OPINION

published: 23 June 2020

doi: 10.3389/fimmu.2020.01531

Nanobodies: Prospects of Expanding

the Gamut of Neutralizing Antibodies
Against the Novel Coronavirus,
SARS-CoV-2
Rocktotpal Konwarh 1,2*
1
Department of Biotechnology, Addis Ababa Science and Technology University, Addis Ababa, Ethiopia, 2 Centre of
Excellence-Nanotechnology, Addis Ababa Science and Technology University, Addis Ababa, Ethiopia

Keywords: COVID-19, SARS-CoV-2, neutralizing antibody, nanobodies, spike protein

With more than 6.9 M confirmed cases and ∼400 K deaths as on June 8, 2020 (1), COVID-19,
ushered in by the SARS-CoV-2 has projected itself as a microscopic-holocaust, much more sinister
than those portrayed in the SciFi movies. Asymptomatic transmission of the virus has been
projected as the Achilles’ heel in the context of the current control strategies of the pandemic (2, 3).
Reports on undiagnosed deep vein thrombosis among patients, succumbing to the viral assault
(4) and demonstration of direct infection of human blood vessel and kidney organoids (5) have
triggered huge hue and cry. The extreme high transmissibility of the virus, bracketed together with
current absence of population immunity and occurrence of stark clinical consequences projects
the swift advancement in effective therapeutic stratagems as the need of the hour. Needless to
say, researchers, across the globe, are beavering to devise appropriate diagnostic and therapeutic
strategies. The various nucleic acid based detection-approaches like PCR, isothermal nucleic acid
amplification-based methods, CRISPR/Cas platforms as well as immunoassay based point-of-care
lateral flow tests are marked with respective pros and cons (6, 7). On the other hand, strategies of
Edited by: inhibiting the viral fusion/entry, disrupting the replication pathway, suppressing the inflammatory
Abdul Qader Abbady,
response, using convalescent plasma treatment and vaccine development have been at the forefront
Atomic Energy Commission
of Syria, Syria
of recent research (8). The success lies in our comprehensive understanding of the “biochemically
and genetically guileful” virus. At this juncture, it is relevant to mention that long-term development
Reviewed by:
Serge Muyldermans,
of appropriate antibody and other protein therapeutics to effectively bind and neutralize the viral
Vrije University Brussel, Belgium infection is imperative. This would be significant in case the researchers need to buy excess time
to ensure befitting vaccine discovery and development. Such therapeutics could possibly provide
*Correspondence:
Rocktotpal Konwarh
an alternative/additional way to assist those people who might show unresponsiveness to vaccines
[email protected]; (as, exemplified by many in the elderly population) or do not obtain vaccine. Amidst the current
[email protected] hay-wired situation, the recent communiqué from Israeli Defense Minister Natfali Bennet about
the successful isolation of a “monoclonal neutralizing antibody” with potency to “neutralize [disease]
Specialty section: inside carriers” bodies’ by the scientists in the Israel Institute for Biological Research has ushered in
This article was submitted to new waves of hope (9).
Vaccines and Molecular Therapeutics, Prior to getting ahead, it would be prudent to recapitulate the general aspects of the
a section of the journal
lifecycle of the highly pathogenic human coronaviruses (CoVs) (10) (Figure 1A). Talking
Frontiers in Immunology
about the viral pathogenesis, the receptor binding domains (RBD) of the spike (S)
Received: 12 May 2020 glycoprotein interact with the human angiotensin-converting enzyme 2 (ACE2)- the receptor
Accepted: 10 June 2020
that invites SARS-CoV and SARS-CoV-2 into human cells (Figure 1Ba). The presence
Published: 23 June 2020
of a furin cleavage site at interfacial zone of the S1 /S2 subunits of the SARS-CoV-2 S
Citation: glycoprotein demarcates the virus from SARS-CoV and SARS-related CoVs (13). Precise
Konwarh R (2020) Nanobodies:
understanding of the SARS-CoV-2 S ectodomain trimer is envisaged to be instrumental
Prospects of Expanding the Gamut of
Neutralizing Antibodies Against the
in developing vaccines, therapeutic antibodies and diagnostics. The prospective targets of
Novel Coronavirus, SARS-CoV-2. neutralizing antibodies (nAbs) against human pathogenic CoVs are depicted in Figure 1Bb.
Front. Immunol. 11:1531. Monoclonal antibodies (mAbs), functional antigen-binding fragment (Fab), single-chain
doi: 10.3389/fimmu.2020.01531 variable region fragment (scFv), and single-domain antibodies (nanobodies or Nbs) have

Frontiers in Immunology | www.frontiersin.org 1 June 2020 | Volume 11 | Article 1531

