SARS-CoV-2 and its impact on the cardiovascular and digestive systems – The interplay between new virus variants and human cells

Abstract

Since infection with the novel coronavirus SARS-CoV-2 first emerged in Wuhan, China, in December 2019, the world has been battling the pandemic COVID-19. Patients of all ages and genders are now becoming infected with the new coronavirus variant (Omicron) worldwide, and its subvariants continue to pose a threat to health and life. This article provides a literature review of cardiovascular and gastrointestinal complications resulting from SARS-CoV-2 infection. COVID-19 primarily caused respiratory symptoms, but complications can affect many vital organs. SARS-CoV-2 binds to a human cell receptor (angiotensin-converting enzyme 2 – ACE2) that is predominantly expressed primarily in the heart and gastrointestinal tract, which is why we focused on complications in these organs. Since the high transmissibility of Omicron and its ability to evade the immune system have raised worldwide concern, we have tried to summarise the current knowledge about its development from a structural point of view and to highlight the differences in its binding to human receptors and proteases compared to previous VOC.

Graphical Abstract

1. Introduction

The novel coronavirus SARS-CoV-2, which emerged in Wuhan in the end of 2019, has reached all continents and is still causing COVID-19 in populations of almost every country in the world. The mortality rate of COVID-19 is still higher than that of seasonal influenza, to which it is often compared [1], in addition, hospital indicators continue to rise and, at least in Europe, increased COVID-19 transmission and mortality continue to be reported among residents of long-term care facilities [2]. The new coronavirus disease continues to pose a serious threat to populations worldwide.The SARS-CoV-2 genome encodes non-structural proteins that form the replicase complex, accessory proteins and, like other coronaviruses, four structural proteins: S (spike), E (envelope), M (membrane) and N (nucleocapsid). The S protein accounts for the interaction of the virus with the cellular receptor angiotensin-converting enzyme 2 (ACE2) and the entry of the virus into the cell [3]. The spike protein consists of subunits S1 and S2, which are responsible for binding the virus to the ACE2 receptor (S1) and fusing the SARS-CoV-2 membrane to the host cell membrane (S2) [3]. Enzymes such as transmembrane serine protease 2 (TMPRSS2) [4] and cellular cathepsin L, which can compensate for the absence of TMPRSS2 in some cells, are required for the fusion of the S protein with ACE2 [5]. These enzymes proteolytically cleave the S1/S2 site [5,6]. In 2021, lineages B.1.351 (first identified in South Africa in V2020), B.1.1.7 (first identified in the UK in IX2020), P.1 (first identified in Brazil in XI2020) and B.1.617.2 (first identified in India in X2020) were designated variants of concern (VOC) by World Health Organization (WHO) [7]. On 26 XI 2021, the WHO added a fifth variant of concern, the Omicron variant, identified in Botswana and South Africa, previously known as B.1.1.529. During the course of the pandemic, people of all ages were found to be susceptible to SARS-CoV-2 infection, with the average age at infection being around 50 years. The clinical course is not gender-specific, but there is an association with the age of patients [8]. Men over 60 years of age, even if they have no concomitant disease, are more likely to develop acute respiratory disease requiring hospitalisation as a result of SARS-CoV-2 infection than younger people, most of whom develop mild or asymptomatic illness. Children among those infected with SARS-CoV-2 showed fewer symptoms than adults. Among young people who tested positive for SARS-CoV-2, 73 % of those tested experienced symptoms such as fever, cough and shortness of breath, compared to 93 % of those aged 18–64. The number of hospital admissions was 14 % in people younger than 17 years and 15–62 % in the rest [3]. Of the 72,314 reported cases in China, 81 % were categorised as mild, 14 % as severe, requiring hospitalisation, and 5 % as critical, i.e. with apnoea, septic shock and/or multiple organ dysfunction [8]. The virus has been shown to be transmitted by droplet infection, i.e. talking, coughing or sneezing, but the possibility of infection through mucosal contact with a contaminated surface has also been demonstrated [9]. The most prevalent symptoms of infection were fever, dry cough and dyspnoea, which occurred in 83 %, 82 % and 31 % of patients, respectively [5]. Rarely, the presence of sputum, headache, haemoptysis, sore throat, chest pain, chills, diarrhoea, nausea and other stomach problems have been reported. Abnormalities of the sense of smell and taste have also been reported by patients. Over time, it has been found that COVID-19 is not only responsible for respiratory illnesses, but also affects other systems, causing long-term complications there. These include the nervous, cardiovascular, muscular and gastrointestinal systems [8].

