Chitosan and its derivatives as polymeric anti-viral therapeutics and potential anti-SARS-CoV-2 nanomedicine

The coronavirus pandemic, COVID-19 has a global impact on the lives and livelihoods of people. It is characterized by a widespread infection by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), where infected patients may develop serious medical complications or even face death. Development of therapeutic is essential to reduce the morbidity and mortality of infected patients. Chitosan is a versatile biomaterial in nanomedicine and exhibits anti-microbial, anti-cancer and immunomodulatory properties. This review highlights the progress in chitosan design and application pertaining to the anti-viral effects of chitosan and chitosan derivatives (hydroxypropyl trimethylammonium, sulfate, carboxymethyl, bromine, sialylglycopolymer, peptide and phosphonium conjugates) as a function of molecular weight, degree of deacetylation, type of substituents and their degree and site of substitution. The physicochemical attributes of these polymeric therapeutics are identified against the possibility of processing them into nanomedicine which can confer a higher level of anti-viral efficacy. The designs of chitosan for the purpose of targeting SARS-CoV-2, as well as the ever-evolving strains of viruses with a broad spectrum anti-viral activity to meet pandemic preparedness at the early stages of outbreak are discussed.

Introduction
The COVID-19 pandemic is marked by a widespread Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) infection. Infected individuals may develop serious medical complications such as acute respiratory distress syndrome or even face death, especially patients with cancer, immunosuppression or comorbidities who may experience increased risks of cardiovascular disorders, thromboembolic stroke, Guillain-Barre syndrome, rhabdomyolysis, brain, hepatic and acute kidney injury (Bourgonje et al., 2020;Murugan et al., 2021;Sanyaolu et al., 2020). With lockdown and safety measures for the prevention of virus spread (Donthu & Gustafsson, 2020), the summative incidences of SARS-CoV-2 infection have a great impact on lives and livelihoods, which warrants the advancement of anti-SARS-CoV-2 therapeutics.
Severe medical conditions and death of COVID-19 patients are mainly brought about by excessive inflammatory response that SARS-CoV-2 induces in its hosts. It is hypothesised that patients with COVID-19 hyperinflammation have a dysregulated level of macrophage subtypes, reduced level of tissue-resident alveolar macrophages and a spike in the number of inflammatory monocyte-derived macrophages (Merad & Martin, 2020). These inflammatory macrophages may promote fibrosis production and stimulate an interferon signature that induces macrophage hyperactivation (Merad & Martin, 2020).
Vaccines for SARS-CoV-2 are being developed at a rapid pace due to the urgent need to control the morbidity and mortality of COVID-19 (Gallagher, 2020). There are more than 50 COVID-19 vaccine candidates on trials. On 11th December 2020, the United States Food & Drug Administration (FDA) approved the first mRNA vaccine developed by Pfizer Biotech under the purview of emergency use authorization (EUA) to control the COVID-19 pandemic (Mahase, 2020). Following the same month, FDA approved the second COVID-19 vaccine developed by Moderna company (Cohen, 2020). Other examples of vaccines have also been developed by Cansino Biologics and Sinovac Biotech, China, and Gamaleya Research Institute of Epidemiology and Microbiology, Russia (Burki, 2020;Kaur & Gupta, 2020). However, the design and manufacture of vaccine for global supply require a considerable period of time. This is further challenged by SARS-CoV-2 mutating into new strains within a short time, thus affecting the vaccine effectiveness (Gallagher, 2020). On this note, concurrent development of other pharmacological therapeutics is imperative to ensure effective therapeutics are available in the shortest possible time.

Treatment strategies for COVID-19
COVID-19 patients have been treated with repurposed medications such as favipiravir (Avigan) and hydroxychloroquine that are antiinfluenza and anti-malarial drugs respectively (Jean, Lee, & Hsueh, 2020). Four treatment modes have been proposed in search of drug candidates: virus removal or weakening, blocking specific host receptors or enzymes, preventing RNA synthesis and replication, and restoring the host's innate immunity.
Drug candidates upon binding to viruses, are able to remove viruses or inhibit virus-host cell binding. Nanosponges, coated with human cell membranes containing ACE2 & CD147 viral entry receptors, have been designed to bind and remove SARS-CoV-2, preventing it from attaching to human host cells . Peptides with structural similarity to the ACE2 receptor and complementary conformation that binds strongly to the receptor binding domain of SARS-CoV-2, have been computationally designed. These peptides have been proposed to serve as multivalent binding sites of nanoparticles for SARS-CoV-2 removal (Han & Král, 2020). Umifenovir blocks the trimerisation of SARS-CoV-2 S-protein needed in host cell viral entry and inhibits membrane fusion between host cell membrane and viral envelope (Vankadari, 2020). Also, Emodin commonly found in Chinese herbs has been shown to bind to SARS-CoV-1 and inhibit the interaction of SARS-CoV-1 S-protein with ACE2 receptor (McKee et al., 2020).
Clinically, the efficacy of many other drugs such as arbidol, baricitinib, camostat mesilate, darunavir, lopinavir, nafamostat mesilate, oseltamivir, recombinant human angiotensin converting enzyme 2, recombinant interferon-α2β, interferon-β1, ribavirin and ritonavir as well as immunomodulatory drugs such as ravulizumab, ruxolitinib, sarilumab and sirolimus have been evaluated against SARS-CoV-2 (Murugan et al., 2021;Tammam et al., 2021). Solid lipid nanoparticles, nanostructured lipid carriers, nanoemulsion, polymeric nanoparticles, metallic nanoparticles, dendrimer, PEGylated cationic liposome, immunoliposome, carbon dots and carbon nanotubes are examples of nanocarrier developed for the delivery of anti-viral drugs (Shah et al., 2021). Nanoformulation of such drugs is envisaged to bring about the following critical advantages: increased drug solubility, prolonged drug residence/circulation time, reduced off-target drug exposure, sustained drug retention or transportation across biological barriers, flexibility in downstream delivery system design, decoratable with targeting ligand for effective binding to host cells and virus, enhanced cellular uptake particularly by the immune cells, and nanoscale geometry sufficient to restrict virus mobility and block its fusion with host (Al-Hatamleh et al., 2021;Tammam et al., 2021;Zhou, Krishnan, Jiang, Fang, & Zhang, 2021).
Lipid nanoparticles have been developed as the carrier of mRNAbased vaccine, which encodes for a full length, prefusion stabilized S protein of SARS-CoV-2 (Al-Hatamleh et al., 2021;Shah et al., 2021). Nanoformulation of remdesivir with PEGylated dendrimer is translated to sustained drug release behaviour and reduced frequent drug dosing (Tammam et al., 2021). Nanoformulation of dexamethasone, administered via injection or inhalation, has been developed to improve anti-COVID-19 treatment efficacy (Lammers et al., 2020). It directs the potent corticosteroid drug to target phagocytic cells that initiate and propagate inflammation in lung, blood, myeloid and lymphoid tissues, which aids to control macrophage activation syndrome and cytokine storm that are responsible for COVID-19 related fatalities. Nanoformulation may potentiate the anti-edema and anti-fibrotic effects of dexamethasone by increasing drug bioavailability and activity in the inflamed lungs. SARS-CoV-2 is believed to gain access through ciliated cells and this was attributed to the high expression of ACE-2 receptors by the cells (Tammam et al., 2021). With reference to nasal application, nanoparticles can be functionalised with cilia binding ligand to promote targeted drug action on the infected ciliated cells. The anionic nanoparticles with diameters greater than 300 nm have been shown to increase cilia beating frequency upon their deposition on the cell target. The surface charge and size of nanoparticles can be tailored to promote viral clearance via the ciliary action. The diffuse alveolar damage in COVID-19 patients is characterized by the formation of fibrin-rich hyaline membranes (Tammam et al., 2021). The fibrin clot in the alveoli, with collagen accumulation, leads to the development of a fibrotic lung. Chitosan nanoparticles have been shown to display an intrinsic collagen binding affinity. Cys-Arg-Glu-Lys-Ala peptide-decorated nanoparticles are endowed with fibrin-fibrinogen binding ability. They are both potentially useful in targeted drug delivery to the infected lung and combat against the SARS-CoV-2.
Chitosan was presented as a promising anti-SARS-CoV-2 therapeutic in recent studies (Mohd Rasul et al., 2020). N-(2-hydroxypropyl)-3-trimethylammonium chitosan chloride has been shown to strongly interact with the recombinant ectodomain of Human Coronavirus (HCoV) S protein (Ciejka, Wolski, Nowakowska, Pyrc, & Szczubiałka, 2017). Importantly, chitosan was reported to have a high binding affinity for the SARS-CoV-2 S protein trimer cavity (Kalathiya et al., 2020). Chitosan nanoparticles were found to exert anti-inflammatory effects on lipopolysaccharide-inflamed Caco-2 cells where lipopolysaccharideinduced production of tumor necrosis factor-α, interleukin-8 and monocyte chemoattractant protein-1 was inhibited, unlike nanoparticles of cationic polyamidoamine dendrimer where cellular injury of lung was induced via deregulation of renin-angiotensin system owing to their ability to bind to the ACE2 receptors (Tammam et al., 2021).