Konwarh Nanobodies Against SARS-CoV-2

been assessed against various human CoVs (14–19). Jiang et al. susceptibility to steric hindrances (that may otherwise obstruct
(10) have recently reviewed the development of SARS-CoV- optimal binding) as well as ability to display antigenic affinity
and MERS-CoV-specific nAbs, while literature reports on nAbs and specificity at par with conventional antibodies (11, 30–34).
against SARS-CoV-2 are comparatively scanty. Previous studies Prospects of genetically linking to Fc-domains, peptide tags,
on neutralization with anti-SARS-CoV-1 RBD and anti-MERS- or other nanobodies as well as site-specific chemical fusion
CoV RBD antibodies had unveiled a premature switching from with nanoscale materials, radionuclides, photosensitizers, etc.
the pre-fusion to post-fusion conformation following a closure widen the spectrum of their applications. Furthermore, the
of the receptor binding site and trapping the RBD in “up” expedient attributes of nanobodies and human Fc domains may
conformation (20–22). The structure of CR3022, an antibody be combined in chimeric nanobody-heavy chain antibodies, half
derived from a convalescent SARS patient, in complex with the the size of the conventional antibodies, as mentioned before (11).
RBD of the S protein at a resolution of 3.1 Å was recently Post perusal of the afore-stated, harnessing VHHs as
reported (23). Interestingly, a cross-reactive interaction between therapeutics against various viral infectious agents seems to be
SARS-CoV-2 and SARS-CoV was evinced by the elucidation an interesting proposition (35). In this respect, use of VHH
that a highly conserved but cryptic, epitope, distal from the against dengue virus (36); hepatitis C virus (37); multiple VHH
receptor binding site is targeted by CR3022. However, at least monovalent candidates against poliovirus (38) and norovirus
two RBDs on the trimeric S protein in the “up” conformation (39); anti-CXCR4 monovalent and bivalent (40) as well as
and slight rotation are prerequisites to access the binding epitope anti-p24 monovalent and bivalent (41) nanobodies against
by CR3022. The authors proposed that albeit, the CR3022 HIV; VHH bivalent/albumin-linked nanobody against rabies
fails to neutralize SARS-CoV-2 in vitro, the epitope could virus (42) and anti-VP6 VHH as an effective prophylactic
plausibly confer in vivo protection. On a similar vein, researchers treatment against rotavirus A-associated diarrhea (43) have been
have resorted to the use of SARS-CoV-2 S murine polyclonal documented. Investigations on the application of nanobodies
antibodies for the inhibition of SARS-CoV-2 S mediated entrance against respiratory pathogens has also gained pace in recent years.
into cells (13). The study vouched that vaccination could elicit Use of H5N1-HA bivalent nanobody against influenza virus (44),
cross-neutralizing antibodies, targeting the conserved S epitopes. as well as the application of multi-domain antibody MD3606
At this juncture, the germaneness of antibody engineering (generated using diverse camelid single-domain antibodies to
may be comprehended in the context of continual search for influenza virus hemagglutinin) to protect mice against influenza
high-affinity antibodies, effective against conserved targets as well A and B infection post intravenous administration or expression
as novel therapeutics with attributes like better tumor and tissue using recombinant adeno-associated vector (32), merit special
penetration and efficient launching of immune effector functions mention. Similarly, two llama-derived single-domain antibodies
(24). Particularly, in the context of antitumor therapeutics, with human respiratory syncytial virus (RSV)–neutralizing
Bannas et al. (11) had raised concerns about the large-size (150 action have been reported to selectively bind to RSV fusion
kDa) dictated practical snag of in vivo delivery of conventional protein (F) in its pre-fusion state with picomolar affinity (45).
antibodies to tumor cells. On the other hand, aggregation Delivering a trimeric nanobody, ALX-0171 (that interacted with
and/or mispairing of V-domains due to lower stability and antigenic site II of RSV F protein at subnanomolar affinity),
solubility of engineered antibodies- a consequence of intrinsic prophylactically or therapeutically directly to lungs of cotton rats
hydrophobic interactions of VH and VL domains (that constitute was effective in down-scaling both nasal and lung RSV titers
the antigen binding fragment (Fab) of IgG antibodies) have (46). Stalin Raj et al. (47) had resorted to direct cloning and
been another pertinent issue. As plausible solutions, nanobodies expression of VHHs of HCAbs from the bone marrow of MERS-
(15 kDa) and nanobody based human heavy chain antibodies CoV–infected Arabian camels and identified several MERS-
(75 kDa) (11) have instigated considerable research impetus. CoV–specific VHHs or nanobodies. With a prolonged half-life
Besides conventional antibodies, camelids produce heavy-chain- in serum, camel/human chimeric HCAbs were efficacious in
only antibodies (HCAbs) with a single variable domain as endowing protection to mice against MERS-CoV challenge. In
the target recognition module (25, 26). This single variable a similar vein, the efficacy to target MERS-CoV S RBD using
domain without an effector domain functions as a single-domain novel neutralizing Nb (NbMS10) and its human-Fc-fused version
antibody, VHH, or nanobody (Nb) (Figure 1C). Although the (NbMS10-Fc) has been documented (48). Remarkably, the Nbs
prospects of using nanobodies as research and diagnostic tools were able to cross-neutralize infections caused by diverse MERS-
have been critically and comprehensively assessed (27, 28) and CoV strains isolated from humans and camels. The Fc-tagged Nb
a plethora of nanobodies are currently being placed under pre- was able to confer complete protection of humanized mice from
clinical or clinical assessments for various diseases like brain lethal MERS-CoV assault.
tumors, inflammation, lung diseases, as well as autoimmune A concerted effort of biologist Michael Rout and chemist
diseases, paralleling the performance of classical antibodies with Brian Chait has been directed toward selecting high affinity and
nanobodies for therapeutic applications could be bit fiddly (29). effective neutralizing nanobodies, interacting with the various
Nevertheless, studies have attested the advantages of nanobodies non-overlapping target-epitopes of SARS-CoV-2 S (49). The
in contrast to conventional antibodies with respect to the researchers envisage to set-up the appropriate nanobodies as
former’s smaller size, amenability for processing into multiple increased level multimers to augment affinity and eventually tune
formats, desirable thermal and chemical stability, high solubility, them at the molecular level to better their neutralizing potency.
commendable in vivo tissue penetration and targeting, lower Similarly, researchers from Protein Production UK, a project

