Strategies for the Management of Spike Protein-Related Pathology

In the wake of the COVID-19 crisis, a need has arisen to prevent and treat two related conditions, COVID-19 vaccine injury and long COVID-19, both of which can trace at least part of their aetiology to the spike protein, which can cause harm through several mechanisms. One significant mechanism of harm is vascular, and it is mediated by the spike protein, a common element of the COVID-19 illness, and it is related to receiving a COVID-19 vaccine. Given the significant number of people experiencing these two related conditions, it is imperative to develop treatment protocols, as well as to consider the diversity of people experiencing long COVID-19 and vaccine injury. This review summarizes the known treatment options for long COVID-19 and vaccine injury, their mechanisms, and their evidentiary basis.

LNPs are a new technology that was not used in vaccine delivery until the emergency use authorization (EUA) of the Pfizer/BioNTech BNT162b2 and Moderna mRNA-1273 COVID- 19 vaccines [4]. This was also unprecedented in the approval process, being the fastest for any vaccine [5], leaving many concerns with regard to long-term safety [6], which was difficult to evaluate due to the unblinding of the initial clinical trials [7].
Whilst the delivery technology of LNPs have previously been used to deliver small molecules, it has only recently been used to deliver RNA. LNPs are advantageous for targeting brain tissue, as they can cross the blood-brain barrier (BBB) [8,9]. The first drug used and LNP to deliver RNA was a small interfering RNA (siRNA)-based drug, known as Onpattro (Alnylam Pharmaceuticals), first approved in 2018 for the treatment of polyneuropathies [10].
Given both the novelty of the technology and the paucity of data on which approval was based (which was also subject to data integrity issues [11]), long-term effects cannot be definitively ruled out, especially because many of the foundational claims on which approval was based have been contested by recent experiments [12][13][14]. For example, in contrast to claims that the injection stayed at the injection site [15], and that spike protein would only be expressed for a short period of time (based on the lability of nonpseudouridylated RNA [16]), the contents and products of the COVID-19 vaccines have been found in the blood stream of most vaccinees studied within hours to days [12].

Methods
This review begins by summarizing the mechanisms of harm from spike protein, either from COVID-19 illness or form COVID-19 vaccination. We also cover the clinical aspects, which can affect the course of the disease. The review then moves to therapeutic mechanisms, which can address the spike protein via different pathways.
For therapeutic interventions for these conditions (long COVID-19 and vaccine injury) with a plausible mechanism of action against spike protein, these are shown in the results section. Relevant clinical trials are added, and any direct evidence or proxy evidence for efficacy (such as efficacy against original COVID-19 illness) is included in the rightmost column.
Additionally, we include clinical trials on long COVID-19 and vaccine injury in Table S1. A search for clinical trials for the condition "Long COVID OR Long COVID-19" in ClinicalTrials.gov revealed 317 studies. A search for clinical trials on vaccine adverse events revealed that one study used rutin and glycoside-rich mulberry juice to reduce adverse events to C19 injection [44]. Other studies, while not specifically treating the immune response, administer therapy alongside vaccination to observe changes in response. These include spermidine [ It is a difficult task to assess the evidentiary basis for each type of intervention, as few meta-analyses have been carried out. For example, a search in the Cochrane Collaboration Library for "Post Acute COVID-19" yields one relevant review on remedying olfactory dysfunction, finding limited evidence for the usefulness of proposed therapies [59]. Furthermore, 46 relevant completed studies for the search term "Long COVID" exist on ClinicalTrials.gov (8 January 2023). As few systematic reviews exist, we aim to summarize the evidentiary basis of the known interventions currently in clinical trials for the treatment of long COVID-19 and COVID-19 vaccine injury are shown in Table S1. There is a single review on treating COVID-19 vaccine injury that could be found, which is included in Table S1.

