Synthetic Monoclonal Antibody Designed for Novel SARS-nCoV-2 Spike-S1 Protein Antigenic Targeted Epitope of Receptor Binding Domain Inhibit to Prevent Viral Entry

Severe acute respiratory syndrome (SARS) is developing disease caused by novel coronavirus (COVID19). This situation has urgently prompted many pharma and R&D research companies and public research health sectors to concentrate their efforts on research for effective therapeutics. SARS-nCoV-2 as a spike (S) protein was a targeted for the development of monoclonal antibody and therapeutics for the prevention and treatment. We developed monoclonal antibody by deep mutational scanning to characterize the effects of mutations in an antibody variable fragment on its based on expression levels, specicity, stability, and anity for antigenic specic epitope to the Spike-S-RBD. Further to make this antibody multipoint core mutations to improve contacts between specic Fv light and heavy chains to the targeted antigen of RBD. This antibody combined enhancing mutations yielding with higher binding anity and substantially improved stability in between RBD and antibody. Overall, this antibody may well prevent into the cell interaction with RBD-hACE2 to viral entry and prevention. SARS-nCoV-Spike-S monoclonal antibodies potently inhibited SARS-nCoV-2-Spike-S mediated entry into cells, indicating that cross-neutralizing antibodies targeting conserved S epitopes can be elicited based upon vaccination.


Introduction
The occurrence of coronavirus disease in December 2019 (COVID-19) is a respiratory illness that can spread from person to person. The virus that causes COVID-19 is a novel coronavirus that was rst identi ed during an investigation into an outbreak in Wuhan, China. A newly identi ed novel coronavirus SARS-CoV-2, formerly known as 2019-nCoV is causing pneumonia-associated respiratory syndrome (Wu et al., 2020). After analysis of genome sequences of SARS-CoV-2 samples obtained from different infected patients, SARS-CoV-2 shares high sequence identity with SARS-CoV (Zhou et al., 2020 and Lu et al., 2020). Compared to SARS-CoV, transmitted from human-to-human of SARS-CoV-2 seems to be greater. As of February 2020, at least 25 countries reported >70,000 cases of SARS-CoV-2 infection. A Complication of this virus is a Patients with nCoV-2 have had mild to severe respiratory illness with symptoms of fever, cough and shortness of breath also some patients have pneumonia in both lungs, multi-organ failure and in some cases death. At present people with nCoV-2, disease con rmed by molecular testing by reverse transcription polymerase chain reaction: RT-PCR, weak, late or absent antibody responses have been reported (Zhao J  Unfortunately, There are currently no speci c vaccine or speci c antiviral drugs to protect against COVID-19. Researchers across the world including several biotech and pharma R&D companies are investigating preventative approaches and treatment options for nCoV-2, including vaccines, monoclonal antibodies, oligonucleotides, small peptides, interferon type 2 alpha and Beta, and small-molecule drugs (Tang et al., 2008). Since only a way suggests antibody-mediated humoral response is crucial for preventing viral infections. A subset of these antibodies, which reduce viral infectivity by binding to the surface epitopes of viral particles and thereby blocking the entry of the virus to an infected cell, are de ned as neutralizing antibodies (NAbs) (Varghese et al., 2004).
The CoV-2 spike (S1) protein is a key target for urgently needed vaccines, therapeutic antibodies, and diagnostics (Xintian et al., 2020). Spike proteins are the visible protrusions on the surface of COVID19, which give the virus its characteristic, crown-like appearance (Figure 1). These homotrimeric proteins are heavily glycosylated, with each comprising two distinct subunits (S1 and S2). The role of Spike is to act as a molecular key, which it achieves by recognizing and binding to speci c hACE2 cell-surface receptors present on the surface of cells, through the S1 receptor-binding domain. When S1 binds to hACE2, Spike undergoes dramatic structural changes to alter the conformation of hACE2 and mediate entry of the virus into the cell. However, as Spike proteins must project into the external environment to effectively bind cellsurface receptors, they are exposed to recognition by the immune system. This makes Spike the immunodominant coronavirus antigen, causing it to elicit a strong neutralizing antibody response (Ng et al., 2016) that has made it the focal target of diagnostic and vaccine development.
In the method of in-silico design allows for rapid iteration around potential solutions without the unique variables that traditional in vivo subjects may possess. Traditional in vivo methods test for the likelihood of producing an antibody that triggers the body's immune system to ght against pathogens. This techniques can be developed and delivered remotely, and do not require the on-site presence needed for traditional wet labs.
The aim of this study was to describe to construct design of synthetics peptide monoclonal antibody (mAbs) for Spike S1 protein to target of speci c antigenic epitopes present in RBD. The key residues between RBD and monoclonal antibody can be particularly identi ed to provide important implications for the vaccines against nCoV-2. Because spike protein is main antigenic component that is responsible for inducing host immune responses, neutralizing antibodies and protective immunity against virus infection. Spike protein has therefore been selected as an important target for coronavirus vaccine and anti-viral development. Therefore, key residues of interface between an monoclonal antibody and the RBD antigen can be optimized to generate with high a nity.

