A Sars-Cov-2 Neutralizing Antibody Protects from Lung Pathology in a Covid-19 Hamster Model

TLDR: Among 598 human monoclonal antibodies (mAbs) from ten SARS-CoV-2 patients, this article identified 40 strongly neutralizing mAbs, including CV07-209, which achieved IC 50 of 3.1 ng/ml.

Abstract: The emergence of SARS-CoV-2 led to pandemic spread of coronavirus disease 2019 (COVID-19), manifesting with respiratory symptoms and multi-organ dysfunction. Detailed characterization of virus-neutralizing antibodies and target epitopes is needed to understand COVID-19 pathophysiology and guide immunization strategies. Among 598 human monoclonal antibodies (mAbs) from ten COVID-19 patients, we identified 40 strongly neutralizing mAbs. The most potent mAb CV07-209 neutralized authentic SARS-CoV-2 with IC 50 of 3.1 ng/ml. Crystal structures of two mAbs in complex with the SARS-CoV-2 receptor-binding domain at 2.55 and 2.70 A revealed a direct block of ACE2 attachment. Interestingly, some of the near-germline SARS-CoV-2 neutralizing mAbs reacted with mammalian self-antigens. Prophylactic and therapeutic application of CV07-209 protected hamsters from SARS-CoV-2 infection, weight loss and lung pathology. Our results show that non-self-reactive virus-neutralizing mAbs elicited during SARS-CoV-2 infection are a promising therapeutic strategy.

Figures

Fig. 3 | Crystal structures of mAbs in complex with SARS-CoV-2 RBD.

Fig. 4 | Interactions and angle of approach at the RBD-antibody interface.

Fig. 6 | Histopathological analysis of hamsters after SARS-CoV-2 infection.

Fig. 2 | SARS-CoV-2 neutralizing antibodies can bind to murine tissue.

Full text

1
A SARS-CoV-2 neutralizing antibody protects from
lung pathology in a COVID-19 hamster model
Jakob Kreye
1,2,3,4,†,*
, S Momsen Reincke
1,2,3,5,†
, Hans-Christian Kornau
1,6
, Elisa Sánchez-Sendin
1,2,3
,
Victor Max Corman
7
, Hejun Liu
8
, Meng Yuan
8
, Nicholas C. Wu
8
, Xueyong Zhu
8
, Chang-Chun D. Lee
8
,
Jakob Trimpert
9
, Markus Höltje
10
, Kristina Dietert
11,12
, Laura Stöffler
1,3
, Niels von Wardenburg
1,3
, Scott
van Hoof
1,2,3
, Marie A Homeyer
1,3,5
, Julius Hoffmann
1,3
, Azza Abdelgawad
9
, Achim D Gruber
11
, Luca D
Bertzbach
9
, Daria Vladimirova
9
, Lucie Y Li
2,10
, Paula Charlotte Barthel
10
, Karl Skriner
13
, Andreas C
Hocke
14
, Stefan Hippenstiel
14
, Martin Witzenrath
14
, Norbert Suttorp
14
, Florian Kurth
14,15
, Christiana
Franke
3
, Matthias Endres
1,3,16,17,18
, Dietmar Schmitz
1,6
, Lara Maria Jeworowski
7
, Anja Richter
7
, Marie
Luisa Schmidt
7
, Tatjana Schwarz
7
, Marcel Alexander Müller
7
, Christian Drosten
7
, Daniel Wendisch
14
,
Leif E Sander
14
, Nikolaus Osterrieder
9
, Ian A Wilson
8,19
, Harald Prüss
1,2,3,*
1
German Center for Neurodegenerative Diseases (DZNE) Berlin, Berlin, Germany.
2
Helmholtz Innovation Lab BaoBab (Brain antibody-omics and B-cell Lab), Berlin, Germany.
3
Department of Neurology and Experimental Neurology, Charité-Universitätsmedizin Berlin, corporate member
of Freie Universität Berlin, Humboldt-Universität Berlin, and Berlin Institute of Health, Berlin, Germany.
4
Department of Pediatric Neurology, Charité-Universitätsmedizin Berlin, corporate member of Freie Universität
Berlin, Humboldt-Universität Berlin, and Berlin Institute of Health, Berlin, Germany.
5
Berlin Institute of Health (BIH), 10178 Berlin, Germany.
6
Neuroscience Research Center (NWFZ), Cluster NeuroCure, Charité-Universitätsmedizin Berlin, corporate
member of Freie Universität Berlin, Humboldt-Universität Berlin, and Berlin Institute of Health, Berlin, Germany.
7
Institute of Virology, Charité-Universitätsmedizin Berlin, corporate member of Freie Universität Berlin,
Humboldt-Universität zu Berlin, and Berlin Institute of Health, Berlin, Germany, and German Centre for Infection
Research (DZIF), Berlin, Germany.
8
Department of Integrative Structural and Computational Biology, The Scripps Research Institute, La Jolla, CA
92037, USA.
9
Institute of Virology, Freie Universität Berlin, Robert-von-Ostertag-Str. 7-13, 14163 Berlin, Germany.
.CC-BY-NC 4.0 International license(which was not certified by peer review) is the author/funder. It is made available under a
The copyright holder for this preprintthis version posted August 16, 2020. . https://doi.org/10.1101/2020.08.15.252320doi: bioRxiv preprint

