvaxovid) deliver more attenuated inflammatory signals or use other mechanisms that contribute to innate immune activation, not just the mRNA encapsulated in lipid nanoparticles of Pfizer's Comirnaty BioNTech, Moderna's Spikevax, and Curevac. For example, Li et al. (2022) analyse the immunogenicity of the BNT162b2 vaccine (CD8 + T-cell and antibody response), it is not attenuated in the absence of TLR2, TLR4, TLR5, TLR7, protein 3 including leucine-enriched pyrin repeat domain (NLRP3), apoptosis-associated Speck-like protein also containing caspase (CARD/ASC), cyclic GMP-AMP synthetase (cGAS), or the stimulator of interferon genes (STING). They suggest the possibility that mRNA (modified with m1ψ and without dsRNA) or its degradation products may be sensed by the above sensors sending signals, supporting the theory of its function as an adjuvant.^{3,12,16,17,22,26–28,30,31,33,34,36,37,39,41–44,46–54,57,98}

Some of the mechanisms are detailed below:

As mentioned above, a particular sensor is capable of directly and/or indirectly sensing iLNPs or their degradation products. For example, Ndeupen et al. (2021) describe how a polycytidine (non-coding) mRNA-iLNP triggers expression of a necroptosis-associated gene set, which could cause the release of inflammatory damage-associated molecular patterns (DAMPs) from dying cells.^3,56 Recently, Plant-Hately et al. (2022) tested a major pro-inflammatory histamine-like immunoregulatory mediator, IL-1β. It is generally stimulated through the exposure of immune cells to various microbial-associated molecular patterns and DAMPs through inflammasomes such as NLRP3. It has been shown that liposomes containing Moderna's ionisable cationic lipid SM-102 can induce the release of IL-1β from peripheral blood cells, suggesting intracellular pattern recognition through a receptor such as NLRP3.⁵⁷ Also, sensing this mRNA vaccine associated with secondary and tertiary mRNA structures or other elements involved in priming, such as incomplete dsRNA removal, is possible.³ Another innate immune signalling pathway proposed by Holm et al. (2012) involves sensing membrane disturbances caused by fusion of iLNPs with the plasma or endosomal membrane (morphological changes, cationic membrane patches, etc.).^3,99 Inflammasomes include a set of pattern recognition receptors and adaptor proteins that respond to a variety of danger signals. For example, the cytokine IL-β1 has a potent iLNP adjuvant effect, is commonly detected in PBMCs, animals, and humans exposed to empty iLNPs or mRNA-iLNPs.^3,54 It has been shown that iLNPs are designed to be fusogenic and that they are able to penetrate the endolysosomal membrane into the cytosol, a common feature of viral infections.³ Verbeke et al. (2019) state that RNA sensing in bridging innate and adaptive immune responses to viral infections, and also impede the therapeutic role of mRNA vaccines, hindering clinical success by suppressing the synthesis of the encoded antigen and causing adverse reactions.^3,10,13Fig. 2 summarises the innate immune sensing mechanisms of synthetic mRNA and its lipid transport.

Fig. 2.

Innate immune mechanisms involved in the immunogenicity and reactogenicity of mRNA-iLNP vaccines. (A) Uptake of empty iLNPs by innate immune cells and other cell types induces local and systemic inflammation, characterised by the release of pro-inflammatory cytokines such as IL-1β and IL-6. (B) Preparation of synthetic mRNA. The incorporation of modified uridines and purification process of IVT mRNA lowers the recognition of IVT mRNA by TLR3, TLR7, TLR8, and other RNA sensors. These modifications are important to minimise the negative effects of type I IFN-stimulated RNA sensors on protein expression of antigen-encoding mRNA and prevent cytotoxicity. Signalling pathway associated with melanoma differentiation-associated gene 5-interferon type α (MDA5-IFN-α) in the induction of CTL to BNT162b2 in a mouse animal model indicates residual type I IFN activity in the current generation of mRNA vaccines. (C) After administration of the second dose of vaccine, strong boost in T cell responses associated with increased IFN-γ production. Enhanced activation of T cells and myeloid cells after booster vaccine reflects cross-talk between lymphocytes and myeloid cells. Verbeke et al. (2022).^2,3,15,17,57

Fig. 3.

Direct protein–protein interaction network (PPI) using the DEGs, in response to BNT162b2 vaccine on day 22. Hajio et al. (2022).¹⁵⁶

Szebeni et al. (2022) indicate additional adjuvants as favouring immune sensing and antigen presentation through complement activation (CARPA), antigen-depot effects, and/or the possibility that iLNPs could deliver mRNA to specific cell types or subcellular compartments. Lipids and lipid-based nanoparticles induce chemokines, whereas therapeutic nucleic acids (e.g., mRNA) induce interferon responses.¹⁵

Understanding the molecular mechanisms by which the inflammatory signature induced by COVID-19 vaccines, in particular synthetic mRNA and its lipid carrier iLNP, is sensed will help in the design of the next vaccines.

Severe adverse effects after vaccination. Molecular mimicry theory-reverse transcription

Some of the reported adverse effects of COVID-19 vaccines, which may overlap with manifestations reported during post COVID-19 or long COVID-19, include: new-onset neuroimmunological disorders (myasthenia gravis [MG], Guillain-Barre syndrome [GBS] or atypical “pseudo-Guillain-Barré” variant, seizures), varicella zoster virus reactivation, cranial neuropathies (vestibular neuritis, Bell's palsy, abducens nerve palsy, olfactory dysfunction, sensorineural hearing loss), neuromyelitis optica, transverse myelitis (TM), neuromyelitis optica spectrum disorder (NMOSD), longitudinal extensive transverse myelitis (LETM), acute encephalopathy, acute disseminated encephalomyelitis (ADEM), small vessel vasculitis, narcolepsy, neuroleptic malignant syndrome (NMS), chronic fatigue syndrome/myalgic encephalomyelitis (CFS/ME); flares or risk of conversion to multiple sclerosis, myositis, dysautonomia (POTS), temporary headaches (intermittent or persistent); cognitive impairment (subjects report loss or impairment of executive, planning, memory and lexical-semantic functions); haematological autoimmune diseases (secondary immune thrombocytopaenia (ITP), immune thrombotic thrombocytopaenic purpura (iTTP), autoimmune haemolytic anaemia (AIHA), Evans syndrome, aplastic anaemia, antiphospholipid syndrome (APS), catastrophic APS (CAPS), and vaccine-induced thrombotic thrombocytopaenia (VITT); vasculitis (cutaneous, IgA vasculitis, ANCA-associated vasculitis, large vessel vasculitis); cerebrovascular complications (cerebral venous sinus thrombosis, ischaemic stroke, haemorrhagic stroke); other rare clinical neurological conditions (Tolosa-Hunt syndrome, Parsonage-Turner syndrome, small fibre neuropathy), and functional neurological disorder (FND); glomerular disease (immunoglobulin A nephropathy [IgAN]); hearing disorders (tinnitus, progressive loss without remission, vertigo with/without spontaneous nystagmus, and dizziness); intestinal dysbiosis; cardiovascular diseases (myocarditis, pericarditis, perimyocarditis/myopericarditis, heart failure, acute coronary syndrome, arrhythmias, palpitations, haemodynamic instability, and autonomic dysregulation); visual disturbances (referred to as decreased visual acuity, floaters, flashes of light, photopsia, vision-obscuring curtains, visual field defects, greyish spots or blurred vision, proptosis, red eye, scalp pain, ophthalmoplegia, retrobulbar pain, temporal headache, uveitis, optic neuritis, etc.); myeloid/lymphoid neoplasms; menstrual cycle irregularities and specific pregnancy outcomes, including maternal and foetal data (miscarriages, pre-mature births, congenital conditions, maternal and neonatal mortality rates); systemic inflammatory response syndrome (SIRS); anaphylactic and anaphylactoid allergic reactions (type III and IV HSR, fatal fulminant necrotising eosinophilic myocarditis); skin disorders (macrophage activation syndrome, systemic erythema).^{1,12,16,23,24,26–39,41,42,44–53,100–106} These long-term effects will affect physical and mental health, interfering with work activity, therefore these symptoms must be identified immediately and treated as soon as possible to avoid progression of the damage and disability they cause.^107,108 The pathogenesis of these diagnoses is mainly neurological, digestive, and cardiovascular, attributable to the dysfunctional mechanism triggered by the S protein used for immunisation or during infection, which could bind to the neurovascular receptor NRP-1 and act as an antagonist to VEGF binding. It also suggests the “spike effect”, known as “spike intoxication” in the context of saturation and subsequent impairment of angiotensin II-converting enzyme receptor (ACE₂) function.^{12,16,19,21,23,25–29,34,39–43,46,50–53,101,103,107,109,110}Table 1 summarises the current evidence, which supports the hypotheses on molecular mimicry, inflammatory signals attributed to post-vaccination reactogenicity, their systemic spread, and the possibility of a “cumulative effect”, i.e. the relationship between elevated antibody levels (anti-S IgG, anti-RBD IgG) post-vaccination and their temporal persistence, suggesting post-COVID-19 mimicry.

Table 1.

Cellular mechanisms and pathogen/viral, autoinflammatory, autoimmune, and paraneoplastic pathways associated with adverse effects in recipients of COVID-19 vaccines.

