Stem Cells: An Alternative Therapy for COVID-19 and Cytokine Storm

By Subodh Kumar

Stem Cells: An Alternative Therapy for COVID-19 and Cytokine Storm provides mechanistic insights into the role of stem cells to combat COVID-19 outbreak and other pathologies where cytokines storm is the cause of concern for e.g., radiation exposure, multiple organ failure and sepsis. There has been an increase in number of cases of new diseases in the last decade, including mucormycosis, Zika virus, H1N1 influenza virus, among others. These diseases can be characterized by the induction of cytokine storm, which is mainly responsible for morbidity and mortality.

Stem cell therapy has emerged as a potential treatment for viral diseases, including, but not limited to, COVID-19. Interestingly, clinical trials in patients with COVID-19 complications depicted faster recovery in patients post mesenchymal stem cells therapy owing to the decreased cytokines levels, anti-viral effects and regeneration of the infected tissue.

• Evaluates the role of MSCs to combat cytokine storm, the challenges regarding COVID-19 therapy and how they can be countered using stem cells, and the risk of opportunistic infections post COVID-19
• Presents how stem cell therapy has emerged as a potential treatment for viral diseases, including, but not limited to, COVID-19
• Provides a detailed understanding of the novel coronavirus, with an emphasis on therapeutic aspects

• Language: English
• Publisher: Academic Press
• Release date: Nov 29, 2023
• ISBN: 9780323955461

Book preview

Preface

Yogesh Kumar Verma, Neeraj Kumar Satija, Pawan Kumar Raghav, Nishant Tyagi and Subodh Kumar

The SARS-CoV-2 gripped the whole world and brought it to its knees. No available treatment was effective during the ensuing COVID-19 pandemic; even the developed economies saw a wave of morbidity and mortality (as observed during the flu pandemic in the year 1918–19). During that phase, not much information was available on the SARS-CoV-2 virus, its pathogenesis, and treatment. Doctors and researchers together explored all the possible therapies with very little success. Individuals with strong immune systems survived without even manifesting COVID-19 symptoms. The most susceptible were those with the compromised immune systems and comorbidities, in which cytokine storm resulted in multiple organ failure and death. Clinical trials around the globe with mesenchymal stem cells showed promising results in patients infected with COVID-19 and consequent cytokine storm. Many reports and articles were published and being published on the pandemic, scattered around in media, journals, and newspapers; however, no single compilation is available on this subject matter. My department, Stem Cell and Tissue Engineering Group in the Institute of Nuclear Medicine and Allied Sciences (INMAS), India, has been working on developing technologies for Defence applications since the year 2000. During the suffocating time of the pandemic, we explored the possibility of utilizing our expertise to consolidate and disseminate the exhaustive knowledge gathered on stem cells, COVID-19 infection, and cytokine storm to develop new therapies based on stem cells. This gave us the inspiration to write this book.

This book would be beneficial to those who intend to delve into drug discovery based on stem cells in accordance with the regulatory guidelines. This book is likely to be an invaluable guide for undergraduates, postgraduates, researchers, and industry partners working in the field of infectious diseases, immunology, microbiology, stem cells, regenerative medicine, regulatory guidelines, etc. The information contained herein would communicate with the people not having fundamental knowledge in the field of stem cells with reasonable solutions suggested against future emerging threats like COVID-19 and new and mutated pathogens.

We are thankful to Ms. Elizabeth A. Brown, Elsevier, for accepting our proposal on the book and encouraging this project at the initial stages and to Ms. Liz for effective coordination. The sustained support throughout the book publication process by Matthew Mapes is highly acknowledged. We thank support by our parent Institutions for permitting us to edit this important book. We acknowledge the contributions of all the authors for their patience and excellent support, who utilized their precious time to share their learnings with us. We thank our families who were with us through thick and thin during the pandemic and supported our endeavor of compiling this edition. Dr. Yogesh Kumar Verma, in particular, thanks his wife, Sarika, and daughters, Disha and Anushka, for their continuous support during the entire project. Subodh Kumar acknowledges the support of his parents and Stem Cell and Tissue Engineering Research Group, INMAS, Delhi, India for unwavering support.

The views expressed here are solely by editors and authors and not that of the Government of India.

Introduction

Yogesh Kumar Verma, Neeraj Kumar Satija, Pawan Kumar Raghav, Nishant Tyagi and Subodh Kumar

There has been an increase in the number of cases of new diseases in the last decade such as mucormycosis, Zika virus, H1N1 influenza virus, and novel coronavirus (SARS-CoV-2). These diseases can be characterized by the induction of cytokine storm, which is mainly responsible for morbidity and mortality. The advent of SARS-CoV-2 resulted in a global pandemic known as COVID-19. It is also referred to as a disease that should have never happened. The whole world witnessed a massive shortage of medical and other essential supplies needed to combat the virus. Stem cell therapy has emerged as a potential treatment for viral diseases, including, but not limited to, COVID-19. Interestingly, the clinical trials in the patients having COVID-19 complications depicted faster recovery in patients postmesenchymal stem cell (MSC) therapy owing to the decreased cytokines level, antiviral effects, and regeneration of the infected tissue. The MSC therapy restored the levels of cytokines and trophic factors post-COVID-19 infection, underscoring the fact that this therapy could be explored as a complementary therapy to alleviate suffering in COVID-19 patients.

This book is divided into 12 sections containing relevant chapters on the COVID-19 outbreak (one chapter), biology (one chapter), complications (two chapters), therapeutic approaches (seven chapters), opportunistic infections (one chapter), cytokine storm (three chapters), computational approaches for COVID-19 drug development (one chapter), stem cells as therapeutics (two chapters), immunomodulation by stem cells (two chapters), constraints of stem cell therapy (two chapters), their research guidelines (one chapter), and the socioeconomic community’s understanding of stem cell treatment (one chapter). All contributors are recognized internationally as experts in their respective fields, who have reviewed their thoughts, concepts, research, and implications.

The information in this book provides mechanistic insights into the role of stem cells in combating the COVID-19 outbreak and other pathologies where cytokine storm is the cause of concern, e.g., radiation exposure, multiple organ failure, and sepsis. An extensive review has been provided wherein we collated information on SARS-CoV-2 biology, including replication, pathogenesis, and epidemiology, specifically the role of cytokine storm in the progression of the disease. It also discusses the pathogenesis of multiple organ failure, sepsis, and independent pathologies, which further resulted in a synergistic epidemic (syndemic). Preventive measures and potential therapeutics against COVID-19 have been summarized, including brief ongoing clinical trials and several in silico analyses, which would likely provide a better understanding of the subject.

