key: cord-0878778-76v5q04a
authors: Bandaru, Ravi; Rout, Smruti Rekha; Kamble, Omkar; Samal, Sangram K.; Gorain, Bapi; Sahebkar, Amirhossein; Ahmed, Farhan J.; Kesharwani, Prashant; Dandela, Rambabu
title: Clinical progress of therapeutics and vaccines: Rising hope against COVID-19 treatment
date: 2022-04-14
journal: Process Biochem
DOI: 10.1016/j.procbio.2022.04.011
sha: 924cbd3ea9e503a3d137ff2abf5b5c81c9648867
doc_id: 878778
cord_uid: 76v5q04a

Cases of deaths due to COVID-19 (COrona VIrus Disease-19) infection are increasing gradually worldwide. Immense research is ongoing to control this pandemic condition. Continual research outcomes are indicating that therapeutic and prophylactic agents are the possible hope to prevent the pandemic from spreading and to combat this increasing death count. Experience gained from previous coronavirus infections (eg., SARS (Severe Acute Respiratory Syndrome), MERS (Middle Ease Respiratory Syndrome), accumulated clinical knowledge during this pandemic, and research helped to identify a few therapeutic agents for emergency treatment of COVID-19. Thereby, monoclonal antibodies, antivirals, broad-spectrum antimicrobials, immunomodulators, and supplements are being suggested for treatment depending on the stage of the disease. These recommended treatments are authorized under medical supervision in emergency conditions only. Urgent need to control the pandemic condition had resulted in various approaches of repurposing the existing drugs, However, poorly designed clinical trials and associated outcomes do not provide enough evidence to fully approve treatments against COVID-19. So far, World Health Organization (WHO) authorized three vaccines as prophylactic against SARS-CoV-2. Here, we discussed about various therapeutic agents, their clinical trials, and limitations of trials for the management of COVID-19. Further, we have also spotlighted different vaccines in research in combating COVID-19.

agents is a tedious and time-consuming process, so repurposing existing therapeutics for the treatment of COVID-19 is considered the best choice to counteract the consistent increase of infected people [1, 21] . Many of clinical trials are in progress to establish the efficacy of the existing antivirals and other therapeutic agents in this pandemic scenario [1, 4, 9, 22, 23] .

Recent news on new variants of SARS-CoV-2 is panicking people worldwide [24] . For example, a team of experts in the UK (United Kingdom) identified a new variant known as B.1.1.7, which increases the risk of death compared to the initial variant. Furthermore, this variant is known to spread more quickly than the other reported variants [25] . In addition, new strains of coronaviruses, such as N440K and E484K, have been detected in different parts of India. Therefore, to understand the potential of such variants, extensive genome surveillance is required to save humanity from this tiny danger [26] . Thus, in this review, we focused on recent advances in repurposed therapeutic agents and the current progress of vaccine development against COVID-19 to support future researchers with a background of research progress.

SARS-CoV-2 contains non-segmented, single-stranded, positive-sense RNA as genetic material. The genome consists of 5'-terminal end, partially overlapping Open Reading Frames 1a and 1b (ORF 1a and 1b), region encoding structural and accessory proteins, and 3'-terminal. The ORF 1a and 1b occupy two-thirds of the genome and encode PolyProtein (PP) 1a (PP 1a) and PP 1ab ( Figure 2 ) [27] . The remaining genome encodes structural proteins, such as spike protein (S), envelop protein (E), membrane protein (M), nucleocapsid protein (N), and accessory proteins. The virion releases its genetic material into the cytoplasm upon entering the cell via endocytosis. After that, the ORF 1a and 1b are translated into PP 1a and PP 1ab, where these PP 1a and PP 1ab are cleaved by virus proteases, Papain-Like protease (PLpro), and 3C-like serine protease (3CLpro), to generate J o u r n a l P r e -p r o o f Non-Structural Proteins (NSPs). These NSPs include RNA-dependent RNA polymerase (RdRp) and helicase (Hel), which play a lead role in the transcription and replication of the virus genome inside the host cells [27, 28] . SARS-CoV-2 shares 94.4% amino acid sequence identity with the SARS-CoV in the ORF 1ab region of the genome [19] . Thus, RdRp and helicase enzymes are highly conserved across the coronaviruses. Hence, broad-spectrum antivirals, which are capable of inhibiting these enzymes, can probably inhibit SARS-CoV-2.

