Road Map to Understanding SARS-CoV-2 Clinico-Immunopathology and COVID-19 Disease Severity

SARS-CoV-2, a novel coronavirus, was first identified in Wuhan, China in December 2019. The rapid spread of the virus worldwide prompted the World Health Organization (WHO) to declare COVID-19 a pandemic in March 2020. COVID-19 discontinuing’s a global health crisis. Approximately 80% of the patients infected with SARS-CoV-2 display undetectable to mild inflammation confined in the upper respiratory tract. In remaining patients, the disease turns into a severe form affecting almost all major organs predominantly due to an imbalance of innate and adaptive arms of host immunity. The purpose of the present review is to narrate the virus’s invasion through the system and the host’s reaction. A thorough discussion on disease severity is also presented regarding the behavior of the host’s immune system, which gives rise to the cytokine storm particularly in elderly patients and those with comorbidities. A multifaceted yet concise description of molecular aspects of disease progression and its repercussion on biochemical and immunological features in infected patients is tabulated. The summary of pathological, clinical, immunological, and molecular accounts discussed in this review is of theranostic importance to clinicians for early diagnosis of COVID-19 and its management.


Introduction
The coronavirus disease 2019 (COVID-19) is the first pandemic and the third consecutive epidemic caused by severe acute respiratory syndrome coronaviruses (SARS-CoVs) in the last two decades. The earlier epidemics caused by CoVs were SARS-CoV in 2003 and Middle East CoV (MERS-CoV) in 2012. COVID-19 was first reported in Wuhan, Hubei province of China [1,2] and as of 26 November 2020, there have been 59,816,510 confirmed cases, including 1,410,378 deaths as per the World Health Organization (WHO) [3].
COVID-19 exhibits a broad spectrum of clinical manifestations ranging from asymptomatic infection to severe disease (Table 1) [4]. The infection is commonly spread through respiratory droplets released by an infected individual while coughing, sneezing, or speaking. These infectious droplets can remain airborne for hours and travel up to 27 feet [5]. Aerosol transmission occurs on exposure to a large amount of SARS-CoV RNA (7.08 × 10 3 to 6.38 × 10 8 copies/mL), present in the saliva in a relatively closed environment [6,7]. Most infections remain mild (81%), while severe disease is reported for~15% of cases [8]. Mild disease may quickly deteriorate into severe or critical events without immediate care [8][9][10][11]. Identifying risk factors for SARS-CoV-2 infection and developing the severe illness is of paramount interest to scientists worldwide. Reports suggest that manifestations of a severe form of the disease are predominantly found in children below 1 year [12], adults over 65 years [13,14], pregnant women [15], and immunocompromised individuals [7,16]. Additionally, the COVID-19 prevalence in men is higher (51.4 to 73.2%) than that in women [17,18]. Moreover, a higher case-fatality rate is observed in COVID-19 patients with pre-existing comorbidities such as diabetes mellitus (7.3%), respiratory disease (6.5%), cardiovascular disease (10.5%), hypertension (6%), and oncological complications (5.6%) [19]. Of note, obesity is associated with most of the comorbidities mentioned above and has also been reported to influence the host response to SARS-CoV-2 infection. A meta-analysis showed that COVID-19 individuals with BMI > 30 had a high mortality risk (>46.0%), presumably attributed to persistent low-grade chronic inflammation and suppressed immunity [20,21]. The exact mechanisms underlying the observed association between obesity and COVID-19-severity need further research. Furthermore, the association of the ABO blood group with indices of disease severity and multiorgan dysfunction in COVID-19 has been reported [22]. Critically ill COVID-19 patients with blood groups A, B, and AB are at an increased risk for requiring mechanical ventilation and prolonged ICU admission compared with patients with blood group O [22]. Even in blood group O patients with hypertension, which is the most frequent comorbidity among COVID-19 patients, a significantly lower value of prothrombotic indices, and a lower rate of cardiac injury and deaths have been reported [23].
There is no doubt that SARS-CoV-2 infection has led to an unprecedented and challenging public health crisis worldwide. Hence, the present review focuses on elaborating the SARS-CoV-2 characteristics, its interaction with the host immune processes, and major therapeutic interventions to highlight a precise notion about the infection cycle, molecular and clinical aspects of disease pathogenesis. It also provides a comprehensive account of clinical features and biochemical abnormalities in COVID-19 patients that will help the medical and scientific communities to find potential predictors of disease severity. The insight accrued from this article may help in designing the appropriate immune intervention for treatment strategy for COVID-19.  [26,27] The symbol "↑" is denoted as increased/unregulated and "↓" as decreased/reduced expression level for each parameter. # Derived from chest CT findings. Parameters marked with * show a similar trend in the subsequent stage. CRP, C-reactive protein; LDH, Lactate dehydrogenase; ALT, Alanine aminotransferase; AST, Aspartate aminotransferase.
contribute to SARS-CoV-2 infectivity [46]. Polymorphisms and increased expression of TLR7/8 are also correlated with an individual's vulnerability to SARS-CoV-2 infection [47]. Furthermore, evasion of types 1 and 3 interferons initiation gives the virus a replicative head start and allows it to spread within multiple tissues by compromising the antiviral immune system [48].

