Vol 57, No 1 (2023)
Review Article
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Blood-brain barrier function in response to SARS-CoV-2 and its spike protein

Łukasz Suprewicz1, Krzysztof Fiedoruk1, Agata Czarnowska2, Marcin Sadowski3, Agnieszka Strzelecka4, Peter A. Galie5, Paul A. Janmey6, Alina Kułakowska2, Robert Bucki1
Pubmed: 36810757
Neurol Neurochir Pol 2023;57(1):14-25.

Abstract

The typical manifestation of coronavirus 2 (CoV-2) infection is a severe acute respiratory syndrome (SARS) accompanied by pneumonia (COVID-19). However, SARS-CoV-2 can also affect the brain, causing chronic neurological symptoms, variously known as long, post, post-acute, or persistent COVID-19 condition, and affecting up to 40% of patients. The symptoms (fatigue, dizziness, headache, sleep disorders, malaise, disturbances of memory and mood) usually are mild and resolve spontaneously. However, some patients develop acute and fatal complications, including stroke or encephalopathy. Damage to the brain vessels mediated by the coronavirus spike protein (S-protein) and overactive immune responses have been identified as leading causes of this condition. However, the molecular mechanism by which the virus affects the brain still needs to be fully delineated. In this review article, we focus on interactions between host molecules and S-protein as the mechanism allowing the transit of SARS-CoV-2 through the blood-brain barrier to reach the brain structures. In addition, we discuss the impact of S-protein mutations and the involvement of other cellular factors conditioning the pathophysiology of SARS-CoV-2 infection. Finally, we review current and future COVID-19 treatment options.

Review Article

Neurologia i Neurochirurgia Polska

Polish Journal of Neurology and Neurosurgery

2023, Volume 57, no. 1, pages: 14–25

DOI: 10.5603/PJNNS.a2023.0014

Copyright © 2023 Polish Neurological Society

ISSN: 0028-3843, e-ISSN: 1897-4260

Blood-brain barrier function in response to SARS-CoV-2 and its spike protein

Łukasz Suprewicz1Krzysztof Fiedoruk1Agata Czarnowska2Marcin Sadowski3Agnieszka Strzelecka4Peter A. Galie5Paul A. Janmey6Alina Kułakowska2Robert Bucki1
1Department of Medical Microbiology and Nanobiomedical Engineering, Medical University of Bialystok, Bialystok, Poland
2Department of Neurology, Medical University of Bialystok, Bialystok, Poland
3Institute of Medical Sciences, Collegium Medicum, Jan Kochanowski University of Kielce, Kielce, Poland
4Institute of Health Science, Collegium Medicum, Jan Kochanowski University of Kielce, Kielce, Poland
5Department of Biomedical Engineering, Rowan University, Glassboro, NJ, United States
6Department of Physiology and Institute for Medicine and Engineering, University of Pennsylvania, Philadelphia, PA, United States

Address for correspondence: Robert Bucki, Department of Medical Microbiology and Nanobiomedical Engineering, Medical University of Bialystok, Mickiewicza 2C St., 15–222 Bialystok, Poland; e-mail: buckirobert@gmail.com

Received: 30.01.2023 Accepted: 15.02.2023 Early publication date: 22.02.2023

ABSTRACT
The typical manifestation of coronavirus 2 (CoV-2) infection is a severe acute respiratory syndrome (SARS) accompanied by pneumonia (COVID-19). However, SARS-CoV-2 can also affect the brain, causing chronic neurological symptoms, variously known as long, post, post-acute, or persistent COVID-19 condition, and affecting up to 40% of patients. The symptoms (fatigue, dizziness, headache, sleep disorders, malaise, disturbances of memory and mood) usually are mild and resolve spontaneously. However, some patients develop acute and fatal complications, including stroke or encephalopathy. Damage to the brain vessels mediated by the coronavirus spike protein (S-protein) and overactive immune responses have been identified as leading causes of this condition. However, the molecular mechanism by which the virus affects the brain still needs to be fully delineated. In this review article, we focus on interactions between host molecules and S-protein as the mechanism allowing the transit of SARS-CoV-2 through the blood-brain barrier to reach the brain structures. In addition, we discuss the impact of S-protein mutations and the involvement of other cellular factors conditioning the pathophysiology of SARS-CoV-2 infection. Finally, we review current and future COVID-19 treatment options.
Key words: SARS-CoV-2, spike protein, blood-brain barrier, encephalopathy, stroke, cytokine storm, neuroinflammation
(Neurol Neurochir Pol 2023; 57 (1): 14–25)

Introduction

SARS-CoV-2 mainly causes inflammatory lung damage associated with thrombosis and increased pulmonary vascular permeability leading to haemorrhage and oedema [1]. In addition, the cytokine storm during the inflammatory cascade and the direct action of the SARS-CoV-2-derived spike protein affect other organs, including the central nervous system [2–4]. Compelling reports indicate that COVID-19 patients develop neurological symptoms during acute infection. They can also develop a chronic condition, long-COVID or PACS (Post-Acute COVID Syndrome), characterised by fatigue and neuropsychiatric symptoms [5–8]. More than 40% of COVID-19 patients exhibit neurological, potentially lethal, symptoms during SARS-CoV-2, increasing the need to understand the underlying molecular mechanisms and to develop effective countermeasures [9–11].