Since ACE2 is expressed most strongly in the small intestine, followed by the colon, duodenum, gallbladder, kidney, testis and heart [10], [11], and because of the complications mentioned above, we have decided to focus on cardiovascular complications and the effects of SARS-CoV-2 on the gastrointestinal tract in the first chapters. Point mutations, deletions, insertions and recombinations are constantly generating new strains of the SARS-CoV-2 virus that can lead to altered pathogenesis. Mutations in the genes encoding the spike protein play a particular role, as changes in its structure can affect the dynamics of virus transmission and its ability to escape the host immune response. The nature of the interaction with the ACE2 receptor, morbidity and mortality can also change, which is why continuous monitoring is so important [12]. In the last chapter, we try to summarise the current reports on the S protein of Omicron subvariants with ACE 2 interactions.

2. Immune response and cardiovascular complications

The way the body's immune system reacts to an infection with the SARS-CoV-2 virus seems to have an important influence on the severity of the course of the disease. The immune system plays an important role in both the antiviral defence and the pathogenesis of COVID-19. A sufficiently rapid activation of immune cells seems to reduce the likelihood of a severe course of the disease, but at the same time an excessive activation of these cells leads to an intensification of symptoms and is more dangerous for the patient than the viral activity itself in the infected organism [13].

The severity of the disease course of COVID-19 has been shown to correlate closely with the level of proinflammatory cytokines and acute phase proteins in patients – among others, elevated levels of Il-6, Il-1β and ferritin predispose to a severe course and even death from COVID-19. Patients with the observed "cytokine storm" usually show high fever and respiratory failure, and massive lung damage is observed in imaging studies [13]. The hyperinflammatory state may contribute to myocardial injury and increased death rate [14]. Indeed, in a retrospective analysis of 191 COVID-19 patients in China, increased amount of biomolecules that indicate inflammation, including tumour necrosis factor α, C-reactive protein (CRP), ferritin, D-dimer and interleukin 6, were found. In addition, markers of cardiac damage, including elevated troponin T levels, were also found [15].

ACE2 not only functions as a receptor for SARS-CoV-2, but also acts as an antagonist of the enzyme ACE1 in the renin-angiotensin-aldosterone system (RAAS), maintaining angiotensin concentrations in equilibrium II [16]. In this way, it establishes a key relationship between immunity, inflammation, increased tendency to clotting and cardiovascular problems, and contributes to the prevention of some illnesses such as hypertension and heart failure. After glycoprotein S binds to the ACE2 receptor, endocytosis and proteolytic cleavage of the enzyme occurs, impairing its function. As a result, complications occur in the metabolic pathway of angiotensin II, and patients' blood levels increase. In addition, fibrin and thrombin are deposited in the pulmonary microcirculation, leading to ARDS and coagulopathy, and the hypoxia that occurs in severe cases of COVID-19 promotes thrombosis [17]. Many severe cases of COVID-19 often have high plasma levels of inflammatory cytokines and a sustained proinflammatory response leading to extensive tissue damage (Fig. 1) [14]. Deficiency of ACE2 may lead to increased frailty and poor vascular condition and plaque instability, while its overexpression may reduce the extent of left ventricular damage in myocardial infarction.

Fig. 1.

The pathways by which SARS-CoV-2 can damage the heart. Indirect damage: An exaggerated defence reaction (inflammatory response) of the human body can be triggered by the virus. Direct damage: Cardiomyocytes, pericytes and endothelial cells express the ACE2 receptor, which allows the virus to enter these cells. This figure was created using the Servier Medical Art Commons Attribution 3.0 Unported Licence (http://smart.servier.com (accessed 15 January 2023)).

Blood laboratory tests of patients with COVID-19 present elevated levels of cardiac biomarkers. Despite the numerous contradictions in the definition of myocardial injury, researchers agree that an increase in these parameters correlates with the severity of the disease course in these individuals [18], [19].