Repurposing of drugs is a better option to meet the global demand to fight against COVID- 19 . Therefore, WHO and other health agencies recommended repurposing drugs [29] .

Therefore, nucleotide analogues (e.g., Favipiravir, Ribavirin, Remdesivir, Galidesivir) are known to inhibit the RdRp enzyme in a wide range of RNA viruses [30] [31] [32] [33] , several pieces of research are prompted to establish their efficacy.

Based on effective control of COVID-19 in clinical trials, Remdesivir was approved for emergency use for hospitalized patients by Food and Drug Administration (FDA) in October 2020 [20, 34] . HIV protease inhibitors, Lopinavir, and Ritonavir were also considered to inhibit SARS-CoV-2 proteases (PLpro, 3CLpro), but WHO's solidarity clinical trials had shown that treatment with Lopinavir and Ritonavir was no better than standard treatment [35, 36] . Other small molecules that were considered for repurposing are Chloroquine and HydroxyChloroquine. Although these treatments were initiated in emergency conditions, WHO's solidarity clinical trials concluded that Chloroquine and HydroxyChloroquine are not effective in reducing mortality [36] . Various types of existing pharmacological agents can be repurposed for the treatment of COVID-19. Apart from Remdesivir, other drugs were also considered, such as Favipiravir, Elbasvir, Cepharanthine; drugs acting on viral entry, such as Arbidol, Darunavir, Nafamostat; anti-inflammatory agents like Tocilizumab, Barcitinib for their efficacy against COVID-19 [29] . The following J o u r n a l P r e -p r o o f section of the article has been elaborated on different therapeutic agents in the management of COVID-19.

It is a broad-spectrum antiviral agent known to inhibit viral RNA synthesis by impeding the RdRp enzyme of RNA viruses. Favipiravir is a prodrug that undergoes intracellular metabolism to transform to the active parent molecule triphosphate. RdRp identifies this triphosphate as a purine analogue and includes it in the nascent viral RNA strand competitively with adenine and guanine. Inclusion of Favipiravir triphosphate into nascent RNA strand inhibits further growth of RNA strand [37] .

It was originally developed to treat the influenza virus in Japan [37] . It was tested as a treatment option during 2013 against the SARS outbreak, and its therapeutic potential against SARS-CoV was established. Since SARS-CoV-2 conserves the RdRp enzyme, repurposing Favipiravir as a treatment for COVID-19 is justified (Figure 3 ) [30] . Around the world, a considerable number (forty-eight, 48) of clinical trials are in progress to test the efficacy of this drug against COVID-19 (www.clinicaltrials.gov). However, the results of these clinical trials suggest that Favipiravir is not effective in critically ill patients, and there is no evidence that it can reduce the COVID-19 associated mortality [38] . Despite these results, India and Russia have approved this drug for emergency use against COVID-19 [39, 40] .

Remdesivir (GS-5734) was first developed by Gilead Sciences, USA, to treat ebolavirus disease [41] . It is a 1'-cyano substituted adenosine nucleotide prodrug, which interferes with the viral RNA replication catalyzed by RdRp in various RNA viruses like ebolavirus, respiratory syncytial virus (RSV), SARS, MERS, etc. Recent in vitro studies showed that it could inhibit the SARS-CoV-2 (EC 50 = 0.77µM in Vero cells) [42, 43] . Another contemporary animal study on rhesus macaques showed that early administration of J o u r n a l P r e -p r o o f Remdesivir could prevent the advancement of the disease [44] . The mechanism of action of Remdesivir depicted that this antiviral agent can cause termination of premature RNA strands, which inhibits further replication of virus [45] . Similar to Favipiravir, this nucleotide analogue also undergoes intracellular metabolism to form the triphosphate of the parent nucleoside. This Nucleoside TriPhosphate (NTP) is the pharmacologically active form that competes with Adenosine TriPhosphate (ATP) in RNA synthesis. Selectivity of ATP over NTP by RdRp is ~4 folds (Figure 4 ) [43] . Incorporating NTP in the RNA chain does not affect the incorporation of immediately next nucleotide but leads to inhibition of the 5 th nucleotide. This effect leads to the termination of the RNA progression. Hence, the mechanism of action of Remdesivir is considered delayed termination. The 1'-cyano group increases the selectivity towards viral RNA polymerase. Human mitochondrial RNA polymerase can efficiently distinguish between NTP and ATP. The selectivity of ATP over NTP is over 500 folds in the case of human RNA polymerase. Thus, Remdesivir does not interfere with human RNA polymerase [43] .

