The outbreak of coronavirus disease that causes severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) originated in the seafood market at Wuhan, Hubei province in China on 31 December 2019. The World Health Organization (WHO) declared the COVID-19 outbreak to be a global pandemic on 11 March 2020 [1]. Coronavirus is an enveloped positive- single-stranded RNA virus. SARS CoV-2 has spike protein that helps virus entry into the host cells by binding through angiotensin-converting enzyme (ACE). Different types of the mutation have been identified in spike protein at different sites or locations. The most common type of mutations detected is D614G, G614 and N501Y.These mutations have exhibited different infection and neutralization susceptibility to vaccines. The researchers and clinicians have been tirelessly working to develop a specific vaccine against SARS- CoV-2. Based on strains and mutations in spike proteins, different susceptibility to vaccines has been reported [2,3]. Several clinical trials and research programs on SARS-CoV-2 has been developed. In this review, we discuss the structural aspects, infectivity and neutralization status of SARS-CoV-2. The vaccines developed for the three most common strains or mutations of SARS-CoV-2 spike protein (i.e. D614G, G614, and N501Y) are discussed.

SARS-CoV-2 Spike Protein

The SARS-CoV-2 Spike Protein

The spike protein is a type 1/type of transmembrane glycoprotein on the viral envelope of SARS-CoV-2 as a clove shaped glycosylated homotrimer [4]. Spike (S) glycoprotein plays an essential role in virus attachment by the receptor recognition, cell membrane fusion process, and entry into the target cell. Recent research on SARS-CoV-2 has shown that spike glycoprotein binds to human enzyme, ACE2 receptor present on the surface of mucosal cells through S1 subunits and then enters into the target cell and makes multiple copies of itself [5]. S protein is cleaved into the S1 (685 amino acids) and S2(588 amino acids) subunits. The S1 subunit consists of a receptor-binding domain that is responsible for the binding of the virus into the target cells, and the S2 subunits mediate the fusion of the viral membrane to the host membrane [6]. The S protein triggers the formation of neutralizing antibodies. Therefore, it is amenable for developing a vaccine and as a therapeutic target. Vaccines based on the S protein can induce antibodies to block virus binding and fusion or neutralize virus infection of SARS-CoV and MERS-CoV.

Structural Properties of Spike Proteins

Spike proteins are glycosylated homotrimers with the size of 180-200 kDa and are composed of a large ectodomain, single transmembrane anchor, C terminal intracellular tail, and also consists of 21-35 glycosylation sites [7] & [8]. The ectodomain consists of S1subunit that form the globular head and transmembrane domain, and the C terminal intracellular tail is composed of S2 subunit. S2 subunit forms a stalk-like region of S protein. The transmembrane domain consists of a very conserved motif rich in aromatic residues at the 5' end, which plays an essential role in membrane fusion [9]. Crystallographic studies have shown that S1 proteins contain a receptor-binding domain (RBD), which binds with its receptor, angiotensin-converting enzyme (ACE) present on the host cell. Cryo-electron microscopy at the atomic level has identified that the S protein RBD has a closed and open conformation region. RBD binds to the ACE receptor with open form in the S1 subunit, and the open form of RBD is more efficient for receptor binding than the closed-form. Heptapeptide repeat sequence1 (HR1), heptapeptide repeat sequence2 (HR2), a transmembrane domain, and cytoplasmic domain comprises the S2 subunits [7, 8]; [10]. In native state SARS-CoV-2 is inactive form. During viral infection, S protein is activated by the target protease such as trypsin by cleaving the S1/S2 subunits, and this cleavage is essential for the membrane fusion and virus entry in to the host cell [11]. Previous studies have shown that cathepsin L and TMPRSS2 (transmembrane protease serine 2) prime SARS-CoV by cleaving S1-S2 subunits in spike protein that results in membrane fusion [12]. S-protein infectivity and binding efficiency depend on open conformation. SARS-CoV-2 has a more efficient open conformation and is more infective as compared to SARS-CoV-1 [13]. Further, S-protein also contains 66 N-linked glycosylation sites that contribute to modification in the host.