Part 1

Outbreak of novel disease

Outline

Chapter 1 Targets of SARS-CoV-2: therapeutic implications for COVID-19

Chapter 1

Targets of SARS-CoV-2: therapeutic implications for COVID-19

Rajni Chadha¹, Aditya Raghav¹, Basudha Banerjee¹, Anugya Sengar², Manisha Sengar³ and Pawan Kumar Raghav⁴,    ¹BioExIn, New Delhi, India,    ²Army Hospital Research and Referral (R&R), New Delhi, Delhi, India,    ³Department of Zoology, Deshbandhu College, University of Delhi, Delhi, New Delhi, India,    ⁴Stem Cell Facility, DBT-Centre of Excellence for Stem Cell Research, All India Institute of Medical Sciences, New Delhi, India

Abstract

The emergence of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and its subsequent manifestation as coronavirus disease-2019 (COVID-19) marked a global health crisis. The rapid and widespread transmission of COVID-19 prompted the World Health Organization (WHO) to declare it a pandemic, necessitating urgent exploration of its underlying mechanisms and therapeutic options. This chapter delves into the intricate pathogenesis of SARS-CoV-2, focusing on its entry into host cells through the interaction between the viral spike protein (S-protein) and the angiotensin-converting enzyme 2 receptor. The consequences of SARS-CoV-2 infection are severe, particularly in the lungs, where it triggers a cascade of events, including cytokine storms, leading to acute respiratory distress syndrome and mortality. In light of these challenges, this chapter provides a comprehensive overview of potential therapeutic targets within human host cells, shedding light on their interactions with coronaviruses. Furthermore, it explores the current landscape of safe and effective treatment modalities, encompassing potential vaccines, immunomodulatory strategies, cold plasma therapy, low-dose radiation therapy, mesenchymal stem cell interventions, monoclonal antibody therapies, anti-inflammatory agents, and immune-boosting multivitamins, all aimed at bolstering the immune response to combat the formidable threat posed by COVID-19.

Keywords

COVID-19; SARS-CoV-2; angiotensin-converting enzyme 2 (ACE2); WHO; S-protein

1.1 Introduction

In December 2019, a cluster of 40 patients linked to the Wuhan seafood market suffered from pneumonia-like symptoms from a novel coronavirus (2019-nCoV), later named SARS-CoV-2 by the WHO [1]. The first case of coronavirus disease-2019 (COVID-19) caused by severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) was reported in 2019 in China [2]. The Center for Disease Control and Prevention (CDC) and the Hubei Provincial CDC conducted a study. They declared that the epidemic was an outbreak with increased cases and deaths [2]. The SARS-CoV-2 genome enters the host cell following the binding of the spike protein (S-protein) to the cell surface receptor of the host, known as angiotensin-converting enzyme 2 (ACE2), thereby leading to the development of COVID-19.

The three-dimensional (3D) structure S-protein of coronavirus resembles a crown that belongs to orders Nidovirales coronaviruses (CoVs), family Coronaviridae, and subfamily Coronavirinae. The coronaviruses are separated into four subgroups [3]:

1. Alphacoronavirus

2. Betacoronavirus

3. Gammacoronavirus

4. Deltacoronavirus.

Currently, there are seven infectious targets of human coronaviruses (HCoVs): The first two belong to alphacoronaviruses: HCoV-NL63 and HCoV-229E, and the other five come under the subgroup betacoronaviruses: HCoV-HKU1, HCoV-OC43, SARS-CoV, MERS-CoV, and SARS-CoV-2 [4]. The S-proteins of SARS-CoV-2 and SARS-CoV use ACE2 receptors for their cellular entrance and pathogenesis, while MERS-CoV uses dipeptidyl peptidase (DPP4) for their subsequent cell entry. The viral entry is also facilitated by priming via TMPRSS2, followed by the fusion of viral and host cell membranes. As ACE2 plays a pivotal role in the renin-angiotensin system (RAS) mechanism, its downregulation causes an increase in angiotensin II (Ang II), leading to inflammatory responses. Prolonged viral infection causes several adverse effects from mild respiratory illness to deteriorating lung injury such as lower respiratory tract infection, pneumonia, and acute respiratory distress syndrome (ARDS), leading to death. Furthermore, ACE2 has a beneficial impact on several health conditions, such as diabetes, cardiovascular disease, and hypertension, in cases where there is a decrease in protein expression [5].

This chapter delves into the intricate interaction between the S1 subunit of the S-protein and the host's receptor, ACE2. This interaction serves as the gateway for coronaviruses to enter the host's cells, ultimately triggering a cascade of events, including the potentially devastating cytokine storm and various pathological manifestations [6]. Furthermore, cleavage of the S2 protein, followed by priming via the serine protease TMPRSS2 and the S1/S2 sites, facilitates the fusion of viral and host cellular membranes, a process regulated by the S2 subunit (as depicted in Fig. 1.1A). Various potential treatment approaches have been employed to combat COVID-19, as illustrated in Fig. 1.1B. Cold plasma therapy disrupts viral structure and integrity through the use of reactive oxygen and nitrogen species, resulting in viral inactivation. Convalescent plasma therapy transfers neutralizing antibodies from recovered individuals to those currently in need. Immune-modulating treatments and low-dose radiation therapy (LDRT) enhance anti-inflammatory macrophages while reducing pro-inflammatory cells. Mesenchymal stem cell (MSC)-based therapies release molecules that contribute to immune regulation and possess regenerative properties. Potential vaccines stimulate T helper cell responses, B cell responses, and the production of plasma cells and memory cells. Monoclonal antibodies (MAb) such as tocilizumab, meplazumab, bevacizumab, vilobelimab, and eculizumab, along with multiple anti-inflammatory drugs and multivitamins, bolster immune responses. These therapeutic strategies, including the development of effective drugs, vaccines, antibodies, and inhibitors targeting the virus's specific binding domains, have played a pivotal role in significantly enhancing patient survival rates.

Figure 1.1 (A) The pathogenesis of SARS-CoV-2 and subsequent pathological consequences, including severe immune deficiency, organ (liver and kidney) malfunction, secondary bacterial infection, and myocardial infection. (B) Treatment modalities used to combat COVID-19 such as cold plasma therapy, convalescent plasma therapy, immune-modulating treatments, low-dose radiation therapy, mesenchymal stem cell-based therapies, potential vaccines, monoclonal antibodie, multiple anti-inflammatory drugs, and multivitamins.