Clinical trials are in progress to establish the efficacy of Remdesivir toward COVID-19 treatment. There are eighty-nine registered clinical trials to test the efficacy of Remdesivir against COVID-19. However, the results of the clinical trials are sometimes conflicting [46] .

A preliminary data of a Chinese clinical trial reported that Remdesivir treatment is not better than a placebo. However, this clinical trial was not completed because of difficulty in recruiting patients as the infection was subsided in China [47] . The preliminary results from a clinical trial of the National Institute of Allergy and Infectious Diseases (NIAID) showed a 31% faster recovery in Remdesivir treated patients compared to the placebo group. A compassionate study from Gilead Sciences showed a higher recovery rate (68%) in Remdesivir treated patients [48] . Recently, several clinical trials were conducted to evaluate the efficacy of Remdesivir when compared to standard care. The studies were conducted on J o u r n a l P r e -p r o o f moderate [49] (NCT04292730) as well as severe patients [50] (NCT04292899) of COVID-19 to study the anti-viral activity of Remdesivir. The findings from these clinical trials encounter several limitations, such as lack of uniformity in indexing the disease severity in the group. In contrast, some clinical trials had small sample sizes, some open-label studies, and some used concomitant medications. Despite these limitations, most clinical trials showed some improved efficacy against COVID-19 in hospitalized patients [51] . Moreover, there is no hard evidence that Remdesivir does not helpful in treating COVID-19. However, with the supporting evidence, US-FDA approved for emergency use authorization on 22 nd October 2020 [20] .

Use of Remdesivir is associated with adverse effects such as nausea, delayed blood clotting, increased transaminase level, and hypersensitive reactions in some. On the other hand, the patient's liver function and renal function of the patients should be closely monitored before and after administration. Thus, Remdesivir is not recommended for patients whose renal function is disturbed (glomerular filtration rate <30 mL/min) [52] . Coadministration of Chloroquine and HydroxyChloroquine had shown to reduce the antiviral activity of Remdesivir hence concomitant administration is not recommended [53] .

Alternatively, co-administration of corticosteroids did not show any effect [51] . Remdesivir is primarily recommended to hospitalized patients who require supplementary oxygen without any inflammatory signs. The basis for this recommendation is to facilitate viral clearance, as this is important to prevent the disease from progressing to a severe inflammatory stage. Combination therapy of Remdesivir and Dexamethasone (corticosteroid) has been recommended for patients with inflammatory responses who require high flow oxygen support [51] .

J o u r n a l P r e -p r o o f Monoclonal antibodies are suggested for non-hospitalized patients with mild to moderate COVID-19 who are at high risk of progression to severe conditions. Currently, three monoclonal antibody therapies are approved on an Emergency Use Authorisation (EUA) basis, including Bamlanivimab plus Etesevimab, Casirivimab plus Imdevimab, and

Sotrovimab. This category of drugs plays a vital role in neutralizing SARS-CoV-2 by binding to the viral S-protein. However, supportive and symptomatic treatment was suggested for patients who were not hospitalized, devoid of any risk factors for progressing to severe conditions. However, not enough clinical data is available to support any specific treatment in this group of patients [51] .

BLAZE-1 is a placebo-controlled, double-blind, randomized clinical trial to evaluate the efficacy of Bamlanivimab plus Etesevimab in non-hospitalized patients with high-risk factors. Surprisingly, this study showed a 70% relative reduction in the incidence of hospitalization or death. However, the results of the study were neither peer-reviewed nor published. Alternatively, the dose of Bamlanivimab plus Etesevimab utilized in this trial was 2800 mg, and 2800 mg, respectively, which were much higher than that of EUA approved dose (700 mg and 1400 mg, respectively). Additionally, in vitro study results demonstrated that Bamlanivimab possesses less neutralizing activity toward variants that exhibit E484K mutation. However, till now, no variants showed reduced susceptibility towards the combination of Bamlanivimab plus Etesevimab, and the clinical impact of this mutation is not identified [54] R10933-10987-COV-2067 is another randomized placebo-controlled, parallel study to test the safety and efficacy of Casirivimab plus Imdevimab in non-hospitalized patients with high-risk factors. Data from this study showed a 71% relative reduction in hospitalization or death in the Casirivimab plus Imdevimab treated patients compared to the placebo group. However, because of its parallel assessment design, the study design hospitalized patients with high-risk factors. The study showed an 85% relative reduction in the hospitalization or death of the patients [56, 57] .