Variants of SARS-CoV-2 Earlier Dominant Variants

Different variants of SARS-CoV-2 have been discovered by analyzing spike protein sequences. The SARS-CoV-2 spike glycoprotein sequences have been analyzed from different zones (Asia, North America, South America, and Oceania) and compared with the SARS-CoV-2 protein sequences from Wuhan-Hu-1 China [8,14]. A total of 10,333 spike protein sequences have been analyzed. It was identified that 9654 mutations are present at different mutation sites, and there are distinct mutation types according to geographical locations. A study on SARS-CoV-2 spike protein has identified mutations at glycosylation sites. There are 80 variants and 26 glycosylation sites. The mutations have also been reported at glycosylation sites; these sites affect the virus's replication cycle and interaction with the host [15, 16]. [17] had identified mutations and glycosylation site modification in SARS-CoV-2 S protein [17]. The three-dimensional crystal structure of human SARS-CoV-2 RBD interacts with the ACE receptor on the mucosal cells. It is used to identify mutation sites and analyze multiple mutations in a single spike protein. The mutations within RBD interact with the human target cell ACE-2 receptor [18]. The three most common mutations in spike protein from different sites or positions identified are D614G, G614, [19] and N501Y [20]. Ongoing research on spike protein has also identified mutations for the residues Y453, T500, and N501. Y453F mutation has been identified from Europe. T500I and N501Y both detected from Oceania. In February 2020, using a combination of Illumina and Nanopore sequencing, three complete genome sequencing of SARS-CoV-2 has been submitted in GISAID. Korber and colleagues used GISAID data with bioinformatics to identify specific viral variants of the spike protein of SARS-CoV that were becoming increasingly common in specific geographic locations. The group identified that the variants carying the D614G mutation in the Spike protein are rapidly becoming the dominant viral strains across the world [19]; & [20]. D614G mutation in Spike protein at the carboxy-terminal region of the S1 domain of SARS-CoV-2 has been detected as the predominant clade in Europe as well as worldwide and is becoming more infectious [19]; & [21] described that the D614G variant transmits efficiently and influences viral load in SARS-CoV-2 patients and demonstrated with higher titers in pseudo viruses through in vitro analysis. This mutation was found with high frequency (87%) from an Italian SARS-CoV-2 sequenced specimen [19]. It was discovered by epidemiological surveillance [22]. At the time of study on D614G, the identified mutation was found only in one sequence in Germany [22]. The structural bioinformatics analysis identified different phylogenetic clade that suggested that D614G has different structural properties than other spike protein variants. The clinical trials on the D614G variant have shown that cleavage efficiency (into S1/S2 subunits) is higher (93%) than other ancestral S protein (67%) using pseudo type virus-based experiments [23]; & [24]. The interatomic study has identified that the D614G exhibited conformational changes that abolish a hydrogen-bond interaction with T859 from a neighboring promoter of the spike trimer, which allosterically shifts the RBD to an 'up' conformation, promoting binding with the ACE2 receptor leading to enhanced infectivity [19]; & [25].

D614G Structural Properties

D614G is an amino acid mutation in the spike protein of SARS-CoV-2. Amino acid modification is important for stabilizing S proteins through hydrogen bonds [19]. D614G altered structural properties of S protein and effect on transmission and replication through increased affinity to ACE receptor on target cells and can affect its transmission and influence antibody binding [26]. Structural analysis has shown that mutation in spike protein encodes gene and change in amino acid sequence from negatively charged aspartate to glycine residue at D614G on the S1 domain of S protein. This might change the structure of S protein, thus increasing S protein cleavage by furin and increases neutralization susceptibility. The D614G mutation in SARS-CoV-2 has an ADE [27] sequence, which can have an impact on viral pathogenesis and response to the immune system. D614G mutation located outside RBD in the S1 subunit of spike protein is responsible for the conformation changes and flexibility of spike protein. Structural analysis has shown that the D614 side chain can interact with the neighboring protomer T859 amino acids through the hydrogen bond. This makes furin and S2 cleavage sites closer [19]; [28]; & [29]. [23] analyzed the ‗S ‘protein sequences available from the Gen Bank and showed that the D614G mutation correlates with the mutation in Viral nsp3 [23]; & [30] and RdRp proteins. Therefore, the role of the S protein in these observations is unclear [23]; & [28].

Spike Protein D614G Increases SARS-Cov-2 Fitness and Neutralization Susceptibility