1.2 Human targets and pathogenicity of SARS-CoV and SARS-CoV-2

1.2.1 ACE2

ACE2 is a cellular receptor present in humans as well as in bats. It is a carboxypeptidase, a type-1 integral membrane glycoprotein whose expression and activity are significant in most tissues. Primarily, ACE2 is expressed in the kidneys, lungs, and heart [7,8]. The S-protein of coronaviruses interacts with the host cells’ ACE2 receptors, and antibodies acting against the S-protein of SARS-CoV can offer some defense against SARS-CoV-2 (Fig. 1.1A). ACE2 degrades Ang II, formed by the metabolism of angiotensin I (Ang I) to generate Ang 1–7; in that way, it negatively regulates the RAS mechanism. It shares some homology with ACE, but ACE inhibitors do not inhibit ACE2. It cleaves other molecules like vasoactive peptides, neurotensin, kinetensin, and des-Arg-bradykinin [7]. The noncatalytic region at the C-terminal domain of ACE2 displays 47.8% sequence similarity with collectrin that plays a crucial role in the reabsorption of neutral amino acids from the kidney and intestine [9]. The ACE2 cytoplasmic tail contains calmodulin-binding sites, influencing the discharge of the catalytic ectodomain [10]. The ACE2 of humans and domestic animals shared a significant sequence similarity, suggesting that viral domains may interact with a broader spectrum of host cells. Majorly known for regulating the RAS mechanism, it also plays a vital role in various health conditions, including hypertension and the cardiovascular system.

The RAS is a homeostatic regulation system that plays an essential factor in vascular function through signaling pathways [6]. It regulates blood pressure, blood volume, natriuresis, blood flow, and control of trophic responses during various stimuli. RAS contains various effector peptides and regulatory components that help to control vascular function dynamism in normal and abnormal conditions. In the RAS mechanism, carboxypeptidase ACE metabolizes Ang I to convert it into Ang II. Ang II gets further metabolized to Ang 1–7 with the help of ACE2 [7,11,12]. Ang 1–7 elicits anti-inflammatory responses and anti-inflammatory effects and acts as an antagonist to Ang II. They can inhibit atherosclerosis and vascular dysfunction in apolipoprotein E knock-out mice possibly through the Mas receptor and Ang type 2 receptor [13]. Regarding pathology, RAS, ACE, and Ang II are considered the primary targets for clinical interventions, while conventional ACE inhibitors cannot antagonize ACE2 activity.

1.2.1.1 Effect of ACE2 on various diseases

ACE2 in Atherosclerosis: Ang 1–7 might be essential in reducing atherosclerotic plaque [14,15]. ACE2 has a spectrum of antioxidant and anti-inflammatory effects that resist those of the vasculature associated with Ang II. It is also probable to have a crucial role in developing atherosclerotic plaque. However, pro-atherosclerotic states like diabetes have reduced the expression of ACE2 [16]. Replenishment of ACE2 may be a method to reduce atherosclerosis. It could be achieved by suppressing RAS activation with ACE inhibitors.

ACE2 in hypertension: The deficiency of ACE-2 is related to systolic hypertension. A study showed that in response to a higher accumulation of Ang II in kidneys, ACE2 knockout mice developed intensified hypertensive responses [17]. Reportedly, spontaneously hypertensive rates suppress the activity of Ang 1–7. These were under RAS blockade [ACEi and ARBs (AT1R blockers)] and annulled the effect of the antihypertensive response of these agents [18,19]. The genetic ACE2 and RAS are linked in the pathogenesis of central hypertension.

ACE2 in heart failure: Preclinical studies in mice indicate that ACE2-deficient mice exhibited early cardiac hypertrophy [20]. It accelerated ventricular remodeling and opposed postmyocardial infarction in the heart. Cardiac pressure overload was parallel to progressive cardiac fibrosis [21]. The cardiac ACE/ACE2 balance plays a crucial role in driving heightened cardiac dysfunctions within the heart.

ACE2 in chronic kidney disease: A significant ACE2 expression is mainly seen in the proximal convoluted tubule at the intestinal brush border of the kidneys. The differential expression pattern of ACE2 in the glomerulus and tubules may be a determining factor for progressive renal disease. In the healthy individual, ACE2 plays a vital role in RAS but a small role in the regulation of renal development. The Ang II product, Ang 1–7, has minor effects in a normal state while eliciting positive effects in abnormal conditions like diabetic kidney and renal damage [22,23].

ACE2 in the lung: Pulmonary ACE2 plays a crucial role in Ang 1–7/Ang II balance. Dysregulation and the accumulation of Ang II would induce pulmonary vasoconstriction in response to hypoxia, leading to acute lung injury [24]. Higher levels of Ang II would trigger vascular permeability, causing pulmonary edema [25]. RAS helps to regulate oxygenation as overall lung injury that would lead to a complete pulmonary shutdown in ARDS.

1.2.2 TMPRSS2

During the entry of the SARS-CoV-2 genome into the host cell, the S-protein of SARS-CoV-2 undergoes priming by a cellular serine protease known as TMPRSS2 [5,26,27]. It is an endothelial cell surface protein involved in the cleavage of S-protein at two sites: S1/S2 and the S2 site, hence allowing the cellular and viral membranes’ fusion process to be run by the S2 subunit (Fig. 1.1A). TMPRSS2 inhibitors blocks the entry of viruses and may be a potent option for treatment. However, it is unidentified whether SARS-CoV-2 S-protein engages ACE2 and TMPRSS2 to facilitate entry into the host cell. A study reported that using N-0385, a TMPRSS2 inhibitor blocks multiple CoV variants, including B.1.1.7, B.1.351, and B.1.617.2 in mice [28].

1.2.3 Dipeptidyl peptidase 4 (DPP4)

Unlike SARS-CoV and SARS-CoV-2 (having a similar receptor binding domain), DPP4 is the receptor for MERS-CoV in humans. MERS-CoV genomic sequence is closely related to the HKU4 and HKU5 coronaviruses in bats with ~75% nucleotide sequence homology [29]. Although bats are the reservoir hosts of coronaviruses and assist MERS-CoV as the gene pool, humans are affected by MERS-CoV through Arabian camels (one-hump camels) and not directly from bats. The investigation indicated that camels serve as a natural reservoir host for MERS-CoV, and the MERS-CoV originating from Arabian camels closely resembled the MERS-CoV identified in humans [30].

