Trend in Scientific Publications Related to COVID-19

Since the outbreak of COVID-19, this new disease and its causativevirus have drawn major global attention. Scientists and physiciansworldwide have been conducting a major campaign to understand thisnew emergent disease and its epidemiology in an effort to uncoverpossible treatment regimens, discover effective therapeutic agents,and develop vaccines.Figure4 shows the total number of journal articles related to COVID-19or SARS-CoV-2 published each week from the last week of 2019 throughthe week of February 24, 2020. Over 500 journal articles were publishedelectronically or in print during this period, and the number of publishedarticles has increased each week since the week of January 13, 2020.Although a large portion of these articles are about clinical manifestationsand treatment options, an increasing number of studies are focusedon elucidation of virus structure, virus transmission mechanisms/dynamics,as well as identification of antiviral agents and accurate diagnosticsfor virus detection. These trends reflect immense interest and desirefrom the scientific community, including both academic and industrialorganizations as well as clinicians, to identify new methods to haltthe progression of this epidemic disease and to prevent infectionand transmission in the future.

Notable Journal Articles Related to COVID-19 and SARS-CoV-2

Table1 lists somejournal articles published from December 30, 2019 through February 23,2020. These articles were selected based on collective use of factorssuch as journal impact factor, citation, and type of study. For example,the No. 8 article listed about the characterization of the SARS-CoV-2genome has greatly facilitated the global effort to develop a vaccinefor prevention of COVID-19. Also shown in this table are journal articlespertaining to potential antiviral drug candidates such as remdesivir, baricitinib, and chloroquine for the treatment of this disease.

Distributionof patents related to SARS and MERS

As mentioned earlier,COVID-19 is caused by SARS-CoV-2, a new type of coronavirus in thesame genus as SARS-CoV and MERS-CoV. Viral proteins responsible forSARS-CoV-2 entry into host cells and replication are structurallysimilar to those associated with SARS-CoV. Thus, research and developmenton SARS and MERS may offer insights that would be beneficial to thedevelopment of therapeutic and preventive agents for COVID-19. Thisreport identified pertinent data from patents related to these twocoronaviruses.Figure5 shows the distribution of patents in the CAS content collectionrelated to SARS (A) and MERS (B). The number of patents related toSARS is almost 12 times the number related to MERS, probably becausethe SARS outbreak occurred about 10 years before the MERS outbreak.Among SARS patents, about 80% are related to the development of therapeutics,35% are related to vaccines, and 28% are related to diagnostic agentsor methods. Because an individual patent may cover any two or moreareas, the sum of percentage values is greater than 100%. A similardistribution pattern was also observed for patents related to MERS.Thus, for both diseases, more patents have been devoted to the developmentof therapeutic agents as opposed to diagnostic methods and vaccines.

Key Proteinsand Their Roles in Viral Infection

Identification of targetsis important for identifying drugs with high target specificity and/oruncovering existing drugs that could be repurposed to treat SARS-CoV-2infection.Table2 listspotential targets, their roles in viral infection, and representativeexisting drugs or drug candidates that reportedly act on the correspondingtargets in similar viruses and thus are to be assessed for their effectson SARS-CoV-2 infection. 3CLpro and PLpro are two viral proteasesresponsible for the cleavage of viral peptides into functional unitsfor virus replication and packaging within the host cells. Thus, drugsthat target these proteases in other viruses such as HIV drugs, lopinavirand ritonavir, have been explored.¹⁹ RdRpis the RNA polymerase responsible for viral RNA synthesis that maybe blocked by existing antiviral drugs or drug candidates, such asremdesivir.¹⁹ Conceivably, the interactionof viral S protein with its receptor ACE2 on host cells, and subsequentviral endocytosis into the cells, may also be a viable drug target.For example, the broad-spectrum antiviral drug Arbidol, which functionsas a virus-host cell fusion inhibitor to prevent viral entry intohost cells against influenza virus,²⁰ hasentered into a clinical trial for treatment of SARS-CoV-2.^21,22 The protease TMPRSS2 produced by the host cells plays an importantrole in proteolytic processing of S protein priming to the receptorACE2 binding in human cells.¹¹ It has beenshown that camostat mesylate, a clinically approved TMPRSS2 inhibitor,was able to block SARS-CoV-2 entry to human cells, indicating itspotential as a drug for COVID-19.¹¹

ACE2 involvement with coronavirus infection is of further interestsince ACE2 is a potent negative regulator restraining overactivationof the renin-angiotensin system (RAS) that may be involved in elicitationof inflammatory lung disease in addition to its well-known role inregulation of blood pressure and balance of body fluid and electrolytes.^23,24 It catalyzes degradation of angiotensin II to angiotensin (1–7).The balance between angiotensin II and angiotensin (1–7) iscritical since angiotensin II binds to angiotensin AT1 receptor tocause vasoconstriction, whereas angiotensin (1–7) elicits vasodilationmediated by AT2.²⁵⁻²⁷ Although the notion that ACE2 mediates coronavirusinvasion is largely accepted, it remains unclear how the levels oractivities of ACE2, AT1 receptors, and AT2 receptors are altered incoronavirus-induced diseases due to the limited number of studies.^23,24 Therefore, it is yet to be determined whether some drugs or compoundsthat target any of these proteins (e.g., L-163491 as a partial antagonistof AT1 receptor and partial agonist of AT2 receptor) may alleviatecoronavirus-induced lung injury.²⁸

Patents and Potential DrugCandidates Related to Key Protein Targets

The CAS contentcollection contains patents related to coronavirus key proteins listedabove.Table3 liststhe number of patents related to each protein target and associatedtherapeutic compounds with a CAS Registry Number (CAS RN) reportedin these patents. CAS data show that targets 3CLpro and RdRp attractedmore attention than other targets, and more compounds with therapeuticpotential were identified for these targets, probably due to the workdone for SARS-CoV which also contains 3CLpro and RdRp.