Neuroinflammation, which typically accompanies central nervous system (CNS) damage, can be mediated directly by the viral invasion of CNS cells or indirectly by mediators that govern the development of systemic inflammation [3, 12]. Briefly, direct action of the SARS-CoV-2 spike protein (S1-protein), which circulates in the blood after being cleaved by the proteases during the viral invasion, and pro-inflammatory cytokines (IL-1β, IL-6, and TNF-α) released by host immunocompetent cells in response to it, impede the blood--brain barrier (BBB) integrity, resulting in viral entry through the endothelium followed by infection of astrocytes [2, 3–15]. Symptoms can range from mild headache, nausea, impaired consciousness or reduced cognition to severe, potentially lethal conditions including encephalopathy, delirium, and acute disseminated encephalomyelitis [9–11]. Multicentre analyses have revealed that stroke (60%+) and encephalopathy (up to 42%) account for most neurological complications associated with SARS-CoV-2 infection. Encephalitis and Guillain-Barré syndrome are considerably less frequent, with incidence rates of 13% and 9%, respectively [11, 16]. Younger patients are more susceptible to ischaemic stroke associated with recurrent vascular occlusion and greater morbidity for COVID-19 patients than influenza patients. Among all neurological symptoms of SARS-CoV-2, patients with stroke and inflammatory syndromes had the worst prognosis [17, 18]. In the majority of cases, neuroimaging reveals ischaemia with large vessel occlusion, perfusion abnormalities, and haemorrhage in locations not typically associated with hypertension. In addition, post mortem analyses of COVID-19 patients have identified a link between microvascular disease and microhaemorrhages [22, 23].

In conclusion, all of these neurological complications may result directly or indirectly from an impairment of the blood-brain barrier (BBB) caused by SARS-CoV-2 infection.

Role of blood-brain barrier in maintaining central nervous system homeostasis

The blood-brain barrier is a specialised system of brain microvascular endothelial cells that guards the brain from circulating toxins, nourishes brain tissues, and filters harmful compounds from the brain to the bloodstream [19]. The proper functioning of the central nervous system is ensured by close interaction between the brain endothelium and other neurovascular/glial components such as astrocytes, pericytes, neurons, and basement membranes [20, 21]. Passage through the BBB is strictly regulated by physical (intercellular tight and adherens junctions) and metabolic (enzymes, transporters etc.) barriers [22, 23]. Notably, there is a functional difference between the brain endothelial cells’ abluminal and luminal membrane surfaces due to the differential expression of specific transporters, secretory bodies, and the release of enzymes locally [24]. Due to its restricted permeability, the BBB is a major obstacle for delivering therapeutic agents into the CNS [25]. Leakage or disruption of the BBB plays a crucial role in the pathogenesis of numerous brain diseases such as Alzheimer’s Disease, multiple sclerosis, ischaemia, Parkinson’s Disease, and PACS/neuro-COVID-19 [26–30].

Interaction of SARS-CoV-2 with host cells

The crown-like (corona) appearance of the virion is attributable to the spike (S) protein, which is assembled in homotrimer form and inserted in multiple copies into the host membrane. In SARS-CoV-2, the S-protein is cleaved by furin into the S1 and S2 subunits during their biosynthesis within the infected cells. In many other coronaviruses, the S-protein is only cleaved when they reach the target cells. Thus, the S-protein on the mature virion consists of non-covalently linked S1 and S2 subunits. The former subunit binds the cell receptor — angiotensin-converting enzyme 2 (ACE2), while the S2 subunit anchors the S-protein to the membrane. ACE2 engagement by the S1-protein triggers conformational changes in both subunits (S1 and S2) that lead to the formation of a fusion pore by bringing the viral and cellular membranes together [31]. As shown in Figure 1, for SARS-CoV-2, this process exposes the S2 site, followed by its subsequent cleavage by a transmembrane protease, serine 2 (TMPRSS2), resulting in bringing the virus closer to the cell surface and ultimately fusion of the S2 subunit to the membrane. Importantly, the S1 subunit is released from the ACE2 receptor to the bloodstream, where it has been detected in unvaccinated and infected patients, leading to inflammation triggered by direct and indirect action of the S-protein throughout the body [15].

Other cell attachment factors/co-receptors for SARS-CoV-2

ACE2 is the primary receptor responsible for the entry and expansion of the SARS-CoV-2 virus in human cells. Nevertheless, several reports have indicated that ACE2 alone cannot ensure SARS-CoV-2’s entry into cells, and additional co-receptors or attachment factors have been proposed for this process [32, 33]. Blood-brain barrier endothelial cells express several of these factors, linking SARS-CoV-2 with neurological symptoms. Due to the substantial genetic relatedness between SARS-CoV-2 and other coronaviruses, particularly SARS-CoV-1, specific targets by which the virus interacts with human cells have been described (Fig. 2) [34].

