The spike protein of SARS-CoV-2 plays a key role in viral entry and is a major therapeutic target. It uses its spike protein to bind to ACE2 receptors on host cells, allowing viral entry. The spike protein undergoes conformational changes during viral fusion and entry. Mutations in the spike protein can affect its ability to bind to ACE2 receptors or evade antibodies. Several human monoclonal antibodies have been identified that target the spike protein and may be potential COVID-19 treatments.

1. SARS COV2 SPIKE PROTEIN: A MAJOR THERAPEUTIC TARGET
SONU KUMAR
M.S pharmacology and toxicology ,
NIPER KOLKATA

2. CONTENT
• INTRODUCTION
• SPIKE PROTEIN
• VIRAL ATTACHEMENT
• VIRAL FUSION
• VIRUS ENTRY AND REPLICATION
• MUTATION IN SPIKE PROTEIN
• MONOCLONAL ANTIBODIES TARGET SPIKE PROTEIN
• CONCLUSION
• REFERENCE

3. INTRODUCTION
• Coronaviruses (CoVs) are a large family of viruses that are phenotypically and genotypically diverse
• CoVs are enveloped viruses containing single-stranded positive-sense RNA that belongs to Coronaviridae family.
• Phylogenetic analysis of the SARS-associated CoV (SARS-CoV) shows that it is neither a mutant nor a recombinant
of previously characterized CoV and forms a new, distinct group within the genus.
• Two highly pathogenic coronaviruses of zoonotic origin such as SARS-CoV and MERS-CoV were identified earlier
which causes widespread epidemics and fatality in many countries. SARS-CoV-2 is the third known highly
pathogenic human coronavirus infection in the last two decades after MERS-CoV and SARS-CoV. Although it is
believed to be originated from bats, the exact source of SARS-CoV-2, animal reservoir and enzootic patterns of
transmission still remain uncertain.
Fig taken from Leila Jahanshahlu,Nima Rezaei Monoclonal antibody as a potential anti-covid-19 Biomed pharmacother 129(2020) 110337, doi.org/10.1016/j.biopha.2020.11033

4. The genome size of the SARS-CoV-2 varies from 29.8 kb to 29.9 kb and its genome structure followed the specific gene
characteristics to known CoVs; the 5′ more than two-thirds of the genome comprises orf1ab (open reading frame)
encoding a polyprotein which is further processed to generate sixteen non-structural proteins (Nsp1–16), and 9 ORFs
encoding accessory proteins genes : ORF3a, ORF3b, ORF6, ORF7a, ORF7b, ORF8, ORF9b, ORF14 while the 3′ one third
consists of genes encoding structural proteins. The structural proteins such as membrane (M), envelope (E) protein,
nucleocapsid (N) protein and spike protein (S), encoded by 3`end of the viral genome which play a major role in virus entry
and virus replication in the host cell.
Fig taken from Ma’mon M. Hatmal, Walhan Alshaer, et.al Comprehensive Structural and Molecular Comparison of Spike Proteins of SARS-CoV-2, SARS-CoV and MERS-CoV, and Their Interactions with ACE2, Cells 9(12) (2020), 2638, doi:10.3390/cells9122638

5. SPIKE PROTEIN
• 2019-nCoV makes use of a densely glycosylated spike (S) protein to gain entry into host cells. The S protein is a
trimeric class I fusion protein that exists in a metastable prefusion conformation that undergoes a substantial
structural rearrangement to fuse the viral membrane with the host cell membrane . The trimeric S protein
decorates the surface of coronavirus and plays a pivotal role during viral entry.
• The spike protein consist of two subunit S1(N-terminal domain) and S2(C-terminal domain). S1 and S2 comprise
the extracellular domain (ECD; 1 to 1208 amino acids) and a single transmembrane helix and mediate receptor
binding and membrane fusion, respectively. S1 subunit, having 15-680 AA consisting of four core domains S1A-
S1D and S2 subunit having 681-1255AA.
• The RBD is responsible for binding to ACE2, whereas the function of NTD is not well understood. In some
coronaviruses, the NTD may recognize specific sugar moieties upon initial attachment and might play an
important role in the prefusion-to-postfusion transition of the S protein. C-terminal domain is also known as
receptor binding domain (RBD), which is crucial to determine tissue tropism and host ranges.
• RBDs of S1 subunit consist of 193AA (AA 318-510) which have five cysteine residues responsible for expression
and formation of RBDs.