Existing Drugs with PotentialTherapeutic Applications for COVID-19

Since SARS-CoV-2 isa newly discovered pathogen, no specific drugs have been identifiedor are currently available. An economic and efficient therapeuticstrategy is to repurpose existing drugs. On the basis of genomic sequenceinformation coupled with protein structure modeling, the scientificcommunity has been able to rapidly respond with a suggested list ofexisting drugs with therapeutic potential for COVID-19.Table4 provides a summary of suchdrugs together with potential mechanisms of actions for their activities.Barcitinib was proposed because of its anti-inflammatory effect andpossible ability to reduce viral entry.³⁵ A fixed dose of the anti-HIV combination, lopinavir–ritonavir,is currently in clinical trials with Arbidol or ribavirin.²² Remdesivir, developed by Gilead Sciences Inc.,was previously tested in humans with Ebola virus disease and has shownpromise in animal models for MERS and SARS.The drug is currently being studied in phase III clinical trialsin both China and the USA. Favipiravir, a purine nucleosideleading to inaccurate viral RNA synthesis,³⁶ was originally developed by Toyama Chemical of Japan, and has recentlybeen approved for a clinical trial as a drug to treat COVID-19.³⁰ Chloroquine, an antimalarial drug, has proveneffective in treating coronavirus in China.³² In addition to the above-mentioned, many other antiviral drugs arealso listed.

Selected Patents Relatedto Promising Small Molecule Drug Candidates

Table5 shows selected patents associatedwith the aforementioned potential drugs, together with patents disclosingsmall molecules for treatment of SARS or MERS. The selection was basedon the presence of important terms in CAS-indexed patents as wellas the presence of the synthetic preparation role assigned by CASscientists during document indexing. Patent applications WO2009114512and WO2014028756 disclose preparation of compounds active as JAK inhibitors,one of which was later named as baricitinib and developed for reducinginflammation in rheumatoid arthritis. Patent application JP5971830discloses preparation of polycyclic pyridone compounds and their useas endonuclease inhibitors. Patent applications US20160122374 and US20170071964 disclosepreparation of the nucleotide analog drug remdesivir that was laterdeveloped as a therapeutic agent for Ebola and Marburg virus infections(Patent US20170071964). Because of its promising results in at leasttwo COVID-19 patients, remdesivir has now entered into phase III clinicaltrials.

Patent application WO2013049382 discloses both structuresand syntheses of compounds from various structure classes (peptidylaldehydes, peptidyl α-ketoamides, peptidyl bisulfite salts,and peptidyl heterocycles), as well as certain formulation compositions,developed to inhibit viral 3C protease or 3C-like protease (i.e.,3CLpro).

Patent application WO2018042343 presents both preparationmethods and biological assay results for compounds capable of inhibitingthe SARS virus proteases. These compounds appeared to exhibit goodenzyme-inhibiting activity (pIC₅₀ ≈ 7 or IC₅₀ ≈ 0.1 μM) and antiviral activity, which wasassessed by host cell viability using cultured human lung fibroblastMRC-5 cells infected with a specified virus (e.g., MERS virus) expressingthe viral S protein. Drug administration routes were also mentionedin this patent.

Small Molecule Compounds in Research andDevelopment with Potential Effects on Key Protein Targets for HumanCoronavirus-Induced Diseases

Besides various commercializedantiviral drugs, there are also small molecule compounds currentlyin research and development that have shown significant inhibitoryeffects on many key proteins from similar coronaviruses such as SARS-CoVand MERS-CoV (Table6). These drug candidates mostly inhibit viral enzymes including proteasesand components for RdRp. Since 3CLpro protease has a high level ofsequence homology between SARS-CoV and SARS-CoV-2, inhibitors against3CLpro of SARS-CoV may also be applicable to SARS-CoV-2. Compounds,including benzopurpurin B, C-467929, C-473872, NSC-306711 and N-65828,which may inhibit the activity of viral NSP15, poly(U)-specific endoribonuclease,were tested for reduced SARS-CoV infectivity in cultured cells withIC₅₀ of 0.2–40 μM.³⁸ Compound C-21 and CGP-42112A are two AT2 agonists, whereas L-163491has dual functions as a partial agonist for AT2 receptor and a partialantagonist of AT1 receptor. Since AT1 and AT2 are important effectorsin the RAS system to which ACE2 belongs, it has been speculated thatthese compounds may be used to adjust the balance between AT1 andAT2, which may be affected by coronavirus infection and to alleviateviral-induced lung injury during the infection.²⁴

Small Molecules Identified by Structure Similarity, Lipinski’sRule of 5, and CAS-Indexed Pharmacological Activity and/or TherapeuticUsage

Besides the aforementioned antiviral drugs, there maybe additional small molecule compounds with therapeutic or pharmacologicalpotential against viruses such as SARS-CoV and MERS-CoV. Compoundslisted inTables4 and6 were subjected to a Tanimoto similarity searchin CAS REGISTRY using CAS proprietary fingerprints.a Those substances with at least 60% structural similaritymatch and meeting Lipinski’s rule of 5 were identified.Table7 lists selected compoundsthat were also identified to have a pharmacological activity or therapeuticusage role. Compound name and CAS RN are provided for each compound.The second column lists the number of compounds that met the structuresimilarity and Lipinski’s rule criteria. Although more workremains to be done in this regard, the methodology and results mentionedhere point to a strategy that may help streamline the process of drugdiscovery for COVID-19.

Distribution of Biologics Patents Relatedto SARS and MERS

The new coronavirus SARS-CoV-2 related toSARS and MERS viruses is causing serious and ongoing epidemiologicalproblems around the world. Since there is limited clinical and basicresearch information at this time, treatment options for COVID-19currently comprise investigational drugs and management of symptoms.As biologics have the potential to broaden the spectrum of the treatmentoptions for coronavirus-induced diseases, leveraging prior knowledgeand practices used to address the SARS and MERS outbreaks providesa practical strategy for developing new target-specific therapeuticagents for SARS-CoV-2. To this end, an analysis of biologics frompatents contained in the CAS content collection was performed. Thepatent analysis included information related to therapeutic antibodies,cytokines, interfering and other therapeutic RNAs, and vaccines forpotential treatment and/or prevention of SARS-related diseases frompatents published from 2003 to the present.Figure6 shows more than 500 patents that disclosethe use of these four biologics classes to treat and prevent SARSand MERS. Of these patents, vaccine development was the largest class(363), followed by therapeutic antibodies (99), interfering RNAs (35),and cytokines (22). Given the indispensable role of vaccines in viraldisease prevention, detailed analysis of vaccines will be presentedin a later section.

