Repurposing Drugs, Ongoing Vaccine, and New Therapeutic Development Initiatives Against COVID-19

As the COVID-19 is still growing throughout the globe, a thorough investigation into the specific immunopathology of SARS-CoV-2, its interaction with the host immune system and pathogen evasion mechanism may provide a clear picture of how the pathogen can breach the host immune defenses in elderly patients and patients with comorbid conditions. Such studies will also reveal the underlying mechanism of how children and young patients can withstand the disease better. The study of the immune defense mechanisms and the prolonged immune memory from patients population with convalescent plasma may help in designing a suitable vaccine candidate not only for the current outbreak but also for similar outbreaks in the future. The vital drug candidates, which are being tested as potential vaccines or therapeutics against COVID-19, include live attenuated vaccine, inactivated or killed vaccine, subunit vaccine, antibodies, interferon treatment, repurposing existing drugs, and nucleic acid-based vaccines. Several organizations around the world have fast-tracked the development of a COVID-19 vaccine, and some drugs already went to phase III of clinical trials. Hence, here, we have tried to take a quick glimpse of the development stages of vaccines or therapeutic approaches to treat this deadly disease.


INTRODUCTION
Severe Acute Respiratory Syndrome (SARS) caused by SARS Coronavirus (SARS-CoV) initially occurred in China (November 2002) and then quickly spread to 29 countries, resulted in 8,096 cases with 774 fatalities (mortality rate 9.6%). SARS was officially contained in July 2003, about eight months since its first outbreak (WHO, 2003;Peiris et al., 2004). MERS (Middle East Respiratory Syndrome) caused by MERS-CoV (MERS Coronavirus) has resulted in a similar outbreak by spreading into 26 countries with 2519 infected cases and 866 deaths (mortality rate 34.4%) after its first report on June 2012 in Saudi Arabia (Assiri et al., 2013;World Health Organization, 2019). The current outbreak of COVID-19 (Coronavirus Disease 2019) caused by SARS-CoV-2, which was first reported in the Wuhan (China) on December 2019 (Hubei province), now gradually spilled over 213 countries and territories resulted in over 16.3 million infected cases with and more than 650,000 deaths (4% mortality rate) as of July 26, 2020 (Wang et al., 2020a). On January 30, 2020, WHO announced the current coronavirus outbreak as a world health emergency, and on March 11, 2020, reclassified it as a pandemic (World Health Organization, 2005;Chakraborty et al., 2020c;WHO, 2020). The virus was initially named Novel Coronavirus 2019 (2019-nCoV), and later it was changed to SARS-CoV-2 (Gorbalenya, 2020). The WHO entitled the disease as COVID-19 on February 11, 2020(World Health Organization, 2020. The SARS-CoV-2 was found to be infectious as it spreads via respiratory droplets and aerosols when an infected individual comes in contact with a healthy person (Chan et al., 2020b;. The virus incubates for about 2-14 days within humans and subsequently resulted in various mild to severe symptoms like fever, dry cough, dyspnea, severe respiratory issues, pneumonia, etc Chan et al., 2020b;Huang et al., 2020;Lauer et al., 2020;Zu et al., 2020).
The cell membrane attached ACE2 converts the vasoconstrictor peptide angiotensin II to angiotensin 1-7 (vasodilator peptide), and it protects the heart and blood vessels (Jiang et al., 2014). ACE2 is found in the heart, lung, kidney, endothelium, etc. and known to reduce the adverse effects of other RAS (Renin-Angiotensin System) components by reducing the concentration of angiotensin II and increasing the concentration of angiotensin 1-7 and regulates the blood pressure in the body. ACE2 also found to express in intestinal epithelial cells where it helps to absorb nutrients from the food particles and was predicted as one of the entry sites that may have been used initially by SARS-CoV-2 upon the consumption of contaminated food from Wuhan seafood market (Hashimoto et al., 2012;Zhang et al., 2020a). Similarly, ACE2 is also found to express on the mucosa of the oral cavity and the epithelial cell of the tongue, making these other entry routes for SARS-CoV-2 . Interestingly, a small subset of type II alveolar cells (AT2) was found to express the ACE2 receptor and several other genes that positively regulate viral reproduction and transmission, making the lung more susceptible to the virus. The ACE2 expressing cells in the lung triggers an immune response, which may overreact to damage the lung cells by filling up the air sacs with fluid instead of gas, causing pneumonia. Patients with a severely damaged lung can develop acute respiratory distress syndrome (ARDS), where breathing becomes difficult . As ACE2 expresses in an array of organs, SARS-CoV-2 can attack several organs, which results in multi-organ failure often observed in patients who died of COVID-19 (Wang T. et al., 2020). Patients with chronic cardiovascular diseases often take drugs that block the angiotensin receptor or inhibit the angiotensinconverting enzyme, which in turn increases the expression of ACE2 receptors in cells. Therefore, COVID-19 patients who regularly take these medications might have an increased hazard of SARS-CoV-2 infection (Diaz, 2020).
Like other coronaviruses, SARS-CoV-2 also consists of two types of protein structural proteins and non-structural. Structural proteins comprise of E (envelope) protein, S (spike) protein, M (membrane) protein, and N (nucleocapsid) protein . The spike protein (S) of SARS-CoV-2 is a trimeric class I type of fusion protein that helps the virus to enter host cells (Bosch et al., 2003;Walls et al., 2020). The spike protein has two subunits, S1 (required for receptor recognition) and S2 (required for membrane fusion). The C-terminal RBD (receptor-binding domain) of the first subunit (S1 subunit) of spike protein directly interacts with the ACE2 receptor (Yuan et al., 2020). Upon the fusion of the S protein, which exists in a metastable prefusion state, with the ACE2 receptor, the S protein undergoes a conformational rearrangement. The binding to the ACE2 destabilizes the prefusion trimer, which results in the discharge of the S1 subunit. This allows the transition of the S2 subunit of S protein to a steady postfusion state (de Wilde et al., 2017). A cellular serine protease TMPRSS2 plays a pivotal role in this S protein priming (Hoffmann et al., 2020;Wrapp et al., 2020). The host cell-mediated S protein priming is an essential step for the virus to move into the host cells (Hoffmann et al., 2018). Once inside of the host cell, SARS-CoV-2 follows the typical life cycle of a positive-sense RNA virus as was found with MERS-CoV and SARS-CoV (Figure 1) (Fehr and Perlman, 2015).