The fusion protein belonging to trimeric class-I, known as the S-protein, occurs in a metastable prefusion conformation. Upon activation, binding the S1 subunit to the cell receptor in the host induces a significant structural rearrangement. This cascade begins when the S1 subunit interacts with the cellular receptor of the host. The prefusion trimer gets destabilized by S1 subunit shedding and conformation changes in the S2 subunit. After occupying a cell receptor in the host, the S1 RBD goes through conformational movements that momentarily either expose or hide the RBD. These two states are denoted as the up and down conformations, where they determine the receptor’s accessibility or inaccessibility, respectively. Antibody-mediated neutralization can be accomplished using the S-protein. Its structure elucidated at the atomic level would help in designing vaccines.

1.3 Treatment of COVID-19

The rapid and widespread outbreak of COVID-19 has placed significant demands on healthcare systems worldwide. Typically, individuals with mild to moderate symptoms do not necessitate specialized treatment. However, those with severe cases require tailored therapeutic interventions, taking into account any underlying health conditions. Currently, there are multiple treatment modalities accessible for combating COVID-19 (Fig. 1.1B).

1.3.1 Immunological role against SARS-CoV-2 and SARS-CoV

As SARS-CoV-2 and SARS-CoV share a close resemblance, studying the defensive activity of cell-mediated and humoral immune reaction pathways against SARS-CoV could be used to design new vaccines for SARS-CoV-2. Studies on mouse models indicate the protective function of antibodies against the S-protein of SARS-CoV [31–33]. Furthermore, numerous observations indicate a high immune response triggered by antibodies against the N protein in SARS-CoV-infected patients [34]. However, these antibodies are short-lived, while the effectiveness of T-cell responses is long term about 11 years post-infection [35–40]. This has invited the attention of designing the SARS-CoV vaccine [41]. Cytotoxic T cells play a pivotal role in eradicating respiratory viruses and contribute to establishing enduring cellular immunity. The most prominent T cell epitopes are predominantly located in three key structural proteins of SARS-CoV-2: the S, M and N proteins [42,43]. It was also observed that the T and B lymphocyte epitopes against SARS-CoV are identical without any mutations to SARS-CoV-2 sequences and might trigger a cross-reactive response against SARS-CoV-2.

Furthermore, the analysis of SARS-CoV-2 at the atomic level could help to enhance the protein expression and its antigenicity for designing vaccines. The 3D structural data can help simplify the assessment of S-protein alterations due to genetic drift, identify surface-exposed residues, and map sites of known antibody epitopes for other coronavirus S-proteins. Additionally, the atomic-level part would help to identify small molecules that could be potent fusion inhibitors.

1.3.2 Suppression of the HCoV-229E virus by interferon (IFN)

The practice of IFN in the treatment of HCoV-infected began in 1960. The direction of IFN use improved the seriousness of symptoms in HCoV-229E affected volunteers. This implies that IFN consequently inhibited the replication of HCoV-229E and plays a vital role in the defense mechanism [44,45]. Since IFN can profitably hinder the early stages of viral replication, downregulation of IFN production and signaling can aggravate the disease conditions.

1.3.3 Cold plasma treatment

The cold plasma treatment can serve as a novel strategy for the inactivation of the virus. The reactive oxygen and nitrogen species (RONS) are the primary factor for the cold plasma technique, while UV radiation and temperature fluctuation contribute a minimal factor or have no effect. Using high-powered cold plasma can disrupt the viral integrity at cellular and genomic levels, where the RONS can change or damage the conformation of capsid proteins, leading to the inactivation of the virus [45]. Cold plasma treatment damages the capsid protein and degrades the nucleic acid of viruses such as T4 bacteriophage, λ-phage, Newcastle Disease virus (NDV), and Feline calicivirus (FCV) [46–49]. The damage to nucleic acid and capsid of the influenza virus had variations in the components of lipids in the envelope [50]. Nucleic acid degradation was also observed in tobacco mosaic virus (TMV), potato virus (PVY), and respiratory syncytial virus (RSV) [51–54]. Hence, the cold plasma technique is environment-friendly and is observed to be effective in both plant- and animal-infecting viruses.

1.3.4 Convalescent plasma therapy

Convalescent plasma therapy for COVID-19 is based on transferring specific antibodies from recovered individuals to those currently infected, a concept historically successful in managing various viral illnesses [55]. Convalescent plasma contains neutralizing antibodies (NAbs) that specifically target critical regions of the SARS-CoV-2 spike protein, effectively blocking viral entry and replication [56]. Additionally, various immune pathways may contribute to its therapeutic effects, such as antibody-dependent cellular cytotoxicity (ADCC), phagocytosis, and complement activation [57,58]. The ongoing quest to determine the efficacy of convalescent plasma in treating COVID-19 remains characterized by mixed results despite extensive and well-designed randomized trials involving more than 21,000 patients [59]. While passive immunotherapy, notably exemplified by monoclonal-antibody therapy, exhibits promise in specific clinical scenarios, mainly when administered early in the disease course or to individuals with inadequate antibody responses, the effectiveness of convalescent plasma continues to elude a clear definition. Several factors contribute to this persistent need for more clarity. Due to their substantially higher antibody content compared to convalescent plasma, monoclonal antibodies appear to yield more favorable outcomes. However, emerging SARS-CoV-2 variants have displayed resistance to monoclonal antibodies, potentially leading to increased transmissibility and virulence [60,61]. Notably, convalescent plasma derived from donors recovering from natural infection does not confer significant benefits to unselected patients in emergency departments or hospital settings. Moreover, fully vaccinated individuals derive minimal advantage from convalescent plasma [62]. Although clinical trials suggest a potential role for convalescent plasma in outpatient settings, caution is essential before adopting it as standard care, given the lack of replication in other trials and the unique biological effects associated with control plasma used in these studies [62,63]. The two ongoing trials, such as COVID-19 (NCT05271929) and REMAP-CAP (NCT02735707), hold promise in shedding more light on convalescent plasma’s utility, particularly in specific patient populations and using higher-titer plasma. International collaboration will be imperative to swiftly and comprehensively assess convalescent plasma’s effectiveness during future pandemics. The intent of clarity regarding convalescent plasma's effectiveness in COVID-19 treatment endures, underscoring the importance of evidence-based therapies to secure the best patient outcomes and the responsible utilization of this treatment option.

