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Distinct evolution of infection-enhancing and
neutralizing epitopes in the spike protein of SARS-
CoV-2 variants (from alpha to omicron) : a structural
and molecular epidemiology study
Patrick GUERIN
OpenHealth
Nouara YAHI
Aix-Marseille University
Fodil AZZAZ
Aix-Marseille University
Henri CHAHINIAN
Aix-Marseille University
Jean-Marc SABATIER
Aix-Marseille University
Jacques FANTINI ( jm.fantini@gmail.com )
Aix-Marseille University
Research Article
Keywords: SARS-CoV-2 variants, vaccine, facilitating antibodies, neutralizing antibodies, molecular
epidemiology
Posted Date: December 13th, 2021
DOI: https://doi.org/10.21203/rs.3.rs-1054360/v2
License: This work is licensed under a Creative Commons Attribution 4.0 International License.
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Distinct evolution of infection-enhancing and neutralizing epitopes in the
spike protein of SARS-CoV-2 variants (from alpha to omicron) : a
structural and molecular epidemiology study
Patrick Guérin1, Nouara Yahi2, Fodil Azzaz2, Henri Chahinian2, Jean-Marc Sabatier3 & Jacques Fantini2
1OpenHealth – CP 130 – 56038 Vannes Cedex, France ; 2Aix-Marseille Univ, INSERM UMR_S 1072,
Marseille, France; 3Aix-Marseille Univ, CNRS, INP, Inst Neurophysiopathol, Marseille, France.
Correspondence: Jacques Fantini, Aix-Marseille University, France.
E-mail: jm.fantini@gmail.com
Abstract
Infection-enhancing antibodies may limit the efficiency of Covid-19 vaccines. We analyzed the evolution of
neutralizing and facilitating epitopes in 1,860,489 SARS-CoV-2 genomes stored in the Los Alamos database
from June to November 2021. The structural dynamics of these epitopes was determined by molecular modeling
of the spike protein on a representative panel of SARS-CoV-2 variants. D614, which belongs to an antibody-
dependent-enhancement (ADE) epitope common to SARS-CoV-1 and SARS-CoV-2, has mutated to D614G in
2020, which could explain why ADE has not been detected following mass vaccination. A second epitope
located in the N-terminal domain (NTD), specific of SARS-CoV-2, is highly conserved among most variants. In
contrast, the neutralizing epitope of the NTD showed extensive variations in SARS-CoV-2 variants. The balance
between facilitating and neutralizing antibodies is in favor of neutralization for the Wuhan strain, alpha and beta
variants, but not for gamma, delta, lambda, and mu. The recently emerging omicron variant is atypic as its
mutational profiles affects both neutralization and ADE epitopes. Overall, our data reveal that the evolution of
SARS-CoV-2 has dramatically affected the ADE/neutralization balance. Future vaccines should consider these
findings to design new formulations adapted to SARS-CoV-2 variants and lacking ADE epitopes in the spike
protein.
Key words: SARS-CoV-2 variants; vaccine; facilitating antibodies; neutralizing antibodies; molecular
epidemiology.
2
Introduction
Cytotoxic T-cells and neutralizing antibodies play a key role in the control of viral infections,
especially in the case of respiratory viruses [1, 2]. However, virus-specific antibodies can also
promote pathology, a phenomenon referred to as antibody-dependent enhancement (ADE)
[3]. ADE of virus infection is generally due to virus-specific antibodies that enhance the entry
of virus into host cells, and in some cases, virus replication in monocytes, dendritic cells and
macrophages through antibody binding to Fc receptors [4]. In addition, alternative
mechanisms of ADE involving the complement component C1q have been reported [5]. ADE
has been observed in two typical situations: i) reinfection with a virus variant after primary
infection with a different strain [6] or a cross-reactive virus [7], and ii) as the result of viral
infection in vaccinated people [8]. The ADE phenomenon was initially discovered in
flaviviruses in the late 1960’s [9] and experimentally demonstrated in the early 1970’s [10]. It
concerns a broad range of viruses including dengue [11], Ebola [12], Zika [13], HIV [14],
influenza [15], and various animal and human coronaviruses [16].
