Emergence of novel coronavirus and progress toward treatment and vaccine.

In late December 2019, a group of patients was observed with pneumonia-like symptoms that were linked with a wet market in Wuhan, China. The patients were found to have a novel coronavirus genetically related to a bat coronavirus that was termed SARS-CoV-2. The virus gradually spread worldwide and was declared a pandemic by WHO. Scientists have started trials on potential preventive and treatment options. Currently, there is no specific approved treatment for SARS-CoV-2, and various clinical trials are underway to explore better treatments. Some previously approved antiviral and other drugs have shown some in vitro activity. Here we summarize the fight against this novel coronavirus with particular focus on the different treatment options and clinical trials exploring treatment as well as work done toward development of vaccines.

INTRODUCTION

Coronaviruses are a form of positive‐strand non‐segmented RNA viruses distributed among birds, mammals and humans that cause respiratory and neurological illnesses. There are six different types that can cause disease among humans. Four of these (HKU1, 229E, NL63 and OC43) cause only the common cold in patients, while two others cause severe acute respiratory syndrome (SARS‐Coronavirus) and Middle East respiratory syndrome coronavirus (MERS‐CoV). SARS‐CoV caused severe respiratory illness in China during its outbreak in 2002‐2003, while the MERS‐CoV outbreak started in the Middle East in 2012. Due to the broad genetic variation and diversity of coronaviruses and higher chances of animal to human spread they are likely to emerge periodically in future.

During December 2019, an outbreak of pneumonia‐like symptoms occurred in patients that were linked to the seafood market in Wuhan China. Investigation identified a new strain of coronavirus called SARS‐CoV‐2.

The detection of this novel coronavirus is key to confirm the cases and proceed to treatment. In an early method for detecting SARS‐CoV‐2, the samples from bronchoalveolar‐lavage fluids were collected, centrifuged to remove debris and inoculated onto human epithelial cells of airway origin. About 20 000 sequences from each sample were obtained and genome matches showed more than 85% identity with SARS‐like betacornavirus. Results were also obtained from real‐time PCR, and isolated viruses were named SARS‐CoV‐2. To further characterize SARS‐CoV‐2, the de‐novo sequence was obtained by using nanopore sequencing and Illumina methods. The airway epithelial cell cultures were suitable for visualization and identification of the novel coronavirus.

TREATMENT OPTIONS FOR CORONAVIRUS

Currently, no treatment is approved for SARS‐CoV‐2, but various already approved drugs are being tried in different clinical trials to check efficacy against this virus. The possible options include nucleoside analogs, interferon that can act as immune modulators and approved antimalarials having antiviral activities such as chloroquine and hydroxychloroquine.

Antiviral drugs

The nucleoside analogs such as ribavirin, favipiravir, galidesivir and remdesivir target RNA polymerase and block the synthesis of viral RNA. Favipiravir was effectively used against influenza and has the potential to inhibit viral RNA synthesis and a new study also supports its activity against SARS‐CoV‐2. Different clinical trials are underway where patients are being recruited to evaluate the efficacy of a combination of favipiravir and interferon‐α and a combination of favipiravir and baloxavir marboxil is being evaluated. Ribavirin is a guanine derivative antiviral drug approved for the treatment of HCV and RSV, but it causes anemia at higher doses that limit its use and its efficacy against coronavirus is uncertain.

Remdesivir is an adenine derivative antiviral drug. It has activity against a variety of viral strains such as SARS and MERS and a recent study also supports its activity against SARS‐CoV‐2. A recent patient in US with SARS‐CoV‐2 has shown recovery with intravenous administration of remdesivir. Recently, Phase‐III clinical trials have been started in USA to evaluate the efficacy of IV remdesivir as 200 mg OD or 100 mg OD for 9 days. A recent RCT apparently reported no significant efficacy, but we await the published findings.

Some protease inhibitors like lopinavirand ritonavir have shown activity against SARS and MERS coronaviruses. , Clinical trials have been started to test the efficacy of lopinavir and ritonavir combination against SARS‐CoV‐2. These drugs are known to inhibit the chymotrypsin‐like protease of MERs and SARS coronaviruses. A study in vitro using Vero E6 cells infected with SARS‐CoV‐2 assessed efficacy of different therapies by quantitative analysis using PCR (qRT‐PCR). Remdesivir and chloroquine showed promising results. Remdesivir has previously shown efficacy against MERS coronavirus and Ebola virus and is currently under clinical trial for treatment of Ebola. The results showed that EC90 value of remdesivir against SARS‐CoV‐2 was 1.76 μM.