According to some mathematical models, the transmission of the disease may quickly rebound if we relax measures like lockdown and social distancing (Yamey et al., 2020). In the absence of effective prophylactic treatment, such eruptions may leave the health system overburdened. The absence of a potential drug or vaccine against SARS-CoV-2 has already resulted in a pandemic situation . The designing and development of the COVID-19 vaccine that can be used globally is, therefore, the utmost priority for ending the current pandemic (Prompetchara et al., 2020). It was observed that both SARS-CoV-2 and SARS-CoV use the same mechanism to enter target cells has vital significance for our understanding of the SARS-CoV-2 pathogenesis and transmissibility. To fight this pandemic, various government and private organizations have sped up their development of vaccines and treatment procedures. In this review article, we have discussed the testing of various existing drugs that are now being repurposed and targets against which various vaccine developments are going on for COVID-19.

HOST IMMUNE RESPONSE TO VIRAL INFECTIONS
Upon viral infection, the host cell initially activates the innate immune response via PRRs (pattern-recognition receptors) that recognizes viral particles (Takeuchi and Akira, 2009). Host cells release a group of signaling proteins called Interferons (IFNs) that play a significant role in host antiviral defense. INFs belong to a group of peptides and proteins called cytokines responsible for transferring signals by binding to the receptors on the surface of appropriate immune cells for triggering host immune response against pathogens. INFs are triggered by the activation of host PRRs. Four types of PRRs are known-TLRs, RLRs, NLRs, and CLRs although during viral infection mainly three types of PRRs are activated-RLRs, TLRs, and NLRs (Gonzaĺez-Navajas et al., 2012;Fehr and Perlman, 2015;Nan et al., 2018;Zhang et al., 2020b). PRRs recognize several viral components including DNA, ssRNA, dsRNA, RNA with 5′-triphosphate ends, and proteins. Detection of viral particles by PRRs activates signaling pathways that release type I INFs, different types of cytokines such as proinflammatory cytokines (primarily IL-1, IL-6, TNF-a), chemokines, and costimulatory molecules like CD40, CD80, and CD86 that results in inflammation and subsequent engagement of innate and acquired immune cells to eliminate viral infection (Gonzaĺez-Navajas et al., 2012;Khan et al., 2012;Nan et al., 2018).
Three types of INFs have been characterized till now-type I IFNs (mainly IFN-a/b), type II IFNs (IFN-g), and type III (INF-l) (Stanifer et al., 2019). INFs-a/b is secreted by all viral-infected cells including pDCs (plasmacytoid dendritic cells) which is a vital cell type for INF-a secretion during viral infection. IFN-g, secreted by NK (natural killer) cells and immune cell-like T cells, plays a vital role in host adaptive and innate immunity. It also regulates the expression of several genes that are affected by type I IFNs. INF-l is mainly secreted by epithelial cells in response to the viral infection at mucosal sites (Zanoni et al., 2017). INFs protect host cells by activating signaling pathways, mainly the JAK/ STAT pathway (Schindler et al., 1992;Darnell et al., 1994), which subsequently trigger the expression of ISGs (IFNstimulated genes) that controls the viral infection (Katze et al., 2002). The activated STAT proteins (STAT1, STAT2, and STAT3) in response to INF stimulation are vital for transferring signals that subsequently activate ISGs (Levy and Darnell, 2002;Tsai et al., 2019). Type I INFs induced during innate immune response also upregulate several ISGs whose expression restricts viral replication (Kane et al., 2016).
Activation of the innate immune cells is critical for setting up adaptive immune responses during the re-infection by the same virus. Activation of adaptive immunity takes a few days to weeks to become established. APCs (antigen-presenting cells; e.g., dendritic cells, B cells and macrophages), that live at the site of viral infection, binds to viral particles (antigens) and present them on major histocompatibility complex (MHC) class II to be recognized by the T cell receptor on CD4 + T cells in presence of co-stimulatory molecules (Rosendahl Huber et al., 2014). The activated CD4 + T cells release a wide range of cytokines and chemokines that helps to differentiate CD4 + T cells into several cell subtypes, mainly T helper cells (such as Th1, Th2, Tfh, etc.) as well as regulatory T cells (Treg). Th1 and Th2 cells release several cytokines (Th1-INF-g; Th2-IL-4, IL-13, IL-5, etc.) to trigger B cell differentiation and activate macrophages (Rosendahl Huber et al., 2014). T follicular helper cells (Tfhs) also helps to activate B cells to produce specific antibodies against foreign pathogens (Crotty, 2014). Treg cells do several regulatory functions, especially controlling immunopathology (Crotty, 2014). Activated CD4 + T cells by its interaction with the APCs through CD40-CD40L upregulate expression of CD80/CD86 markers on APCs which interacts with the CD28 on the CD8 + T cells. The APCs presents viral particles on the MHC class I molecules that bind to the TCRs on the CD8 + T cells through the CD80/CD86-CD28 interactions and activate CD8 + T cells. The activated cells proliferate and differentiate into CTLs (cytotoxic T lymphocytes) which releases cytotoxic molecules, and activates the production of cytokines (e.g., TNF-a, IL-2, IFN-g, etc.) that promotes apoptosis of virally infected cells (Crotty, 2014).
Both innate and adaptive immunity (humoral and cellmediated) are equally important to control viral infections. Innate immunity mounts host defenses to control viral infection at the early phases by releasing proinflammatory molecules and also activates adaptive immunity by upregulating co-stimulatory molecules. In adaptive immunity, B cells (humoral immunity) and T cells (cell-mediated immunity) are activated that prevent further viral infections. Immunoglobulins (IgG, IgM, and IgA) produced by activated B lymphocytes bind to viruses to block viral spread and also eliminate virus-infected cells via ADCC (antibody-dependent cytotoxic cells) or complement-mediated pathways. CTLs differentiated from activated CD8 + T cells kill the virus-infected cells by releasing cytotoxic cytokines that trigger apoptosis of the target cells. Some of these immune cells (T cells and B cells) are converted into memory cells that prevent further infections and provide long-term immunity (Klimpel, 1996).