Cellular mechanisms and hypotheses associated with serious adverse events following vaccination	Cellular mechanisms	Association with reported adverse effects of COVID-19 vaccines			References
INVERSE TRANSCRIPTION	Aldén et al. (2021) present the first study on the effect of the COVID-19 mRNA vaccine BNT162b2 on the human liver cell line Huh7 in vitro. They show rapid entry of BNT162b2 into cells (6 h after exposure to BNT162b2) and subsequent intracellular reverse transcription of BNT162b2 mRNA into DNA, they indicate a possible mechanism for reverse transcription via endogenous reverse transcriptase with the long interspersed nuclear element 1 (LINE-1), and nucleus distribution of the LINE-1 protein being elevated by BNT162b2 and leading to robust expression of the BNT162b2 antigen^9	They suggest studying whether liver cells also present the vaccine-derived SARS-CoV-2 spike protein, which could make liver cells targets for pre-primed spike-reactive cytotoxic T cells consistent with reported cases of autoimmune hepatitis following BNT162b2 vaccination.At this stage of reverse transcription, the authors are concerned about BNT162b2-derived DNA integrating into the host genome and affecting the integrity of genomic DNA, which would lead to genotoxicity.^9 In the toxicity report of BTT162b2, neither genotoxicity nor carcinogenicity studies have been provided,^72 the authors point out that they do not know whether the DNA reverse transcribed from BT162b2 integrates into the cell genome.^9	Further studies are needed to verify the effect of BNT162b2 on genomic integrity, including whole genome sequencing of cells exposed to BNT162b2 and tissues from human subjects receiving BNT162b2 vaccine. They also indicate endogenous LINE-1 expression and/or elevated levels associated with viral infection, including SAR-CoV-29 infection.^9 The exact regulation of LINE-1 activity in response to BNT162b2 should be further studied	The cell model used in this study is a carcinoma cell line with active DNA replication that differs from non-dividing somatic cells, showing significantly different expression of proteins and genes. However, cell proliferation is also active in several human tissues such as: bone marrow, basal layers of the epithelium and during embryogenesis. In addition, they report effective retrotransposition in non-dividing and terminally differentiated cells, such as human neurons.^9 Pfizer's evaluation report also showed that BNT162b2 distributes in the spleen, adrenal glands, ovaries, and testes.^27 Therefore, the effect of BNT162b2 on genomic integrity under such conditions needs to be investigated. No data on placental transfer of BNT162b2 are available in the EMA assessment report on Pfizer	^9^,^27^,^72
HYPERSENSITIVITY RESPONSES TO COVID-19 VACCINES	Plant-Hately et al. (2022) in their study discussed above, related to the cellular mechanisms of basophils, showed that cell surface proteins (CD63, CD203c, and CD164) have upregulated expression during immune system activation in response to allergen recognition^111	It is useful to look at the level of expression of inflammatory biomarkers. In this case, the 3 selected basophil activation markers exhibit distinct expression patterns that may not be mutually exclusive, suggesting several activation pathways. These mechanisms occur in eosinophils during granulation.^111	–	–	^111
	Szebeni et al. (2022) describe another useful model in the evaluation of hypersensitivity responses to mRNA-based SARS-CoV-2 vaccines. They describe all components of mRNA-iLNP vaccines (mRNA, carrier, excipients and expressed antigen), which have diverse immunostimulatory effects on a broad spectrum of effector and target cells (antigen presenting cells [APCs], T and B lymphocytes, platelets, NK cells, and myocytes) and biochemical pathways (complement and coagulation), all of which are necessary to achieve vaccine efficacy. The same components also contribute to hypersensitivity reactions (HSRs) and other types of immune-mediated adverse effects (IMAEs) through their wide inter-individual variability in both quality (spectrum of inflammatory mediators) and quantity (complement split products, cytokine levels, tryptase and induced and/or pre-existing antibodies)^15	Anaphylaxis is an IMAE in the type-1 immediate-type hypersensitivity category (ITH) category. Classical ITH leads to the release of preformed and newly synthesised mediators such as histamine, tryptase, interleukins, prostaglandins, and leukotrienes, among others. Although cytokines are produced by activated immune cells during ITH and other types of HSR, some authors separate anaphylaxis from cytokine release syndrome, they also highlight the function of biomarkers to define the genotypes that underlie the different phenotypes of anaphylaxis. It is known that mediator binding to tissue and cellular receptors induces local and systemic symptoms that affect the skin, respiratory, gastrointestinal, and cardiovascular systems^1^,^2^,^15^,^16^,^35-39^,^41^,^42^,^45-53^,^82^,^86^,^88^,^110^,^112-114	Anaphylaxis can occur without immunoglobulin E (IgE) through newly described (IgG) G-coupled protein receptors, by direct activation of mast cells and/or basophils and by activation of the complement system (anaphylactoid reaction or CARPA pseudoallergy). The enzymatic cleavage of complement will create anaphylatoxins that act as cytokines and activate immune cells to stimulate secondary inflammation mediators. Anaphylatoxins can also activate mast cells, both IgG and allergen-specific immunoglobulin M (IgM) are involved in the activation of the complement system^1^,^2^,^15^,^41^,^42^,^48^,^50^,^52^,^82	Reported delayed reactions to COVID-19 vaccines suggest type III and IV HSR by their timing of onset and symptoms, most have been reported in the context of IMAE to SARS-CoV-2 vaccines, especially mRNA.^1^,^2^,^15^,^41^,^42^,^48^,^50^,^52^,^82Mast cell activation through IgE and non-IgE mechanisms must be taken into account, this condition occurs in people with clonal and non-clonal mast-cell disorders, including asthma, mastocytosis, acute myeloid leukaemia, and myelodysplastic syndrome. They may have high levels of proteases (tryptases), histamine, heparin, and cytokines that are associated with strong mast cell activation. It could be considered that they could develop an increased predisposition to HSR to SARS-CoV-2 mRNA vaccines without symptoms of anaphylaxis, a pre-vaccination risk assessment is required^2^,^15	^1^,^2^,^15^,^16^,^35-39^,^41^,^42^,^45-53^,^82^,^86^,^88^,^110^,^112-114
	In another study, Yoshida et al. (2023) confirm the potential risk of experiencing more adverse reactions after vaccination with BNT162b2^2^,^115	The authors suggest that people with allergic disease, such as asthma, hay fever, allergic rhinitis, atopic dermatitis, food allergies and/or intolerances, who are potentially susceptible to COVID-19, are associated with the development of systemic adverse reactions and a worsening of their chronic disease after vaccination with BNT162b2, which is also a risk factor for anaphylactic shock and HSR^115	–	–	^2^,^115
	Granados Villalpando et al. (2022) conclude that the BNT162b2 and ChAdOX1 nCOV-19 vaccines are strongly associated with the occurrence of allergic reactions, with OR of 1.6 (95% CI; 1.18–2.3) and 1.87 (95% CI; 1.35–2.6), respectively. They also associate a higher likelihood of developing adverse effects and/or allergic reactions associated with COVID-19 vaccines with female sex, older age, and prevalence of comorbidities after vaccination and COVID-19 infection after vaccination. They report a significantly high prevalence of asthma (7.8%) in this setting, already assessed in other studies^75^,^116	Another variable to consider is the genetic diversity of the human population that influences the great inter-individual variation in cytokine responses. One dose of the same immune adjuvant produces an optimal cytokine response in one subject, which may be too strong or too weak for another subject. In summary, innate and adaptive immune responses vary between individuals due to their cellular and biochemical components. In addition, the SARS-CoV-2 spike protein contains the sequence and structural motif of a superantigen, causing the hyperinflammatory syndrome that involves direct T-cell stimulation and excessive cytokine hyperproduction^1^,^2^,^12^,^15^,^16^,^20^,^24^,^26^,^28^,^29^,^31^,^35-39^,^41^,^44^,^45^,^47-52	These cytokines interact with complement systems which, in addition to producing inflammation and cross-talk between innate-adaptive immunity, activate the coagulation system and increase the permeability of the endothelial layer in a time- and dose-dependent manner, in turn promoting their distribution to the systemic circulation, resulting in elevated inflammation^1^,^2^,^12^,^15^,^16^,^20^,^24^,^26^,^28^,^29^,^31^,^35–39^,^41^,^44^,^45^,^47–52	In fact, the fall in RANTES (regulated upon activation, normal T cell expressed and secreted) is known to increase the risk of cardiovascular events in patients with atherosclerosis. The low plasma concentration of RANTES may reflect its increased deposition in the vascular endothelium of areas with atherosclerosis which in turn leads to an increase in its human CC chemokine receptor type 5 (CCR5) and worsens inflammatory organ damage. These data would explain the recruitment of IFN-gamma-secreting T cells to the vessel wall (confined to the adventitia and intima) and reinforce the suggestion that it may be a site of immune privilege, i.e., recruitment, retention, and infiltration of IP-10 and RANTES chemokines. The CCR5 receptor is a G-protein-coupled receptor (GPCR) that plays an important role in inflammation and is involved in cancer, HIV, and COVID-19^117–119	^1^,^2^,^12^,^15^,^16^,^20^,^24^,^26^,^28^,^29^,^31^,^35-39^,^41^,^44^,^45^,^47-52^,^75^,^116-119
PLATELET ACTIVATION	Both L5NP-mRNA and the spike protein activate platelets^15^,^20^,^24^,^26–28^,^39^,^41^,^47–50^,^52	This study specifies the involvement of platelets in inflammation, proposed as a biomarker of anaphylaxis, CARPA, and HSR through the release of biologically active molecules (ATP, thromboxane, and chemokines) and lipid inflammatory molecules such as platelet activating factor (PAF), which triggers the degranulation of perivascular mast cells and the release of thromboxane and serotonin, also causing inflammatory responses and tissue injury^15^,^20^,^24^,^26–28^,^39^,^41^,^47–50^,^52	–	–	^15^,^20^,^24^,^26-28^,^39^,^41^,^47-50^,^52
OXIDATIVE STRESS	Another study attributes oxidative stress to nanoparticle-mediated toxicity of some drugs^15	Related to HSR, it inhibits the negative regulation of complement and induces complement-mediated toxicities. Individuals with common variable immunodeficiency (CVID) may develop HSR to SARS-CoV-2 mRNA vaccines. They also discuss the variation of human leukocyte antigens (HLA) associated with an increased risk of reactions mediated by T cells and likelihood of reactions to SARS-CoV-2 mRNA vaccines for the development of HSR^1^,^15	–	–	^1^,^15
NEUROVASCULAR CROSS-TALK MEDIATED BY VASCULAR ENDOTHELIAL GROWTH FACTOR (VEGF) AND INDUCED BY THE SARS-COV-2 SPIKE PROTEIN	Neurological and cardiovascular adverse effects simulating prolonged COVID-19 have been reported in recipients of COVID-19.^1^,^12^,^16^,^21^,^24^,^26-28^,^30^,^34^,^35^,^37-39^,^41^,^42^,^44-47^,^49-52^,^103 The clinical similarity is due to the shared pathogen of the SARS-CoV-2 spike (S) protein used for immunisation and produced by the virus. The study by Talotta (2022) describes the impaired vascular endothelial growth factor (VEGF)-mediated neurovascular cross-talk induced by SARS-CoV-2 spike protein associated with the side effects of COVID-19 vaccines and explaining prolonged COVID-19 symptoms^107	The S protein can bind to neuropilin 1 (NRP-1), which normally functions as a co-receptor for vascular endothelial growth factor A (VEGF-A). By antagonising the docking of VEGF-A to NRP-1, the S protein could disrupt physiological pathways involved in angiogenesis and nociception. The consequence would be an increase in unbound forms of VEGF that could bind to other receptors. People vaccinated and/or infected with SARS-CoV-2 exhibit elevated plasma levels of VEGF during convalescence and acute illness, which could be responsible for diffuse neurological and microvascular damage. Other studies are indicated suggesting that serum VEGF may also be a potential biomarker for COVID-19 vaccines, and prolonged COVID infection^1^,^16^,^22^,^27^,^107^,^104	More research is needed to better understand the clinical course, pathogenesis, and treatment of adverse effects following vaccination. Recent research suggests that the adverse effects of COVID-19 vaccines are the result of a profound endotheliopathy caused by both direct viral damage and hyperactivation of the immune response. In addition to favouring viral entry, the SARS-CoV-2 S protein interferes with the physiological function of bound receptors, e.g., NRP-1/VEGF-A-mediated pathways, resulting in dysfunctional complications and thus producing symptoms, even in the absence of organ damage^16^,^27^,^28^,^30^,^35^,^39^,^41^,^44-47^,^49^,^51^,^53^,^104^,^107^,^120	But the evidence in favour of this mechanism is scant given the lack of experimental data	^1^,^12^,^16^,^21^,^22^,^24^,^26-28^,^30^,^34^,^35^,^37-39^,^41^,^42^,^44-47^,^49-53^,^103^,^104^,^107^,^120
CALPROTECTIN, NEUTROPHIL EXTRACELLULAR TRAPS (NET) AND SYNDECAN LEVELS AS PREDICTORS OF SEVERITY	The study by Hetland et al. (2022) recently demonstrated that levels of neutrophil extracellular traps (NETs) correlate with the severity of side effects of the ChAdOx1 vaccine. Calprotectin, NETs, and syndecans are key inflammatory markers with physiological effects involved in innate immune activation and vascular endothelial damage, respectively. Calprotectin is the main cytosolic protein in neutrophil granulocytes, used as a sensitive faecal marker in gastrointestinal diseases, and has antimicrobial and cytotoxic properties through zinc binding. It has been associated as a predictor of severe prognosis in COVID-19^46	NETs are webs of DNA surrounded by histones and granular proteins, which are expulsed from neutrophils to trap and destroy invading pathogens in the extracellular space. Elevated levels of NETs have been demonstrated in post-COVID-19 and NETosis has been associated with thrombosis formation in patients with vaccine-induced immune thrombotic thrombocytopaenia (VITT). Circulating NETs are removed from the blood by extracellular DNAases that digest free DNA. Syndecan-1 is a glycocalyx marker for damaged endothelium in the vasculature, elevated levels of syndecan-1 have been demonstrated in COVID-19^46	Vascular endothelial damage and dysfunction is suggested as a prognostic indicator for post-COVID-19, a disease in which there is a direct effect of immune response dysregulation on endothelial damage, which, as discussed in this study, is an essential pathological response to infection that in severe cases results in certain side effects of vaccination^46	Therefore, if elevated NET and calprotectin levels can be detected early after vaccination with ChAdOx1, these biomarkers may be useful predictors for a severe outcome of vaccine complications. They could be used after vaccination with the Janssen vaccine, which is also an adenoviral vector vaccine with the potential to trigger VITT^46	^46
LOSS OF ACE2RECEPTOR ACTIVITY	Angelini et al. (2022) note that spike proteins produced after vaccination have the native-like mimicry of the SARS-CoV-2 spike protein's receptor binding functionality and prefusion structure. COVID-19 vaccines increase endogenous synthesis of SARS-CoV-2 spike proteins. Once synthesised, the free-floating spike proteins released by the destroyed cells previously targeted by the vaccines circulate in the blood and interact with ACE2 receptors (a zinc metalloproteinase) expressed by other cells, promoting ACE2 internalisation and degradation, mimicking the pathological features of SARS-CoV-2^121	This mechanism can increase the imbalance between angiotensin II (Ang II) overactivity and Ang1–7 deficiency through loss of ACE2 receptor activity, triggering inflammation, thrombosis, increased blood pressure and other adverse reactions, the mechanism known as the “spike effect” of COVID-19 vaccines. Also, the detrimental effects of deficiency of other angiotensins (prolyl oligopeptides[POP] and prolyl carboxypeptidases [PRCP]) on blood pressure, thrombosis, and inflammation have been well studied^121^,^101	The authors compare the relationships between different mechanisms of Ang II cleavage and accumulation, offering the possibility of closing the pathophysiological loop between the risk of progression to severe forms of COVID-19 and adverse reactions to SARS-CoV-2 vaccination^101^,^121	The hypothesis of loss of ACE2 receptor activity due to interaction between these receptors and free-floating spike proteins can be observed at all stages of cardiovascular disease progression. They add that increased catalytic activity of POP and PRCP is not common in the young population, but is more frequent in the elderly with comorbidities or previous cardiovascular events. Therefore, reported adverse reactions with Ang II accumulation associated with COVID-19 vaccination will be more likely in young, previously healthy subjects^101^,^121	^101^,^121
NEUROINVASION AND NEUROIMMUNE CROSS-TALK AS INDUCERS OF NEUROLOGICAL SYMPTOMS PRODUCED BY SARS-CoV-2 VACCINATION	The above-mentioned study by Talotta (2022) details the physiopathological cross-talk between NRPs and VEGFs, reflecting the interconnection between vessels and nerves from both an anatomical and functional perspective, their role in tumour progression and invasiveness of several types of cancer has been extensively studied due to the mitogenic effects of VEGF in NRP-overexpression in malignant cells and in multiple sclerosis lesions^43^,^107^,^122^,^123	Impaired NRP-1 activity can cause dysfunctional neurological symptoms, expressed in a variety of cells such as endothelial cells, neurons, glial cells, and immune cells. NRP-1 is involved in cell proliferation, migration, survival, and invasion during embryogenesis and carcinogenesis. It is involved in immunological synapse formation, B-cell differentiation, and immunotolerance^18^,^91^,^107^,^124^,^125	VEGF-A is closely associated with systemic inflammation. Neuropilins 1 and 2 (NRP-1, NRP-2) act as co-receptors enhancing the binding of VEGF-A to VEGF receptor 2 (VEGF-R2) in blood vessels and the binding of VEGF factor C (VEGF-C) to VEGF receptor 3 (VEGF-R3) in lymphatic vessels, respectively.^107^,^125-128 The physiological functions of NRP-2 is less well studied, this receptor can bind to other types of semaphorin and VEGF ligands and participate in the development of the lymphatic system during embryogenesis. NRP-2 controls functions such as phagocytosis, chemotaxis, and antigen presentation, as it is abundant in immune cells^107^,^129^,^130	The author describes a single route for the pathogenesis of the NRP-1/VEGF-A pathway, the NRP-1-dependent entry route for human T-cell lymphotropic virus type 1 (HTLV-1), a human retrovirus able to infect CD4 + and CD8 + T lymphocytes, monocytes/macrophages, and DCs. Infection causes lymphoproliferative, inflammatory disorders and/or neuromyelopathies, including neuroinflammatory alteration, and alter the blood–brain barrier, which may explain the development of the adverse effects cited above.^1^,^16^,^33-39^,^41^,^45^,^51^,^114^,^122^,^123^,^131 In addition, like other viral infections, VEGF-A variations may reflect changes in circulating pro-inflammatory mediators, such as tumour necrosis factor alpha (TNF-α), IL-1β, IL-6, IL-8, monocyte chemotactic protein 1 (MCP-1, also known as chemokine ligand 2 [CCL2]), and IFN-γ-inducible protein 10 (IP-10). Searching for VEGF-A in plasma samples could accurately reflect the actual concentrations of this mediator in peripheral blood, it is known that the concentration of VEGF in serum samples could be affected by the number of platelets, platelets being a major source of VEGF-A^3^,^107^,^112^,^127	^1^,^3^,^16^,^18^,^33-39^,^41^,^43^,^45^,^51^,^91^,^107^,^112^,^114^,^122-131
	Chen et al. (2022) propose neuroinvasion and neuroimmune cross-talk as inducers of neurological symptoms produced by SARS-CoV-2 vaccination. On the one hand, the shared SARS-CoV-2 pathogen can infect the brain directly, via haematogenous propagation and retrograde axonal transport, except for the olfactory neuron pathway. On the other hand, it can induce indirect neurotoxicity through immune-mediated pathogenesis and gastrointestinal infection^132	They report 13 cases of classical GBS associated with vaccination (infection), with mild pulmonary involvement and negative SARS-CoV-2 tests in cerebrospinal fluid. They conclude that not all neurological symptoms require direct infection of the nervous system; indirect neurotoxicity secondary to immune-mediated pathogenesis may occur. They also report autoantibodies binding to endothelial and epithelial cells, which could induce some of the cell lyses^107^,^132	Similarly, in patients with post-COVID-19 infection, antibodies to the virus may attack antigens of endothelial cells in cerebral vessels. Gastrointestinal infection also threatens the nervous system by increasing the transport of aberrant proteins like alpha-synuclein (α-synuclein), which are associated with neurodegenerative diseases^132	They highlight the importance of aggravated brain damage caused by nerve injury leading to abnormal intestinal blood flow and intestinal dysmotility, which could further promote entry of metabolites and bacterial components into the blood and brain parenchyma^132	^107^,^132
OREXINERGIC SYSTEM. ASSOCIATION BETWEEN REDOX IMBALANCE, INFLAMMATION, AND ENERGY METABOLISM	Garrido Suárez et al. (2022) postulate the hypothesis of the orexinergic (Ox) system linked to inflammatory signals, specifically its involvement in reactogenic somnolence following peripheral activation of the innate immune system by COVID-19 vaccines. They call for their consideration by pharmacovigilance and, further understanding of possible additional long-term inflammatory mechanisms in future studies to preserve confidence in these vaccines. Further research identifying biomarkers linked to the reactogenicity of COVID-19 vaccines is needed to better understand their immunogenicity^133	They suggest a mechanistic link between reactogenic inflammatory parameters and hypothalamic circuits regulating the sleep–wake cycle. They propose that pro-inflammatory cytokines INF-γ, TNF-α, and IL-1β may propagate a peripheral inflammatory response after vaccination, activating a subset of lateral hypothalamic area neurotensin-expressing GABAergic neurons (LHA Nts), as well as inhibitory neurons from sleep-inducing areas providing inhibitory input to Ox neurons that that control wakefulness. The adenosinergic modulation of sleep–wake signals could also be involved.^133^,^134 In this case, immunity and sleep are bidirectionally linked, but the cellular and molecular mechanisms underlying these interactions are limitless and not entirely understood	In some cases, somnolence, narcolepsy, or other sleep disorders (prolonged sleep deficiency) are reported as a moderate adverse effect of vaccination, most commonly reported in Pfizer-BioNTech COVID-19 vaccination. Prolonged sleep deficiency can lead to chronic low-grade systemic inflammation, associated with several diseases that share an inflammatory component (neurodegeneration, cancer, diabetes, and atherosclerosis)^12	This would indicate that the vaccine is effective because it may produce immunological memory, in response to pronounced immune activation. The sleep-modulating effect is triggered by pathogen components and decomposition products, including the pathogen-associated molecular patterns of PAMPs (endotoxins), peptides, lipids, and viral double-stranded RNA discussed above. These factors are recognised in macrophages or dendritic cells resident in various tissues, as well as downstream pathways like the nuclear factor kappa-light-chain-enhancer of activated B-cells (NF-κB), controls transcription and inflammasomes, which induce cytokine and prostaglandin expression. Vaccine antigens as potential pathogens causing autoimmune reactions through the mechanisms of molecular mimicry and T-cell activation are recognised, further research is warranted^1^,^2^,^12^,^13^,^16^,^24^,^26-29^,^31^,^35-39^,^41^,^44^,^47^,^48^,^50-53^,^105^,^135	^1^,^2^,^12^,^13^,^16^,^24^,^26-29^,^31^,^35-39^,^41^,^44^,^47^,^48^,^50-53^,^105^,^133-135
	Other mediators involved in the neurobiological bases and regulation of sleep are hormones of the hypothalamus–pituitary–adrenal (HPA) axis, neuropeptides such as orexin, the hormone melatonin, and classical neurotransmitters (γ-aminobutyric acid [GABA], glutamate, serotonin, acetylcholine, histamine, noradrenaline, and dopamine). These molecules are imbalanced in the functional interaction between the immune system and the nervous system^135^,^136	In addition, dense projections from Ox neurons in spinal, diencephalic, and cortical regions are involved in regulating diverse physiological functions. Therefore, Ox neurons not only regulate eating, energy metabolism, and the sleep–wake cycle, but also closely regulate cardiovascular control, cognition and mood, nociception, reproduction, stress, reward and addiction^18	Recent studies support the elevated co-expression of the leptin receptor (LepRb) in some subpopulations of Ox neurons, which are activated by the adipose tissue-derived hormone leptin,^137 and also specify the role of galanin (GAL) as a potential mediator of leptin to modulate nutrient reward by inhibiting orexin neurons via GAL receptor 1 (GalR1).^138 They speculate that following vaccination with COVID-19 vaccines, pro-inflammatory cytokines may propagate an inflammatory response to the hypothalamus causing sleep disorders through 2 pathways: leptin, which has pro-inflammatory properties and up-regulates the secretion of multiple inflammatory cytokines and the subset of GABAergic sleep-inducing neurons. In both cases, the action of pro-inflammatory brain cytokines may be mediated by prostaglandins or nitric oxide produced in endothelial cells of cerebral blood vessels and perivascular macrophages^133	Another potential biomarker is myeloperoxidase (MPO), a pro-inflammatory enzyme that triggers oxidative stress and brain neuroinflammation during the acute and chronic phases of COVID-19. Oxidative stress from MPO occurs by promoting the production of reactive oxygen and nitrogen species, which in turn causes damage to the lungs (chronic hypoxia) causing some of the symptoms seen in the adverse effects of COVID-19 vaccines and in long COVID. In turn, the damage caused by this factor to the blood–brain barrier leads to neuroinflammation, by activation of microglia, also leading to a drop in cerebral perfusion which generates, among other typical symptoms, chronic fatigue and mental confusion^86^,^139^,^140	^18^,^86^,^133^,^135-140
POTENTIAL BIOMARKERS THAT INCREASE NEUROVASCULAR, CARDIOVASCULAR, HEPATIC (HEPATOCELLULAR DAMAGE), AND INTESTINAL MUCOSAL INFLAMMATION WITH REGIONAL IMMUNE SYSTEM AND NEURO/MYOENTERIC DYSFUNCTION	Other potential biomarkers have been studied and could be proposed to investigate adverse effects of COVID-19 vaccines. Growth differentiation factor 11 (GDF-11) is expressed in several organs and tissues, including skeletal muscle, intestine, pancreas, heart, nervous system, olfactory system, retina, and kidneys. It is also highly concentrated in platelets.^135^,^141^,^142 Low plasma concentration of this bioprotective factor has been shown to be associated with ageing in mice losing their reported actions to promote skeletal muscle regeneration, muscle dysfunction.^141^,^143 Findings from subsequent studies show that GDF-11 may cause skeletal muscle atrophy rather than regeneration^13^,^141^,^144	However, in humans, results related to circulating plasma levels are variable, showing a decrease, increase/trend to an increase or no change with ageing.^141^,^135,136,145 Falling plasma levels of GDF-11 in the context of adverse effects of COVID-19 vaccines could contribute to myalgia and chronic fatigue/asthenia or symptoms referred to as functional component fibromyalgia in patients, some of which are reported by pharmacovigilance systems, such as myalgic encephalomyelitis/chronic fatigue syndrome (ME/CFS). Although it should first be ensured that myalgia symptoms (muscle pain without elevated creatine phosphokinase levels) are not caused by statin treatment (up to 10% of statin-treated patients have myalgia)^141	Therefore, the GDF-11 factor may be involved in the metabolism, regulation of inflammation and may be involved in thyroid pathophysiology.^141 Recently, BMP11 target genes were found to be down-regulated by Reference microRNA (miRNA) and YAE1 and RSU1 by the SARS-CoV-2 Delta variant, a diagnostic predictor validated through the differentially expressed gene (DEG) dataset).^58^,^146 Falling plasma levels of fibroblast growth factor 21 (FGF21) contribute to glucose intolerance, higher blood insulin levels, and the development of fatty liver. FGF21 promotes weight loss through increased fatty acid oxidation and decreases triglyceridaemia and blood glucose by improving insulin sensitivity. This peptide functions as a hormone with anti-inflammatory, anti-diabetic, and anti-obesity effects. It is produced in the liver, adipose tissue, skeletal muscle, and pancreas, although its endocrine action depends mainly on the liver. FGF21 is induced in situations of muscle stress, particularly in mitochondrial myopathies^58	A fall in both factors could increase neurovascular, cardiovascular, and hepatic vulnerability (hepatocellular damage), leading to intestinal mucosal damage and inflammation, and regional immune system and neuro/myenteric dysfunction. The disorders reported by pharmacovigilance systems suggest the persistence of these symptoms over time and are experienced as: gastrointestinal disorders (intestinal dysmotility, abdominal inflammation and pain, dyspepsia), musculoskeletal pain (myalgia), cognitive impairment (difficulty concentrating, loss of memory, executive functions, and lexical-semantic functions), headaches, sleep disorders, and chronic disabling fatigue.^1^,^16^,^42^,^49^,^51^,^58^,^141^,^146 The association between redox imbalance, inflammation, and energy metabolism must be an urgent objective of the therapeutic plan	^1^,^13^,^16^,^42^,^49^,^51^,^58^,^135^,^136^,^141-146
DYSFUNCTION BETWEEN THE INTESTINAL BARRIER AND THE IMMUNE SYSTEM	Data from clinical studies in animal models indicate that the composition of the gut microbiota plays an essential role in modulating immune responses to vaccines, but the mechanisms by which the gut microbiota modulates immunogenicity to different vaccines in various populations are not clear. The addition of natural adjuvants to enhance responses to vaccination is suggested.^147-149 The intestinal barrier plays a role in protecting the intestinal mucosal layer tissues and circulatory system from exposure to pro-inflammatory molecules, such as micro-organisms, toxins, antigens and is vital for the maintenance of health and well-being. Gut barrier dysfunction has been implicated in several diseases such as food allergies, microbial infections, irritable bowel syndrome, inflammatory bowel disease, coeliac disease, metabolic syndrome, non-alcoholic fatty liver disease, diabetes, and septic shock^96^,^148^,^150^,^151	In the samples studied, the decrease in Bacteroides thetaiotaomicron and cellulosilyticus is very negative for maintaining the integrity of the intestinal wall (intestinal homeostasis), which favours an increase in intestinal permeability due to a deterioration of tight junctions, allowing the passage of intestinal contents into the bloodstream, with its serious consequences such as increased hepatic toxic load and increased antigenic load, among others.^148-150 Ng et al. (2022) describe the composition of the gut microbiota by shotgun metagenomic sequencing in stool samples from 138 vaccinees (37 with CoronaVac and 101 with BNT162b2) in relation to immune responses and adverse effects in adults who received inactivated CoronaVac vaccine or the mRNA vaccine Pfizer's BNT162b2 BioNTech Comirnaty^147	They observe both spike and neutralising antibody levels 1 month after the first and second doses with Pfizer positively correlated with the efficacy of the mRNA vaccine. They suggest a longitudinal assessment of the gut microbiota profile and long-term (longer than 1 month) antibody response after inoculations. This study reinforces the theory of vaccine immune response in the context of HSR.^1^,^147 The protective bacterial microbiota provides the microenvironment that prevents overgrowth of proteolytic bacteria and pathogens. The balance between the resident bacterial species confers stability to the overall microbial population. The barrier effect is due to the ability of certain bacteria to secrete anti-microbial substances (bacteriocins), which inhibit the proliferation of other bacteria, and to competition between bacteria for resources in the system, either nutrients or ecological space^149^,^150	Bifidobacterium adolescentis when abundant, is usually due to the use of probiotics of this bacterial species. Probiotics should always be used under the supervision of a physician. Some experiments have been reported where protein metabolites of Bifidobacterium adolescentis could cause hepatotoxicity in cells of human origin (THLE-2). Therefore, monitoring with liver function test would be advisable.^148–151Bacteroid thetaiotaomicron degrades essential plant polysaccharides in the human gut, stimulates angiogenesis in the gut. It also mediates the formation of the intestinal mucosal barrier, which protects against pathogen invasion through regulation of the expression of species-specific antibiotic proteins^150^,^151	^1^,^96^,^147-151