Dataset preparation and selection
Numbers of antibody structures was currently around more than 2,000 depositions are available in NCBI and protein data bank (PDB). Based on these data's, the comparative model of monoclonal antibody onto the viral surface antigen that can be predicted. We performed selected 500 antibodies sequence retrieved from the NCBI and PDB server to validate and ltered by molecular dynamics (MD) simulation (Wallraven et al., 2020) according to the best correlation between calculated and measured binding a nities in between the target of nCoV-2 Spike-S1 RBD epitopes speci c antigen to monoclonal antibody.
Identi cation of antigen and antibody sequence A vaccine trains the body's immune system to recognize some viral antigen signatures that were present in Spike-S1 protein speci c antigen in the RBD. The nCoV-2 is a recombinant antigen, which contains normal amino acids 408-470 and 540-573 of the Spike-S1 protein immunodominant region. Since we identi ed speci c antigenic epitopes of small synthetic peptide are regions of protein surface in RBD to the target of antibody by two different programs the immune epitope database (IEDB) (Jespersen et al., 2017) and Support Vector Machine (SVM) Tri-peptide similarity and Propensity (SVMTriP) (Yao et al., 2012). We identi ed consists of antibody sequence in two chains (VL/VH) after ltered by MD simulation in out of 500 from NCBI and PDB. In . Models are not separated to light and heavy chains and are numbered according to Chothia numbering for the prediction by using sequence homology, conducts a Monte Carlo-based search for lowenergy combinations of backbone conformations to yield accurate and unstrained antibody structures. The RMSD estimates above 90% sequence identity were calculated by using the entire set of full dataset sequences as well using TM-align did calculation. Further targeted antigen structure carried out by PDB ID: 6VW1 structure of 2019-nCoV-2 receptor-binding domain (RBD) complexed with its receptor human (ACE2).

Protein-to-protein interactions and structures validation
To conduct initial sampling of antigen and monoclonal antibody was used by the protein-to-protein docking server ClusPro 2.0 (Vajda et al., 2017), which is based on a docking program called PIPER2 (Kozakov et al., 2006). Which was more effective docking algorithm for antibody-protein antigen complex prediction is an important rst step toward design of biologics and vaccines. PIPER is an FFTbased docking program that uses a structure-based pairwise potential as one component of its energy function. The total energy was the sum of terms representing shape complementarity, electrostatic and desolvation contributions by the pairwise potential (Chuang et al., 2008). For result assessment, we consider the model a hit if ligand of antigen atoms within 10A ̊ of the receptor of monoclonal antibody are within 10A ̊ RMSD in the model to ignore the motion of that part of the ligand which is not participating in the interaction. To evaluate our docking of the receptor and ligand potentials was incorporated as the pairwise component of the energy function used in PIPER, followed by clustering of the top 1000 results for evaluate (Kozakov et al., 2006), as implemented in our protein-protein docking server ClusPro (Comeau et al., 2004) that was here in more advantage.

Prediction of protein-protein interaction interfaces hot spots
The amino acid residues in the binding region of proteins is not uniform rather, they contain critical residues, called hot spot. Hot spots are primary targets of therapeutic agents, because designing a molecule that will bind to hot spots may lead to the disruption of a protein-protein interaction. We predict hot spots for our both antigen and antibody used by PRISM Concepts (Baspinar et al., 2014 andTuncbag et al., 2011). In experimentally, hot spots are determined by alanine scanning mutagenesis where if the contribution of the mutated residue to the binding is more than 2.0 kcal/mol these residues are labeled as hot spots. If the distance of any two atoms between residues was less than their sum of van der Waals radii plus 0.5 Angstrom, both residues were registered as interfaced residues. When an assigned interface residue was less than a 10 residues, an arbitrary but reasonable number to re ect the minimum requirement of contact, the interface was considered as a result of crystal packing force.