As early as in June 2020, at a time when Covid-19 vaccines had just entered clinical
evaluation, Akiko Iwasaki and Yexin Yang from Yale University School of Medicine alerted
that “ADE should be given full consideration in the safety evaluation of emerging candidate
vaccines for SARS- CoV-2” [17]. A similar warning on vaccine safety due to potential risks of
ADE was independently published by Shibo Jiang [18]. In contrast, several authors
considered the risk to be null or minimal in the case of SARS-CoV-2 [19] [20] [21] [22].
However, several pieces of evidence strongly argue in favor of an ADE issue for SARS-CoV-
2. i) ADE has been reported for animal coronaviruses such as feline infectious peritonitis
virus [23]. In the most dramatic cases, kittens previously vaccinated with a recombinant virus
containing the spike protein gene succumbed of early death after a coronavirus challenge [24].
ii) ADE epitopes were characterized in the spike protein of this feline coronavirus [25]. iii)
ADE epitopes have also been found in human coronaviruses related to SARS-CoV-2, i.e.
SARS-CoV-1 [26] and MERS-CoV [27] [28]. The case of SARS-CoV-1 is particularly
interesting since its spike protein displays a linear ADE epitope, 597-LYQDVNC-603
(recognized by the monoclonal antibody 43-3-14) [26] that is fully conserved in the SARS-
CoV-2 spike protein sequence used for mRNA Covid-19 vaccines. iv) ADE antibodies
directed against the N-terminal domain (NTD) of the spike protein have been detected and
characterized in convalescent Covid-19 patients [29] [30]. v) ADE antibodies are suspected to
3
be particularly efficient in vaccinated Covid-19 patients infected with the delta variant [31]
[32]. In this context, we recently reported that facilitating anti-spike antibodies targeting the
NTD have a higher affinity for the delta variant than for the initial Wuhan strain. We also
reported that the main neutralizing epitope of the NTD is almost lost in δ variants [31]. This
finding is of critical importance since ADE infection of coronaviruses is known to be induced
by the presence of sub-neutralizing levels of anti-spike antibodies [33]. Overall, our data
suggested that the balance between neutralizing and facilitating antibodies may greatly differ
according to the virus strain.
In the present study, we analyzed a panel of representative SARS-CoV-2 variants including
alpha, beta, gamma, delta, lambda, mu as well as the most recent South-Africa strains C.1.2
(with no attributed Greek letter at the time of submission) and omicron. We used multiple
amino acid sequence alignment methods combined with structural and molecular modeling
approaches to determine the variability of ADE and neutralizing epitopes and the impact of
this variability on antibody-spike protein interactions. Our main objectives were i) to decipher
the evolution of neutralizing and facilitating epitopes since the beginning of the Covid-19
pandemic, and ii) to predict for each SARS-CoV-2 variant which way the balance between
neutralization and facilitation is tipping.
Methods
Molecular modeling studies were performed with Hyperchem (http://www.hyper.com) and
Deep View/Swiss-Pdb viewer (https://spdbv.vital-it.ch) programs, as described in previous
studies [34] [35] [36] [37]. The energy of interaction (∆G) of each antibody-spike protein
complex was calculated with Molegro Molecular Viewer (http://molexus.io/molegro-
molecular-viewer). The cluster of gangliosides GM1 in a typical lipid raft organization was
generated as described previously from CHARMM-GUI Glycolipid Modeler [38] and
submitted to several minimization steps with the Polak-Ribière algorithm [39].
Results
Description of two distinct ADE epitopes in SARS-CoV-2 spike protein
The mutational patterns and geographic origins of the SARS-CoV-2 variants analyzed in this
study are summarized in Table 1. All variants have a dual nomenclature (lineage and Greek
letter) except for C.1.2 which, at the date of submission of this article, had no Greek letter
4
attributed. Our analysis is focused on the NTD and on the rod-like domains of the spike
protein. Other ADE and neutralization epitopes do exist in the RBD, but during the complex
process of viral adhesion to target cells, this domain is involved at later step [36] [37].
Clearly, the NTD is key to understand how SARS-CoV-2 initially interacts with the plasma
membrane of host cells.