Neuraminidase inhibitors are recognized antiviral agents for the treatment of influenza. The treatment with oral oseltamivir has been given to suspected patients in China. In past, oseltamivir has shown efficacy against MERS‐COV.

Chloroquine and hydroxychloroquine

Small molecular weight drugs such as chloroquine have shown inhibitory effects against SARS‐CoV‐2 (EC50 = 1.14 μM in Vero E6 cells) and is under evaluation in open‐label trials.. Chloroquine (CQ) is a recognized anti‐malarial drug but also has antiviral activity. The antiviral activity of chloroquine was first noticed in the late 1960s. Chloroquine and its analog hydroxychloroquine both have inhibitory effects against various viruses including SARS, enterovirus and Zika virus. Chloroquine inhibits the virus by increasing endosomal pH and so reducing viral cell fusion and also interferes with cellular receptor glycosylation. The EC90 value of chloroquine against SARS‐CoV‐2 is 6.90 μM that can be achieved with administration of 500 mg dose. Remdesivir and chloroquine have shown activity in in vitro studies and can easily be tested in patients with SARS‐CoV‐2.

In another recent study, Gao et al found that chloroquine phosphate reduced the symptoms of pneumonia in SARS‐CoV‐2 patients and shortening the duration of disease. The guidelines for the treatment of SARS‐CoV‐2 were revised six times since its issuance on 15 January 2020. The recent guidelines also include IFN‐α, remdesivir, ribavirin, ritonavir and chloroquine. The mode of administration of IFN‐α is through inhalation at a dose of 5 million units diluted with water for injection. The dose of ritonavir is 100 mg BD for adults. Ribavirin may be given in combination with IFN‐α or ritonavir at a dose of 500 mg BD or TDS. Chloroquine phosphate should be administered at a dose of 500 mg BD. Arbidol can be given three times a day at 200 mg.

Hydroxychloroquine (HCQ) is an approved disease‐modifying antirheumatic drug that also has immunomodulatory effects and prevents organ damage. HCQ alters endosomal pH and interrupts the biding between RNA/DNA and toll‐like receptors that leads to suppression of TLR signaling. , , Inside the cytoplasm, HCQ also interferes with the interaction between nucleic acid sensor cyclic GMP and cytosolic DNA. These two mechanisms lead to increase production of IL‐1, TNF and type‐1 interferon. Such mechanism supports the idea that HCQ suppress the cytokine release storm (CRS) which is due to SARS‐CoV‐2 triggered overreaction of immune system. In a recent study, HCQ was found to be more effective than chloroquine; a loading dose of 400 mg twice daily and maintenance dose of 200 mg twice a day is recommended for SARS‐CoV2 infection.

The mechanism involves in antiviral role of HCQ and CQ is inhibition of receptor binding and fusion of cell membrane. These two are crucial steps that are required for cell entry of SARS‐CoV‐2. Chloroquine interferes with the glycosylation of ACE‐2 (angiotensin‐converting enzyme receptors) receptors that are considered as cellular receptors for SARS‐CoV‐2 and block the fusion of SARS‐CoV‐2 with host cell. Thus, the binding of virus is blocked and infection is prevented. The HCQ and CQ after entry into the cell tend to concentrate in lysosomes and endosomes. The SARS‐CoV‐2 use endosome as a tool for cellular entry. HCQ and CQ increase the pH of endosomes and create a negative influence on the binding of SARS‐C0v‐2 with endosomes. Lysosomal protease helps in viral fusion with cell membrane. Increase lysosomal pH prevents the action of protease and fusion and replication of virus is blocked . The mechanism of action of chloroquine and hydroxychloroquine is represented as (Figure 1).

FIGURE 1

Mechanism of action of HCQ and CQ by blocking binding of virus with ACE‐2 receptors and increasing endosomal pH and preventing fusion with the cell

Chymotrypsin‐like inhibitors

Cinanserin is an antagonist of serotonin receptors. It can inhibit chymotrypsin‐like (3C‐like) protease and has shown promising results against SARS coronavirus. The 3C‐protease was also investigated to be encoded in SARS‐CoV‐2.

Flavonoids such as chalcones, flavones and isoflavones produce antioxidant effects but they also have antiviral effects. A study has found that flavonoids can inhibit the entry of hepatitis‐C virus. Some flavonoids have activity against MERS coronavirus, presumably due to inhibition of 3C‐like protease.