Chitosan
Chitin is a natural polysaccharide produced from arthropod shells (Chandra Hembram, Prabha, Chandra, Ahmed, & Nimesh, 2016;Wang et al., 2019). Chitosan is a deacetylated form (> 50%) of chitin, that comprises repeating N-acetyl-D-glucosamine and D-glucosamine units (El Knidri, Belaabed, Addaou, Laajeb, & Lahsini, 2018). Chitosan and chitin have amino and hydroxyl moieties which provide sites for chemical modification to produce the desired physicochemical properties for anti-viral applications (El Knidri et al., 2018). The typical molecular weight of chitin and chitosan is 1030-2500 kDa and 100-500 kDa respectively (Ravi Kumar, 2000). Chitosan possesses a lower acetylation degree and no inter-sheet hydrogen bonds, and is thus more aqueous-soluble than chitin (El Knidri et al., 2018). Chitosan is soluble at pH < 6.5 due to the protonation of amino moieties conferring polycationic characteristics (El Knidri et al., 2018), but is insoluble at pH > 7 (Chandra Hembram et al., 2016). Chemical modification of chitosan increases its aqueous solubility in a broader pH range. This facilitates the use of chitosan as drug carrier and vaccine adjuvant.
The chitosan and chitin have identical molecular structures. They however exhibit different physicochemical attributes. The presence of polycationic moieties allows chitosan to form complexes with heavy metals and polyelectrolytes, undergo specific chemical modifications, and exhibit biological properties (Piegat, Goszczyńska, Idzik, & Niemczyk, 2019). Broadly, chitosan has been reported to exhibit antiinflammatory, anti-oxidative, anti-fungal, anti-cancer, anti-bacterial and anti-viral activities (Sharma et al., 2019). The anti-viral potential of chitosan and its derivatives is reflected by their affinity to interact with the cell walls of microorganisms as a function of the state of amino protonation, molecular weight and degree of acetylation of chitosan (Dimassi, Tabary, Chai, Blanchemain, & Martel, 2018;Fernandes et al., 2010;Sharma et al., 2019), free radical-scavenging and anti-oxidative activity (Younes et al., 2014), and ability to induce chemotaxis of mononuclear and polymorphonuclear cells (Kumar, Muzzarelli, Muzzarelli, Sashiwa, & Domb, 2004), anti-inflammatory cytokine production (Tripathi & Singh, 2018) and immunomodulation (Adhikari & Yadav, 2018). Chitosan is mucoadhesive (Bonam et al., 2021). It readily adheres to the mucus of the host for a sufficient period of time to exert its anti-viral activity.
While there is a strong research interest on chitosan in drug delivery system development, few studies have focused on chitosan and its derivatives as an anti-viral therapeutic. With reference to COVID-19, inhalable nanovaccine comprising of chitosan and SARS-CoV-2 spike protein has been designed to provide a strong spike protein-specific antibody immune response and augment local mucosal immunity in bronchoalveolar lavage and lungs (Zhuo et al., 2021). Nonetheless, there is very limited knowledge about the relationship of the chitosan derivatives with their application as an anti-SARS-CoV-2 therapeutic. In this critical review, (1) developmental studies using chitosan and its derivatives as anti-viral therapeutics are identified, and the (2) characteristics of these anti-viral chitosan and derivatives are assessed along with their potential to translate into nanomedicine and for SARS-CoV-2 treatment.

Anti-viral activities of chitosan and its derivatives
The anti-viral activities of chitosan and its derivatives are affected by three main characteristics-molecular weight, degree of acetylation and degree of substitution-among others. This paper reports and evaluates the effects that the characteristics of chitosan-based therapeutics have on anti-viral activity, in relation to the mechanism of action of chitosanbased anti-viral therapeutics. Chitosan and its derivatives may exhibit anti-viral effects via acting on the virus or by host immunomodulation, where the host refers to the virus-infected organism.
Increasing the molecular weight of chitosan has different effects on anti-viral activity against different virus types. While the inhibitory effect against MS2 increases with the molecular weight of chitosan (Davis, Zivanovic, Davidson, & D'Souza, 2015;Su, Zivanovic, & D'Souza, 2009), the growth inhibition of feline calicivirus F-9 and phiX174 is less dependent on the molecular weight variation of chitosan (Davis et al., 2012;Davis et al., 2015). Anti-TMV activity is greater with lower molecular weight chitosan at 15-17 kDa below which, there is no further increase in the anti-viral activity (Davydova et al., 2011).
Compared to molecular weight, the degree of acetylation of chitosan weakly affects the anti-viral activity. Generally, the anti-viral influence of chitosan is mediated via an enhancement of direct and/or complement-mediated chemotactic activities of macrophages and polymorphonuclear cells when chitosan of larger molecular weight and/ or lower degree of acetylation is concerned (Chirkov, 2002;He et al., 2016). With reference to NDV, β-chitosan (3.375-10.491 kDa, < 10% degree of acetylation) shows a higher anti-viral activity than α-chitosan (He et al., 2016), possibly due to its parallel orientation contributing to a less compact intermolecular hydrogen bond network (Dimassi et al., 2018). This in turn increases its aqueous solubility and enhances its contact with the virus. Chitosan can inhibit neuraminidase, an enzyme involved in the release of influenza virus from the infected host cells (Park, Park, Jo, & Lee, 2010), and is thus potentially useful for negating virus replication. Moderately large to large molecular weight (50-1900 kDa) chitosan inhibits neuraminidase (Park, 2010). Chitosan oligosaccharides, composed of a few monomer units of chitosan derived from chitosan degradation (Guo et al., 2018;Naveed et al., 2019), show greater inhibition toward neuraminidase activity in vitro than hetero-oligosaccharides of lower molecular weight and lower degree of acetylation .
Additionally, MS2, phiX174 and enteric virus surrogates (murine norovirus-1, feline calicivirus F-9) have viral capsid proteins with negatively charged functional groups that positively charged chitosan binds to (Zhu et al., 2020). This binding may damage viral structures such as capsid, tail sheath and tail fibres (Davis et al., 2015). Chitosan is reported to chelate trace metals, inhibit enzymatic activity, expose DNA and affect nucleic acid synthesis, thereby inactivating viral infection and replication.

Sulfated chitin
Sulfated chitin is more hydrophilic than chitin. Low molecular weight (16-58 kDa) sulfated chitin exhibits anti-human immunodeficiency virus (HIV) activity (Nishimura et al., 1998) (Fig. 1). The site of sulfation exerts a greater influence on the anti-HIV activity than the total degree of substitution of sulfated chitin (Nishimura et al., 1998). Sulfated chitin 3S with a lower degree of sulfate substitution shows a greater anti-HIV-1 activity than 6S. Sulfation of the N-acetylglucosamine residue at the O-6 position seems to reduce the anti-viral activity of sulfated chitin against HIV-1. It is postulated that the sulfate moieties at O-3 and/or O-2 of chitin sulfate associate with the gp120 of HIV-1 in a specific manner (Nishimura et al., 1998). The binding of negatively charged HIV gp120 to positively charged T lymphocyte receptors via electrostatic forces triggers HIV entry into T lymphocyte host cells (Briz, Poveda, & Soriano, 2006). Through complexing HIV gp120 with 2,3-Osulfonated chitin, the infection of T lymphocytes by HIV can be negated (Vo & Kim, 2010).
The sulfation of chitosan is important in imparting effective and selective viral inhibition against HIV and other enveloped viruses. The anti-viral activity of sulfated chitosan and sulfated chitin are affected by the sulfation site, while that of sulfated chitosan is also enhanced by a higher sulfation degree (Ishihara et al., 1993;Karthik et al., 2016;Nishimura et al., 1998;Stepanov et al., 2012). Using aqueous-soluble β-chitosan as the polysaccharidic backbone may increase the reactivity of the chitosan derivative (Dimassi et al., 2018). Chitosan derivatives with richer sulfate content and higher molecular weight (100 kDa) display greater anti-viral activity than those of lower molecular weights (25 kDa) (Stepanov et al., 2012), except those that mediate their antiviral action via immunomodulation. In the latter, the influence of molecular weight on nitric oxide production has mixed reports. Yang et al. (2018) reported that sulfated β-chitosan (5.030 kDa) induces the greatest rise in nitric oxide production in hosts compared to those with higher (14.481 kDa) or lower (3.169 kDa) molecular weights. However, low molecular weight sulfated chitosan oligosaccharides (< 1 kDa parent molecule) decrease nitric oxide production in another study . The conflicting results may have been confounded by the sulfation degree, position of substituted sulfate group and preparation method of sulfated chitosan, among other factors (Yang et al., 2018). Nitric oxide exhibits anti-viral activity against viruses such as herpes virus, RNA viruses and SARS coronaviruses (Akaike & Maeda, 2000;PeÑarando, Aranda, & RodrÍguez-Ariza, 2019). It inhibits viral replication and respiratory tract infection via altering essential viral proteins and viral RNA synthesis (PeÑarando et al., 2019;Xu, Zheng, Dweik, & Erzurum, 2006). While nitric oxide modulates host immune response, excessive nitric oxide levels may suppress T-cell-mediated immune responses, and contribute to respiratory tract inflammation and injury (PeÑarando et al., 2019). The molecular weight of sulfated chitosan could possibly be adjusted to modulate nitric oxide levels and thus immune response, specific to the inflammatory state of patients.