Frontiers in Immunology | www.frontiersin.org 2 June 2020 | Volume 11 | Article 1531

Konwarh Nanobodies Against SARS-CoV-2

FIGURE 1 | Life cycle of highly pathogenic human coronaviruses (CoVs) and specific neutralizing antibodies (nAbs) against these coronaviruses. (A) Life cycle of highly
pathogenic human CoVs. These CoVs enter host cells by first binding to their respective cellular receptors [angiotensin-converting enzyme 2 (ACE2) for severe acute
(Continued)

Frontiers in Immunology | www.frontiersin.org 3 June 2020 | Volume 11 | Article 1531

Konwarh Nanobodies Against SARS-CoV-2

FIGURE 1 | respiratory syndrome (SARS)-CoV-2 or SARS-CoV and dipeptidyl peptidase 4 (DPP4) for Middle East respiratory syndrome (MERS)-CoV] on the
membranes of host cells expressing ACE2 (e.g., pneumocytes, enterocytes) or DPP4 (e.g., liver or lung cells including Huh-7, MRC-5, and Calu-3) via the surface
spike (S) protein, which mediates virus–cell membrane fusion and viral entry. Viral genomic RNA is released and translated into viral polymerase proteins. The negative
(–)-sense genomic RNA is synthesized and used as a template to form sub-genomic or genomic positive (+)-sense RNA. Viral RNA and nucleocapsid (N) structural
protein are replicated, transcribed, or synthesized in the cytoplasm, whereas other viral structural proteins, including S, membrane (M), and envelope (E), are
transcribed then translated in the endoplasmic reticulum (ER) and transported to the Golgi. The viral RNA-N complex and S, M, and E proteins are further assembled
in the ER–Golgi intermediate compartment (ERGIC) to form a mature virion, then released from host cells. (B) Potential targets of nAbs against SARS-CoV-2 and other
pathogenic human CoVs. (a) Human CoV receptor binding and membrane fusion process. The CoV first binds a viral receptor (ACE2 or DPP4) through the
receptor-binding domain (RBD) in the S protein, followed by fusion of the virus with cell membranes via the formation of a six-helix bundle (6-HB) fusion core. NTD,
N-terminal domain. (b) Potential targets of nAbs on the S protein of human CoVs. Monoclonal antibody (mAb), antigen-binding fragment (Fab), single-chain variable
region fragment (scFv), or single-domain antibody [nanobody (Nb) or VHH derived from camelid heavy chain antibody (HcAb)] binds to the RBD, S1 subunit (non-RBD,
including NTD), or S2 of the viral S protein, blocking binding between the RBD and the respective receptor (for RBD-targeting nAbs), interfering with the conformational
change of S (for S1-targeting nAbs), or hindering S2-mediated membrane fusion (for S2-targeting nAbs), leading to the inhibition of infection with pathogenic human
CoVs in the host cells. The figure was created using BioRender (https://biorender.com/). [Reproduced from (10), under the provisions of Creative Commons License,
CC BY 4.0, Copyright © 2020 The Author(s). Published by Elsevier Ltd.]. (C) Advantageous features of camelid heavy chain antibodies. Heavy chain antibodies are
composed of two heavy chains. The target-binding module is composed of a single VHH domain. A recombinant VHH domain, designated nanobody (Nb) is highly
soluble and does not show any tendency to associate with other hydrophobic protein surfaces. Conventional antibodies are composed of two heavy and two light
chains. The target-binding module is composed of two non-covalently associated variable domains VH and VL. In intact antibodies, the proper orientation of these
domains is mediated by a hydrophobic interface and is further stabilized by the disulfide-linked CL and CH1 domains. A pair of VH and VL domains can be linked
genetically into a single-chain variable fragment (scFv) in which the proper orientation of domains is mediated alone by the hydrophobic interface between the two
V-domains. [Reproduced from (11), under the provisions of Creative Commons Attribution License (CC BY). Copyright © 2017 Bannas, Hambach and Koch-Nolte].
(D) Targeting of diverse epitopes within the SARS-CoV-2 spike protein receptor binding domain (RBD) by human single-domain antibodies, potential therapeutic
candidates for COVID-19. [Reproduced from (12) Copyright ©2020 Elsevier Inc., based on the reuse-provisions of Elsevier’s COVID-19 Resource Centre].