6. VIRAL ATTACHMENT
• After entry into the host body SARS virus get attached with ACE2 receptor of epithelial cell, through their receptor
binding domain of spike protein.
• Receptor binding domain of S1 subunit have concave surface which attached with the tip of ACE2 receptor,
known as receptor binding motif (RBM).
• Similar to SARS CoV, SARS CoV-2 uses CTD as the receptor binding domain (RBD), however some beta coronavirus
for example the HCoV-0C43 and HCoV-HKV1 binds with NTD by recognising 9-Oacetylated sialic acid moieties of
glycosylated cell surface receptor, while MERS utilized both dipeptidyl peptidase-4 (DPP-4) receptor and NTD.
• Whenever SARS virus get attached with ACE2 receptor then, RBDs of S1 subunit show hinge like conformational
movement either one receptor binding domain in the “up” or “open” conformation which leads to receptor
accessible state, thought to be less stable conformation or all RBDs in the “down” or “closed” conformation leads
to receptor inaccessible state.
• Fig taken from Balamurugan Shanmugaraj Konlavat,Siriwattananon et.al Perspectives on monoclonal antibody therapy as potential therapeutic intervention for Coronavirus disease-19 (COVID19) , Asian pacific journal of Allergy
immunology,38(1) 2020,10-18, DOI 10.12932/AP-200220-0773

7. VIRAL FUSION
• During infection, the S protein is cleaved into the N-terminal S1 subunit and C-terminal S2 subunit by host
proteases such as TMPRSS2 and changes conformation from the prefusion to the postfusion state.
• The S2 domain consist of putative fusion peptide and two conserved 4,3-hydrophobic repeated domains or helical
regions HR1(HR-N) & HR2(HR-C) which formed coiled-coil structures that upon cleavage by the endosomal
protease cathepsin L form the six helix bundle fusion core and are separated by an intervening stretch of 140
amino acid residues called the interhelical domain.
• S2 subunit involved in membrane fusion, coronavirus S2- fusion protein shows three different states during viral
entry State-1(native):- during these state both S1 and S2 are remain intact to each other ,State -2 (intermediate
state ):- the N-terminal (S1) domain is dissociate (shedding) to expose the fusion peptide(FP) region and State-
3(collapsed 6-helix bundle or fusion active state):- The collapsed S2 domain draws the viral and cellular
membranes together causing fusion and release of the viral nucleocapsid into cell.
• Fig1 and 2, taken from Fei Yu, Xiang et.al, Receptor-binding domain-specific human neutralizing monoclonal antibodies against SARS-CoV and SARS-CoV,nature communication 5(1),2020,212, doi.org/10.1038/s41392-020-00318- 0
• Fig3 taken from Brian Tripet, Megan W. Howard et.al Structural Characterization of the SARS-Coronavirus Spike S Fusion Protein Core, the journal of biological chemistry,279(20),(2004),20836-20849, DOI 10.1074/jbc.M400759200.

8. VIRUS ENTRY AND THEIR REPLICATION
• After attachment of S1 subunit spike protein undergoes proteolytic cleavage which is catalysed by several host
proteases, such as furin, TMPRSS2 and cathepsin-L, which activate spike protein and allow viral-host membrane
fusion and entered into cells via endocytosis process, in the host cell.
• The virus liberates its nucleocapsid containing viral genome into the cytoplasm, then the viral RNA is first
translated to synthetize the non-structural proteins (Nsps) responsible for the RNA-dependent RNA
polymerization, which in turn, synthetize both new genomic RNAs and also the sub genomics mRNAs necessary
for the translation of the structural proteins (S, E, M, N: assembly of new viral particles) and another accessory
proteins (ORF3a-ORF10).
• In particular, the Nsp factors form the replicase–transcriptase machinery, which include the enzymatic activity
essential for viral RNA replication (Nsp12, Nsp7, Nsp8, Nsp13, Nsp14) and viral protein translation (Nsp14 and
Nsp16). With the help of host cells machinery its produce its on genetic material and viral component followed by
assembling of viral particle and released by exocytosis leading to causing further infection to another health cells.
• Nsps may act also as virulence factors inhibiting the host immune system defenses (Nsp1, Nsp15).
• Fig taken from Ma’mon M. Hatmal, Walhan Alshaer, et.al Comprehensive Structural and Molecular Comparison of Spike Proteins of SARS-CoV-2, SARS-CoV and MERS-CoV, and Their Interactions with ACE2, Cells 9(12) (2020), 2638,
doi:10.3390/cells9122638