Sulfated 6-O-carboxymethyl-chitin (SCM-chitin)
SCM-chitin (430 kDa, 0.0766-1.69 degree of sulfate substitution, 0.56 degree of carboxymethyl substitution) significantly inhibits Friend murine leukemia helper virus (F-MuLV) and herpes simplex virus-1 growth (Ishihara et al., 1993) (Fig. 1). Viruses may infect host cells via specific binding to host cell receptors. Since there is no common host cell adsorption receptor between F-MuLV and herpes simplex virus, SCM-chitin is likely to exhibit its anti-viral effect via non-specific binding to the viruses. A high molecular weight (430 kDa) and/or the presence of sulfate moiety in SCM-chitin could be important contributor (s) to the anti-viral activity, since its non-sulfated counterpart-CM-chitin-of low molecular weight (63 kDa) has no inhibitory effect against F-MuLV and herpes simplex virus (Ishihara et al., 1993).

N-carboxymethylchitosan N,O-sulfate (NCMCS)
NCMCS is shown to potently inhibit HIV-1 replication in human T helper cells and Rausher murine leukemia virus (Sosa et al., 1991). The anti-viral NCMCS has a molecular weight of 7.4 kDa, 16% degree of acetylation, and 0.18 degree of N-carboxymethyl substitution (Sosa et al., 1991) (Fig. 1). Ampholytic polymer, NCMCS, varies in its charge distribution depending on the degree of sulfate and carboxymethyl substitution, and the pH and ionic strength of the medium (Sosa et al., 1991). The zwitterionic characteristic of NCMCS could enhance its binding to the cell or the virus, and cellular permeability. Moreover, the anti-viral activity of NCMCS is postulated to be affected by the site of sulfation with O-2 and O-3 sulfated derivatives being the most active (Boroumand et al., 2021;Vo & Kim, 2010).
The highly negatively charged and ampholytic NCMCS is electrostatically attracted to and binds to the gp120 of HIV-1 (Sosa et al., 1991). HIV gp120 is a major viral coat glycoprotein receptor of the HIV-1 virus, and is important for HIV-1 entry into host cells (Chirkov, 2002). By binding to the gp120, NCMCS inhibits HIV-1 viruses from adsorbing to specific target CD4+ cell receptors (Sosa et al., 1991). NCMCS also competitively inhibits the virus-specific reverse transcriptase (Chirkov, 2002). Overall, NCMCS is likely to inhibit HIV-1 infection via blocking the fusion of viral and cell membranes and/or inhibition of reverse transcription of the viral genome.

6-Deoxy-6-bromo-N-phthaloyl chitosan (6BrNPC)
6BrNPC with a molecular weight of 1160 kDa and 0.5471 degree of bromine substitution shows anti-viral activity against NDV (He et al., 2019) (Fig. 1). It is more active than the non-brominated chitosan derivatives (N-phthaloyl chitosan and 6-aminoethylamino-6-deoxy-Nphthaloyl chitosan). 6BrNPC increases the expression level of tumor necrosis factor-α and interferon-β in hosts; these molecules exert an immune response against NDV, and inhibit virus transcription (He et al., 2019). It was also reported that bromination of chitosan alters its electron density and this greatly improves its anti-viral activity (He et al., 2019). Similar to bromine drugs such as ambroxol, bromocriptine and brivudine, its exact mechanism remains unknown.

Chitosan-sialyllactose conjugate
Chitosan-sialyllactose conjugate interacts with influenza viruses via high affinity binding with hemagglutinin of the virus through its sialyllactose component, and this binding between the chitosansialyllactose conjugate and the virus in turn, inhibits viral binding to the host cells and infection (Li, Wu, Gao, & Cheng, 2011). The chitosansialyllactose conjugate is characterized by a molecular weight of 219.6 kDa and 0.156-0.623 degree of sialyloligosaccharide substitution (Fig. 1). Sialic acid analogues in the form of ligands alone do not successfully inhibit influenza virus activity via hindering its binding to viral receptors of the hosts (Umemura et al., 2008). The monomeric 6'sialyllactose-chlorin e6 binds to viral hemagglutinin of influenza in a weaker manner than 6 ′ -sialyllactose-chitosan-chlorin e6 (SCC), which has a higher ligand content (Jeong et al., 2020). The interaction between the sialyllactose ligand and the hemagglutinin of influenza virus depends on their binding affinity and the accessibility of the sialyllactose ligand to the hemagglutinin of influenza virus (Umemura et al., 2008). A higher degree of sialyllactose substitution promotes a greater probability of ligands binding to the hemagglutinin of influenza virus, which results in a greater inhibition of influenza virus infection.
It was reported that SCC displays 23% and 50% higher anti-viral effects on influenza A and B respectively than oseltamivir, the conventional anti-viral drug (Jeong et al., 2020). SCC gives full protection from influenza virus infection in mice subjected to laser irradiation (Jeong et al., 2020). Here, chitosan serves as a backbone for conjugation with multiple 6 ′ -sialyllactose molecules which influenza hemagglutinin recognises and binds to (Jeong et al., 2020). The chlorin e6 moiety produces reactive oxygen species that permanently damage the viral membrane when excited by light irradiation, thus causing irreversible inactivation of the influenza virus (Jeong et al., 2020). Therein, 6 ′ -sialyllactose-chitosan-chlorin e6 possesses both prophylactic and anti-viral potential.
To prevent influenza virus infection, the length of chitosan backbone-governed by its polymerisation degree-should exceed the virus particle diameter (100 nm) to introduce a physical barrier between the virus and the host (Umemura et al., 2008). As the polymerisation degree of sialylglycopolymer-conjugated chitosan increases from 20 to 1430, its strength of inhibition against three strains of human influenza viruses (H1N1, H3N2 and B/Shanghai/ 361/2002) becomes greater. Aqueous solubility is lowered with an increase in the polymerisation degree of chitosan. Chitosan with optimal aqueous solubility and anti-viral activity has been found to associate with a polymerisation degree of 500, 0.15 degree of sialyloligosaccharide substitution and a molecular weight of 219.6 kDa (Umemura et al., 2008). At 500 polymerisation degree, the "tail" of sialyloligosaccharide-conjugated chitosan is sufficiently long to prevent the chitosan-bound virus particles from approaching the host cell surfaces.

Chitosan-peptide conjugate
Chitosan oligomers (< 1 kDa, 10% degree of acetylation), conjugated with tripeptides containing tryptophan, methionine and glutamine, have been reported to show anti-HIV-1 activity (Karagozlu, Karadeniz, & Kim, 2014). Chitosan-peptide conjugates bind to viral glycoprotein gp41 and thus inhibit gp41 from interacting with the receptors on CD4 host cell membrane, impeding HIV-1 fusion with infected and uninfected cells. The study also reported that chitosan-peptide conjugates protect cells from lysis and inhibit HIV-triggered syncytia formation in a co-culture assay. The type, sequence and number of specific amino acid attached to the oligochitosan backbone could be key factors in the antiviral effect displayed by chitosan-peptide conjugates. Notably, the peptide sequence was found to be similar to a strong HIV-1 fusion inhibitor (CP621-652), which inhibits the protein binding domain of the HIV-1 gp41 (Karagozlu et al., 2014).