Tissue andOrgan Damages
The pathogenesis of SARS-CoV-2 infection begins when the virus passes through the nasal and larynx mucosal membranes and enters the lungs through the upper respiratory tract. The earliest symptoms include fever, cough, myalgia, headache, and loss of smell (anosmia/hyposmia) and taste (hypogeusia) ( Table 2) [14]. One of the possible factors causing anosmia includes direct or indirect damage of the receptor neurons located in the olfactory epithelium by a virus-induced cytokine storm and infiltration of immune cells [49].
Although the lungs are the primary organ affected in SARS-CoV-2 infection, other organs, namely the heart, gastrointestinal tract, kidney, liver, brain, spleen, lymph nodes, and testes, are not spared ( Table 2). Increased ACE2 expression in these tissues correlates with the disease severity and propensity of multiorgan failure [50,51]. One of the reasons that COVID-19 patients with cardiovascular complications are susceptible to heart attack is that thepericytes and capillary endothelial cardiac cells express higher levels of ACE2 [52]. ACE2 is a crucial part of the renin- Figure 1. SARS-CoV-2 life cycle and pathogenesis. SARS-CoV-2 enters through direct or indirect contact with infectious respiratory droplets. After infiltration from the upper respiratory tract to the alveoli, the spike proteins bind to ACE2 receptors. Endocytosis or direct fusion of the viral envelope with the host plasma membrane takes place. With the assistance from host proteolytic enzymes, such as transmembrane protease serine 2 (TMPRSS2), the virus particle is uncoated to release single-stranded RNA (ssRNA) genome into the cell cytoplasm. A multiprotein replicase-transcriptase complex synthesizes genomic and sub genomic RNA by the replication and transcription processes respectively. The structural and accessory proteins are translated inside the rough endoplasmic reticulum (RER). The assembly of viral structural-spike (S), envelope (E), and membrane (M) with nucleocapsid (N) proteins occurs at the endoplasmic-reticulum-Golgi intermediate compartment (ERGIC). New virions are assembled by budding into intracellular membranes followed by vesicle-mediated exocytosis and advanced infection of the vital organs.

Tissue and Organ Damages
The pathogenesis of SARS-CoV-2 infection begins when the virus passes through the nasal and larynx mucosal membranes and enters the lungs through the upper respiratory tract. The earliest symptoms include fever, cough, myalgia, headache, and loss of smell (anosmia/hyposmia) and taste (hypogeusia) ( Table 2) [14]. One of the possible factors causing anosmia includes direct or indirect damage of the receptor neurons located in the olfactory epithelium by a virus-induced cytokine storm and infiltration of immune cells [49].
Although the lungs are the primary organ affected in SARS-CoV-2 infection, other organs, namely the heart, gastrointestinal tract, kidney, liver, brain, spleen, lymph nodes, and testes, are not spared (Table 2). Increased ACE2 expression in these tissues correlates with the disease severity and propensity of multiorgan failure [50,51]. One of the reasons that COVID-19 patients with cardiovascular complications are susceptible to heart attack is that thepericytes and capillary endothelial cardiac cells express higher levels of ACE2 [52]. ACE2 is a crucial part of the renin-angiotensin system and counterbalances ACE1 and angiotensin-II. Angiotensin-II is a pro-inflammatory and vasoconstrictive factor that promotes organ damage. The depletion of ACE2 by SARS-CoV-2 tilts the balance in favor of the fatal ACE1/angiotensin-II axis and promotes tissue injury leading to hypercoagulability [53]. Approximately 40.1% of SARS-CoV-2patients develop acute kidney injury (AKI) with a mortality rate of 71.1% and 2 to 10% of COVID-19 patients manifest gastrointestinal symptoms such as vomiting, diarrhea, and abdominal pain [54][55][56]. The presence of SARS-CoV-2 in fecal samples in a single-cell transcriptomic study provides evidence that the digestive system may serve as a potential route for transmission of SARS-CoV-2 [14,57]. Abnormal liver functions are frequently reported as an extra pulmonary clinical feature among COVID-19 patients [58]. Chen Y et al. have demonstrated that the SARS-CoV-2 can directly infect lymph nodes and spleens to reduce lymphocytes through enhancing IL-6 (interleukin, IL)-mediated Fas up regulation [59]. Consequently, in the early infection stage, the peripheral white blood cell (WBC) count declines and lymphopenia is observed, that affects the antibody production [14,60]. Detailed clinic pathological information conducted in a large population is required to clarify the transmission, pathogenesis, and disease severity caused by SARS-CoV-2.

