Introduction
Seven coronaviruses are known to cause infection/disease in humans. Of these, human
coronavirus (HCoV)-229E and HCoV-NL63 are alphacoronaviruses (α-CoVs), whereas OC43,
HKU1, Severe Acute Respiratory Syndrome Coronavirus (SARS-CoV), Middle East Respiratory
Syndrome Coronavirus (MERS-CoV), and Severe Acute Respiratory Syndrome Coronavirus
2 (SARS-CoV-2) are betacoronaviruses (β-CoVs) (V’kovski et al., 2021). The recently
emerged, highly mutated, and transmissible Omicron (B.1.1.529) and its variants have
now become the dominant circulating SARS-CoV-2 variants worldwide with recombinant
SARS-CoV-2 variants XD, XE, and XF also causing increased infection in humans (Elbe
and Buckland-Merrett, 2017). Most of the concerning mutations are being reported in
the spike protein of SARS-CoV-2 which is primarily responsible for the entry of the
virus into the host cells, which it does through binding of its receptor-binding domain
(RBD) to angiotensin-converting enzyme 2 (ACE2) receptor present on the host cells
(V’kovski et al., 2021). The spike protein contains two subunits (S1 and S2) and two
cleavage sites that are present at S1/S2 and S2’ sites. The S1/S2 site is cleaved
by the endogenous enzyme furin whereas S2’ site is cleaved by the membrane enzyme
TMPRSS2 (V’kovski et al., 2021). The S1 subunit sheds after RBD binding to ACE2 and
the S2’ site is cleaved which leads to a conformational rearrangement of the S2 subunit
for the insertion of fusion peptide into the host cell membrane. The spike protein
is therefore a primary target of neutralizing antibodies (nAbs) towards COVID-19 therapy
(Corti et al., 2021; Kumar et al., 2021; Kyriakidis et al., 2021). SARS-CoV-2 nAbs
provide an attractive alternate strategy for immediate therapy or prophylaxis to COVID-19,
especially in immunocompromised patients, unvaccinated, vaccine-hesitant patients,
and also in situations where vaccines are less effective against a particular variant
(Siemieniuk et al., 2021; Boeckel et al., 2022; Gentile and Moriello, 2022; Gupta
et al., 2022). Therapeutic nAbs primarily function by blocking the entry of the virus
into the host cells, and perhaps also facilitate the elimination of infected host
cells by Fc-mediated effector functions and reducing viral load in vivo (Kumar et al.,
2021). When administered as prophylaxis or during the early stage of a natural infection,
nAbs have been reported to reduce the incidence of hospitalizations and mortality
(Kumar et al., 2021). NAb therapy is not specified for severe COVID-19 cases requiring
hospitalization (Corti et al., 2021). Except a few minor side-effects (e.g. diarrhea)
reported in ~1% of patients after infusion of nAb based therapy in COVID-19 individuals,
no major side-effects have been observed (Corti et al., 2021; Gupta et al., 2022).
Overall, these findings suggest that broadly neutralizing antibodies (bnAbs) based
therapies are generally safe and effective for COVID-19 treatment.
Recent Progress in Broadly Neutralizing Antibodies Against SARS-CoV-2 Variants and
Other Human Coronaviruses
Presently, 8524 SARS-CoV-2 specific monoclonal antibodies (mAbs) have been reported
(Raybould et al., 2021; Wang et al., 2022) (
Figure 1A
). A large proportion of mAbs target RBD, N-terminal domain (NTD), and S2 domains
of the SARS-CoV-2 spike protein (Wang et al., 2022). A total of 2639/5406 (RBD-specific),
475/511 (NTD-specific), and 845/976 (S2-specific) mAbs have been tested in the SARS-CoV-2
neutralization assay. Of these, 56.76% (1498/2639), 21.68% (103/475), 5.44% (46/845)
are neutralizing mAbs targeting RBD, NTD and S2 respectively (
Figure 1B
) (Raybould et al., 2021). The global consortium study has underscored four major
classes (I, II, III, and IV) of potent RBD-specific nAbs (Barnes et al., 2020) as
RBD1-7 types (Hastie et al., 2021). RBD class I-II or RBD1-4 specific nAbs overlap
with ACE2 binding regions and thus as expected are not very effective against major
variants of concern (VOCs) and variants of interest (VOIs) where mutations have occurred
in the ACE2 binding contacts (Hastie et al., 2021) in the receptor binding motif (RBM)
region of the RBD. In contrast, nAbs belonging to class III-IV or RBD5-7 targeting
relatively conserved least mutated outer/inner RBD regions thus far effectively neutralizing
most VOCs and VOIs (Hastie et al., 2021; Greaney et al., 2022) (
Figure 1C
). Before the emergence of Omicron VOC, RBM directed mAbs with gene usage VH1-58 were
among the most potent mAbs (e.g. S2E12, COV2-2196) which were highly effective against
all SARS-CoV-2 variants (Tzou et al., 2020). Eight nAbs targeting the RBD region were
approved by the US-FDA under emergency use authorization for COVID-19 treatment (EUA)
(Kumar et al., 2021; Corti et al., 2021). Among these, only Sotrovimab (S309) neutralizes
Omicron (BA.1) variant but poorly neutralizes the BA.2 variant (Cao et al., 2022).