S protein plays an essential role in receptor binding and membrane fusion. Thus, vaccines based on the S protein can induce antibodies to block virus binding and fusion or neutralize virus infection. Therefore, S protein has been selected as an important target for vaccine and anti-viral drug development [31]; & [32]. [33] showed that a DNA vaccine encoding the full-length S protein SARS- CoV Urbani strain (HKU39849) could induce T-cell and neutralizing-antibody responses as well as protective immunity in a mouse model [33]. D614G mutation present on the outer surface of the virus's spike protein sustains a lot of consciousness from the human immune system and could have an effect on the ability of the SARS-CoV-2 to evade vaccine-induced immunity. D614G enhances viral replication in the upper respiratory tract and increases neutralization susceptibility [24]; & [29]. D614G mutation in SARS-CoV-2 affects neutralizing antibody sensitivity and the ADE activity as observed in the SARS-CoV study. D614G could utilize an intermediate antibody escape mechanism that makes individuals more susceptible to second infections [34]; & [35]. In the ADE mechanism, antibodies bind to virus particles, which fail to neutralize the interaction of virus–antibody immune complexes with Fcγ and/or complement receptors on host cells. This process leads to viral membrane fusion and entry into the monocytes, B Cells, and macrophages, and reducing viral clearance by affecting the viral generation. ADE also forms a complex that may trigger the cytokine storm and can be involved in the release of inflammatory cytokines [36]. Evidence suggests that D614G is not located in the RBD but in the interface between individual spike promoters, which stabilize mature trimeric form on virus outer surface through hydrogen bonding. This interference could result in the loss of promoter hydrogen bonds and also changes the glycosylation sites [37]. These changes could alter infectivity and the effect on the immunogenicity of RBD epitopes. This is thought to be necessary for antibody neutralization. The antibodies generated from infection with SARS-CoV-2also suggest that D614G mutation is unlikely to have an enormous impact on the efficiency of vaccines and its protection against SARS-CoV-2. Most SARS-CoV- 2vaccines currently in clinical trials are based on the original D614 spike sequence [30]; & [38]. Previous clinical trials observed neutralization titers of a panel of sera collected from hamsters that were infected with D614G SARS-CoV-2 [39] and then analyzed that D614G mutation may confer higher susceptibility to serum neutralization. The experiments on RBD mAbs reported that D614G mutation might change spike protein conformation to affect mAb neutralization in an epitope-specific manner [40]. The clinical trials have also shown that the neutralizing potency of some monoclonal antibodies could be affected by the D614G mutation [39] [40]. Most SARS-CoV-2 vaccines were initially designed with the D614 modification of the Spike protein, which was located in the first sequence of SARS-CoV-2 from Wuhan [41]; [42]; & [43]. Over twenty vaccines are currently in Phase 3 clinical trial. In these vaccines, the immunogens are derived from the D614 variant in the Spike protein [41]; & [42]. Due to the effect ofD614G on spike protein, its function and viral membrane fusion are unclear [42]. Therefore, the impact of this mutation on the vaccine and therapeutic entry inhibitors is unknown. No current evidence suggests that D614G mutation would interfere in restorative designs such as monoclonal antibodies.

G614 Variant is More Infectious than D614 Variant

G614 is more infectious variant of SARS-CoV-2 and might influence vaccine development [42]. The G614 variant has a higher level of viral RNA load in the respiratory tract than the D614G variant and first detected in late January 2020 [42]. Investigation on spike protein mutation of SARS-CoV-2 in the sera from mice, rhesus macaques and humans immunized with nucleoside-modified mRNA-LNPs encoding RBD and full-length spike immunogens observed that another spike protein variant G614 is neutralized at a higher level than the D614 variant. The two phenotypes S (D614) and S(G614) variant of SARS-CoV-2 were characterized and compared in one study. It was observed that retroviruses pseudotyped with S (G614) infected hACE2-expressing cells significantly and more efficiently than those with S(D614) [39]; & [44]. The group reported that S(G614) has higher stability due to less S1 shedding and greater incorporation of the S protein into pseudovirion. [27]; [38] Studies on SARS-CoV-2 has identified that spike protein variant (G614) mediates more efficient ACE2-mediated transduction of cells by S-pseudotyped vectors and are more efficient for infection of cells and animals by live SARS-CoV-2 [42]. The investigators also identified 3-4 folds higher viral load in patients infected with S(G614) virus compared to those infected with S(D614) virus. Maloney murine leukemia virus (MLV) pseudoyped with S(G614) versus S(D614) resulted in significantly higher transduction of ACE2-overexpressing HEK293T cells with or without TMPRSS2 overexpression. The VLPs with S(G614) had a higher density of incorporated S protein than S(D614) Upon visualization of live SARS-CoV-2 G614 variant by cryo-electron microscopy, much greater spike density was observed than reported in prior studies with D614 viruses. In a survey on spike protein in the PV and VLP experimental models, more muscular retention of the S1 subunit on the S(G614) compared to S(D614) variant was observed [45]; & [46]. It has also been observed that infection of primary human nasal epithelial cells and large airway epithelial cells infected with G614 SARS-CoV-2 resulted in higher titers than those infected with an equal amount of isogenic D614 virus [45]; [47]. Researchers infected Syrian hamsters and observed a modestly more significant weight loss in G614 virus-infected hamsters than in D614 [39]. HEK293T cells overexpressing S protein have shown stronger S1 versus full-length S signal in cells transfected with S (G614) than in those transfected with S(D614). It has been observed that protease cleavage at the furin site is more efficient in S (G614) than S(D614) [42]; & [48]. The S(G614) was more resistant to cleavage as determined by the amount of uncleaved S in cell lysates and upon digestion with furin in vitro than S (D614) [42]. The spike proteins produced virus-like particles (VLPs) that comprised of SARS-CoV-2 nucleoprotein (N), membrane protein(M), an envelope protein (E), and S(D614) or S(G614) [41]; & [49].