Poly-quaternary phosphonium oligochitosan (PQPOC)
PQPOC with 0.2855 degree of poly-quaternary phosphonium substitution shows greater anti-viral activity against feline calicivirus, hepatitis A virus, and Coxsackievirus B4 than that of lower substituent content (Sofy et al., 2019) (Fig. 1). PQPOC is constituted of a permanently positive charged moiety in the polymer chain and exhibits a higher ionic character than the zwitterionic chitosan. The positively charged PQPOC is likely to interact with negatively charged viral capsids, which may block viral replication via causing structural injury to the viral capsids. The chitosan backbone in PQPOC could hinder the virus propagation by promoting ribonuclease activity in hosts, which increases viral RNA degradation, and therefore, hinders virus transcription and translation.

Insights into anti-viral action of chitosan-based therapeutics
Overview of the literature indicates that the chitosan derivatives that inhibit viral replication via binding to enveloped viruses have a molecular weight of 1-430 kDa, 14-16% degree of acetylation, 0.18-0.56 degree of carboxymethyl substitution, 0.0766-1.69 degree of sulfate substitution and 0.62-0.77 degree of N-(2-hydroxypropyl)-3-trimethylammonium substitution. The chitosan derivatives that inhibit enveloped NDV via stimulation of the host immune response are generally characterized by a molecular weight of 1-1160 kDa, < 10% degree of acetylation, and 0.5471 degree of bromine substitution.
The anti-viral effects of chitosan can be virus-specific and are dependent on the molecular weight, degree of acetylation, and/or conjugate type and its substitution site and extent. Generally, chitosanbased therapeutics express positive charges that are favourable to interact with the protein domain of the virus or negatively charged virus. The positive charges promote aqueous solubility at physiological pHs (Chandra Hembram et al., 2016) and this enhances their contact with the target. Chitosan-based therapeutics are characterized by a polymerisation degree that enables them to protect the viral binding site of the host from the virus. The relationship of the chemical descriptor with the anti-viral activity, however, may not be linear. A rise or reduction in the molecular weight of a polymeric therapeutic may increase its anti-viral effects. Each chitosan-based therapeutic is likely to have an optimal molecular weight, specific to the virus used in the study. The extent of conjugate substitution may not be critical to combat the virus, when compared to its substitution site or vice versa, probably depending on the target variant of virus and choice of parent polymer. The choice of substituent to be conjugated with chitosan can be critical in the eradication of a specific virus. A study reported that while some sulfonated non-chitosan polymers exhibited anti-H3N2 influenza virus activity, N-sulfonated chitosan did not . A change in chemical characteristics, such as molecular weight, may affect the anti-viral mechanisms of the therapeutics. Chitosan-based therapeutics that directly interact with virus particles tend to be of larger molecular weight. Conversely, the chitosan-based therapeutics that activate and increase the host immune response had mixed reports on their molecular weight effects. Fig. 2 summarizes the general anti-viral mechanisms of actions of the chitosan and chitosan derivatives. Broadly, chitosan-based therapeutics act on non-enveloped viruses (murine norovirus-1, MS2, feline calicivirus F-9, human papillomavirus, hepatitis A virus, Coxsackievirus B4, phi X174) through viral capsid interaction and (human papillomavirus) through host immunomodulation. One or more mechanisms of action may take place. The positively charged chitosan has been postulated to bind to viral nucleic acid and reduce the viral genome integrity (Zhu et al., 2020). With respect to enveloped viruses, chitosanbased therapeutics may stimulate the immune system (to inhibit NDV), bind to envelope glycoprotein to inhibit viral entry (of HIV-1, influenza) and/or inhibit the virus growth (of herpes simplex virus) in a nonspecific manner. The anti-viral actions of such therapeutics and their respective physicochemical constructs can be exploited to eradicate the SARS-CoV-2, another variant of enveloped virus.
SARS-CoV-2 infection is characterized by pulmonary mucus hypersecretion and mucus plugging in COVID-19 patients (Kumar, Binu, Devan, & Nath, 2021). The mucin gene is overexpressed under the influence of pro-inflammatory cascades involving interferon-γ and interferon-β which activate aryl hydrocarbon receptor signaling thereby leading to mucus hypersecretion, impaired mucociliary clearance, airway obstruction and respiratory distress. Mucus targeting is foreseeably a viable approach in the delivery of drugs to a site where a high viral load is reflected by its pathophysiological conditions. The chitosan is a mucoadhesive (Gulati, Dua, & Dureja, 2021). As an anti-viral therapeutic, the high affinity for mucus enables it to interact with the host and/or virus in a close proximity and exert the biological action. The chitosan derivatives adhere to mucus via hydrogen bonding and/or electrostatic attraction . Quaternization of chitosan at C 2 -N (HTCC) or attaching a phosphonium moiety to the chitosan (PQPOC) provides permanent positive charges that electrostatically bind to the negatively charged sialic acid of the mucus. Carboxymethylation and sulfation could promote chitosan-mucus interaction via the formation of hydrogen bonding. With reference to 6BrNPC and SCC that active against the enveloped viruses, the introduction of phthaloylation and sialyllactose is expected to reduce the intermolecular binding between the chitosan molecules, thus facilitating the chitosan to bind to the mucus. Overall, the binding affinity of chitosan derivatives for mucus is governed by a complex interplay of chemistry of substituents and their degree of substitution, molecular weight and chain flexibility of derivatives, as well as ionic and pH environments of mucus as a function of the disease progression (Collado-Gonzalez, Espinosa, & Goycoolea, 2019).

Manufacturability of chitosan-based therapeutics into nanomedicine
Chitosan is widely employed as the excipient of choice in the Fig. 2. General anti-viral mechanisms of actions of the chitosan and chitosan derivatives (Created with BioRender.com). development of nanomedicine for a wide range of diseases (Alhajj et al., 2020;Calderon et al., 2013;Loutfy et al., 2020;Meng, Sturgis, & Youan, 2011;Xue et al., 2015;Zhao et al., 2014). With reference to anti-viral application, several attempts to formulate chitosan nanoparticles for the treatment of HIV, and hepatitis B and C viruses have been initiated (Loutfy et al., 2020;Meng et al., 2011;Xue et al., 2015). Chitosan derivatives of molecular weight of 3-1000 kDa, 5-27.8% degree of acetylation and 0.07-1.48 degree of conjugate substitution, have been used in nanoparticle development for pharmaceutical applications (Table 1). The synthesized nanoparticles exhibit a wide range of size (5.1-1279 nm), polydispersity index (0.034-0.780), and zeta potential (− 50.5 to +55.2 mV) that are influenced by choice of pH and/or substituent. They are spherical or cuboidal, and are generally presenting with smooth surfaces. While chitosan is usually partially crystalline, drug-loaded chitosan nanoparticles are typically amorphous with a few exceptions. Drug loading into chitosan nanoparticles has varied or no effect on the size, polydispersity and surface charge of chitosan, depending on its formulation and preparation method.
The molecular characteristics of chitosan that are used in the nanomedicine development resemble those of chitosan derivatives that show positive anti-viral effects and can potentially be developed as anti-SARS-CoV-2 polymeric therapeutics. It is hypothesised that the chitosan-based therapeutics, with proven anti-SARS-CoV-2 if any, can be easily processed into nanomedicine that can possibly bring about a higher interaction propensity with the viral transmembrane spike glycoprotein. As the chitosan derivatives are largely ionic in nature, their required aqueous solubility for nanoparticle preparation can be met via simple solution pH modulation or solubilization process. The nanofabrication of chitosan-based anti-SARS-CoV-2 therapeutics may be introduced with an additional anti-viral agent to synergistically promote the therapeutic effect. An example is PQPOC, which synergistically stabilises silver nanoparticles that have the ability to bind to the viral envelope glycoprotein, through the formation of a PQPOC-silver nanoparticle biocomposite (Karagozlu et al., 2014;Sofy et al., 2019). This prevents the virus from binding and entering into the host cell to a greater extent than the chitosan therapeutics alone.
Polymersomes are artificial vesicles that are constituted of amphiphilic block or grafted copolymers (Al-Hatamleh et al., 2021). They exhibit a high colloidal stability and ease of being decorated with targeting ligand. Lately, the polymersomes have been advocated as an antiviral drug carrier. In comparison to liposomes (3-5 nm), the introduction of polymers provides a resultant bilayer thickness of 5-50 nm. The polymersomes are physicochemically more stable than liposomes. With reference to anti-viral chitosan therapeutics that are polymeric in nature, their use in the development of polymersomes is expected to lead to the formation of a strong and mucoadhesive nanostructure with prolonged residence time and biological action in the nanoscale. Combination of chitosan therapeutic with anti-viral drug in polymersomes can have their anti-viral actions synergized with chitosan controlling drug release and activity.

