Abstract
Since the WHO declared coronavirus disease 2019 (COVID-19) as a pandemic, huge efforts were made to understand the disease, its pathogenesis, and treatment. COVID-19 is caused by severe acute respiratory syndrome (SARS) coronavirus-2 (SARS-CoV2), which is closely related to SARS-associated coronavirus (SARS-CoV). This article attempts to provide a timely and comprehensive review of the coronaviruses over the years, and the epidemics they caused in this century with a focus on the current pandemic COVID-19. It also covers the basics about the disease immunopathogenesis, diagnosis, prognosis, and treatment options. Although almost every single week new clinical findings are published, which change our understanding of COVID-19, this review explores and explains the disease and the treatment options available so far. In summary, many therapeutic options are being investigated to treat and/or ameliorate the symptoms of COVID-19, but none is registered and no sufficient data to support immune-based therapy beyond the context of clinical trials. For that, strengthening our immune system is the best defense at this time.
Introduction
On the last day of the 2019 year, December 31, China reported a cluster of pneumonia cases of unknown etiology to the WHO office in China. The cases were reported from the city of Wuhan in Hubei province, China. With time, the disease spread in China and other countries affecting thousands of people (117). In January, the Chinese authorities identified the virus as a novel coronavirus (2019-nCoV) and sequenced its genome. Later, the virus was officially named SARS-CoV2.
On January 30, 2020, the WHO considered the outbreak as “Public Health Emergency of International Concern” (117) and on February 11, the disease caused by the virus was named COVID-19, short for “coronavirus disease 2019” (119). Within 3 months, the disease spread to 117 countries and territories, affecting more than 125,000 patients and claimed the lives of more than 4,600 persons. This had urged the WHO on March 12 to declare the disease a pandemic (118). By the mid of August 2020, more than 21 million people were affected by the disease and the deaths surpassed 760,000 (World Health Organization. COVID-19 Dashboard.
The aim of this review is to provide a timely and comprehensive review of human coronaviruses over years, and the epidemics they caused in this century with a focus on the current pandemic, COVID-19. Also, to cover the basics about the disease immunopathogenesis, diagnosis, prognosis, and treatment options. Due to the variable clinical findings in the clinical data of infected patients, we hope, in this review, to help in exploring and explaining the disease and the treatment options available so far.
Coronaviruses Background, Origin, and Reservoirs
Coronaviruses are a group of viruses that belong to Coronaviridae family. The name corona comes from the resemblance to solar corona or the crown. This halo appearance is given by the S protein spikes projecting from the envelope when visualized by electron microscope (42). Members of this family are enveloped nonsegmented positive-sense single-strand RNA viruses, with large RNA genome. According to the International Committee on Taxonomy of Viruses (ICTV), this family has two subfamilies, Orthocoronavirinae and Letovirinae (57). The Orthocoronavirinae previously known as Coronavirinae has four genera, Alphacoronavirus, Betacoronavirus, Gammacoronavirus, and Deltacoronavirus. Orthocoronavirinae has the largest identified RNA genome containing about 32 kb (42,89).
Viruses in the genera Alphacoronaviruses and Betacoronaviruses infect mainly mammals, Gammacoronavirus infects avians, and viruses belonging to the Deltacoronavirus were found in both mammals and avian species (89). In the past it was thought that coronaviruses of animals cannot be transmitted to humans. But the outbreaks and pandemics in the last two decades by these viruses showed that they are potential zoonotic pathogens, where a spillover occurs from animals to humans. This made the animals as reservoir hosts. Numerous animal species can harbor these viruses such as bats, rodents, lagomorphs, and other vertebrates (8).
Human coronaviruses
Human coronaviruses (HCoV) were first identified in 1962 (59). HCoV-229E and HCoV-OC43 were the first to be identified. Later, in the early years of the 21st century, another two HCoV were identified, HCoV-NL63 and HCoV-HKU1 (42,57). 229E and NL63 are alphacoronaviruses, whereas OC43 and HKU1 are betacoronaviruses (42,57,76). These viruses were found to be globally distributed and circulating worldwide, that is, they are endemic to human population (42). They cause mild-to-moderate upper respiratory tract infections and considered the second most common cause of coryza or common cold, where 15–30% of respiratory tract infections are linked to them (42). In some cases, lower respiratory tract infections such as bronchitis or pneumonia may occur but these are linked to immunocompromised patients and elderly (60). The prevalence of these human species varies depending on the time of the year and the geographic region.
In the last two decades three new HCoV emerged: SARS-CoV (the beta coronavirus that causes severe acute respiratory syndrome), MERS-CoV (the beta coronavirus that causes Middle East Respiratory Syndrome), and the most recent SARS-CoV2 (the novel beta coronavirus that causes coronavirus disease 2019, or COVID-19). Unlike the previous viruses, these three species caused severe respiratory illnesses that affected the lower respiratory tract and resulted in fatalities. They spread worldwide causing epidemics and the current pandemic. Since the emergence of these epidemics, attention has been paid to coronavirus ability to transfer from wildlife animals to domestic animals or to humans (79).
SARS outbreak, symptoms, and epidemiology
In the mid November 2002, severe cases of respiratory illnesses appeared in Guangdong Province, China. In February 2003, an official report was received by WHO about atypical pneumonia affecting 305 persons and resulted in 5 deaths (122). WHO named the outbreak as SARS and issued emergency travel recommendations to alert health authorities and the public to a worldwide threat to health (122). Within a few months, the illness spread to more than 25 countries in North America, South America, Europe, and Asia, including Canada, which was considered a hotspot of the illness outside Asia (76). The outbreak affected 8,098 patients worldwide and claimed the lives of 774 with a mortality rate of 9% (19).