Analysis of the laboratory tests of 273 patients hospitalised for COVID-19 in Wuhan in 2020 showed an increase in cardiac troponin I (ultra-TnI) in 10 % of patients, N-terminal fragment of B-type natriuretic peptide (NT-proBNP) in 12.5 % and myoglobin (MYO) in 10.6 % of patients. In addition, patients with severe and critical course were found to have higher NT-proBNP and MYO levels than patients with mild course, with no statistically significant difference between them. The increase in ultra-TnI was higher in patients with severe course than in those with mild course, but the correlation of the level of this parameter with critical state was not demonstrated. Another study conducted in 112 patients in Wuhan showed an increase in cardiac troponin levels in 66.1 % of patients, of whom 42.2 % had a 3-fold increase. Compared to the group of patients with a mild course, statistically significant increases in cardiac troponin and NT -proBNP were found in the patients with a severe course. There were also large differences in the lactate dehydrogenase and creatine kinase values within these patient groups. 12.5 % of patients studied died, and every patient had elevated levels of cardiac markers before death, with a peak cardiac troponin I level in the week before death [19], [20]. There are many other reports here suggesting that there are signs of myocardial damage in patients with COVID-19, the severity of which may depend on the severity of the disease course.

Heart failure (HF) is an important risk factor for the severe clinical course and higher mortality associated with COVID-19 and may develop as a complication of the disease [21]. SARS-CoV-2 can directly attack cardiomyocytes from human pluripotent stem cells, which is dependent on ACE2 capability [14]. Immune cells appear at the site of infection, and the entry of the virus into the cells leads to cell swelling, which promotes the development of HF [22].

20–30 % of COVID-19 patients develop myocarditis, which can lead to dilated cardiomyopathy. The development of HF is related to the age of the patient, the presence of comorbidities, and the severity of the myocardial lesions. The administration of medications that reduce pain and inflammation as well as diuretics seems appropriate given the need to reduce inflammatory processes and the associated myocardial oedema as a mechanism of HF formation [22]. It has been suggested that cardiomyocytes are more susceptible to invasion by Omicron leading to myocardial damage because it is not dependent on TMPRSS2 like earlier variants [23]. However, the clear influence of Omicron on heart damage needs further research.

Case reports have also been published of patients who suffered acute myocardial infarction while undergoing COVID-19. A 40-year-old female patient with hypertension, hyperlipidaemia, diabetes mellitus and schizophrenia was admitted to hospital for COVID-19 pneumonia and to rule out myocardial infarction as she reported stenocardial pain and dyspnoea. On the second day of hospitalisation, the woman reported increasing chest pain. Electrocardiography (ECG) revealed tachycardia and coronary angiography showed an extensive thrombus in the left anterior descending artery [24]. A 60-year-old man with no cardiovascular risk factors also presented with ST-Elevation Myocardial Infarction (STEMI) on day 7 of hospitalisation due to pneumonia with SARS-CoV-2 aetiology. Coronarography showed a thrombus blocking flow through the right coronary artery [25].

A study conducted between 30 February and 30 March 2020 in Lombardy, Italy, found 28 cases of ST-Elevation Myocardial Infarction (STEMI) in patients infected with SARS-CoV-2. STEMI was defined by the presence of ST -segment elevation (89.3 % of patients) or the diagnosis of new left bundle branch block (10.7 % of patients) and co-existing characteristic symptoms. In 85.7 % of them, STEMI was the first manifestation of COVID-19. Angiography of these patients revealed obstruction due to thrombus in a coronary vessel [26].

Studies show that due to the systemic inflammation and hypercoagulable state in the course of viral diseases, including COVID-19, there is an increased risk of plaque damage or thrombus formation that occludes the coronary vessel, which may increase the risk of developing acute myocardial infarction [27], [28].

According to some reports, cardiac arrhythmias are one of the most widespread cardiovascular complications in COVID-19 infected patients, as they are reported with a frequency of up to 16.7 %. They have also been observed to be more common in ICU patients [29]. They most commonly occur in the form of symptomatic or asymptomatic tachycardia, and less frequently in the form of bradycardia. In addition, they may be associated with myocarditis or myocardial ischaemia and may occur in critically ill patients with hypoxia or shock. It has been suggested that factors such as hypokalaemia, chloroquine, hydroxychloroquine or azithromycin therapy can cause QT interval prolongation and the development of polymorphic ventricular tachycardia (VT), leading to the development of arrhythmias in COVID-19 [30]. Another common form of cardiac dysfunction in COVID-19 was atrial fibrillation (AF). Systemic infections, direct viral damage to cardiomyocytes, hypoxaemia, old age, race, comorbidities, but also overactivity of the sympathetic nervous system are considered to be mechanisms that may have contributed to their development [31]. Interestingly, pre-existing AF has been shown to be common in COVID-19 patients admitted to Italian hospitals in 2020, but had no effect on the risk of death [32]. A recent study showed that new-onset AF was common (5.4 %) in patients with COVID-19 who were hospitalised from January 2020 to March 2021. Almost half of the patients with new-onset AF died during their hospital stay [33].