Extracellular vimentin

A link between extracellular vimentin and SARS-CoV-2 infection has been established by several studies [33, 35–38]. Vimentin (Vim) is a type III intermediate filament-forming protein and an essential cytoskeleton element. In addition, Vim exists in the extracellular environment and on the surface of various cells regardless of its expression due to its secretion by immune cells triggered by inflammation [44] and by endothelial cells in tumours [39]. Recent studies have shown that Vim directly interacts with S-protein, and that blocking Vim on the cell surface significantly reduces the entry of SARS-CoV-2 into host cells [33]. Specifically, antibodies targeting the vimentin C-terminal domain block entry of the pseudovirus containing S-protein by up to 80% in cells expressing extracellular vimentin and ACE2 [33] in vitro, and anti-vimentin antibodies show potential therapeutic value in vivo [40]. Similarly, an inhibitory effect on viral uptake has been recorded for other vimentin-blocking compounds [36]. Moreover, limiting the concentration of vimentin in the blood suppresses SARS-CoV-2 replication and, in turn, virus-triggered inflammation [33, 38].

Figure 1. ACE2- and TMPRSS2-mediated SARS-CoV-2 cell entry indicates a possible explanation for presence of S1 protein in free form in circulation (A). Scheme shows SARS-CoV-2 Spike S1 protein-driven disruption of blood-brain barrier. Direct action of S-protein and cytokine storm causes tight junctional complex rupture and enhances autocrine downstream proinflammatory signalling in endothelial cells (B). Illustration created in Biorender
Neuropilin-1

Neuropilin-1 (NRP1) is another putative attachment protein or co-receptor for SARS-CoV-2 since its selective inhibitors or anti-NRP1 antibodies reduce the virus entry to host cells [41]. NRP1 physiologically has the ability to bind all of the furin-cleaved proteins and acts as an inhibitor for neuronal demyelination and a suppressor of inflammatory signalling that preserves BBB integrity [41].

Heparan sulfate

Heparan sulfate (HS), a polysaccharide attached to various cell surfaces or extracellular matrix proteins, is another compound associated with SARS-CoV-2 uptake by host cells. HS participates in several biological processes, including cell adhesion, signalling, and angiogenesis [42]. Interestingly, the S-protein interacts with cellular HS through a domain adjacent to its receptor-binding domain (RBD) [43]. Furthermore, HS inhibitors, such as heparin derivatives and mitoxantrone, may directly block the interaction between HS and S1 protein, inhibiting the uptake of both pseudotyped and authentic SARS--CoV-2 [44]. Similar effects have been observed for sunitinib and BNTX, drugs that indirectly inhibit HS-mediated viral entry by rearrangement of the actin network [49].

Sialic acid

Sialic acids on the surface of BBB-forming endothelial cells play a vital role in the transport kinetics between the blood and the brain, hence appearing to be naturally associated with the invasion of CNS by SARS-CoV-2. Indeed, the direct binding of S-protein to sialic acid has been observed with electron microscopy and in silico studies [45]. However, due to conflicting conclusions, the role of sialic acid in the process of SARS-CoV-2 penetration is not clearly defined [46, 47]. For instance, ACE2 glycosylation inhibition studies with neuraminidase indicate that sialic acids on the ACE2 receptor prevent ACE2/spike protein interaction [46]. On the other hand, it has been reported that gangliosides could serve as ligands for the receptor-binding domain of the SARS-CoV-2 spike protein, thus improving viral uptake [47].

Scavenger receptor class B, type I

Scavenger Receptor Class B, Type I (SR-B1) on immune cells is involved in processes of the innate immune response. On the BBB, SR-B1 acts as a major high-density lipoprotein (HDL) receptor that modulates reverse sterol transport. Since the S1-subunit attaches to cholesterol and possibly HDL components, improving viral uptake in vitro, SR-B1 may be implicated in this process. For example, the SARS-CoV-2 infection of the cells overexpressing SR--B1 has been shown to be enhanced, while the viral uptake in SR-B1 knockout cells is reduced [48]. Similarly, in a study with cholesterol-poor synthetic biological HDL targeting SR-B1, 80% inhibition of SARS-CoV-2 pseudovirus uptake was observed [49].