SARS is presented with high fever (more than 38°C), headache, body aches, and general feeling of discomfort. Some patients show mild respiratory symptoms. Within a week, patients develop dry cough then the severe disease progresses rapidly to respiratory distress, which requires intensive care. Most of these patients develop severe pneumonia and 10–20% develop diarrhea (19,121).
In March 2003, one laboratory each in Hong Kong, Germany, and the Centers for Disease Control and Prevention (CDC), USA, almost simultaneously were able to isolate the virus that caused the SARS (65). The virus genome was sequenced and revealed that it is a new HCoV (35,65). The virus was named SARS-associated coronavirus (SARS-CoV), which is a betacoronavirus.
Members of betacoronaviruses are clustered into four lineages, A, B, C, and D. SARS-CoV belongs to B lineage. It is thought to be originated from bat coronaviruses, which spilled over to humans (55,121). Bats were found to host several strains of coronaviruses and many of these are genetically related to SARS-CoV. Their spike proteins are similar to those of SARS-CoV and they use the same receptors to enter host cells (121). However, it is thought that there are intermediate hosts between bats and humans. Civet cats were suspected as an intermediate host and transmission host. When the outbreak started from a wet market in Guangdong Province, SARS-CoV was isolated from civet cats. Chinese health officials ordered massive culling of these cats and other mammals to contain the spread of the outbreak (87).
MERS outbreak, symptoms, and epidemiology
Ten years after the emergence of SARS, in June 2012, a man in Saudi Arabia died from severe pneumonia and renal failure (31). A novel coronavirus was isolated from his sputum. In the meantime, several other cases were retrospectively identified from patients in Jordan, which had occurred in April 2012. The virus was called MERS coronavirus (MERS-CoV) (31,123). Within one year, the virus affected eight countries, the majority in the Middle East and some European countries. During that year, 64 laboratory confirmed cases were reported, with 60% mortality (123). All the cases were linked to people traveling or residing in the Arabian Peninsula or countries near it. In 2015, the largest outbreak outside the Arabian Peninsula occurred in the Republic of Korea.
Until now, the disease is still under surveillance. By November 2019, 27 countries have reported cases of this disease and the total number of cases reached 2,494, with 858 deaths (34.3%) (124). Although some of the infected people are asymptomatic, most of the patients presented with fever, cough, and shortness of breath. Some also had diarrhea and nausea or vomiting. The symptoms take 2–14 days to appear. Many patients develop severe complications, such as pneumonia and kidney failure (18,56). Patients with severe illness require support in intensive care and those who had respiratory failure require mechanical ventilation. More than 30% of the patients died. The majority of those who died had pre-existing medical conditions, such as diabetes, cancer, chronic lung disease, chronic heart disease, and chronic kidney disease (18,56).
Several studies suggested that the disease is transmitted through animal–human contact and less frequently through human–human contact (8). The later was mostly seen in hospitals. In general, the infected people had close contact with ill people (124).
After reporting cases and isolating the virus, the search for the virus reservoirs commenced. Bats were suspected and surveyed with many other animals. MERS-CoV was genotypically linked to the betacoronavirus lineage C viruses identified in bats (8). But, no decisive evidence was found to prove that bats are the natural reservoirs of the virus. However, serological screening showed that dromedary camels have high MERS-CoV antibodies. In addition, MERS CoV and other coronaviruses related to MERS-CoV lineage were detected in many cases in dromedary camels in the Middle East and many parts in Africa (18,26).
Coronavirus disease-2019
Until this date (August, 2020), COVID-19 is still spreading in the globe at different rates. In some countries the number of new cases is declining, in others it is ascending. The disease presents with variable signs and symptoms and the symptoms range from no symptoms or mild flu-like symptoms to acute respiratory distress and multiple organ dysfunction. This will be discussed in more details in immunopathogenesis and clinical presentation of SARS-CoV2 section.
The origin of the of SARS-CoV2 is still under investigation. Two scenarios were proposed for its origin, (i) Natural selection in an animal host followed by zoonotic transfer or (ii) Zoonotic transfer followed by natural selection in humans (4). Full-length genome sequencing of the viruses isolated from five patients in China showed that the viruses are identical and share 80% nucleotide sequence to SARS-CoV. Phylogenetic analysis of the virus showed that it is a lineage B betacoronavirus closely related to a SARS-like coronavirus in bats. The resemblance was 96% in nucleotide sequence, which suggests that SARS-COV2 could have been emerged from bats, which makes bats as a likely reservoir host for the virus, however, it is not known yet if there is an intermediate host for this virus (132).
To date, it is known that the virus can be transmitted mainly through small respiratory droplets (sneezing or coughing) or through close contact (nearly 6 feet) (17). The respiratory droplets are either inhaled or landed on surfaces that are contacted by people who touch their eyes, nose, or mouth (17,36). The virus seems to be transmitted easily and sustainably from human to human (community spread) through the respiratory routes. Recently, growing evidence suggests the possible transmission of the virus through aerosols, which was seen in crowded and closed settings with inadequate ventilation (80,81). This has driven the WHO to urgently request more investigational studies about this route of transmission (120).
Role of Angiotensin-Converting Enzyme 2 in COVID-19 Pathogenesis
Angiotensin-Converting Enzyme 2 (ACE2) is a type I transmembrane zinc metalloenzyme and carboxypeptidase with homology to ACE that is an essential regulator of heart function (30). It was found to be highly expressed in many cells like alveolar epithelial type II cells of lung, esophagus upper and stratified epithelial cells, nasal epithelial cells, colon, myocardial cells, kidney proximal tubule cells, epithelia of the small intestine, testes, and bladder urothelial cells (127,131,135).