Other therapeutic nAbs have been ineffective towards Omicron, mainly due to the presence
of extensive mutations in their epitopes, remodeling of the antigenic surface of the
spike trimer and dominance of RBD closed state of Omicron spike protein (
Figure 1C
) (Cao et al., 2022; Gobeil et al., 2022; Guo et al., 2022; Zhang et al., 2022). Therefore,
for the treatment of Omicron infected patients, US-FDA has provided emergency use
authorization (EUA) to a class III or RBD-5 targeting bnAb Bebtelovimab (or LyCoV1404)
in Feb 2022 that maintains its potency and neutralizes all SARS-CoV-2 VOCs (Westendorf
et al., 2022). Currently, majority of the RBD class III mAbs (Bebtelovimab, 002-S21F2
and Sotrovimab) binding the outer RBD region have shown highest effectiveness against
Omicron variants (Kumar et al., 2022; Zhou T. et al., 2022). Interestingly, though
Omicron mutations are present in the epitope regions of Bebtelovimab and 002-S21F2
SARS-CoV-2 bnAbs, no loss in potency have been reported (Kumar et al., 2022; Westendorf
et al., 2022). This has been explained in their structural studies showing that Omicron
mutations in their targeted regions are either favoring the binding by making favorable
interaction or not a part of epitope residues targeted by Bebtelovimab and 002-S21F2
bnAbs (Kumar et al., 2022; Zhou T. et al., 2022). This suggests that understanding
the structural basis of bnAbs mediated neutralization mechanism of immune evading
viruses can provide blueprints to guide structural-based therapeutics and vaccine
design by employing affinity maturation through directed evolution, CDRH3 swapping
and Reverse Vaccinology 2.0 approaches, respectively (Burton, 2017; Kadam et al.,
2017; Traboulsi et al., 2021; Zhao et al., 2021; Zupancic et al., 2021). Moreover,
RBD mAbs from hybrid immune individuals dominantly target class III and IV epitopes
with potent broad sarbecoviruses neutralizing potential (He et al., 2022; Muecksch
et al., 2022). These SARS-CoV-2 bnAbs exhibit unique immunogenetic features like presence
of ‘YYDRxG’ motif in their CDRH3 region, enrichment of IGHV3-30, IGHV1-46, IGHV1-69
germline V-genes, IGHD2-15 and IGHD3-22 germline D-genes, moderately higher somatic
hypermutation (SHM) >5% (He et al., 2022; Muecksch et al., 2022). On the other hand,
nAbs targeting NTD have not been ideal for clinical use development due to recurring
mutations in the epitopes they target, resulting in lower effectiveness against the
majority of the VOCs and VOIs (Hastie et al., 2021). By contrast, few RBD conserved
region-directed nAbs (e.g. S2X324, S2K146, S2X259 and S2H97) have shown broad in vitro
cross-neutralization potential and in vivo protective efficacy at low doses against
sarbecoviruses (Starr et al., 2021; Tortorici et al., 2021; Park et al., 2022; Park
et al., 2022). Interestingly, a rare class of nAbs (e.g. S2P6, CV3-25, and CC40.8)
targeting the pan-β-CoVs conserved hydrophobic regions (stem-helix region) involved
in the fusion machinery neutralizes not only all SARS-CoV-2 variants but also pan-β-CoVs
(Pinto et al., 2021; Zhou et al., 2022b; Hurlburt et al., 2022). Though these stem-helix
specific nAbs neutralize pan-β-CoVs at a higher concentration (IC50 >1μg/ml), these
bnAbs have shown in vivo protective efficacy at low-dose against all 3 major human
infecting coronaviruses that include SARS-CoV, MERS-CoV and SARS-CoV-2 (Zhou et al.,
2022a) suggesting antibody effector functions via their Fc-region may be playing a
role in virus clearance. Lastly, clinical evaluation is awaited for recently identified
a new class of conserved fusion peptide-directed antibodies (COV44-62 and COV44-79)
with the broadest neutralization potential against both α-CoVs and β-CoVs (Dacon et al.,
2022).