	The genera Bifidobacterium, Enterococcus, and Lactobacillus are part of the mucosa-associated microbiota and play an important role in pathogen containment and protective function. Low levels of these genera may compromise their function.^151Bifidobacteria reduce intestinal lipopolysaccharide (LPS) levels, decrease levels of pro-inflammatory cytokines, improve intestinal motility, and may reduce inflammatory status. When their abundance is decreased, it complicates this function.^149^,^151 The genus Enterococcus comprises lactic acid bacteria (LAB) members of the commensal flora of the human colon. Enterococci can produce bacteriocins, antimicrobial compounds, which limit the proliferation of pathogens, although they may act as pathogens in certain diseases. Enterobacteriaceae have the ability to secrete LPS, which can act as an endotoxin, increasing inflammation in the gut^149^,^151Collinsella aerofaciens at physiological levels can deconjugate bile acids and this ability to modify bile acids modulates the virulence and pathogenicity of enteric pathogens. Bile acids under physiological conditions are associated with inflammatory processes and carcinogenesis of the digestive tract^147	Bacteria of the genus Streptomyces (phylum Actinobacteria) can be found both in soil and in the gut. Although there are pathogenic species, it is also true that they produce anti-proliferative, anti-inflammatory, immunosuppressive, and antibiotic compounds. These substances are good allies against allergy and autoimmunity, as well as inflammatory bowel diseases.^151 The study by Ng et al. (2022) suggests that the loss of the intestinal mucosa and the disruption of this immunomodulatory microbiota also make it possible for inflammatory processes to occur continuously over time in the intestinal tract, and situations of intestinal permeability may occur under conditions of an altered ecosystem (intestinal dysbiosis) and certain diseases. When levels are elevated in the sample, they may be producing a remarkably complex array of pro-inflammatory neurotoxins including surface lipopolysaccharides (BF-LPS) and toxic proteolytic peptides and pro-inflammatory cytokines. Factors released by intestinal epithelial cells (IECs), such as retinoic acid (RA) and transforming growth factor beta (TGF-β), promote the development of antigen-presenting cells (APCs), both DCs and macrophages, with tolerogenic properties, in the lamina propria.^149^,^151	APCs stimulated by signals such as flagellin release IL-23, which also promotes IL-22 production by innate lymphocytes type 3 (ILC3). IL-22 stimulates the release of RegIIIγ, an antimicrobial peptide (AMP) produced by IECs, which is retained in the mucus layer. Innate lymphocytes type 2 (ILC2) indirectly control microbiota colonisation by secreting IL-13, a cytokine that drives the differentiation of intestinal epithelial stem cells into goblet cells, which in turn produce mucin glycoproteins.^149 These antimicrobial mechanisms are constitutively engaged by the immune system to prevent overgrowth of colonising microbes and to monitor the resident microbiota through immune homeostasis.^149 Tolerance of the normal gut microbiota is crucial for gut homeostasis, which requires an extensive network of immune regulatory cells, including regulatory T cells (Tregs) and tolerogenic dendritic cells. Detection of commensal microbiota through TLR-MyD88 signalling is one method applied by the immune system to maintain host microbial homeostasis. The expression of pattern recognition receptors (PRRs) varies in different gut tracts, as does the composition of the resident microbiota. This results in cross-talk between the microbiota and the immune system.^150^,^152	Regional immuno-inflammatory activation has been demonstrated in the lungs, nervous system, gastrointestinal (GI) tract, and liver following administration of COVID-vaccines.^1^,^16^,^35^,^41^,^86^,^124 As a consequence of SARS-CoV-2 GI infection, viral particles may infect the liver, causing hepatocellular damage and an immune-inflammatory response that may persist over time	^1^,^16^,^35^,^41^,^86^,^124^,^147^,^149-152
OTHER BIOMARKERS PREDICTING SYSTEMIC INFLAMMATION AND DRUG INDUCED INJURY	Lyoumi et al. (1998) and Vlasakova et al. (2022) evaluated 2 biomarkers of tissue remodelling and inflammation, α2-macroglobulin (A2M), and α1-acid glycoprotein (AGP) as potential biomarkers in blood of drug-induced tissue injury (DITI), drug-induced vascular injury (TIMP-1) and systemic inflammatory response (SIR). They observed an increase in hepatic acute-phase reactant proteins (AGP, A2M) with central nervous system (CNS) toxicity^153^,^154	Although plasma increases in AGP and A2M may have beneficial effects in relation to the systemic antimicrobial response, their inflammatory basis has causes that need to be investigated. Indeed, the hepatic response suggests the persistence of viral antigens in the GI tract with alterations in the permeability of the intestinal barrier and activation of pathophysiological responses with a neuro-immuno-inflammatory basis, such as dyspepsia and irritable bowel. In this regard, it should be recalled that the persistence of viral antigens in the GI tract leads to alterations of intestinal physiology, which in turn generate alterations in intestinal permeability, with neuroinflammatory and neurocognitive consequences in the patient's brain via the hepato-pulmonary pathophysiological response^1^,^4^,^16^,^51^,^86^,^153^,^154	On the other hand, pathophysiological communication through the brain–hepatopulmonary axis is bidirectional and involves the parasympathetic nervous system, represented by the vagus. As a mixed interoceptive nerve, with afferent (80%) and efferent (20%) fibres, it constantly monitors gut health, sensing metabolites from the microbiota and regional immune system, whose information it transmits centrally to activate an anti-inflammatory cholinergic response that dampens GI inflammation and modulates the composition of the microbiota, helping to regularise altered intestinal permeability. However, when abnormal brain activation via the vagal route coincides with an alteration of the blood–brain barrier, excessive activation of microglia occurs, which has neuroinflammatory effects, in turn altering neuronal signalling pathways with possible neuropsychiatric and neurocognitive consequences that must be prevented and treated^1^,^4^,^16^,^51^,^86^,^153^,^154	High levels of A2M interfere with the effects of leptin, contributing to hyperleptinaemia with consequences for the enteric nervous system.^155 The mechanisms that may affect vaccination need to be assessed. They are complex and include factors related to COVID-19 vaccines (nature of the antigen, delivery system, adjuvants, and immunomodulators), the host immune system, and the gut microbiota. This “endogenous adjuvant potential” has been demonstrated in the significantly impaired antibody responses to these vaccines.^1^,^2^,^13^,^25–29^,^31^,^35^,^38^,^39^,^43^,^46–52 Ng et al. (2022) offer a model to study how the immunogenic potential of the microbiota could be exploited to improve the immunogenicity of COVID-19 vaccines	^1^,^2^,^4^,^13^,^16^,^25-29^,^31^,^35^,^38^,^39^,^43^,^46-52^,^86^,^153-155
SYSTEMS BIOLOGY EFFECTS OF mRNA VACCINES AGAINST SARS-CoV-2	Hajjo et al. (2022) analyse the systems biology effects of COVID-19 mRNA vaccines to assess their safety and putative side effects. Large-scale simultaneous vaccination of the global population will reveal heterogeneity in immune responses, as well as predisposition to develop adverse effects post-vaccination, especially in vulnerable subjects. They apply a systems biology workflow, based on transcriptomics data for BNT162b2 from Pfizer-BioNTech in vaccinated subjects. No publications were available for Moderna mRNA-1273 at the time they evaluated these results. However, they generalise them to other mRNA vaccines based on evidence from the biomedical literature^57^,^71^,^156^,^157	They analyse the chemogenomic effects of vaccines and their composition at the systems biology level to generate testable hypotheses about the effects of vaccines on biological systems. Her group and others have successfully validated and used this theory to study the systems biology effects of small-molecule drugs and the various COVID-19 vaccines^17^,^74^,^92^,^101^,^111^,^156^,^158-161	The results show that the mRNA-based BNT162b2 vaccine affects immune response pathways related to interferon and cytokine signalling, which should lead to vaccine success, but may also produce adverse effects post vaccination. They also point to cardiac adverse effects related to mRNA vaccines, specifically, the effects of BT162b2 on calcium homeostasis. Their findings are significant for drug and medical device regulatory agencies, clinicians, policy makers, and the general public^156	They use multiple transcriptional gene signatures to study the pharmacological effects of mRNA vaccines. Their results indicate that BNT162b2 produces a strong immune response 21–22 days after receiving the first dose and subsequent booster in vaccinated individuals.^156	^17^,^57^,^71^,^74^,^92^,^101^,^111^,^156-161
	The biological pathways stimulated are the immune response pathways necessary to induce the adaptive immune response triggered by vaccines. They highlight the role of innate and inflammatory immune mechanisms associated with IFN-gamma, interleukin, and protein C signalling. They also point to the clinical efficacy of the vaccine in stimulating the innate immune response, however, IFN-gamma signalling triggers inflammatory responses with perverse consequences including the involvement of female and male hormones in some of the inflammatory processes^1^,^22^,^24^,^28^,^35^,^38^,^45^,^51^,^104^,^156	These data should be used as testable hypotheses for the presence of putative functional links of the BNT162b2 vaccine. In turn, they compare vascular and cardiovascular diseases in COVID-19 patients with the top 50 enriched diseases by DEGs prioritised from single-cell transcriptomics data of BNT162b2, especially in the context of mRNA COVID-19 vaccines and myocarditis^26^,^156	Their results indicate that the selected 76 DEGs are involved in interferon signalling, cytokine signalling, interferon alpha/beta signalling, antiviral mechanisms by interferon-stimulated genes and interferon gamma signalling (Fig. 3). They point out that genes with a greater effect on transcription, such as transcription factors, or those with greater aberrations in response to treatment, exert greater effects on modulating the underlying biology, including the immune dysregulation shown. For example, IP10 and CCL2, both genes are linked to SARS-CoV-2 infection and thromboinflammation. They suggest that IP10 can be used as a biomarker for induced disease severity^156	In this study, they rank all DEGs according to their expression levels and generate 3 gene lists to identify compounds inducing transcriptional signatures similar to the BNT162b2 mRNA vaccine. If the query gene lists share few or no common genes they would be ranked in similar lists, this increases their confidence that the results are statistically significant and biologically relevant. Overall, they recognise 13 high confidence hits that belonged to 6 pharmacological classes: 4 ATPase inhibitors (38.46%), 3 protein synthesis inhibitors (30.76%), 1 Bruton's tyrosine kinase (BTK) (7.69%), 1 apoptosis stimulant (7.69%), 1 ribonucleoside reductase inhibitor (7.69%), and 1 guanylate cyclase activator (7.69%). They highlight that 12 of the top positive compound connections they report are known modulators of the immune system through their effects on innate immune pathways, such as stimulating apoptosis and NLRP3 inflammasome pathways^156	^1^,^22^,^24^,^26^,^28^,^35^,^38^,^45^,^51^,^104^,^156
OVEREXPRESSION OF TRANSMEMEMBRANE SERINE PROTEASE PROTEINS	Another finding to consider is the overexpression of several members of the transmembrane serine protease subfamily (TMPRSS) 21–22 days after vaccination. TMPRSS proteins are required for haemostasis, promotion of some cancers, and entry of respiratory viruses into human cells. Activation of the SARS-CoV-2 spike protein by TMPRSS2 and TMPRSS4 has been studied. In this study TMPRSS4, TMPRSS6, TMPRSS7, and TMPRSS9 were overexpressed in response to the BNT162b2 vaccine. Therefore, they suggest that the adverse effects of BNT162b2 on TMPRSS expression may favour SARS-CoV-2 infection.^156	They note, if COVID-19 is characterised by an increased inflammatory response and a decreased immune response that trigger common vascular diseases, mRNA vaccines stimulate immune pathways to fight off SARS-CoV-2, leading to the production of particular antibodies and the induction of inflammatory responses, which could trigger rare vascular diseases. This study and others have validated these hypotheses, also finding that BT162b2 triggers immune responses similar to LPS-induced platelet activation^4^,^156	The role of platelets in haemostasis, host defence mechanisms and their modulatory effect on T- and B-cell communication for proper acquired immunity has been discussed previously. Platelets can recognise pathogens, select host defence peptides such as platelet factor 4 (PF4), a platelet-derived CXC chemokine, and together with leukocytes construct thrombotic immune complexes. Cases of vaccine-induced immune thrombotic thrombocytopaenia (VITT) have been reported following administration of adenovirus vector and mRNA vaccines^1^,^75^,^156^,^162–168	Therefore, this study indicates that mRNA COVID-19 vaccines can act as immunogens by encoding for the SARS-CoV-2 spike protein and also as adjuvants through their LNP content or through the intrinsic immunostimulatory properties of the modified and/or non-modified RNA	^1^,^4^,^76^,^156^,^162-168
MORTALITY CAUSED BY COVID-19 VACCINATION	Murata et al. (2022) present 4 cases who died at home after receiving mRNA COVID-19 vaccine. Three cases vaccinated with 2 doses of Moderna mRNA, and 1 case received 2 doses of Pfizer-BioNTech mRNA vaccine. The time from administration of the second dose to death was 1–10 days.^102	In the case of the Pfizer-vaccinated subject, post-vaccination adverse effects such as headache and fever were not observed on the day before death, whereas in the other cases, they were observed the day before death. The post-mortem interval inferred from post-mortem phenomena and the coroner's rectal temperature measurements estimated high body temperatures for all cases at the time of death. They use RNA sequencing and identify genes that were differentially expressed in the post-vaccination cases and the control group that died from blood loss and strangulation^102	They identify 390 up-regulated genes associated with neutrophil degranulation and cytokine signalling and 115 genes were down-regulated in post-vaccination cases compared to controls. They suggest causality of death with temporal occurrence due to immune dysregulation after vaccination, systemic immune response syndrome (SIRS).^102 SIRS must meet at least 2 of the following criteria: fever < 38 °C or hypothermia < 36 °C, tachycardia > 90 beats/min, tachypnoea > 20 breaths/min, leukocytosis > 12 × 10^9/l, or leukopaenia < 4 × 10^9/l.^19 SIRS has been reported to be induced by several factors, such as infection (including COVID-19 infection), ischaemia, surgery, and trauma.^169	Autopsies of these patients did not reveal cause of death in any patient and pathological analysis showed findings of sudden death, such as primary organ congestion without hypoperfusion, and no information about cause of death, including myocarditis. In this study, they estimate the cause of death by sequencing RNA obtained from peripheral blood. They obtain an average of 39.1150-base pair (bp) paired-end (PE) reads per sample. Of the 43 751 genes with non-zero total read counts, 505 genes were significantly differentially expressed between individuals with unknown cause of death (vaccinated subjects) and the control group.^102 They define COVID-19 vaccination as pseudoinfection with SARS-CoV-2 and as an inducer of SIRS. In the 4 deceased patients, immune function was sensitised by the first dose and the second dose caused SIRS and death in the more susceptible patients. They warn of the need to detect this aberrant cytokine response, such as the analysis of single nucleotide polymorphisms to prevent such cases ^1^,^13^,^16^,^37^,^39^,^52^,^82^,^102^,^114^,^170	^1^,^13^,^16^,^19^,^37^,^39^,^52^,^82^,^102^,^114^,^169^,^170
	Ittiwut et al. (2022) reported 13 cases of sudden unexplained death (SUD) following COVID-19 vaccination in Thailand, aged 23–72 years; 10 (77%) were male and 3 (23%) female, 12 of Thai origin and 1 Australian. They reported underlying diseases in 5 patients, without arrhythmia. No case had a history of ultrasensitive troponin UST in family members. Eight (61%), 4 (31%), and 1 (8%) died after the first, second, and third vaccine doses, respectively. SUD was triggered after all types of COVID-19 vaccines administered in Thailand, 7 (54%), 2 (15%), 2 (15%), 2 (15%), 1 (8%), and 1 (8%) deaths after receiving ChAdOx1 nCoV-19 (AstraZeneca), BBIBP-CorV (Vero Cells) from Sinopharm (Beijing), CoronaVac (Sinovac), BNT162b2 (Pfizer/BioNTech), and mRNA-1273 (Moderna), respectively^171	Eight (61%) died after receiving the first dose of AstraZeneca's ChAdOx1nCoV-19 vaccine.^171 Fever was self-reported in 3 cases. Ten (77%) and 11 (85%) died within 24–72 h after vaccination. In 6 cases (46%), autopsy identified no explainable causes of death, they recorded another 6 cases with cardiac symptoms, including coronary atherosclerosis in 4 cases (31%), arrhythmogenic right ventricular dysplasia in one case (8%), and one case of dilated cardiomyopathy (DCM) (8%), the remaining case of the sample had thalassaemia (haemochromatosis and liver cirrhosis). Whole exome sequencing analysis identified 5 cases with SCN5A gene variants, coincident in patients diagnosed with Brugada syndrome, with a frequency of 38% (5 out of 13 cases)^171	A significantly higher rate compared to recorded cases of SUD in Thais occurring after vaccination with COVID-19 (between 8 and 30 days), in a Thai SUD cohort studied before the pandemic (12%, 3 of 25 cases) and in their in-house exome database (12%, 386 of 3231 cases). All cases tested negative for SARS-CoV-2 by reverse transcriptase polymerase chain reaction (RT-PCR). Patients confirmed to have died naturally were referred for genetic studies^171	The authors suggest the hypothesis of an association between SCN5A variants and SUD within 7 days after COVID-19 vaccination, 13 variants in 7 genes in 11 of 13 cases (85%). They show that the vaccine had a direct effect and causes myocarditis in the right ventricular outflow tract area, with pre-existing aberrant conduction in patients with SCN5A and leads to death. Regardless of vaccine type, number of doses and the presence of underlying diseases, or post-vaccine fever^171	^171
NEOPLASTIC EFFECTS OF mRNA-BASED COVID-19 VACCINES	Çinar et al. (2022) discuss the role of COVID-19 itself and the spike protein produced endogenously by mRNA vaccines and their immunological, microenvironmental, prothrombotic, and neoplastic effects. They show 4 cases of myeloid neoplasms following administration of Pfizer's BNT162b2 mRNA-based COVID-19 vaccine^172	They have used immunophenotyping to understand the impact of B-cell dysfunction on the serological response to COVID-19 vaccination in primary antibody deficiencies^172	They reinforce the theory of probable pathobiological mechanisms and causalities of spike protein-related toxicity and clonal myeloid disorders^172	They call for objective pharmacovigilance-based follow up to establish the clinical–biological correlation between spike-mRNA vaccines and leukaemogenesis. Meanwhile, all haematological adverse events must be closely monitored and reported.^172	^172
IMMUNE RESPONSES IN THE HUMAN FEMALE REPRODUCTIVE TRACT AND POTENTIALLY HARMFUL EFFECTS ON FERTILITY AND LONG-TERM HEALTH IN WOMEN FROM COVID-19 VACCINES	Finally, the systematic review by Nazir et al. (2022) summarises the main predictors and rates of menstrual cycle changes after COVID-19 vaccination, including 78 138 patients who received mRNA-based vaccines (Pfizer-BioNTech [BNT162b2] and Moderna [mRNA-1273]), inactivated whole virus vaccines (Sinopharm/BBIBP-CorV and Sinovac Biotech) and recombinant adenoviral vector vaccines (Johnson & Johnson/Janssen/Ad26.COV2.S, Oxford-AstraZeneca/ChAdOx1nCov-19 and Sputnik V). Of this sample, 39 759 (52.05%) participants had menstrual abnormalities (menorrhagia, oligomenorrhoea, polymenorrhoea, dysmenorrhoea, and metrorrhagia) following COVID-19 vaccination, a significant sample, indicating a large heterogeneity in the occurrence of these adverse effects. The overall rate of adverse effects following COVID-19 inoculations in this study varied from 83% to 90.9%. There are few studies reported in the literature addressing the causal relationship between SARS-CoV-2 vaccination status and menstrual irregularities^172	They point to 3 studies that establish increase in female age (ages 18–30) as a causal predictor of menstrual problems. The authors add other predictors such as: history of pregnancy, first dose and subsequent boosters, systemic side effects of COVID-19, and smoking. Most of the cases reported were cross-sectional, questionnaire-based studies and had to be associated with the above predictors. Notably, 10 retrospective studies report a proportion of women with menstrual irregularities that could be associated with COVID-19 vaccination, for example, in a Norwegian cross-sectional survey based on 5756 women recruited through random sampling, 39%–41% reported these adverse events.^173^,^21 They were most frequently experienced after the second dose, with 2 studies recording 3–6 menstrual cycles following vaccination. Symptoms reported are heavier bleeding, dysmenorrhoea, longer and/or shorter than normal cycles, and unexpected breakthrough bleeding, also in patients receiving contraceptive treatment.^20^,^21^,^173^,^174 Pre-vaccination infection with SARS-CoV-2 also resulted in menstrual disturbances ^103^,^173^,^175	These findings allude to a cumulative immune effect due to repeated antigen exposure, as discussed above evidenced by the “spike effect”. They also recognise the need to follow more cycles for longer periods to study the long-term consequences, as well as the likelihood of pregnancy.^173 They found gynaecological disorders such as menorrhagia, endometriosis, polycystic ovarian syndrome, fibroids, and adenomyosis that caused participants to have increased menstruation after vaccination mostly of short duration, but suggest that it may predispose to these changes. Most studies published to date conclude and repeat that the effect of COVID-19 vaccines on the menstrual cycle is temporary, mild, and without long-term consequences. However, it is known that the particular antigen that elicits this immune response is debatable, both the spike protein shared with SARS-CoV-2 and the adjuvants used in vaccination have been found to cause the intense immune response after administration of COVID-19 vaccines, and similarly after SARS-CoV-2 infection as a potential stressor that changes the activity of the hypothalamic–pituitary–ovarian axis that regulates the onset and duration of the menstrual cycle^113^,^173	The menstrual cycle and restoration of the endometrium are mediated by innate immune cells that reside in the endometrium. Activation of these immune cells would trigger heavier, irregular, and unexpected bleeding over time. Hormone levels involved in the menstrual cycle could be affected by vaccine-induced immune dysfunction.^173^,^176 Other possibilities attributable to vaccination could occur through changes in thyroid and VITT, effects already observed in other vaccines (measles, diphtheria, and hepatitis).^173^,^177–179 Another finding to consider is the 2-dose dosing schedule (21 and 28 days) for Pfizer-BioNTech and Moderna, respectively; the first dose would be administered in the early follicular phase. Thus, it would affect the development of the dominant follicle during the follicular phase and consequently the cycle length.^1^,^98^,^156^,^173 A contradictory finding that could permanently disturb the endocrinological homeostatic regulation of the menstrual cycle would be a severe and chronic systemic disease like long COVID, an adverse effect to be evaluated in the context of COVID-19 vaccination.^173 This study evidences the complex interplay between inflammatory markers and hormonal disruptions due to COVID-19 infection, including mRNA-based vaccination, warning of its potential for damaging the long-term health and fertility of women, especially those diagnosed prior to vaccination.^176^,^177^,^180^,^181 Tolerability and safety must be ensured for better acceptance of COVID-19 vaccination. Further cohort studies are needed to identify the temporal link between menstrual cycle changes. The authors call for population-based studies, which record variance in epigenetic findings due to genetic polymorphisms in ethnic and at-risk communities ^173	^1^,^20^,^21^,^98^,^103^,^113^,^156^,^173-181
	Data on the safety of COVID-19 vaccines administered during, before, and after pregnancy are scarce. Moro et al. (2022) evaluated Vaccine Adverse Event Reporting System (VAERS) reports of 3462 adverse event reports in pregnant women who received a dose of COVID-19 vaccines: 1831 (52.9%) with BNT162b2, 1350 (39.0%) with mRNA-1273, and 275 (7.9%) with Ad26.COV2.S, and 6 of unknown manufacturer. Reports indicating that vaccination was given before the last menstrual period or during the post-partum period were excluded. Eight maternal deaths and 12 neonatal deaths were reported. Six hundred and twenty-one (17.9%) reports were coded as serious. They included: 878 (25.4%) spontaneous abortions (< 20 weeks) (SAB), 101 (2.9%) episodes of vaginal bleeding, 76 (2.2%) preterm deliveries (< 37 weeks), 62 (1.8%) stillbirths (≥ 20 weeks), and 33 (no calculated rate) cases of babies with major birth defects or a chromosomal abnormality, 18 of the 33 cases with BNT162b2 vaccine, and 15 of the 33 cases with mRNA-1273^182	They recorded 107 infant conditions including all 12 neonatal deaths, indicating prematurity as the cause of death. The crude reporting rates for preterm deliveries and stillbirths, as well as maternal and neonatal mortality rates were compared with the background rates of published sources. They (did not) note disproportionate reporting. They estimated 3 173 387 live births in the USA during their study period (December 2020–October 2021)^182	According to the National Centre for Health Statistics methods referenced include foetal deaths at 20 weeks' gestation or more determined by obstetric estimate, among women aged 15–44 years, excluding foreign residents. They report 18 945 foetal deaths during the study period. Most SABs, (81.3%) 490 out of 603 cases with gestational age data, occurred at < 12 weeks gestation and (39.3%) 338 out of 859 cases the pregnant woman was aged ≥ 35 years. Among the 849 SAB cases reporting onset interval (period of time from vaccination to presentation of adverse effects) were: 0–3 days (26.4%), 4–7 days (12.0%), 8–14 days (15.5%), and ≥ 15 days (46.1%)^182	They report the 8 maternal deaths as pulmonary embolism, amniotic fluid embolism, eclampsia with peripartum cardiomyopathy, and acute cerebellar intraparenchymal haemorrhage secondary to severe persistent thrombocytopaenia due to acute myelogenous leukaemia in 4 cases, respectively. Two others had sudden clinical deterioration leading to death. There were no medical reports for 2 of the 8 cases.^182 All findings should be interpreted with caution	^182
	In a recent study, Monin et al. (2020) discuss immune responses in the human female reproductive tract. They provide a detailed review of the current knowledge of the immune responses of the female reproductive tract (FRT) and their heterogeneity within the organ and between compartments. In the commensal-rich vagina, the immune system must allow the growth of beneficial microbes, whereas the purpose of the uterus is to allow the growth of the foetus^183	In both compartments, these objectives must be balanced with the activity of eliminating pathogens. This will not only facilitate interventions to prevent the spread of sexually transmitted diseases, but in the context of the “spike effect” will allow for improved fertilisation, implantation, and pregnancy outcomes for mothers and babies, as infections are among the common causes of infertility and late pregnancy failure^183	The authors point out that the uterine immune system also changes with the menstrual cycle and pregnancy. It is now beginning to be appreciated that cyclical changes also occur in the vagina and that these may have an impact on susceptibility to disease. They reiterate and indicate the composition of maternal immune cells (innate lymphoid cells and innate T cells in the vagina and uterus, in addition to APCs) in the blood and decidua, and prior to pregnancy, the frequency of immune cells in the endometrium varies during the menstrual cycle^183	In the female reproductive tract (FRT), antibodies contribute to pathogen clearance through mechanisms including pathogen neutralisation, opsonisation, and complement-driven lysis. IgG- and IgA-secreting plasma cells can be detected in the lamina propria (LP) of both the cervix and vagina, although compared to other mucosal sites, IgG, rather than IgA, is the predominant antibody isotope in the lower FRT^183^,^184	^183^,^184
	Recently, Mansour et al. (2023) linking menstrual disturbances following COVID-19 vaccination and the role of immune and endocrine pathways (hypothalamic–pituitary–thyroid axis, hypothalamic–pituitary–adrenal axis, and hypothalamic–pituitary–ovary axis), focus on adverse effects in women of reproductive age. They repeat and evidence the detrimental effects of cleaved S protein in COVID-19 vaccines, including SARS-CoV-2 entry into target cells, endothelial damage, pro-inflammatory cytokine release, Toll-like receptor (TLR) activation, stimulation of microglia, molecular mimicry with chaperon and heat shock proteins (HSP), and activation of SARS-CoV-2 S protein and its binding to angiotensin-converting enzyme 2 (ACE2)^185	It can also induce hyper-stimulation of the immune system and synthesis of multiple autoantibodies. Thus, they demonstrate that the normal menstrual cycle can be disrupted by impairment of any of these pathways. These findings suggest that disrupting the monocyte–endothelial–platelet axis may restore the immune dysregulation observed in all adverse effects secondary to administration of COVID-19 vaccines and could be a potential therapeutic alternative in the near future.^27–29^,^46^,^49^,^185–187 Post-vaccination menstrual disorders are common and can occur with different vaccines, such as the human papillomavirus (HPV) vaccine ^185^,^188	During the follicular phase, vaccination may prolong the menstrual cycle and/or arrest/delay ovulation and disrupt the reproductive cycle. These adverse effects can be attributed to individual variation, and are very similar after the first and second dose.^185^,^189 A specific profile could be defined in the female sex, young age (second dose with BNT162b2: SD = 38.9 years; third dose with BNT162b2: SD = 45.9 years) and without comorbidities as risk factors for vaccine-induced immune dysregulation.^190 Incomplete inactivation of the X chromosome in females may be implicated in vaccine efficacy.^185^,^190^,^191	This inactivation is not uniform among all immune cells and there is a probability of biallelic expression of genes. X chromosomes come from different parents and offer more immune diversity in females. Females have a double copy of immune genes due to their XX chromosomes; this chromosome involves the immune-related genes in the human genome. The X chromosome regulates different components of the immune system such as TLR7, TLR8, CXCR3, and CD40L receptors, which are overexpressed in women and actively influence the response to vaccination.^185^,^190	^27-29^,^46^,^49^,^185-191
	Women respond to vaccination through increased antibody production and a Th2-cell-dominant immune response, which produces anti-inflammatory cytokines.^185^,^192 TLR7, a frequent TLR in females, is strongly associated with vaccine efficacy and sex differences in autoimmunity. It can escape X-chromosome inactivation and lead to increased expression of innate immune cell and T-cell associated genes, which induces production of type I IFN and activates the adaptive immune response. It would explain the higher frequency of adverse effects in women due to the stronger immediate response to the antigen, modulated through the innate immune system^25^,^53^,^185^,^187	Testosterone has a suppressive effect on innate and adaptive immune response, confirming that sex hormones are involved in the mechanism of increased vaccine reactogenicity in women. For example, genetic factors could also be involved in vaccine reactogenicity, such as the ACE2 gene and the angiotensin type 2 (Ang-II) receptor through interaction with sex hormones, a key peptide in vascular and renal damage^131,187,193–195	The differential activity of X-linked genes and polymorphisms of the ChrY (sex-differentiated immunity) gene, which are regulated by escape from X-chromosome inactivation and epigenetic mechanisms, respectively, also play a role. Thus, they may affect global gene expression in immune cells^131,187,193–195	Together, the sex chromosome complement, the activity of regulatory elements on sex chromosomes, and the expression of sex chromosome genes contribute to sex differences in immunity to viral infections.^131,187,193–195 Intersectional approaches will be needed to better understand the adverse effects of COVID-19 vaccines, as well as treatments to decrease antiviral adaptive immunity, virus replication, and inflammation, taking into account that there are sex differences.^193	^25^,^53^,^131^,^185^,^187^,^192-195