Antibody structural annotation of CDR's for RBD and estimated accuracy
Our targeted antibody sequence are Chothia-numbered by ANARCI. The numbered sequences was Chothia-aligned with more-than 2,000 high quality antibodies with known structure from SAbDab. For each dataset sequence from an Ig-seq, the alignment identi es the best structural templates for the full variable region and framework independently. FREAD then identi es, if any, the most suitable template structures for the CDRs. CDRs are germline to obtain a more comprehensive structural interpretation of the Ig-seq CDRs for which FREAD do not nd direct matches to the PDB, but predicted the loop conformation from the targeted of our antibody sequence only. Well over 50 % of the unique sequences of non-H3 CDRs can be accurately structurally predicted. This appears to recapitulate on a large scale the canonical shape phenomenon of non-H3 CDRs. In the case of H3, even though there were only a handful of direct PDB matches H3 sequences, according to FREAD estimates to produce structural models for 65 % of all non-redundant and 75 % of redundant H3 loops ( Table 1).
The antibody structure that the speci c physicochemical con guration, which modulates the molecule's speci city and a nity. Binding shape biases in antibody repertoires was insight into the strategies of the immune system for tackling arbitrary RBD antigen. For instance, sequence similarities can be indicative of shared RBD antigen speci city. CDRs identi ed the physicochemical properties of the paratope, identifying potential RBD antigen-speci c of our antibody. Such structural annotations were accurate as more paired Ig-seq datasets as based on these data allow the entire Fv regions as followed the full frameworks VH: (H1, H2, H3) and VL (H1, H2, H3) was modeled rather than separate heavy and light chains ( Figure 2). Our monoclonal antibody for targeted RBD of speci c antigenic epitope annotations was employed in immunodiagnostics to nd antibody-markers of known viral diseases. Therefore, employing structural information provides novel ways to study the diversity of the immune system. The estimated accuracy of modelled VH and VL domains procedure has been benchmarked. Result was estimated each region of the model was likely to have been predicted 75 % of VH framework structures with sequence identity of 80 % +/-2.5 % have a backbone RMSD of 1Å or better. Therefore the model has been predicted based on our antibody sequence with 80 % sequence identity to the target we have a con dence of 75 % that the VH framework was modelled with 1Å RMSD.
Antibody designed by another program AbPredict for binding a nity in between VH/VL comparison. For this modeling same consists with a random combination of four backbone fragments a two fragments for the VL and VH, which comprise CDRs 1 and 2 and the light and heavy chain framework regions, respectively, and two fragments for LCDR3 and HCDR3, it then our antibody sequence on the backbone fragments and performs a simulated annealing Monte-Carlo search over all conformational degrees of freedom. In each conformational move, the method randomly samples from pre-computed databases a backbone conformation belonging to the VL, VH, LCDR3, HCDR3 followed by combinatorial side-chain packing and side-chain and backbone minimization to reduce stereochemical strain. At the end of each trajectory, the lowest-energy structure sampled during the trajectory. The result of antibody Fv region accuracy and stereochemical strain in models produced for eight antibodies that were part of the AMA-II blind benchmark of monoclonal antibody structure modeling (Figure3). Both programs estimated that were mostly given best antibody deviated by <1.2 Å rmsd over backbone-carbonyl atoms and exhibited stereochemical quality that is expected of structures at resolutions <1.2 Å. The 500 resulting models were clustered by carbonyl rmsd and the nally lowest-energy structures from the top-three clusters.
Antigenic epitope of nCoV2-Spike-S2-RBD binding interaction with monoclonal antibody The nCoV-2 assortment was re ected in the variable spike proteins (S proteins), which have evolved into forms differing in their receptor interactions and their response to various environmental triggers of virus-cell membrane fusion. It was mainly infect the human respiratory epithelial cells through interaction with the hACE2 receptor. Indeed, the recombinant Spike protein can bind with recombinant hACE2 protein. The S1 contains two subdomains, a N-terminal domain (NTD) and a C-terminal domain (CTD) both are able to function as receptor binding domains (RBDs) and bind variety of proteins and sugars. The S2 domain was typical of a class I viral fusion proteins. Heptad repeats comprise a repetitive heptapeptide with being hydrophobic residues characteristic of the formation of coiled-coil that participate in the fusion process. The nCoV-S1 complexed with human hACE2 provided the ssionable view of coronavirus S (Figure 4), which contains two subdomains as core structure and RBD. A ve-stranded anti-parallel βsheet (β1-β4 and β7) that connects with three short α-helices (αA-αC) constitutes