Antibodies

Ninety-ninepatents containing information about antibodies with therapeutic and/ordiagnostic potential for SARS and MERS were identified. Of these,61 patents claimed preparation of SARS-specific antibodies (23), MERS-specificantibodies (17), or antibodies with diagnostic application (21). Similarto SARS-CoV, the receptor-binding domain (RBD) in the S protein ofSARS-CoV-2 binds to human ACE2 receptor in order to gain access intohost cells.⁴² In viral infection, the Sprotein, but not the other structural proteins, M, E, and N in SARS-CoV,elicits an immune response.⁴³Table8 shows the targetanalysis of patents related to development of therapeutic antibodiesfor SARS. Over 90% of these antibodies are directed against S proteinincluding its RBD. The data indicate that the S protein is a putativetarget for SARS-CoV-2 antibody development.

An additional 38 patents containedinformation pertaining to other types of antiviral antibodies thatwere useful for SARS and MERS therapies. These included neutralizingantibodies or antibodies designed to target proteins such as IL-6/IL-6R,TLR3 (Toll-like receptor 3), CD16, ITAM (immunoreceptor tyrosine-basedactivation motif), DC-SIGN (dendritic cell-specific intercellularadhesion molecule-grabbing nonintegrin), ICAM-3 (intercellular adhesionmolecule 3), or IP-10/CXCL10 (interferon γ-inducible protein10). Cytokine storm has been reported to correlate with disease severityin SARS-CoV-2 infection. Patients admitted to an ICU had higher concentrationsof proinflammatory cytokines and chemokines, particularly G-CSF, IP-10/CXCL10,MCP1 (monocyte chemoattractant protein 1), and TNFα, as wellas elevated cytokines from T helper 2 cells such as IL-4 and IL-10.⁴⁴ Patent application WO2005058815 discloses humananti-IP-10 antibodies, including bispecific molecules and immunoconjugatesthat bind to IP-10 with high affinity, for treating inflammation,autoimmune disease, neurodegenerative disease, bacterial infection,and viral infection. Patent application WO2017095875 discloses thepreparation of human antibodies and immunoconjugates specificallytargeting chemokine IP-10, including an anti-IP-10 antibody shownto suppress free serum IP-10 in about 3 days at 0.5 mg/kg and in approximately10 days at 10 mg/kg in Cynomolgus macaques.

In addition, DC-SIGN/CD209,a type II transmembrane adhesion molecule with C-type lectin function,is mainly expressed on interstitial dendritic cells and lung alveolarmacrophages.⁴⁵ DC-SIGN functions as anentry cofactor in transferring SARS-CoV to susceptible cells suchas pneumocytes.⁴⁶ Patent application WO200505824claims the production of a humanized anti-DC-SIGN antibody that interferedwith the interaction of DC-SIGN with its receptor, ICAM-3. The antibodyeffectively blocked viral binding, infection, and transmission forviral infections/diseases, including SARS.

Cytokines

Cytokines are low-molecular-weightproteins that act as chemical signals in the immune response to pathogeninvasion. The production of various cytokines in response to an invadingpathogen such as a virus contributes to the host organism’sability to eliminate the pathogen. Specific types of cytokines, includingchemokines, interferons (IFNs), interleukins, and lymphokines, havebeen reported and characterized in the literature over the past 40years. By early 2020, the CAS Lexicon contained over 700 terms forspecific types of cytokines associated with 76 724 documents,including 11 837 patents.

During a viral infection, themost prominent cytokines produced are IFNs, which interfere with viralreplication. IFNs are classified as type I (IFN-α, IFN-β,IFN-δ, IFN-ε, IFN-κ, IFN-ν, IFN-τ, andIFN-ω), type II (IFN-γ), or type III (IFN-λ) basedon the receptor complex used for signaling as well as sequence homology.⁴⁷ Because of their ability to interfere with viralreplication, interferons and interferon fusion proteins have beenutilized as therapeutic agents for treatment of viral infections forthe past 20 years. A few patents disclosing these proteins and theiruse for treating SARS are described below.

RNA Therapies

RNA interference (RNAi) is a biological process wherein small complementaryRNA duplexes target and neutralize specific mRNA molecules,resulting in inhibition of gene expression or genetic translation.Interfering RNAs include microRNAs and small interfering RNAs (siRNAs)that are generally about 21–25 nucleotides in length. Shorthairpin RNAs (shRNAs) are artificial synthetic RNA molecules designedto fold into a tight hairpin conformation that allows them to silencetheir target genes, and can serve as precursors of siRNAs. The expressionof shRNAs in cells is typically accomplished by their delivery viaplasmids or viral or bacterial vectors.⁴⁸ Although microRNAs are noncoding and naturally found in plants,animals, and some viruses, synthetic versions are currently beingused to silence a variety of genes.⁴⁹ Theability to chemically synthesize modified analogues of microRNAs aswell as siRNAs, which are capable of altering disease-related geneexpression or inhibiting pathogen gene expression, has created a hostof new therapeutic options.⁵⁰

Incontrast to the microRNAs and siRNAs, antisense RNAs are single-strandedRNAs which are naturally occurring or synthetic and usually around19–23 nucleotides in length with a sequence complementary tothat of a protein coding mRNA, allowing it to hybridize and blockprotein translation.⁴⁸

Since thediscovery of RNAi in the late 1990s, it has become a well-known methodfor silencing/suppressing target genes associated with virulence andpathogenesis. Thirty-five patents in the CAS content collection disclosethe use of RNAi in treating SARS, with 28 patents using siRNA molecules,three patents using antisense oligonucleotides, two patents usingRNA aptamers, one patent using a ribozyme, and one patent using amicroRNA inhibitor. Supporting InformationTable S1 provides a high-level view of these 35 patents includingthe specific RNAi targets. A few of these patents are further discussedbelow.