The term “safety profile” refers to all adverse effects that could be caused, triggered, and/or worsened at any time after vaccination, such as anaphylactic reactions, diseases diagnosed after vaccination and autoimmune events.¹⁹⁶ The WHO defines 5 types of adverse events following immunisation (AEFI), recorded in a 30-day window following vaccination, which are discussed in the current published literature and some reports of spontaneous adverse events: (1) vaccine product-related reaction; (2) vaccine quality defect-related reaction; (3) immunisation error-related reaction; (4) immunisation anxiety-related reaction; and (5) coincidental event.¹⁹⁷ The first and third type of adverse effect are likely in parallel with COVID-19 vaccine technology, the current biomedical literature has evaluated more data for: (a) LNP components, (b) modified and unmodified mRNA, (c) spike protein produced by COVID-19 vaccines, not excluding their co-occurrence.

The present systematic review discusses, based on published evidence, the innate and adaptive immune stimulation induced by COVID-19 vaccines, based on the activation of biomarkers of inflammation and their effect on immunogenicity and reactogenicity will drive the design of effective approaches to manage the disease induced by these new vaccine platforms against SARS-CoV-2. To this end, we conducted a review of the published literature on the innate immune mechanisms of this new therapy. We also studied other variables such as unknown serious adverse effects and/or atypical variants of other post-viral syndromes, their clinical and epidemiological patterns, inflammatory signatures, and temporal occurrence associated with the presence of post-vaccine recognition antibodies such as anti-RBD IgG, anti-S1 IgG, anti-S2 IgG, and anti-N (nucleocapsid) in the context of infection by contagion. Clinical characteristics that, in principle, make this drug an ideal candidate to prevent the disease and/or the deleterious consequences as a secondary prevention system in the form of a vaccine.