In severe cases, COVID-19 may lead to coagulopathy, including disseminated intravascular coagulation (DIC). Patients infected with SARS-CoV-2 have a coagulation profile suggestive of DIC with platelet insufficiency, prolonged prothrombin time (PT) and elevated D-dimers. In contrast to DIC associated with sepsis, thrombocytopenia is not as pronounced in SARS-CoV-2 infection, while D-dimer levels reach higher values [17].

In a study of 183 patients (85 women and 98 men) with confirmed COVID-19 at Tongji Hospital (Wuhan, China), 21 patients (11.5 %) died. Among them, 15 people (71.4 %) developed DIC. In comparison, only one person (0.6 %) who survived met the criteria for DIC [34].

Coagulopathy, which occurs in COVID-19, is closely related to increased mortality and is not a good prognosis. It can lead to embolism of a thrombus in an artery, deep vein thrombosis (DVT), pulmonary embolism (PE), limb ischaemia, stroke and myocardial infarction [35]. The increased risk of cardiovascular disease, which includes DVT and PE, is connected with patient immobilisation, excessive inflammation from infection and the development of DIC in COVID-19 [36].

A meta-analysis published in September 2020 showed that VTE affected ∼ 30 % of COVID-19 patients, deep vein thrombosis ∼ 20 % of patients and pulmonary embolism ∼ 18 % [37]. From 1 March to 30 April 2020, 2943 patients with confirmed COVID-19 were treated at the University Hospital 12 de Octubre (Spain), of whom 261 were in the intensive care unit. 106 patients (67.92% were male) were diagnosed with symptomatic arterial or venous thrombosis, and 11 patients had multivessel thrombosis at different sites and of different types. Twenty patients (18.86 %) developed PE, and the most common vascular event was symptomatic PE (in 58 patients - 54.71 %) [38]. COVID-19-associated coagulopathy (CAC) brings the threat to multiple tissue and organ sites, causing apart from myocardial infarction, also skin swelling and rashes (also known as 'COVID toes') or dysfunctions of the neurological system [39].

Recently, it has been shown that the coagulation parameters in COVID-19 caused by Omicron variant are significantly higher than those of control subjects, but lower than those observed in previous variants of concern. Humans infected with Omicron variants were found to have less pronounced formation of microclots in platelet-poor plasma compared to carriers of the earlier variants of concern. [40].

3. SARS-CoV-2 and gastrointestinal tract

It is known that SARS-CoV-2 mainly gets in host lung cells and that respiratory symptoms are common in patients with COVID-19. Nevertheless, SARS-CoV-2 can also infect and replicate in the intestinal tract. Indeed, the data from a few public databased were taken and the transcriptome records were analysed revealing that the highest expression of ACE2 mRNA was in the colon [41]. An interesting study suggests that susceptibility to gastrointestinal SARS-CoV-2 infection may be correlated with age-related changes in ACE2 expression in the gut [42]. SARS-CoV-2 RNA has been detected in the faeces of about half of infected individuals [43]. The presence of viral RNA was found more frequently in anal swabs from patients infected with the Omicron variant than in patients infected with other variants of SARS-CoV-2 [44]. On the other hand, gastrointestinal symptoms are relatively rare in patients with variant Omicron [44].

Gastrointestinal symptoms in patients infected with SARS-CoV-2 are commonly reported in the literature and include diarrhoea, nausea, vomiting, anorexia, loss of appetite, abdominal pain, abdominal distension and ageusia [45], [46]. The frequency of these symptoms ranged from 2 % to ∼ 79 % [47]. Diarrhoea was the most commonly reported symptom [48]. Most gastrointestinal symptoms observed in patients with COVID-19 are mild and may occur with or before respiratory symptoms [49]. Nevertheless, patients with COVID-19 with gastrointestinal symptoms tend to have a worse disease course with a higher risk of acute respiratory distress syndrome and liver damage [50], [51].