A recent study raised concerns of antigenic drift, suggesting that vaccines targeting S (D614) may have limited protection against viruses with S (G614). The neon green gene was at the open-reading-frame 7 of the SARS-CoV-2 genome and exhibited 1.4- to 2.3-folds higher neutralization titers (mean 1.7-folds) against the G614 virus than those against D614. Experimental data about the neutralization of S (G614) and S (D614) pseudoviruses have also demonstrated similar neutralization of S (D614) and S (G614) PVs by antibodies or patient antisera directed at an S protein variant. Therefore, the difference between S (D614) and S (G614) is unclear [50]; 7 [29]. In this review, we have discussed that S(G614) is more infectious than S (D614) and effective for vaccine design. Studies have shown that S (G614) is neutralized at a higher level and high stability due to less S1 shedding and 3-4folds higher viral load than S (D614) variant. The protease cleavage at the furin site of S (G614) is also more efficient than S (D614). The characteristics ofG614 and D614 variants are summarized in table 1 below.

Table 1: Characteristics of G614 and D614 Variants

Recent transcriptional analysis revealed that there was an imbalanced response with minimal levels of interferons and a remarkable increase of chemotactic and inflammatory response characterized by expression of IL‐1β, IL‐6, TNF, and CXCLs. The excessive formation of pro inflammatory cytokines, such as IL‐6 and IL‐1β, may result in a higher risk of vascular hyper permeability and respiratory failure. Recent research progress on mechanistic studies of SARS‐CoV‐2 and clinical trials of neutralizing antibodies and vaccine development is one giant leap. Antibodies that block the interaction between S protein and angiotensin‐ converting enzyme 2 (ACE2) may effectively prevent the infection of SARS‐CoV‐2 [4]; & [5].

Newer Emerging Variants of SARS-CoV-2

Newer variants of SARS-CoV-2 are emerging in India, UK and South Africa with mutations particularly affecting RBD domain. Two of the most common identified variants are N501Y and E484K.

N501Y Mutation Structure

A new mutation has been identified in the UK, and their infection detected in the south east in December 2020. It was identified by the random genetic sequencing of positive SARS-CoV-2-19 samples around the UK [51]. N501 mutation in the receptor-binding domain of the S protein results in a change of asparagine to tyrosine residue at position 501. It affects binding affinity of ACE receptor. Different mutation at position 681, P681H, which directly associate to the furin cleavage site and enhanced hydrolysis by TMPRSS2 protease and influence the viral fusion [52]. The newer UK variant is important since it contains both N501Y and P681H in combination. Studies has also shown that N501Y exhibit a deletion mutation in another viral gene, ORF8, and this viral gene associated with a less clinical infection and less post-infection inflammation. Studies have shown that N501Y exhibit a doubling of the occupancy of the open conformation state related to decrease of the closed state of spike protein. Therefore, N501Y mutation has higher transmission. N501Y variant may also build up the antibody neutralization of the virus due to doubling occupancy of the open state [53]. It is strong concern that D614 receptor-binding domain of old variant is beneficial for immune system. Due to the more open conformation of its RBD, which potentially contribute these new variants make more immunogenic. It has been identified that deletion mutation of ORF8 in the most recent UK variant N501 [3] has less infectivity and less post-infection inflammation as reported [54].

Neutralization Susceptibility

Despite having the mutation in RBD region, studies have shown conflicting neutralization susceptibility of this mutation to common vaccines in different regions of world. Recent studies in UK showed preserved neutralization of Y501 virus in sera of individuals who received BNT162b2 vaccine [55]. Contrary to this, in South African population, a significantly reduced titre of protective neutralization antibodies to SARS-CoV-2 has been observed in convalescent plasma of patients with N501Y variant. This is a new mutation that makes viruses more infectious but does not necessarily make them more dangerous. There is no enough evidence about this new strain causing severe illness [56].

E484K Variant Structure

A new variant was detected on 01February 2021 in South Africa. In this new variant, glutamate (E) is substituted by lysine (K) at position 484 (E484K) in the receptor binding domain (RBD) of the spike protein and rapidly spreading variants of concern belonging to the B.1.351 and P.1 lineages. This variant has less probability to escape. Studies suggest thatE484K is one of the most frequent mutations to escape monoclonal antibodies. GIASID data suggest that E484K is the most common RBD mutation. It has been shown by experimental trials that E484 being intended by antibodies which derived from IGHV3-53 and jointly to IGHV3-66 genes. Further, the most common germ lines for antibodies have been directed against the RBD. Bioinformatics study on spike protein has shown that N3 and N5 exhibited insertion and deletion which prevent binding of neutralizing antibodies [56]; [54]; [57]; & [58].