Objectives

The research objectives were as set out below: to systematically review the literature on adverse effects following administration of COVID-19 vaccines to demonstrate their therapeutic potential in the treatment and/or prevention of the disease, and to demonstrate the association of causality and temporal occurrence.

The specific objectives were:

• 1.

  To identify articles publishing information on the immunogenicity and reactogenicity of COVID-19 vaccines that induce long-lasting expression of the SARS-CoV-2 spike protein with systemic effect and that mimic long COVID.

• 2.

  To identify and describe methodological aspects of work reporting potential inflammatory biomarkers that could predict cellular and humoral immune dysregulation.

• 3.

  To assess the association between adverse effects and molecular mimicry, the production of particular autoantibodies, and the role of certain adjuvants.

• 4.

  To demonstrate the mechanisms of exposure and subsequent dissemination to the cell line, integration into the human genome (genotoxicity), VEGF-mediated vascular cross-talk induced by the spike protein, and involvement of the gut microbiota composition.

• 5.

  To verify the reactogenicity of COVID-19 vaccines with higher post-vaccination antibody levels.

Method

A systematic search of the scientific literature reporting data on the adverse effects of vaccination against SARS-CoV-2.

The study was conducted in 2 phases: in the first phase, a systematic review was conducted of scientific articles published in the literature addressing the issue of immunogenicity and reactogenicity after vaccination.

The most relevant health science databases were used: MEDLINE (through PubMed and Web of Science); Web of Science, selects other bases: Web of Science (WOS) main collection, Current Contents Connect (CCC), Derwent Innovations Index (DIIDW), KCI - Korean Journal Database (JKD), MEDLINE, Russian Science Citation Index (RSCI), and SciELO Citation Index; Scopus (Elsevier).

The following MeSH (Medical Subject Headings) terms were used as search descriptors¹⁹⁸: vaccine COVID-19, adverse events, as well as the same in Spanish, with their corresponding Boolean formulation (AND) and the truncation term for some of them. The search filters used for the PubMed-MEDLINE search platform were adapted to the Scopus and Web of Science databases. To locate articles more precisely, a search was conducted as to whether the term existed in the thesauri of the databases and when this was not possible, it was used as free text.

The limits of the search to select the documents in the databases were: documents in article format, published in scientific journals, without language restrictions, open access. It was limited to the titles of the articles and their abstracts.

The search period was taken as the last 3 years of each database until 11 July 2023. In addition, a reverse search was conducted by analysing the literature references of the selected articles.

The inclusion criteria in selecting articles were that their objectives and hypotheses should address potential mechanisms of unknown and severe immune reactivity of COVID-19 vaccines. Also, the putative plausibility of molecular mimicry between viral spike peptides and the patient's own peptides resulting in an event (study design was considered for inclusion); articles published in the last 3 years (2021–2023); provide primary data, i.e., data obtained directly by the authors.

Exclusion criteria were articles without available abstracts, editorials, conference papers, book reviews, correspondence, and social media, or unauthorised comments on COVID-19 vaccines, studies with methodological problems that condition the quality of the reported data, and articles reporting common spontaneous adverse events with a mild prognosis and no torpid course, poor reporting (under-diagnosis and/or half-diagnosis), no clear follow-up, laboratory results of positive nucleocapsid antibodies (SARS-CoV-2 anti-nucleocapsid IgG) that only appear from contact with the virus and not from vaccination, pre-existing medical conditions requiring immunosuppressive treatment at the time of vaccination and chronically due to immune-stimulatory underproduction.

Texts were initially selected based on the relevance of the title and abstract. For those that were selected in this way, the full text of the articles was analysed to be considered for inclusion in the review.

Articles included in the review had to meet the quality criteria established by the Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA) statement.^170,199

The Spanish Critical Appraisal Skills Programme (CASPe)^114,184 was used to assess the quality of the studies, and the methodological quality of the review was assessed according to the criteria established in the AMSTAR (Assessing the Methodological Quality of Systematic Review) tool.²⁰⁰

Information of interest for the study was extracted using an ad hoc form that collected information on the general characteristics of the studies, which analysed the main results.

The following variables were determined for each article: authors, year of publication, study design, study participants, sample size, time from vaccination to symptom onset (counted in days), recommendations (post-vaccination antibody titre, therapeutic alternatives/future treatments for adverse reactions caused), risk of bias-limitations (not peer-reviewed, sample size), biomarkers of inflammation-immune T-cell activation, clinical and epidemiological patterns.

Data were extracted and independently verified by the investigator.

For further analysis, the data were entered into a database table in Excel and then into a database created with SPSS Statistics IBM version 29.

In the second phase, the articles identified in the review were grouped and analysed according to the clinical and epidemiological characteristics associated with serious adverse events. Given the length of the systematic review, only the data referring to the inflammatory signature derived from reactogenicity are presented in this project.

For the variables included in the analysis, frequency measures for qualitative variables were calculated using SPSS Statistics IBM version.

After conducting the systematic review and establishing the existing evidence in the literature, it was decided whether the existence of causality and temporal occurrence derived from COVID-19 vaccines is valid and reliable for the prevention of the disease.