hosted by the Rosalind Franklin Institute in association with (encompassing ribosome and phage display), the task of selecting
Diamond Light Source, UK, have made nanobodies (exhibiting the sybodies was accomplished within 12 days. Six of the selected
high affinity to the S protein of the SARS-CoV-2), available to sybodies displayed double-digit nanomolar binding affinity with
scientist at the University of Oxford for deeper delving into the the viral spike while five of them could inhibit RBD interaction
structure of the virus (50). On a stimulating note, scientists from with ACE2. Furthermore, the researchers identified a pair of anti-
the University of Texas (UT) at Austin, the National Institutes RBD sybodies that could concomitantly interact with the RBD.
of Health and Ghent University in Belgium have documented It would be interesting to peruse the outcomes of the authors’
the isolation of two potently neutralizing VHHs, targeting the previously reported NestLink technology (52) based delving of
SARS-CoV-1 and MERS-CoV RBDs, respectively (34). Wrapp the selection pools to unearth unique sybodies with little off-
et al. (34) had resorted to sequential immunization of a llama rates and capacity to identify rare epitopes. The authors are
subcutaneously multiple times with SARS-CoV-1 S and MERS- upbeat about plausible therapeutic exploitation of the sybodies
CoV S protein. Two sequential rounds of panning were executed for the development of an inhalable drug as useful prophylaxis
by phage display using either SARS-CoV-1 S or MERS-CoV against COVID-19.
S proteins to procure VHHs directed against the S proteins. To speak about yet another development, Beroni Group
The researchers successfully isolated seven unique MERS-CoV (an international biopharmaceutical enterprise) in concert with
S and five SARS-CoV-1 S specific VHHs post-sequencing of the Tianjin University in China has recently identified 24 types
positive clones, multiple sequence alignment, and phylogenetic of nanobodies (post-screening a library with one billion-plus
analysis. Following expression in Pichia pastoris and purification nanobody sequences) for prompt detection and treatment of
from yeast medium, the interaction of the purified VHHs with SARS-CoV-2 (53). Eight of them are directed against the S
the perfusion-stabilized MERS-CoV S and SARS-CoV-1 S was protein while sixteen of them target the nucleocapsid (N)
attested by ELISA. Pertinently, the SARS-CoV-1 RBD-directed protein- the latter could find application as a marker in diagnostic
VHH could cross-react with the SARS-CoV-2 RBD. A fascinating assays. Based on approaches of structural biology, computational
dimension to the work was the neutralization of the SARS-CoV- biology, and protein engineering, the researchers are gearing
2 S pseudotyped viruses by the cross reactive VHH, engineered up to optimize the properties of the nanobodies besides
as a bivalent human IgG Fc-fusion. The plausible scaled up endeavoring to reduce their immunogenicity and augment the
production of the VHH-Fc fusion was attested in a commercial- therapeutic efficiency by humanizing them. By the same token,
standard CHO cell system. The MERS VHH-55, SARS VHH-72 researchers from Fudan University and Biomissile Corporation,
and VHH-72-Fc, exhibiting desirable biophysical attributes and China have directed their endeavors toward the development
potent neutralization potency, could be prospective therapeutic of a phage-displayed single-domain antibody library based on
candidates. However, appropriate in vivo experimentations as embedding naive complementarity-determining regions (CDRs)
part of preclinical studies are prerequisite. into framework sites of a human germline immunoglobulin
Retrieval of information from the preprint at BioRxiv evinces heavy chain variable region (IGHV) allele (12). Their study,
the successful endeavors of Swiss researchers Walter et al. (51) in encompassing the library-biopanning against SARS-CoV-2 RBD
identifying 63 unique anti-RBD synthetic nanobodies or sybodies, and S1 subunit led to the revelation of fully human single-
interacting in the context of the full-length SARS-CoV-2 spike domain antibodies, displaying low-nanomolar/subnanomolar
ectodomain. Assisted by a prompt in vitro selection platform range affinities toward five distinct epitopes on SARS-CoV-2