Hyper-Inflammation and Organ Injury
In COVID-19, the chance of developing a severe disease depends on the interplay between SARS-CoV-2 virulence and host resistance [82]. In mild infection, the host has moderate resistance, and thus the disrupted homeostasis has a higher chance of recovery. However, in severe cases, the host resistance becomes hyperactive and mounts an excessive inflammatory reaction popularly referred to as cytokine storm [83]. In vitro studies suggest that a cytokine storm is correlated directly with tissue injury and an unfavorable COVID-19 prognosis. SARS-CoV-2 infects respiratory epithelial cells, dendritic cells, and macrophages to amplify cytokines to limit the viral infection [76]. Following primary exposure, progeny viruses released from these cells infect alveolar macrophages [84]. These cells secrete low levels of the antiviral interferons (IFNs) and high levels of pro-inflammatory cytokines IL-6, IL-8, IL-1β, granulocyte macrophage colony-stimulating factor (GM-CSF), ROS, tumor necrosis factor (TNF), and chemokines, such as C-C motif chemokine ligand: CCL2, CCL5, CCL3, and IFNγ-induced protein 10 (IP-10) [62,85]. In COVID-19 patients, the serum levels of IL-2R and IL-6 are positively correlated with the disease severity [52]. The delayed release of IFNs in the early stages of viral infection obstructs the antiviral response of the host. Consequently, in the later stages, the rapidly increased cytokines and chemokine molecules attract neutrophils and monocytes, resulting in excessive infiltration of inflammatory cells into lung tissues [86]. Rapid viral replication and vigorous pro-inflammatory immune response induce apoptosis-mediated destruction of lung epithelial and endothelial cells.
Pro-thrombotic alteration of the hemostatic system due to systemic hyper inflammation accounts for one of the pathophysiological aspects of COVID-19-associated coagulopathy. This may involve Virchow's triad that includes (i) diffuse endothelial cell injury, (ii) abnormal blood flow dynamics, and (iii) uncontrolled platelet activation [87]. Coagulopathy is characterized by an exuberant increase in D-dimer, deranged fibrin degradation product due to complete/partial shutdown of fibrinolysis of clots, thrombocytopenia, and prolongations of activated partial thromboplastic time (aPTT), or prothrombin time (PT) [88]. Activation of the neutrophil extracellular trap (NET), which serves as a template for binding activated platelets, accumulation of platelets, and von Willebrand factor multimers (within the microvasculature), impairs vascular integrity and causes organ injury [87,89,90]. The SARS-CoV-2infectiondamages the pulmonary microvascular and alveolar epithelial cell barriers causing vascular leakage and alveolar edema, eventually leading to hypoxia in the body [91,92].
Typical chest computerized tomography (CT) images of COVID-19 patients have contained a ground-glass opacification in the peripheral or central location of the lungs. The mortality of elderly patients with acute respiratory distress syndrome (ARDS) is significantly high (67.3%) [11]. The dysfunctional immune response in elderly and patients with comorbidities may fail to eradicate the pathogen. One of the possible reasons could involve altered dendritic cell maturation and migration to the lymphoid organs in the aging lung microenvironment causing defective T cell activation [93]. Elevated cytokine levels such as IL-1-βand TNF-αcause septic shock, myocardial damage, and circulatory failure [94]. The studies mentioned above suggest that the dysregulated cytokine storm leads to ARDS or multiple-organ failure, and it is a decisive factor that causes COVID-19 exacerbation or even death [95].