the core, whereas a two-stranded β-sheet (β5 and β6) forms the loop. N* and C* represent the amino and carboxyl termini of RBD. The RBD gently concave outer surface to bind hACE2. The base of this concave surface was a short, two-stranded antiparallel β-sheet, and two ridges are formed by loops. The ectodomain of hACE2 contains a membrane-distal peptidase domain and a membrane-proximal collecting domain. The RBD of S protein contains multiple conformation-dependent with different epitopes and the main domain that induces neutralizing antibody. We identi ed entire regions antigenic epitope of small peptide even single amino acid in the RBD that was taken place from predict liner antigenic epitopes (SVMTrip). It was  (Table 2). A calculation of a high rate of the binding a nity between RBD and antibody was -12.1kcal/mol. We identi ed two amino acids Asn479 and Thr487 of the RBD are important for the interaction higha nity overtone of S protein with hACE2. Particular mutations at Arg441, and Asp454 of the RBD disrupt the antigenic structure and binding activity of RBD to hACE2. Accordingly boosted our antibody stability and a nity in to Fv framework regions (CDRs) which are typically in direct contact with the antigenic epitope for instance, improved electrostatic complementarity with the antigen of RBD a nity of monoclonal antibody by which altered amino acids in hydrophobic surface patch. There are four epitopes regions of the RDB were identi ed but since only three are the best epitopes by rank were interacts with the monoclonal antibody ( Figure 5). This monoclonal antibody were interacting regions of RBD in out of three the best-selected rank one that are main of our target. This same region of RBD binding with the hACE2 receptor and neutralize infection. Monoclonal antibody of VH chain 1 were interact antigenic epitope binding hotspot residues of the RBD (Pro38-Val505, Tyr58-Asn437, Phe109-Val503, Phe109-Gly502, Phe110-Gly502, Phe110-Tyr505, Phe109-Tyr505, Tyr64-Arg439, Tyr64-Asn440, Ser57-Arg439, Phe109-Gly504, Asn113-Thr500, Tyr66-Arg439, Asn113-Gly502 and Tyr66-Pro499) as for VL chain 2 were interact antigenic epitope binding hotspot residues of the RBD (Glu109-Gln498, Glu109-Thr500, Asn108-Thr500 and Trp114-Thr500) ( Table 3). In due to the interactions direct H-bonds across the epitope to paratope interfaces could contribute to the speci city of the antibody to antigen recognition that was short-chain hydrophilic side chains are particularly suitable for this interaction because of the smaller side-chain conformational entropy penalty in the interface.
Optimizing Lead antibody of a nity, speci city, and stability The mutated VH and VL domain combinations by developed a new antibody (>108 additional diversity) with prede ned antigen speci city. Compared to the hit discovery, it can be used for in-vitro selection under more stringent conditions to identify antibodies with improved characteristics. The most important antibody properties relate to their natural functions, such as they're high binding a nity and speci city mediated by their complementarity determining regions of monoclonal antibody (CDRs) within the variable VL/VH chains. Other key natural antibody properties include their effector functions in both dependent cell mediated of complement and dependent cytotoxicity. We created designed library of variants of RBD-monoclonal antibody interface. These structures contain a complex of two proteins, one in "RBD: Chain F" and the other in VH: "Chain H" and "Chain L". The proteins interact through an interface comprises a conserved core, known as the interaction hotspot ( Additionally, the periphery of the binding interface, make important contributions to speci city. Inspired by this modularity of binding interfaces, while conserving the interaction hotspot and optimizing the rigidbody orientation and sequence of other interfacial regions for further conformation periphery of the binding interface, the main implicit that backbone designs that retained the structural geometry would allow the remainder of the our complex proteins, including the essential hotspot, to fold to the native conformation, thus maintaining high-a nity binding in between monoclonal antibody and RBD.
The association between the light and heavy domains in immunoglobulins, and these can be speci cally associated with key residues in our monoclonal antibody sequence. Belong the sequence of CDRs rest on a conserved framework, which provides the biochemical properties of structural stability. Structural diversity in the Fv framework region was experimental primarily in the CDRs, which vary in length, backbone conformation, and amino acid sequence. The rigid-body orientation of the light relative to the heavy chain was another important that affects the conformation of the RBD antigen-binding site. Furthermore con rmation of protein stability effect of salts dependent and it was a complex balance of the multiple mechanisms by which the ionic salt interacts with protein molecules, shielding charged solvent exposed residues and then potentially decreasing protein-protein long-range electrostatic interactions.