2
10
Institute of Integrative Neuroanatomy Berlin, Charité-Universitätsmedizin Berlin, corporate member of Freie
Universität Berlin, Humboldt-Universität zu Berlin, and Berlin Institute of Health, Berlin, Germany.
11
Institute of Veterinary Pathology, Freie Universität Berlin, Robert-von-Ostertag-Str. 15, 14163 Berlin,
Germany.
12
Veterinary Centre for Resistance Research, Freie Universität Berlin, Robert-von-Ostertag-Str. 8, 14163
Berlin, Germany.
13
Department of Rheumatology and Clinical Immunology, Charité-Universitätsmedizin Berlin, corporate member
of Freie Universität Berlin, Humboldt-Universität Berlin, and Berlin Institute of Health, Berlin, Germany.
14
Department of Infectious Diseases and Respiratory Medicine, Charité-Universitätsmedizin Berlin, corporate
member of Freie Universität Berlin, Humboldt-Universität Berlin, and Berlin Institute of Health, Berlin, Germany.
15
Department of Tropical Medicine, Bernhard Nocht Institute for Tropical Medicine and I. Department of
Medicine, University Medical Center Hamburg-Eppendorf, Hamburg, Germany
16
Center for Stroke Research Berlin, Charité-Universitätsmedizin Berlin, corporate member of Freie Universität
Berlin, Humboldt-Universität Berlin, and Berlin Institute of Health, Berlin, Germany.
17
Excellence Cluster NeuroCure Berlin, Charité-Universitätsmedizin Berlin, corporate member of Freie
Universität Berlin, Humboldt-Universität Berlin, and Berlin Institute of Health, Berlin, Germany.
18
German Centre for Cardiovascular Research (DZHK), partner site Berlin, Charité-Universitätsmedizin Berlin,
corporate member of Freie Universität Berlin, Humboldt-Universität Berlin, and Berlin Institute of Health, Berlin,
Germany.
19
The Skaggs Institute for Chemical Biology, The Scripps Research Institute, La Jolla, CA 92037, USA.
†
These authors contributed equally.
* Correspondence to:
Jakob Kreye & Harald Prüss
German Center for Neurodegenerative Diseases (DZNE) Berlin
c/o Charité – Universitätsmedizin Berlin
CharitéCrossOver (CCO), Charitéplatz 1, 10117 Berlin, Germany
Email: jakob.kreye@dzne.de; harald.pruess@dzne.de.
.CC-BY-NC 4.0 International license(which was not certified by peer review) is the author/funder. It is made available under a
The copyright holder for this preprintthis version posted August 16, 2020. . https://doi.org/10.1101/2020.08.15.252320doi: bioRxiv preprint