Figure 2. Host molecules interacting with S-protein of SARS-CoV-2. Visualisation performed using ChimeraX v.1.4 software [98] with proportional dimensions, based on 3D model of SARS-CoV-2 virion [99] and 3D models of proteins predicted using AlphaFold v2.2.2 tool [100] as well as heparan sulfate (octamer) and sialic acid molecules
Phosphatidylserine receptors

Phosphatidylserine (PS) receptors (PSR) namely TIM (TIM-1 and TIM-4), and TAM (AXL) have been reported as factors that enhance SARS-CoV-2’s attachment to cells, promote uptake of virions, and boost ACE2-dependent infection of SARS-CoV-2. Wang et al. indicated that AXL interacts directly with the N-terminal domain of the SARS--CoV-2 S-protein [50]. But contradicting that, Bohan et al. stated that AXL does not bind to S-protein but rather to PS, which is abundant on the surface of the SARS-CoV-2 virion [51]. Furthermore, bemcentinib, an AXL inhibitor, significantly inhibited SARS-CoV-2 viral uptake into Vero 6 cells.

Transferrin receptor

Transferrin receptor (TfR) is highly expressed in brain capillary endothelial cells, but not the peripheral endothelium. TfR is responsible for the transport of iron into the brain parenchyma to maintain iron homeostasis, which is crucial for proper brain function, e.g. metabolism and neural conductivity. TfR is another protein involved in viral uptake by direct interaction with Spike S1 RBD. An anti-TfR antibody shows a potent inhibitory effect on SARS-CoV-2 infection in mice [52]. In vitro studies have indicated that low doses of ferristatin II reduce the uptake of different variants of concern (VOCs) of SARS-CoV-2 [53].

Blood-brain barrier and SARS-CoV-2

Given the unique properties of the BBB, viruses such as SARS-CoV-2 are unable to enter the CNS easily. However, numerous case reports of patients with neurological complications indicate that nearly 60% of all COVID-19 patients have had disrupted BBB, resulting in increased permeability [54]. Post-mortem histopathological examinations indicate the presence of SARS-CoV-2 in different types of glial and endothelial cells within the brain [55]. Due to the large number of co-receptors/attachment factors other than ACE2 that may play a role in viral invasion, the mechanisms utilised by SARS--CoV-2 to enter the brain and cause neurological disorders still need to be fully understood.

So far, two major routes for virus entry into the CNS have been identified: retrograde neuronal transport, and hematogenous transmission including immune cells as vectors in a mechanism known as the ‘Trojan horse’. The neuroinvasive route involves direct or indirect contact with the oral mucosa and eyes, and also implicates the olfactory, vagus, and trigeminal nerves as the primary cranial nerves involved in viral entry into the CNS [61]. Other mechanisms involve the indirect action of S-protein and the cascade of events associated with cytotoxicity and systemic inflammatory responses (the cytokine storm) that lead to the deterioration of the BBB. However, this review focuses on the hematogenous route, which involves the brain endothelium more profoundly.

Direct interaction of SARS-Cov-2 with BBB

Although initial in vitro studies have shown that SARS--CoV-2 is incapable of infection and replication in endothelial cells, numerous post mortem reports of patients who died from COVID-19 have demonstrated the presence of virus particles within the brain vasculature, pericytes, and neurons [55, 56]. These inconsistencies indicate that BBB infection is more complex and requires further examination. As mentioned previously, SARS-CoV-2 can infect endothelial cells by interacting with ACE2 on their surface [14]. However, the degree of infection is substantially less pronounced than in the primary target lung cells of SARS-CoV-2 — type II pneumocytes. On the other hand, overexpression of ACE2 in the endothelium of the cerebral circulation occurs in patients with hypertension and dementia and positively correlates with the severity of neurological symptoms [2, 57]. In addition, the S-protein may interact directly with the BBB, and S1, S1RBD, and S2 subunits exhibit pro-inflammatory effects, resulting in increased BBB permeability via damage to tight junctions (TJs) but not adherens junctions (AJs) [2, 58].

Trojan horse

One of the hallmarks of virus-induced neuropathogenesis is the breakdown of the BBB, which leads to uncontrolled infiltration of the virus-infected immune cells into the CNS via the ‘Trojan horse’ mechanism [59]. Using monocytes and macrophages as vectors, the viruses exploit the physiological process of CNS infiltration by immune response cells passing through the BBB. This process of permeation through endothelial cells, known as extravasation, consists of a series of consecutive steps, i.e. tethering, rolling, adhesion, and transmigration/diapedesis [60]. During this process, immune cells interact with adhesion molecules (ICAM, VCAM, E-Selectin) on the surface of the endothelium. Expression of these adhesion molecules is increased during SARS-CoV-2 infection, further contributing to the severity of neurological symptoms [58, 61]. Once the infected cells enter the CNS, the released virions can further infect glial cells, especially astrocytes and pericytes, whose impaired function further disrupts BBB homeostasis, leading to the loss of its function.

Adsorptive transcytosis

Direct passage of the virus across the BBB via interaction with receptors and co-receptors that facilitate infection, as well as the use of immune response cells as transporters, is not the only mechanism exploited by SARS-CoV-2 in order to penetrate endothelial barriers. Rhea et al. showed that iodine-labelled S-protein from SARS-CoV-2 can cross endothelial barriers through adsorptive transcytosis [3]. During this process, glycopeptides, i.e. spike protein with a positive charge circulating in the bloodstream, interact with endothelial cells’ negatively charged surface [62]. Subsequently, the interaction causes invagination of the cell membrane and vesicle formation. Vesicles containing S-protein can now enter the intercellular space and may interact with the parenchyma side of the BBB.