1.3.5 Immune-modulating treatment

Formerly conversed immune evasion, infection mechanisms, and innate and adaptive immune response dysregulation are significant concerns among patients undergoing immune-modulating therapies. These patients were mostly associated with systemic or malignant autoimmune diseases. A small COVID-19 cohort study showed that the potential risk factors leading to poor outcomes were old age, the existence of comorbidities, male sex, obesity, coronary heart ailment, kidney ailment, and chronic obstructive pulmonary ailment [64]. There may also be a chance of secondary infection like bacterial pneumonia in COVID-19 patients as the disease is associated with lymphopenia. At the same time, other immune-modulating medicines may protect from viral infections. Additionally, immune-modulating medicines (classical and biologic DMARDs, antimalarial, and other drugs) can avert and regulate cytokine storm syndromes. Unregulated termination of such treatment may result in inflammatory, autoimmune conditions or recurrence of malignancies and organ refusal in transplant patients, which may enhance the chances of viral infection. The ACR, EULAR, and other national and international societies have recommended treatments for asymptomatic conditions under the supervision of clinical services (ACR. Coronavirus Disease; EULAR. EULAR Guidance for patients COVID-19) [65,66]. There is a requirement for international collaboration to monitor, audit, and safely assess the risk factor of any individual in these vulnerable patient groups.

1.3.6 Low-dose radiation therapy

X-ray was used initially to treat pneumonia by Edsall and Pemberton in 1907 [67]. The death range in COVID-19 patients from pneumonia and ARDS ranges from 2% to 15% [68–70]. The result of a pilot study of 10 patients on the efficacy of LDRT in COVID-19 patients with moderate to severe risk showed success with a 90% recovery rate. This revealed that LDRT administered before the cytokine storm improves the survival outcome of the patients [71]. LDRT (<100 cGy) has been known to increase anti-inflammatory macrophages and T cells. Inversely, it decreases pro-inflammatory macrophages, which counterattacks the cytokine storm caused due to COVID-19 and reduces death risk [72].

1.3.7 Mesenchymal stem cells in COVID-19 treatment

MSCs can be isolated from several sources like bone marrow, wharton's jelly, amniotic fluid, umbilical cord (UC), adipose tissue, and dental pulp [73–75]. According to the International Society for Cellular Therapy (ISCT), an ideal MSC should have the adherence properties to plastic during in vitro culture, the presence of specific cell surface markers CD105, CD90, and CD73, and the ability to differentiate into mesodermal cell lineage [76,77]. Mesenchymal stem cells (MSCs) secrete a variety of molecules that play roles in differentiation, immune regulation, and regenerative properties. These secreted molecules have a positive impact on type II alveolar epithelial cells [78].

Soluble human leukocyte antigen-G (HLA-G) secreted by MSCs is recognized for its ability to reduce T-cell proliferation and dendritic cell activity. This, in turn, inhibits the production of cytokines such as GM-CSF, TNF-α, IL-9, IL-8, IL-7, and IL-6. On the contrary, HLA-G promotes the secretion of TGF-β and IL-10, which have an inhibitory effect on the proliferation of cytotoxic T cells (Tc) [79]. The allogeneic transplantation of MSCs is considered safe due to MHC class II negative cells, which can bypass the need for immunosuppressive treatments [80].

Previously, MSCs have shown positive results in treating idiopathic pulmonary fibrosis, and pulmonary hypertension [81]. In COVID-19, once SARS-CoV-2 enters the respiratory tract, it triggers immune cell infiltration and a cytokine storm, which subsequently result in pathological consequences. A reduction in the proportion of immune cells infiltrating the lung, facilitated by stem cell infusion, led to the repair of lung damage. The intravenous administration of MSCs resulted in enhanced conditions for COVID-19 patients [82]. In another study, UC-MSCs treated group received intravenous infusions without any serious adverse events. The treatment led to reduction on inflammatory molecules including IL-6, TNFα, GM-CSF. Importantly, there was no significant difference in viral load at baseline between the treatment and control groups, confirming the safety of UC-MSCs infusions in COVID-19 ARDS [83].

MSC transfusions are also associated with risks of impaired MSC proliferation, contamination of products, infections, tumors, and thrombus development. Critical COVID-19 patients are at risk of thromboembolism and disseminated intravascular coagulation [84,85]. TF/CD142 expressed by MSC products is a procoagulant tissue factor that stimulates prothrombotic activity in COVID-19 [86]. The long-term culture of MSCs includes higher passaging and decreased telomerase activity. In such cases, MSCs show morphological alterations and dysregulated differentiation properties [87]. Clinical applications of MSCs require a set of proper standard guidelines regulating treatment efficacy and success rates [88]. Factors such as the source of MSCs, their state (fresh or frozen), the timing of administration, co-treatment approaches, and the method of delivery must all be carefully considered when implementing any procedure [89].

1.3.8 Potential vaccines

Currently available COVID-19 vaccines are categorized into several types, including inactivated vaccines such as CoronaVac and COVAXIN, which generate antibodies against multiple epitopes [90,91]. These inactivated vaccines, however, require higher biosafety levels due to their use of live viruses. COVAXIN, in phase III clinical trials (NCT04641481), showed a response rate of 80.6%, while CoronaVac (NCT04456595) induced an 84% immune response in patients requiring medical attention and 51% in normal cases [92]. Viral vector vaccines like AZD1222 (AstraZeneca—University of Oxford), Ad26.COV-2-S (Johnson & Johnson), and Covishield (Serum Institute of India) do not contain live virus but instead include viral polypeptides to recruit T helper cells (Th1) [92]. Ad26.COV-2-S demonstrated an efficacy of 66.3% in phase III clinical trials (NCT04614948) [93]. AstraZeneca's vaccine completed phase III trials in the UK (NCT04536051) and phase II/III trials in India (CTRI/2020/08/027170) with efficacy rates of 76% and 90%, respectively [94–96].

Covovax and Novovax represent protein subunit vaccines that enhance immune responses by recruiting T helper 1 (Th1) cells [97]. Notably, Novovax exhibits a superior ability to elicit antibodies capable of effectively neutralizing the SARS-CoV-2 virus compared to inactivated or viral vector vaccines [98]. Phase III clinical trial data (NCT04611802) reveal an impressive efficacy of 96% against the wild strain and 86% against the B.1.1.7 (alpha) variants [99–101].

BNT162b2 (Pfizer) and mRNA-1273 (Moderna) represent mRNA vaccines that employ viral proteins encapsulated in vectors renowned for their capacity to induce robust immune responses. These vaccines are particularly adept at stimulating germinal center B-cell responses and memory cell production, including plasma cells and memory cells, in addition to eliciting the conventional Th1 cell response [102,103]. In phase III clinical trials, BNT162b2 exhibited an impressive efficacy of 95%, while mRNA-1273 demonstrated strong efficacy at 94.1% [101,104]. mRNA vaccines offer the advantage of rapid production as they solely require protein synthesis. Nonetheless, they are linked to myocarditis risks [105,106] and necessitate stringent storage conditions due to mRNA instability. It's important to note that the high mutation rate of the virus has impacted vaccine efficacies [107].