Mechanisms of Harm
As mentioned previously, while it was expected that the LNP-encapsulated synthetic mRNAs would remain at the injection site and rapidly degrade, there is substantial evidence that they enter the bloodstream [ Ontologically, both infection and vaccination express the spike protein, though some subtle differences exist between the vaccine-generated and the infection-generated spike protein. Importantly, the spike protein encoded by vaccines is static and does not undergo evolution, whereas the spike protein produced by infection evolves as the virus evolves [76,77]. There is one exception to this, and that is when the vaccine is updated, as it is in the bivalent boosters of Pfizer and Moderna, which express the spike protein of both the B.1.1.529 (omicron) BA.5 sublineage and the ancestral WA1/2020 strain [78]. The other important distinction between vaccine spike and infection spike is the stabilized pre-fusion state in the vaccine spike, which results in an increased ACE2 binding affinity compared to spike proteins generated via SARS-CoV-2 infection [79]. The difference in the circulating (in the population) SARS-CoV-2 spike protein to the spike protein (either vaccine or infection generated) of one's initial immune imprinting has important implications for immune escape [77,80] and immune-mediated damage [81]. Immune escape is demonstrated in population studies showing waning vaccine efficacy [82].
In 2021, a comprehensive investigation revealed consistent pathophysiological alterations after vaccination with COVID-19 vaccines, including alterations of immune cell gene expression [83].

Clinical Observations
Although no official definition exists for 'post-COVID-19-Vaccine Syndrome,' a temporal correlation between receiving a COVID-19 vaccine and the beginning or worsening of a patient's clinical manifestations is sufficient to make the diagnosis of a COVID-19 vaccine-induced injury when the symptoms are unexplained by other concurrent causes. It should, however, be recognized that there is a significant overlap between the symptoms and features of the long COVID-19 syndrome [84] and the post-COVID-19-Vaccine Syndrome [85]. However, a number of clinical features appear to be distinctive of the post-COVID-19 vaccine syndrome; most notably, severe neurological symptoms (particularly small fiber neuropathy) appears to be more common following vaccination [86][87][88]. To complicate matters further, patients with long COVID-19 are often vaccinated [89], making the issue of definition more difficult.
Unfortunately, only post mortem examination to date can prove causal relationship when tissues damaged demonstrate the presence of spike protein and absence of nucleocapsid protein (SARS-CoV-2 only) [90].
The true magnitude of post-COVID-19-Vaccine Syndrome is unknown, as data are limited to short duration clinical trials. From a survey of vaccinated individuals, approximately 1% required medical attention immediately following vaccination [91]. A nationwide cohort study of U.S. veterans reported adverse reactions in 8.5% of recipients of the Pfizer vaccine and 7.9% of those receiving the Moderna vaccine [92].
A number of factors are associated with an increased risk of adverse events; these include: • Genetics: first-degree relatives of people who have suffered a vaccine injury appear to be at a very high risk of vaccine injury. People with a methylenetetrahydrofolate reductase (MTHFR) gene mutation [93] and those with Ehlers-Danlos type syndromes, may be at an increased risk of injury. Increased homocysteine levels have been linked to worse outcomes in patients with COVID-19 [94,95]. Increased homocysteine levels may potentiate the microvascular injury and thrombotic complications associated with spike protein-related vaccine injury [96,97]. • mRNA load and quantity of spike protein produced: this may be linked to specific vaccine lots that contain a higher concentration of mRNA due to variances in manufacturing quality, as well as heterogeneity within the vial [98]. • Type and batch of vaccine: variances in the levels of adverse reactions were observed, depending on the manufacturer of the vaccine [91]. • Number of vaccines given: the risk of antibody enhancement (ADE) increases with each exposure to the virus or a vaccine. A negative inverse correlation of dosages given, as well as effectiveness, was also observed [99]. • Sex: the majority of vaccine-injured people are female [100], and vaccines historically have sex-specific effects [101].
• Underlying nutritional status and comorbidities: certain preexisting conditions may likely have primed the immune system to be more reactive after vaccination [102]. This includes those with preexisting autoimmune disorders [103].