Cytokine storm

In severe cases, COVID-19 is accompanied by a systemic inflammatory response, referred to as a cytokine storm, contributing to the disease’s lethality. Patients with a poor prognosis frequently have elevated serum levels of cytokines and chemokines, mainly IL-1, IL-6, IL-8, IL-12, and TNF-α [63].

These pro-inflammatory mediators exhibit a positive feedback-like effect that stimulates neighbouring cells to produce even more cytokines, thereby promoting the infiltration of other immune response cells into the site of infection. In addition, S-protein can interact with Toll-like receptors 2 and 4 (TLR2, TLR4) and, in turn, activate signalling pathways involving PI3K, AKT, MAPK, and NF-κB [64, 65]. Subsequently, translocation of NF-κB into the cell nucleus results in increased production of pro-inflammatory cytokines, as well as adhesion factors such as ICAM, VCAM, and E-Selectin. As the aforementioned cytokines exert either direct or indirect effects that increase vascular permeability, the severity of inflammation correlates closely with BBB damage. For example, IL-1β indirectly increases the expression of matrix metalloproteinase 9 (MMP-9), which breaks down TJS-proteins (ZO-1, occludin, and claudin-5) [66]. Similarly, IL-6, IL-8, IL-12, and TNF-α lead to BBB breakdown [67]. Moreover, S-protein stimulates the production of VEGF, one of the most potent inducers of vascular permeability [68, 69]. The increased vascular permeability significantly contributes to virus entry into the CNS and glial cell infection, resulting in neurological disorders.

TJ disruption through cytoskeleton and glycocalyx involvement

Tight junctions, i.e. close cell-cell connections formed by complexes of proteins, i.e. cytoplasmic scaffolding: ZO-1, -2, -3, and transmembrane proteins: occludins, and claudins, play a critical role in maintaining BBB homeostasis by restricting paracellular permeability [19]. These junctional complexes require the involvement of actin cytoskeleton rearrangement in processes associated with the formation and maturation of cell-cell contacts. Recently, RhoA activation has been associated with SARS-CoV-2-mediated barrier breakdown, indicating that the signalling mechanism involves cytoskeletal components. RhoA is a key regulator of endothelial cytoskeleton and TJ complex dynamics; its activation has been shown to disrupt vascular integrity. SARS-CoV-2 S1 protein triggered activation of RhoA in a 3D microfluidic model of the BBB. The use of C3 transferase, a RhoA inhibitor, mitigated the negative effect of S1 on BBB permeability and TJ degradation [4].

Another study has implicated the interaction between hyaluronic acid (HA) and CD44 in the endothelial barrier breakdown during COVID-19 [70]. This work analysed circulating glycosaminoglycans levels and their degradative enzymes’ activities among SARS-CoV-2-infected patients [70], and found that the glycocalyx was severely damaged, which corresponded with disease outcome. Moreover, the circulating HA fragments and hyaluronidase level strongly correlated with organ failure assessment scores and hyperinflammation. In conclusion, the authors suggested that HA fragments are released into blood circulation due to COVID-19-mediated dysregulation of HA biosynthesis and degradation, which leads to direct endothelial barrier disruption in a ROCK- and CD44--dependent manner, indicating a role for HA in the vascular pathology of COVID-19.

Coagulopathy

Since the beginning of the COVID-19 pandemic, numerous reports have indicated an increased incidence of thrombotic events in patients with SARS-CoV-2. The syndrome of life-threatening clotting complications has been named COVID-19--associated coagulopathy (CAC) [71]. Cohort studies have shown that the incidence of ischaemic stroke as a complication of thromboembolic events oscillated around 2% [72, 73]. In most cases, elevated levels of fibrinogen, D-dimers, C-reactive protein, and P-selectin are recorded in the plasma of patients with CAC [74].

Endothelial cells have an antithrombotic system that determines platelet aggregation (NO, prostacyclin) and coagulation (TPFI, EPCR, thrombomodulin). In addition, the heparan sulfate in the glycocalyx layer exhibits anticoagulant activity [75]. During SARS-CoV-2 infection, there is a state of hyperactivation of the immune response, which results in activation of the complement system, increased release of neutrophil extracellular traps, and overproduction of cytokines, chemokines, and reactive oxygen species [76].