The ACE2 gene is located on chromosome Xp22, spans 39.98 kb of genomic DNA, encoding a protein of 805 amino acids, and contains 20 introns and 18 exons (107). Its expression was found to be associated with age, but not sex or race. Its expression was positively correlated with age among middle-aged and older adults, whereas no significant difference was found among individuals of East Asian, African or European ancestry (24).
The ACE2 gene exhibits a high degree of genetic polymorphism (15,74). A very recent study by Cao et al., investigated the allele frequency (AF) differences between East Asian, European, African, South Asian, and admixed American (15). Their findings suggested that the genotypes of ACE2 gene polymorphism among East Asian population may be associated with higher expression levels of ACE2. Also, moderate difference was shown in AFs of genetic analysis of expression quantitative trait loci (eQTLs) between South Asians and East Asians, which suggests the potential difference of ACE2 expression in different populations and ethnicities in Asia and the diversity of ACE2 expression pattern in populations (15).
ACE2 has been found to be a receptor for the entry of the novel human pathogenic coronaviruses SARS-CoV and SARS-CoV2 with an increasing evidence of its role in its pathogenesis (88,132). The suggested mechanism for the viral entrance involves binding of its spike proteins, S-protein, which are located on the coat of the virus, with ACE2 receptors, which are normally found on the epithelial cells of different organs (66). This binding is proteolytically processed by type 2 transmembrane protease, TMPRSS2, which would lead to the cleavage of ACE2 and the activation of the spike protein, thus, facilitating virus entry and replication (53).
Many similarities were found between the original SARS-CoV and the new virus (SARS-CoV2). Both share 76.5% identity in amino acid sequences with a high degree of homology (66,125). Although studies showed that SARS-CoV spike protein has a strong binding affinity to human ACE2, recent studies suggested that SARS-CoV2 recognizes human ACE2 more efficiently, increasing the ability of SARS-CoV2 to transmit from one person to another (85,125).
Lung appears to be the most vulnerable target organ for SARS-CoV2 and according to Zhang and his team, they recently indicated that it could be due to the vast surface area of the lung making the lung highly susceptible to inhaled viruses in addition to other biological factors (128). Zhao et al. (131) demonstrated that 83% of ACE2-expressing cells in adults were in alveolar epithelial type II cells (AECII), which can serve as a reservoir for viral invasion. Their gene ontology enrichment analysis showed that the ACE2-expressing AECII have high levels of multiple viral process-related genes, including regulatory genes for viral processes, viral life cycle, virion assembly, and regulation of viral genome replication, suggesting that the ACE2-expressing AECII facilitate coronaviral replication in the lung (131).
In a previous study on SARS-CoV infection, ACE2 expression was found to be positively correlated with the differentiation state of human airway epithelia. Undifferentiated cell expression ACE2 was poorly infected with SARS-CoV, whereas the well-differentiated cells expressed more ACE2 and were readily infected (135).
Studies suggested that the susceptibility, symptoms, and outcome of COVID-19/SARS-CoV2 infection might be correlated with the state of cell differentiation of human airway epithelia, expression level, and expression pattern of human ACE2 in different tissues (15,135,128). The expression level of ACE2 receptors was found to be increased in hypertension and diabetic patients treated with ACE inhibitors and angiotensin II type-I receptor blockers (44,66,110).
Several hypotheses were published regarding the relationship between diabetes and hypertension treatment with ACE2-stimulating drugs, and the increase in ACE2 expression, and their role in increasing the risk of developing severe and fatal COVID-19. However, limited evidence showed changes in ACE2 levels expressed in serum or pulmonary samples (88). These hypotheses were recently refuted by the European Society of cardiology indicating that there is no scientific base or evidence to support (91).
Other very recent studies found an ACE2 genetic predisposition for the increased risk of SARS-CoV2 infection. ACE2 genetic polymorphisms have been linked to diabetes mellitus, cerebral stroke, and hypertension (39). A previous study found a high degree of genetic heterogeneity among ACE2 polymorphisms that are linked to type 2 Diabetes (70).
Smoking, which in the developed world is the primary etiological factor behind chronic obstructive pulmonary disease (COPD), is considered as a factor to increase vulnerability to respiratory viruses like SARS-CoV2 (10). Smoking was found to upregulate ACE2 receptors on the airway epithelium. Guoshuai Cai (2020) recently reported significantly a higher ACE2 gene expression in smoker samples compared with nonsmoker (11). Consequently, the increased number of ACE2 is expected to facilitate infection with SARS-CoV2 (10,15).
Differences in ACE2 coding variants among different populations suggest that the diverse genetic basis might affect ACE2 functions among populations which could affect the association between ACE2 and S-protein in SARS-CoV2. Therefore, the state of cell differentiation and ACE2 expression levels are both important determinants of the susceptibility of human airway epithelia to infection. The expression level and expression pattern of human ACE2 in different tissues and populations might be critical for the susceptibility, symptoms, and outcome of SARS-CoV2 infection. In summary, differences in immunity, ACE2 gene expression, age, or even genetic background may contribute to the different susceptibility to and severity of SARS-CoV2 infection.
Immunopathogenesis and Clinical Presentation of SARS-CoV2
The immunopathology of the novel SARS-CoV2 is still under investigation. Although, it can activate both innate and adaptive immunity, an uncontrolled and impaired immune response might occur leading to harmful tissue damages locally and systemically. Since the start of the pandemic, several hypotheses have been suggested describing the interaction of the immune system with the virus in the development of the disease and its most severe forms, where cytokine storm had the most important role (6,86). While trying to restore hemostasis after infection, inflammation happens, which can be very harmful if not controlled (6,86).