Distributionof Patents Related to SARS and MERS among Vaccine Types

Antiviralvaccines generally fall into one of the following types: inactiveor live-attenuated viruses, virus-like particle (VLP), viral vectors,protein-based, DNA-based, and mRNA-based vaccines. There are 363 patentsin the CAS content collection related to vaccine development to preventviral disorders/diseases, including SARS and MERS. Of these, 175 patentsdisclose vaccines for non-coronaviruses that may have relevance toSARS and MERS, while 188 patents are directly associated with anti-SARSand anti-MERS vaccines with a demonstrated immune response. SupportingInformationTable S2 contains additionalinformation on these SARS/MERS vaccine-related patents.

Figure7 reveals the distributionof patents among these vaccine types related to SARS and MERS. Ascan be seen, 15 patents disclose information about inactive and live-attenuatedvirus vaccines, 28 patents describe DNA vaccines, 21 patents discloseinformation on viral vector vaccines, 13 patents disclose informationon VLP vaccines, and three patents are focused on mRNA vaccines.

It was reported that viral S protein subunit vaccines produced higherneutralizing antibody titers and more complete protection than live-attenuatedSARS-CoV, full-length S protein, and DNA-based S protein vaccines.⁵¹ Unsurprisingly, about half of the patents focusedon protein vaccines comprising the S protein subunit vaccine and vaccinesspecifically targeting the receptor binding domain (RBD) of the S1subunit of the viral S protein. Collectively, S protein/gene is thepreferred target site in SARS/MERS vaccine development, and the samestrategy can be potentially useful in developing SARS-CoV-2 vaccines.A condensed report on several patents that describe vaccines for generatingimmunity to SARS and MERS follows.

Attenuated Virus Vaccines

Patent application US20060039926discloses live attenuated coronavirus or torovirus vaccines. Introductionof a mutation (Y6398H) into the Orf1a/b polyprotein (p59/nsp14/ExoN)was shown to completely attenuate virulence of mouse coronavirus (MHV-A59).The attenuated MHV virus exhibited reduced replication in mice atday five following intracerebral inoculation.

DNA-Based Vaccines

Patent application WO2005081716 discloses compositions and methodsfor inducing/enhancing immune responses, particularly antigen-specificCD8+ T cell-mediated responses, against antigens of the SARS coronavirus.An enhancement of the immune response involving particularly cytotoxicT cell immune responses is induced in vivo by chimeric nucleic acidsthat encode an endoplasmic reticulum chaperone polypeptide (e.g.,calreticulin) linked to at least one antigenic polypeptide or peptidefrom SARS-CoV. Using gene gun delivery of DNA-coated gold particles,vaccination of mice against a calreticulin–nucleocapsid fusionprotein resulted in potent nucleocapsid-specific humoral and T cell-mediatedimmune responses. Vaccinated animals were capable of significantlyreducing the titer of a challenging vaccinia vector expressing theN protein of the SARS virus.

Patent application WO2015081155discloses immunogens, which comprise consensus proteins derived fromthe MERS-CoV spike protein, for use in DNA-based vaccines targetingMERS-CoV. The consensus spike protein significantly induced both humoraland cellular immune responses, including increased titers of IgG andneutralizing antibodies. The induced cellular immune response involvedincreased CD3+CD4+ and CD3+CD8+ T cell responses that produced IFN-γ,TNF-α, IL-2, or both IFN-γ and TNF-α. On March 3,2020, Inovio Pharmaceutical, Inc. announced they had designed theDNA vaccine called INO-4800 to be planned for human trials in theUnited States in April.⁵⁷

Protein-BasedVaccines

Patent application WO2010063685 by GlaxoSmithKline(GSK) discloses a vaccine capable of provoking a protective immuneresponse against SARS. The vaccine comprises an S protein immunogenand an oil-in-water emulsion adjuvant. An engineered ectodomain immunogen(soluble S protein), in combination with the emulsion adjuvant, GSK2,induced high levels of anti-SARS-CoV IgG2a or IgG2b antibody responsesand neutralizing antibody responses in animal models. In late February2020, GSK announced a collaboration with Chinese firm Clover Biopharmaceuticalsto assess a coronavirus (COVID-19) vaccine candidate.⁵² This collaboration will involve the use of Clover’sprotein-based coronavirus vaccine candidate (COVID-19 S-Trimer) withGSK’s adjuvant system. By applying their Trimer-Tag technology,Clover has manufactured an S-Trimer subunit vaccine using a rapidmammalian cell culture-based expression system. The Trimer-Tag isan advanced drug development platform, which enables the productionof novel, covalently trimerized fusion proteins that can better targetprevious undruggable pathways.

Patent application US20070003577discloses immunogenic compositions and vaccines associated with theS protein of SARS coronavirus. A TriSpike SARS coronavirus vaccinewas prepared from a recombinant full-length trimeric S protein. Therecombinant protein was shown to (1) exhibit native antigenicity asshown by reactivity with convalescent SARS patient sera; (2) exhibitspecific binding to soluble ACE2 receptor; (3) promote antibody-dependentviral entry in otherwise refractory human Raji B cells; and (4) elicitprotection against a challenge infection in an animal model.

Patent application US20060002947 (Antigen Express, Inc., a subsidiaryof Generex) discloses the preparation of hybrid peptides composedof three elements, including (a) an invariant chain (Ii) key peptidefor antigen presentation enhancing activity, (b) a chemical structurelinking the Ii to the antigenic epitope, and (c) an antigenic epitopethat binds to a MHC class II molecule. The methodology was used tocreate Ii-Key/MHC II SARS hybrids. Recently, Generex announced thatit is developing a COVID-19 vaccine following a contractual agreementwith a Chinese consortium comprised of China Technology Exchange,Beijing Zhonghua Investment Fund Management, Biology Institute ofShandong Academy of Sciences, and Sinotek-Advocates InternationalIndustry Development. The company will utilize its Ii-Key immune systemactivation technology to produce a COVID-19 viral peptide for humanclinical trials.⁵³