Studies comparing the efficacies between monoclonal antibodies are not done yet.

Hence one of the above monoclonal antibodies is suggested for treatment against COVID-19 in non-hospitalized patients with high-risk factors. The criteria of considering risk factors is not uniform in the above-mentioned clinical trials; hence it is difficult to compare these treatment methods. Treatment with monoclonal antibodies is not recommended hospitalised patients due to the severity of COVID-19 [51] .

Dexamethasone is a corticosteroid, which has been approved and enlisted by WHO as an essential medicine for its immunosuppressant and anti-inflammatory properties [58] . Usually, Dexamethasone is used to treat conditions like rheumatism, allergies, skin problems, respiratory issues, ulcerative colitis, cancer, liver fibrosis, etc. [59, 60] . As COVID-19 is affecting the lives of a large population, Dexamethasone has shown the potential to rescue the lives of some critically ill patients. A clinical study at Oxford University, UK, J o u r n a l P r e -p r o o f -RECOVERY‖ (Randomized Evaluation of COVid 19 thERapY) had established the promising role of Dexamethasone. In the study, patients treated with Dexamethasone and standard treatment were compared with the outcome of patients who received only standard care [61, 62] . From the reported results of the clinical trial, it was demonstrated that the cotreatment of Dexamethasone and standard treatment to the patients on ventilator resulted in the reduction of mortality rate to one-third compared to the patients who received standard care. On the other hand, the mortality was reduced to one-fifth in patients who required oxygen support [61] . The results are quite promising to incorporate in reducing the death toll due to COVID-19 infection.

Moreover, Dexamethasone is inexpensive and can be easily accessible worldwide. However, it is not suitable for asymptomatic patients with mild symptoms [62] . Dexamethasone exhibits high affinity towards glucocorticoid receptors [63, 64] . This binding inhibits the enzyme phospholipase A2, which, in turn, is responsible for preventing the secretion of arachidonic acid. Such action of Dexamethasone further suppresses the formation of cytokines [65] . It has been well documented that increased production of cytokines results in ‗cytokine storm,' which can affect healthy cells leading to inflammation [66] . Mortality in COVID-19 is due to the uncontrolled inflammatory response of the immune system [67] .

Concurrently, another randomized trial of this drug was performed comparing with and without standard intensive care. This clinical trial revealed the reduction in duration of mechanical ventilation in patients with COVID-19 [22] . Dexamethasone can suppress immune responses in critically ill patients. Thus, it is being repurposed as a treatment option for hospitalized COVID-19 patients.

Furthermore, the dosing regimen of this corticosteroid is highly essential. A high dose of this corticosteroid can cause harm to the patient rather than a beneficial deed. The usage of this drug can be fatal if there is a low level of inflammation and replication of the virus is fast J o u r n a l P r e -p r o o f [68] . Patients receiving the dose of a corticosteroid (e.g., Dexamethasone) may face minor risks of secondary infections, including invasive fungal and bacterial infection or worsening the pre-existing condition [69] .

A total of fifty-five clinical trials are registered (www.clinicaltrials.gov) to establish the efficacy of Dexamethasone in COVID-19, where a few of the studies are already completed.

Most of the studies are at recruiting stage of COVID-19 patients in establishing its positive potential. To specify, these studies are primarily conducted on moderate to severe cases of COVID-19 cases (NCT04603729) [70] or in conditions with ARDS (NCT04395105) [71] . Furthermore, clinical trials using different doses of Dexamethasone are also performed with standard intensive care. According to the study, the dosage of Dexamethasone should be high (20mg) during the initial five days and low (10mg) during the next five days, which could shorten the duration of respiratory system failure, hence, can be used for the treatment of SARS-CoV2 [72] . Also, investigation on this steroidal agent is ongoing in severe hypoxic patients at a lower dose to establish its prominent role (NCT04509973) [73] .