Frontiers in Immunology | www.frontiersin.org 4 June 2020 | Volume 11 | Article 1531

Konwarh Nanobodies Against SARS-CoV-2

RBD (Figure 1D). Amongst the groups of A, B, C, D, and into biomolecular mechanisms and interactions. This could be
E neutralizing antibodies, the group D members, n3088 and of immense relevance to mine information on SARS-CoV-2
n3130 could target a “cryptic” epitope, positioned in the pathogenesis. Most importantly, highly stable VHHs could
spike trimeric interface, resulting in effective neutralization be nebulized and exploited for the development of inhalable
of SARS-CoV-2. The researchers are buoyant about the apt prophylactic formulations, thereby ensuring straight delivery to
application of these, either alone or in synergy with other SARS- the lungs- the combat zone. Another merit lies in the plausibility
CoV-2 neutralizing antibodies, especially the ACE2-competing of stockpiling the VHHs without trade-off in their stability even
neutralizing antibodies. They may also be employed as integrant after extended storages and using them as therapeutic choices in
for creating bispecific or multispecific antibodies (12). Previously, case of disasters like COVID-19. To conclude, I do hope that the
He et al. (54) had demonstrated an augmented efficacy of incessant and concerted research endeavors would surely pave
oligomeric nanobodies, relative to monomeric nanobodies the way to a safer world, liberated from the grasp of SARS-CoV-2
against MERS coronavirus RBD. Investigating the potential of and akin.
such oligomeric nanobodies in the case of SARS-CoV-2 would
be attention-grabbing.
These studies spark obvious anticipations and hopes for the AUTHOR CONTRIBUTIONS
potential application of nanobodies against COVID-19. The
attributes of small size (almost one-fourth of the size of human RK reviewed the literature, critically analyzed it and authored
antibodies) and simple structure, ease and comparatively lower the article.
cost, low immunogenicity and ability to display high affinity have
endowed them with a special niche in the realm of therapeutics ACKNOWLEDGMENTS
and rapid point-of-care diagnostics. Nanobodies seem to be quite
efficient in trapping and stabilizing conformation-switchable A bouquet of gratitude is extended to all the researchers and
targets in specific conformations, facilitating greater insight frontline warriors, engaged in the battle against COVID-19.

REFERENCES 11. Bannas P, Hambach J, Koch-Nolte F. Nanobodies and nanobody-based human