SARS-CoV and other coronaviruses are sensitive to IFN-a/b. Some of these viruses are also very pathogenic. It might be attributed to their ability to modulate an effective host immune response. The nucleocapsid protein of SARS-CoV can evade host interferon responses (Spiegel et al., 2005;Kopecky-Bromberg et al., 2007;Lu et al., 2011). It was reported that EV71 (Liu et al., 2014) and Ebola virus infections can downregulate the JAK-STAT pathway mediated by type-I IFNs, and promote viral replication and proliferation within the host (Okumura et al., 2010). Several antibodies, for example, MCA1, CSCC5, CDC-C2, CDC-A10, CDC-A2, MERS-GD27, etc., isolated from recovered MERS-CoVinfected patients have been found useful in controlling the disease (Chen et al., 2017;Niu et al., 2018a;Niu et al., 2018b). Recognition mechanisms involving the surface proteins of virus and the receptors of host are vital for an understanding of the crossspecies transmission and host tropism to establish animal models for effective vaccine development (Ahn et al., 2020).
Some COVID-19 patients with severe symptoms experience a sudden surge of cytokines in the body, released by the immune cells in response to the viral infection, commonly referred to as 'cytokine storm' . The excessive release of the cytokines or cytokine release syndrome (CRS) is a major determinant in inducing ARDS in COVID-19 patients. The excessive secretion of proinflammatory cytokines (e.g., IL-6, IL-1, TNF-a, etc.) with the help of the innate immune system within the body leads to several lung complications like pneumonitis and ARDS which can cause multi-organ failure and death (Nicholls et al., 2003;Mahallawi et al., 2018;Ragab et al., 2020). Among various proinflammatory cytokines, IL-6 plays a major role in inducing ARDS as an increase in the concentration of IL-6 in the plasma was found to be linked with ARDS in COVID-19 patients (Ragab et al., 2020). Association of IL-6 to mIL-6R (membrane-bound IL-6 receptor) and gp130 activates the JAK-STAT3 pathway which contributes toward CRS. Besides, at high concentrations, IL-6 binds to sIL-6R (soluble form of IL-6 receptor) and gp130, and activates JAK-STAT3 pathway in cells that do not express mIL-6R which again induces cytokine storm by releasing several cytokines and chemokines (e.g., VEGF, IL-6, MCP-1/CCL2, etc.), and by reducing E-cadherin production that leads to ARDS (Magro, 2020;Ragab et al., 2020). Therefore, preventing the occurrence of cytokine storm by drugs that inhibits the release of cytokines may help in alleviating severe COVID-19 symptoms.

VIRAL AND HOST PROTEIN TARGETS
Vaccines SARS-CoV-2 expresses four structural proteins, N (nucleocapsid), E (envelope), S (spike) protein, and M (membrane) similar to SARS-CoV. These proteins are potential antigens to induce nAbs (neutralizing antibodies) and provide protective functions Chan et al., 2020a;Shang et al., 2020). So, the finding of a protein that has the dominant neutralizing epitopes should be the first step of the investigation. Before this identification, the inactivated virus can also be used as a firstgeneration vaccine because it is probably easier to generate than the whole-killed virus particles. Whole-cell killed or live-attenuated vaccines represent all the antigens present in a pathogen like proteins, nucleic acids, polysaccharides, lipids, and some other components capable of inducing a potent immune response (Sharma et al., 2011). Several studies have shown that SARS-CoV inactivated through an agent such as formaldehyde, bpropiolactone and UV light can also instigate virus-neutralizing antibodies in immunized animals (He et al., 2004;Xiong et al., 2004;Jiang et al., 2005;Qu et al., 2005;Te-hui et al., 2005). So in principle, inactivated SARS-CoV-2-based vaccines can also be used. However, upon identification of the neutralizing epitopes, the vaccines that are made based on fragments containing neutralizing epitopes should be used, as they are safer and more effective than the inactivated virus vaccine. Several organizations are using viral deoptimization techniques to synthesize more effective vaccines such as live-attenuated vaccines . Though, attenuated vaccine mimics the natural course of infection to stimulate the toll-like receptors e.g. (TLR-3, TLR-4, TLR-7, TLR-8, and TLR-9) and provide long-term immunity, ensuring low or no pathogenicity is always a major concern (Chakraborty et al., 2020d). Also, killed vaccines show difficulty in maintaining consistency in quality (Chen W. H. et al., 2020).