Disease Tolerance in Asymptomatic Carriers
A significant proportion (40-60%) of SARS-CoV-2-infected patients remain asymptomatic. This jeopardizes disease control at a population level. Asymptomatic cases are diagnosed by the presence of viral nucleic acid and the absence of anytypical COVID-19 symptoms, such as fever, respiratory symptoms, and abnormal chest radiographs [96]. The appearance of symptoms in COVID-19 usually depends on a person's immunity, comorbidity, and age [14]. Of note, it is unclear why the younger age group remains mostly asymptomatic. Possible reasons include the absence of comorbidities and efficient immune systems that provide sufficient time to mount a significant T cell response, hence, not allowing any signs/symptoms to arise [97]. Detailed investigations are required to understand the dynamics of the immune response in asymptomatic cases that might open up new therapeutic and disease management strategies [18].

Acquired Immunity
Clinico-immunological progression of COVID-19 is categorized into acute (flu-like illness), critical (accelerated inflammatory response), and recovery phases [98]. If the immune function of patients in the early phase removes the virus efficiently, the recovery phase is achieved. Recovering COVID-19 patients harbor high and sustained levels of S-protein-specific neutralizing antibodies (NAbs) [99]. Most patients with COVID-19 have virus-specific IgM, IgA, and IgG responses following infection. The kinetics of Sprotein-specific antibodies in SARS-CoV-2-infected patients follows the order that IgA antibodies are produced in the first week, followed by IgM which subsequently wanes 18 days after the infection. IgG titers are increased during the first 3 weeks [100]. SARS-CoV-2-specific IgA in serum was shown to have a stronger and more persistent response than IgM [26,27,101]. Cellular immunity to SARS-CoV-2 has been shown to comprise CD4+ T-cell and CD8+ T-cell responses which increase within the first 1 to 2 weeks of the onset of infection and wane after 20 days. Th1 cytokines are mainly generated [28]. Studies also suggest that COVID-19 patients can develop specific T-cell memory responses in the absence of specific antibodies, indicating the importance of cellular immunity in containing the disease [101][102][103]. The mutual contribution of cellular and humoral immunities in protection against COVID-19 is not currently clear. A balanced immune response consisting of high titers of neutralizing antibodies and Th1-biased T cells are expected to be optimal in mild disease and recovering cases [102]. Recently convalescent plasma transfusion (CPT) has been suggested to be an effective treatment of COVID-19 as the donor's plasma contained specific IgG and IgM anti-SARS-CoV-2 antibodies that can neutralize the virus [104,105]. Early transfusion of convalescent plasma raises the chance of preventing disease progression in elderly patients with COVID-19 [106,107]. Nonetheless, implementing a CPT program to tackle this pandemic in a large population might need comprehensive planning [108]. There are risks associated with the passive administration of convalescent sera, and that includes changes of blood infections from the recovered patient's plasma and/or antibody-dependent enhancement of infection to another viral strain [109]. Human monoclonal antibody (mAb) has been, thus introduced as an alternative approach. A majority of mAbs such as 47D11 (human), m396, and S230.15 (human) neutralize viral spike proteins and dampen the course of virus action in the host [110].

Correlates of COVID-19 Disease Progression
Apart from the characteristic chest radiograph, there are other clinical parameters reflecting disease severity in symptomatic patients [111,112]. The use of RT-PCR methods based on the detection of viral S and N genes is considered as a gold standard method for the detection of SARS-CoV-2 infection [113]. Serological identification of viral antigens is favored for false-negative RT-PCR results and vice versa. ELISA-based detection of viral Sand N-protein-specific antibodies have also been successfully developed [1].
To detect SARS-CoV-2, nasopharyngeal and oropharyngeal swabs are primarily collected [114]. Of note, the CoV-2 virus has also been detected in no respiratory specimens including stool, ocular secretions, and semen, albeit in a lower frequency [115][116][117]. In particular, SARS-CoV-2 RNA or live viruses have been identified in stool specimens, even after viral RNA was no longer detectable from the upper respiratory specimen [117,118]. Nonetheless, fecal-oral transmission has not been clinically confirmed at present [119]. Surveys from the Netherlands, China, and the USA have shown the presence of SARS-CoV-2 RNA in sewage samples indicating a widespread circulation of the virus in the population [120].