With regard to ethical aspects, the systematic review was conducted on based on the analysis of primary and secondary sources, in no case was there any experimentation with groups of patients or animals. The design and execution of the research was in accordance with the specified AMSTAR methodological criteria to guarantee the quality and veracity of the information collected, facilitating its replication in the future.

With the support of the PRISMA statement and the CASPe tool, a clear and organised structure was achieved for interpretation of the review. Inclusion and exclusion criteria were considered, providing rigour to the review.

Results

The systematic review identified a total of 2033 records which, after a screening process according to the inclusion criteria and the elimination of duplicate papers, papers with methodological problems, and papers without open access, were reduced to 58 articles, as described in the PRISMA flowchart of the review (Fig. 4); of these, 50 articles on human models and 2 on cellular models were identified. Tables A1 and A2 summarise the most relevant information from the articles/reports included in the review (study participants and time from vaccination to symptom onset counted in days, as well as the characteristics of all included studies).

Fig. 4.

PRISMA diagram of the systematic review.

Analysis of articles found

Based on the study participants, it can be seen that 50 articles (93.5%) are from human model studies; 2 (2.2%) from cell model studies.

Fig. 5 shows the temporal distribution of the articles according to the year of publication.

Fig. 5.

Temporal distribution of articles according to year of publication.

Graph 5 shows that the number of publications is not equal, in ascending order: 2 articles (3.4%) were found in 2021; 5 (8.6%) in 2023; 51 (87.9%) in 2022. Most of the articles are a year old.

A total of 58 research articles have been included, after discarding repeated or duplicate studies, 15 are descriptive studies (14 [15.1%] prospective and 1 [1.1%] retrospective), as well as 12 case reports (12.9%), 7 case series (7.5%), 1 case report with systematic review (1.1%), 1 systematic review with meta-analysis (1.1%), 8 systematic reviews (8.6%), 11 narrative reviews (11.8%), 1 randomised clinical trial (1.1%), and 2 experimental trials (2.2%), on human liver cell line Huh7 in vitro and in vivo, respectively. Forty-three articles record the sample size of each study, the smallest being one case report (12.9%) and the largest having 1 057 000 participants (1.1%).

The studies analysed serious adverse events based on histological analysis, human species, study participants, ethnically differentiated populations, time from vaccination to symptom onset (counted in days), clinical characteristics, follow-up period of the disease process, form of treatment, deaths, temporal association, identification of the inflammatory signature, design of the analyses performed in the studies, and measurement of outcomes.

Tables A1 and A2 summarise the common current evidence associated with adverse events induced after administration of COVID-19 vaccines, based on the spike effect of COVID-19 vaccines and the SARS-CoV-2 virus spike protein, as well as related variables.

The studies included in this review demonstrate the relevance of a systems biology approach to correlate the positive regulation of genes associated with innate immunity, cytokine production, and responses to virus infection, specifically IFN-inducible genes, with observed adverse effects. Most adverse effects were reported after vaccination with BNT162b2 (37 of 58 articles), ranking in descending order: 15 of 58 articles with mRNA-1273, 12 of 58 articles with ChAdOx1, 7 of 58 articles with Ad.26.COV2.S, 4 of 58 articles with CureVac, 3 with Sinovac, 1 with AZD1222, and 1 case with BBIBP-CorV.

Table A1 shows that the number of days to symptom onset varied greatly, in descending order: 31 articles (70.8%) were found between 0 and 365 days; 8 articles (8.8%) between 0 and 15 days; 6 articles (6.4%) between 1 and 7 days; 4 articles (4.3%) for 2 days.

Here, we review the current understanding that COVID-19 vaccines induce innate and adaptive immune activation. We discuss the innate immune recognition of mRNA vaccines and iLNP technology at the cellular level and consider the contribution of mRNA and LNP components to their immunogenicity. In this context, they act as adjuvants for this type of vaccine platform.³Table 2 summarises the immunological effects of adverse events caused by SARS-CoV-2 vaccines.

Table 2.

Summary of immunological effects of adverse events resulting from SARS-CoV-2 vaccines.