Discussion
The Spike's receptor binding domain is the primary determinant of a coronavirus's ability to infect a host species and its tropisms. SARS-CoV-2's genome shows that its closest human-infecting relative is the Severe Acute Respiratory Syndrome Coronavirus (SARS-CoV), with a sequence identity of approximately 79 % (Roujian et al., 2020). Identity between the spike proteins is 76.47 %, with slightly lower sequence identity between the RBDs (73 %) (Xu et al., 2020). Within SARS-CoV-2's 193 residues RBD, a large proportion of mutations are highly accumulated at the C-terminal region that includes the receptorbinding motif (Yushun et al., 2020). The RBD in S1 is responsible for virus binding to host cell receptors (Wong et al., 2004, Li 2003. Neutralizing antibodies and T-cell immune responses can be raised directly against several nCoV-2 proteins (S, E, M and N) (Wang 2020, Zhou 2018, Du2016, See 2008), but mainly target the S protein (Zakhartchouk et al., 2007), suggesting that to induced speci c immune responses on the surface of the viral particle plays key roles in the binding of the cell receptor and membrane fusion (Cui et al., 2029). The hACE2 is expressed and active in various cell types including alveolar epithelial cells, surface enterocytes of the small intestine, and heart and kidney endothelial cells (Sims et al., 2005). Essentially the S protein of nCoV-2 is involved in the hACE2 receptor recognition, as well as virus attachment and entry it represents one of the most important targets for the development of nCoV-2 vaccines and therapeutics.
In the study, we choose the spike protein, as a candidate target to monoclonal antibody way to stop the virus infection is to block the RBD with hACE2 interaction. We designed antibody in each conformational passage, the method randomly samples from pre computed dataset a backbone conformation belonging to the antibody of VL, VH, L-CDR3, H-CDR3 in Fv framework by combinatorial Sidechain packing and Sidechain and backbone minimization to reduce stereochemical strain (Lapidoth et al., 2015). Based on we analyzed the accuracy and stereochemical strain in models produced for eight antibodies that were part of the AMA-II blind benchmark of antibody structure modeling methods (Almagro et al., 2014). Our top models exhibited stereochemical quality that was expected of structures at resolutions <1.2Å rmsd. The 500 resulting antibody models are clustered by carbonyl rmsd and the lowest-energy structures from the top-three clusters.
Our monoclonal antibody was highly interacting binding with a speci c antigenic epitope to the RBD. During the interaction of RBD with the receptor, RBD presents a concave surface for the N terminus of the receptor peptidase, on which amino acids 441-451: NIDATSTGNYN broadcasters the entire receptor binding loop of RBD core. These loop amino acids 473-483: YQAGSTPCNGV of the RBD, which makes directly complete contact with the antibody. The RBD region also contains multiple cysteine residues that are linked by di-sulphide bonds . These two amino acid regions in particular, those at positions 441-451 and 473-483, determine nCoV-2 disease progression . Any residues changes in these two positions might therefore develop animal-to-human and human-to-human transmission (Arcilla et al., 2017). Further to the structure of RBD, residue 498 GLN in nCoV-2 was located very near virus-binding hot spot 500 Thr (i.e., hot spot 109Glu and 110 Phe) on antibody. Hot spot 353 consists of a salt bridge between Lys353 and Asp38 also buried in a hydrophobic environment (Wu et al., 2012).
The RBD are important for the high-a nity association of S protein with hACE2 . A point mutation at Arg441, Asp454 of the RBD disrupts the antigenic structure and binding activity of RBD to hACE2 (He et al., 2006). Our antibody structure improved interactions across the VL-VH interface result in substantial optimization of a range of essential parameters for antibody development, including impressibility, stability, and a nity which more highly capable interact with antigenic epitope to the RBD which mainly antibody core alter according to the structure of antigen-binding site, these interactions contribute the majority of the binding energy for the antibody to protein complexes as hot spots in the interfaces. The designed mutations cooperate with surface mutations identi ed through conventional antibody to increased a nity and stability. Finally, taken together, this study provides developed monoclonal antibody model and together of essential function for the development of nCoV-2 RBD for therapeutics to completely prevent vial infection.