Most of the subunit vaccines against coronaviruses depend on mounting immune responses against the spike protein by preventing its binding to the host ACE2 receptor (Jiang et al., 2012). One way to block access to the entry receptor, i.e., human ACE2 receptor is to use the spike protein RBD (receptor-binding domain) of SARS-CoV-2 that has been shown to attach to the ACE2 receptor (Lan et al., 2020). Spike protein's RBD from SARS-CoV has been shown to block the virus from accessing the ACE2 receptor in cell culture . Besides, the RBDs of spike proteins in both SARS-CoV-2 and SARS-CoV were found to interact similarly with the ACE2 receptor (Lan et al., 2020). Other researchers have proposed that the RBDs on the spike proteins of other coronaviruses like MHV (mouse hepatitis virus), TGEV (transmissible gastroenteritis virus), HCoV-229E, SARS-CoV, etc. contain key antigenic determinants that can induce production of neutralizing antibodies (Godet et al., 1994;Kubo et al., 1994;Bonavia et al., 2003;He et al., 2004). As spike proteins of coronaviruses are the most important antigenic determinants known to trigger neutralizing antibodies, spike proteins can be used as antigens for developing vaccines (Saif, 1993;Schmidt et al., 2006;Bhattacharya et al., 2020a;Bhattacharya et al., 2020b). Spike protein RBD sequences are relatively conserved. So, this may possible to find the neutralizing epitopes present into the SARS-CoV-2 spike protein for designing and developing of effective, safe vaccine against this virus. How spike protein RBD can activate extremely effective neutralizing antibodies against this virus has been elucidated by the mAbs (monoclonal antibodies) which was isolated from the inactivated virus-immunized human and mice antibody libraries (Sui et al., 2004;He et al., 2005). Thus, the RBD of this virus S protein is not only a functionally important domain for receptor binding of this virus but also a significant neutralization determinant element of SARS-CoV-2. So, the proteins that contain the RBD region or vectors encoding the spike protein RBD can be utilized for developing a highly effective vaccine candidate (Table 1). Therefore, the RBD alone could block access to ACE2 for SARS-CoV-2. Alternatively, single-domain antibodies (sdAbs) or nanobodies based on the RBD can also block the ACE2 receptor effectively (Arbabi-Ghahroudi, 2017). Researchers are developing virus-like nanoparticles based on the expression of recombinant spike protein, which can act as a potent immunogen. Others have developed subunit vaccines consisting of the RBD from SARS-CoV S protein (Chen W. H. et al., 2020). However, certain limitations of subunit vaccines exist, for example, the requirement of multiple booster shots and suitable adjuvants .
During the vaccine candidate development against SARS-CoV-2, one may have to consider the possibility of antibodydependent enhancement (ADE) triggering in vaccinated individuals where instead of mounting protection against the virus infection the virus-bound antibody bind to the host cell receptors to facilitate the cellular entry of the virus. Activation of ADE has been observed in vaccines against several diseases, e.g., Ebola, HIV, Dengue, feline coronavirus, etc (Takada and Kawaoka, 2003;Halstead, 2017;Takano et al., 2019). Human and rodent antibodies produced against the SARS-CoV S protein also shown to induce ADE in vitro . However, ADE was not observed in several pre-clinical studies done in rhesus monkeys using a SARS-CoV vaccine (Luo et al., 2018). Besides, in a pre-clinical study using an inactivated SARS-CoV-2 vaccine did not show any evidence of ADE (Gao Q. et al., 2020).
Therapeutics SARS-CoV-2 does not use receptors that are utilized by other coronaviruses, for example, APN (aminopeptidase N; used by  Frontiers in Pharmacology | www.frontiersin.org August 2020 | Volume 11 | Article 1258     HCoV-229E), DPP4 (dipeptidyl peptidase 4; used by MERS-CoV), or O-acetylated sialic acid receptor (used by HCoV-OC43 and HCoV-HKU1) (Yeager et al., 1992;Krempl et al., 1995;Raj et al., 2013;Huang et al., 2015). It uses the human ACE2 cell receptor to enter the host cell, similar to SARS-CoV and HCoV-NL63 (Hofmann et al., 2005;Ge et al., 2013;Wrapp et al., 2020). So, soluble human ACE2 protein can also be a potential competitor for the ACE2 cell surface receptor, but it can only be achieved when the gene expression of soluble ACE2 is higher than the gene expression of cell surface ACE2 receptor. However, an increase in the concentration of soluble ACE2 in blood found to be associated with chronic cardiac dysfunction (Epelman et al., 2008;Epelman et al., 2009;Ortiz-Peŕez et al., 2013). SARS-CoV was found to downregulate ACE2 by binding to it by its spike protein and inflicting severe lung damage (Kuba et al., 2005). Therefore, overexpressed soluble ACE2 may help in neutralizing SARS-CoV-2 by competitively binding to it and free the cellular ACE2 to perform its normal function. A recombinant human ACE2 (APN01) was found to decrease the levels of angiotensin II and plasma IL-6 in different patients diagnosed with ARDS (acute respiratory distress syndrome) may also be utilized for inhibiting SARS-CoV-2 from accessing cellular ACE2 receptor (Zhang et al., 2020a). Soluble human ACE2 protein was shown to bind SARS-CoV with an affinity close to the affinities of monoclonal antibodies and blocks the virus from accessing cellular ACE2 receptor in cell culture (Li et al., 2003;Sui et al., 2004). Interestingly, membrane-anchored metalloproteinase ADAM17 cleaves ACE2 to release the soluble ACE2 domain, which was predicted to have some adverse effects on the heart (Jiang et al., 2014). Another strategy is to develop anti-ACE2 antibodies that would bind to the human ACE2 protein and block this viral entry, as was shown in SARS-CoV (Li et al., 2003). Unfortunately, there are problems with generating antibodies or protein fragments against the cellular ACE2 as it plays several important roles in controlling cardiovascular diseases including heart attack, diabetes, kidney problems, high blood pressure, etc. Therefore, inactivating the cellular ACE2 receptor is probably not a viable solution.
Alternatively, an ACE2-Fc fusion protein can also increase the lifespan of the soluble ACE2 protein in circulation and inhibit the virus from accessing the cellular ACE2 receptor. Similarly, in a study, the extracellular ACE2 domain fused to the human IgG1 domain was shown to neutralize the SARS-CoV in vitro (Gu et al., 2016), which shows that the use of ACE2-Fc could be a viable solution to block SARS-CoV-2 from infecting human cells. However, this strategy may induce ADE and therefore a thorough investigation is needed to eliminate any adverse effects. The spike protein RBD could also be attached to a human IgG Fc fragment to increase its immunogenicity and stability (Zhang et al., 2009;Li et al., 2011;Du et al., 2013b), as was done in MERS-CoV (Du et al., 2013a). The MERS-CoV spike protein RBD-Fc fusion was found useful in blocking viral cell surface receptor from accessing it by the virus and also stimulated the host immune response against the viral protein domain in mice (Du et al., 2013a). Here one has to consider the mutation of the Fc domain that eliminates its cellular Fc receptor (FcgR) binding ability and triggering of cytotoxic effects Kang and Jung, 2019). The binding of the Fc region to FcgR would activate immune cells to trigger the ADCC pathway and release proinflammatory cytokines, which may lead to cytokine storm . Therefore, the Fc fusion strategy requires a thorough investigation of toxicity and efficacy, followed by the engineering of the Fc fragment for immune silencing and increasing effectiveness (Kang and Jung, 2019).