REVERSE TRANSCRIPTION	Two studies looking at the in vitro effect of BNT162b2 mRNA vaccine in the human liver cell line (Huh7) Aldén et al. (2022) and Mouliou et al. (2022) confirm the rapid entry of BNT162b2 into cells and subsequent intracellular reverse transcription of BNT162b2 mRNA into DNA. It is likely that liver cells also present the vaccine-derived SARS-CoV-2 spike protein, explaining that liver cells are targets for pre-primed spike protein reactive cytotoxic T cells. The authors suggest in vivo models in future studies to better understand the effects of BNT162b2 on liver function. They show that BNT162b2 is reverse transcribed into DNA in the Huh7 cell line, they observe that BNT162b2-derived DNA integrates into the host genome and affects the integrity of genomic DNA, leading to genotoxicity as an adverse effect	It is unclear whether DNA reverse transcribed from BNT162b2 is integrated into the cellular genome, including whole genome sequencing of cells exposed to BNT162b2, as well as systemic distribution in tissues from human subjects who received BNT162b2 vaccination, which is of great concern. Different expression of genes and proteins has also been demonstrated, in addition to certain up-regulated proteins involved in RNA metabolism	Aldén et al. (2022) using a carcinoma cell model to verify effective retrotransposition of LINE-1, with active DNA replication which differs from non-dividing somatic cells, report active cell proliferation in several tissues, such as bone marrow or basal epithelial layers and during embryogenesis, as well as in non-dividing, terminally differentiated cells, e.g., human neurons. As discussed, the advantage of mRNA-iLNP platforms is that they do not require the addition of adjuvants to produce protective immune responses against pathogens, however, mRNA-iLNP vaccines are not immunosilent	(Aldén et al. 2022, Mouliou et al. 2022)
RECOGNITION OF VACCINE ANTIGENS AS A POTENTIAL PATHOGEN CAUSING ADVERSE EVENTS	Vaccine antigens are recognised as potential pathogens causing autoimmune reactions by the mechanisms of molecular mimicry and T-cell activation in 26 articles	We must remember that innate and adaptive immune responses vary between individuals due to their cellular and biochemical components. The SARS-CoV-2 spike protein in COVID-19 vaccines is confirmed to contain the sequence and structural motif of a superantigen, causing the hyperinflammatory syndrome that involves direct T-cell stimulation and excessive cytokine production in certain subjects	–	(Saluja et al. 2022, Garrido Suárez et al. 2022, Mouliou et al. 2022, Castaldo et al. 2022, Verbeke et al. 2021, Morikawa et al. 2022, Lagousi et al. 2022, Zagorec et al. 2022, Piras et al. 2022, Abu Serhan et al. 2022, Mohseni Afshar et al. 2023, Lee et al. 2022, Hoffmann et al. 2022, Magen et al. 2022, Pisani et al. 2022, Ekobena et al. 2022, Caliskan et al. 2022, Aliasin et al. 2022, Ahmed et al. 2022, Finsterer et al. 2021, Mingot-Castellano et al. 2022, Watanabe et al. 2022, Chow et al. 2022, Zlotnik et al. 2022, Ameratunga et al. 2022, Duijster et al. 2023)
PEAK EFFECT IN THE CONTEXT OF CUMMULATIVE EFFECT AND LOSS OF RECEPTOR ACTIVITY OF ACE2	In addition, 19 studies observed the “spike effect”, known as “spike intoxication “, in the context of saturation and subsequent impairment of angiotensin II-converting enzyme receptor (ACE2) function	They propose the mechanisms: (1) cross-reaction between foreign antigens and self-antigens, (2) over-activation of antigen-presenting cells and subsequent autoimmune response, and (3) polyclonal activation of B cells or bystanders leading to cytokine synthesis and activation of autoreactive T cells. Studies show that spike proteins produced after vaccination have the native-like mimicry of the SARS-CoV-2 spike protein's receptor binding functionality and prefusion structure.	COVID-19 vaccines increase endogenous synthesis of SARS-CoV-2 spike proteins. Once synthesised, the free-floating spike proteins released by the destroyed cells previously targeted by the vaccines circulate in the blood and interact with ACE2 receptors (a zinc metalloproteinase) expressed by other cells, promoting ACE2 internalisation and degradation, mimicking the pathological features of SARS-CoV-2, a mechanism known as the spike effect of COVID-19 vaccines	(Talotta 2022, Lee et al. 2022, Castaldo et al. 2022, Saluja et al. 2022, Namiki et al. 2022, Levy et al. 2022, Piras et al. 2022, Abu Serhan et al. 2022, Mohseni Afshar et al. 2023, Lee et al. 2022, Tamborska et al. 2022, Aliasin et al. 2022, Azzolini et al. 2022, Ahmed et al. 2022, Kim et al. 2022, Angelini et al. 2022, Zlotnik et al. 2022, Ameratunga et al. 2022, Duijster et al. 2023)
RELATIONSHIP WITH HYPOTHESES ON MOLECULAR MIMICRY, INFLAMMATION SIGNATURE, SYSTEMIC DISTRIBUTION, PERSISTENCE, AND CONTINUED HIGH AVAILABLITY OF ANTIBODIES POST-VACCINATION	Six studies relate testable hypotheses on molecular mimicry, inflammatory signals attributed to post-vaccination reactogenicity, their systemic spread, temporal persistence, and the possibility of a “cumulative effect” of the spike protein with post-vaccination IgG anti-S and IgG anti-RBD antibodies, suggesting post-COVID-19 mimicry	These features offer high and sustained antigen availability, favouring more robust antibody responses. Both spike-encoding mRNA and spike protein are detectable 60 days after the second dose of BNT162b2 and mRNA-1273 in vaccinated humans	It is therefore possible that SARS-CoV-2 vaccines composed of viral and recombinant glycoprotein vectors deliver more attenuated inflammatory signals or by other mechanisms that contribute to innate immune activation, not just mRNA encapsulated in lipid nanoparticles	(Mouliou et al. 2022, Levy et al. 2022, Pisani et al. 2022, Azzolini et al. 2022, Ahmed et al. 2022, Kim et al. 2022)
ENDOGENOUS ADJUVANT MECHANISM INVOLVED IN INNATE IMMUNE ACTIVATION	Endogenous adjuvant potential can be observed in significantly impaired antibody responses with these vaccines	–	–	(Mouliou et al. 2022, Verbeke et al. 2021, Morikawa et al. 2022, Namiki et al. 2022, Levy et al. 2022, Lee et al. 2022, Magen et al. 2022, Pisani et al. 2022, Azzolini et al. 2022, Abbasi et al. 2022)
PATHOGENIC HYPOTHESIS ASSOCIATED WITH THE NEOPLASTIC, INFLAMMATORY, NEURO-IMMUNE/VASCULAR EFFECTS PRODUCED BY SARS-CoV-2	The “pseudoinfection” proposed in this review is more associated with lymphoproliferative, inflammatory disorders and neuromyelopathies, including neuroinflammatory alteration with alteration of the blood–brain barrier, which explains the development of the adverse effects cited above	–	–	(Mouliou et al. 2022, Castaldo et al. 2022, Mohseni Afshar et al. 2023, Hoffmann et al. 2022, Kim et al. 2022, Tamborska et al. 2022, Magen et al. 2022, Pisani et al. 2022, Ekobena et al. 2022, Caliskan et al. 2022, Aliasin et al. 2022, Ahmed et al. 2022, Umezawa et al. 2023, Zlotnik et al. 2022)
CELLULAR MECHANISMS AND PATHOGENIC/VIRAL, AUTOINFLAMMATORY, AUTOINMUNE AND PARANEOPLASTIC HYPOTHESIS ASSOCIATED WITH ADVERSE EFFECTS IN RECIPIENTS OF SARS-cov-2 VACCINES	Studies show the cytokine storm to be caused by uncontrolled infection (pathogenic/viral hypothesis, e.g., neurotropism), autoantibodies or autoreactive T cells associated with predisposing germline mutations (autoimmune hypothesis), germline mutations in genes that regulate inflammation (autoinflammatory hypothesis) and/or somatic mutations in monoclonal lymph node cells resulting in ectopic cytokine secretion (paraneoplastic hypothesis) in certain cases	Most of the included articles verify potent innate immune activation in reports of local and systemic adverse effects of post COVID-19 vaccination in humans, 56 articles (97.8%) and 46 articles (87%) for molecular mimicry	–	(Mouliou et al. 2022, Hajjo et al. 2022, Verbeke et al. 2022, Castaldo et al. 2022, Saluja et al. 2022, Chen et al. 2022, Morikawa et al. 2022, Lagousi et al. 2022, Pirani et al. 2022, Zagorec et al. 2022, Levy et al. 2022, Piras et al. 2022, Abu Serhan et al. 2022, Mohseni Afshar et al. 2023, Lee et al. 2022, Hoffmann et al. 2022, Wong et al. 2022, Magen et al. 2022, Pisani et al. 2022, Ekobena et al. 2022, Caliskan et al. 2022, Aliasin et al. 2022, Ahmed et al. 2022, Finsterer et al. 2021, Umezawa et al. 2023, Hetland et al. 2022, Mingot-Castellano et al. 2022, Watanabe et al. 2022, Abbasi et al. 2022, Chow et al. 2022, Zlotnik et al. 2022, Ameratunga et al. 2022)
BIOMARKERS THAT RECOGNISE AND DIAGNOSE SEVERE FORMS OF ADVERSE REACTIONS CAUSED BY COVID-19 VACCINES	It can be seen that several biomarkers were used for the recognition and diagnosis of some severe forms of SARS-CoV-2 vaccine adverse reactions, the most representative being: 3 studies used vascular endothelial growth factor (VEGF) (Talotta 2022, Mouliou et al. 2022, Abu Serhan et al. 2022); 9 studies used human type 1 interferons: the proteins IFN-α, IFN-β, IFN-γ, MDA5-IFN- α (Verbeke et al. 2022, Garrido Suárez et al. 2022, Hajjo et al. 2022, Mouliou et al. 2022, Shafiq et al. 2022, Watanabe et al. 2022, Szebeni et al. 2022, Castaldo et al. 2022, Morikawa et al. 2022)	Five studies used plasmacytoid dendritic cells (pDCs), such as: CD123 + y BDCA2 +(Verbeke et al. 2022, Watanabe et al. 2022, Szebeni et al. 2022, Castaldo et al. 2022, Morikawa et al. 2022); 14 studies used B-cells: immunoglobulin specific (IgG anti-S and IgG anti RBD) post-COVID-19 vaccination (Mouliou et al. 2022, Levy et al. 2022, Pisani et al. 2022, Azzolini et al. 2022, Ahmed et al. 2022, Kim et al. 2022, Verbeke et al. 2022, Mohseni Afshar et al. 2023, Hoffmann et al. 2022, Finsterer et al. 2021, Ameratunga et al. 2022, Szebeni et al. 2022, Castaldo et al. 2022, Morikawa et al. 2022);	Eleven studies used draining lymph nodes and a set of cell types, T cells: monocytes, macrophages, dendritic cells (DC), follicular helper T cells (Tfh), cytotoxic T lymphocyte (CTL), natural killer (NK) cells and granulocyte-macrophage colony-stimulating factor (GM-CSF) (Verbeke et al. 2022, Mohseni, Mouliou et al. 2022, Mohseni Afshar et al. 2023, Hoffmann et al. 2022, Magen et al. 2022, Ekobena et al. 2022, Finsterer et al. 2021, Ameratunga et al. 2022, Szebeni et al. 2022, Castaldo et al. 2022, Morikawa et al. 2022)	Eleven studies used the cytokines: tumour necrosis factor alpha (TNF-α) and the interleukins IL-1, IL-1β, IL-4, IL-5, IL-6 (Zlotnik et al. 2022, Garrido Suárez et al. 2022, Ameratunga et al. 2022, Szebeni et al. 2022, Castaldo et al. 2022, Morikawa et al. 2022, Verbeke et al. 2022, Mohseni Afshar et al. 2023, Piras et al. 2022, Hoffmann et al. 2022, Umezawa et al. 2023); neuropilin-1 (NRP-1) (Talotta 2022); transmembrane serine protease 2 protein (TMPRSS2) (Aliasin et al. 2022)
	Four studies used angiotensin converting enzyme 2 (ACE2) receptors (Castaldo et al. 2022, Piras et al. 2022, Aliasin et al. 2022, Ameratunga et al. 2022); calprotectin in blood (Hetland et al. 2022); other proteins: syndecan-1 and histone H3-neutrophil extracellular trap (NET)- (H3-NETS) (Hetland et al. 2022); natriuretic peptides (NT-proBNP) (Lee et al. 2022); prostaglandin (PG) E2 (Zlotnik et al. 2022)	Specific antibodies: 2 studies used antibodies to anti-aquaporin 4 (AQP4-IgG) (Umezawa et al. 2023, Caliskan et al. 2022), antibodies against myelin oligodendrocyte glycoprotein (MOG-IgG) (Umezawa et al. 2023), 3 studies used antibodies against platelet factor 4 (anti-PF4) (Mouliou et al. 2022, Mingot-Castellano et al. 2022, Abbasi et al. 2022), antibodies against leucine-rich glioma inactivated 1 (anti-LGI1) (anti-LGI1)) (Zlotnik et al. 2022), antibodies against cyclin citrullinated peptide (CEP) (Morikawa et al. 2022) and anti-ganglioside antibodies (Mohseni Afshar et al. 2023)	Two studies used C-reactive protein (Morikawa et al. 2022, Hajjo et al. 2022); γ-aminobutyric acid (GABA) neurotransmitter (Garrido Suárez et al. 2022); 6 studies used Toll-like receptors: TLR3, TLR7, and TLR8 (Verbeke et al. 2022, Watanabe et al. 2022, Szebeni et al. 2022, Castaldo et al. 2022, Morikawa et al. 2022, Rahimi Mansour et al. 2023); other laboratory findings suggestive of VITT, such as low platelets, low fibrinogen, and high D-dimer, together with elevated anti-PF4 titres, are classical findings (Abbasi et al. 2022).	These cytokines interact with complement systems which, in addition to producing inflammation and cross-talk between innate-adaptive immunity, activate the coagulation system and increase the permeability of the endothelial layer in a time- and dose-dependent manner, in turn promoting their distribution to the systemic circulation, resulting in elevated inflammation. (Umezawa et al. 2023, Pisani et al. 2022, Abu Serhan et al. 2022, Mouliou et al. 2022, Levy et al. 2022)
	Several studies propose platelets as a biomarker of inflammation, we know that both LNP mRNA and spike protein activate platelets. The identification of VEGF in plasma samples accurately measures the actual concentrations of this mediator in peripheral blood, like other viral infections, alterations in VEGF cause changes in circulating pro-inflammatory mediators such as TNF-α, IL-1β, IL-6, and IFN-γ-inducible protein 10 (IP-10	Studies show that the concentration of VEGF in serum samples could be affected by the number of platelets, platelets being a major source of VEGF	Three studies identify elevated plasma levels of VEGF in SARS-CoV-2 vaccinees responsible for diffuse neurological and microvascular damage. The authors test the hypothesis that the adverse neurological and cardiovascular effects of COVID-19 vaccines could be caused by both direct viral damage and hyperactivation of the immune response. In turn, they mediate the prolonged symptoms of adverse events	(Talotta 2022, Verbeke et al. 2022, Abu Serhan et al. 2022)
HYPERSENSITIVITY RESPONSES TO COVID-19 VACCINES	Also, non-anaphylactic allergic reactions, mostly HSR types III and IV classified by timing of onset and symptoms, can be observed, and were reported in the context of IMAE to SARS-CoV-2 vaccines, especially with mRNA vaccines	Ten studies confirm alterations in platelets together with activation of the complement system in mediating anaphylaxis, CARPA, and HSR through the release of biologically active molecules (ATP, thromboxane, and chemokines) and lipid inflammatory molecules such as PAF, which triggers degranulation of perivascular mast cells and release of thromboxane and serotonin, also causing inflammatory responses and tissue injury	They include systemic and local symptoms affecting the skin, gastrointestinal, respiratory, and cardiovascular systems	(Yoshida et al. 2023, Szebeni et al. 2022, Granados Villalpando et al. 2022, Mouliou et al. 2022, Morikawa et al. 2022, Piras et al. 2022, Pisani et al. 2022, Ahmed et al. 2022, Son et al. 2022, Ameratunga et al. 2022)
GUT MICROBIOTA AND IMMUNE SYSTEM DYSFUNCTION AND DAMAGE	Three studies assess immunomodulatory gut microbiota with COVID-19 vaccines	They suggest intestinal mucosal damage and inflammation and regional immune system and neuro/myenteric dysfunction system leading to gastrointestinal disorders, persistence of viral antigens in the gastrointestinal tract with alterations in the permeability of the intestinal barrier and activation of pathophysiological responses with neuroinflammatory and neurocognitive consequences	This hypothesis reinforces the theory of the immunogenicity-mediated role of HSR triggered by the vaccine and how it may induce indirect neurotoxicity through immune-mediated pathogenesis and gastrointestinal infection	(Ameratunga et al. 2022, Ng et al. 2022, Chen et al. 2022)
COEXISTENCE OF ADVERSE EFFECTS CAUSED BY COVID-19 VACCINES WITH THE ABSENCE OF ORGAN DAMAGE	Three studies confirm the absence of organ damage producing symptoms following administration of COVID-19 vaccines	This has been studied in inflammatory disorders that produced impairment without accompanying structural disease. But the evidence in favour of these findings is sparse given the lack of published data	–	(Zlotnik et al. 2022, Chen et al. 2022, Finsterer et al. 2021)
ASSOCIATION OF GENES INVOLVED WITH TRANSCRIPTIONAL EFFECTS OF mRNA VACCINES (Pfizer's BNT162b2)	Six articles evaluate genes associated with the cell cycle and transcription, they look at diseases related to genes involved in the transcriptional effects of BNT162b2, such as: immune system diseases, viral diseases, autoimmune diseases, musculoskeletal, rheumatological, joint, skin and connective tissue, connective tissue, lacrimal apparatus, xerostomia, and Sjögren's syndrome (Hajjo et al. 2022)	Magen et al. (2022) performed partial mapping of the BNT162b2 vaccine spike protein mRNA sequence in a patient's sample, showing an unusual pattern of vaccine mRNA expression in blood cells, namely, “chopped” parts of the mRNA vaccine molecules from the BNT162b2/Pfizer vaccine, indicating that the exogenously expressed mRNA was stable enough to persist over time	Lagousi et al. (2022) adding the hypothesis that in susceptible individuals the immune response to mRNA may not reduce, they associated younger age with greater alterations in monocyte, inflammatory response, and platelet-related gene expression shortly after the second dose of the BNT162b2 vaccine. It is likely that the amount of mRNA antigen contained in each vaccine formulation may also be a factor in mRNA reactogenicity	(Murata et al. 2022, Mouliou et al. 2022, Morikawa et al. 2022, Lagousi et al. 2022, Magen et al. 2022, Hajjo et al. 2022)
	Çinar et al. (2022) encourage undertaking expanded immunophenotyping studies to detect B- and T-cell dysfunction in the serological response to COVID-19 vaccination	–	–	–
MORTALITY CAUSED BY COVID-19 VACCINES	Data on mortality are limited, this review includes 3 articles. Murata et al. (2022) define COVID-19 vaccination as SARS-CoV-2 pseudoinfection and as inducing SIRS. They present 4 patients who died after COVID-19 vaccines. They identify 390 up-regulated genes associated with neutrophil degranulation and cytokine signalling; 115 genes were down-regulated in post-vaccination cases compared with controls. They note the need to detect this aberrant cytokine response, as well as analysis of single nucleotide polymorphisms to prevent such cases	Ittiwut et al. (2022) describe 13 cases of sudden unexplained death (SUD) following COVID-19 vaccination	Moro et al. (2022) reported 8 maternal deaths and 12 neonatal deaths, 621 (17.9%) reports were coded as serious. They included: 878 (25.4%) spontaneous abortions (< 20 weeks) (SAB), 101 (2.9%) episodes of vaginal bleeding, 76 (2.2%) premature deliveries (< 37 weeks), 62 (1.8%) stillbirths (≥ 20 weeks), and 33 (no calculated rate) cases of babies with major birth defects or a chromosomal abnormality, which were 18 of 33 cases with BNT162b2 vaccine and 15 of 33 cases with mRNA-1273. They recorded 107 infant conditions including the 12 neonatal deaths, indicating prematurity as the cause of death	(Murata et al. 2022, Ittiwut et al. 2022, Moro et al. 2022)
ASSOCIATION OF CELL APOPTOSIS AND LEUCEMOGENESIS CAUSED BY VACCINATION AGAINST SARS-CoV-2	Five studies comparing the endogenous adjuvant potential in vaccination associated with apoptotic cells and leukemogenesis are noted (Verbeke et al. 2021, Mouliou et al. 2022), such as: malignant neoplastic events related to the pathogenic role of the spike protein in COVID-19 vaccines (clonal myeloid disorders) (Çinar et al. 2022), uterine fibroids (non-malignant) (Nazir et al. 2022), lymphoproliferative disorders associated with neuropilin (NRP-1), this mediator may disrupt physiological pathways involved in angiogenesis, nociception, embryogenesis, carcinogenesis, inflammatory disorders and/or neuromyelopathies, and alter the blood–brain barrier (Talotta 2022)	–	–	(Verbeke et al. 2021, Mouliou et al. 2022, Çinar et al. 2022, Nazir et al. 2022, Talotta 2022)
DIFFERENTIATED IMMUNITY WITH THE HIGHEST INCIDENCE OF ADVERSE EVENTS IN FEMALES	Sex-differentiated immunity is observed in 6 articles, the higher frequency of adverse effects in women could be explained by the stronger immediate response to the antigen, modulated through the innate-adaptive immune system	Differential activity of X-linked genes and ChrY gene polymorphisms are regulated by escape from X-chromosome inactivation and epigenetic mechanisms, respectively. Testosterone depresses the innate and adaptive immune response, confirming that sex hormones are involved in the mechanism of increased vaccine reactogenicity in women	The articles included confirm that women respond to vaccination through increased production of antibodies and anti-inflammatory cytokines	(Moro et al. 2022, Green et al. 2022, Namiki et al. 2022, Rahimi Mansour et al. 2023, Nazir et al. 2022, Mouliou et al. 2022)
DRUG TREATMENTS TRIED AFTER VACCINATION AND OTHER THERAPEUTIC OPTIONS	Immunosuppressive and/or immunomodulatory treatment was administered after vaccination in 19 articles: oral systemic steroids in intermittent short courses (Saluja P et al. 2022); intravenous methylprednisolone (1 g/24 h) for 5 days, with gradual tapering (Chen et al. 2022); intravenous methylprednisolone 1000 mg/24 h for 5 days, followed by a tapering dose of oral prednisone for the next 10 days (Pirani et al. 2022); ramipril and methylprednisolone pulses for 2 months (Zagorec et al. 2022)	Intravitreal anti-VEGF (n = 39 cases, 30.4%), followed by corticosteroids, 18 cases (14.2%). They include other treatments (thrombolytic, antiplatelet, or anticoagulant) in addition to surgery (Abu Serhan et al. 2022); antivirals and steroids have been frequently tried as treatment and accelerate recovery in Bell's palsy and transverse myelitis, intravenous immunoglobulin and plasmapheresis can be considered effective treatment for Guillain-Barre syndrome. High-dose methylprednisolone (1 g/24 h for 3–7 days) started immediately in all cases of transverse myelitis improved neurological function and accelerated recovery (Mohseni Afshar et al. 2023)	Oral prednisolone 0.3 mg/kg/day (20 mg), colchicine (0.6 mg/12 h), and low-molecular weight heparin (enoxaparin 40 mg/day) were started. Prednisolone was tapered over 2 months after discontinuation of all medications and colchicine was given for 3 months. Colchicine may be helpful in treating multisystem inflammatory syndrome (MIS) or preventing the recurrence of hyperinflammation with moderate doses of systemic corticosteroids. At 2-month follow-up laboratory results were in the normal range (Lee HJ et al. 2022); interleukin-6 blockade with siltuximab. Six months after completion of treatment, the patient experienced no signs of idiopathic multicentric Castleman disease (iMCD) or inflammation (Hoffmann et al. 2022)	The same treatment is proposed for adverse effects related to other drugs or inflammatory processes (steroids, antivirals, acetylsalicylic acid, and/or cochlear implant) (Pisani et al. 2022); same treatment for other adverse effects related to COVID-19 vaccines (steroids, antivirals, antihistamines) (Ekobena et al. 2022); partial clinical improvement after treatment with intravenous methylprednisolone, scheduled long-term treatment with rituximab (Caliskan et al. 2022). The immediate use is recommended of steroids for sudden onset tinnitus following COVID-19 vaccination due to its underlying immunosuppressive mechanism (Ahmed et al. 2022).
	The case showed improvement 28 days later after treatment with high-dose glucocorticosteroids: 2 cycles of therapy, each of 1000 mg intravenous methylprednisolone for 3 days (first cycle was started 21 days after vaccination, the second cycle was started at 28 days), continued for 16 days with 40 mg oral prednisone and a tapering dose for 2 more weeks (Umezawa et al. 2023). Intravenous immunoglobulins (IVIG) (n = 13 patients), steroids (n = 3 patients) or no therapy (n = 3 patients). Two patients required plasmapheresis because IVIGs were ineffective, 6 patients required artificial ventilation, 1 case received pregabalin for dysaesthesia. Only 1 case recovered completely and partially recovered in 9 cases (still disabled). Outcome was not reported in 9 patients (Finsterer et al. 2021).	Case 1: topical steroids and oral antihistamines. Case 2: high-dose intravascular immunoglobulin therapy and intravenous methylprednisolone (Watanabe et al. 2022); apixaban as anticoagulant and intravenous immunoglobulin (IVIG) (1 g/kg for 2 days), platelets improved. For long-term treatment, apixaban was prescribed for 6 months and recanalisation of previously thrombosed veins (Abbasi et al. 2022); prednisone (Chow et al. 2022)	Initially treated with high-dose methylprednisolone (1 g/24 h for 5 days), with favourable response, the case continued treatment with oral prednisolone (1 mg/kg), showing marked improvement in temporal orientation, short-term memory, and language, although executive skills remained impaired. Serum sodium levels normalised within 3 weeks of symptom onset. While continuing to improve gradually 4 months later, the patient still has some difficulties with executive skills (Zlotnik et al. 2022). All cases receiving immunosuppressive treatment with systemic corticosteroids showed clinical improvement, but not complete cure	Patterson et al. (2023) propose that disruption of the monocytic–endothelial–platelet axis with maraviroc 300 mg/12 h and pravastatin 10 mg/24 h to restore the immune dysregulation observed in post-acute sequelae of COVID (PASC) could be a therapeutic alternative in the near future for COVID-19 vaccine adverse events. This provides the framework for a future randomised, double-blinded, placebo-controlled trial to further investigate the efficacy of immunosuppressive drugs, especially antiviral drugs, statins, and corticosteroids compared to other immunomodulators such as colchicine

Discussion

This systematic review has met the initial objectives set out to assess knowledge of the topic under investigation.