Virus-like Particle Vaccines

In 2015, patent application WO2015042373 by Novavax disclosed animmunogenic composition composed of MERS-CoV nanoparticle VLPs containingat least one trimer of a S protein, produced by baculovirus overexpressionin Sf9 cells. This VLP preparation induced a neutralizing antibodyresponse in mice and transgenic cattle, when administered along withtheir proprietary adjuvant Matrix M (RN 1235341-17-9). In addition,preparations of sera from vaccinated cattle (SAB-300 or SAB-301) wereinjected into Ad5-hDPP4 transduced BALB/c mice prior to challengewith MERS-CoV. Both SAB-300 and SAB-301 were able to protect thesemice from MERS-CoV infection with a single prophylactic injection.Novavax announced on February 26, 2020⁵⁴ that it was beginning animal testing on potential COVID-19 vaccinecandidates due to their previous experiences working with other coronaviruses,including both MERS and SARS. Their COVID-19 candidate vaccines targetingthe S protein of SARS-CoV-2 were developed using their recombinantnanoparticle vaccine technology along with their proprietary adjuvantMatrix-M.

mRNA-Based Vaccines

The potential advantages of anmRNA approach to prophylactic vaccines include the ability to mimicnatural infection to stimulate a more potent immune response as wellas the ability to combine multiple mRNAs into a single vaccine. Patentapplication WO2017070626 by Moderna discloses mRNA vaccines composedof mRNAs encoding antigenic viral full-length S, S1, or S2 proteinsfrom SARS-CoV and MERS-CoV virus, formulated in cationic lipid nanoparticles.They show that mice vaccinated with mRNA encoding coronavirus full-lengthS protein generated much higher neutralizing antibody titers comparedto mRNA encoding the S protein S2 subunit. New Zealand white rabbitsimmunized with MERS-CoV mRNA vaccine encodingthe full-length S protein reduced more than 90% of the viral loadin the lungs of the rabbits and induced a significant amount of neutralizingantibody against MERS-CoV. Moderna announced on February 24, 2020⁵⁵ that it has released the first batch of mRNA-1273against SARS-CoV-2 for use in humans, prepared using methods and strategiesoutlined in their previous patents. Vials of mRNA-1273 have been shippedto the National Institute of Allergy and Infectious Diseases (NIAID),a division of the National Institutes of Health (NIH), to be usedin the planned Phase 1 study in the United States. Moderna reportsthat mRNA-1273 is an mRNA vaccine targeting aprefusion-stabilized form of the S protein associatedwith SARS-CoV-2, which was selected by Moderna in collaboration withinvestigators at the NIAID Vaccine Research Center. Manufacture ofthis batch was funded by the Coalition for Epidemic Preparedness Innovations.

Patent application WO2018115527 describes vaccines comprising mRNAencoding at least one antigen of a MERS coronavirus, preferably aS protein or a S protein fragment (S1), an envelope protein (E), amembrane protein (M), or a nucleocapsid protein (N), all of whichwere effective in inducing an antigen-specific immune response. Intradermaladministration into mice of a lipid nanoparticle (LNP)-encapsulatedmRNA mixture encoding MERS-CoV S proteins was shown to result in translationin vivo and induction of humoral immune responses.