This cascade of events results in an imbalance between fibrinolysis and coagulation. Furthermore, due to damage to the glycocalyx and a reduction in the production of thrombomodulin, nitric oxide, and prostacyclins, the anti-thrombogenic fibrinolytic activity of the endothelium decreases. Additionally, an increase in free fibrin, tissue factors, and P-selectin is noted, which is associated with an increase in the prothrombogenic activity of the endothelium. Thrombin, fibrinogen, and plasminogen levels increase in CAC and directly or indirectly damage the endothelial barrier. Thrombin increases BBB permeability by indirectly damaging TJ-forming proteins through phosphorylation of MMPs and TJs and upregulation of VEGF by Src kinase [77]. Fibrinogen at pathologically high levels destroys the junctions between actin filaments and TJ, which widens intercellular junctions, impairing endothelial integrity [78]. Ventricular administration of tissue plasminogen activator results in dose-dependent increased BBB permeability, suggesting plasminogen’s direct involvement in endothelial injury [79].

ACE2, which is the master receptor for SARS-CoV-2, has a dual function, i.e. it regulates the renin-angiotensin-aldosterone system as well as the kallikrein-kinin system. Kinins are peptides with the ability to increase vascular permeability. An occupied ACE2 receptor during SARS-CoV-2 infection can cause dysregulation of kinin degradation, resulting in what has been called a ‘kinin storm’, leading to increased vascular permeability, inflammation, and ultimately organ damage, including to the brain [80].

Lung hypoxia increases BBB permeability

COVID-19 is a predominantly pulmonary disease that causes infection of the alveoli, impairing their function, and patients with SARS-CoV-2 are chronically hypoxic due to pathological changes in the lungs. Many studies have described so-called ‘happy hypoxia’ in SARS-CoV-2 infected patients, i.e. hypoxemia without signs of shortness of breath, which leads to chronic mild hypoxia (CMH) [81].

Hypoxia, encephalitis, and stroke are the leading long-term causes of neurocognitive symptoms in COVID-19 patients [88]. Multiple mechanisms of chronic hypoxia can result in BBB impairment. Hypoxia can induce cell death in endothelial and glial cells via the apoptosis pathway by activating hypoxia--inducible factor 1 (HIF-1) [89]. In addition, CMH has been shown to cause a temporary vascular leak in brain blood vessels, along with microglial activation and infiltration around damaged vessels. CMH-induced increased endothelial permeability exerts a regional preference, with considerably higher prevalence within the brainstem and olfactory bulb compared to the cerebellum and cerebral cortex [82]. These regions have also been reported to have the most significant levels of angiogenic development, with a peak in VEGF production that may additionally influence hypoxic-induced BBB disruption. Furthermore, in vitro coculture models of endothelial cells and astrocytes have shown that hypoxia causes a decrease in ZO-1 expression, which correlates with the rearrangement of activated filaments resulting in increased permeability to molecular tracers [83].

Mutations and their impact on severity of neurological complications

The high transmissibility and genetic diversity of SARS--CoV-2 have led to the emergence of mutated virus strains, some of which have been termed ‘variants of concern’ (VOCs) due to their increased infectiousness. VOC strains have critical point mutations within spike proteins that affect the virus’s ability to survive in harsh conditions and evade the host’s immune response. The most dangerous VOCs hold several common mutations.

For example, the D614G and P681R/H mutations in the S-protein increase the transmission and infectivity of the virus due to the increased affinity of the S1 subunit to the ACE receptor [84]. Remarkably, certain VOCs with a higher virulence can eliminate from circulation less pathogenic variants. Such displaced variants are then referred to as de-escalated variants if they meet at least one of the following criteria: (i) the variant is no longer circulating; (ii) although the strain has circulated for a long time in the population, it has no impact on the epidemiological situation; or (iii) the variant does not have specific, more virulent, properties based on scientific evidence.

SARS-CoV-2 is still evolving, and the emergence of more pathogenic mutants in the future is possible. For this reason, freshly discovered variants are closely monitored and listed as Variants of Interest (VOI) or Variants under Monitoring (VUM). A summary of the most common mutations, along with their impact on transmissibility and induction of the immune response, is set out in Table 1.

Recently, Taquet et al., in a massive two-year cohort study including almost 1.3 million patients, analysed risk trajectories of neurological and psychiatric outcomes after SARS--CoV-2 infection [85]. They compared the risk for adverse effects within CNS in cohorts (a minimum of 39,845 patients per group) of patients diagnosed with different VOCs of SARS-CoV-2, e.g. alpha (B.1.1.7), delta (B.1.617.2), and omicron (B.1.1.529) comparing them to control cohorts, before the emergence of those VOCs. Only minor changes were observed for the alpha variant in the six-month hazard risk of neurological and psychiatric events. In contrast, patients infected with the delta variant were at a significantly higher risk of anxiety, insomnia, cognitive disorders, seizures, and ischaemic stroke incidents, but at a lower risk of dementia. Mortality among patients diagnosed with the delta strain was also higher. The omicron variant caused an increased risk of dementia, as well as mood, nerve, and plexus disorders. However, the death rate was significantly lower than among patients before the emergence of omicron.