The first ADE epitope studied is the 611-617 motif with the original amino acid sequence
LYQDVNC recognized by the 43-3-14 antibody [26]. This ADE epitope is common to human
coronaviruses SARS-CoV-1 and SARS CoV-2. Interestingly, this epitope is centered on
position 614 which is an aspartic acid residue in the original Wuhan strain but has rapidly
evolved to the ultra-dominant D614G during the first months of 2020 [40]. The localization of
this epitope on the spike protein (Wuhan strain) is shown in Figure 1A (epitope colored in
yellow, except for D614 highlighted in red). It is well exposed on the protein surface so that it
can be recognized by facilitating antibodies generated during previous coronavirus infections
in humans, especially in geographic areas previously exposed to SARS-CoV-1. The second
ADE epitope targeted by facilitating antibodies is divided in two parts (both colored in blue in
Figure 1A): one in the NTD (27-32, 64-69 and 211-218 segments) and the other one in the
rod-like domain (600-607, 674-677 and 689-691 segments) of the spike protein. Antibodies
directed against this epitope have been detected in sera from convalescent Covid-19
patients[30]. Although the two parts of this ADE epitope seem to be spatially distant, both are
close to a flexible 20-amino acid residue loop (621-640) that is unresolved in PDB files but
was added by molecular modeling in the structures shown in Figure 1. It is interesting to note
that this loop (highlighted in green) is ideally located to connect the NTD and the RBD, but
also to provide a conformational link between both ADE epitopes (Figure 1B).
Once the NTD is bound to the cell membrane of the host cell, a conformational change
unmasks the RBD which becomes available for a functional interaction with a viral receptor,
chiefly ACE2 [37]. This spatial reorganization leads to the open, fusion-compatible
conformation of the trimeric spike protein [41]. In the Wuhan strain, the closed conformation
of the trimer [42] is stabilized by a hydrogen bond between D614 of one subunit and T859 of
its neighbor (respectively chains B and C in Figure 2A). The global spreading of the
pandemic during the first months of 2020 has been associated with the breakthrough of the
first SARS-CoV-2 variant with a unique mutation in spike protein, D614G. As shown in
Figure 2B, this mutation induces the loss of the hydrogen bond that stabilized the closed
conformation. Thus, we analyzed the status of this hydrogen bond in the complex between the
5
facilitating 1052 antibody and the spike protein trimer. As shown in Figure 2C, the antibody
has a long range conformational effect on both D614 and T859, which renders impossible the
formation of this hydrogen bond. It is likely that the 621-640 loop, which conformationally
connects the 1052 and the 611-617 epitopes, mediates this distal effect. In this respect, it is
interesting to note that this facilitation can be induced by two distinct mechanisms: i) the
replacement of aspartic acid by a glycine at position 614 (D614G mutation), or ii) binding of
the ADE antibody 1052 to the original Wuhan spike protein displaying an aspartic acid
(D614) at this position.
Analysis of amino acid sequence variations in ADE and neutralizing epitopes during the
global spreading of the Covid-19 pandemic
Then, we analyzed the evolution of the amino acid sequence of ADE epitopes among SARS-
CoV-2 variants (Figure 3). The 611-617 epitope (lower left panel), which is common to
SARS-CoV-1 and SARS-CoV-2, has a unique signature in all variants, i.e. the D614G
mutation. As position 614 is central to the epitope, this epitope is probably no longer
recognized by ADE antibodies generated by previous coronavirus infections in humans. The
second ADE epitope is formed by several distinct areas in the NTD and in the rod-like regions
of the spike protein (Figure 3, upper panel). In the NTD, the epitope is divided in three linear
segments that represent ca. 80% of the total energy of interaction of the 1052 antibody-NTD
complex (as calculated from PDB: 7LAB): 27-32, 64-69 and 211-219 (accounting
respectively for 12, 19 and 51 % of the energy of interaction). The complex is further
stabilized by auxiliary contacts with the rod-like region of the spike protein (chiefly 600-607,
674-677 and 689-691). Overall, the whole epitope appeared to be extremely well conserved,
except at two amino acid residues positions: H69 and D215. Indeed, a deletion (∆H69) is
found in the alpha variant, and D215 is mutated in D215G in the beta and the more recent
C1.2 variants (Figure 3, upper panel). Surprisingly, the recently emerging omicron variant
does not seem to follow this general rule as its ADE epitope is heavily affected by a
combination of single point mutations (A67V, L212I), two deletions (∆H69, ∆N211), and a 3-
aminoi acid insertion (between R214 and D-215).