Conclusion and future perspectives
The present review highlights that chitosan is a viable biopolymer for the development of broad-spectrum anti-viral therapeutics. Structural modification of chitosan through conjugating with hydroxypropyl trimethylammonium, sulfate, carboxymethyl, bromine, sialyllactose, peptide and phosphonium moieties grants or greatly improves the antiviral activity of the parent polymer. Chitosan conjugates-such as HTCC, sulfated chitosan, SCM-chitin, NCMCS, 6BrNPC and SCC -exhibit broad-spectrum anti-viral activity against human coronavirus, HIV, herpes simplex virus, influenza virus, NDV, human papillomavirus and F-MuLV. Their anti-viral effectiveness depends on the interplay of molecular weight, degree of acetylation, degree and site of conjugate substitution. One or more substituents may be introduced to a chitosan monomeric residue. The resultant conjugate may be blended with alternative anti-viral agents such as silver nanoparticles in the advancement of a more efficacious nanomedicine (Sofy et al., 2019). Further, it is envisaged that the anti-viral chitosan therapeutics can be processed into nanomedicine as their physicochemical characteristics are similar to those of chitosan and derivatives used in nanomedicine design. The nano-enabling process is foreseen to promote the anti-viral activity of the chitosan therapeutics to a greater extent.
Chitosan can potentially inhibit SARS-CoV-2 via binding with the SARS-CoV-2 spike glycoprotein trimer cavity (Kalathiya et al., 2020). SARS-CoV-2, as an enveloped virus, may be treated with chitosan-based therapeutics developed with due consideration to the structure-activity relationship of chitosan derivatives substituted with carboxymethyl, sulfate, bromine, N-(2-hydroxypropyl)-3-trimethylammonium, phthaloyl and sialyllactose moieties that are active against other enveloped virus variants. Future studies may also research on the optimal characteristics-molecular weight, degree of acetylation, degree and site of conjugate substitution-of chitosan which will provide the greatest antiviral effect against SARS-CoV-2. Further, conjugating chitosan with optimal anti-viral characteristics by other anti-SARS-CoV-2 ligands, may confer additive or synergistic effects, and greater effectiveness in inhibiting SARS-CoV-2 replication.
The current pandemic preparedness planning is focused on expediting vaccine research and repurposing the use of approved anti-virals and other drugs (Cho & Glenn, 2020). Such an approach may be insufficient in promptly controlling the spread of contagious viral infections such as COVID-19. It has been highlighted that investing time and resources to develop an anti-viral therapeutic for a single virus may not be worthwhile (Cho & Glenn, 2020). Therefore, an effective broadspectrum anti-viral strategy will be the next generation solution to work against various viruses, including COVID-19 and other novel ones. Such an approach is foreseeable to enable potential viable therapeutics to greatly improve pandemic preparedness at early stages of outbreaks. The anti-viral chitosan derivatives described herein are characterized by a broad spectrum anti-viral activity. They can be the potential lead in the development of anti-SARS-CoV-2 therapeutics and novel ones in future.

CRediT authorship contribution statement
Rebecca Shu Ling Tan: Validation, Formal analysis, Investigation,

Declaration of competing interest
There are no conflicts of interest to declare.


Computational design of a neutralizing antibody with picomolar binding affinity for all concerning SARS-CoV-2 variants

ABSTRACT Coronavirus disease 2019, caused by SARS-CoV-2, remains an on-going pandemic, partly due to the emergence of variant viruses that can “break-through” the protection of the current vaccines and neutralizing antibodies (nAbs), highlighting the needs for broadly nAbs and next-generation vaccines. We report an antibody that exhibits breadth and potency in binding the receptor-binding domain (RBD) of the virus spike glycoprotein across SARS coronaviruses. Initially, a lead antibody was computationally discovered and crystallographically validated that binds to a highly conserved surface of the RBD of wild-type SARS-CoV-2. Subsequently, through experimental affinity enhancement and computational affinity maturation, it was further developed to bind the RBD of all concerning SARS-CoV-2 variants, SARS-CoV-1 and pangolin coronavirus with pico-molar binding affinities, consistently exhibited strong neutralization activity against wild-type SARS-CoV-2 and the Alpha and Delta variants. These results identify a vulnerable target site on coronaviruses for development of pan-sarbecovirus nAbs and vaccines.

Introduction
Zoonotic coronaviruses have caused three major disease outbreaks in the past two decades: the SARS outbreak caused by severe acute respiratory syndrome coronavirus 1 (SARS-CoV -1) in 2002-2004, the Middle East respiratory syndrome (MERS) outbreaks caused by MERS-CoV since 2012, and coronavirus disease 2019 (COVID-19) caused by SARS-CoV-2 since December 2019. 1,2 The three viruses that caused these outbreaks are positive-sense single-stranded RNA viruses 3 belonging to the genera Betacoronavirus. SARS-CoV-1 and SARS-CoV-2 are members of the subgenus Sarbecovirus (lineage B), while MERS-CoV belongs to the subgenus Merbecovirus (lineage C). 1,4 SARS-CoV-2 is divergent from SARS-CoV-1, residing in its own distinct phylogenetic clade. 5 As of October 2021, the ongoing global COVID-19 pandemic has caused more than 240 million infections and claimed at least 4.8 million lives worldwide. With unprecedented speed, several vaccines were developed and licensed for use in a massive global vaccination program. 6 Since the beginning of the pandemic, however, SARS-CoV-2 has undergone significant antigenic drift, resulting in the emergence of several more virulent variants. The Alpha variant was first found in the United Kingdom, but it quickly became a dominant strain. 7 Alpha was then outcompeted by the Delta variant, which arose in India and spread to become the most prevalent strain in the world. 8 Mutations can confer increased infectivity, resistance to neutralizing antibodies (nAbs), and higher replication efficiency. [9][10][11] As an example of this process, the key mutations (K417N and E484K) that distinguish the Beta and Gamma variants were found to abrogate binding to and neutralization by the two receptor-binding domain (RBD)-binding antibody families most frequently elicited by the spike glycoprotein or RBD derived from the Wuhan strain, thus reducing the effectiveness of current vaccines. 12 Rising concern about viral escape triggered efforts to develop a more broadly protective second-generation of coronavirus vaccines 13,14 or another novel vaccination strategy. 15 While the deployment of preventive vaccines has been the most effective approach to curb the spread of SARS-CoV-2, nAbs can also provide immediate solutions for the treatment of COVID-19 patients. To date, more than 10 monoclonal antibodies (mAbs) have been developed for COVID-19 therapy or prophylaxis, and five of them are approved for clinical use. 16,17 Most of these mAbs prevent the RBD from binding the ACE2 receptor, thus blocking SARS-CoV-2 entry into cells. These nAbs were based on the original virus strain, and four clinical-stage nAbs (etesevimab, bamlanivimab, casirivimab, and regdanvimab) show reduced neutralization potency against SARS-CoV-2 variants of concern (VOCs) (Alpha/B.1.1.7, Beta/B.1.351, Gamma/P.1, Delta/ B.1.617.2, DeltaPlus/AY.1, AY.2, AY.3). [16][17][18][19] This underscores the need for new nAbs that protect against all currently circulating variants, ideally all sarbecovirus clades. Several broadly neutralizing mAbs were discovered in blood samples from COVID-19 convalescent individuals or vaccinated individuals. 15,[19][20][21] Herein, we report a computational approach with additional experimental affinity enhancement that we used to develop a highly potent nAb that broadly targets all currently circulating SARS-CoV-2 variants, as well as SARS-CoV-1 and pangolin coronavirus.