1. Gorbalenya A. E.et al. The species severe acute respiratory syndrome-related coronavirus:classifying 2019-nCoV and naming it SARS-CoV-2. Nature Microbiology 2020, 10.1038/s41564-020-0695-z [DOI] [PMC free article] [PubMed] [Google Scholar]
2. Kupferschmidt K.; Cohen J.Will novel virus gopandemic or be contained?. Science2020, 367 (6478), 610–611. 10.1126/science.367.6478.610. [DOI] [PubMed] [Google Scholar]
3. Coronavirus Disease(COVID-2019) Situation Reports 1–45; World Health Organization, 2020.https://www.who.int/emergencies/diseases/novel-coronavirus-2019/situation-reports.
4. Coronavirus is now expected to curb globaleconomic growth by 0.3% in 2020.https://www.forbes.com/sites/sergeiklebnikov/2020/02/11/coronavirus-is-now-expected-to-curb-global-economic-growth-by-03-in-2020/#5de149ad16da.
5. Anthony S. J.; Johnson C. K.; Greig D. J.; Kramer S.; Che X.; Wells H.; Hicks A. L.; Joly D. O.; Wolfe N. D.; Daszak P.; Karesh W.; Lipkin W. I.; Morse S. S.; Mazet J. A. K.; Goldstein T.Global patternsin coronavirus diversity. Virus Evol2017, 3 (1), vex012. 10.1093/ve/vex012. [DOI] [PMC free article] [PubMed] [Google Scholar]
6. Su S.; Wong G.; Shi W.; Liu J.; Lai A. C.K.; Zhou J.; Liu W.; Bi Y.; Gao G. F.Epidemiology, genetic recombination, and pathogenesisof coronaviruses. Trends Microbiol.2016, 24 (6), 490–502. 10.1016/j.tim.2016.03.003. [DOI] [PMC free article] [PubMed] [Google Scholar]
7. Zhu N.; Zhang D.; Wang W.; Li X.; Yang B.; Song J.; Zhao X.; Huang B.; Shi W.; Lu R.; Niu P.; Zhan F.; Ma X.; Wang D.; Xu W.; Wu G.; Gao G. F.; Tan W.A novel coronavirus from patients with pneumonia in China, 2019. N. Engl. J. Med.2020, 382 (8), 727–733. 10.1056/NEJMoa2001017. [DOI] [PMC free article] [PubMed] [Google Scholar]
8. Tang B.; Bragazzi N. L.; Li Q.; Tang S.; Xiao Y.; Wu J.An updated estimationof the risk of transmission of the novel coronavirus (2019-nCov). Infect Dis Model2020, 5, 248–255. 10.1016/j.idm.2020.02.001. [DOI] [PMC free article] [PubMed] [Google Scholar]
9. Du L.; He Y.; Zhou Y.; Liu S.; Zheng B.-J.; Jiang S.The spike protein of SARS-CoV - A target for vaccine and therapeuticdevelopment. Nat. Rev. Microbiol.2009, 7 (3), 226–236. 10.1038/nrmicro2090. [DOI] [PMC free article] [PubMed] [Google Scholar]
10. Wrapp D.; Wang N.; Corbett K. S.; Goldsmith J. A.; Hsieh C.-L.; Abiona O.; Graham B. S.; McLellan J. S.Cryo-EM structure of the 2019-nCoV Spike in the prefusionconformation. Science2020, eabb2507. 10.1126/science.abb2507. [DOI] [PMC free article] [PubMed] [Google Scholar]
11. Hoffmann M.; Kleine-Weber H.; Schroeder S.; Kruger N.; Herrler T.; Erichsen S.; Schiergens T. S.; Herrler G.; Wu N.-H.; Nitsche A.; Muller M. A.; Drosten C.; Pohlmann S.. SARS-CoV-2Cell Entry Depends on ACE2 and TMPRSS2 and Is Blocked by a ClinicallyProven Protease Inhibitor. Cell 2020, 10.1016/j.cell.2020.02.052. [Epub ahead of print]. [DOI] [PMC free article] [PubMed] [Google Scholar]
12. Gorbalenya A. E.; Snijder E. J.; Ziebuhr J.Virus-encoded proteinases and proteolytic processingin the Nidovirales. J. Gen. Virol.2000, 81 (4), 853–879. 10.1099/0022-1317-81-4-853. [DOI] [PubMed] [Google Scholar]
13. Baez-Santos Y. M.; St. John S. E.; Mesecar A. D.The SARS-coronaviruspapain-like protease: structure, function and inhibition by designedantiviral compounds. Antiviral Res.2015, 115, 21–38. 10.1016/j.antiviral.2014.12.015. [DOI] [PMC free article] [PubMed] [Google Scholar]
14. Lee T.-W.; Cherney M. M.; Huitema C.; Liu J.; James K. E.; Powers J. C.; Eltis L. D.; James M. N.G.Crystalstructures of the main peptidase from the SARS coronavirus inhibitedby a substrate-like aza-peptide epoxide. J.Mol. Biol.2005, 353 (5), 1137–1151. 10.1016/j.jmb.2005.09.004. [DOI] [PMC free article] [PubMed] [Google Scholar]
15. Lu R.; Zhao X.; Li J.; Niu P.; Yang B.; Wu H.; Wang W.; Song H.; Huang B.; Zhu N.; Bi Y.; Ma X.; Zhan F.; Wang L.; Hu T.; Zhou H.; Hu Z.; Zhou W.; Zhao L.; Chen J.; Meng Y.; Wang J.; Lin Y.; Yuan J.; Xie Z.; Ma J.; Liu W. J; Wang D.; Xu W.; Holmes E. C; Gao G. F; Wu G.; Chen W.; Shi W.; Tan W.Genomic characterizationand epidemiology of 2019 novel coronavirus: implications for virusorigins and receptor binding. Lancet2020, 395, 565–574. 10.1016/S0140-6736(20)30251-8. [DOI] [PMC free article] [PubMed] [Google Scholar]
16. Morse J. S.; et al. Learning from the past: possible urgent prevention and treatmentoptions for severe acute respiratory infections caused by 2019-nCoV. ChemBioChem2020, 21 (5), 730–738. 10.1002/cbic.202000047. [DOI] [PMC free article] [PubMed] [Google Scholar]
17. Chan J. F.-W.; Kok K.-H.; Zhu Z.; Chu H.; To K. K.-W.; Yuan S.; Yuen K.-Y.Genomic characterizationof the 2019 novel human-pathogenic coronavirus isolated from a patientwith atypical pneumonia after visiting Wuhan. Emerging Microbes Infect.2020, 9 (1), 221–236. 10.1080/22221751.2020.1719902. [DOI] [PMC free article] [PubMed] [Google Scholar]
18. Dong N., et al. Genomic and protein structure modelling analysis depicts the originand infectivity of 2019-nCoV, a new coronavirus which caused a pneumoniaoutbreak in Wuhan, China, bioRxiv 2020, 10.1101/2020.01.20.913368. [DOI] [Google Scholar]