As the virus name indicate, SARS-CoV2 affects the lungs leading in some cases to SARS. On the other hand, it had a probable asymptomatic incubation period between 2 days and 2 weeks, where the virus can be transmitted (23,44,111). In some cases a period of 2–3 weeks was observed between developing symptoms and the final clinical outcome (108). According to the clinical studies of hospitalized COVID-19 patients, most frequently they show symptoms associated with viral pneumonia, fever, sore throat, cough, myalgia, and fatigue (23,51). In addition to that, a few cases showed severe-to-critical complications, where most of them did not develop severe clinical manifestations in the early stages of the disease, but at the later stage. They showed respiratory failure requiring mechanical ventilation, septic shock, or other organ dysfunction or failure requiring intensive care at the hospital (108).
By the end of the third month of the pandemic, a group of European physicians submitted a letter to the American Journal of Respiratory and Critical Care Medicine, showing discrepancies in patients with COVID-19 presenting an atypical form of Acute Respiratory Distress Syndrome (ARDS) recommending to avoid jumping to unnecessary mechanical ventilation (46).
Recent clinical reports described destructive damages in the cardiovascular system, kidneys, gut, and brain (109). Significant observations were also reported showing the tendency of the patients to form clots in blood leading to vessel obstruction, which might result in pulmonary embolism or large vessel ischemic stroke, in addition to forming ischemic fingers and toes (109).
A recent study indicated that the chance of survival following SARS-CoV2 infection in people 60 years of age and older is ∼95% in the absence of comorbid conditions. But the chance considerably decreased in patients with health conditions and continues to decrease with age beyond 60 years (94,96,103). From the experience of scientist and clinicians in COVID-19, not all people exposed to SARS-CoV2 are infected and relatively a small proportion of infected patients develop severe respiratory syndrome (51).
According to Wang et al. SARS-CoV2 infection can be divided into three stages: an asymptomatic stage with or without viral detection, where the patients might spread the virus unknowingly, a nonsevere symptomatic stage with viral presence and a severe respiratory symptomatic stage with high viral load, which occurs in about 15% of the confirmed cases (45,111). However, transmission of the virus might occur from presymptomatic patients, where they are infected but did not develop symptoms yet (114). This variation in response to the viral infection is one of the main concerns to scientists and clinicians where the overall immunity of the infected patients cannot explain the differences in disease presentation.
Innate and adaptive immunity in COVID-19-infected patients
Limited information is available regarding the immunological response to SARS-CoV2 in infected patients. Thus, to understand its immunological response, we have returned to previous studies on SARS-CoV in addition to the current findings about SARS-CoV2 patients. Generally, the innate immune system is the first to respond after viral infection. It recognizes the virus through pattern recognition receptors (PRRs) like Toll-like receptor (TLR) (2,73). This leads to the activation of different pathways where the virus induces the expression of inflammatory factors and the synthesis of type I interferons (IFNs) limiting its spread and accelerating macrophage phagocytosis of viral antigens (2). However, the nucleocapsid protein (N) of SARS-CoV can help the virus to escape immune responses (73).
Recently lots of efforts and studies were done trying to understand the immune response to SARS-CoV2. Innate immunity was linked with the development of the cytokine storm and found to be responsible for boosting more severe forms of the disease (6). When the virus infects the upper and lower respiratory tract it either causes a mild or a highly acute respiratory syndrome. This critical and fatal outcome is due to the release of interleukin (IL)-1β and IL-6 proinflammatory cytokines after SARS-CoV2 binds to TLRs leading to the activation of the immune response (6,86). Those stimulated proinflammatory cytokines in addition to tumor necrosis factor (TNF) mediates lung inflammation, fever, and fibrosis (86). Therefore, harmful tissue damage both at the site of virus entry and at systemic level might occur.
The adaptive immune response activates CD4+ and CD8+ T lymphocytes, which play a critical role in clearing the virus by eliminating virus-infected cells. CD4+ helper T cells can direct the cytotoxic T cells and B cells and enhance their function to eliminate the virus (78,133). Activated CD8+ cytotoxic T cells will directly kill infected cells by secreting molecules like granzymes, perforin, and IFN-γ to eradicate the virus, whereas CD4+ T cells will stimulate B cells to produce antibodies specific to the virus (105).
The early stage defense antibodies produced after almost 1 week of SARS-CoV2 viral infection are the neutralizing IgM antibodies. They are relatively efficient in fully blocking viral entrance into the host cells, limiting the infection, and are considered as a critical immune player for protection against viral diseases. Then, the class switch, where high-affinity IgG antibodies are developed for the long-term immunity and immunological memory (67). Subsequently, cellular immunity will be mediated by T lymphocytes. This adaptive immune response is directed by T helper cells, where cytotoxic T cells will then play a vital role in the destruction and clearance of the viral infected cells (67). However, T cell exhaustion might be induced due to viral persistent stimulation that leads to the loss of the cytokine production ability and reduced functions (43,83).
On the other hand, in patients with severe SARS-CoV infection, a delayed adaptive immune response development and prolonged viral clearance were noticed (14). Also, in a previous study measuring the T cell responses in patients with MERS-CoV infection, all tested MERS survivors developed both CD4+ and CD8+ T cell responses, and patients with mild or subclinical illness developed prominent virus-specific CD8+ T cell response (129).
In this rapidly evolving field, researchers reported that individuals with positive SARS-CoV2 polymerase chain reaction (PCR) results were able to develop IgM, IgA, and IgG antibodies against SARS-CoV2 surface spike and nucleocapsid proteins within 1–2 weeks after having symptoms and continue elevation after initial viral clearance (97). According to Seow et al., patients with severe COVID-19 symptoms showed higher levels of antibodies compared with those with mild disease (97). They also showed that their levels began to decline 20–30 days post onset of symptoms and sometimes nearly to undetectable levels in less than 2 months after symptoms appeared.
The length of the antibody-specific response to SARS-CoV2 is still unknown. On the other hand, the antibodies produced in patients infected with SARS-CoV or MERS-CoV coronaviruses were found to wane over time ranging between 12 and 52 weeks from the onset of the disease, while homologous reinfections were detected (58).