Therapeutic Interventions
There are several non-specific means of counteracting the effects of long-COVID-19 and post-COVID-19 vaccine injury. These include nutritional support for general immune regulation and for overall health [104], as well as more specific, spike protein-specific therapeutics.
Non-specific therapeutic moieties include nutritional optimization, as diet-related pathologies, including obesity [105] and type 2 diabetes [106], were associated with worse outcomes from COVID-19 infection. Additionally, high blood glucose facilitates several steps of the viral lifecycle and infection progression [107], motivating the reduction in sugar and refined carbohydrate intake, which are associated with increases in blood sugar. Furthermore, adoption of a whole-food, plant-based diet is associated with decreased oxidative stress and inflammation [108] and better cardiovascular conditions. These positive impacts are attributed to their nutrient profiles, consisting of antioxidants, vitamins, minerals, and phytochemical-containing phenolic compounds, which can exert antioxidant, anti-inflammatory, and other beneficial effects [109,110].
The microbiota plays a fundamental role in the induction, training, and function of the host's immune system and thus shape the responses to its challenges [111]. Gut microbiome composition was significantly altered in patients with COVID-19 compared with non-COVID-19 individuals, irrespective of whether patients had received medication [112]. The researchers said patients with severe illness exhibit high blood plasma levels of inflammatory cytokines and inflammatory markers. Additionally, given altered gut microbiota composition in SARS-CoV-2 infected subjects, there is substantial involvement of the GI tract during infection. These results suggest that gut microbiota composition is associated with the magnitude of immune response to COVID-19 and subsequent tissue damage and thus could play a role in regulating disease severity. The scientists also found that, because a small subset of patients showed gut microbiota dysbiosis, or imbalance, even 30 days after recovery, this could be a potential explanation for why some symptoms persist in long COVID-19 [113].
Given the intricate influence of gut microbiota (GM) on host immune effectors and subsequent inflammatory profile, GM composition and function might contribute to explaining the individual resilience/fragility with respect to COVID-19 and/or the response to therapeutics (vaccines), which deserve further research [114]. Microbial diversity can be improved by consuming many prebiotics and probiotics, such as sauerkraut and kimchi.
The design and discovery of spike protein inhibitors have followed a typical drug repurposing process. Given the structural similarity of the SARS-CoV-2 spike protein to other coronaviruses [115,116], compounds that work for these could potentially be repurposed for SARS-CoV-2 spike inhibition.
Typically, once a prospective compound for repurposing has been identified, it is tested using a ligand-binding assay (LBA) [117]. These assays can provide information on binding affinity and kinetics, as well as binding stoichiometries and even cooperative effects [117].
The next level of verification may be an in vitro assay for viral inhibition in cell culture, where cells are infected with a virus, and viral levels or titre (concentration) are measured by counting viral plaques [118] or measuring viral nucleic acid (NA) levels [119]. Control cells are compared with treated cells. Though the approach has limitations, in not considering the whole-body dynamics of a virus [120], it can serve as a useful starting point.
In vivo studies are a further level of verification, which show the impact of the intervention in an animal model. Beyond in vivo studies, there are clinical studies, which are typically of two design types: observational and randomized control trials (RCTs) [121].
To date, little to no guidance has been provided by health authorities on how to manage spike protein related disease, leaving it up to independent scientists and doctors to develop.
Regarding the COVID-19 Vaccine induced Thrombotic Thrombocytopenia Syndrome (TTS), a 2021 review made suggestions on management, including intravenous immunoglobulin, anticoagulants, and plasma exchange in severe cases [122]. These compounds are nutritional supplements and natural products, with some repurposed pharmaceuticals (Tables 1 and 2).
This list points to the available evidence on each therapy and advances them for further investigation. The following therapeutics work through different mechanisms, but we largely focus on those proteins that bind directly with the spike protein for improved clearance. Here, we summarize studies with different levels of evidence for their respective efficacies, from in silico predictions, which can be based on binding predictions or systems biological associations, to those showing activity in an in vitro or cell-free assay, in vivo studies, and any clinical or epidemiological evidence.
Given the many uncertainties around the duration of spike protein production and the variables determining production, adopting a preventive approach seems sensible, provided the proposed interventions are safe. It remains unknown whether full recovery from COVID-19 Vaccine Injury is possible. However, we suggest targeting several different processes to reduce symptoms associated with both vaccine injury and long COVID-19. These include: (1) Establishing a healthy microbiome (2) Inhibiting spike protein cleavage and binding (stopping ongoing damage) (3) Clearing the spike protein from the body (clearing the damaging agents) (4) Healing the damage caused by the spike protein (restoring homeostasis and boosting the immune system) These categories are not clearly separate, as compounds binding to the spike can both inactivate it by preventing its binding to ACE2, as well as aid in its clearance. There are many biological pathways through which a given effect can occur. To inhibit the harmful effects of the spike protein, it is possible to target the furin cleavage, either by directly binding to the furin cleavage site itself [123][124][125] or by interfering with the serine protease reaction [126][127][128] to block the interaction by binding to ACE2 [129], downregulating ACE2 expression [130], inhibiting the transition to the active conformation of S protein [131], or binding the RBD of spike protein and allosterically inhibiting interaction with ACE2 [132] ( Figure 1). Clearing of spike proteins can also be accomplished by increasing autophagy, which clears proteins and recycles their amino acids [133].
In vivo studies are a further level of verification, which show the impact of the intervention in an animal model. Beyond in vivo studies, there are clinical studies, which are typically of two design types: observational and randomized control trials (RCTs) [121].
To date, little to no guidance has been provided by health authorities on how to manage spike protein related disease, leaving it up to independent scientists and doctors to develop. Regarding the COVID-19 Vaccine induced Thrombotic Thrombocytopenia Syndrome (TTS), a 2021 review made suggestions on management, including intravenous immunoglobulin, anticoagulants, and plasma exchange in severe cases [122]. These compounds are nutritional supplements and natural products, with some repurposed pharmaceuticals (Tables 1 and 2).
This list points to the available evidence on each therapy and advances them for further investigation. The following therapeutics work through different mechanisms, but we largely focus on those proteins that bind directly with the spike protein for improved clearance. Here, we summarize studies with different levels of evidence for their respective efficacies, from in silico predictions, which can be based on binding predictions or systems biological associations, to those showing activity in an in vitro or cell-free assay, in vivo studies, and any clinical or epidemiological evidence.
Given the many uncertainties around the duration of spike protein production and the variables determining production, adopting a preventive approach seems sensible, provided the proposed interventions are safe. It remains unknown whether full recovery from COVID-19 Vaccine Injury is possible. However, we suggest targeting several different processes to reduce symptoms associated with both vaccine injury and long COVID-19. These include: (1) Establishing a healthy microbiome (2) Inhibiting spike protein cleavage and binding (stopping ongoing damage) (3) Clearing the spike protein from the body (clearing the damaging agents) (4) Healing the damage caused by the spike protein (restoring homeostasis and boosting the immune system) These categories are not clearly separate, as compounds binding to the spike can both inactivate it by preventing its binding to ACE2, as well as aid in its clearance. There are many biological pathways through which a given effect can occur. To inhibit the harmful effects of the spike protein, it is possible to target the furin cleavage, either by directly binding to the furin cleavage site itself [123][124][125] or by interfering with the serine protease reaction [126][127][128] to block the interaction by binding to ACE2 [129], downregulating ACE2 expression [130], inhibiting the transition to the active conformation of S protein [131], or binding the RBD of spike protein and allosterically inhibiting interaction with ACE2 [132] (Figure 1). Clearing of spike proteins can also be accomplished by increasing autophagy, which clears proteins and recycles their amino acids [133]. The process of spike protein cleavage into S1 and S2 subunits and subsequent binding of the S1 receptor binding domain (RBD) to the angiotension converting enzyme2 (ACE2) receptor on Figure 1. The process of spike protein cleavage into S1 and S2 subunits and subsequent binding of the S1 receptor binding domain (RBD) to the angiotension converting enzyme2 (ACE2) receptor on host cells. Each of the different subprocesses present opportunities for interference in spike binding to ACE2, as well as a potential means of treating spike protein related pathology.
Microbiome diversity and richness can be improved through a diet rich in prebiotic fiber and probiotics, particularly fermented foods, which can subsequently lower inflammation [151].