Computational design approach to discover an antibody against the wild-type SARS-CoV-2 RBD
We initially aimed to discover nAbs against wild-type SARS-CoV -2 (Wuhan strain) by computational design of mAbs that bind to the SARS-CoV-2 RBD. We used four available crystal structures of the SARS-CoV-1 RBD bound to the antigen-binding fragment (Fab) of four nAbs against different surfaces of the RBD of SARS-CoV-1 (m396, 22 F26G19, 23 80 R, 24 S230 25 ) (Figure 1(a)). By superposing the SARS-CoV-2 RBD in complex with human angiotensin-converting enzyme 2 (hACE2) 26 on these structures, we were able to extract four models of the variable fragment (Fv) bound to the SARS-CoV-2 RBD. Of note, none of these four antibodies showed any cross-reactivity with the SARS-CoV-2 spike glygcoprotein. 27,28 Assuming it was possible, with these Fab docking positions, to shift the antigen-binding specificity to the SARS-CoV-2 RBD, we performed a sequence design on the complementarity-determining regions (CDRs) using the Rosetta software suite. 29,30 From ~1000 outputs, we selected 55 designs based on criteria including shape-complementarity, buried solvent-accessible surface area, and number of unsatisfied polar atoms. We then reformatted the selected designs into their full IgG1 form and produced them in Chinese hamster ovary (CHO) cells. One mAb, designated D27, exhibited outstanding binding with the SARS-CoV-2 RBD in a biolayer interferometry (BLI) experiment (Figure 1(b)). When we quantified their interaction, however, we measured a weak dissociation constants (K D ) of only 177 nM (Figure 1(c)), which is even weaker than the interaction between hACE2 and the RBD (K D < 100 nM), as reported in the literature. 31,32 Of note, D27 was derived from m396, and contains 16 amino acid substitutions relative to m396, all in the CDRs. Computational design approach to discover mAbs against the SARS-CoV-2 RBD. (a) The anti-SARS-CoV-1 RBD Fvs derived from the four indicated structures were modeled on the SARS CoV-2 RBD and then subjected to a sequence design protocol. Only CDR sequences were designed, with 55 out of 1000 generated Fv designs ultimately selected for experimental validation. (b) Screening. 55 Fvs were converted into their full IgG format, produced in culture, and tested for binding to the SARS-CoV-2 RBD by BLI. One antibody, D27, showed outstanding binding signal. (c) Quantification of the binding affinity of D27. The SARS-CoV-2 RBD concentration ranged from 126-1000 nM. The BLI experiment was technically duplicated with similar results, and representative sensorgrams are shown.

Structural validation of the computational design
We next determined the crystal structure of the Fab of D27 (D27-Fab) bound to the SARS-CoV-2 RBD at 2.2 Å resolution (Table 1, PDB ID: 7VYR). This structure was closely superposable onto the D27 design, validating the design was largely correct (Figure 2(a)). In comparison to the original m369, a number of amino acid residues substituted in the sequence design are involved in favorable interactions. For example, S92F L and S93L L on light-chain CDR 3 (CDRL3) are involved in hydrophobic interactions with V407 and Y506, as well as the aliphatic portions of T376 and K378 on the RBD (Figure 2(b) and Figure S1b; Region 1). Likewise, S31R H on CDRH1 approaches a cavity of the RBD and forms a hydrogen-bonded network involving backbone atoms of Y369, S371, and F374 (Figure 2(b) and Figure S1b; Region 2).

Structure-based CDR3 loop extension enhanced the binding affinity by 25 fold
With the high-resolution D27-Fab-SARS-CoV-2 RBD structure in hand, we sought to improve the binding affinity between the two proteins. One way to accomplish this is saturation mutagenesis either of all CDR residues or at least those in contact with the target antigen. We chose not to take this approach because closer examination of the structure revealed that the CDRL3 and the CDRH3 loops are rather loosely packed against the RBD (Figure 3(a)). Thus, extension of the tips of these loops by one and two residues, respectively, would lead to better occupation of the interfacial space between D27 and the RBD, likely strengthening their intermolecular interactions. To find the best possible sequences, we used random DNA libraries in the form of two sets of oligonucleotides to convert F93L (at the tip of CDRL3) into two randomized residues, as well as E101H and Q102H (at the tip of CDRH3) into four randomized residues (Figure 3(a)). Subsequently, these oligonucleotide libraries were incorporated into template DNA encoding the single-chain Fv (scFv) of D27. The resulting scFv library (~6.4 x 10 7 diversity) was expressed and displayed on the surface of yeast (Figure 3(b)). We performed two rounds of magnetic-activated cell sorting (MACS) and three rounds of fluorescence-activated cell sorting (FACS) with increasing stringency. This allowed us to enrich and select scFvs with increased binding affinity for the RBD (Figure 3(c)). Of the nine scFvs we selected, the one with strongest binding affinity was reformatted in its full IgG form (Figure 3(d) and Table 2). We then found that this mAb, designated D27LE, bound the SARS-CoV-2 RBD with a KD of 7.12 nM (Figure 3(e)), representing a binding affinity roughly 25-fold tighter than that of D27.  shows key hydrophobic interactions of L92 and F93 (on CDRL3) with a hydrophobic pocket formed by the side chains of the RBD residues T376, K378, V407, R408, and Y508. Region 2 shows key hydrogen-bonding interactions of the guanidine group of R31H (on CDRH1) inserted into a pocket formed by backbone atoms Y369, S371, and F374, plus two water molecules. This hydrogen-bonded network in the crystal structure was not recapitulated by the sequence design because it does not include explicit water molecules.

Computational affinity maturation for the SARS-CoV-2 Alpha variant
By the time we had developed D27LE, the Alpha variant was a serious concern, having spread to many countries. This variant includes the N501Y mutation, which increases RBD binding affinity for the human ACE2 receptor by several fold relative to the wild-type RBD. 12,33 On our crystal structure, N501 is close to four residues: one on CDRH1 (S33) and three on CDRH2 (D52, S54, R55) (Figure 4(a)). After modeling the N501Y mutation on the crystal structure, we sought to optimize local interactions with Y501 using a sequence design protocol confined to these four nearby residues in hopes of increasing binding affinity for variants with this mutation. We selected 10 of 100 resulting designs and expressed their scFv forms in E.coli. Eight of the 10 designs exhibited improved binding affinity for the N501Y mutant RBD target, and one design (Y4.2) showed outstanding binding affinity (K D of 4.87 nM) (Figure 4(b-c) and Table 3). Notably, its IgG form bound the N501Y RBD target with ultra-high binding affinity (K D < 1 pM) (Figure 4(c)). Sequencing showed that two of the four designed CDR residues, D52 H and S54 H , were changed to A52 H and G54 H . The resulting Ab, named D27LEY, demonstrates that significant affinity maturation against the results of antigenic drift can be achieved quickly by using extensive computational amino acid sampling for a small set of specific residues on a mother antibody. Interestingly, we found D27LE has higher binding affinity for the N501Y mutant RBD (K D = 0.54 nM) than for the wild-type RBD (K D = 7.12 nM) ( Figure S2a). Thus, computational affinity maturation for the N501Y mutant RBD enhanced binding affinity > 500 fold. Because N501 is remote from the CDRL3 and CDRH3 loops (extended in D27LE), the affinity enhancement we observed for D27LE must be attributed to the N501Y mutation itself. It is likely that a tyrosine residue at this position provides an even stronger interaction with D27LE than the wild-type asparagine residue because of its hydrophobicity and bulkiness. Of note, the two-residue changes in D27LEY (in comparison with D27LE) that optimized binding to the N501Y mutation increased rather than decreased the binding affinity for the wild-type RBD, with the K D falling from 7.12 nM to 1.14 nM ( Figure S2b). These results indicate that the original interface design was suboptimal, at least in part due to inaccuracies in the initial Ab-RBD model. We suspect saturation mutagenesis of CDR residues would have led us to similar results.

D27LEY potently binds to the RBDs of SARS-CoV-2 variants, SARS-CoV-1 and pangolin CoV
As antigenic drift has continued, new SARS-CoV-2 VOCs have emerged as follows: Beta (B.1.351) and Gamma (P.1) with K417N/ T and E484K mutations in addition to N501Y, Delta (B.1.617.2) having key L452R and T478K mutations, and DeltaPlus (AY.1) with an additional K417N mutation. Two SARS-CoV-2 variants of interests (VOIs) (Lambda/C.37, Mu/B.1.621) and a dozen of variants under monitoring (VUMs), including Epsilon (B.1.427) and Kappa (B.617.1), have also emerged. The Lambda variant has key T76I and L452Q mutations. Of note, the K417N and E484K mutations abrogate the RBD binding and neutralization capacity of the two most frequently elicited SARS-CoV-2 antibody families (IGHV3-53/3-66 and IGHV1-2). 12 They also reduce the neutralizing activity of sera isolated from convalescent plasma and from vaccinated individuals. 34 The T76I and L452Q mutations seem to be responsible for conferring higher infectivity and resistance to nAbs induced by mRNA vaccination. 35,36 Remarkably, of all these mutations, only N501Y is located on the D27LEY-binding surface of the RBD ( Figure 5(a)). This suggests that D27LEY might not be affected by these key mutations, and thus may bind to the variant RBDs similarly as it binds the wild-type RBD. Indeed, we found D27LEY bound to the RBD of the Beta and Gamma variants (which carry N501Y) with a K D of less than 1 pM ( Figure 5 We tested whether D27LEY might exhibit cross-reactivity against the RBD of SARS-CoV-1 (Tor 2) and pangolin CoV (PCoV-GD1). Indeed, D27LEY binds to the SARS-CoV-1 RBD with a K D of <1 pM and to the PCoV-GD1 RBD with a K D of 0.66 nM ( Figure S3). Thus, D27LEY has RBD-binding potency across sarbecovirus clades Ia and Ib. The sub-picomolar binding affinity of D27LEY for the SARS-CoV-1 RBD is intriguing because its binding interactions were optimized for the SARS-CoV-2 RBD and for its tyrosine substitution of N501, which is a threonine residue in the SARS-CoV-1 RBD. It is possible that the extended CDR3 loops created "hot spot" interactions for the SARS-CoV-1 RBD, and the minor sequence variation in the binding epitopes between the two RBDs, as discussed below, allowed parallel affinity enhancement.