Liver involvement is shown by an elevation of serum levels of alanine aminotransferase(ALT), aspartate aminotransferase (AST), glutamyltransferase (GGT), and total bilirubin. [48], [52], [53]. Kaafarani et al. found a 7.5- and 12-fold increase in aspartate aminotransferase and alanine aminotransferase levels, respectively, compared to physiological levels [53]. Wang et al. demonstrated that SARS-CoV-2 infection directly contributed to liver impairment in patients with COVID-19. The coronavirus particles were discovered in the cytoplasm of hepatocytes by ultrastructural examination. In addition, the hepatocytes infected with SARS-CoV-2 showed a striking swelling of the mitochondria, an expansion of the endoplasmic reticulum and a lower level of glycogen in the cytosol. [54]. Patients with gastrointestinal symptoms were found to have significantly higher levels of C-reactive protein, lactate dehydrogenase and α-hydroxybutyrate dehydrogenase [47]. In addition, gastrointestinal imaging results showed thickening of the bowel wall, sometimes with hyperaemia and mesenteric thickening, a fluid-filled colon and rarely intestinal pneumatosis and ischaemia [52]. Rarely, patients develop acute pancreatitis, acute appendicitis, intestinal obstruction, intestinal ischaemia, peritoneal haematoma or abdominal compartment syndrome [49]. Similarly, histopathology of excised gastrointestinal specimens in patients with COVID-19 revealed superficial ischaemic colitis of the mucosa, transmural ischaemic colitis and associated lesions suggestive of a rare disease - pneumatosis cystoides intestinalis [55].

Miyakawa et al. used human small intestinal organoids to investigate the replication efficiency of the Wuhan virus strain and the Delta and Omicron SARS-CoV-2 strains in a mini-intestinal model. It was found that the replication rate of the Delta strain was about 4–6 times higher than that of the wild-type Wuhan strain, while the replication capacity of the Omicron strain in the intestinal model was low [56].

Rahban et al. reported that ACE2 expression is about 100-fold higher in the gastrointestinal tract, especially the colon, than in alveolar cells of the lung and respiratory system [57].

This makes it possible for SARS-CoV-2 to directly attack the cells of the gastrointestinal tract [58]. The possible mechanisms of damage to the gastrointestinal tract during COVID-19 infection can be divided into direct and indirect. SARS-CoV-2 can directly cause a cytopathic effect through cell entry via ACE2 and indirectly lead to a systemic inflammatory response to the virus, which disrupts the balance of the gut microenvironment and results in excessive inflammation of the gut and lungs, which can lead to a cytokine storm [47].

Recently, it has been reported that the long prevalence of the SARS-CoV-2 virus in the gastrointestinal tract may cause the formation of a viral reservoir and the multiple release of viral proteins across the intestinal epithelium after some time. This in turn can activate the immune system through a SARS-CoV-2 protein motives which are very similar to those of a bacterial superantigen.Consequently, superantigen-mediated activation of immune cells may be the cause of the multisystemic inflammatory syndrome seen in children. In addition, it has been proposed that children previously infected with SARS-CoV-2 who have a viral reservoir that is subsequently infected with an intestinal trophic adenovirus exhibit immunopathology that explains cases of acute and severe hepatitis [59].

The digestive tract is inhabited by a large number of bacteria. The intestinal microbiota lives in close association with humans and is very important to the host. The microbiota determines the absorption of nutrients, influences the defence against pathogens, activates peripheral immune cells and ultimately keeps the gut in good condition [60]. Viral infection increases the permeability of the wall of the gastrointestinal tract to foreign pathogens and consequently leads to impaired absorption by enetrocytes [61]. In addition, increased levels of faecal calprotectin have been found in individuals infected with COVID-19. There is evidence that elevated levels of this protein are a biomarker for intestinal inflammation caused by SARS-CoV-2 [45].

Recently, many studies have shown a connection between COVID-19 and dysbiosis of the gut microbiota, as summarised by Farsi at al. [62]. The most important change in bacterial configuration in humans infected with SARS-CoV-2 was a decrease in beneficial bacteria such as Bifidobacterium, Alistipes, Eubacterium, Faecalibacterium, Ruminococcus, Roseburia, Fusicathenibacter and Blautia, and the augmentation of Bacteroides, Clostridium, Streptococcus, Rotia, Eggerthella, Actinomyces, and Collinsella [62].

As previous literature shows, the distribution of the ACE2 receptor in the body is not fully related to the symptoms and severity of the disease course of Covid. Furthermore, although Omicron spreads more quickly, it seems to cause milder symptoms [63].