Neutralization Susceptibility

A reduction in the neutralization susceptibility by 0.81- to 1.46-folds in the Pfizer vaccine directed against E484K variant has been reported. Newer strains of natural variants have been reported in the United Kingdom and South Africa. It is suggested that SARS- CoV-2 has the prospective to escape an effective immune response. The vaccines and antibodies able to control N501Y and E484Kvariants will help in fighting the pandemic in an effective manner [57]; & [58].

Double Mutant Variant B.1.617 with Two Mutation E484Q and L452R

Researchers have identified a new variant with two mutation E48Q and L452R in receptor binding domain of S protein. These double mutant has been detected in India, primarily in states of Maharashtra, Delhi, West Bengal and Chhattisgarh. One of the mutation, E484Q bears similarity to E484K strain (lineage B.1.1.7) which was detected in United Kingdom earlier this year and it also possessed some similarity with South African variant (lineageB.1.351). Another mutation of double mutant, L452R mutation has been identified to possess structural similarity to fast spreading variants identified in California (lineage B.1.427 and B.1.429). This double mutant has been shown to influence the binding affinity of spike proteins with ACE2 receptors on human cells thereby making it more transmissible. L452R mutation has also been shown to influence replication potential of the virus [46]; [57]; & [58].

Neutralization Susceptibility of Double Mutant Virus

E484Q and L452R have been observed to influence immune escape mechanisms and was shown to reduce neutralizing antibody titers in convalescent plasma by more than four times. This may be a cause of potential concern for the current vaccination drive worldwide. Immune selection pressure from a previously exposed population has been suggested to be a plausible explanation for emergence of newer strains [58].

Summary

Covid-19 continuing to affect many countries. Different strains of SARS-COV-2 have been identified in different region of the world. Spike proteins in SARS-COV-2 helps in binding virus to host cell. The older Mutant variants (mutations outside the RBD of spike protein) are D414G, G614, N501Y, E484K and the newly Emerging Indian double mutant strain with two mutation E484Q and L452R are the resent variants in India, UK, and South Africa.

Trimetric spike protein seems to be a potentially favorable site for mutation in various variants of SARS- CoV-2. Earlier dominant variants primarily had mutations in region adjacent to RBD like D614G and G614.These variants showed increased infectivity potential. Despite the possibility of their interaction with RBD by forming hydrogen bond, studies have shown equivocal neutralization susceptibility for common vaccines. Newer emerging variants seems to primarily harbor mutations in RBD region and have shown higher infectivity potential. For example, N501Y showed equivocal neutralization susceptibility in UK. However, a reduced response was observed in South African population. On the contrary, newer variants like E484K, E484Q and L452Rshowed reduction in neutralization susceptibility in both experimental models as well as human sera to common vaccines. Epidemiological studies with larger number of patient‘s data are required before any conclusion can be drawn about effectiveness of common vaccine designed against different variants of SARS-CoV-2.

1. Cucinotta, Domenico, and Maurizio Vanelli. "WHO Declares COVID-19 a Pandemic." Acta Bio Medica: Atenei Parmensis, vol. 91, no. 1, 2020, pp. 157. doi:10.23750/abm.v91i1.9397.

2. Won, Jung-Hyun, and Howard Lee. "The Current Status of Drug Repositioning and Vaccine Developments for the COVID-19 Pandemic." International Journal of Molecular Sciences, vol. 21, no. 24, 2020, 9775. doi:10.3390/ijms21249775.

3. Rathnasinghe, Raveen, et al. "The N501Y Mutation in SARS-CoV-2 Spike Leads to Morbidity in Obese and Aged Mice and Is Neutralized by Convalescent and Post-Vaccination Human Sera." MedRxiv, 2021. doi:10.1101/2021.05.21.21257521.

4. Sternberg, Ariane, and Cord Naujokat. "Structural Features of Coronavirus SARS-CoV-2 Spike Protein: Targets for Vaccination." Life Sciences, vol. 257, 2020, 118056. doi:10.1016/j.lfs.2020.118056.

5. Astuti, Indwiani. "Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2): An Overview of Viral Structure and Host Response." Diabetes & Metabolic Syndrome: Clinical Research & Reviews, vol. 14, no. 4, 2020, pp. 407-412. doi:10.1016/j.dsx.2020.04.020.

6. Kim, Myung Hee, Hyun Jik Kim, and Jun Chang. "Superior Immune Responses Induced by Intranasal Immunization with Recombinant Adenovirus-Based Vaccine Expressing Full-Length Spike Protein of Middle East Respiratory Syndrome Coronavirus." PLOS ONE, vol. 14, no. 7, 2019, e0220196. doi:10.1371/journal.pone.0220196.

7. Fraguas Bringas, Conchita, and David Booth. "Identification of a SARS-Like Bat Coronavirus That Shares Structural Features with the Spike Glycoprotein Receptor-Binding Domain of SARS-CoV-2." Access Microbiology, vol. 2, no. 11, 2020, e000166. doi:10.1099/acmi.ac2020.po0062.