In marked contrast with the conservation of the 1052 ADE epitope in most variants, the main
neutralizing epitope of the NTD showed extensive amino acid sequence variations (Figure 3,
lower right panel). The changes included deletions, insertions and single point mutations that
6
are distributed among two key regions, 144-158 (N3 loop) and 242-249 (N5 loop) that
constitute the three-dimensional site recognized by the neutralizing 4A8 antibody [43]. The
localization of the neutralization epitope of the NTD at the virus/host cell interface is
consistent with this high variability as it is submitted to a strong pressure of selection for
SARS-CoV-2 variants. Conversely, the ADE epitope, which is on the lateral side of the NTD,
is not facing the plasma membrane of the host cell and for this reason is not subjected to such
a high selective pressure.
The frequency of amino acid sequence variations of the ADE and neutralizing epitopes was
analyzed by specific queries of the Los Alamos database over the last six-month period
(2021-06-01 to 2021-11-27) (Table 2). All the epitopes listed in Figure 3 were analyzed in
1,860,489 genomes. The ADE epitope of the NTD is highly conserved (>98% for all
segments) except for the 64-69 motif at position H69 (variation of 5.46% with 1 mutation),
mostly reflecting the alpha variant [44]. The ADE epitope 611-617 displays 1 mutation in
98.70% of cases, consistent with the worldwide dominance of the D614G mutant [45]. The
situation of the neutralization epitope of the NTD is by far more complex, in particular for the
144-158 segment which shows high amino acid sequence variability (frequency of the Wuhan
sequence <0.05%). Remarkably, 92.04% of the sequences have 2 mutations and some viruses
with 3, 4 and even 5 mutations are currently detected. The second linear segment (242-249) is
more conserved (99.22% of sequences are identical to the Wuhan strain), but viruses with up
to 4 mutations have been characterized. Interestingly, the amino acid variations of the
neutralization epitope are concentrated on positions that are associated with the variants
analyzed in the present study: Y144, E156, F157 and R158, in the N3 loop, R246 in the N5
loop (Figure 3).
Estimating the risks of the facilitation phenomenon depending on the variant concerned:
a molecular modeling approach
Finally, we used molecular modeling approaches to determine how mutations in ADE
epitopes could impair the binding of facilitating antibodies. In our analysis of ADE epitopes
in SARS-CoV-2 variants (Figure 3), we detected two essential mutations that can potentially
suppress the facilitation phenomenon: ∆H69 and D215G. Thus, we studied the localization of
H69 and D215 in the molecular complex between ADE antibody 1052 and the spike protein
(Figure 4A, left panel). Both H69 and D615 appeared critical for the 1052 antibody binding
7
site on the NTD of the spike protein. These positions are fully conserved in the gamma, delta,
mu and lambda SARS-CoV-2 variants, which are still recognized by the ADE antibody 1052.
An illustration of the efficiency of this antibody to facilitate the infection by the variant is
shown in Figure 4A (right panel). The plasma membrane of the host cell is represented by a
cluster of gangliosides GM1 to figure the lipid raft that acts as a landing platform for the NTD
[36]. In line with previous data from our group [31], once the 1052 antibody is bound to the
NTD of the delta spike protein, a global conformation change involving both the NTD and the
antibody allows the formation of a highly energetic trimolecular complex (antibody-NTD-
lipid raft) with an obvious geometric complementarity of all partners. Then, we compared the
structure of the delta variant NTD with the mu, lambda, and C.1.2 variants (Figure 4B).
Except for C.1.2 which displays a D215G mutation, and the highly divergent omicron (Figure
3) all other variants have both H69 and D215 accessible on the NTD surface.
In line with these data, the energy of interaction of the C.1.2 variant spike protein with the
1052 antibody was less than half the value calculated for the Wuhan strain (-229 kJ.mol-1),
whereas it reached -246 kJ.mol-1 for the delta variant [31], -236 kJ.mol-1 for mu and -228
kJ.mol-1 for lambda variants. Thus, the conservation of H69 and D215 (in gamma, delta, mu
and lambda variants) is critical for virus infectivity as it favors the ADE phenomenon by
allowing an optimal binding of the 1052 antibody to the spike protein. In contrast, the ADE
epitope is affected as soon as at least one of these positions is mutated (as it the case for alpha,
beta, C.1.2 and omicron variants).