9. MUTATION IN SPIKE PROTEIN
• Till now several genomic sequences show variation in SARS CoV2 isolates. One of these variations encodes an S-
protein mutation, D614G, in the carboxy (C)-terminal region of the S1 domain. This region of the S1 domain
directly associates with S2 . The mutant virus with glycine at the residue 614 (G614), replacing aspartic acid
(D614), has been shown to rapidly dominate in many locales where it is found.
• The rate of evolution of SARS-CoV-2 from December 2019 to October 2020 was consistent with the virus acquiring
approximately two mutations per month in the global population.
• The D614G mutation indeed increases infectivity and that reduced S1 shedding and increased S-protein density in
the virion correlate with this increased infectivity.
• The most noticeable difference between G614 and D614 is markedly higher amounts of the S1 and S2 bands of
G614 compared to D614,as well as densitometric analyses showed there was 4.7 times more S1 + S2 band in G614
compared to D614.
• Another RBM amino acid change, Y453F — associated with increased ACE2-binding affinity — received
considerable attention following its identification in sequences associated with infections in human.
• The B.1.1.298 lineage also has Δ69–70, an amino-terminal domain (NTD) deletion that has emerged several times
across the global SARS-CoV-2 population, including in the second N439K lineage, B.1.258. Δ69–70 is predicted to
alter the conformation of an exposed NTD loop and has been reported to be associated with increased infectivity
and considered as variant of concern.

10. MUTATION IN SPIKE PROTEIN
Fig taken from Helene Banoun, Evolution of SARS-CoV-2: Review of Mutations, Role of the Host Immune System,nephron,45(1),(2021),392-403, DOI: 10.1159/000515417

11. MECHANISM OF ANTIGENIC CHANGES
Amino acid substitutions that alter the epitope:-a change in the biophysical properties of an epitope residue
directly diminishes antibody binding. For example, the neutralizing antibody 4a8 forms salt bridges with spike
protein residues K147 and K150, and therefore substitutions at these residues are likely to inhibit binding.
Increasing receptor-binding avidity:-substitutions that individually increase receptor-binding affinity can shift the
binding equilibrium between glycoprotein and neutralizing antibodies in favor of a higher avidity interaction
between glycoprotein and the cellular receptor. the spike amino acid substitution N501Y, which increases ACE2-
binding affinity, has been described as emerging in individuals treated with convalescent plasma, potentially as a
means of immune escape.
Changes in glycosylation:-Glycans are bulky sugar molecules that may shield epitopes from antibody binding. N-
linked glycans are typically prominent in glycan shielding of virus surface glycoprotein epitopes, although O-linked
glycans can also contribute. a substitution can introduce an additional N-linked glycosylation motif. the acquisition
of epitope-masking glycans during the evolution of human influenza viruses is well described.
Deletions and insertions:-the deletion or insertion of residues has the potential to alter epitope conformation,
diminishing antibody binding. several deletions in the spike amino-terminal domain (NTD) that affect recognition by
neutralizing antibodies have been described. in laboratory experiments, a multiresidue insertion in the spike NTD
has been described as emerging and contributing to escape from polyclonal antibodies in convalescent plasma.
Allosteric structural effects:-similarly to deletions or insertions, an amino acid substitution outside an epitope
footprint may affect antibody binding by changing the protein conformation in such a way that an epitope is altered
or differently displayed. in the spike NTD, changes to disulfide bonds are thought to reduce binding by multiple
monoclonal antibodies through this mechanism.