Unfortunately, despite extensive research into the pathogenicity and molecular mechanisms of SARS-CoV-2 infection, little is known about the direct impact of mutations in the S-protein on the integrity of the BBB. Most in vitro BBB studies are concerned with the effects of spike protein subunits derived from the Wild Wuhan strain of SARS-CoV-2.

SARS-CoV-2 treatment

Since the beginning of the COVID-19 pandemic, concerted efforts by researchers worldwide have led to the development of therapies, some using previously developed broad-spectrum drugs, and some that have been newly developed and are specifically placed to treat SARS-CoV-2. Implementing vaccination against COVID-19, using, among others, innovative mRNA vaccine technology, has been a great success, which significantly reduced severe adverse events (SAE), the frequency of hospitalisations, and consequently, mortality [86, 87].

Table 1. Summary of key, emerging mutated variants of SARS-CoV-2 and its impact on immune response and severity of infection compared to Wild Wuhan strain of SARS-CoV-2. Its current status, according to World Health Organisation, is denoted as either VOC (Variant of Concern), VUM (Variant under Monitoring), VOM (Variant of Interest), or DEV (De-escalated Variants). Table created according to current (as of January 2023) reports of WHO (https://www.who.int/emergencies/diseases/novel-coronavirus-2019/situation-reports)

WHO label
and lineage

WHO
status

Spike mutations
of interest

First detection date
and place

Transmissibility

Impact on
immunity

Severity

Alpha B.1.1.7

DEV

N501Y, D614G, P681H

September 2020, United Kingdom

Comparable

Beta B.1.351

DEV

K417N, E484K, N501Y, D614G, A701V

September 2020, South Africa

Gamma P.1

DEV

K417T, E484K, N501Y, D614G, H655Y

December 2020, Brazil

Delta B.1.617.2

DEV

L452R, T478K, D614G, P681R

December 2020, India

Omicron BA.1

DEV

A67V, Δ69-70, T95I, G142D, Δ143-145, N211I, Δ212, ins215EPE, G339D, S371L, S373P, S375F, K417N, N440K, G446S, S477N, T478K, E484A, Q493R, G496S, Q498R, N501Y, Y505H, T547K, D614G, H655Y, N6 79K, P681H, N764K, D796Y, N856K, Q954H, N969K, L981F

November 2021, South Africa and Botswana

Omicron BA.2

VOC

142D, N211I, Δ212, V213G, G339D, S371F, S373P, S375F, T376A, D405N, R408S, K417N, N440K, S477N, T478K, E484A, Q493R, Q498R, N501Y, Y505H, D614G, H655Y, N679K, P681H, N764K, D796Y, Q954H, N969K

November 2021, South Africa

Omicron BA.3

DEV

A67V, Δ69-70, Δ143-145, N211I, Δ212, G339D, S371F, S373P, S375F, D405N, K417N, N440K, G446S, S477N, T478K, E484A, Q493R, Q498R, N501Y, Y505H, D614G, H655Y, N679K, P681H, D796Y, Q954H, N969K

November 2021, South Africa

No evidence

No evidence

No evidence

Omicron BA.4

VOC

As BA.2 + L452R, F486V, R493Q

January 2022, South Africa

No evidence

No evidence

Omicron BA.5

VOC

As BA.2 + L452R, F486V, R493Q

February 2022, South Africa

No evidence

Unclear

Omicron BA.2.75

VOI

W152R, F157L, I210V, G257S, D339H, G446S, N460K, Q493

May 2022, India

No evidence

No evidence

Omicron XBB

VOI

As in BA.2.75 + N460K, F490S

Unclear

No evidence

No evidence

Omicron BA.2.3.20

VUM

K444R, L452M, N460K

Unclear

No evidence

No evidence

No evidence

Omicron XBC

VUM

N440K, F486P

Unclear

No evidence

No evidence

No evidence

Vaccination — current status

As of January 2023, nearly 5.6 billion people, 69.1% of the world’s population, have received at least one dose of a COVID-19 vaccine. 13.19 billion doses have been administered globally, and 2.2 million are administered daily. Almost 26% of people in low-income countries have received at least one dose. In Poland, over 57.8 million doses have been administered so far, and 22.6 million people are considered fully vaccinated, with at least two doses administered [88]. There are several types of vaccines against SARS-CoV-2 [89]. mRNA-based vaccines use mRNAs developed in the laboratory that are encapsulated in liposome lipid envelopes that enable entry into human cells, where they stimulate ribosomes to produce viral S-protein or its fragment, which further triggers antibody production by plasmocytes conditioning a specific immune response. Other vaccines trigger an immune response using weakened, modified, or inactivated viruses. Viral vector vaccines contain a modified version of a vector virus that includes fragments of SARS-CoV-2 but cannot cause infection. In contrast, protein subunit vaccines contain the spike protein fragment but without vector virus. Additionally, protein subunit vaccines contain an adjuvant that modulates immune system responses in the future when the human body meets viral particles during infection again. Due to the high ability of SARS-CoV-2 to mutate, vaccine manufacturers regularly update their formulations to maintain a high level of effectiveness in preventing COVID-19, which requires repeated booster doses.