D27LEY strongly or potently neutralizes SARS-CoV-2 variants
The sub-picomolar binding affinity between D27LEY and the N501Y mutant RBD (of the Alpha variant) is at least 10,000fold higher than the affinity between hACE2 and the N501Y mutant RBD (K D = 22 nM). 37 D27LEY also binds the RBD of wild-type SARS-CoV-2 and that of the Delta variant with reduced but still potent affinities (K D = 1.14 nM and 0.32 nM, respectively) ( Figure 5(c) and Figure S2b). We examined whether these binding affinities measured in vitro correlate with virus neutralization potency against wild-type SARS-CoV-2 and the SARS-CoV-2 Alpha and Delta variants. In a Vero E6 cell-based focus reduction neutralization test (FRNT), D27LEY exhibited the most potent neutralizing activity against the Alpha variant with a neutralization constant 50 (NC 50 ) of 0.10 nM ( Figure 6). D27LEY exhibited similarly strong neutralization activity against the wild-type virus and the Delta variant with NC 50 values of 8.3 nM and 12.2 nM, respectively ( Figure 6). Given that the Delta variant infects host cells more efficiently than wild-type SARS-CoV-2, 38 the neutralization potency of D27LEY correlates nicely with its potency of binding to each respective RBD. Presumably, D27LEY is likely to exhibit similar neutralization activity against most, if not all, circulating SARS-CoV-2 variants and related sarbecoviruses that may emerge in the future.  , and its bivalent binding affinity was quantified in triplicate at D27LEY concentrations ranging from 15.6-125 nM. The K D value of this interaction is less than 1 pM, which is lower than the instrument sensitivity. Very recently, three potent and broadly nAbs, S2X259, S2X35 and DH1047, were reported. A structural comparison shows that they target the same side on the RBD containing site IIa, which is actually the surface recognized by S2X35 39 and is partly shared by D27LEY, S2X259 and DH1047 (Figure 7  (a-b)). The D27LEY-binding surface is closer to the RBM than is the other binding surfaces. Consequently, the summed binding surface spans a fairly wide patch on this side of the RBD (Figure 7(b)). This surface does not include the key mutations sites found in many SARS-CoV-2 variants except N501, which is an epitope residue for D27LEY. According to a multiple sequence alignment, apart from N501, the epitope residues recognized by D27LEY are all invariable in the SARS-CoV-2 VOCs, VOIs, and VUMs (Figure 7(c)). This explains the broad and potent binding of D27LEY to currently circulating SARS-CoV-2 variants. The alignment also shows that only three epitope residues for D27LEY are variable in sarbecovirus clade Ia, which includes SARS-CoV, and in clade Ib, which includes pangolin CoV. Moreover, these three residues exhibit limited variation among clades Ia and Ib (Figure 7(c)), explaining the cross-reactivity of D27LEY for their RBDs. Similar conclusions can be drawn to explain the broad neutralization activities of the other three nAbs. Importantly, this comparison of the binding epitopes convincingly defines a vulnerable surface on the RBD that can be targeted for developing broadly protecting nAbs and vaccines.

Discussion
De novo computational antibody design involves two steps: global docking of scaffold antibodies to antigens, followed by the design of CDR sequences. Yet, the success rate of de novo antibody design is low, with computational approaches remaining mostly limited to affinity maturation based on antibody-antigen co-crystal structures. This indicates that obtaining "plausible" antibody-antigen docking models remains a significant challenge. 40 Recently, a computational method using a positionspecific structure-scoring method led to the discovery of distinct CDRH3 sequences that exhibit structural homology to the CDRH3 of the original antibody against influenza H1 hemagglutinin. In this approach, the antibodyantigen docking problem is precluded. 41 In our study, we constructed docking models for Ab-SARS-CoV-2 RBD based on four known structures of human mAbs bound to the SARS-CoV-1 RBD. This approach was successful, providing a lead antibody from among only 55 designs. It should be noted, however, that only one model for Ab-SARS-CoV-2 RBD using m396 gave a hit. In retrospect, the other three mAbs (F26G19, 80 R, S230) used for the initial docking models were in contact with a structurally most variable region between the SARS-CoV-1 and SARS-CoV-2 RBDs. This uncertainty was probably the main reason why the three docking models were not productive, although insufficient sampling might have been responsible for the failure.
The computational sequence design of CDRs involving the Monte Carlo search for amino acid sequence is analogous to the experimental phage display biopanning. It is notable that recent experimental approach led to the discovery of two nAbs against the SARS-CoV-2 RBD by using a ~ 10 9 -diversity phage display library constructed based on the anti-SARS-CoV-1 mAbs (m396, 80 R, CR3022). 42 In this study, experimental affinity enhancement based on structural validation at high-resolution increased the binding affinity for the wild-type RBD by more than 20-fold, while computational affinity maturation for the N501Y mutant RBD increased affinity much further. This was all accomplished while also enhancing the antibody's affinity for the wild-type RBD. Whereas D27LEY exhibits ultrapotent binding affinity for SARS-CoV-2 variants with the N501Y mutation, its binding affinities for other variants lacking this mutation are lower, but still potent. It is likely that saturation mutagenesis at other selected sites could further enhance binding affinity for all variants.
In conclusion, D27LEY appears to be a strong candidate for development into a therapeutic or prophylactic mAb for COVID-19. Our work here also reveals a rational path toward vaccine design aimed at broad and effective protection against SARS-CoV-2 and other related viruses. While all the broadly protective nAbs were discovered from COVID-19 patients or vaccinated individuals, D27LEY was discovered and developed via a computational antibody design. This suggests our approach can also be used as a general method for developing mAbs with ultrapotent binding affinities for a given antigen.

Sequence design of the binding interface
All computations were performed with Rosetta. 29 Four pseudo-complexes between the SARS-CoV-2 RBD and a Fv derived from four human nAbs isolated from SARS-CoV-1 convalescent plasma were constructed by aligning the hACE2-SARS-CoV-2 RBD structure (PDB entry: 6LZG) with each of four Fab-SARS-CoV-1 RBD structures (PDB entry: 2DD8, 2GHW, 3BGF, 6NB8). These four complex models were first refined with the Rosetta FastRelax protocol and then subjected to a sequence design protocol in which the CDR residues within 7 Å of the RBD were allowed to change and those within 7-9 Å of the RBD were restricted to repacking in the PackRotamersMover and FastDesign protocols in Rosetta (Supplementary data 1). Water molecules were placed at the Fv-RBD interface in the output design models using ExplicitWaterMover 43 so they could be used for counting buried unsatisfied polar atoms (buried_unsat). Finally, 55 of 1000 design models were selected based on multiple filtering criteria, such as buried_unsat, buried solvent accessible surface area, pack score and shape complementarity.

Production and purification of designed antibodies
The DNA fragments encoding CL of the қ-type light chain or CH1-CH2-CH3 of the gamma heavy chain were inserted into the pCEP4 vector (Invitrogen). The resulting vectors were designated pCEP4(k-light) and pCEP4(heavy), respectively.
For each design, DNA fragments encoding VL and VH were synthesized (IDT) and cloned into either pCEP4(k-light) or pCEP4(heavy), respectively. Each resulting pair of vectors was introduced into CHO-S cells (Gibco) at a density of 6 × 106 cells/ml using ExpiFectamine (Gibco) to express full-length mAbs. The cells were grown in ExpiCHO expression medium (Gibco) for 12 d. Then, the culture supernatant was collected by centrifugation at 4,000 g for 30 min at 4°C, filtered through a 0.45 µm filter (Millipore), diluted by half with the addition of protein A binding buffer (150 mM NaCl, 20 mM Na2HPO4, pH 7.0), loaded onto an open column containing Protein A resin (Sino Biological), and eluted with Protein A elution buffer (0.1 M glycine, pH 3.0). The eluent was then immediately neutralized with Protein A neutralizing buffer (1 M Tris-HCl, pH 8.5). The antibodies were then further purified using a HiLoad 26/60 Superdex 200 gel-filtration column (Cytiva) equilibrated with buffer A (20 mM Tris-HCl, 150 mM NaCl, pH 7.5).