19. Sheahan T. P.; Sims A. C.; Leist S. R.; Schafer A.; Won J.; Brown A. J.; Montgomery S. A.; Hogg A.; Babusis D.; Clarke M. O.; Spahn J. E.; Bauer L.; Sellers S.; Porter D.; Feng J. Y.; Cihlar T.; Jordan R.; Denison M. R.; Baric R. S.. Comparative therapeutic efficacy of remdesivir andcombination lopinavir, ritonavir, and interferon beta against MERS-CoV Nat. Commun. 2020, Ahead of Print. 10.1038/s41467-019-13940-6 [DOI] [PMC free article] [PubMed] [Google Scholar]
20. Kadam R. U.; Wilson I. A.Structural basisof influenza virus fusion inhibition by the antiviral drug Arbidol. Proc. Natl. Acad. Sci. U. S. A.2017, 114 (2), 206–214. 10.1073/pnas.1617020114. [DOI] [PMC free article] [PubMed] [Google Scholar]
21. Therapeutic options for the 2019 novel coronavirus(2019-nCoV).https://www.nature.com/articles/d41573-020-00016-0. [DOI] [PubMed]
22. The Efficacy of Lopinavir Plus Ritonavirand Arbidol Against Novel Coronavirus Infection (ELACOI).https://clinicaltrials.gov/ct2/show/NCT04252885.
23. Glowacka I.; Bertram S.; Herzog P.; Pfefferle S.; Steffen I.; Muench M. O.; Simmons G.; Hofmann H.; Kuri T.; Weber F.; Eichler J.; Drosten C.; Pohlmann S.Differential downregulation of ACE2by the spike proteins of severe acute respiratory syndrome coronavirusand human coronavirus NL63. Journal of Virology2010, 84 (2), 1198. 10.1128/JVI.01248-09. [DOI] [PMC free article] [PubMed] [Google Scholar]
24. Wu Y.Compensation of ACE2 functionfor possible clinical management. Virol. Sin. 2020, 10.1007/s12250-020-00205-6. [Epub ahead of print]. [DOI] [PMC free article] [PubMed] [Google Scholar]
25. Donoghue M.; Hsieh F.; Baronas E.; Godbout K.; Gosselin M.; Stagliano N.; Donovan M.; Woolf B.; Robison K.; Jeyaseelan R.; Breitbart R. E.; Acton S.A novel angiotensin-converting enzyme-related carboxypeptidase (ACE2)converts angiotensin I to angiotensin 1–9. Circ. Res.2000, 87 (5), 426. 10.1161/01.RES.87.5.e1. [DOI] [PubMed] [Google Scholar]
26. Imai Y.; Kuba K.; Rao S.; Huan Y.; Guo F.; Guan B.; Yang P.; Sarao R.; Wada T.; Leong-Poi H.; Crackower M. A.; Fukamizu A.; Hui C.-C.; Hein L.; Uhlig S.; Slutsky A. S.; Jiang C.; Penninger J. M.Angiotensin-converting enzyme 2 protects from severeacute lung failure. Nature2005, 436 (7047), 112–116. 10.1038/nature03712. [DOI] [PMC free article] [PubMed] [Google Scholar]
27. Tipnis S. R.; Hooper N. M.; Hyde R.; Karran E.; Christie G.; Turner A. J.A human homologof angiotensin-converting enzyme. Cloning and functional expressionas a captopril-insensitive carboxypeptidase. J. Biol. Chem.2000, 275 (43), 33238–33243. 10.1074/jbc.M002615200. [DOI] [PubMed] [Google Scholar]
28. De Witt B. J; Garrison E. A; Champion H. C; Kadowitz P. J.L-163,491 is a partial angiotensin AT(1) receptor agonistin the hindquarters vascular bed of the cat. Eur. J. Pharmacol.2000, 404 (1-2), 213–219. 10.1016/S0014-2999(00)00612-9. [DOI] [PubMed] [Google Scholar]
29. Guo D.Old weapon for new enemy: drugrepurposing for treatment of newly emerging viral diseases. Virol. Sin. 2020, 10.1007/s12250-020-00204-7. [Epub ahead of print]. [DOI] [PMC free article] [PubMed] [Google Scholar]
30. Maxmen A.More than 80 clinical trials launchto test coronavirus treatments. Nature2020, 578 (7795), 347–348. 10.1038/d41586-020-00444-3. [DOI] [PubMed] [Google Scholar]
31. Arabi Y. M; Shalhoub S.; Mandourah Y.; Al-Hameed F.; Al-Omari A.; Al Qasim E.; Jose J.; Alraddadi B.; Almotairi A.; Al Khatib K.; Abdulmomen A.; Qushmaq I.; Sindi A. A; Mady A.; Solaiman O.; Al-Raddadi R.; Maghrabi K.; Ragab A.; Al Mekhlafi G. A; Balkhy H. H; Al Harthy A.; Kharaba A.; Gramish J. A; Al-Aithan A. M; Al-Dawood A.; Merson L.; Hayden F. G; Fowler R.. Ribavirin and Interferon Therapyfor Critically Ill Patients With Middle East Respiratory Syndrome:A Multicenter Observational Study. Clin. Infect.Dis. 2019, DOI: 10.1093/cid/ciz544. [Epub ahead of print]. [DOI] [PMC free article] [PubMed] [Google Scholar]
32. Wang M.; Cao R.; Zhang L.; Yang X.; Liu J.; Xu M.; Shi Z.; Hu Z.; Zhong W.; Xiao G.Remdesivir and chloroquine effectively inhibit the recently emergednovel coronavirus (2019-nCoV) in vitro. CellRes.2020, 30, 269. 10.1038/s41422-020-0282-0. [DOI] [PMC free article] [PubMed] [Google Scholar]
33. Kangji Xinguangzhuang Bingdu Feiyan ZhuanliXinxi Yanbao.https://tech.sina.cn/2020-02-17/detail-iimxxstf2046715.d.html.
34. Warren T. K.; Wells J.; Panchal R. G.; Stuthman K. S.; Garza N. L.; Van Tongeren S. A.; Dong L.; Retterer C. J.; Eaton B. P.; Pegoraro G.; Honnold S.; Bantia S.; Kotian P.; Chen X.; Taubenheim B. R.; Welch L. S.; Minning D. M.; Babu Y. S.; Sheridan W. P.; Bavari S.Protection Against Filovirus Diseasesby a Novel Broad-Spectrum Nucleoside Analogue BCX4430. Nature2014, 508 (7496), 402–405. 10.1038/nature13027. [DOI] [PMC free article] [PubMed] [Google Scholar]
35. Richardson P.; Griffin I.; Tucker C.; Smith D.; Oechsle O.; Phelan A.; Stebbing J.Baricitinibas potential treatment for 2019-nCoV acute respiratory disease. Lancet2020, 395 (10223), e30–e31. 10.1016/S0140-6736(20)30304-4. [DOI] [PMC free article] [PubMed] [Google Scholar]
36. Mifsud E. J.; Hayden F. G.; Hurt A. C.Antiviralstargeting the polymerase complex of influenza viruses. Antiviral Res.2019, 169, 104545. 10.1016/j.antiviral.2019.104545. [DOI] [PubMed] [Google Scholar]
37. Zeldin R. K.Pharmacologicaland therapeutic properties of ritonavir-boosted protease inhibitortherapy in HIV-infected patients. J. Antimicrob.Chemother.2003, 53 (1), 4–9. 10.1093/jac/dkh029. [DOI] [PubMed] [Google Scholar]
38. Ortiz-Alcantara J.; et al. Small molecule inhibitors of the SARS-CoV NSP15 endoribonuclease. Virus Adapt. Treat.2010, 2, 125–133. 10.2147/VAAT.S12733. [DOI] [Google Scholar]