Immunological Factors
Recent data indicate that elevated neutrophils drive the increase in WBCs. At the same time, lymphocytes, monocytes, and eosinophil levels are reduced in complete blood counts (CBC), signifying a poor clinical outcome [124]. In the early stages, a marked depletion in CD4+ and CD8+ T cells is noted [117,126]. In COVID-19 patients, an elevated D-dimer and prolonged erythrocyte sedimentation rate serve as a clinical predictor of severity. Besides, a marked increase in inflammatory factors such as IL-2, IL-6, IL-7, IL-10, GCSF (granulocyte colony-stimulating factor), IP10 (interferon gamma-induced protein 10), MCP1 (monocyte chemotactic protein 1), MIP1 (macrophage inflammatory protein), and TNF-α together define the "cytokine storm", potentially driving acute lung injury and other organ failures [55,127]. It is also suspected that these inflammatory markers cause exaggerated secondary infections and septic shocks [61]. Cardiac and muscle injury biomarkers such as troponin are elevated in severe COVID-19 patients. A significant elevation in liver enzymes (alanine aminotransferase (ALT) and aspartate aminotransferase (AST), renal biomarkers (blood urea nitrogen, creatinine), and coagulation measures (prothrombin time) indicate COVID-19-related fatality (Table 3). Table 3. Summary of various immuno-metabolic and clinical markers used for COVID-19 detection.

Managing the Disease Severity
The mainstay of management of COVID-19 is still based on classical interventions such as antipyretics administration, oxygen supplementation, ventilation, and fluid management [133]. An overview of SARS-CoV-2 detection biomarkers and an outline of the therapeutic approaches, many of which are based on repurposing of drugs initially used against other viral infections and inflammatory diseases, is summarized (Table 4) [134,135]. COVID-19-related research is a rapidly evolving field. The safety and efficacy of the agents mentioned in this article need an appropriate and time-bound evaluation.
Prevention of viral entry and replication and suppression of immune over-reaction are some of the strategies to tackle the disease severity [136]. The RNA polymerase of SARS-CoV-2lacks proofreading that enables it to accumulate mutation sat a faster rate. This is anticipated to pose a challenge for vaccine intervention. Nevertheless, several new therapeutic drugs and preventive vaccines are in the pipeline, awaiting approval from the Food and Drug Administration (FDA). As pro-inflammatory molecules are deregulated during the disease progression, several immune modulators also show the potential to inhibit the symptoms arising due to cytokine release. Specific immune modulators such as antagonists for IL-1 and IL-6 and their receptors (e.g., anakinra, tocilizumab, sarilumab), Janus kinase (JAK) inhibitors (e.g., baricitinib, ruxolitinib), and antitumor necrosis factor-α (e.g., adalimumab) show promising results. Nonspecific immunomodulators include human immunoglobulin, corticosteroids (e.g., Methylprednisolone), interferons, statins, and angiotensin pathway modulators (hydroxychloroquine and chloroquine). Other alternatives, such as CPT, recombinant antagonists, and combinational antiviral drugs are also under consideration for COVID-19 management and treatment. Although most infected cases report mild symptoms, elderly individuals and those with comorbidities are at higher risk of developing a severe disease, which may lead to death. Therefore, early prediction of disease severity using relevant biomarkers is critical for COVID-19-related fatality.

Future Perspectives
COVID-19 is still a new disease as far as our knowledge is concerned. The scientific community is working to understand its nature, transmission, pathophysiology, and remedy [159]. Researchers have developed diverse formulations of vaccines, such as nucleic-acid-based, viral-vector, and inactivated or recombinant protein to prevent SARS-CoV-2 infection [159]. Vaccination of specific age groups, such as children and young adults with a high level of immunity, would help to induce herd immunity and indirectly safeguard the elderly, immunocompromised, or unvaccinated population [25]. As the development of long-term natural immunity to SARS-CoV-2 is still under question, artificial induction of herd immunity, which depends on the effectiveness of the immune response and vaccine stability, needs serious consideration. A study estimated that the herd immunity could only be achieved if the population contained 50-66% of immune individuals, as per present infection rates [160,161]. Findings have suggested that NAbs against SARS-CoV-2 remain high for a few weeks after infection and begin to diminish thereafter, hence, even a potential vaccine may require regular boosters to achieve long-term immunity [162]. Therefore, the concerns about the immune response duration, seasonal outburst, emergence of new mutant strains, and efficacy of viral epitope-vaccine interaction need to be addressed critically. It is anticipated that immunity to SARS-CoV-2 may extend beyond antibodies. Therefore, the general public's psycho-social preparedness against COVID-19 infection should be nurtured.

Acknowledgments:
We appreciate the scientific vigor and efforts of all research scholars working in SSG, BL, and JC groups during this pandemic for obtaining the funding to study SARS-CoV-2.

Conflicts of Interest:
The authors declare no conflict of interest.