The other alternative strategy would be to generate antibodies or protein-fragments that would bind to the virus itself and protect the cellular ACE2 receptor from binding the virus . If a protein or peptide fragment that can mimic the binding domain of ACE2 cell receptor and induce similar changes in conformation, as the receptor likely does, then also it can compete with the ACE2 cell receptor. Recently a 23-mer peptide designed from the ACE2 a1 helix has shown a specific binding affinity toward RBD of S protein from SARS-CoV-2, which shows that the development of a peptide-based therapeutics is possible that blocks of this virus interaction with human ACE2 and protecting the cell from virus entry .
A recent report has shown that murine polyclonal antibodies generated against SARS-CoV spike protein were capable enough to inhibit spike protein-mediated cellular entry of SARS-CoV-2 (Walls et al., 2020). Also, a human monoclonal antibody (47D11), which interacts with a conserved epitope on RBD of spike protein, was found to cross-neutralize with both SARS-CoV-2 and SARS-CoV (Wang et al., 2020b). Another antibody having neutralizing property (antibody CR3022) previously isolated from the SARS-CoV infected patient was found to interact with the S protein RBD of SARS-CoV-2 at a site different from the ACE2 binding site indicating cross-reactivity of the antibody for having similar structural regions on the spike proteins of both the viruses (Yuan et al., 2020).
SARS-CoV-2 nucleocapsid protein (N) is another vital protein having several critical roles, including viral genome replication, transcription, etc., and therefore is an attractive drug target. Recently a 3D structure (x-ray crystallography) of the aminoterminal RNA-binding domain of this virus N protein has been elucidated, indicating drug targets (Kang et al., 2020). Broadspectrum antiparasitic drug nitazoxanide has been shown to inhibit the expression of nucleocapsid protein in MERS-CoV and other coronaviruses (Rossignol, 2016). Nitazoxanide also found to suppress proinflammatory cytokines, including IL-6 in mice (Rossignol, 2016). The viral M protein is also highly conserved in evolution among different species (Neuman et al., 2011), and hence, may also be used as a candidate for developing the SARS-CoV-2 therapeutics ( Table 2).
Human monoclonal antibody-based drug sarilumab which inhibits IL-6 receptor is now being tested against COVID-19 (Lamb and Deeks, 2018). Monoclonal antibody-based rheumatoid arthritis drug tocilizumab which is also an inhibitor of IL-6 receptor found to be effective in critically ill COVID-19 patients with cytokine storms and elevated IL-6 levels (Venkiteshwaran, 2009;Chakraborty et al., 2020e;Luo et al., 2020;Saha et al., 2020b). Another monoclonal antibody-based drug leronlimab (PRO 140) known to bind to the CCR5 receptor on the CD4 + T lymphocytes is now being tested in COVID-19 clinical trials (Pugach et al., 2008). The proinflammatory chemokine such as C-C motif chemokine ligand 5 (CCL5) also recognized as regulated through activation, normal T cell expression, and secretion (RANTES), binds to its receptor C-C chemokine receptor type 5 (CCR5) and activates inflammatory responses by directing immune cells to the inflammation site (Vangelista and Vento, 2018). Blocking of CCR5 by leronlimab found to reduce serum IL-6 levels, which is linked with cytokine storm, in critical COVID-19 patients (Patterson et al., 2020). Interleukin-6 (IL-6) plays a vital role in inducing cytokine storm in critical COVID-19 patients and a reduction in IL-6 levels by antiinflammatory drugs is expected to ease CRS and reduce viral loads .
Anti-inflammatory corticosteroid drug dexamethasone has been suggested recently to treat severe COVID-19 patients with CRS. Dexamethasone reduces the production of cytokines but is also known to inhibit the protective functions of T cells and B cells. Therefore, the drug may be used selectively in some severe COVID-19 cases, but its general usage in other COVID-19 patients may cause more harm by increasing the viral load in patients due to the inhibition of protective antibody production (Lee et al., 2004;Russell et al., 2020). A recent clinical trial has shown that dexamethasone reduced the death rate among severe COVID-19 patients who needed oxygen support ( Table 2). A recent study with severe COVID-19 patients found a direct link between C-reactive protein (CRP) and inflammation where higher CRP levels in the blood show greater inflammation. The study also showed that dexamethasone should only be used in severe COVID-19 patients with CRP levels above 20 mg per deciliter of blood, and the use of dexamethasone should be avoided in COVID-19 patients (under ventilator support) with CRP level below 10 as it may turn out to be fatal (Keller et al., 2020).
Anti-inflammatory rheumatoid arthritis drug baricitinib was found to reduce the levels of cytokines, including IFN-g in severe COVID-19 patients . High levels of proinflammatory cytokines and chemokines including INF-g in the plasma causes inflammatory cytokine storm that may lead to the occurrence of ARDS in virus-infected patients, therefore use of antiinflammatory drugs in COVID-19 may help in the reduction of severe symptoms (Ye et al., 2020). Another rheumatoid arthritis drug anakinra is known to block the IL-1 receptor and reduce the inflammatory effects of IL-1. Survival rate within patients with hyperinflammatory conditions was found to increase when treated with anakinra (Shakoory et al., 2016).