Discussion
Vaccine strategies against viral diseases are confronted to the risk of antibody facilitation
(ADE), especially when the strain used for the immunization protocol is distinct from
circulating viruses [46]. In the past, ADE has been evidenced for a broad range of human
RNA viruses including HIV, influenza, filoviruses, and coronaviruses [4]. Although ADE
antibodies have been consistently characterized in the serum from Covid-19 convalescent
patients [29] [30], the risk of ADE linked to vaccination with spike protein-based vectors
(either mRNA or adenovirus) has not been considered as critical. As a matter of fact, it has
been generally assumed that ADE antibodies exhibited SARS-CoV-2 infection enhancement
in vitro but not in vivo [30]. However, a potential caveat of these studies is that SARS-CoV-2
variants have not been specifically assessed. Moreover, surprising higher incidence rates in
8
vaccinated vs. unvaccinated individuals in the 0-14 days after the first dose were recently
reported in long-term care facility residents and health-care workers, which resulted in
significant negative vaccine efficiency estimates of -37% and -113%, respectively [47]. To
which extent this apparent enhancement of SARS-CoV-2 infection is due (or not due) to an
imbalance between vaccine-induced (and/or pre-existing) neutralizing and facilitating
antibodies warrants further investigation. Moreover, a recent report revealed that there is no
clear relationship between the percentage of fully vaccinated individuals and new Covid-19
cases in 68 countries including Israel, a pioneer in mass vaccination against SARS-CoV-2
[48]. Taken together, these observations suggested that ADE, or more specifically the
ADE/neutralization balance, could pose a problem for Covid-19 vaccine strategies, especially
during the outbreak of SARS-CoV-2 variants. Finally, it is worth noting that ADE has been
suspected to increase the severity of Covid-19 symptoms in selected geographic areas [49].
The objective of the present study was thus to assess the potential risk of ADE in vaccinated
individuals challenged with SARS-CoV-2 variants. To this end, we studied the amino acid
sequence variability of ADE and neutralizing epitopes in the NTD and rod-like regions of the
spike protein. Then we used our target-based molecular modeling strategy to interpret these
data at the level of the three-dimensional structure of the spike proteins.
We focused our attention on two distinct ADE epitopes: one linear epitope common to SARS-
CoV-1 and SARS-CoV-2 (611-617 in the rod-like region of the spike protein, recognized by
the 43-3-14 antibody) [26] and a complex three-dimensional NTD epitope (recognized by the
1052 antibody) [30].
Both epitopes are present on the spike protein generated by mRNA vaccines as the original
formulas are based on the Wuhan strain [50]. Therefore, it is of high importance to determine
whether these epitopes are still expressed and accessible on SARS-CoV-2 variants. The 611-
617 epitope has probably escaped facilitating antibodies because the D614G variant has
rapidly replaced the original strain [45]. Although in the initial study of the D614G mutation
the authors mentioned the presence of D614 in a conserved ADE epitope, they did not
comment further this important issue [40]. Our modeling approaches revealed a common
molecular mechanism leading to enhanced infectivity for the D614G variant and for ADE
antibodies with the Wuhan strain (Figure 2). In both cases, the loss of a stabilizing hydrogen
bond between amino acid residues 614 and 859 of two vicinal spike protein chains relaxes the
trimer and facilitates the conformational change that unmasks the RBD. A major outcome of
our study is the identification of the 621-640 loop, which is missing in PDB files, as the
9
conformational transmitter that allows the 1052 antibody to induce distant effects on amino
acid residue 614. In this respect, the enhancement of infection provided by this ADE antibody
involves two distinct Fc-independent mechanisms: a long range conformational effect and a
stabilization of the NTD bound to a lipid raft [31].