As the antibody levels declined with time, it was important to identify antigen-specific T cell responses in COVID-19 patients. Recently, physicians and scientists were able to detect and characterize some of SARS-CoV2 T cell responses in humans (98). Nevertheless, they are still uncertain about the role of the adaptive immunity in the disease, whether it is protective or pathogenic or both depending on the time, composition, or magnitude of the adaptive response. According to Blanco-Melo et al. an early CD4+ and CD8+ T cell response against SARS-CoV2 can be protective, but it is difficult to have an early response due to the efficient innate immune evasion mechanisms of SARS-CoV2 in humans (7). On the other hand, in the presence of sustained high viral load in lungs, late T cell response may increase the pathogenic inflammatory outcomes (52).
Cytokine storm mechanism in COVID-19
Although cytokines are known to play an important role in immunopathogenesis during viral infection, overreactive and dysregulated immune responses were observed in SARS-CoV2 patients leading to organ damages (6,86). This was also observed in the previous human coronaviruses, SARS and MERS, where they might enhance an over-reactive immune response leading to the production of cytokine storm that can cause severe damages to the lungs causing ARDS and to other organs, which might lead to multiorgan failure and even death (20).
Previous in vitro studies at the early stage of SARS-CoV infection showed a delay in the release of cytokines and chemokines at the respiratory epithelial cells, dendritic cells, and macrophages (25,64). Whereas at later stages, cells secrete low levels of the antiviral factors, interferons (IFNs), and high levels of proinflammatory cytokines [IL-1β, IL-6, and TNF and chemokines (C-C motif chemokine ligand (CCL)-2, CCL-3, and CCL-5)] (28,67), where they contribute to the occurrence of ARDS in SARS-CoV patients (14). These findings supported the view that high viral load and cytokine/chemokine dysregulation can cause the cytokine storm, which is accompanied with immunopathological changes in the lungs.
The severe systemic elevation of the proinflammatory cytokines leading to cytokine storm in COVID-19 infections was mostly noticed in elderly critical adults (77). Meftahi et al. reported that balance between proinflammatory and anti-inflammatory immune responses is needed to limit the progression of COVID-19 infection. Healthy adults showed this balance in the cytokine network and were able to shut down immune activity at the right time. Whereas, elderly patients did not show the same balanced immune response as a young adult (77).
Laboratory findings in COVID 19
In the previous studies for the acute phase of SARS-CoV infection, lymphocytopenia, mainly T lymphocytes, was observed, and both CD4+ and CD8+ T lymphocytes were decreased (68). In SARS-CoV2-infected patients, studies showed a normal leukocyte, platelet count, and lymphocytopenia at the early stage of the disease. Upon admission, lymphocytopenia, thrombocytopenia, and leukopenia were observed (51,111). Diao et al., showed that CD4+ and CD8+ T cells were dramatically reduced in COVID-19 patients, especially among elderly patients and those needed the intensive care unit (33). According to Zhou et al., age, lymphopenia, leucocytosis, and elevated ALT, lactate dehydrogenase, high-sensitivity cardiac troponin I, creatine kinase, d-dimer, serum ferritin, IL-6, prothrombin time, creatinine, and procalcitonin were associated with death in severe cases of COVID-19 patients (128).
Selected Potential Pharmacotherapeutic Options for Treating COVID-19
To this date, there are no drugs that have proven effective for the management of COVID-19 (38,126). So, there is a crucial need to look for therapeutic options to control virus entry, replication and/or spread. Developing new drugs that target various steps of the viral infection would be the optimum choice, however, the later would not be practical in lieu of urgency to find a treatment for this pandemic infection. Therefore, screening of the existing drugs with potential antiviral effect can hopefully create new therapeutic options, while bypassing much of the costs and time involved with having a new drug available in the market.
We have done an online search on PubMed and Web of Science with the keywords of SARS, MERS, and coronaviruses, treatment, and prevention. The followings summarize some of the proposed therapeutic options available for the treatment of this novel coronaviruses.
Small molecules
Chloroquine/hydroxychloroquine and other antimalarial agents
As mentioned in the Role of Angiotensin-Converting Enzyme 2 in COVID-19 Pathogenesis section, ACE2 receptor is the site of binding of SARS-CoV, which mediates its entry into the cell through binding with spike (S) protein (66). Thus, blocking the binding of S protein to ACE2 would be a key for the treatment of SARS-CoV2 infection.
Chloroquine interferes with the glycosylation of cellular receptor of SARS-CoV2, ACE2, and thus prevents viral entry (72). It also works by increasing the pH in mammalian cell lysosomes, which will subdue viral replication (72). Recently, it was shown that chloroquine and hydroxychloroquine can reduce the production of various proinflammatory cytokines, such as IL-1, IL-6, interferon-α, and tumor necrosis factor, which are involved in the cytokine storm. These immunomodulatory effects potentiate the antiviral effects of these agents in the treatment of COVID-19 (130).
It was anecdotally reported that this antimalarial drug has antiviral effect probably due to its ability to inhibit viral entry and/or replication. Chloroquine showed in vitro activity against SARS-CoV and MERS-CoV (112), in addition, its less toxic derivative, hydroxychloroquine (HCQ), showed an in vitro activity against the novel SARS-CoV2 (72). Clinically, however, there is still a controversy about the clinical efficacy and safety of chloroquine or HCQ. Cortegiani et al. summarized 23 clinical trials in China that have pending approval or already recruiting patients (27), which are using chloroquine with or without other agents.