CONVALESCENT PLASMA Therapeutics
Convalescent plasma (CP) therapy is another procedure now being tested for COVID-19. This therapy is very simple yet effective, where the serum from the COVID-19 recovered persons can treat new patients (Mire et al., 2016). Recovered patients who have suffered from COVID-19 should have an elevated amount of polyclonal antibodies raised by the immune system to prevent new rounds of infection by SARS-CoV-2. Therefore, the plasma harvested from the recovered patients can be transfused to the patients who have contacted the virus (Marano et al., 2016). As the application of convalescent plasma is a well-known procedure and has been utilized before by medical practitioners, it should not be too difficult to apply this procedure to SARS-CoV-2 infected patients. Convalescent plasma has been used previously during the Ebola outbreak in 2014 and was found to be effective in treating Ebola patients (Kraft et al., 2015). A recent report has shown that CP acquired from recovered patients was effective in treating new COVID-19 infected persons (Duan et al., 2020). One problem using CP therapy is the significant variability of potency that has been found in the sera of recovered patients in neutralizing the antigen, making it a less viable option in the treatment of patients (Marano et al., 2016). Also, if the number of infected patients is much higher than the recovered patients, it would be tough to get enough CP for transfusion. Although CP therapy is being considered or used for

INTERFERON THERAPY Therapeutics
Type I interferons (IFN-I) stimulate the immune system upon viral infection by activating macrophages, natural killer cells, etc. and are expected to hinder SARS-CoV-2 infection (Samuel, 2001;Belhadi et al., 2020;Martinez, 2020). IFN-I is secreted by several cells when the pattern recognition receptors (PRRs) binds viral particles (Liu, 2005). IFN-I is recognized by the interferon-a/b receptor (IFNAR) in the plasma membrane. Upon binding of IFN-I, IFNAR induces the phosphorylation of several transcriptional factors, including STAT1. Once localized in the nucleus, STAT1 activates interferon-stimulated genes (ISGs), including PRRs, which further helps in decreasing membrane fluidity that inhibits viral entry through the membrane (Totura and Baric, 2012;Schneider et al., 2014). Although interferon treatment against SARS-CoV and MERS-CoV has shown variable efficiency (Stockman et al., 2006), the IFNb subtype appears to work well in COVID-19 treatment if administered in the early stages of infection (Sallard et al., 2020). The side effects of interferon treatment could be toxic to a patient, especially when the patient is at critical stages of infection. Therefore, it is recommended to use this therapy in the early stages of infection.

MEMBRANE FUSION INHIBITORS Therapeutics
Well-known antimalarial drugs chloroquine and its less toxic derivative hydroxychloroquine, both known to elevate the pH of endosomes/lysosomes that blocks membrane fusion and inhibits viral infection (Mauthe et al., 2018). Also, chloroquine found to impede glycosylation of the ACE2 receptor, which may inhibit the virus from receptor binding (Vincent et al., 2005). Both of these drugs helped inhibit this virus in the in vitro assays Wang M. et al., 2020). However, some studies have raised concerns about the effectiveness of chloroquine/hydroxychloroquine in treating COVID-19 patients as these repurposed drugs were found to possess several side effects (Chary et al., 2020;Gautret et al., 2020;Kamp et al., 2020). Current reports suggested that the influenza drug umifenovir is effective in reducing symptoms of COVID-19 (Zhang J. N. et al., 2020). Umifenovir (Arbidol) intercalates with the membrane lipids to inhibit the fusion between the virus particle and host membrane, which blocks the entrance point of the virus inside the host cell (Villalaí n, 2010; Blaising et al., 2014). Another influenza drug oseltamivir, which reduces infection in the respiratory system by blocking viral neuraminidase and inhibits viral particles from escaping host cells, was found to be effective in the COVID-19 outbreak in China (Uyeki, 2018;Wang D. et al., 2020).
Coronaviruses use several modes of endocytosis (clathrin-or caveolin-mediated, or by the formation of lipid rafts) depending on the virus and cell type, and therefore, blocking of the endocytic pathways could be a promising strategy for the development of antiviral drugs (Glebov, 2020;Yang and Shen, 2020). Several anti-endocytotic drugs (e.g., chlorpromazine, bafilomycin, etc.) that are known to inhibit clathrin-or caveolin-mediated endocytosis proposed to have therapeutic activities against coronaviruses including SARS-CoV-2 (Yang and Shen, 2020). In lung AT2, alveolar epithelial cells, AAK1 regulates endocytosis, and baricitinib inhibits AAK1 with high affinity. Therefore, researchers argue that baricitinib could be one of the potential drugs against COVID-19 (Richardson et al., 2020). However, others argue that baricitinib also inhibits the JAK-STAT mediated signaling pathway which affects the interferon-mediated immune response. It might have a fatal effect on COVID-19 patients (Favalli et al., 2020). Clinical trials are currently underway to find out whether the drug has any positive effect in treating COVID-19 patients.

Human Protease Inhibitors (Therapeutics)
Proprotein convertases (PCs) are essential for turning precursor proteins into their active forms, e.g., furin and other proteases that control viral host cell entry and infectivity (Yamada et al., 2018;Izaguirre, 2019). Host proteases cleaved the coronavirus S proteins, including furin, TMPRSS2 (transmembrane protease serine protease 2), trypsin, cathepsin, etc., and the availability of these proteases in the infected cells are important for subsequent host cell entry (Ou et al., 2020). Furin or trypsin dependent proteolytic cleavage of the viral (SARS-CoV) S protein at two distinct sites was found to be essential for priming and subsequent membrane fusion with the host cell (Belouzard et al., 2009). MERS-CoV spike protein was also found to be activated by furin cleavage (Millet and Whittaker, 2014). Similarly, the S protein of SARS-CoV-2 has a putative cleavage site (furin) between S1 and S2 subunits, but whether it is cleaved during the priming event remains elusive (Ou et al., 2020). Another serine protease TMPRSS2 was found to be crucial for S protein priming in both SARS-CoV-2 and SARS-CoV (Matsuyama et al., 2010;Shulla et al., 2011;Iwata-Yoshikawa et al., 2019;Hoffmann et al., 2020). For SARS-CoV, it is the availability of specific proteases that appears to be the determinant factor to choose whether it enters the host cell via the cell surface or by using the endosomal cathepsin L-mediated pathway for viral entry. So, non-appearance of the host proteases within the cell surface, SARS-CoV invade host cells though a pathway (endosomal pathway) where cathepsin L activates the spike protein, allowing the association of the viras particle and endosome membranes (Simmons et al., 2004;Kam et al., 2009;Chan et al., 2013). Previous studies have shown that the dual treatment of an inhibitor of TMPRSS2-camostat mesylate and an inhibitor of cathepsin L efficiently blocked host cell entry of SARS-CoV. This competent inhibition could be attributed to the double barrier of entry for SARS-CoV from the surface of a cell and through the endosomal pathway (Kawase et al., 2012). Serine protease inhibitor camostat mesylate was found to block TMPRSS2-mediated priming of spike protein and inhibits COVID-19 infection in lung cells in vitro (Hoffmann et al., 2020). Another TMPRSS2 inhibitor drug nafamostat mesylate was found to inhibit the membrane fusion of MERS-CoV and expected to have similar effects on this virus (Yamamoto et al., 2016;Hoffmann et al., 2020). These observations suggest that this protease inhibitor, camostat mesylate, and a cathepsin inhibitor can be used as antiviral drugs to prevent cathepsin L and TMPRSS2 -mediated SARS-CoV-2 infection.