From an epidemiologic point of view, we can propose a scenario according to which the first
cases of SARS-CoV-2 infections in China could have been facilitated by ADE antibodies
directed against the 611-617 epitope in individuals previously infected by SARS-CoV-1 or
similar coronaviruses. This notion is supported by the recent demonstration that non-
neutralizing antibodies directed against SARS-CoV-1 and recognizing the SARS-CoV-2 spike
protein may persist for at least 15 years [51]. Then the global extension of SARS-CoV-2 has
probably levied this constraint by selecting the D614G variant in SARS-CoV-1 free
populations. This scenario is consistent with the rapid raise and long-term maintenance of
D614G worldwide. It is also consistent with the discrepancy between the severity of Covid-19
cases observed in the Hubei province of China and those occurring elsewhere in the world at
the beginning of the pandemic [49]. Moreover, it explains why ADE has not been detected
during the first months following mass vaccination, since ADE antibodies directed against the
611-617 epitope are no longer active on D614G variants. The observation that anti-SARS-
CoV-1 antibodies isolated from a convalescent patient could enhance virus infection mediated
by civet virus spike proteins [52] also supports this notion. Retrospectively, it is important to
note the statement of Helen Pearson in a Nature editorial commenting these data in 2005: “a
jab against one strain might even aggravate an infection with SARS virus from civets or
another species” [53].
After the first wave of D614G, several other SARS-CoV-2 variants have emerged until the
rise of the delta variant which is now by far the most common strain worldwide. Indeed, key
variations in the ADE epitope at positions 69 and 215 have probably protected patients
infected with alpha or beta strains from the ADE risk (Figure 3). Nevertheless, these variants
also showed significant variability of the neutralizing epitope, which could have decreased
vaccine efficiency [54]. The situation is more dramatic for the delta variant. Indeed, several
studies converged to alert on the potential risk of ADE when a delta SARS-CoV-2 variant
infects a vaccinated individual [31] [32]. Our study confirms this possibility and further
extends it to other circulating variants, including lambda and mu, for which the
neutralization/facilitation balance is unfavorable. A useful approach to anticipate such ADE
risk in face of any variant is to analyze both the ADE and neutralizing epitopes of the NTD, as
10
developed in Figure 3. At first glance, one can determine the balance between neutralization
and facilitation and assess the risks of virus escape, ADE and/or both. Our molecular
modeling approaches confirmed that hot mutational spots in ADE and neutralizing epitopes of
the NTD give reliable information on antibody recognition of the spike protein, allowing us to
determine which way the balance between neutralization and facilitation is tipping.
We recently hypothesized that the delta variant is dominating because its electrostatic surface
potential of the NTD region that faces the host cell membrane has evolved to a large
electropositive flat area that is complementary to the electronegative surface of lipid raft
gangliosides [37]. The electrostatic potential surface value, which reflects the kinetics of virus
infection, is a key parameter of a mathematic formula giving the transmissibility score (T-
index) of any SARS-CoV-2 strain. This original and straightforward approach, which has
recently received experimental confirmation for both enhanced transmissibility and faster
infection kinetics [55] [56], allowed us to correctly predict the rapid emergence of the delta
variant (T-index >10) over alpha (T-index <4) even though both variants display a similar
affinity for ACE-2 [37]. At present, the T-index of the delta variant is still higher than all
other circulating variants (including omicron, T-index <5). Proposing a third and potentially a
fourth jab to improve vaccine efficiency to face the threat of the delta variant may not be a
good idea as it may further increase the amount of ADE antibodies without significant gain in
neutralizing activity. Instead, we believe that it is critical to design new vaccine formulations
able to induce neutralizing antibodies against this strain and, most importantly, lacking ADE
epitopes in the NTD. Molecular epidemiology surveillance of SARS-CoV-2 coupled with
structural analysis of variant spike proteins will certainly help to reach this goal.
Transparency declaration
Funding. No external funding was received.
Acknowledgements. We thank Dr. Coralie Di Scala (ORCID 0000-0003-0655-7056)
and Dr. Helene Banoun (ORCID 0000-0001-8391-7989) for helpful discussions and
critical advice.
Contribution. All authors contributed equally to this study. J.F. and F.A., molecular
modeling; N.Y., sequence data analysis; H.C. molecular analysis of protein-protein
complexes; PG and JMS, ADE analysis of animal and human virus infections.
Conflict of interest. The authors declare that they have no conflict of interest.
11
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Figure 1. Localization of ADE epitopes on the spike protein.
A. Three distinct views of the SARS-CoV-2 spike protein (Wuhan strain). The ADE epitopes
recognized by the 1052 antibody are colored in blue. The common coronavirus ADE epitope
is colored in yellow, with amino acid residue D614 in yellow. The 621-640 loop that is
missing in PDB: 7LAB is in green. B. ADE antibody 1052 (in cyan) bound to the monomeric
spike (left panel) or to the trimeric spike protein (right panel). The N-terminal domain (NTD)
and receptor-binding domain (RBD) are indicated in all models.