heavy chain antibodies as antitumor therapeutics. Front Immunol. (2017)
1. World Health Organization. WHO Coronavirus Disease (COVID- 8:1603. doi: 10.3389/fimmu.2017.01603
19) Dashboard. (2020). Available online at: https://covid19.who.int/? 12. Wu Y, Li C, Xia S, Tian X, Kong Y, Wang Z, et al. Identification of human
gclid=Cj0KCQjww_f2BRC-ARIsAP3zarEfohyPnXxXij4jnp412SSC_ single-domain antibodies against SARS-CoV-2. Cell Host Microbe. (2020)
7kKj9J3pEly4tpE21VVLydZtb3KaZsaAlH-EALw_wcB (accessed June 9, 27:891–8.e5. doi: 10.1101/2020.03.30.015990
2020). 13. Walls AC, Park YJ, Tortorici MA, Wall A, McGuire AT, Veesler D. Structure,
2. Day M. COVID-19: four fifths of cases are asymptomatic, China figures function, and antigenicity of the SARS-CoV-2 spike glycoprotein. Cell. (2020)
indicate. BMJ. (2020) 369:m1375. doi: 10.1136/bmj.m1375 181:281–92.e6. doi: 10.1016/j.cell.2020.02.058
3. Gandhi M, Yokoe DS, Havlir DV. Asymptomatic transmission, the Achilles’ 14. Zhou Y, Yang Y, Huang J, Jiang S, Du L. Advances in MERS-CoV vaccines
heel of current strategies to control COVID-19. N Engl J Med. (2020) and therapeutics based on the receptor-binding domain. Viruses. (2019) 11:60.
382:2158–60. doi: 10.1056/NEJMe2009758 doi: 10.3390/v11010060
4. Wichmann D, Sperhake JP, Lütgehetmann M, Steurer S, Edler C, Heinemann 15. Zhu Z, Chakraborti S, He Y, Roberts A, Sheahan T, Xiao X, et al.
A, et al. Autopsy findings and venous thromboembolism in patients Potent cross-reactive neutralization of SARS coronavirus isolates by human
with COVID-19: A prospective cohort study. Ann Intern Med. (2020). monoclonal antibodies. PNAS. (2007) 104:12123–8. doi: 10.1073/pnas.07010
doi: 10.7326/M20-2003. [Epub ahead of print]. 00104
5. Monteil V, Kwon H, Prado P, Hagelkrüys A, Wimmer RA, Stahl M, 16. Rockx B, Corti D, Donaldson E, Sheahan T, Stadler K, Lanzavecchia A,
et al. Inhibition of SARS-CoV-2 infections in engineered human tissues et al. Structural basis for potent cross-neutralizing human monoclonal
using clinical-grade soluble human ACE2. Cell. (2020) 181:905–13.e7. antibody protection against lethal human and zoonotic severe acute
doi: 10.1016/j.cell.2020.04.004 respiratory syndrome coronavirus challenge. J Virol. (2008) 82:3220–35.
6. Bai H, Cai X, Zhan X. Landscape coronavirus disease 2019 test (COVID-19 doi: 10.1128/JVI.02377-07
test) in vitro- A comparison of PCR vs Immunoassay vs CRISPR-based test. 17. Sui J, Li W, Murakami A, Tamin A, Matthews LJ, Wong SK, et al. Potent
OSF [Preprints]. (2020). doi: 10.31219/osf.io/6eagn neutralization of severe acute respiratory syndrome (SARS) coronavirus by
7. Shen M, Zhou Y, Ye J, AL-maskri AAA, Kang Y, Zeng S, et al. Recent advances a human mAb to S1 protein that blocks receptor association. PNAS. (2004)
and perspectives of nucleic acid detection for coronavirus. J Pharm Anal. 101:2536–41. doi: 10.1073/pnas.0307140101
(2020) 10:97–101. doi: 10.1016/j.jpha.2020.02.010 18. He Y, Li J, Li W, Lustigman S, Farzan M, Jiang S. Cross-neutralization of
8. Li H, Zhou Y, Zhang M, Wang H, Zhao Q, Liu J. Updated approaches human and palm civet severe acute respiratory syndrome coronaviruses by
against SARS-CoV-2. Antimicrob Agents Chemother. (2020) 64:e00483–20. antibodies targeting the receptor-binding domain of spike protein. J Immunol.
doi: 10.1128/AAC.00483-20 (2006) 176:6085–92. doi: 10.4049/jimmunol.176.10.6085
9. Perper R. Israel’s Government Research Agency Says it Successfully Isolated 19. Tian X, Li C, Huang A, Xia S, Lu S, Shi Z. Potent binding of
a Key Coronavirus Antibody, Paving the Way for a Possible Breakthrough 2019 novel coronavirus spike protein by a SARS coronavirus-specific
Treatment. (2020). Available online at: https://www.pulselive.co.ke/bi/ human monoclonal antibody. Emerg Microbes Infect. (2020) 9:382–5.
politics/israels-government-research-agency-says-it-successfully-isolated-a- doi: 10.1080/22221751.2020.1729069
key-coronavirus/fyfyz3q (accessed May 8, 2020). 20. Hwang WC, Lin Y, Santelli E, Sui J, Jaroszewski L, Stec B, et al.
10. Jiang S, Hillyer C, Du L. Neutralizing antibodies against SARS-CoV- Structural basis of neutralization by a human anti-severe acute respiratory
2 and other human coronaviruses. Trends Immunol. (2020) 41:355–9. syndrome spike protein antibody, 80R. J Biol Chem. (2006) 281:34610–6.
doi: 10.1016/j.it.2020.03.007 doi: 10.1074/jbc.M603275200

Frontiers in Immunology | www.frontiersin.org 5 June 2020 | Volume 11 | Article 1531