Future Directions:
Overall we successfully designed new monoclonal antibody for novel coronavirus based on S protein to the targeted of speci c antigenic epitope to the RBD to inhibit the host viral entry. Future studies to understand immunologic factors and antibody pathways associated with the successful development of antibodies may provide insights for the design of improved immunogens and immunization strategies.

Declarations
Acknowledgement: I thanks the World Health Organization (WHO) continue to update the Covid19 platform with new data sets from national and sub-national level, as well as showing data of clinical trials, country-speci c public health measures, and other important indicators which is highly supported to my research work. We collaborate our research analysis was partially performed at the Center for High-Performance Computing, the regional research laboratory, Ministry of Health, Makkah, The Kingdom of Saudi Arabia.

Disclaimer:
The study sponsor had no role in study design, data collection, data analysis, data interpretation, or writing of the report. The rst and corresponding author's had full access to all study data and had nal responsibility for the decision to submit for publication.     The antibody structure and altered energy minimization. The accuracy and stereochemical strain in models produced for eight query antibodies that were part of the antibody modeling assessment (AMA-II) blind benchmark of antibody structure modeling manners. A models exhibited stereochemical quality of structures at resolutions <1.2 Å by clustered by carbonyl rmsd and the lowest-energy structures resulting of top-eight models Fv regions were in CDR C=0 H1, H2, H3, CDR C=0 L1, L2, L3, and aligned with CDRs and antibody VL/VH model complex. nCoV2 spike proteins are consists of two domains: a N-terminal domain named S1 that was responsible for receptor binding and a C-terminal S2 domain responsible for fusion. The spike S1 protein was a class I fusion protein α-helical coiled-coil structure was characteristic of this class of fusion protein, which contain in their C-terminal part regions predicted to have an α-helical secondary structure and to form coiled-coils with Horizontal angle 50 Å, and perpendicular angle. The S2 subunit was the most conserved region of the protein, whereas the S1 subunit diverges in sequence of a single coronavirus. The S1 contains two subdomains, a N-terminal domain (NTD) and a C-terminal domain (CTD). Both are able to function as receptor binding domains (RBDs) and bind variety of proteins and sugars. The nCoV diversity was re ected in the variable spike proteins (S proteins), which have evolved into forms differing in their receptor interactions and their response to various environmental triggers of virus to cell membrane fusion. It's been infect the human respiratory epithelial cells through interaction with the hACE2 receptor (red) and RBD (green). Indeed, the recombinant spike protein binds with recombinant hACE2 protein. (D) Antibody VL (blue) /VH (half white) binding interaction to the speci c epitope with the RBD (red) which interact binding a nity was -12.1kcal/mol.

Figure 5
Interactions interface between antigenic epitopes and antibody binding site. Epitope was consecutive fragment from the amino acid sequence, and composed of several fragments scattered along the amino acid sequence, which form the antigenic regions of RBD-binding interface. Such predictions based on the amino acid properties were including hydrophobicity, solvent accessibility, secondary structure, exibility, and antigenicity. (A). There are three antigenic epitopes regions was predicted based on rank1, which was highlighted color in red to a direct contact of RBD to the antibody with interacting high binding a nity other blue are non-epitope. (B) A rank2 antigenic epitopes was predicted regions are (red: epitope and blue: non-epitope) present at a lower of RBD. (C) A rank3 antigenic epitopes was predicted regions are (red: epitope and blue: non-epitope) which conserved at the forward-facing portion of the RBD to interact with antibody. (A-C) In all the segments of the RBD antigenic epitopes speci c amino acids are listed a near the model of protein structure.