Biolayer interferometry
BLI experiments were performed using an Octet QKe384 system (ForteBio). Biotinylated or 6x(His)-tagged RBD and its variants (Acrobio Systems) at 5 nM was loaded onto streptavidin or Ni-NTA biosensor tips (ForteBio), respectively, in Kinetics Buffer (ForteBio) for 40 s. For screening, the designed mAbs at 450 nM went through an association step for 120 s and a dissociation step for 120 s before their binding kinetics were analyzed with the BLItz Pro software (ForteBio). For measuring KD, the designed mAbs at four to five different concentrations were subjected to BLI runs with 240-s association steps and 480s dissociation steps before the binding kinetics were analyzed with the Octet DataAnalysis 10.0 software package (ForteBio).

Production and purification of D27-Fab
DNA fragments encoding VL and CL or VH and CH1 with an 8xHis tag were cloned separately into the pCEP4 vector. CHO-S cells were then co-transformed with the two resulting vectors. The expression and purification procedures we followed were virtually identical to those used for full-length mAbs, except that we used HisPur Ni-NTA resin (Thermo Scientific) instead of Protein A resin.

Production of the wild-type SARS-CoV-2 RBD and truncated hACE2
The SARS-CoV-2 RBD and the N-terminal hACE2 peptidase domain were expressed using the Bac-to-Bac® Baculovirus system (Invitrogen, USA). A DNA fragment encoding the RBD (residues R319-F541) with an N-terminal gp67 signal peptide and a C-terminal HRV3C cleavage site, a 10x(His) tag, and a FLAG tag was cloned into the pFASTbac1 vector (GenScript, USA). The resulting construct was expressed in Spodoptera frugiperda (Sf9) insect cells at 27°C for 3 days. The culture medium was collected, filtered, and loaded onto a HisTrap excel column (Cytiva, USA). The column was then washed with buffer A containing 20 mM imidazole. Bound RBD was eluted with buffer A containing 250 mM imidazole. After cleaving the C-terminal tags with HRV3C protease, the RBD was further purified using a HiLoad® 26/600 Superdex 200 pg column (Cytiva) equilibrated with buffer A containing 1 mM dithiothreitol. The N-terminal hACE2 peptidase domain (residues S19-N615) was cloned, expressed, and purified according to the same protocol used for the SARS-CoV-2 RBD.

Production and purification of RBDs
DNA fragments encoding the RBD of the Alpha, Delta, DeltaPlus, or Epsilon variants were prepared by site-directed mutagenesis and cloned into the pCEP4 vector. The DNA fragment encoding the RBD of PG-GD1 was synthesized (IDT) and cloned into the pCEP4 vector. The protein production and purification procedures were virtually identical to those used for D27-Fab. The RBDs of the Beta and Gamma variants, however, were purchased from Acrobio Systems and the RBD of SARS-CoV-1 was purchased from R&D Systems.

Crystallization, structure determination, and refinement
Purified RBD (9.8 mg/ml) and D27-Fab (12.3 mg/ml) were mixed in a 1:1.1 molar ratio. Crystals were obtained using the hanging-drop vapor diffusion method at 20°C in a solution containing 20% (v/v) PEG 3350 and 200 mM NH 4 Cl. For cryoprotection, the crystal was briefly immersed in the mother liquor containing an additional 7.5% (v/v) glycerol. X-ray diffraction data were collected on beamline 11 C at the Pohang Accelerator Laboratory and processed with HKL2000. 44 Phases were determined by the molecular replacement protocol in PHENIX 45 using the structures of a kappa light chain (PDB ID: 4MIK), SARS-CoV-2 RBD (PDB ID: 7JMO), and the computational design model of the heavy chain as search models. Model building and structure refinement were carried out using COOT 46 and PHENIX. 45

Construction of the CDR3 loop-extended D27-scFv library
The CDR3 loop-extended D27-scFv library was constructed using DNA oligonucleotides and a standard overlapping polymerase chain reaction (PCR) method. To randomize and extend the E101H and Q102H sequence in D27 to 4 residues and F93L to 2 residues, the synthetic oligonucleotides contained NNMNNMNNMNNM or NNMNNM, respectively, where "N" stands for any base and "M" stands for A or C. A DNA oligomer was prepared to link VH and VL of D27 via a (G4S)4 linker. Its sequence was as follows: 5ʹ-GGCGGAGGTGGTTCAGGTGGCGGCGGTTCTGG AGGAGGGGGCAGTGGTGGCGGAGGTTCA-3ʹ After PCR, 6 µg of the resulting scFv library and 2 µg of linearized pCTCON2 vector (New England Biolabs) containing a TRP selectable marker were introduced into the S. cerevisiae EBY100 strain using an Electroporator 2510 (Eppendorf) according to a published protocol. 47

Yeast surface display and cell sorting
After transformation, the yeast cells were cultured in Trpdeficient SD-CAA medium. Then, 3 × 106 cells/ml were resuspended in galactose-replete SGCAA medium and cultured for another 20 h at 20°C to induce the expression of Aga2p-scFv containing a C-terminal Myc tag. To label the Aga2p-scFv fusion protein produced in yeast, the resulting cells (10 7 cells/ 100 µL) were washed two times in phosphate-buffered saline (PBS), resuspended with PBSF (PBS with 1% bovine serum albumin (BSA)), and incubated with 0.2 µL of phycoerythrin (PE)-or PE-Cyanine7 (PE-Cy7)-labeled anti-c-Myc antibody (Cell Signaling Technology; Cat # 3739, Cat # 36271) for 1 h at 4°C. For fluorescence labeling of the RBD, 6x(His)-tagged RBD was incubated with fluorescein isothiocyanate (FITC)labeled anti-6x(His) antibody (Abcam; Cat # ab1206) at a 1:1 molar ratio for 1 h at 4°C. The cells were then incubated with labeled RBD at different concentrations for 1 h at 4°C and washed two times before cell sorting. Two rounds of MACS were performed with FITC-labeled RBD (5 nanomoles for the 1st round and 1 nanomole for the 2nd round) to deplete the clones with weak target-binding affinity. The cells were then collected using anti-FITC microbeads (Miltenyi Biotec) and subjected to three rounds of FACS using a S3e cell sorter (Bio-Rad) in the presence of 100, 10, and 5 nM FITC-labeled RBD. The resulting sorted cells were plated on an SDCAA plate, and individual plasmids were prepared for 9 clones. The scFv sequences were determined by DNA sequencing (Cosmogenetech, Korea).

Focus reduction neutralization test
Virus neutralization was determined using the FRNT assay. 48 In triplicate, mAbs were serially diluted in Eagle's minimum essential medium (EMEM) containing 2% fetal bovine serum. They were then incubated with 8 × 10 3 plaque forming units (PFU) of SARS-CoV-2 for 1 h at 37°C in a final volume of 50 μL. The mAb-virus mixture was added to 4 × 10 4 Vero E6 cells on a 96-well plate. After incubation for 8 h at 37°C, the cells were washed with PBS and fixed with 3.7% formaldehyde. The fixed cells were permeabilized with 100% methanol and incubated with a blocking buffer (1% BSA, 0.5% goat serum, 0.1% tween-20 in PBS) for 30 min. The cells were then stained with a mAb against the nucleocapsid of SARS-CoV-2 (40143-R001, Sino Biological) for 1 h and incubated with a goat anti-rabbit IgG (H + L)-HRP (1706515, Bio-Rad) for 1 h. Using TMB Stabilized Substrate (W4121, Promega), viral infection foci were counted with an ImmunoSpot analyzer (2001726, CTL). NC 50 s were calculated by nonlinear, doseresponse regression analysis using GraphPad Prism 8.

List of abbreviations:
nAbs Neutralizing antibodies mAbs Monoclonal antibodies hACE2human angiotensin-converting enzyme 2 RBD Receptor-binding domain SARS-CoV-2Severe acute respiratory syndrome coronavirus-2 VOCs Variants of concern VOIs Variants of interests VUMs Variants under monitoring CDRs Complementarity-determining regions