Awaiting the efficacy and safety results from these trials, scrutiny should be exercised upon clinically utilizing this drug (27). Two debatable French studies by Gautret et al. (47,48) showed a reduction of viral load when HCQ used in combination of hydroxychloroquine with azithromycin in patients with COVID-19 25% (74) at day 6 of treatment, and 93% of patients had negative PCR test after 8 days. In spite of promising results, those studies suffered from small sample size (48) and the lack of a control group (47). Conversely, a recent study from China in 30 individuals with COVID-19 found no difference in the rate of virologic clearance nor clinical improvement after 5 days of using HCQ compared with placebo (22).
Even more, a phase 2b randomized controlled trial (RCT) in 81 patients with severe COVID-19 infection showed that lethality rate was significantly higher in the high-dose group when compared with low-dose group (39% vs. 15%, respectively), which warranted the cessation of the high dose immediately (CloroCovid-19) (9). Due to inconsistencies in the abovementioned results, timely results from studies with larger sample size, and in patients with severe COVID-19 are pivotal to dictate the drug's utilization worldwide.
A total of 1,542 patients were randomized to hydroxychloroquine and compared with 3,132 patients randomized to usual care alone. Interestingly, preliminary (unpublished) results showed that there was no significant difference in the 28-day mortality (25.7% hydroxychloroquine vs. 23.5% standard care), as well as the lack of beneficial effects on hospital stay duration or other outcomes (63,104). In addition, the interim results from solidarity trial, which was established by the WHO, showed that hydroxychloroquine and lopinavir/ritonavir did not provide a significant reduction in the mortality of hospitalized COVID-19 patients when compared with standard care. Therefore, WHO discounted both treatment arms immediately (116). These preliminary results dampened the initial enthusiasm toward hydroxychloroquine.
Another antimalarial drug, arbidol, was also tested against other antiviral agents (32,69,115,134). One study retrospectively analyzed data from fifty patients who were randomized to receive arbidol (n = 17) versus the antiviral lopinavir/ritonavir (LPV/r) (n = 34) (134). In the arbidol arm, no viral load could be detected in pharyngeal swab after 2 weeks of treatment, whereas it was found in 44.1% of patient in the LPV/r group. Furthermore, a cohort study showed that LPV/r combined with arbidol versus LPV/r alone showed more favorable outcomes in terms of absence of viral load after weeks, as well as improvement of chest CT scan (32).
Despite these promising results, the latest observational cohort study (32) showed that there was no evidence to prove that LPV/r and arbidol could shorten the negative conversion time of novel coronavirus nucleic acid in pharyngeal swab nor improve the symptoms of patients. Furthermore, the combination usage of LPV/r and arbidol has no benefit of improving the disease, while posing more adverse effects when compared with conventional therapy (115).
Remdesivir
This drug was originally developed for Ebola, as well as other single-strand RNA viruses, including coronavirus-related viruses, SARS (112) and MERS (49). Remdesivir (RDV) gets incorporated into viral RNA, and targets RNA polymerase thereafter, and thus inhibits viral replication. RDV has shown to inhibit coronavirus replication in cell culture and animal models (16,112). Accordingly, it is very plausible that this drug would work in clinical settings (3,16). There are still pending multiple clinical trials with larger sample size, with a target of thousands patients. Until data from such trials are published, we need to interpret results about RDV efficacy and safety very cautiously (37,84,102).
Presently, there have been published results about the compassionate use of RDV in treating patients hospitalized for COVID-19 (50). Clinical improvement in oxygen-support class was observed in 68% (36/61) of patients, 47% (25/61) of patients were discharged, and mortality was 13% (7/61). While results from this study is promising, this study lacked a control arm and is underpowered (49). Therefore, the efficacy and safety of RDV should be further evaluated with randomized, placebo-controlled trials.
On a larger scale, a multicenter, placebo-controlled RCT, which recruited 237 patients with severe COVID-19 symptoms, recently evaluated the efficacy and safety of RDV (113). Patients were randomized in a 2:1 ratio either to receive IV RDV (200 mg on Day 1 followed by 100 mg for days 2–10) versus placebo. There was no statistical difference in time to clinical improvement in both groups, as well as adverse events and mortality rate. However, more patients in the RDV group discontinued the drug due to adverse events (12%) compared with placebo (5%), and the trial was therefore stopped before the intended sample size is reached (113). While the later study was a well-designed RCT to evaluate efficacy and safety of RDV, it is still too early to judge the success or failure of RDV from this study. First, the intended sample size was 453 and since the trial was stopped early, this lead to underpowered results. Second, there is no consensus on what is minimally accepted clinical improvement difference, since this was the first RCT evaluating RDV.
Favipiravir
Favipiravir, another RNA polymerase inhibitor, marketed as Avigan, developed by Fujifilm, Toyama, in Japan is being tested for efficacy and safety in China. Previous studies showed that favipiravir, the anti-influenza drug, inhibits SARS-CoV2 in some cultured cells and protects mice against Ebola (101). So far, favipiravir was compared with the anti-HIV combination lopinavir in 80 patients in a nonrandomized trial. On day 1, the intervention arm took 1,600 mg of favipiravir twice (in two separate doses), in addition to inhaled interferon. On day 2 until the end, the dose was reduced to 600 mg twice daily, and they kept taking inhaled interferon. The control group received lopinavir/ritonavir for 14 days at a dosage of 400 mg, then 100 mg, twice daily, with inhaled interferon (12).
The preliminary results showed that favipiravir may potentially be superior to lopinavir/ritonavir, as evidenced by better chest X-ray images and thus lung improvement in the intervention group, an effect that was interestingly independent of viral load reduction effect. No significant adverse reactions were noted in the favipiravir treatment group, and it had significantly fewer adverse effects than the lopinavir/ritonavir group. In addition, a recent preprint showed that this drug is superior to arbidol in terms of latency to relief fever and cough, but did not significantly improve clinical recovery rate after 1 week of treatment (21).