8. Huang, Yuan, et al. "Structural and Functional Properties of SARS-CoV-2 Spike Protein: Potential Antivirus Drug Development for COVID-19." Acta Pharmacologica Sinica, vol. 41, no. 9, 2020, pp. 1141-1149. doi:10.1038/s41401-020-0370-1.

9. Örd, Mihkel, Ilona Faustova, and Mart Loog. "The Sequence at Spike S1/S2 Site Enables Cleavage by Furin and Phospho-Regulation in SARS-CoV2 but Not in SARS-CoV1 or MERS-CoV." Scientific Reports, vol. 10, no. 1, 2020, 16944. doi:10.1038/s41598-020-73721-5.

10. Tang, Tiffany, et al. "Coronavirus Membrane Fusion Mechanism Offers a Potential Target for Antiviral Development." Antiviral Research, vol. 178, 2020, 104792. doi:10.1016/j.antiviral.2020.104792.

11. Hilgenfeld, Rolf, et al. "Structural Proteomics of Emerging Viruses: The Examples of SARS-CoV and Other Coronaviruses." Structural Proteomics and Its Impact on the Life Sciences, edited by J. Sussman and I. Silman, 2008, pp. 361-433.

12. Davidson, Anne M., Jan Wysocki, and Daniel Batlle. "Interaction of SARS-CoV-2 and Other Coronaviruses with ACE (Angiotensin-Converting Enzyme)-2 as Their Main Receptor: Therapeutic Implications." Hypertension, vol. 76, no. 5, 2020, pp. 1339-1349. doi:10.1161/HYPERTENSIONAHA.120.15466.

13. Yuki, Koichi, Miho Fujiogi, and Sophia Koutsogiannaki. "COVID-19 Pathophysiology: A Review." Clinical Immunology, vol. 215, 2020, 108427. doi:10.1016/j.clim.2020.108427.

14. Van Dorp, Lucy, et al. "Emergence of Genomic Diversity and Recurrent Mutations in SARS-CoV-2." Infection, Genetics and Evolution, vol. 83, 2020, 104351. doi:10.1016/j.meegid.2020.104351

15. Crispin, Max, and Katie J. Doores. "Targeting Host-Derived Glycans on Enveloped Viruses for Antibody-Based Vaccine Design." Current Opinion in Virology, vol. 11, 2015, pp. 63-69. doi:10.1016/j.coviro.2015.10.005.

16. Guruprasad, Lalitha. "Human SARS CoV‐2 Spike Protein Mutations." Proteins: Structure, Function, and Bioinformatics, vol. 89, no. 5, 2021, pp. 56. doi:10.1002/prot.26046.

17. Li, Qianqian, et al. "The Impact of Mutations in SARS-CoV-2 Spike on Viral Infectivity and Antigenicity." Cell, vol. 182, no. 5, 2020, pp. 1284-1294. doi:10.1016/j.cell.2020.07.012.

18. Chi, Xiangyang, et al. "A Neutralizing Human Antibody Binds to the N-Terminal Domain of the Spike Protein of SARS-CoV-2." Science, vol. 369, no. 6504, 2020, pp. 650-655. doi:10.1126/science.abc6952.

19. Groves, Danielle C., Sarah L. Rowland-Jones, and Adrienn Angyal. "The D614G Mutations in the SARS-CoV-2 Spike Protein: Implications for Viral Infectivity, Disease Severity and Vaccine Design." Biochemical and Biophysical Research Communications, vol. 538, 2021, pp. 104-107. doi:10.1016/j.bbrc.2020.10.065.

20. Parray, Hilal Ahmad, et al. "Identification of an Anti–SARS–CoV-2 Receptor-Binding Domain–Directed Human Monoclonal Antibody from a Naïve Semisynthetic Library." Journal of Biological Chemistry, vol. 295, no. 36, 2020, pp. 12814-12821. doi:10.1074/jbc.ac120.014357.

21. Lau, Siu-Ying, et al. "Attenuated SARS-CoV-2 Variants with Deletions at the S1/S2 Junction." Emerging Microbes & Infections, vol. 9, no. 1, 2020, pp. 837-842. doi:10.1080/22221751.2020.1773106.

22. Berrio, Alejandro, Valerie Gartner, and Gregory A. Wray. "Positive Selection within the Genomes of SARS-CoV-2 and Other Coronaviruses Independent of Impact on Protein Function." PeerJ, vol. 8, 2020, e10234. doi:10.7717/peerj.10234.

23. Korber, Bette, et al. "Tracking Changes in SARS-CoV-2 Spike: Evidence That D614G Increases Infectivity of the COVID-19 Virus." Cell, vol. 182, no. 4, 2020, pp. 812-827. doi:10.1016/j.cell.2020.06.043.