Preventing Spike Protein Damage Inhibiting Spike Protein Cleavage
The furin cleavage site on SARS-CoV-2 has been suggested as a reason for its increased infectivity relative to SARS-CoV [152], which had a higher fatality rate, which was much less infectious [153]. Cleavage of the full-length spike protein into S1 and S2 subunits is essential for SARS-CoV-2 entry into human lung cells [126,[154][155][156]. The full-length spike is present in both SARS-CoV-2 infection, as well as vaccination, and it is the only protein common to SARS-CoV-2 infection and vaccination (it is the only protein present in vaccination) [157].
These dual proline mutations featured in the mRNA vaccines stabilize the pre-fusion state, though some cleavage still occurs [162,165,166], and, interestingly, the mutations produce an unknown cleavage product of 40 kDa, where typical cleavage products for the wild-type spike protein are 80 kDa [166]. As such, targeting the cleavage of spike protein is likely to make a difference in long COVID, as well as vaccine injury from the vaccines encoding the full-length wild-type spike protein (AstraZeneca, SinoVac and others), though this may have less of an impact in vaccines encoding the pre-fusion-stabilized spike protein (Pfizer, Moderna, Johnson & Johnson, NovaVax and others).
Notably, targeting cleavage has also been identified as a therapeutic modality in the context of acute COVID-19 [167,168], which can take place via at least three distinct pathways: cleavage by furin, trypsin, or trans-membrane serine protease [167][168][169].