39. Zhou Y.; Vedantham P.; Lu K.; Agudelo J.; Carrion R.; Nunneley J. W.; Barnard D.; Pohlmann S.; McKerrow J. H.; Renslo A. R.; Simmons G.Protease inhibitors targeting coronavirusand filovirus entry. Antiviral Res.2015, 116, 76–84. 10.1016/j.antiviral.2015.01.011. [DOI] [PMC free article] [PubMed] [Google Scholar]
40. Kumar V.; Shin J. S.; Shie J.-J.; Ku K. B.; Kim C.; Go Y. Y.; Huang K.-F.; Kim M.; Liang P.-H.Identification and evaluation of potent Middle Eastrespiratory syndrome coronavirus (MERS-CoV) 3CLPro inhibitors. Antiviral Res.2017, 141, 101–106. 10.1016/j.antiviral.2017.02.007. [DOI] [PMC free article] [PubMed] [Google Scholar]
41. Anand K.Coronavirus Main Proteinase (3CLpro)Structure: Basis for Design of Anti-SARS Drugs. Science2003, 300 (5626), 1763–1767. 10.1126/science.1085658. [DOI] [PubMed] [Google Scholar]
42. Li F.Structureof SARS Coronavirus Spike Receptor-Binding Domain Complexed with Receptor. Science2005, 309 (5742), 1864–1868. 10.1126/science.1116480. [DOI] [PubMed] [Google Scholar]
43. Bisht H.; Roberts A.; Vogel L.; Subbarao K.; Moss B.Neutralizing antibody and protectiveimmunity to SARS coronavirus infection of mice induced by a solublerecombinant polypeptide containing an N-terminal segment of the spikeglycoprotein. Virology2005, 334 (2), 160–165. 10.1016/j.virol.2005.01.042. [DOI] [PMC free article] [PubMed] [Google Scholar]
44. Huang C.; Wang Y.; Li X.; Ren L.; Zhao J.; Hu Y.; Zhang L.; Fan G.; Xu J.; Gu X.; Cheng Z.; Yu T.; Xia J.; Wei Y.; Wu W.; Xie X.; Yin W.; Li H.; Liu M.; Xiao Y.; Gao H.; Guo L.; Xie J.; Wang G.; Jiang R.; Gao Z.; Jin Q.; Wang J.; Cao B.Clinical features of patients infectedwith 2019 novel coronavirus in Wuhan. Lancet2020, 395, 497–506. 10.1016/S0140-6736(20)30183-5. [DOI] [PMC free article] [PubMed] [Google Scholar]
45. Yang Z.-Y.; Huang Y.; Ganesh L.; Leung K.; Kong W.-P.; Schwartz O.; Subbarao K.; Nabel G. J.pH-dependent entryof severe acute respiratory syndrome coronavirus is mediated by thespike glycoprotein andenhanced by dendritic cell transfer throughDC-SIGN. J. Virol.2004, 78, 5642–5650. 10.1128/JVI.78.11.5642-5650.2004. [DOI] [PMC free article] [PubMed] [Google Scholar]
46. Marzi A.; Gramberg T.; Simmons G.; Moller P.; Rennekamp A. J.; Krumbiegel M.; Geier M.; Eisemann J.; Turza N.; Saunier B.; Steinkasserer A.; Becker S.; Bates P.; Hofmann H.; Pohlmann S.DC-SIGN and DCSIGNR interact with the glycoproteinof Marburg virus and the S protein of severe acute respiratory syndromecoronavirus. J. Virol2004, 78, 12090–12095. 10.1128/JVI.78.21.12090-12095.2004. [DOI] [PMC free article] [PubMed] [Google Scholar]
47. The overview of Interferon.https://www.cusabio.com/c-20629.html.
48. Zeng J.Cross Kingdomsmall RNAs among Animals, Plants and Microbes Cells 2019, 8( (4), ) 371, 10.3390/cells8040371. [DOI] [PMC free article] [PubMed] [Google Scholar]
49. Yan B.microRNAs in Cardiovascular Disease:Small Molecules but Big Roles. Current Topicsin Medicinal Chemistry2019, 19 (21), 1918–1947. 10.2174/1568026619666190808160241. [DOI] [PubMed] [Google Scholar]
50. Liu J.; Guo B.RNA-based therapeuticsfor colorectal cancer: Updates and future Directions. Pharmacol. Res.2020, 152, 104550. 10.1016/j.phrs.2019.104550. [DOI] [PMC free article] [PubMed] [Google Scholar]
51. Buchholz U. J.; Bukreyev A.; Yang L.; Lamirande E. W.; Murphy B. R.; Subbarao K.; Collins P. L.Contributions ofthe structural proteins of severe acute respiratory syndrome coronavirusto protective immunity. Proc. Natl. Acad. Sci.U. S. A.2004, 101 (26), 9804–9809. 10.1073/pnas.0403492101. [DOI] [PMC free article] [PubMed] [Google Scholar]
52. GlaxoSmithKline press release on 2/24/20.https://www.gsk.com/en-gb/media/press-releases/clover-and-gsk-announce-research-collaboration-to-evaluate-coronavirus-covid-19-vaccine-candidate-with-pandemic-adjuvant-system.
53. Generex press release on 2/27/20.https://storage.googleapis.com/wzukusers/user-26831283/documents/5e57ed391b286sVf68Kq/PR_Generex_Coronavirus_Update_2_27_2020.pdf.
54. Novavax press release on 2/26/20.http://ir.novavax.com/news-releases/news-release-details/novavax-advances-development-novel-covid-19-vaccine.
55. Moderna press release on 2/24/2020.https://investors.modernatx.com/news-releases/news-release-details/moderna-ships-mrna-vaccine-against-novel-coronavirus-mrna-1273.
56. Modjarrad K.; Roberts C. C; Mills K. T; Castellano A. R; Paolino K.; Muthumani K.; Reuschel E. L; Robb M. L; Racine T.; Oh M.-d.; Lamarre C.; Zaidi F. I; Boyer J.; Kudchodkar S. B; Jeong M.; Darden J. M; Park Y. K; Scott P. T; Remigio C.; Parikh A. P; Wise M. C; Patel A.; Duperret E. K; Kim K. Y; Choi H.; White S.; Bagarazzi M.; May J. M; Kane D.; Lee H.; Kobinger G.; Michael N. L; Weiner D. B; Thomas S. J; Maslow J. NSafety and immunogenicity of an anti-Middle East respiratorysyndrome coronavirus DNA vaccine: a phase 1, open-label, single-arm,dose-escalation trial. Lancet Infect. Dis.2019, 19 (9), 1013–1022. 10.1016/S1473-3099(19)30266-X. [DOI] [PMC free article] [PubMed] [Google Scholar]
57. InovioAccelerates Timeline for COVID-19 DNA Vaccine INO-4800.http://ir.inovio.com/news-and-media/news/press-release-details/2020/Inovio-Accelerates-Timeline-for-COVID-19-DNA-Vaccine-INO-4800/default.aspx.

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