The WHO has identified various variants including B.1.1.7, B.1.351, P.1, B.1.617.2, and B.1.1.529, each influencing vaccine efficacy differently. Implementing advanced strategies to enhance vaccine effectiveness is crucial for reducing infection risk and improving overall patient survival. Moderate decrease or negligible difference in nAb activity (1.7 to 6.0 fold) was provided by mRNA vaccine BNT 162b2 against B.1.1.7 virus. Interestingly, the nAB activity was reduced for P.1 (5.1 fold) and B.1.617.2 (1.4 to 3.0 fold) variants and significantly decreased (6.5 to 10.4 fold) against B.1.351 variants. Another mRNA based vaccine, mRNA-1273 also showed negligible to very less difference in nAB activity against B.1.1.7 but a significant reduction (6.4 fold) was observed against B. 1.351 variant. The nAB activity significantly decreased in P.1 and B.1.427/429 variants when compared to B.1.351. Notably, mRNA vaccines have shown reduced efficacy against the B.1.351 and P.1 variants [108,109]. The introduction of nanoparticle vaccines has demonstrated enhanced neutralizing effects [110]. Additionally, the use of adjuvants like alum [111], Matrix-M [112], and MF59 [113] has proven effective in boosting immune responses.

1.3.9 Monoclonal antibodies

COVID-19 has been associated with elevated levels of IL-6, a crucial player in immune responses, inflammation, and the development of cytokine storms [114]. The severity of the disease correlates with the level of IL-6, with mild cases exhibiting lower IL-6 levels and severe cases having higher levels. Initially, corticosteroids were employed to manage cytokine storms, but their high dosages and associated side effects prompted the use of tocilizumab (TCZ), a monoclonal antibody targeting IL-6 receptors. Recent research has assessed TCZ's efficacy among moderately, severely, and critically ill patients [115]. The findings suggest that critically ill patients require multiple TCZ doses, as a single dose often fails to improve their condition. The extended half-life of TCZ allows for reduced dosing frequency. TCZ binds to IL-6 receptors, leading to an initial surge in serum IL-6 levels, followed by a later decline, indicative of improved clinical outcomes [116]. Another study indicates that patients experience decreased oxygen requirements, peripheral blood lymphocyte counts, and C-reactive protein levels within five days of TCZ administration [117]. Multiple studies consistently highlight the high efficacy of TCZ in the treatment of COVID-19.

Other monoclonal antibodies (MAbs) like meplazumab, bevacizumab, vilobelimab, and eculizumab have also been introduced. Meplazumab, an anti-CD147 MAb, reduces inflammation by preventing the virus from entering host cells. Bevacizumab, an anti-vascular endothelial growth factor (VEGF) agent, lowers elevated VEGF levels associated with COVID-19. It has been administered to COVID-19 patients experiencing aggravated acute respiratory distress syndrome (ARDS) and pulmonary edema. Vilobelimab operates by inhibiting the C5 protein in the complement system, resulting in reduced inflammation [118].

1.3.10 Other treatments

A variety of inhibitors targeting human receptors have proven to have a significant improvement in patient survival conditions. ACE2 inhibitors (ACEI) prevent hypertension by blocking the ACE2 receptors. FDA-approved drugs captopril and ramipril are ACEI that target and show competitive binding with ACE2 receptors that may prevent viral invasion. Similarly, recombinant human ACEs mimicking the ACE2 receptors can be used. APN01 prevents the binding of the spike protein to the ACE2 receptor and prevents viral entry. MPRSS2 serine protease inhibitors, camostat mesylate, and nafamostat mesylate prevent the interaction of TMPRSS2 with spike proteins. This blocks the viral entry into host cells and decreases the risk of infection [118].

Anti-inflammatory compounds are known to deal with the extreme inflammation caused by a cytokine storm in COVID-19. Anti-inflammatory drugs such as dexamethasone, prednisone, and methylprednisolone, suppress the release of IL-6, IL-8, and IL-12. These drugs control inflammation and combat the cytokine storm [119]. The WHO recommends that patients with severe or critical COVID-19 cases follow a corticosteroid regimen.

Multivitamins are boosters of the immune system that strengthen the immune responses. Zinc is known for its antioxidative and anti-inflammatory properties, significantly reducing SARS-CoV inflammation. Zinc also acts on the viral entry points and modulates ACE2 and TMPRSS2 functions [120]. Several other therapies exist for human cells or stem cells to eradicate COVID-19 [121,122].

1.4 Conclusion and future perspectives

In conclusion, the COVID-19 pandemic, triggered by the emergence of SARS-CoV-2, has ushered in an era of unprecedented challenges for global health. Understanding the intricate interplay between the viral spike protein (S-protein) and the host receptor ACE2 has shed light on the pathogenesis of the disease, providing crucial insights. Therapeutic advancements have been diverse, from innovative treatments like cold plasma therapy, low-dose radiation therapy, and mesenchymal stem cell-based therapies to developed vaccines and monoclonal antibodies. These interventions have significantly improved patient outcomes and bolstered immune responses. Beyond COVID-19, the interaction between ACE2 and SARS-CoV-2 has far-reaching implications for various health conditions, including hypertension, cardiovascular disease, kidney disease, and heart failure. This has opened new avenues for research into the renin-angiotensin system (RAS) and ACE2 modulation. Looking ahead, ongoing research into the molecular mechanisms of SARS-CoV-2 infection and the development of targeted therapies, vaccines, and inhibitors holds the promise of further enhancing patient survival rates. Robust prevention strategies and global preparedness for potential future pandemics are also crucial. Moreover, exploring immunological responses, structural analysis, interferon therapy, convalescent plasma treatment, and immunomodulating treatments continue to be vital in the fight against COVID-19. The advent of multiple vaccines and the management of virus variants demand ongoing research and development. In summary, the landscape of COVID-19 treatment is dynamic, evolving through international collaboration and rigorous research. As we progress, these efforts offer hope in the battle against COVID-19 and our readiness to face future global health challenges.

References

1. Bogoch II, Watts A, Thomas-Bachli A, Huber C, Kraemer MU, Khan K. Pneumonia of unknown aetiology in Wuhan, China: potential for international spread via commercial air travel. J Travel Med. 2020;27 taaa008.

2. Truscott J, Fraser C, Cauchemez S, et al. Essential epidemiological mechanisms underpinning the transmission dynamics of seasonal influenza. J R Soc Interface. 2012;9(67):304–312.