Antiviral drugs

Despite the high vaccination prevalence in all age groups, many people have yet to receive a full dose. In addition, among people with impaired immunity, such as the elderly or those undergoing hematological treatment, the response to vaccination has been insufficient. Therefore, it became necessary to look for antiviral therapies. Several drugs have been found useful and approved by global organisations that control the approval of medicines for COVID-19 treatment. Examples of such antiviral drugs used in treating SARS-CoV-2 are remdesivir, molnupiravir, nirmatrelvir, and ritonavir.

Remdesivir is approved for COVID-19 treatment in adults and pediatric patients who are at least 28 days of age and weigh at least 3 kilograms. Remdesivir is used both in clinical practice and at home by people diagnosed with mild to moderate SARS--CoV-2 at high risk for severe disease progression. Remdesivir is a nucleoside analogue that inhibits RNA-dependent RNA polymerase (RdRp) of different coronaviruses. Incorporation by RdRp results in highly limited RNA synthesis [90].

Molnupinavir, like remdesivir, is a nucleoside analogue with a broad antiviral spectrum. It also inhibits the replication of other coronaviruses, i.e. SARS-CoV-1, SARS-CoV-2, and MERS, as well as viruses from different families, RSB, HCV, and Ebola [91].

Nirmatrelvir and ritonavir are used in concurrent therapy because of their synergistic effect [92]. Nirmatrelvir is an oral protease inhibitor active against mPRO, a viral protease involved in the replication process.

Ritonavir is a cytochrome p450 (CYP) 3A4 inhibitor that enhances the pharmacokinetics of protease inhibitors. Coadministration is required to increase nirmatrelvir concentration to the therapeutic range. Molnupinavir, nirmatrelvir, and ritonavir, unlike remdesivir, have yet to receive authorisation from the US Food and Drug Administration (FDA). However, the FDA granted Emergency Use Authorisation (EUA) status for these formulations in late 2021.

Monoclonal antibodies

SARS-CoV-2 targeting monoclonal antibodies (mAbs) are artificial antibodies that target specific epitopes within S-protein that act as neutralising agents limiting viral entry. Bebtelovimab and tixagevimab co-packaged with cilgavimab are SARS-COV-2-targeting mAbs authorised for use through a EUA [93, 94]. However, the COVID-19 Treatment Guidelines Panel recommends against using anti-SARS-CoV-2 mAbs to treat SARS-CoV-2 because the currently dominant Omicron variant is unlikely to be susceptible to mAbs, given its high variability within the S-protein sequence.

Immunomodulatory agents

Unlike previously mentioned compounds, immune modulators are therapeutic agents that do not target virus-infected cells but instead activate, suppress or boost immune responses. During SARS-CoV-2, immune modulators are especially useful during the cytokine storm due to their ability to suppress hyperinflammation. Several immunomodulatory drugs have been authorised to treat COVID-19, including anakinra, baricitinib, and tocilizumab.

Anakinra is an IL-1 receptor agonist used to treat hospitalised adults with SARS-CoV-2 requiring supplemental oxygen [95]. Baricitinib is a Janus kinase (JAK) inhibitor, preventing downstream activation of signalling pathways leading to the secretion of various interleukins, interferons, and growth factors [96]. Baricitinib treats COVID-19 in pediatric patients requiring oxygen supplementation, mechanical ventilation, or extracorporeal membrane oxygen (ECMO). Tocilizumab inhibits IL-6 action by competitive blockage of its receptor IL6-R, which inhibits downstream signal transduction that leads to infiltration of immune cells, e.g. B and T cells [97]. Tocilizumab has found use among pediatric patients requiring mechanical ventilation and ECMO, with concomitant administration of corticosteroids.

Conclusions

SARS-CoV-2 remains a substantial threat to global health and the global economy. Neurological and psychiatric disorders involve disruption of blood-brain barrier integrity, and their longitudinal effects are unknown. Despite the introduction of effective vaccines, continued research toward a deeper understanding of the intricate and complex processes involved in the pathophysiology of SARS-CoV-2 infections is still needed due to its high genetic variability and evolutionary dynamics. In particular, understanding the effects of S-protein from different VOCs on the integrity of the BBB is essential if we are to recognise the neurological consequences of COVID-19. Equally important is the education of medical professionals and patients about the aetiopathology, prevention, treatment, and mechanism of action of SARS-CoV-2.

Conflicts of interest: None.

Funding: This work was supported by the National Science Centre, Poland, under Preludium 21 Grant UMO-2022/45/N/

/NZ6/01454 (to Ł.S.), Medical University of Bialystok SUB/1/
/DN/22/006/112
2 (to K.F.) and the Polish National Agency for Academic Exchange PPN/STA/2021/1/00003/U/00001 (scientific internship at Rowan University, NJ, USA to Ł.S.).

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Neurologia i Neurochirurgia Polska