Based on various clinical trials, WHO and National Institute of Health (NIH) have encouraged and appreciated the use of Dexamethasone alone or combined with Remdesivir to treat COVID-19 [58] . There is a rationale for suggesting the combination of Remdesivir and Dexamethasone. Corticosteroid delays the viral clearance; hence if Dexamethasone alone was administered it would take long time for viral clearance. Thus, co-administration of Remdesivir is suggested. However, for critically ill patients, Dexamethasone is only suggested [51] . One of the subgroup in clinical trials RECOVERY and ACTT-2 tested the efficacy of Barcitinib and Remdesivir and Tocilizumab and Remdesivir combination therapies instead of Dexamethasone on a small subgroup of patients who were hospitalized and receiving oxygen therapy [74] . Study results showed the benefit of using Barcitinib and Tocilizumab. However, these studies are underpowered. Hence, there is insufficient data to J o u r n a l P r e -p r o o f suggest Barcitinib and Tocilizumab in the treatment of COVID-19. Some experts suggested useing of Barcitinib and Tocilizumab when Dexamethasone is contraindicated, where these drugs should be given with Remdesivir [51] . For critically ill patients, Dexamethasone might be the only therapeutic option because corticosteroids play an important role in suppressing the body's inflammatory responses.

Although WHO has declared through the solidarity clinical trials that HydroxyChloroquine is unable to produce benefits in terms of reducing mortality, however, there are two hundred sixty-two studies registered for human trials. Amongst, ten studies were suspended, fourteen were terminated, and nineteen were withdrawn due to safety reasons. Around one hundred seventy-nine studies are in the active stage of recruiting or will be recruiting.

HydroxyChloroquine is a USFDA approved drug for treating malaria, rheumatoid arthritis, and lupus. It was initially thought to be a treatment option for patients with COVID- 19 . This agent's predicted mechanism of action to control COVID-19 demonstrated an increase in endosomal pH to restrict fusion of the viral particle to the cells at the respiratory epithelium ( Figure 5 ) [75] [76] [77] . It has also been reported that the endocytosis of the SARS-CoV-2 could be interfered by the action of this agent [78] , and also known to block sialic acid receptors and finally prevent the formation of cytokine storm. The major limitation of this agent includes QT interval prolongation, retinopathy, and gastrointestinal complications [76] . Some clinical studies were carried out that raised the question of the efficacy of HydroxyChloroquine in COVID-19 (NCT04340544) [79, 80] . After observing a large group of randomized clinical trials and retrospective observational studies, NIH (National Institute of Health) suggested that Chloroquine and HydroxyChloroquine are not recommended for the treatment of COVID-19 patients either alone or in combination with Azithromycin [51, 81] .

J o u r n a l P r e -p r o o f

Clinical progress towards the treatment of COVID-19 has not been limited to the above-mentioned therapeutics. Several other therapeutic agents have also been considered for clinical research. A recent study started recruiting patients in October 2020 to evaluate the efficacy of immune modulators in combating COVID-19 [82] . It has been shown positive response in controlling pathological immune response in patients. It has been reported that administration of Infliximab to COVID-19 patients with concomitant inflammatory bowel disease had shown marked improvement in the symptoms associated with COVID-19 [83, 84] . Thus, clinical trials are in the process of recruiting patients to establish the efficacy of Infliximab in COVID-19 [82] . Baricitinib, a Janus kinase (JAK) inhibitor, is also known to suppress immune responses. The clinical trial data [74, 85] of this drug showed that it is effectively reduces mortality in hospitalized patients who require non-invasive ventilation.

US-FDA issued a EUA for this agent. Furthermore, NIH recommended Barcitinib or Tocilizumab (JAK inhibitors) in combination with Remdesivir when corticosteroids are contraindicated [51, 86] .

Several study reports demonstrate the efficacy of the combination of baricitinib and Remdesivir in the treatment of COVID-19. The clinical outcome of combination therapy in patients has revealed better results than the patients who received Remdesivir alone.

Reduction in recovery time was observed when the combination of these two drugs was used [87] . Thus, combination drug therapy is being encouraged to increase the effectiveness of individual drugs. Therefore, the combination of HydroxyChloroquine and nitazoxanide is also expected to have efficacy against COVID-19. Hence, clinical trials are in progress to test the efficacy of this combination [88, 89] Patients infected with SARS-CoV-2 face several other complications, such as gut microbiota dysbiosis, pulmonary fibrosis, venous thromboembolism (VTE), etc. [90, 91] . A J o u r n a l P r e -p r o o f meta-analysis report depicted a 14.1% prevalence of VTE in hospitalized COVID-19 patients. COVID-19 associated VTE is characterised by an increase in fibrin and fibrin degradation products, D-dimers in the blood. Based on the meta-analysis of pooled data and results obtained from randomized clinical trials, NIH recommended using a prophylactic dose of anticoagulation therapy for hospitalised and critically ill COVID-19 patients [51, 92] .