Figure 1
Description of SARS-CoV-2 monoclonal antibodies, their contact epitopes and information
on spike conserved regions. (A) Distribution of total SARS-CoV-2 mAbs identified so
far as documented in CoV-AbDab database and by Wang et al. (Raybould et al., 2021;
Wang et al., 2022). (B) All mAbs are classified based on the region specificity and
distribution of neutralizing (green) vs non-neutralizing (gray) mAbs. Frequency of
mAbs targeting the stem-helix region (0.08%) and fusion peptide region (0.06%) are
not visible in the plot due to their very low frequency. The mAbs for which neutralization
data was unavailable were removed. (C) Epitope contacts of RBD specific SARS-CoV-2
mAbs are shown. Targeted NAb epitopes of RBD specific class I to class IV and therapeutic
NAbs (marked in asterisks) along with ACE2 contacts, RBD mutations and Omicron mutations
are highlighted. Information of contact residues for each mAb shown here was obtained
from previously reported studies (Greaney et al., 2022; Kumar et al., 2022; Zhou T.
et al., 2022). (D) Mutations identified among the key SARS-CoV-2 variants from Alpha
to Omicron documented in CoVariants database (Elbe and Buckland-Merrett, 2017) is
represented as a Shannon entropy plot. Shannon entropy, is a measure of diversity
and hence conservation at each amino acid residue, was calculated using R package
Bio3d. The x-axis shows the amino acid residues while the y-axis shows the Shannon
entropy value at each residue. The value of Shannon entropy ranges from 0 (no amino
acid variation at that position) to 4.32 (all amino acids are equally represented
at that position). NTD, RBD, S2 subunit, fusion peptide region and stem-helix region
are highlighted in green, red, cyan, pink, and blue respectively. (E) Mutations in
fusion peptide region and stem-helix region of β-CoVs (SARS-CoV-2, SARS-CoV, HCoV-HKU1,
HCoV-OC43 and MERS-CoV) and α-CoVs (HCoV-229E and HCoV-NL63) are shown.
SARS-CoV-2 Broadly Neutralizing Antibodies Target Conserved Regions on the Spike Protein
Viral variants can arise due to increasing and persistent immune pressure from both
B and T cell compartments on a particular region of the virus (Harvey et al., 2021).
Several mutations arise during the normal course of viral replication and many of
these mutations may not affect the virus, or some may even be harmful to the virus
and thus go unnoticed (Upadhyay et al., 2021). However, during this process of viral
propagation/replication, certain mutations can result in increased viral fitness.
For example, the generation of viral mutants acquires the ability to escape host immune
responses (Harvey et al., 2021; Upadhyay et al., 2021). Mutations reported in the
SARS-CoV-2 VOCs and VOIs revealed that these mutations primarily occur in the NTD
region, ACE2 contact regions of the RBD, and furin cleavage site region of the S2
domain with few minor exceptions to other residues of S1 and S2 domains (
Figure 1D
) (Tzou et al., 2020; Harvey et al., 2021; Upadhyay et al., 2021). One plausible reason
behind this pattern might be the high immune selection pressure elicited by nAbs targeting
these key regions during natural infection in the host or vaccinated individuals (Garcia-Beltran
et al., 2021). In contrast, no mutation is reported in the broadly conserved fusion
peptide and stem-helix regions of the S2 domain of SARS-CoV-2 VOCs/VOIs (Pinto et al.,
2021; Dacon et al., 2022; Hurlburt et al., 2022; Zhou et al., 2022b) (
Figure 1E
) which are also recognized by the host’s immune system during natural infection (Wang
et al., 2021). The sequence of the fusion peptide region is identical in all SARS-CoV-2
variants and highly conserved across human coronaviruses (Dacon et al., 2022) (
Figure 1E
). NAbs targeting these two regions block fusion machinery by preventing S2 domain
refolding from the pre- to the post-fusion state. Limited exposure of these regions
to the host immune response or critical requirement of their conserved sequences may
be responsible for low antibody-mediated immune pressure, hence limiting the occurrence
of mutations in these epitopes (Harvey et al., 2021; Pinto et al., 2021; Upadhyay
et al., 2021). Further, both stem-helix and fusion peptide directed nAbs are rarely
elicited in convalescent individuals, but their frequencies are higher, with enhanced
affinity and breadth in vaccinated individuals who were previously infected (Pinto
et al., 2021; Zhou et al., 2022a). For example, Zhou et al. screened 247 SARS-CoV-2
specific mAbs and found that 16% (40/247) mAbs exhibited cross-reactive binding to
stem-helix peptides from at least two different β-CoVs (Zhou et al., 2022a). Dacon
et. al., screening identified only 2% of 211 mAbs showed broad reactivity to at least
two other non-SARS β-CoVs, suggesting that frequency of fusion peptide targeted mAbs
are lower than stem-helix mAbs (Dacon et al., 2022). Overall, these studies suggest
that nAb cocktails that include relatively conserved least mutated RBD regions, fusion
peptides, and stem-helix regions targeting nAbs could be effective against evolving
SARS-CoV-2 variants and human coronavirus outbreaks that may arise in the future.