The findings of this review are relevant to the general public, clinicians, drug and medical device regulatory agencies, and policy makers. The main enriched biological pathways are the immune response pathways, which are essential for inducing adaptive immune response. The results of enrichment in process networks highlight inflammatory and innate immune mechanisms associated with signalling of IFN-γ, cytokines, VEGF, platelet activation, B cells (specific immunoglobulins IgG anti-S and IgG anti-RBD following COVID-19 vaccination), the set of cell types (T cells: monocytes, macrophages, dendritic cells [DC], follicular helper T cells [Tfh], cytotoxic T lymphocyte [CTL], NK cells, and granulocyte-macrophage colony-stimulating factor [GM-CSF]), procalcitonin, and C-reactive protein.^{3,15,22,46,107,156,201} This confirms that vaccines produce the desired effect of stimulating the immune response, however, some of the stimulated innate immune processes can trigger inflammatory processes and cause adverse effects following COVID-19 vaccination, mainly IFN-γ signalling. The results confirm that COVID-19 vaccines induce a strong immune response in subjects vaccinated within the first 4 h (i.e., on the same day of receiving the first and/or booster dose) for up to 365 days (70.8%), compared to reactogenicity responses assessed by clinical and gene expression at 0, 1, 2, 3, 4, 5, 6, 7, 8, 10, 12, 13, 14, 15, 17, 20, 21, 22, 30, 37, 39, 42, 60, 62, 90, 117, 120, and 180 days.

These results should be seen as testable hypotheses for the presence of putative functional links between COVID-19 vaccines and the diseases caused, and should not be considered proof that what the vaccine will produce will depend on the individual's particular immunity. Vascular and cardiovascular diseases are considered known risk factors in patients infected with COVID-19. Hajjo et al. (2022) identified 50 diseases enriched by DEGs from single-cell transcriptomics data of BNT162b2, the authors associate the functional link between mRNA COVID-19 vaccines and cardiovascular effects.

It can be observed that mRNA vaccines, especially BNT162b2 and mRNA-1273, induce immune responses similar to platelet activation triggered by lipopolysaccharide (LPS). Platelets, in addition to mediating communication between T and B cells (acquired immunity), regulate haemostasis and mechanisms of pathogen recognition and recruitment of host defence peptides, such as platelet factor 4 (PF4), a platelet-derived CXC chemokine. In turn, platelet activation may develop thrombotic adverse effects, such as VITT following administration of adenovirus vector vaccines and the less common mRNA vaccine involving antibodies against platelet factor 4 (anti-PF4). Platelets and leukocytes build immune thrombotic complexes, a classic sign of disseminated intravascular coagulation.^47,49,156

Much of the innate immune activation depends on the iLNP component of nucleoside-modified iLNP-mRNA vaccines, as empty iLNPs stimulate a variety of cytokines (TNF-α, IL-1, IL-1β, IL-4, IL-5, IL-6, GM-CSF) and chemokines.^{9,16,55,56,76} In relation to the mRNA component, modification of the nucleoside m1ψ would improve the tolerability of mRNA-iLNP vaccines in humans by attenuating innate immune recognition that would promote translation efficiency and adaptive immunity, including licenced SARS-CoV-2 mRNA vaccines, capable of inducing a type 1 IFN response, raising the possibility of complementary, even synergistic, innate immune activation by the mRNA component.^9,16,87 NK cells and T cells express a progressive and continuous increase in the presentation of genes associated with cell cycle and transcription. Another observation to note is the MDA5-IFN-α signalling pathway, which is required for T-cell responses, e.g., CD8 +, may have implications for the development of mRNA vaccines for other infectious diseases and cancer, where CD8 + T cells are critical for effective immunity.³ The up-regulation of these immune pathways appears to underlie immune-mediated diseases, especially in young women, due to overexpression of X-linked genes by the required antiviral response and stimulation of the immune system by immune and endocrine pathways (hypothalamic–pituitary–thyroid axis, hypothalamic–pituitary–adrenal axis, and hypothalamic–pituitary–ovarian axis); the focus is on adverse effects in women of reproductive age.^1,182,185

Cationic LNPs, similar to those used in mRNA vaccines, have been shown to act as adjuvants, can activate Toll-like receptors (TLR2, TLR3, TLR4, TLR5, TLR7, and TLR8) and NLRP inflammasome pathways.^9,156 This finding confirms that COVID-19 mRNA vaccines produce immunogenicity by encoding the SARS-CoV-2 spike protein and act as adjuvants because of their LNP content and/or the intrinsic immunostimulatory characteristics of unmodified RNA (used in CureVac) and nucleoside-modified RNA (BNT162b2, mRNA-1273). This explains the differences in immunogenicity and reactogenicity between the COVID-19 vaccines. Prolonged duration of biopersistence of adjuvants, or the ability of adjuvant particles to move and accumulate slowly in lymphoid organs and other tissues are important safety concerns.¹

A recent study confirmed that BNT162b2 is rapidly taken up in the human liver cell line Huh7 in vitro, causing changes in LINE-1 expression and distribution after exposure to BNT162b2, the mRNA of BNT162b2 is intracellularly reverse transcribed into DNA from 4 to 24 h, the regulation of LINE-1 activity in response to BNT162b2 mRNA vaccines should be studied. Questions arise as to whether vaccine-derived mRNA can integrate into the human genome, which would cause genotoxicity.^1,59

These features not only offer high and sustained antigen availability, but also favour stronger antibody responses, a condition that resembles a vaccine overdose, under specific circumstances. Reactogenicity has been associated with higher levels of antibodies following administration of COVID-19 vaccines, which reinforces the cumulative effect hypothesis, i.e., the relationship between elevated post-vaccination antibody levels (anti-S IgG, anti-RBD IgG) and their temporal persistence, which seems to mimic that of post-COVID-19. These data associate them with the cumulative immune effect due to repeated exposure and sensitisation to the antigen with each vaccine boost.^{25,36,40,41,43}

Mediators have been identified in ACE₂ and TMPRRS2 that affect the expression of receptors related not only to PASC but also in the adverse effects of COVID-19 vaccines.^121,156 The spike effect of COVID-19 vaccines is recognised, evidencing that the SARS-CoV-2 spike protein contains the sequence and shares the native-like structural and functional mimicry of the antigen, causing the inflammatory syndrome involved in direct T-cell stimulation and excessive cytokine production in some subjects. These cytokines interact with complement systems which, in addition to producing inflammation and cross-talk between innate–adaptive immunity, activate the coagulation system and increase the permeability of the endothelial layer in a time- and dose-dependent manner, in turn promoting their distribution to the systemic circulation, which causes elevated inflammation and regulates persistent symptoms of adverse effects. The resemblance of vaccine antigens as potential pathogens causing adverse reactions is verified by the mechanisms of molecular mimicry and T-cell activation.

The production of the spike protein could be studied in liver cells, the persistence of viral antigens in the GI tract causing alterations in the permeability of the intestinal barrier and the activation of neuro-immuno-inflammatory pathophysiological responses, with neuroinflammatory and neurocognitive consequences, could occur through the hepato-pulmonary pathophysiological response, which could in turn alter neuronal signalling pathways. Mouliou et al. (2022) associate the cross-reactivity of the SARS-CoV-2 spike protein with strong interactions with transglutaminases, myelin basic proteins, mitochondria, nuclear antigens, myosin, collagen, claudin 5/6, and S100B, among other proteins, including proteins involved in synaptic vesicle trafficking, endocytosis, axonal transport, neuronal transmission, thrombosis, inflammation, the blood–brain barrier, and mitochondrial barrier, as well as protein growth factors.

The cross-talk between the human gut microbiota and the immune system has been demonstrated in recent years, and the context of COVID-19 vaccines is no exception, it can also modulate the immune response to vaccination and microbial disturbance and has an impact on the immune response to COVID-19 vaccines. Microbial imbalance, a state called “dysbiosis”, predisposes the body to disease. Many studies highlight the role of the gut microbiota in inflammatory bowel diseases, Clostridium difficile infection, metabolic and neuropsychiatric disorders, autoimmune diseases, and of the non-intestinal microbiota in diseases such as central nervous system disorders, chronic obstructive pulmonary and periodontal disease, cystic fibrosis, and chronic rhinosinusitis. Currently, understanding dysbiosis is challenging because of the intravariability and dependent intervariability of the human microbiota that make it difficult to identify distinct abnormal and normal microbial communities. Understanding the underlying dynamics of the interactions between the microbiota and the immune system is necessary to clarify the potential implications for human health following vaccination.^147–152 The immune system is built by a set of innate and adaptive immune mechanisms aimed at enhancing microbiota containment, barrier immunity, and tissue repair unlinked to inflammation. Tolerance of the normal gut microbiota is essential for gut homeostasis, which requires a large network of immune regulatory cells, including regulatory T cells (Tregs) and tolerogenic dendritic cells. Detection of the commensal microbiota through the TLR-MyD88 signalling pathway is one strategy used by the immune system to maintain host microbial homeostasis.^147–152 Combined approaches will identify the gene expression pattern, gene function, regulation, and physiology of bacteria in a community to improve characterisation of the microbiome. DNA sequencing-based diagnostics do not distinguish between live and metabolically active, damaged, or dead bacterial cells, or free DNA, giving an incomplete picture of the functional profile of a microbial community. RNA sequencing analyses can identify living and metabolically active cells and inform about the transcriptional activity of the community. A detailed understanding of the transcriptome of a given organism will make it easier to recognise cellular processes and proteins that may or may not be expressed and/or produced under current culture assay conditions.¹⁵⁰

Due to the increasing number of cases with symptoms following COVID-19 vaccination and the lack of approved studies and therapies, many of those affected have received different therapies. This is reflected in case series participants and single cases who had received immunosuppressive and immunomodulatory treatments, such as antiviral drugs, statins, systemic corticosteroids, and colchicine. This suggests that the pathophysiology of adverse effects may be the monocyte–endothelial–platelet axis.¹⁸⁶ Most manifest in the brain, colon, heart, lungs, along with endothelial cells and astrocytes. Stimulated platelets also release RANTES, which bind to endothelial cells and stimulate monocyte adhesion to inflamed endothelial tissues and function as a chemotactic agent for inflammatory cells. Activated platelets and endothelial cells also secrete VEGF which induces microvascular hyperpermeability and angiogenesis. VEGF is a known diagnostic biomarker for pro-inflammatory, prothrombotic, and vasculitic neuropathy. Patterson et al. (2023)¹⁸⁶ target treatment for vascular endothelialitis and effects associated with PASC consistent with the effects caused by SARS-CoV-2 vaccines that require assistance with activities of daily living (neurological symptoms, severe headache, cognitive impairment, neurological symptoms): severe headaches, cognitive impairment, neuropathy and weakness; autonomic dysfunction such as postural orthostatic tachycardia syndrome [POTS] consisting of unexplained tachycardia, dizziness, light-headedness, fainting, and abdominal pain, along with sensitivity to light; cardiorespiratory complaints causing exertional intolerance and limitation of physical activity [these include basic activities for daily living]; fatigue along with myalgias. They use the combination of maraviroc's protective role on the endothelium with pravastatin, targeting CCR5 and fractalkine receptors. Their hypothesis has been studied, it is described how CD14 + and CD16 + monocytes transmigrate across the blood–brain barrier and play an important role in central nervous system (CNS) immune surveillance, these monocytes act as reservoirs of human immunodeficiency virus (HIV) in the CNS causing neuroinflammation, neuronal damage, and cognitive impairment. Both maraviroc and statins cross the blood–brain barrier, and maraviroc specifically has been proposed as a treatment for Parkinson's disease, stroke, and cognitive impairment. The authors reported improved neurological function and capacity. Their findings correlated with a statistically significant decrease in VEGF (r = 0.4, p = .02) and sCD40L (r = 0.42, p = .01), suggesting that treatment targeting cytokines associated with vascular endothelialitis correlated with improvement in neurological symptoms. It is possible that antagonism of these receptors may also inhibit autonomic effects, suggesting that pro-inflammatory macrophage activation may trigger vascular endothelialitis, the authors associated elevations in sCD40L, already associated with sympathoadrenal activation. Many patients with cardiac, neurological, and pulmonary symptoms have been studied with electrocardiogram, echocardiogram, stress test, pulmonary function tests, cerebrospinal fluid puncture, computed tomography, and magnetic resonance imaging, which have not detected structural abnormalities, or disease. Symptoms have only been treated with anti-arrhythmics, bronchodilators, alpha-adrenergics, anti-inflammatory drugs, and anti-depressants, rather than treating the underlying cause. The proposed treatment improved exercise tolerance, mobility, and respiratory symptoms. Both maraviroc and statins may reduce cardiovascular risk by protecting the endothelium from infiltration by pro-inflammatory macrophages (IL-2, IL-8, and TNF-α).

Clinical trials will need to be conducted to understand the variation in length of treatment and adapt them to the personal clinical conditions of each patient. CD4/CD8 ratios and the amount of post-vaccine antibodies should also be studied before and after treatment.

It should be noted that COVID-19 vaccines do not contain live virus and therefore should not cause disease in the vaccinated person without prior exposure to the virus. Paradoxically, the results of this study show that COVID-19 vaccines may expose some people to an increased risk of immune dysregulation. This likelihood is confirmed by recent evidence from the published biomedical literature linking immune dysregulation, the spike effect of COVID-19 vaccines, and the temporal occurrence with the adverse effects caused. These findings are testable, not confounded by SARS-CoV-2 infection, concurrent or prior autoimmune diseases, or effects of host genetic background on susceptibility to reactogenicity. However, cases of systemic adverse reactions have been reported in patients with allergic diseases, such as asthma, hay fever, allergic rhinitis, atopic dermatitis, food allergies, and/or intolerances, who are potentially susceptible to COVID-19 and worsening of their chronic diseases after vaccination with BNT162b2, such as intestinal dysbiosis.¹¹⁵ Some reports posit the onset autoimmune diseases after vaccination ruling out that autoimmune conditions were diagnosed at baseline or recurred after vaccinations, including infections. In post-mortem investigations of post-vaccination deaths, the causal association was established, all sudden deaths should be disclosed and investigated to determine clear evidence about the death following COVID-19 vaccination,^102,171 and maternal and neonatal mortality rates should be compared with baseline rates from actual published reports. Close and continuous surveillance for adverse effects in pregnant women who received SARS-CoV-2 vaccines should be performed because of the risk of spontaneous abortion, foetal growth retardation, and congenital abnormalities caused.

Most of the studies included have small sample sizes, some were single case reports, this could be related to a limited observation period as well as inconsistent population adherence to mass vaccination. The most complete data on adverse events were published in follow-up reports obtained from online questionnaires, pharmacovigilance systems, or case series, some of these reports might not be clinically verified as they are subject to recording and reporting biases by pharmacovigilance systems.

It is concerning that a vaccine may cause immune dysregulation that can lead to the disease against which it was developed, this observation warrants further investigation by vaccine manufacturers, the Centres for Disease Control and Prevention (CDC), and the Spanish Agency for Medicines and Health Products (AEMPS). Cellular and humoral immune dysregulation following SARS-CoV-2 vaccination is likely to be predicted.

This analysis proposes that personalised studies should be undertaken using patient-specific genetic signatures for future vaccine genomics research. The application of systems biology methods to analyse the pharmacological responses of COVID-19 vaccines strengthens the understanding of adverse events following vaccination.¹⁵⁶ They provide testable hypotheses alongside clinical data on adverse events.

Conclusions

This study provides a framework for future clinical trial designs to further investigate the efficacy, utility, and especially the potential cure of adverse events caused by COVID-19 vaccines.

A better understanding of the nature of reactogenicity will allow the design of next generation COVID-19 vaccines to stimulate more tolerable adaptive immune responses while mitigating or eliminating serious adverse reactions experienced by those affected by the vaccine. The final suggestion is that further, more accurate, laboratory diagnostic tests should be performed, with clinical evidence establishing the correct causality of case reports. Clinical and laboratory information should be used to assess the current and past medical condition of each case to understand the adverse events.

Funding

This research received no specific grant from any funding agency in the public, commercial, or not-for-profit sectors.

Conflict of interest

The author declares no conflict of interest related to the topic of the article.

Acknowledgements

I would like to thank my family, who have supported me throughout the process.