Currently, only some subvariants of Omicron are marked as VOC [64]. Therefore, we decided to take a closer look at the latest data on the binding of Omicron subvariants to the ACE2 receptor and the methods of their entry into the human body.

4. Structural and functional impact of Omicron spike mutations

The invasion of the human host by SARS-CoV-2 virions begins with the binding of the spike (S) protein to the angiotensin-converting enzyme 2 (ACE2) receptor (Fig. 2 A and 2 B) and subsequent cleavage into the S1 and S2 protein parts by host cell proteases. Adaptive mutations in the genome of SARS-CoV-2 have raised concern because they can alter the pathogenicity and transmissibility of the virus. The previous four VOC ( Alpha, Beta, Gamma and Delta) had a few mutations within the RBD (Fig. 2 C) and throughout the whole S protein. The most prevalent variant at the end of 2021 year (Delta) has shown higher transmissibility and immune evasion than the previous VOC. The increased transmissibility of the delta variant was most likely related to crucial mutations such as D614G, L452R, P681R and T478K in the S protein [65].

Fig. 2.

The structures of A) WT, B) the Omicron variant of SARS-CoV-2 spike receptor binding domain (PDB number 6M0J and 7WBL, respectively) bound to ACE2 receptor (ACE2 in green),visualised by USCF Chimera [67]; C) the mutations in the RBD domain of five known VOC up to date. The mutations in red are present in the receptor binding motif (RBM).

The new SARS-CoV-2 variant (Omicron, which emerged in Botswana and South Africa in November 2021) has a total of about 50 mutations, 30–35 of which are in the S protein [66], [68]. So far, the Omicron variant is the predominant variant in circulation worldwide. Of the 107,678 sequences reviewed between 24 September and 24 October 2022, 99.7 % belonged to the Omicron variant of concern (VOC) [64]. In recent months, several Omicron subvariants have circulated globally, including BA.1, BA.2, BA. 2.75, BA. 2.12.1, BA.3, BA.4, and BA.5 [68], [69], [70]. BA.5 lineage remains predominant in October 2022 with a prevalence of 77.1 %, followed by BA.4 ancestral lineage with a prevalence of 5.4 %. BA.2 ancestral lineage has increased in prevalence, accounting for 4.3 % of the sequences in the same reporting period [64]. Trends in phylogeny suggest that Omicron subvariants will continue to predominate in the future [68].

Structural studies and molecular dynamics simulations revealed an increased number of hydrophobic interactions, salt bridges and hydrogen bonds between hACE2 and RBD of Omicron variant than RBD of WT [70], [71], [72]. This is the result of 15 mutations in the receptor binding domain (RBD) compared to the WT strain, 10 of which are specific for the receptor binding motif (RBM) [73], [74] (Fig. 2 C). Many reports showed that N501Y mutation enhances RBD interaction with hACE2 [75], [76]. Mutations that significantly impact the affinity between the S1 protein of the virus and its human host receptor include the residues at positions 417 (e.g. K417), 484 (e.g. K484), 493 (e.g. R493) and 498 (e.g. R498) [77]. Some of them decrease the strength of ACE2 binding, e.g. K417N which abolishes a salt bridge to ACE2 residue D30 [78]. On the other hand, some new bonds between RBD of Omicron and ACE2 have been shown to increase the affinity of these two proteins [73]. These include the salt bridge R498-D38 (residues of the RBD and ACE2 proteins, respectively) and the hydrogen bond between the RBD residue S496 and the ACE2 residue K353 [79].

Interestingly, studies with Cryo-EM have shown that Omicron spike molecules exist primarily in the open conformation, with a vertical RBD poised for receptor binding. It was concluded that new mutations in the Omicron strain enhance cross-domain packing and open conformation of the S protein [74]. Moreover, Cui at al. detected Omicron S trimer at both 5.5 and 7.5 pH in a one conformational state with one "up" RBD and two "down" RBDs. In contrast, WT, Alpha, and Delta S trimers have been shown to have two different conformational states. One is a closed form, with all three RBDs “down”, and the second is open, with one RBD pointing upwards [77], [80]. Most reports indicate that the Omicron variant RBD had higher affinity than the WT or other four variants of concern [71], [72], [77], [81], [82], [83]. Nonetheless, there are also studies showing that it binds the ACE2 receptor with comparable affinity to some other VOC [84] or even lower affinity than Delta [85] or all four VOC [79]. In the study by Han et al., using the SPR methodology [79], ACE2 was coated to the chip in random orientations, what can be the reason for differences in affinity.It is also noteworthy that successful receptor binding by SARS-CoV-2 does not necessarily result in successful replication in the host. Omicron's fusogenic abilities are apparently reduced, as many studies have shown [86], [87], [88], [89]. The situation with its S protein seems to follow the hypothesis that a severely mutated virus must compromise its fitness in order to acquire the ability to bypass host immunity.