Other treatment options

Pirfenidone is under clinical trial for the treatment of idiopathic pulmonary fibrosis resulting from SARS‐CoV‐2 infection. It also has an anti‐inflammatory effect and serves as an anti‐oxidant. The results obtained during SARS 2003 were promising and now beneficial effects could be obtained in patients with severe pneumonia from SARS‐CoV‐2.

Nitric oxide (NO) is a biological gas produced from arginine. NO after reaction with superoxide forms peroxynitrite which has cytotoxic bactericidal action. Nitric oxide is also known to regulate airway function and reduce inflammation of airways. The beneficial effect of NO in SARS patients was observed in some studies. NO can also inhibit the synthesis of RNA and viral protein. A study has found that organic nitric oxide could inhibit the replication cycle of SARS‐CoV‐2.

Αlpha‐lipoic acid (ALA) is used in hepatic disorders and polyneuropathies. ALA is an antioxidant that protects cells against oxidative stress by increasing the glutathione level. It was reported that individual oxidative stress plays a role in coronavirus infection and G6llPD deficiency was a crucial factor in SARS‐CoV‐2 patients. So, ALA could be an option to decrease the oxidative stress in patients with SARS‐CoV‐2 infection.

Mucroporin‐M1 is a peptide derived from scorpion venom with broad spectrum antiviral activity against various infections like influenza H5N1 and SARS‐CoV.

The use of convalescent plasma for the treatment of SARS‐CoV‐2 is also under consideration with varying hope regarding efficacy. The pros of using plasma from recovered patients include easy availability and it can also be used as prophylactically in healthcare professionals who are at high risk of exposure to SARS‐CoV‐2 infection. The cons include that not all recovered patients have enough antibodies. Lack of availability of validated SARS‐CoV‐2 assay for detection of neutralizing antibodies may cause hindrance in identification of suitable donor. A large scale randomized clinical trials are needed to establish the efficacy of convalescent plasma.

Some studies support the role of angiotensin‐converting enzyme (ACE) inhibitors. This is based on the hypothesis that ACE‐2 serves as receptor for SARS‐CoV‐2. , So, ACE inhibitor could potentially compete for site binding and reduce the mortality and morbidity associated with SARS‐CoV‐2.

The various ongoing clinical trials are summarized in Table 1.

TABLE 1

Clinical trials for various treatment options for SARS‐CoV‐2

Clinical Trial Registration Number	Trial information	Date	Type	References
ChiCTR2000030897	The efficacy of Newgen beta‐gluten probiotic powder in patient with 2019‐nCOV.	16 March 2020	Interventional	57
ChiCTR2000030894	The efficacy of combination of Favipiravir + Tocilizumab in patient with pneumonia (COVID‐19).	16 March 2020	Interventional	58
ChiCTR2000030892	A prospective study on Pirfenidone in the treatment of Coronavirus Pneumonia (COVID‐19) Fibrosis.	16 March 2020	Interventional	59
ChiCTR2000030883	The efficacy of traditional Chinese qingfei detoxification decoction (mixture) in coronavirus pneumonia (COVID‐19).	16 March 2020	Observational	60
ChiCTR2000030857	A study for bronchoscopic alveolar lavage in patients undergoing trachea intubation with new coronavirus pneumonia (COVID‐19).	16 March 2020	Observational	61
ChiCTR2000030853	The study of protective effect of dexmedetomidine in severe novel coronavirus pneumonia (COVID‐19) patients.	16 March 2020	Interventional	62
NCT04252885	The combination of Lopinavir + Ritonavir and Arbidol against 2019‐nCOV.	28 January 2020	Interventional	63
NCT04273763	The study of bromhexine hydrochloride along with standard treatment in patients with novel coronavirus pneumonia (COVID‐19).	18 February 2020	interventional	64
NCT04280588	The use of Fingolimod in patient with COVID‐19.	21 February 2020	Interventional	65
NCT04288713	The efficacy of Eculizumab (Soliris) in Covid‐19 Infected Patients (SOLID‐C19).	28 February 2020	Interventional	66

