Human Coronavirus 2019-nCoV Variants
SARS-CoV-2 (severe acute respiratory syndrome coronavirus-2), the causative agent of COVID-19, was first reported in Wuhan (China) and has rapidly disseminated around the globe . Only one year after the pandemic outbreak, scientists have developed efficient vaccines and promising therapeutic interventions, such as monoclonal antibodies (mAbs). However, SARS-CoV-2 variants exhibiting increased transmission have emerged. Some are raising concern as they might develop resistance to natural immunity and therapies . The escalating number of "variants of concern" may jeopardize worldwide control of the disease. The race is on: epidemiologists and immunologists are working hand-in-hand to detect, track, and predict SARS-CoV-2 mutations for adjusted countermeasures.
SARS-CoV-2 variants are defined as viruses accumulating mutations, including single nucleotide polymorphisms (SNPs), that confer them with a selection advantage such as higher infectivity within the host and transmissibility between hosts . Variants of concern are defined as viruses that have acquired antigenic changes altering the immune response and/or drug efficacy. Genome sequencing allows visualization of the virus's ongoing evolution. While mutations have been reported throughout the SARS-CoV-2 genome, the most concerning ones have occurred in the Spike (S) encoding gene.
The Wuhan-Hu D614 isolate/strain was the first described early in the pandemic, in December 2019  and belongs to clade 19A. In 2020, over 20,000 mutations and some insertions/deletions have been detected in SARS-CoV-2 strains. In April 2020, the original variant was globally replaced by the D614G variant (clade 20) . The D614G variant has since developed numerous mutations allowing increased transmission of the virus. Clusters of the D614G variant have occurred in specific geographical areas, before spreading around the globe. These new variants are often named after the country they first appeared in (e.g. United-Kingdom (U.K) variant, South-Africa (S.A) variant, Brazil (BRA) variant, and California (CAL; USA).
While a single mutation (D→G in position 614 of the Spike protein) characterizes the D614G variant, a minimal number of specific mutations classifies a variant to the U.K (5 of 17) , S.A (5 of 9) , BRA (10 of 17) , or CAL lineages.
The terminology of viral variation can be confusing as scientists often use the terms variant, strain, and lineage interchangeably. In a virus phylogenetic tree, a new variant emerges when specific (sets of) mutations are selected. If these mutations produce a virus with distinct phenotypic characteristics, the variant is co-termed a strain. A lineage, or clade, is a phylogenetic cluster of variants associated with an epidemiological event (i.e. rapid geographical spread). Although efforts are being made to meet a standardized nomenclature for new variants , the two most cited platforms sharing SARS-CoV-2 genomes, Nextstrain and PANGO, use a distinct terminology. For more clarity, we hereby use the term variant with the Nextstrain nomenclature. The PANGO lineage equivalent is mentioned in brackets.
Mutations of concern in the Spike protein
SARS-CoV-2 Spike protein is assembled at the virus membrane as a clove-shaped trimer. Each protomer's ectodomain contains two subunits, S1 and S2. The S1 portion is implicated in viral attachment to the target cell. S1 is comprised of the N-terminal domain (NTD), the ACE2 receptor-binding domain (RBD), and the C-terminal domain (CTD). NTD and RBD are two superantigenic regions, within which mutations have been found to impact both infectivity and immunity. The S2 portion contains elements, such as the fusion peptide (FP), that are critical for viral genome delivery into the target cytosol. To date, mutations in the S2 subunit have not been linked to the virus's spread, nor escape from immunity.
Mutations in Spike RBD and S1-CTD:
Structural studies suggest that the D614G substitution increases the ability to shift the RBD into the "up" position required for ACE2 receptor interaction . Consistent with this, D614G has been shown to increase SARS-CoV-2 infection and transmissibility [10, 11]. This mutation is found in all current variants of concern.
Position 501 in the S protein is one of the six contact amino acid (a.a.) residues with ACE2. The N501Y substitution has been shown to improve the binding affinity of SARS-CoV-2 to ACE2 . Adaptation experiments of SARS-CoV-2 with the N501Y mutation confers the virus capacity for replication in mice, whose ACE2 does not readily engage the S protein . The antigenic impact of this substitution is very minor, with no pronounced effects on the in vitro neutralizing activity of convalescent plasma or vaccinee sera . N501Y is prominent in most current variants of concern.
The substitution at K417 to a N (Asparagine) in S.A variants or to a T (Threonine) in the BRA variant changes a polar a.a. to a neutral one. A mouse adaptation study has suggested that the K417N mutation improves the Spike interaction with ACE2 .