Indeed, Omicron S has been shown to require a much higher concentration of ACE2 than all other variants for efficient invasion of the host membrane and cells. The same study also implicates that mutations N679K and P681H in the Omicron S protein, which occurred near the furin cleavage site, do not enhance cleavage of this protein and virus entry into human cells [90].

It is worth mentioning that the multibasic site (RRAR) placed at the S1–S2 junction differentiate SARS-CoV-2 from SARS-CoV [91], enabling cleavage by furin protease. S protein processing can occur independently of furin, but the presence of this protease greatly enhances cleavage [92]. Arora at al. have shown that polymorphisms at the S1/S2 site can modulate S protein processing and entry into the host cell [93]. Recently identified cathepsin L (CTSL) cleavage sites in the S protein are highly conserved in SARS-CoV-2 variants, suggesting its indispensability during infection of all SARS-CoV-2 variants that have occurred until now (including the recent Omicron variant) [94] In addition, CTSL-enhanced entry of SARS-CoV-2 virus has been shown to be independent of furin cleavage [94]. Moreover, SARS-CoV-2 S has been shown to utilise the matrix metalloproteinases (MMPs) MMP2/9 to initiate cell invasion in the absence of membrane-bound serine proteases [95]. Therefore, drugs aiming at metalloproteinases, cathepsins and proteases involved in S protein cleavage and processing may be useful in reducing SARS-CoV-2 infection and disease severity COVID-19.

It is noteworthy that many of the mutations that occurred in the Omicron S protein allowed the virus to escape the neutralising antibodies known at the time of the appearance of this variant [84], [96], [97], [98]. It has been shown that even the serum of individuals who had been vaccinated and had received a booster dose of mRNA-based vaccines had significantly reduced neutralising activity against B.1.1.529 [98]. Studies by Cao et all revealed that 85 % of the tested 247 human anti-RBD neutralising antibodies were escaped by Omicron [96].

The greatest ability to escape neutralisation has so far been presented by the Omicron subvariants BA.2 and BA.4/5 [69], [99], [100]. Studies by Kimura at al. on a hamster model suggest that BA. 4/5 is more pathogenic than BA.2 and carries a more fusogenic S protein [100]. Such mutations as follows: K417N, S477N, T478K, E484A, F486V (in Omicron BA. 4/5), Q493R and N501Y have been found to cause resistance to some neutralising antibodies in many different studies, either together or individually [77], [94], [95], [97], [98].

It is worth noting that some clinical reports and experimental results show that the non-spike protein mutations can also affect viral fitness and the course of COVID-19 illness [101], [102].

5. Summary and outlook

The SARS-CoV-2 continues to mutate. Earlier variants not only caused acute respiratory distress syndrome, but were also responsible for other health problems, including problems with the cardiovascular and digestive systems. This is mainly related to the expression of the ACE2 receptor in these organs. However, the relationship between the virus and ACE2 is not so simple. SARS-CoV-2 also requires some other proteins for successful entry and replication. Recently, new cleavage enzymes have been proposed to be responsible for this function. Many research groups report that the binding of ACE2 is enhanced by the Omicron RBD, but the pathogenicity of this new VOC is lower. There is a possibility that viruses with lower fusogenicity also have lower pathogenicity. However, compared to other VOC, the Omicron variant carries an increased risk of reinfection. So there is a possibility that millions of people will soon have a serious health problem after an Omicron infection, as reinfection dramatically increases this risk. Therefore, any new mutations, especially in the S protein, must be monitored and their impact on the interaction of the virus with the human body studied in order to prevent the next pandemic. This is the race in which we are all involved.

CRediT authorship contribution statement

Angelika Szpulak, Urszula Garlak, Hanna Ćwirko: Conceptualization, Writing – original draft. Bogusława Witkowska: Writing – original draft, Writing – review & editing, Visualization: Danuta Witkowska and Agnieszka Rombel-Bryzek: Writing – review & editing, Supervision.

Conflicts of Interest

None.