Prodrug EIDD-2801
Base-modified nucleoside analog, beta-D-N(4)-hydroxycytidine (NHC), has shown potent antiviral activity against wide spectrum of viruses, such as Venezuelan equine encephalitis virus (VEEV), respiratory syncytial virus (RSV), influenza A virus (IAV), influenza B virus (IBV), chikungunya virus (CHIKV), and CoVs, plausible through inducing mutagenesis in the viral RNA (92).
The novel broad-spectrum antiviral drug was tested in mouse pathogenesis models of SARS-CoV2 and MERS-CoV for both prophylactic as well as therapeutic use (100). This drug was shown to reduce viral load in the lungs and to improve the pulmonary function, provided its use within 24–48 h of the symptoms' initiation. Very recently, this drug passed phase 1 trial and was found to be safe, and therefore Ridgeback biotherapeutics, a majority-women-owned biotechnology company, announced the commencement of two phase 2 trials to test the efficacy of EIDD-2801 as an antiviral medication for COVID-19 in newly diagnosed and hospitalized patients, respectively (95).
Biologics
It was found that patients who had higher concentrations of proinflammatory cytokines and chemokines, were more likely to be admitted to an intensive care unit, namely, G-CSF, IP-10/CXCL10, monocyte chemoattractant protein 1 (MCP1), and TNFα, as well as elevated cytokines from T helper 2 cells such as Interleukin (IL)-4 and reduced IL-10 (34,71). Therefore, it is tempting to assume that interfering with these inflammatory pathways can potentially ameliorate symptoms of COVID-19.
IL-6 inhibitors
Genentech®, with collaboration with the Food and Drug Administration (FDA), commenced a randomized, double-blind, placebo-controlled phase 3 clinical trial to examine the safety and efficacy of tocilizumab in addition to standard care in hospitalized adult patients with severe COVID-19 pneumonia compared with placebo plus standard care. The primary and secondary endpoints of the study include clinical status, mortality, mechanical ventilation, and ICU variables (41). Two trials are held to test the efficacy and safety of tocilizumab in the management of COVID-19. A 150-patient unblinded RCT is assessing tocilizumab in combination with favipiravir, compared against each drug alone, with time clinical cure rate as the major outcome (NCT04310228) (40).
Another Chinese multicenter RCT trial with 188 patients is assessing tocilizumab alone versus standard care in patients with severe COVID-19 and elevated IL-6 levels (ChiCTR2000029765) (1). An open-label, noncontrolled, nonpeer-reviewed study was conducted in China in 21 patients with severe respiratory symptoms related to COVID-19. In addition to standard care (including lopinavir and methylprednisolone), patients received a single dose of intravenous infusion with 400 mg tocilizumab. There was a clinical improvement as patients had less oxygen requirements, normalized lymphocyte counts, and 19 patients were discharged after about 2 weeks of tocilizumab treatment (82). It is noteworthy that this study had a small sample size and no control arm, therefore, results should be interpreted with extreme caution.
Sarilumab, and leronlimab are other IL-6 inhibitors, which are being evaluated in critically and severely ill COVID-19 patients. Sarilumab is tested in a phase 2/3 trial, which is multicenter, double-blind, trial with an adaptive design, which is anticipated to enrol around 400 patients. The evaluation of outcomes will occur on two stages. In the first one, the effect of sarilumab on fever and the need for supplemental oxygen will be examined. The second, will assess longer-term outcomes, such as mortality and the need for mechanical ventilation, supplemental oxygen, and/or hospitalization (93).
Leronlimab, a chemokine receptor type 5 antagonist, will also be evaluated in a phase 2b/3 trial (106). As of mid-April, one severely ill COVID-19 patient and 15 mild/moderate patients were enrolled. The mortality rate will be assessed at 2 and 4-week intervals. An interim analysis will be performed on the first 50 patients. This agent has been granted a Fast Track designation from the FDA for two potential indications of COVID-19. The drug was chosen based on preliminary results, in eight severely ill COVID-19 patients who demonstrated a significant improvement in several important immunologic biomarkers after a 3-day course of Leronlimab. Patient test data reveal reduction in cytokines, IL-6, and a pattern of normalization of the CD4/CD8 ratio (106).
Interferon-beta
Interferon-beta (INF-β) is an endogenous protein, which coordinates the antiviral response. Studies showed that deficiency in IFN-β production increases the odds of a more severe lower respiratory tract disease during respiratory viral infections, especially in those at risk. Interestingly, viruses, including coronaviruses such as SARS-CoV (5) and MERS-CoV, (99) have developed mechanisms, which suppress endogenous INF-β production that augments the virus's ability to escape the innate immune system. It is therefore expected that the addition of exogenous INF-β before or during viral infection of the lower respiratory system would either prevent or greatly diminish cell damage and viral replication, respectively.
Synairgen®, a respiratory drug development company, developed IFN-β-1a for direct delivery to the lungs through nebulization. It is pH neutral, and is free of mannitol, arginine, and human serum albumin, making it suitable for inhaled delivery direct to the site of action. With collaboration of the National Institute of Health (NIH), Synairgen have shown that INF-β could protect against SARS-COV2 infection of lung cells in vitro (75). Very recently, a double-blind, placebo-controlled Phase 2 trial in COVID-19 patients is taking place. It is anticipated to recruit 100 COVID-19 patients, will take place across a number of NHS trusts, and has been adopted by the NIH Research (NIHR) Respiratory Translational Research Collaboration, which consists of leading centers in respiratory medicine in the UK (2).