12. IMPACT OF CORONAVIRUS ON IMMUNITY
• After entering into epithelial lung cells SARS-CoV2 release their RNA that senses by endosomal toll-like receptors
(TLR3,TLR7,TLR8.&TLR9) ,RIG1, melanoma differentiation - associated gene5(MDA5), and cGAS
(nucleotidyltransferase cyclic GMP-AMP synthase)in the cytoplasm.
• Interaction between SARS-CoV2 and alveolar cells, trigger downstream signalling pathway via TIR-domain-
containing adaptor inducing interferon-(TRIF) and stimulator of interferon genes (STING) adaptor molecules
leads to triggering MyD88 adaptor molecule, causing activation of the NF-κB and interferon regulatory factor
3(IRF3).
• Resulting in stimulation of production of proinflammatory mediator including IFN-,IFN-,IFN-, IL-1, IL-2, IL-4, IL-
7, IL-10, IL-12, IL-13, IL-17, TNF-α, macrophage inflammatory protein1(MIP-1α),interferon-inducible protein( IP-
10), GCSF,monocyte chemoattractant protein1( MCP-1), MCSF, and hepatocyte growth factor (HGF) ,leads to the
lung injuries in covid-19 patients.
• According to different studies ,different subsets of lymphocyte including CD4+ & CD8+ T-lymphocyte ,as well as
natural killer cells are decreased in covid-19 patients with severe cases.
• SARS-CoV2 can also recognise C-type lectin protein receptor, these proteins have carbohydrate recognition
domains, which mediate cell-cell, cell-pathogen interaction. C-type lectin proteins are highly expressed on
immune cells, including monocyte, macrophages and dendritic cells, which were implicated in SARS-CoV2
pathogenesis. due to extra hACE2 binding site SARS-CoV2 can infect another organ like cardiovascular system,
gastrointestinal tract and kidneys and provide an alternative infection route.

13. • On the basis of Cryo-electron microscopy and crystal structure of RBD and ACE2 receptor ,it was reported that
some human monoclonal antibodies was screened out which target SARS-CoV2 RBD in immunized
mice,rabbit,non-human primates(NHP) against SARS-CoV2.They may target different site of SARS CoV2.
1.RBD-specific human monoclonal antibodies against SARS-CoV2:(a). monoclonal antibodies bind with RBM of RBD
which functionally mimic ACE2 and block ACE2-RBD-binding.
(b)Second monoclonal antibodies partially bind with epitope, which present on RBD and indirectly block ACE2-RBD
binding without functionally mimicking ACE2 to bind RBD.
(c) Third monoclonal antibodies that bind to the epitope present outside of ACE2 binding site on RBD and could not
block ACE2-RBD binding.
2.Prevent fusion of protein by inhibiting conformational changes
3. Destabilize to release viral nucleic acid outside from the cells
On the basis of cryo-electron microscopy structure of RBD and S-protein as well as screening of memory B-cells
using peripheral blood mononuclear cells collected from infected individual with both SARS-CoV and SARS-CoV2,
few human monoclonal antibodies were identified which can be possible treatment for SARS-CoV2.
1.47D11:-Target a conserved epitope in the SARS-CoV2-S1B domain. 2.B38, H4:-its bind with SARS-CoV2 receptor
binding domain with IC50 value of 0.177 µg/ml. 3. ab1:-it blocks RBD-ACE2 binding by competing with ACE2 for
binding to RBD.4:-CB6 ,CA1, .S303, S304, S309 ,S315, REGN10989, REGN10987, REGN10933, REGN10934,
REGN10977, REGN10964, RE.GN10954, REGN10984, REGN10986, H014, CoV2-2196, and CoV2-2130 etc
MONOCLONAL ANTIBODIES TARGET SPIKE PROTEIN

14. CONCLUSION
• Coronaviruses cases increases exponentially from December 2019 to till now and it become health of
concern.
• There was a number of vaccine are available in the market which was approved by both WHO and
FDA,but many of country are failed to control the cases of coronaviruses, this is due to its rapid
mutation in their genome.
• Mutation in SARS CoV2 spike protein has great impact on failure of vaccination as well as monoclonal
antibody therapy, mutation in genome of spike protein leads to increased its affinity towards ACE2
receptor as well as enhanced its infectivity.
• Hence in future its required to develop such kind of monoclonal antibodies which target specific part of
spike protein and which is resist for mutation.
• Its also required to developed such kind of vaccine which target or generate such amount o antibodies
,which neutralized all possible type of variants.
• We can also generate soluble ACE2 receptor ,which are able to bind free circulating SARS CoV2 virus,
but its have a drawback that in such situation SARS CoV2 can utilized another pathway for cell
attachment.

15. REFERENCE
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