24. Hu, Jie, et al. "D614G Mutation of SARS-CoV-2 Spike Protein Enhances Viral Infectivity." BioRxiv, 2020. doi:10.1101/2020.06.03.130113.

25. Gong, Yu-Nong, et al. "SARS-CoV-2 Genomic Surveillance in Taiwan Revealed Novel ORF8-Deletion Mutant and Clade Possibly Associated with Infections in Middle East." Emerging Microbes & Infections, vol. 9, no. 1, 2020, pp. 1457-1466. doi:10.1080/22221751.2020.1822724.

26. Erol, Adnan. "Are the Emerging SARS-CoV-2 Mutations Friend or Foe?" Immunology Letters, vol. 230, 2021, pp. 63. doi:10.1016/j.imlet.2021.02.004.

27. Gavor, Edem, et al. "Structural Basis of SARS-CoV-2 and SARS-CoV Antibody Interactions." Trends in Immunology, vol. 41, no. 11, 2020, pp. 1006-1022. doi:10.1016/j.it.2020.09.007.

28. Mohammad, Anwar, et al. "Higher Binding Affinity of Furin for SARS-CoV-2 Spike (S) Protein D614G Mutant Could Be Associated with Higher SARS-CoV-2 Infectivity." International Journal of Infectious Diseases, vol. 103, 2021, pp. 611-616. doi:10.1016/j.ijid.2020.11.081.

29. Plante, Jessica A., et al. "Spike Mutation D614G Alters SARS-CoV-2 Fitness." Nature, vol. 592, no. 7852, 2021, pp. 116-121. doi:10.1038/s41586-020-2338-2.

30. Fernandes, Jason D., et al. "The UCSC SARS-CoV-2 Genome Browser." Nature Genetics, vol. 52, no. 10, 2020, pp. 991-998. doi:10.1038/s41588-020-00796-3.

31. Ovsyannikova, Inna G., et al. "The Role of Host Genetics in the Immune Response to SARS‐CoV‐2 and COVID‐19 Susceptibility and Severity." Immunological Reviews, vol. 296, no. 1, 2020, pp. 205-219. doi:10.1111/imr.12830.

32. Du, Lanying, et al. "The Spike Protein of SARS-CoV—a Target for Vaccine and Therapeutic Development." Nature Reviews Microbiology, vol. 7, no. 3, 2009, pp. 226-236. doi:10.1038/nrmicro2100.

33. Yang, Zhi-Yong, et al. "A DNA Vaccine Induces SARS Coronavirus Neutralization and Protective Immunity in Mice." Nature, vol. 428, no. 6982, 2004, pp. 561-564. doi:10.1038/nature02463.

34. Mansbach, Rachael A., et al. "The SARS-CoV-2 Spike Variant D614G Favors an Open Conformational State." Biophysical Journal, vol. 120, no. 3, 2021, pp. 298a. doi:10.1016/j.bpj.2020.11.402.

35. Nidom, Reviany V., et al. "Investigation of the D614G Mutation and Antibody-Dependent Enhancement Sequences in Indonesian SARS-CoV-2 Isolates and Comparison to Southeast Asian Isolates." Syst Rev Pharm, vol. 11, no. 8, 2020, pp. 203-213. doi:10.5530/srp.2020.8.31.

36. Smatti, Maria K., Asmaa A. Al Thani, and Hadi M. Yassine. "Viral-Induced Enhanced Disease Illness." Frontiers in Microbiology, vol. 9, 2018, 2991. doi:10.3389/fmicb.2018.02991.

37. Mathavan, Sarmilah, and Suresh Kumar. "Evaluation of the Effect of D614G, N501Y and S477N Mutation in SARS-CoV-2 through Computational Approach." 2020. doi:10.2139/ssrn.3596697.

38. Weissman, Drew, et al. "D614G Spike Mutation Increases SARS-CoV-2 Susceptibility to Neutralization." Cell Host & Microbe, vol. 29, no. 1, 2021, pp. 23-31. doi:10.1016/j.chom.2020.11.015.

39. Zhou, Bin, et al. "SARS-CoV-2 Spike D614G Variant Confers Enhanced Replication and Transmissibility." BioRxiv, 2020. doi:10.1101/2020.07.07.190830.

40. Hou, Yixuan J., et al. "SARS-CoV-2 D614G Variant Exhibits Enhanced Replication Ex Vivo and Earlier Transmission In Vivo." BioRxiv, 2020. doi:10.1101/2020.08.04.236016.

41. Tripathi, Kanchan, et al. "Newer Strains of SARS-CoV-2 Evolving from RBD of Spike Protein: A Potential Concern for Neutralizing-Antibody Response." 2021. doi:10.2139/ssrn.3718890.

42. Daniloski, Zharko, et al. "The Spike D614G Mutation Increases SARS-CoV-2 Infection of Multiple Human Cell Types." Elife, vol. 10, 2021, e65365. doi:10.7554/eLife.65365.