Conclusions and Future perspectives
Lessons learned with the emergence of Omicron and its sublineages there is an anticipation
that more evolving variants may emerge in the future and there is also a potential
for future coronavirus outbreaks. This provides an impetus for urgent discovery and
development of exceptionally potent bnAbs-based cocktails comprising bnAbs targeting
distinct conserved epitopes on spike protein which are effective against pan-SARS-CoV-2
variants and a broad range of human coronaviruses. RBD-specific bnAbs targeting the
ACE2 binding region could also be effective against highly mutated variants when an
affinity matured bnAb of much higher affinity with RBD than the host ACE2 receptor
is developed e.g. a SARS-CoV-2 specific multabody neutralized the virus at extremely
low picomolar (pM) concentration (Rujas et al., 2021). Recently, an affinity matured
RBD specific nAb CAB-A17 targeting the ACE2 binding region have been shown to neutralize
highly mutated Omicron without losing the potency (Sheward et al., 2022). Interestingly,
two RBM specific ACE2 blocking SARS-CoV-2 bnAbs S2K146 and S2X324 with cross-reactivity
to other SARS-related viruses have been reported to potently neutralize broad SARS
related viruses and Omicron variants (Park et al., 2022; Park et al., 2022). Considering
all that is known about the structure-function relationship between antibodies and
SARS-CoV-2, the following strategies could be effective to combat highly mutated SARS-CoV-2
variants and any coronavirus outbreak(s) in the future: (1) by developing more potent
bnAbs targeting RBD conserved regions (e.g. class III/IV or RBD5-7), fusion peptide
and stem-helix regions. (2) by enhancing the potency of existing bnAbs, especially
directed against the fusion peptide and stem-helix regions, through engineering them
with a directed evolution or other antibody engineering approaches, e.g. a non-nAb
CR3022 was engineered to a nAb using the directed evolution approach (Zhao et al.,
2021). (3) by generating multi-specific bnAbs targeting distinct conserved regions
of the RBD, fusion-peptide and stem-helix regions e.g. Bi-specific, Tri-specific,
and Multabody approaches as these have shown higher potency, affinity, and breadth
against SARS-CoV-2 variants and human CoVs (De Gasparo et al., 2021; Rujas et al.,
2021). (4) understanding the structural/molecular antibody-antigen interaction mechanisms
of bnAbs effective against pan-SARS-CoV-2 variants, α-CoVs and β-CoVs are critically
important for the development of effective therapeutic bnAb cocktails and structure-guided
universal vaccine design. (5) by designing effective vaccine candidates based on epitopes
targeted by RBD class III bnAbs (Bebtelovimab, 002-S21F2 and Sotrovimab) as these
bnAbs can overcome the mutational plasticity of the spike protein in Omicron variants.
Structural footprints of these highly potent Omicron neutralizing bnAbs can potentially
guide rational vaccine design focused to steer B cells to elicit such bnAbs. Epitope
focused vaccines to elicit HIV-1 bnAbs VRC01 and PGT121 have been successfully developed
which are currently in clinical trials (Jardine et al., 2015; Steichen et al., 2016).
Moreover, in-vivo half-life of the potential bnAbs can be increased by incorporating
LS-mutation in the Fc domain (Zalevsky et al., 2010; Ko et al., 2014). Additionally,
cost-effective bnAbs with persistent in-vivo expression can be developed through DNA,
mRNA and viral vector-based therapeutics (Patel et al., 2020; Gardner, 2020; Deal
et al., 2021). A combination of any of these approaches could potentially dilute immune
pressure across the conserved regions of the spike and not specifically target the
ACE2 binding region that encourages the generation of viral escape mutants. These
efforts will also empower the rapid selection of therapeutic bnAb-based cocktails
to alleviate the destructive effects of evolving SARS-CoV-2 variants and outbreak(s)
of any human coronavirus in the future.
Author Contributions
Conceptualization: SK and AC; Writing original draft: SK, PB, VS, and AC; Reviewing
and editing of the manuscript: SK, PB, and AC. All authors contributed to the article
and approved the submitted version.
Funding
SK is supported by the DBT/Wellcome Trust India Alliance Early Career Fellowship grant
IA/E/18/1/504307.
Conflict of Interest
The authors declare that the research was conducted in the absence of any commercial
or financial relationships that could be construed as a potential conflict of interest.
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