Inhibiting Spike Protein Binding
One of the most direct therapeutic mechanisms is to seek compounds which disrupt the ACE2/Spike interface, either through binding ACE2 or spike in isolation, or disrupting the interface itself. This problem is a steric and conformational problem, for which computational prediction using structural models is highly amenable. A great many computational studies of the spike protein and ACE2 binding compounds have been performed, and some of these hits have further been developed through LBAs, in vitro studies, in vivo studies in animal models, and, lastly, clinical trials with human subjects. Few of the compounds reach the final stage, though several with this mechanism of action have been investigated. Most promising were ivermectin and quercetin, as computational prediction showed these bind to the spike. If the spike is bound in the receptor binding domain (RBD), the interaction with ACE2 receptors, by which spike protein exerts its inflammatory effect, is also inhibited.
Similarly, compounds which bind to the ACE2 receptor can also antagonistically compete with the spike protein for a limited number of receptor sites. For example, the diabetes medication metformin has been identified as a potential long COVID-19 therapeutic agent due to this mechanism of action. Decreasing the level of spike actively binding to ACE2 has therapeutic implications.

Clearing Spike Protein
So far, we have discussed ways to inhibit the impacts of the spike protein on the host's system. Importantly, to progress beyond this, it is necessary to clear out the spike protein. This can be accomplished through upregulation of the protein degradative pathways in the body through upregulation of autophagy. Autophagy can be upregulated by fasting [170] and calorie restriction [171], especially if protein is reduced [172]. Autophagy in many instances does not require the complete cessation of food intake (protocols are available at https://COVID19criticalcare.com/treatment-protocols/, accessed on 15 April 2023). Sharply decreasing protein intake can upregulate autophagy pathways [173], and this can be accomplished while still eating, which makes this more approachable as a protocol. Regular fasting was also associated with better outcomes from acute COVID-19 [174].