43. Ortiz-Prado, Esteban, et al. "Clinical, Molecular, and Epidemiological Characterization of the SARS-CoV-2 Virus and the Coronavirus Disease 2019 (COVID-19), A Comprehensive Literature Review." Diagnostic Microbiology and Infectious Disease, vol. 98, no. 1, 2020, 115094. doi:10.1016/j.diagmicrobio.2020.115094.

44. Dearlove, Bethany, et al. "A SARS-CoV-2 Vaccine Candidate Would Likely Match All Currently Circulating Variants." Proceedings of the National Academy of Sciences, vol. 117, no. 38, 2020, pp. 23652-23662. doi:10.1073/pnas.2008237117.

45. Long, S. Wesley, et al. "Molecular Architecture of Early Dissemination and Massive Second Wave of the SARS-CoV-2 Virus in a Major Metropolitan Area." MBio, vol. 11, no. 6, 2020, pp. 10-1128. doi:10.1128/mBio.02707-20.

46. Jackson, Cody B., et al. "Functional Importance of the D614G Mutation in the SARS-CoV-2 Spike Protein." Biochemical and Biophysical Research Communications, vol. 538, 2021, pp. 108-115. doi:10.1016/j.bbrc.2020.11.076.

47. Cloutier, Maryse, et al. "ADE and Hyperinflammation in SARS-CoV2 Infection-Comparison with Dengue Hemorrhagic Fever and Feline Infectious Peritonitis." Cytokine, vol. 136, 2020, 155256. doi:10.1016/j.cyto.2020.155256.

48. Sharun, Khan, et al. "Antibody-Based Immunotherapeutics and Use of Convalescent Plasma to Counter COVID-19: Advances and Prospects." Expert Opinion on Biological Therapy, vol. 20, no. 9, 2020, pp. 1033-1046. doi:10.1080/14712598.2020.1823804.

49. Duan, Liangwei, et al. "The SARS-CoV-2 Spike Glycoprotein Biosynthesis, Structure, Function, and Antigenicity: Implications for the Design of Spike-Based Vaccine Immunogens." Frontiers in Immunology, vol. 11, 2020, 576622. doi:10.3389/fimmu.2020.576622.

50. Alouane, Tarek, et al. "Genomic Diversity and Hotspot Mutations in 30,983 SARS-CoV-2 Genomes: Moving Toward a Universal Vaccine for the 'Confined Virus'?" Pathogens, vol. 9, no. 10, 2020, 829. doi:10.3390/pathogens9100829.

51. Wise, Jacqui. "Covid-19: New Coronavirus Variant Is Identified in UK." 2020. BMJ, vol. 371, n. 7002. doi:10.1136/bmj.m4857.

52. Duan, Liangwei, et al. "The SARS-CoV-2 Spike Glycoprotein Biosynthesis, Structure, Function, and Antigenicity: Implications for the Design of Spike-Based Vaccine Immunogens." Frontiers in Immunology, vol. 11, 2020, 576622. doi:10.3389/fimmu.2020.576622.

53. Gómez, Carmen Elena, Beatriz Perdiguero, and Mariano Esteban. "Emerging SARS-CoV-2 Variants and Impact in Global Vaccination Programs Against SARS-CoV-2/COVID-19." Vaccines, vol. 9, no. 3, 2021, 243. doi:10.3390/vaccines9030243.

54. Abdullahi, Idris N., et al. "Implications of SARS-CoV-2 Genetic Diversity and Mutations on Pathogenicity of the COVID-19 and Biomedical Interventions." Journal of Taibah University Medical Sciences, vol. 15, no. 4, 2020, pp. 258-264. doi:

55. Xie, Xuping, et al. "Neutralization of Spike 69/70 Deletion, E484K, and N501Y SARS-CoV-2 by BNT162b2 Vaccine-Elicited Sera." Preprint, 2021. doi:10.1101/2021.01.14.426949.

56. Wibmer, Constantinos Kurt, et al. "SARS-CoV-2 501Y.V2 Escapes Neutralization by South African COVID-19 Donor Plasma." Nature Medicine, vol. 27, no. 4, 2021, pp. 622-625. doi:10.1038/s41591-021-01285-x.

57. Deng, Xianding, et al. "Transmission, Infectivity, and Antibody Neutralization of an Emerging SARS-CoV-2 Variant in California Carrying a L452R Spike Protein Mutation." MedRxiv, 2021. doi:10.1101/2021.02.05.21250889.

58. Focosi, Daniele, and Fabrizio Maggi. "Neutralising Antibody Escape of SARS‐CoV‐2 Spike Protein: Risk Assessment for Antibody‐Based Covid‐19 Therapeutics and Vaccines." Reviews in Medical Virology, vol. 31, no. 6, 2021, e2231. doi:10.1002/rmv.2231.