Konwarh Nanobodies Against SARS-CoV-2

21. Walls AC, Xiong X, Park YJ, Tortorici MA, Snijder J, Quispe J, et al. 41. Gray ER, Brookes JC, Caillat C, Turb,é V, Webb BL, Granger LA, et al.
Unexpected receptor functional mimicry elucidates activation of coronavirus Unravelling the molecular basis of high affinity nanobodies against HIV
fusion. Cell. (2019) 176:1026–39. doi: 10.1016/j.cell.2018.12.028 p24: in vitro functional, structural, in silico insights. ACS Infect Dis. (2017)
22. Wang L, Shi W, Chappell JD, Joyce MG, Zhang Y, Kanekiyo M, 3:479–91. doi: 10.1021/acsinfecdis.6b00189
et al. Importance of neutralizing monoclonal antibodies targeting multiple 42. Terryn S, Francart A, Rommelaere H, Stortelers C, Van Gucht S. Post-
antigenic sites on the Middle East respiratory syndrome coronavirus spike exposure treatment with anti-rabies VHH and vaccine significantly improves
glycoprotein to avoid neutralization escape. J Virol. (2018) 92:e02002–17. protection of mice from lethal rabies infection. PLoS Neglect Trop D. (2016)
doi: 10.1128/JVI.02002-17 10:e0004902. doi: 10.1371/journal.pntd.0004902
23. Yuan M, Wu NC, Zhu X, Lee CCD, So RT, Lv H, et al. A highly conserved 43. Maffey L, Vega CG, Miño S, Garaicoechea L, Parreño V. Anti-VP6 VHH: an
cryptic epitope in the receptor-binding domains of SARS-CoV-2 and SARS- experimental treatment for rotavirus A-associated disease. PLoS ONE. (2016)
CoV. Science. (2020) 368:630–3. doi: 10.1101/2020.03.13.991570 11:e0162351. doi: 10.1371/journal.pone.0162351
24. Saeed AF, Wang R, Ling S, Wang S. Antibody engineering for pursuing a 44. Ibanez LI, De Filette M, Hultberg A, Verrips T, Temperton N, Weiss
healthier future. Front. Microbiol. (2017) 8:495. doi: 10.3389/fmicb.2017.00495 RA, et al. Nanobodies with in vitro neutralizing activity protect mice
25. Hamers-Casterman CTSG, Atarhouch T, Muyldermans S, Robinson G, against H5N1 influenza virus infection. J Infect Dis. (2011) 203:1063–72.
Hammers C, Songa EB, et al. Naturally occurring antibodies devoid of light doi: 10.1093/infdis/jiq168
chains. Nature. (1993) 363:446–8. doi: 10.1038/363446a0 45. Rossey I, Gilman MS, Kabeche SC, Sedeyn K, Wrapp D, Kanekiyo M,
26. Muyldermans S. Nanobodies: natural single-domain antibodies. Annu Rev et al. Potent single-domain antibodies that arrest respiratory syncytial
Biochem. (2013) 82:775–97. doi: 10.1146/annurev-biochem-063011-092449 virus fusion protein in its prefusion state. Nat Commun. (2017) 8:14158.
27. De Meyer T, Muyldermans S, Depicker A. Nanobody-based products doi: 10.1038/ncomms14158
as research and diagnostic tools. Trends Biotechnol. (2014) 32:263–70. 46. Detalle L, Stohr T, Palomo C, Piedra PA, Gilbert BE, Mas V, et al. Generation
doi: 10.1016/j.tibtech.2014.03.001 and characterization of ALX-0171, a potent novel therapeutic nanobody
28. Steeland S, Vandenbroucke RE, Libert C. Nanobodies as therapeutics: big for the treatment of respiratory syncytial virus infection. Antimicrob Agents
opportunities for small antibodies. Drug Discov Today. (2016) 21:1076–113. Chemother. (2016) 60:6–13. doi: 10.1128/AAC.01802-15
doi: 10.1016/j.drudis.2016.04.003 47. Stalin Raj V, Okba NM, Gutierrez-Alvarez J, Drabek D, van Dieren B, Widagdo
29. Jovčevska I, Muyldermans S. The therapeutic potential of nanobodies. W, et al. Chimeric camel/human heavy-chain antibodies protect against
BioDrugs. (2019) 34:11–6. doi: 10.1007/s40259-019-00392-z MERS-CoV infection. Sci Adv. (2018) 4:eaas9667. doi: 10.1126/sciadv.aas9667
30. De Vlieger D, Ballegeer M, Rossey I, Schepens B, Saelens X. Single-domain 48. Zhao G, He L, Sun S, Qiu H, Tai W, Chen J, et al. A novel nanobody
antibodies and their formatting to combat viral infections. Antibodies. (2019) targeting Middle East respiratory syndrome coronavirus (MERS-CoV)
8:1. doi: 10.3390/antib8010001 receptor-binding domain has potent cross-neutralizing activity and protective
31. Govaert J, Pellis M, Deschacht N, Vincke C, Conrath K, Muyldermans S, et al. efficacy against MERS-CoV. J Virol. (2018) 92:e00837–18. doi: 10.1128/JVI.
Dual beneficial effect of interloop disulfide bond for single domain antibody 00837-18
fragments. J Biol Chem. (2012) 287:1970–9. doi: 10.1074/jbc.M111.242818 49. Rockefeller University. Research Program on COVID-19/SARS-CoV-2 (2020).