Healing the Damage
After the damage process has been attenuated, it is necessary to heal the damage that has occurred. The healing stage requires normalizing the immune response, reducing lingering inflammation (such as by targeting interleukin 6 [189]), and addressing any acute damage in affected tissues, particularly cardiovascular damage [69-71]. Damage reduction may also mean reducing the level of blood clotting if clotting is present and repairing any organ damage, if relevant. The stage of healing requires normalizing the immune response, reducing lingering inflammation (such as by targeting interleukin 6 [189]), and addressing any acute damage in whatever affected tissues, which, for our purposes, includes blood. Micro-clots are a possible etiological factor in long COVID-19 [190][191][192], as well as COVID-19 vaccine injury [193]. Damage reduction may also mean reducing the level of blood clotting if clotting is present, and repairing any organ damage, if relevant. Sufferers of long COVID-19 have been found to have a higher inflammatory response to the initial COVID-19 infection than those who recover completely from COVID-19 [194], so antiinflammatory and immunomodulatory medications have been identified as potential long COVID-19 therapeutics.
Anti-coagulant medication, such as aspirin, can be useful in alleviating the cardiovascular complications of COVID-19 [195,196], as they have a long history of use in improving blood flow and reducing coagulopathies [197][198][199].
Another useful compound for breaking up blood clots is nattokinase, which is a fibrinolytic found in fermented soybeans (bacterial species Bacillus subtilis var. natto) [200,201]. Experiments have demonstrated that it potently degrades spike protein [202,203], which is an added benefit in addition to its fibrinolytic and anti-coagulant properties [204].

Potential Therapeutics
In Table 1, we grouped the therapeutics by mechanism and stage (as per our above definitions) and included information on their origins. Our categorization for sources is based on the classification of natural products (NP) or pharmaceutical drugs (PD). For natural products, we included the most common source organism(s) based on its scientific name for consistency. The pharmaceutical compounds with plausible applicability for the treatment of long COVID-19 and post-vaccine syndrome are listed in Table 1. An amount of 42% relative decrease in long-COVID incidence after treatment of initial C19 infection [224] Likewise, natural compounds and supplements with plausible applicability for the treatment of long COVID-19 and post-vaccine syndrome are listed in Table 2. Table 2. Natural compounds and supplements with plausible mechanisms of action against spike protein-related pathologies.

Discussion
The amelioration of symptoms and recovery of large numbers of people worldwide from both long COVID and post-vaccine syndrome and injury requires the use of noninvasive, integrative therapies that can be scaled and administered in a decentralized fashion. It is important to disseminate this knowledge to the lay public so that they can mitigate their individual risks and those of their loved ones. While it is difficult to enumerate the true scale of post-vaccination or post-COVID clotting disorders, there has been an appreciable rise in cardiac incidents [29], strokes (inter-cerebral hemorrhages [338]), and non-COVID excess mortality [339,340]. A significant increase in total mortality due to a vaccine is not unprecedented, as the DTP vaccine administered in Guineau-Bissau in the 1980s increased child mortality by four times compared to unvaccinated mortality [341].
While the magnitude of the impact of both long COVID-19 and post-COVID-19 Vaccine Syndrome or injury is unclear, it is important to prepare for the potential consequences by having information ready for dissemination, as well as to perform research on promising therapeutics to relieve the damage caused by spike protein and other potential mechanisms of harm, such as DNA integration [342]. One limitation of this study is that it focuses on spike-protein related pathology and can leave out other possibilities, such as allergies to vaccine components, or other disease etiologies. Long COVID-19 and post-COVID-19 vaccine syndrome are multifaceted disorders, with highly varied manifestations; as such, the development of objective diagnostics is important in treating patients. The therapies discussed in this review have a varying evidentiary basis and may serve as starting points for the development of therapies to relieve spike protein-related pathologies in the coming years.
Further research requires validating the treatments outlined in this review by randomized control trial (RCT), observational studies, and laboratory studies of biological mechanism. Furthermore, integration of the current research on spike-protein related disorders is helpful. One possibility is the application of systems biology tools to describe the perturbations to different biological pathways influenced by the spike protein. When such a model exists, it is possible to treat the acute manifestations of the disease while still clearing spike protein form the body.
Governments and national health services are beginning to come to terms with the sheer magnitude of the task in front of them. This review outlines some of the most promising therapies form an evidentiary and biological mechanistic perspective. We hope that this article be used in the construction of treatment protocols to treat these highly related conditions in their many disease manifestations, prioritizing not only safety and efficacy, but cost and availability to large numbers of people.