Interferons (alfa, beta) are a group of cytokines that show antiviral properties. These were also tested in various clinical trials for their efficacy against COVID-19 in hospitalized and critically ill patients. Some clinical trials [93] showed a reduction in the severity of COVID-19 disease but the sample size of the study was not sufficient to derive a clinical conclusion. In contrast, an opposite outcome was reported based on another study's clinical trial data [94] . Furthermore, another clinical study showed the benefit of survival using interferon. However, the interferon utilized in that study was not USFDA approved [95] .

Hence, there is no sufficient data suggesting interferons as a treatment option for COVID-19.

Moreover, interferons produce toxic effects when administered in high doses [51] .

It has been well understood that the severe stage of COVID-19 includes an unregulated immune response which may cause inflammation and sepsis. Interleukin inhibitors are a group of immunosuppressants, prone to suppress interleukins (1 and 6).

Interleukin-1 (IL-1) inhibitor (Anakinra) was tested for its efficacy against COVID-19 in hospitalized patients with inflammatory indications. According to NIH, clinical data is not sufficient to recommend Anakinra as a treatment option because of the poor design of clinical trials [51, 96, 97] . As discussed earlier, Tocilizumab is an anti-IL-6 receptor monoclonal antibody, approved by the FDA for rheumatoid arthritis and other immune-related disorders.

This was also tested as a treatment option for COVID-19 in combination with corticosteroid in hospitalised patients [29, 98, 99] .

Along with the antiviral and immunomodulatory agents, other adjunctive therapeutic agents like vitamins (C and D) [100] [101] [102] and minerals (Zinc) [103] are also considered for the treatment against COVID-19. Clinical trials to test the efficacy of these adjunctive agents are mostly open-label, small sample size, heterogeneous study population. Hence there is no rigorous evidence to suggest them as a treatment option for COVID-19 [51] . Therefore, many clinical trials are in progress to establish the efficacy of those agents.

An antiparasitic drug, ivermectin, was also thought to be a possible treatment for COVID-19. It is an FDA-approved antiparasitic drug for helminthiasis, onchocerciasis, and scabies. Despite its in vitro evidence, ivermectin was not approved for treatment against COVID-19 because of the high dose requirement. Based on in vitro studies, the effective dose to achieve antiviral activity is 100 fold more than the safe dose prescribed in humans. In Lopinavir/Ritonavir are retroviral protease inhibitors, which were also investigated for COVID-19 treatment. The clinical trial data [35, 106, 107] from the solidarity trial and RECOVERY trial [108] revealed that these drugs are ineffective in reducing the mortality or progression to severe disease. A pharmacodynamic study demonstrated that the plasma concentration of these drugs achieved through oral administration is very low compared to in vitro concentration required to inhibit SARS-CoV-2 [109] effectively. Hence, Lopinavir and Ritonavir are not recommended for the treatment against COVID-19.

Overall, scientists worldwide are in search of therapeutic agents against this pandemic causing viral particles to save humanity. Connecting section of the review has been focused on the progress of vaccines against this coronavirus.

A vaccine is a preparation that induces an immune response towards a particular disease-causing pathogen by exposing specific antigens to the body's adaptive immune system. After that the adaptive immune system develops antigen-specific immune responses, such as cell mediate, and anti-body-mediated immune responses. These responses protect the person from future infection of that particular pathogen [110] . Generally, vaccines are prophylactic agents, where vaccination is considered the best strategy to stop a pandemic from spreading, particularly when there are no specific therapeutic agents available [111] . found that this variant is more transmissible and causes less severe disease [116] . All the variants exhibit mutations in different regions of the viral genome. Most mutations are observed in the spike protein region [24] . Variants present a challenge to vaccines and therapeutic agents for their prevention and efficacy against this viral particle. It is uncertain that the approved vaccines will show their effectiveness towards the new mutants or variants.