15
Figure 2. How the D614G mutation and the ADE antibody 1052 enhance SARS-CoV-2
infectivity.
A. Hydrogen bond between D614 (chain B) and T859 (chain C) stabilizing the trimeric spike
protein (PDB: 6VSB). B. The D614G mutation renders impossible the formation of the
hydrogen bond and facilitate the conformational change inducing the demasking of the RBD
(PDB: 7BNM). C. Binding ADE of ADE antibody 1052 breaks the hydrogen bond between
D614 and T859 (PDB: 7LAB). The arrow in panels B and C illustrates the lack of contact
between vicinal spike protein monomers in the context of the trimeric association.
16
Figure 3. Amino acid sequence alignments of ADE and neutralizing epitopes in SARS-
CoV-2 variants.
Amino acid residue variations are highlighted in yellow. -, identity; ∆, deletion. Note that the
144-158 neutralizing epitope of the µ variant displays a threonine residue (T, in red) inserted
after Y144, then two mutations after this insertion (colored in blue). The insertion induces a
shift of the amino acid sequence (highlighted in grey).
17
Figure 4. Critical amino acid residues control the binding of the ADE antibody 1052 to
variant spike proteins.
A. Binding of the 1052 antibody (ADE mAb) to the Wuhan spike protein (PDB: 7LAB) with
the NTD and RBD indicated. In the left panel, the light and heavy chains of the antibody are
represented in standard secondary structures (red, -helix, blue, -strand). H69 (in blue) and
D215 (in yellow) are highlighted. In the right panel, a surface representation illustrates the
geometric complementarity of the spike protein-antibody complex bound to a cluster of
gangliosides GM1 figuring a lipid raft on the plasma membrane of a host cell. Note that the
1052 antibody binds simultaneously to the NTD of the spike protein and to the edge of the
lipid raft. B. Molecular modeling of the NTD of several SARS-CoV-2 variants showing
different levels of surface exposure of H69 (in blue) and D215 (in yellow) amino acid
residues.
18
Table 1. Mutations in SARS-CoV-2 variants.
Virus strain NTD Rod
Alpha
B.1.1.7 (UK)
∆H69 ∆V70
∆Y144
D614G P681H T716I
S982A D1118H
Beta
B.1.351 (S_Afr)
L18F D80A D215G
∆L242 ∆A243 ∆L244
D614G A701V
n/a
C.1.2 (S_Afr)
P9L C136F ∆Y144 R190S
D215G ∆A243 ∆L244
D614G H655Y N679K
T716I T859N
Gamma
P.1 (Brazil)
L18F T20N P26S
D138Y R190S
D614G H655Y T1027I
V1176F
Delta
B.1.617.2 (India)
T19R T95I G142D
∆E156 ∆F157 R158G
D614G P681R D950N
Mu µ
B.621 (Columbia)
T95I +143T Y144S Y145N D614G P681H D950N
Lambda
C.37 (Peru)
G75V T76I R246N ∆S247
∆Y248 ∆L249 ∆250
∆P251 ∆G252 ∆D253
D614G T859N
Omicron
B.1.1.529 (S_Afr)
A67V H69 ∆V70 T95I G142D
∆V143 ∆Y144 ∆Y145 L212I
+214EPE
D614G H655Y N679K
P681H N764K D796Y
N856K N954K L981F
Mutations patterns in the NTD and rod-like regions of the SARS-CoV-2 spike
protein were obtained from the GISAID database
(https://www.gisaid.org/hcov19-variants). Deletions (∆) and insertions (+) are
underscored.
19
Table 2. Frequency of ADE and neutralizing epitope sequences.
The frequency of mutations of each epitope sequence is calculated as the percentage of
identity with the reference amino acid sequence of the SARS-CoV-2 spike protein (Wuhan
strain). The most variable amino acid residues of each epitope are underlined. 1,860,489
sequences were analyzed from 2021-06-01 to 2021-11-27.
The raw data were obtained from the Los Alamos website
(https://cov.lanl.gov/content/sequence/ANALYZEALIGN/analyze_align.html).