One problem with using human protease inhibitors as antiviral drugs is that they might affect the normal physiological processes in the human cells, which may lead to further complications or side effects. Therefore, human protease inhibitors may be used in combinatorial therapies with other antiviral drugs which would allow using a less concentration of protease inhibitors to minimize side effects while keeping stronger efficacy. However, no human protease inhibitor has been approved as of now to use in treating viral infections despite having several experimental reports on their effectiveness as antiviral drugs (Steinmetzer and Hardes, 2018).

Viral Protease Inhibitors (Therapeutics)
In coronavirus, chymotrypsin-like protease (3CL pro or M pro ) is the main protease, and along with papain-like protease (PL pro ) it processes the polyproteins pp1ab and pp1a (Brierley et al., 1989;Gorbalenya et al., 2006). These two proteases are attractive targets for designing drugs to inhibit cleavage functions and render the virus non-functional (Anand et al., 2003;Yang et al., 2003;Ratia et al., 2008;Hilgenfeld, 2014;Arya et al., 2020;Wu C. et al., 2020). The structures of M pro from SARS-CoV-2 and SARS-CoV are known. Hence, the designing of drugs to inhibit the protease has been accelerated (Xue et al., 2008;). An a-ketoamide inhibitor has been identified that blocks SARS-CoV-2 M pro from performing its functions shown in mice . HIV protease inhibitor drug lopinavir/ritonavir was found to be useful in decreasing viral loads in COVID-19 patients (Lim et al., 2020). However, in clinical trials on COVID-19 patients, the HIV drug was found to be ineffective . Another HIV protease inhibitor darunavir is also under clinical trials to find out its efficacy in treating COVID-19 (Santos et al., 2019). In vitro studies have shown that several other antiretroviral protease inhibitors (e.g., nelfinavir, etc.) were highly effective in inhibiting coronaviruses (Yamamoto et al., 2004). However, the failure of Kaletra (lopinavir/ritonavir) has shown that protease inhibitors optimized for HIV are unlikely to be effective against SARS-CoV-2 as the proteases expressed by these two viruses are structurally different. Nonetheless, some efficacy against SARS-CoV-2 has been shown by HIV protease inhibitors under in vitro conditions and some of these inhibitors are also under various clinical trials to confirm their effectiveness against COVID-19 (Table 2). However, protease inhibitors specific for HIV protease (e.g., darunavir, etc.) are doubtful to be effective against SARS-CoV-2 protease because of the structural dissimilarities between them.

REPLICASE INHIBITORS Therapeutics
Another attractive target for drug development is the SARS-CoV-2 RNA-dependent RNA polymerase (RdRp), as this is the main molecule for the replication/transcription complex in coronaviruses. The cryo-EM structure of SARS-CoV-2 RdRp (nsp12) has been elucidated recently, along with cofactors nsp7 and nsp8 . The structure derived using cryo-EM methodology also explained how the drug remdesivir binds to the RdRp . The nucleotide analog remdesivir has been shown to inhibit RdRp in SARS-CoV (Agostini et al., 2018;Saha et al., 2020a), MERS-CoV (Gordon et al., 2020), and SARS-CoV-2 (Holshue et al., 2020;Wang M. et al., 2020). In a recent study, remdesivir was found to provide benefit to the majority of COVID-19 patients who needed oxygen support (Grein et al., 2020). European Medicines Agency (EMA) has given conditional marketing approval to Veklury (remdesivir) for the therapy of critical COVID-19 patients (12 years of age or higher) with pneumonia and under oxygen support. Remdesivir is the first drug to get the required authorization to use in the EU for the treatment of COVID-19 ( Table 2).

NUCLEIC ACID-BASED SOLUTIONS Vaccines
The advantages of nucleic acid-based vaccines are that they can be quickly constructed and can induce strong cell-mediated and humoral immune responses even in the absence of an adjuvant (Du et al., 2009). During the Zika virus outbreak, DNA vaccines were the first to enter clinical trials (Prompetchara et al., 2020). A DNA vaccine is a new and innovative mode of vaccination involved in direct injection of a plasmid encoding the antigens . Certain advancements like the use of electroporation for delivering the plasmid and use of adjuvant further increases the efficacy by invoking better immune response. Several organizations are working for pre-clinical trials of DNA vaccines against COVID-19 (Liu, 2019) ( Table 1). DNA vaccines against COVID-19 mainly encode different forms of the SARS-CoV-2 S protein that was found to stimulate both cellular and humoral immune responses in mice, guinea pigs, and rhesus macaques (Amanat and Krammer, 2020;Smith et al., 2020;Yu et al., 2020). However, there is a risk of integration and mutation of DNA vaccines within the host genome. Being safer, mRNA vaccines stand as a promising alternative to DNA and other conventional vaccine approaches because of its safety and quick development (Liu, 2019). So far, several organizations are working on developing an mRNA-based vaccine for SARS-CoV-2. Small interfering RNA (siRNA) based vaccines are also being developed targeting conserved regions on the SARS-CoV-2 genome, especially 3CL pro , RdRp, and spike protein, to degrade viral mRNAs resulting inhibition of translation .