However, continuous research is going on to test the efficacy of these vaccines against new variants.

Around 120 vaccine candidates have been proposed worldwide for COVID-19 [117] .

These vaccine candidates are based on different approaches; some are conventional, whereas some are relatively new. Fundamentally, vaccines can be classified into 4 categories virusbased, viral-vector based, nucleic-acid based, and protein-based [118] . We have summarised different categories of vaccines hereafter.

This type of vaccine utilizes an inactivated or weakened (attenuated) pathogen (SARS-CoV-2), with no disease-causing ability. It keeps antigenic properties to stimulate the J o u r n a l P r e -p r o o f immune system once injected into the biological system ( Figure 6 ). Polio and measles vaccines were developed by this approach [118] . This is a conventional approach for developing vaccines, where the virus particle is inactivated or killed by applying of heat, solvent treatments (formaldehyde), acidic pH, steam, or a combination of these [119] .

Alternatively, the virus can be weakened by passing through animal or human cells to pick up mutations, which makes the virus's genome weak enough that it would not be able to cause the disease anymore. Table 1 The main advantage of the virus-based vaccine candidates is to mass production of the vaccine upon successful completion of clinical trials. This is possible because of the existing infrastructure as they are licensed platforms for vaccine production. However, the production of inactivated or weakened virus particles requires many pathogenic virus clones.

Extensive safety testing is required before the approval of this type of vaccine candidates [121] .

Developing vaccines based on other vaccine platforms, such as viral vector-based, [recombinant] under EUA [134] . The severity of COVID-19 is particularly high in old and immunocompromised people. Therefore, a non-replicating adenovirus vector is a good choice for immunogenicity. However, booster shots are required to induce long-lasting immunity [135] . CanSino Biological Inc. and Beijing Institute of Biotechnology are jointly developing a COVID-19 vaccine using adenovirus type 5 as a vector. This vaccine candidate has been reached in phase 1/2 clinical trial [117] . This platform has been previously explored to develop a vaccine against the Ebola virus [117, 136] . Similarly, Gamaleya Research Institute is also developing an adenovirus-based vaccine candidate, where they are using human adenovirus types 5 and 26 as vectors [137] . However, this platform is not licensed. A few of the non-replicating viruses, which are at the clinical progress are mentioned in Table 2 . 

This vaccine platform uses nucleic acids (RNA or DNA), which encode pathogen (SARS-CoV-2) proteins. These nucleic acids induce the production of pathogen proteins in vivo, which stimulate the adaptive immune system. This platform is relatively new and currently, a few of these vaccines are licensed based on this technology [128, 131, [139] [140] [141] [142] [143] .

This technology allows the rapid development of vaccines because millions of copies of protein-specific nucleic acids can be generated from a template using the polymerase chain Infectious Diseases. It has shown 94% efficacy to treat patients of COVID-19 [146] . As cells do not accept mRNA, lipid nanoparticles (LNP) are used to deliver the mRNA inside the cells [145] . Few nucleic acid-based vaccines are presented in Table 3 , which are at different stages of clinical research. Similarly, Pfizer-BioNTech has also developed another mRNA vaccine, BNT162b2. It is also LNP formulated nucleoside modified mRNA vaccine encoding PS2 spike protein of SARS-CoV-2. In the preclinical trials, the vaccine showed a good cellbased immune response. The results of conducted clinical trials showed safety and immunogenicity in humans [147] . BNT162b2 and mRNR-1273 are now WHO-approved mRNA vaccines.

Inovio's DNA vaccine candidate, INO-4800, uses the entire S protein gene of SARS-CoV-2 [118, 148] . This S protein gene is introduced into an optimized plasmid, where these plasmids would be introduced into the host cell by electroporation [118, 149] . This DNA will J o u r n a l P r e -p r o o f trigger the cell to synthesize virus spike protein, which will act as an antigen to stimulate the immune system ( Figure 6 ).

Protein-based vaccines are of two types, protein subunit vaccines and virus-like particles (VLP) (Figure 6 ). Protein subunit vaccines use a virus protein, which is specific to the pathogenic virus or a subunit of a protein (epitope) as an immune-stimulating antigen.