32. Laursen NS, Friesen RH, Zhu X, Jongeneelen M, Blokland S, Vermond J, Available online at: https://www.rockefeller.edu/coronavirus/research-
et al. Universal protection against influenza infection by a multidomain program-covid-19-sars-cov-2/ (accessed May 8, 2020).
antibody to influenza hemagglutinin. Science. (2018) 362:598–602. 50. The Rosalind Franklin Institute. Science Minister Hails Nanobody
doi: 10.1126/science.aaq0620 Breakthrough at Franklin. (2020). Available online at: https://www.rfi.ac.
33. Fernandes JC. Therapeutic application of antibody fragments in autoimmune uk/science-minister-hails-nanobody-breakthrough/ (accessed May 8, 2020).
diseases: Current state and prospects. Drug Discov Today. (2018) 23:1996– 51. Walter JD, Hutter CA, Zimmermann I, Earp J, Egloff P, Sorgenfrei M, et al.
2002. doi: 10.1016/j.drudis.2018.06.003 Synthetic nanobodies targeting the SARS-CoV-2 receptor-binding domain.
34. Wrapp D, De Vlieger D, Corbett KS, Torres GM, Wang N, Van BioRxiv [Preprint]. (2020). doi: 10.1101/2020.04.16.045419
Breedam W, et al. Structural basis for potent neutralization of 52. Egloff P, Zimmermann I, Arnold FM, Hutter CA, Morger D, Opitz
betacoronaviruses by single-domain camelid antibodies. Cell. (2020) L, et al. Engineered peptide barcodes for in-depth analyses of binding
181:1–12 doi: 10.1101/2020.03.26.010165 protein libraries. Nat Methods. (2019) 16:421–8. doi: 10.1038/s41592-019-
35. Sroga P, Safronetz D, Stein DR. Nanobodies: a new approach for the diagnosis 0389-8
and treatment of viral infectious diseases. Future Virol. (2020) 15: 195–205. 53. Beroni Group. Beroni Group and Tianjin University Have Made a Significant
doi: 10.2217/fvl-2019-0167 Discovery in Developing a Medical Solution Using Nanobody Technology
36. Fatima A, Wang H, Kang K, Xia L, Wang Y, Ye W, et al. Development of VHH for the Novel Coronavirus. (2020). Available online at: https://www.
antibodies against dengue virus type 2 NS1 and comparison with monoclonal beronigroup.com/2020/05/19/beroni-group-and-tianjin-university-have-
antibodies for use in immunological diagnosis. PLoS ONE. (2014) 9:95263. made-a-significant-discovery-in-developing-a-medical-solution-using-
doi: 10.1371/journal.pone.0095263 nanobody-technology-for-the-novel-coronavirus/ (accessed June 3, 2020).
37. Tarr AW, Lafaye P, Meredith L, Damier-Piolle L, Urbanowicz RA, Meola A, 54. He L, Tai W, Li J, Chen Y, Gao Y, Li J, et al. Enhanced ability of oligomeric
et al. An alpaca nanobody inhibits hepatitis C virus entry and cell-to-cell nanobodies targeting MERS coronavirus receptor-binding domain. Viruses.
transmission. Hepatol. (2013) 58:932–9. doi: 10.1002/hep.26430 (2019) 11:166. doi: 10.3390/v11020166
38. Thys B, Schotte L, Muyldermans S, Wernery U, Hassanzadeh-
Ghassabeh G, Rombaut, B. In vitro antiviral activity of single domain Conflict of Interest: The author declares that the research was conducted in the
antibody fragments against poliovirus. Antivir Res. (2010) 87:257–64. absence of any commercial or financial relationships that could be construed as a
doi: 10.1016/j.antiviral.2010.05.012 potential conflict of interest.
39. Koromyslova AD, Hansman GS. Nanobodies targeting norovirus capsid
reveal functional epitopes and potential mechanisms of neutralization. PLoS Copyright © 2020 Konwarh. This is an open-access article distributed under the
Pathog. (2017) 13:e1006636 doi: 10.1371/journal.ppat.1006636 terms of the Creative Commons Attribution License (CC BY). The use, distribution
40. Jähnichen S, Blanchetot C, Maussang D, Gonzalez-Pajuelo M, Chow KY, or reproduction in other forums is permitted, provided the original author(s) and
Bosch L, et al. CXCR4 nanobodies (VHH-based single variable domains) the copyright owner(s) are credited and that the original publication in this journal
potently inhibit chemotaxis and HIV-1 replication and mobilize stem cells. is cited, in accordance with accepted academic practice. No use, distribution or
Proc Natl Acad Sci USA. (2010) 107:20565–70. doi: 10.1073/pnas.1012865107 reproduction is permitted which does not comply with these terms.

Frontiers in Immunology | www.frontiersin.org 6 June 2020 | Volume 11 | Article 1531