Therapeutics
Double-stranded RNA drug rintatolimod is now being tested for COVID-19, which stimulates the innate immune system by binding to one of the PRRs named TLR-3 found in the endosomal membrane. Once rintatolimod binds to TLR-3, the host cell gets a signal to produce interferons, which lead to various protective systems against pathogenic viruses or bacteria. Rintatolimod predicted to stimulate RNase L enzyme production, which degrades pathogenic RNAs of viruses (Gowen et al., 2007;Pardi et al., 2018).

CONCLUSIONS
There are several new vaccines and novel therapeutic molecules which are currently under development against COVID-19 (Tables 1 and 2). The finding of a safe and attractive target for vaccine development is of utmost importance at this point to prevent further spread of this virus. Unfortunately, the way SARS-CoV-2 is spreading around the world and infected cases increasing exponentially, we may have to witness much bigger devastation before a cure is found. Several promising drug targets have been identified, and several organizations are working relentlessly to develop vaccines against these targets ( Table 1). Different available antiviral drugs (repurposed) are being tested for COVID-19 in large clinical trials, as they have shown some positive effects in initial phases (Table 2; Figure 1). Contradictory reports are also started to pouring in against some antiviral therapies targeted at COVID-19, where although initial reports suggested positive effects, later others showed no effect. For example, hydroxychloroquine treatment, along with azithromycin, has shown a significant reduction of viral load in COVID-19 infected patients (Gautret et al., 2020), but subsequent report refutes that claim and showed no benefit in severe COVID-19 patients by this treatment (Molina et al., 2020). Repurposing existing antiviral drugs against COVID-19 has shown some positive effects, but further scientific results are necessary to prove whether these affect COVID-19 treatment, or we are just looking at the placebo effect which can be dangerous for patients.
Recently, some unproven theories are spreading like wildfires, which may also hinder the actual progress on the vaccine development against COVID-19. One example is the use of the BCG vaccine, which is being advocated as a potential cure for COVID-19. Countries, where people have taken the antituberculosis Bacillus Calmette-Guerin (BCG) vaccine, appear to be immune from COVID-19 compared to countries where BCG vaccination is not a norm, as per some recent non-peerreviewed reports (Hegarty et al., 2020;Miller et al., 2020). Research organizations have already started clinical trials to test the efficacy of the BCG vaccine in COVID-19. It is not clear at this point how and whether BCG vaccination helps in preventing COVID-19 at all; therefore, further research is necessary to find the link between these two.
Several vaccine clinical and pre-clinical trials are currently ongoing (Table 1), and even if some trials finally become successful, a preventive vaccine may not be widely available for at least another 12-18 months. For a vaccine to be successful, much time is needed to conduct proper clinical trials, especially phase III and phase IV trials where the control group is large enough to get a conclusive report (Green, 2020). Therefore, fasttracking of any clinical trial could be potentially dangerous, and comprehensive safety tests are necessary before a vaccine can be marketed. It applies the same to any repurposed drugs that show positive effects in the initial phases of clinical trials. The catastrophic failure of the respiratory syncytial virus (RSV) vaccine in 1966 showed the importance of a proper clinical trial and advocating for fast-tracking any SARS-CoV-2 clinical trials should be avoided at this stage. The RSV vaccine failed due to the lack of antibody affinity maturation, the possibility of which should be thoroughly checked to avoid a similar situation in COVID-19 (Glezen et al., 1986).
Due to the high genome mutation rates in RNA viruses as the viral RNA polymerase (e.g., influenza virus) or reverse transcriptase (e.g., HIV) lacks proofreading activity, and therefore, it is difficult to make an effective vaccine against RNA viruses (Boutwell et al., 2010;Sanjuań et al., 2010). Although the excessive mutation rate in RNA viruses helps them to adapt quickly to the variable environmental conditions, it also makes them vulnerable because of the accumulation of lethal mutations in the essential genes. Interestingly, in SARS-CoV, the nsp14 protein found to contain an exoribonuclease domain (ExoN) that provides proofreading activity and the deletion of the gene results in a reduction of virulence (Hofer, 2013;Pachetti et al., 2020). This information is important as SARS-CoV-2 also contains a similar gene on its genome, and any proofreading activity would ensure low mutational rates during the synthesis of the viral genome, which would be helpful to design and to develop a vaccine candidate against the SARS-CoV-2 virus.
Coronaviruses are known for a long time and an extensive amount of knowledge has been gathered on SARS-CoV, despite that we still do not have a vaccine against it. We still do not have an effective vaccine against HIV or malaria, for example, although these pathogens are known to us for a long time (Boutwell et al., 2010;Rts, 2015;King, 2019). Challenges posed by these pathogens are far more complex and require an extensive investigation that may take several years to complete. Therefore, extensive safety trials in humans with sizable groups of people are needed even if data from the initial phases are encouraging. Any rush at these stages may be catastrophic if upon vaccination to people who never exposed to the virus develop serious side effects.
Reports from the recent clinical trials of two COVID-19 vaccine candidates have shown promise as they were found to be safe for human use and also induced strong immune response against SARS-CoV-2 (Beyrer et al., 2012;Zhu et al., 2020). The vaccine AZD1222 (ChAdOx1 nCoV-19) developed jointly by Oxford University and AstraZeneca provides double protection against COVID-19 by producing both antibodies and T-cells that directly kill infected cells (Beyrer et al., 2012). Another vaccine (Ad5-nCOV) developed by CanSino Biologics, China, also shown to provide protection against SARS-CoV-2 . These reports instill faith that a protective vaccine would be available soon to ease the suffering that the world is facing today because of COVID-19.
The virus has locked up several parts of the world from social and economic activities, and we have no other option but to wait for the development of a vaccine against COVID-19. This situation was envisaged by several scientists earlier, but no one thought we have to witness this disaster in our lifetime. Humanity always prevailed under challenging conditions and the way many research organizations are trying to find a cure one can only hope that we could get a vaccine against COVID-19 sooner than later, but until then social distancing, rigorous testing, and isolation of infected persons in COVID-19 appears to be a potent strategy to contain the spread of the virus.