3. Woo PC, Lau SK, Lam CS, et al. Discovery of seven novel Mammalian and avian coronaviruses in the genus deltacoronavirus supports bat coronaviruses as the gene source of alphacoronavirus and betacoronavirus and avian coronaviruses as the gene source of gammacoronavirus and deltacoronavirus. J Virol. 2012;86(7):3995–4008.

4. Corman VM, Muth D, Niemeyer D, Drosten C. Hosts and sources of endemic human coronaviruses. Adv Virus Res. 2018;100:163–188.

5. Crowley SD, Gurley SB, Oliverio MI, et al. Distinct roles for the kidney and systemic tissues in blood pressure regulation by the renin-angiotensin system. J Clin Investig. 2005;115(4):1092–1099.

6. Glowacka I, Bertram S, Müller MA, et al. Evidence that TMPRSS2 activates the severe acute respiratory syndrome coronavirus spike protein for membrane fusion and reduces viral control by the humoral immune response. J Virol. 2011;85(9):4122–4134.

7. Donoghue M, Hsieh F, Baronas E, et al. A novel angiotensin-converting enzyme–related carboxypeptidase (ACE2) converts angiotensin I to angiotensin 1–9. Circ Res. 2000;87(5):e1–e9.

8. Tipnis SR, Hooper NM, Hyde R, Karran E, Christie G, Turner AJ. A human homolog of angiotensin-converting enzyme: cloning and functional expression as a captopril-insensitive carboxypeptidase. J Biol Chem. 2000;275(43):33238–33243.

9. Zhang H, Wada J, Hida K, et al. Collectrin, a collecting duct-specific transmembrane glycoprotein, is a novel homolog of ACE2 and is developmentally regulated in embryonic kidneys. J Biol Chem. 2001;276(20):17132–17139.

10. Lambert DW, Clarke NE, Hooper NM, Turner AJ. Calmodulin interacts with angiotensin-converting enzyme-2 (ACE2) and inhibits shedding of its ectodomain. FEBS Lett. 2008;582(2):385–390.

11. Turner AJ, Hooper NM. The angiotensin–converting enzyme gene family: genomics and pharmacology. Trends Pharmacol Sci. 2002;23(4):177–183.

12. Rice GI, Thomas DA, Grant PJ, Turner AJ, Hooper NM. Evaluation of angiotensin-converting enzyme (ACE), its homologue ACE2 and neprilysin in angiotensin peptide metabolism. Biochem J. 2004;383(1):45–51.

13. Tesanovic S, Vinh A, Gaspari TA, Casley D, Widdop RE. Vasoprotective and atheroprotective effects of angiotensin (1–7) in apolipoprotein E–Deficient mice. Arteriosclerosis Thrombosis, Vasc Biol. 2010;30(8):1606–1613.

14. Probstfield JL, O'Brien KD. Progression of cardiovascular damage: the role of renin–angiotensin system blockade. Am J Cardiol. 2010;105(1):10A–20A.

15. Ferrario CM, Trask AJ, Jessup JA. Advances in biochemical and functional roles of angiotensin-converting enzyme 2 and angiotensin-(1–7) in regulation of cardiovascular function. Am J Physiol-Heart Circ Physiol. 2005;289(6):H2281–H2290.

16. Tikellis C, Johnston CI, Forbes JM, et al. Characterization of renal angiotensin-converting enzyme 2 in diabetic nephropathy. Hypertension. 2003;41(3):392–397.

17. Gurley SB, Allred A, Le TH, et al. Altered blood pressure responses and normal cardiac phenotype in ACE2-null mice. J Clin Investig. 2006;116(8):2218–2225.

18. Stanziola L, Greene LJ, Santos RA. Effect of chronic angiotensin converting enzyme inhibition on angiotensin I and bradykinin metabolism in rats. Am J Hypert. 1999;12(10):1021–1029.

19. Kakishita M, Nakamura K, Asanuma M, et al. Direct evidence for increased hydroxyl radicals in angiotensin II-induced cardiac hypertrophy through angiotensin II type 1a receptor. J Cardiovasc Pharmacol. 2003;42:S67–S70.

20. Sadoshima JI, Xu Y, Slayter HS, Izumo S. Autocrine release of angiotensin II mediates stretch-induced hypertrophy of cardiac myocytes in vitro. Cell. 1993;75(5):977–984.

21. Vickers C, Hales P, Kaushik V, et al. Hydrolysis of biological peptides by human angiotensin-converting enzyme-related carboxypeptidase. J Biol Chem. 2002;277(17):14838–14843.

22. Li N, Zimpelmann J, Cheng K, Wilkins JA, Burns KD. The role of angiotensin converting enzyme 2 in the generation of angiotensin 1–7 by rat proximal tubules. Am J Physiol-Renal Physiol. 2005;288(2):F353–F362.

23. Kiely DG, Cargill RI, Wheeldon NM, Coutie WJ, Lipworth BJ. Haemodynamic and endocrine effects of type 1 angiotensin II receptor blockade in patients with hypoxaemic cor pulmonale. Cardiovasc Res. 1997;33(1):201–208.

24. Imai Y, Kuba K, Rao S, et al. Angiotensin-converting enzyme 2 protects from severe acute lung failure. Nature. 2005;436(7047):112–116.

25. Matsuyama S, Nagata N, Shirato K, Kawase M, Takeda M, Taguchi F. Efficient activation of the severe acute respiratory syndrome coronavirus spike protein by the transmembrane protease TMPRSS2. J Virol. 2010;84(24):12658–12664.

26. Shulla A, Heald-Sargent T, Subramanya G, Zhao J, Perlman S, Gallagher T. A transmembrane serine protease is linked to the severe acute respiratory syndrome coronavirus receptor and activates virus entry. J Virol. 2011;85(2):873–882.

27. Shapira T, Monreal IA, Dion SP, et al. A TMPRSS2 inhibitor acts as a pan-SARS-CoV-2 prophylactic and therapeutic. Nature. 2022;605(7909):340–348.

28. Ge XY, Li JL, Yang XL, et al. Isolation and characterization of a bat SARS-like coronavirus that uses the ACE2 receptor. Nature. 2013;503(7477):535–538.

29. Adney DR, van Doremalen N, Brown VR, et al. Replication and shedding of MERS-CoV in upper respiratory tract of inoculated dromedary camels. Emerg Infect Dis. 2014;20(12):1999.

30. Yang ZY, Kong WP, Huang Y, et al. A DNA vaccine induces SARS coronavirus neutralization and protective immunity in mice. Nature. 2004;428(6982):561–564.

31. Deming D, Sheahan T, Heise M, et al. Vaccine efficacy in senescent mice challenged with recombinant SARS-CoV bearing epidemic and zoonotic spike variants. PLoS Med. 2006;3(12):e525.