The E484K mutation is shared by S.A variants and the BRA variant. This a.a. change may not only improve binding to ACE2 but also contribute to SARS-CoV-2 escape from mAbs and convalescent COVID-19 patient sera [15, 16]. It has been proposed that the K (Lysine) substitution at position 484 disrupts the hydrogen bonds with clinical mAbs that target the Spike RBM or polyclonal antibodies from vaccinee sera .
The substitution at P681 to a H (Histidine) in the U.K variant is adjacent to the SARS-CoV-2 furin cleavage site (aka S1/S2 cleavage site) . Further studies are required to determine the overall impact of this mutation.
The substitution at L452 to a R (Purine) in the CAL (USA) variant is located within SARS-CoV-2 RBD [18, 19]. While the L452 residue does not directly contact the ACE2 receptor, it is plausible that the L452R mutation causes structural changes promoting the Spike-Ace2 interaction . This conformational change has also been suggested to impact neutralizing antibody binding [18, 19].
Mutations in Spike S1-NTD:
The NTD of the SARS-CoV-2 Spike protein is the least conserved domain . Several substitutions and deletions are found within this region. Notably, 90% of the deletions in Spike occur in four regions within the NTD that are named "recurrent deletion regions" (RDR1 to RDR4) . These RDRs cover external surfaces containing antibody epitopes and therefore contribute to SARS-CoV-2 resistance to neutralizing antibodies .
The substitution of L18 to a F (Phenylalanine) is an SNP found in the BRA variant, as well as in the S.A. B.1.351 v2 variant . The L18F mutation has been predicted to impact mAb binding, based on structural data [19, 25]. Further functional studies are now required to assess this hypothesis.
The loss of two residues at positions 69 and 70 is the most common RDR1 deletion . Del69-70 has been observed in the U.K variant  as well as in a variant that emerged in danish mink farms in April 2020 . Although this deletion is found in a prominent exterior loop of the Spike, it has not been associated with escape from NTD antibodies, using in vitro neutralization assays with convalescent patients or vaccinee sera [23, 24]. Del69-70 is associated with 2-fold increased Spike incorporation into virions but not increased cell-cell fusion. Further studies are required to determine the overall impact of this mutation.
The deletion of one of two adjacent Y (Tyrosine) in position 144/145 is located in RDR2 and is a key signature of the U.K. variant . This mutation is adjacent to glycosylation sites which may play a role in SARS-CoV-2 attachment to target cells or in escaping neutralizing antibodies [17, 20].
The loss of a.a. in the 242-244 stretch within RDR4 is another favored Spike variation. It is one SNP signature of S.A variants and the only one non-surface exposed . Little information is available regarding its impact on SARS-CoV-2 infectivity and immune escape. This mutation belongs to the Spike NTD antigenic supersite targeted by antibodies isolated from convalescent patients . Together with the R246I mutation, del242-4 largely contributes to S.A variants' resistance to most NTD mAbs .
This substitution is in close proximity to del242-4 and is another SNP signature of one of the three reported S.A. variants, B.1.351 v3 [6, 19]. It also belongs to the Spike NTD antigenic supersite and is predicted to facilitate escape from neutralizing antibodies . Together with the 242-4 deletion, R246I largely contributes to the B.1.351 v3 S.A. variant's resistance to most NTD mAbs .
SARS-CoV-2 variants of concern
Starting in April 2020, the 20/D614G variant started to out-compete the original Wuhan-Hu-1 virus, and rapidly, it became the globally dominant variant . It is defined by the unique D614→G substitution in the Spike protein, allowing a better fusion-competent conformation (see above) [9-11]. The 20/D614G variant has not been associated with antibody escape. Yet, it accumulated more mutations, giving rise to new variants of concerns in distinct geographical regions.
20I/501Y.V1 (B.1.1.7) variant – United Kingdom (U.K)
In October 2020, the 20I/501Y.V1 (B.1.1.7) variant emerged in the U.K  before spreading to many countries (https://cov-lineages.org/global_report_B.1.1.7.html). In addition to the D614G mutation, B.1.1.7 carries 8 Spike mutations: del69/70, del144/5, N501Y, A570D, P681H, T716I, S982A, and D1118H . Among those, del69/70, del144/5, N501Y, and P681H are the mutations with the greatest concerns if maintained in future variants (see above). Other mutations occur outside of Spike, in ORF1ab (T1001I, A1708D, I2230T, del3675-77), Orf8 (Q27stop, R25I, Y73C), and Nucleocapsid (D3L and S235F) .