These proteins can be produced by recombinant technology [150] [151] [152] [153] [154] [155] . These protein subunit vaccines need adjuvants for better immunogenicity and multiple doses must be administered [156, 157] . According to WHO (7 th July 2020), three protein subunit-based vaccine candidates are in clinical trials and 51 are in pre-clinical evaluation. All the subunit vaccines in clinical trials use SARS-CoV-2 spike protein or RBD of spike protein [158] [159] [160] [161] [162] .

VLPs are multi-protein structures similar to native virus particles except that they lack genetic material, so they cannot replicate. VLPs can stimulate strong immune responses because the protein structure of VLPs mimics the native virus particle. Therefore, when these are introduced into the body, they can stimulate strong immune responses [163] . These vaccine candidates are safe because of the lack of genetic material thereby they cannot cause any disease; however, these are difficult to manufacture [118] . Medicago Inc. is developing a VLP vaccine, which is currently in phase I clinical trials, where these VLPs are plant-based [164] . Other protein-based vaccines are listed in Table 4 .

Being S protein is a structural component of the SARS-CoV-2 virus particle, it enables the virus to infect the host cells by latching with the ACE2 receptor of host cells.

Similarly, the SARS virus also uses the same receptor; however, SARS-CoV-2 S protein shares only 73% similarity with SARS [165] , and the binding affinity of SARS-Cov-2 S J o u r n a l P r e -p r o o f protein with ACE2 receptor is more than that of SARS S protein [166] . Immunogenic experiments in mice had revealed that the S protein of both SARS-CoV-2 and SARS are known to trigger the immune system to generate specific antibodies. SARS-CoV-2 S proteinspecific antibodies and SARS S protein-specific antibodies did not exhibit any cross neutralisation [167] . This lack of cross-neutralisation between the two spike protein-specific antibodies indicates that SARS-CoV-2 spike protein has unique antigenic properties [167] .

Hence, it is justified to consider SARS-CoV-2 spike protein as a specific antigen for vaccine development.

Persons, who recovered from COVID-19 disease, exhibit SARS-CoV-2 specific antibodies in their plasma. Passive immunization injects the plasma of recovered patients to those critically ill or those with a high chance of infection [168] . Passive immunization is an age-old practice. This practice was followed in 1918 for the H1N1 influenza virus pandemic.

During the outbreak of SARS in 2002-03, this approach was evaluated [169] . Passive immunization is mainly prophylactic rather than a therapeutic option. Convalescent plasma therapy may be effective for those in the early stages of infection or those susceptible to infection [1, 4, 168, 170] . This approach might not provide a good prognosis for those already critically ill. Mechanism of action of passive immunization includes neutralization of virus particles by convalescent antibodies and induction of antibody-dependent cellular cytotoxicity [168] . Serum containing high titre virus-neutralizing antibodies can be administered prophylactically to prevent infection in vulnerable individuals, including healthcare providers susceptible individuals (e.g., hospital staff), or can be used in patients with mild conditions to reduce symptoms and mortality [168] .

The advantage of passive immunization includes ready availability [168] . On the other hand, risks of passive immunization include the possibility of other infections that can J o u r n a l P r e -p r o o f be transferred via plasma constituents, immunological reactions such as serum sickness, transfusion-elated acute lung injury (TRALI), and antibody-dependent disease enhancement (ADE). ADE is a condition in which antibodies fail to neutralize the virus and facilitate disease enhancement; however, there is no evidence for this until now [168] . According to FDA and NIH guidelines, only high titre convalescent plasma therapy is suggested for nonhospitalized patients based on randomised clinical trials, but these clinical trials had some limitations such as small sample size and poor design [51] 

Plasma contains several components along with various antibodies. Hyperimmune serum preparations contain polyclonal SARS-CoV-2 specific immuno-globulins derived from the convalescent plasma [171] . Plasma-derived therapies help treat rare, life-threatening, complex genetic diseases. Convalescent plasma from patients with mild symptoms is collected and tested for adequate titres of virus-neutralizing antibodies [172] . However, the large-scale production of H-Ig preparations is complicated. Till now no hyperimmune serum preparations are available for COVID-19 treatment [51] . Table 5 presents some of the products which are in the developmental stage.

Overall, it can be concluded that the research progress to combat this pandemic situation has brought several leads for different stages of the disease condition. The Declaration of competing interest ☒ The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

☐The authors declare the following financial interests/personal relationships which may be considered as potential competing interests:

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The authors acknowledge ICT-IOC, Bhubaneswar for providing necessary support. Rambabu