32. Graham RL, Becker MM, Eckerle LD, Bolles M, Denison MR, Baric RS. A live, impaired-fidelity coronavirus vaccine protects in an aged, immunocompromised mouse model of lethal disease. Nat Med. 2012;18(12):1820–1826.

33. Lin Y, Shen X, Yang RF, et al. Identification of an epitope of SARS-coronavirus nucleocapsid protein. Cell Res. 2003;13(3):141–145.

34. Wang J, Wen J, Li J, et al. Assessment of immunoreactive synthetic peptides from the structural proteins of severe acute respiratory syndrome coronavirus. Clin Chem. 2003;49(12):1989–1996.

35. Liu X, Shi Y, Li P, et al. Profile of antibodies to the nucleocapsid protein of the severe acute respiratory syndrome (SARS)-associated coronavirus in probable SARS patients. Clin Vac Immunol. 2004;11(1):227–228.

36. Tang F, Quan Y, Xin ZT, et al. Lack of peripheral memory B cell responses in recovered patients with severe acute respiratory syndrome: a six-year follow-up study. J Immunol. 2011;186(12):7264–7268.

37. Peng H, Yang LT, Wang LY, et al. Long-lived memory T lymphocyte responses against SARS coronavirus nucleocapsid protein in SARS-recovered patients. Virology. 2006;351(2):466–475.

38. Fan YY, Huang ZT, Li L, et al. Characterization of SARS-CoV-specific memory T cells from recovered individuals 4 years after infection. Archives of Virol. 2009;154(7):1093–1099.

39. Ng OW, Chia A, Tan AT, et al. Memory T cell responses targeting the SARS coronavirus persist up to 11 years post-infection. Vaccine. 2016;34(17):2008–2014.

40. Liu WJ, Zhao M, Liu K, et al. T-cell immunity of SARS-CoV: implications for vaccine development against MERS-CoV. Antivir Res. 2017;137:82–92.

41. Li CKF, Wu H, Yan H, et al. T cell responses to whole SARS coronavirus in humans. J Immunol. 2008;181(8):5490–5500.

42. Channappanavar R, Fett C, Zhao J, Meyerholz DK, Perlman S. Virus-specific memory CD8T cells provide substantial protection from lethal severe acute respiratory syndrome coronavirus infection. J Virol. 2014;88(19):11034–11044.

43. Higgins PG, Phillpotts RJ, Scott GM, Wallace J, Bernhardt LL, Tyrrell DA. Intranasal interferon as protection against experimental respiratory coronavirus infection in volunteers. Antimicrobial Agents chemother. 1983;24(5):713–715.

44. Mesel-Lemoine M, Millet J, Vidalain PO, et al. A human coronavirus responsible for the common cold massively kills dendritic cells but not monocytes. J Virol. 2012;86(14):7577–7587.

45. Guo L, Xu R, Gou L, et al. Mechanism of virus inactivation by cold atmospheric-pressure plasma and plasma-activated water. Appl Environ Microbiol. 2018;84(17):e00726 -18.

46. Yasuda H, Miura T, Kurita H, Takashima K, Mizuno A. Biological evaluation of DNA damage in bacteriophages inactivated by atmospheric pressure cold plasma. Plasma Process Polym. 2010;7(3-4):301–308.

47. Su X, Tian Y, Zhou H, et al. Inactivation efficacy of nonthermal plasma-activated solutions against Newcastle disease virus. Appl Environ Microbiol. 2018;84(9):e02836 -17.

48. Aboubakr HA, Mor SK, Higgins L, et al. Cold argon-oxygen plasma species oxidize and disintegrate capsid protein of feline calicivirus. PLoS One. 2018;13(3):e0194618.

49. Sakudo A, Misawa T, Shimizu N, Imanishi Y. N2 gas plasma inactivates influenza virus mediated by oxidative stress. Front Biosci (Elite Ed). 2014;6:69–79.

50. Hanbal SE, Takashima K, Miyashita S, et al. Atmospheric-pressure plasma irradiation can disrupt tobacco mosaic virus particles and RNAs to inactivate their infectivity. Arch Virol. 2018;163(10):2835–2840.

51. Filipić A, Primc G, Zaplotnik R, et al. Cold atmospheric plasma as a novel method for inactivation of potato virus Y in water samples. Food Environ Virol. 2019;11(3):220–228.

52. Sakudo A, Toyokawa Y, Imanishi Y, Murakami T. Crucial roles of reactive chemical species in modification of respiratory syncytial virus by nitrogen gas plasma. Mater Sci Eng C. 2017;74:131–136.

53. Yamashiro R, Misawa T, Sakudo A. Key role of singlet oxygen and peroxynitrite in viral RNA damage during virucidal effect of plasma torch on feline calicivirus. Sci Rep. 2018;8(1):1–13.

54. Lai CC, Liu YH, Wang CY, et al. Asymptomatic carrier state, acute respiratory disease, and pneumonia due to severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2): facts and myths. J Microbiol Immunol Infect. 2020;53(3):404–412.

55. Casadevall A, Joyner MJ, Pirofski LA. SARS-CoV-2 viral load and antibody responses: the case for convalescent plasma therapy. The Journal of clinical investigation. 2020;130(10):5112–5114.

56. Nashaat HAH, Anani M, Attia FM. Convalescent plasma in COVID-19: Renewed focus on the timing and effectiveness of an old therapy. Blood research. 2022;57(1):6–12.

57. Rojas M, Rodríguez Y, Monsalve DM, et al. Convalescent plasma in Covid-19: Possible mechanisms of action. Autoimmunity reviews. 2020;19(7):102554.

58. Raghav PK, Mann Z, Ahluwalia SK, Rajalingam R. Potential treatments of COVID-19: Drug repurposing and therapeutic interventions. Journal of Pharmacological Sciences. 2023;152(1):1–21.

59. Estcourt L, Callum J. Convalescent plasma for Covid-19 making sense of the inconsistencies. New England Journal of Medicine. 2022;386(18):1753–1754.

60. Jary A, Marot S, Faycal A, et al. Spike gene evolution and immune escape mutations in patients with mild or moderate forms of COVID-19 and treated with monoclonal antibodies therapies. Viruses. 2022;14(2):226.

61. Ong, SWX, Chiew, CJ, Ang, LW, Mak, TM, Cui, L, Toh, MPH, ... & Young, BE. (2021). Clinical and virological features of SARS-CoV-2 variants of concern: a retrospective cohort study comparing B. 1.1. 7 (Alpha), B. 1.315 (Beta), and B. 1.617. 2