POTENTIAL VACCINES

Vaccine provides immunity against a particular pathogen before exposure of that infectious agent. Several types of vaccines exist that can be nucleic acid based, live attenuated vaccine, subunit proteins or nanoparticle vaccines. Different technologies are being utilized to develop potential vaccine for SARS‐CoV‐2 including DNA and mRNA‐based nanoparticles. Phase‐I trials of potential vaccines focus on safety and immunogenicity, for example, against MERS. Two of the clinical trials on MERS vaccine are expected to be complete by the end of 2020 , in Russia and one in Germany by December 2021. The Inovo pharmaceutical company has tested its vaccine against MERS coronavirus funded by coalition for epidemic preparedness innovation (CEPI) using DNA‐based technology and named as INO‐4700. The University of Oxford recombinant chimpanzee adenovirus has also begun Phase 1 randomized multicenter trials for intramuscular injection of vaccine ChAdOx1 against SARS‐CoV‐2. The 1100 participants have been divided into four groups and they will be observed for any serious adverse event for 6 months.

The ongoing clinical trials for development of vaccines have been summarized in Table 2 and completed vaccine trials for SARS and MERS viruses have been summarized in Table 3.

TABLE 2

Ongoing clinical trials on vaccine development

Company	Technology	Stage	Expected timeline	References
Inovo pharmaceuticals	INO‐4800 DNA based	Pre‐clinical studies funded by Coalition for Epidemic Preparedness Innovations (CEPI)	Human testing in few months	73
Vir Biotechnology	anti‐coronavirus monoclonal antibodies (mAbs), CRISPR‐ based screening	Pre‐clinical	No decided timeline	74
The university of oxford	Adenovirus vector and SARS‐CoV‐2 spike protein	Phase‐1 started on 23rd April in 1100 peoples	Upto 6 mo	75
University of Queensland	Molecular clamp technology	Pre‐clinical	6 mo‐1 y	. 76
Johnson & Johnson	Adenovirus based technology	Pre‐clinical	1 y	77
The University of Saskatchewan's	Not known	Pre‐clinical	3‐4 mo	78
GeoVax and BravoVax	Modified Vaccinia Ankara (MVA) platform technology	Pre‐clinical	Not known	79
Chinese clinical trial center ChiCTR2000030906	Clinical trial for recombinant novel coronavirus (2019‐CoV) vaccine based on adenoviral vector	Phase‐1 clinical trail	Not known	80

TABLE 3

Completed vaccine trials on SARS‐COV and MERS‐COV

Company	Vaccine	Subjects	Results	Conclusion	References
Sinovac Biotech	inactivated SARS coronavirus (SARS‐CoV) vaccine	Thirty six subjects were enrolled. They received 16 units of vaccine two time or 32 units one time	On 56th day 100% of patients were seroconverted receiving 16 unit and 91% patients were seroconverted receiving 32 units one dose.	The inactivated vaccine against SARS‐COV is safe and well tolerated	81
NIH, Vaccine research center	single‐plasmid DNA vaccine encoding the Spike (S) glycoprotein	Ten healthy adults	Eight out of 10 subject develop antibody against SARS‐COV and detected by ELISA	The VRC SARS DN vaccine is safe and well tolerated	82
National Institute of Allergy and Infectious Diseases and Clinical Center, National Institutes of Health (NIH); the National Cancer Institute, NIH (contract number HHSN261200800001E);	Anti‐MERS coronavirus antibody	Double blind phase‐I single dose study including 43 participants	Out of 43 there were 38 eligible participant and 28 received the dose while 10 were placebo. They develop antibodies against MERS	Single dose at 50 mg/kg of SAB infusion appeared to be safe.	83
GeneOne Life Science and Inovio Pharmaceuticals.	GLS‐5300 DNA vaccine	Phase‐I open label, dose escalation study including 75 participants	Out of 75, 25 patients receive 0.67 mg, other 25 receive 2 mg and 3rd group receive 6 mg of GLS‐DNA vaccine 5300.	The immune response was dose‐independent and further trials needed to establish efficacy	84

Development of neutralizing antibodies against coronaviruses

The fusion of coronavirus with a cell occurs after biding of S protein to a target receptor delivers viral nucleocapsid and initiates replication. The S protein causes syncytial formation at the receptor site. A neutralizing antibody that can target the S protein on the surface of SARS‐CoV‐2 could also be an option to produce passive immunity in patients. Recent research on the genome of SARS‐CoV‐2 (MN908947.3) allows scientists to express the S protein as immunogen on the surface and explore options such as yeast libraries for generating antibodies that neutralize the virus. ,

CONFLICT OF INTEREST

The authors have no conflict of interest in this manuscript.