Granulocyte/macrophage colony-stimulating factor alpha monoclonal antibody (Mavrilimumab)
Granulocyte/macrophage colony-stimulating factor alpha (GM-CSFRα) works upstream of interleukin-6 in the pathophysiology of the hyperinflammation associated with severe pneumonia of COVID-19 (71). Mavrilimumab, an investigational fully human monoclonal antibody that inhibits GM-CSFRα, is now being tested in uncontrolled, single-center pilot study (61). Patients with severe pneumonia from COVID-19, defined as acute respiratory distress, fever, and clinical and biological markers of systemic hyperinflammation status were treated with a single intravenous dose of mavrilimumab.
To date, six patients have been treated with this drug. While being well tolerated, all patients showed an early resolution of fever and improvement in oxygenation within 1–3 days. Interestingly, none of these patients has progressed to require mechanical ventilation and half of them were discharged within 5 days (61).
Dexamethasone
Dexamethasone is a synthetic systemic glucocorticoid, which is known for its potent anti-inflammatory effect (62). Dexamethasone acts by suppressing proinflammatory cytokines IL-1, IL-2, IL-6, IL-8, TNF, and IFN-γ, all of which are linked to COVID-19 severity. In vitro studies showed that dexamethasone protected alveolar cells from destruction by proinflammatory cytokines (62).
Preliminary results from the RCOVERY Trial showed that dexamethasone reduced the mortality rate in hospitalized COVID-19 patients when compared with those given usual care (54). Indeed, 454/2,104 (21.6%) patients allocated dexamethasone and 1,065/4,321 (24.6%) patients allocated usual care died within 28 days (age-adjusted rate ratio = 0.83; and 95% confidence interval 0.74–0.92; p < 0.001), which was dependent on the level of respiratory support at randomization. Thus, the significant reduction in mortality in the dexamethasone group was mostly significant among those receiving invasive mechanical ventilation or oxygen at randomization, but not among patients not receiving respiratory support (54).
Plasma of recovered COVID-19 patients
Plasma from patients who recovered from COVID-19 contains the SARS-CoV2-specific neutralizing antibodies, which could infer a passive immunity to the recipients. The later can be utilized to produce two potential therapeutic modalities: convalescent plasma and hyperimmune immunoglobulins. A recent Cochrane living review summarized 20 studies, including one RCT, 3 nonrandomized controlled trials, and 16 non-randomized noncontrolled trials. Collectively, the controlled trials showed that there was a low-certainty evidence about the effectiveness of these therapies in reducing all-cause mortality, time to death, or even improvement of clinical symptoms, as assessed by the need of respiratory support.
For the safety, the majority of evidence comes from the uncontrolled trials. These studies reported side effects, including death, anaphylaxis, dyspnea, and lung injury, but most of them were found to be transfusion related. So until more properly designed studies are available plasma-derived therapies cannot be reliably recommended for the management (90).
Conclusions and Future Insights
In this review we tried to explore and explain the disease in a timely and comprehensive manner over the years. During those years, lots of challenges faced the scientists and researchers. Complete understanding of the viral mechanism of action and the process and mechanism of the immune system in the presence of the virus is needed to determine the potential therapeutic options for SARS-CoV2 and all were and are still being investigated globally.
Future studies should focus on targeting more sites of viral entry and replication, which was the basis for investigating drugs like hydroxychloroquine and RDV, respectively. For instance, it was found that the spike glycoprotein of the new coronavirus SARS-CoV2 contains a multibasic furin-like cleavage site, which was lacking in other related SARS-like coronaviruses (28). These viral fusion proteins are activated by proteases in the host cell, therefore, any modifications in the cleavage site/s with proteolytic activity will alter the spike glycoprotein, which will ultimately dictate viral membrane fusion, entry, and hence the tropism of the host cell (28).
Furin is a convertase enzyme that is highly expressed in lungs, so the novel SARS-CoV2 may successfully exploit this convertase to activate its surface glycoprotein. Thus, the inhibition of this enzyme might represent a novel target for antiviral therapy. Inhibition of furin had shown to inhibit proinflammatory and matrix protein production, such as TGF-β, platelet-derived growth factor, TNF-α-Converting Enzyme, and MMP2 in in vitro models (29) and consequently reducing proinflammatory cytokines (TNF-α, IL-1β), while increasing the anti-inflammatory IL-10 and IL-4, in vivo (29).
Many furin inhibitors have been previously suggested in literature to treat various diseases such as cancer as well as bacterial and viral infections. Nevertheless, while furin can have a role in controlling inflammation through its immunomodulation function, which can be very beneficial when treating immune-related diseases like COVID19, it might also cause long-term adverse effects if used long term. Therefore, more studies should be done to evaluate their safety and efficacy (29).
Furthermore, phenotypic screening strategy could be used, which includes the identification of molecules with particular biological effects in cell-based assays or animal models. This might entail screening large libraries of chemical compounds in automated high-throughput cellular assays that measure the levels of various proteins or effects on characteristics, such as cell proliferation. For example, just recently, the antiparasite ivermectin was found to inhibit SARS-CoV2, when added to Vero-hSLAM cells 2 h postinfection with SARS-CoV2 and cause around ∼5,000-fold reduction in viral RNA at 48 h. These interesting results merit further evaluation of this medication (13).
Thereafter, finding the right animal model would be crucial, which might be a challenge for this virus, which exhibited various responses in different species. For example, dogs and cats seem to handle this virus very well. But either way, this process will take years, so the best we can do is to repurpose existing medications for treatment of COVID-19, while waiting for the vaccines.
In conclusion, according to results from the most recent studies, antiviral and antimicrobial agents have limited use in the management of coronavirus. RDV is only recommended by CDC in severe cases of COVID-19, and the use of high-dose chloroquine is discouraged. Likewise, there are still no sufficient data to support immune-based therapy beyond the context of clinical trials.
Footnotes
Author Disclosure Statement
The authors declare no conflict of interest.
Funding Information
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