The U.K variant is estimated to be ~30-60% more infectious than ancestral strains . In vitro neutralization assays using VSV-based SARS-CoV-2 pseudoviruses containing all 8 Spike mutations of the U.K. variant revealed that B.1.1.7 is refractory to most NTD mAbs and relatively resistant to a number of RBD mAbs . Moreover, neutralization titers against B.1.1.7 by convalescent and Moderna or Pfizer vaccinee sera are modestly reduced (~2 fold), and remain robust [17, 26, 27].
20H/501Y.V2 (B.1.351) variants – South Africa (S.A)
The 20H/501Y.V2 (B.1.351) variant emerged in South Africa in October 2020  and has since spread to all continents (https://cov-lineages.org/global_report_B.1.351.html). Three S.A strains (B.1.351 v1, B.1.351 v2, and B.1.351 v3) have been reported. They all share the same mutations within the RBD: K417N, E484K, and N501Y. The three S.A variants also share the D614G and N501Y Spike mutations with the U.K variant. Thus, it is thought to also have a high transmission potential. Each B.1.351 variant carries an additional and unique set of Spike mutations: D80A, D215G, A701V for B.1.351 v1, L18F, D80A, D215G, A701V for B.1.351 v2, D80A, R246I, A701V for B.1.351 v3 . Some of these mutations have been associated with decreased in vitro neutralization potency by convalescent plasma (~10 fold), and sera from Moderna and Pfizer vaccinees (~5-6 fold) (see above) [17, 19]. Other SNPs in the B.1.351 lineage include P71L in the Envelope protein, T205I in the Nucleocapsid protein, and K1655N in ORF1a .
20J/501Y.V3 (B.188.8.131.52, alias P.1) variant – Brazil (BRA)
The 20J/501Y.V3 (P.1) variant was detected in December 2020 in the Amazonas state, North Brazil [7, 28] before it started spreading to Japan, North America, Europe, and Australia (https://cov-lineages.org/global_report_P.1.html). The BRA variant shares the D614G and N501Y Spike mutations with the U.K and S.A. variants. Thus, it is thought to also have a high transmission potential. It also shares the two RBD mutations of concern, K417N/T and E484K, with the S.A. variants . Importantly, in vitro assays have associated K417N/T and E484K substitutions with increased ACE2 binding  and SARS-CoV-2 escape from mAbs and convalescent COVID-19 patient sera [15-17] (see above). Other SNPs in the P.1 lineage are found in ORF1ab (S1188L, K1798Q, del3675-7, E5666D), Spike (L18F, T20N, P26S, D138Y, R190S, H655Y, T107I, V1116F), Orf8 (E92K, ins28269-28273), and Nucleocapsid (P80R) .
20C/S:452R (B.1.427/B.1.429) variant – CAL (USA)
The 20C/S:425R (aka 20C/L452R or CAL.20C) variant arising from clade 20C emerged in Southern California (United States) in May 2020. Between September 2020 and January 2021, it outcompeted the most reported contemporary variant in this region [18, 29]. The nomenclature for the CAL variant is not well established yet, as it may span two lineages, B.1.427 and B.1.429 . This Californian strain features three key Spike mutations, S13I, W152C, and L452R, and unlike the U.K, S.A, and BRA variants, it does not feature the N501Y mutation [18, 19]. The L452R a.a. substitution within the RBD is associated with increased pseudovirus infection in vitro, suggesting that this mutation increases the CAL variant transmissibility . Importantly, L452R has been associated with decreased in vitro neutralization potency by convalescent plasma (~4-6.7 fold) and sera from Moderna and Pfizer vaccinees (~2 fold) (see above) [18, 19].
Race against SARS-CoV-2 variants
Owing to its proofreading mechanism, SARS-CoV-2 exhibits a low mutation rate compared to the Influenza virus. However, the emergence of variants is raising concerns that immunity developed in COVID-19 recovered patients and vaccinated individuals could become obsolete as the virus mutates. Our SARS-CoV-2 understanding is still ongoing. To keep up with and confront the evolving virus, chasing emerging variants will not suffice. Scientists also need to scrutinize the current variants' overall impact on immunity within populations. Which vaccination method elicits the most robust immunity to prevent re-infection with another variant? What information can we get from convalescent patients who went through mild and severe disease? To answer these questions, antibodies from infected and vaccinated individuals could be screened for SARS-CoV-2 neutralization, using flow-cytometry detection and cellular fusion assays.
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