3
ABSTRACT
The emergence of SARS-CoV-2 led to pandemic spread of coronavirus disease 2019 (COVID-19),
manifesting with respiratory symptoms and multi-organ dysfunction. Detailed characterization of virus-
neutralizing antibodies and target epitopes is needed to understand COVID-19 pathophysiology and
guide immunization strategies. Among 598 human monoclonal antibodies (mAbs) from ten COVID-19
patients, we identified 40 strongly neutralizing mAbs. The most potent mAb CV07-209 neutralized
authentic SARS-CoV-2 with IC
50
of 3.1 ng/ml. Crystal structures of two mAbs in complex with the
SARS-CoV-2 receptor-binding domain at 2.55 and 2.70 Å revealed a direct block of ACE2
attachment. Interestingly, some of the near-germline SARS-CoV-2 neutralizing mAbs reacted with
mammalian self-antigens. Prophylactic and therapeutic application of CV07-209 protected hamsters
from SARS-CoV-2 infection, weight loss and lung pathology. Our results show that non-self-reactive
virus-neutralizing mAbs elicited during SARS-CoV-2 infection are a promising therapeutic strategy.
.CC-BY-NC 4.0 International license(which was not certified by peer review) is the author/funder. It is made available under a
The copyright holder for this preprintthis version posted August 16, 2020. . https://doi.org/10.1101/2020.08.15.252320doi: bioRxiv preprint

4
INTRODUCTION
The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) started emerging in humans in
late 2019, and rapidly spread to a pandemic with millions of cases worldwide. SARS-CoV-2 infections
cause coronavirus disease 2019 (COVID-19) with severe respiratory symptoms, but also pathological
inflammation and multi-organ dysfunction, including acute respiratory distress syndrome,
cardiovascular events, coagulopathies and neurological symptoms (Helms et al., 2020; Zhou et al.,
2020; Zhu et al., 2020). Some aspects of the diverse clinical manifestations may result from a
hyperinflammatory response, as suggested by reduced mortality in hospitalized COVID-19 patients
under dexamethasone therapy (Horby et al., 2020).
Understanding the immune response to SARS-CoV-2 therefore is of utmost importance. Multiple
recombinant SARS-CoV-2 mAbs from convalescent patients have been reported (Brouwer et al.,
2020; Cao et al., 2020; Ju et al., 2020; Kreer et al., 2020; Robbiani et al., 2020; Rogers et al., 2020;
Wec et al., 2020). mAbs targeting the receptor-binding domain (RBD) of the viral spike protein S1 can
compete with its binding to human angiotensin converting enzyme 2 (ACE2) and prevent viral entry
and subsequent replication (Cao et al., 2020; Ju et al., 2020; Walls et al., 2020). Potent virus
neutralizing mAbs that were isolated from diverse variable immunoglobulin (Ig) genes typically carry
low levels of somatic hypermutations (SHM). Several of these neutralizing mAbs selected for in vitro
efficacy showed prophylactic or therapeutic potential in animal models (Cao et al., 2020; Liu et al.,
2020; Rogers et al., 2020; Zost et al., 2020). The low number of SHM suggests limited affinity-
maturation in germinal centers compatible with an acute infection. Near-germline mAbs usually
constitute the first line of defense to pathogens, but carry the risk of self-reactivity to autoantigens
(Lerner, 2016; Liao et al., 2011; Zhou et al., 2007). Although critical for the therapeutic use in
humans, potential potential tissue-reactivity of near-germline SARS-CoV-2 antibodies has not been
examined so far.
.CC-BY-NC 4.0 International license(which was not certified by peer review) is the author/funder. It is made available under a
The copyright holder for this preprintthis version posted August 16, 2020. . https://doi.org/10.1101/2020.08.15.252320doi: bioRxiv preprint

5
Here, we systematically selected 18 strongly neutralizing mAbs out of 598 antibodies from 10 COVID-
19 patients by characterization of their biophysical properties, authentic SARS-CoV-2 neutralization,
and exclusion of off-target binding to murine tissue. Additionally, we solved two crystal structures of
neutralizing mAbs in complex with the RBD, showing antibody engagement with the ACE2 binding
site from different approach angles. Finally, we selected mAb CV07-209 by its in vitro efficacy and the
absence of tissue-reactivity for in vivo evaluation. Systemic application of CV07-209 in a hamster
model of SARS-CoV-2 infection led to profound reduction of clinical, paraclinical and histopathological
COVID-19 pathology, thereby reflecting its potential for translational application in patients with
COVID-19.
.CC-BY-NC 4.0 International license(which was not certified by peer review) is the author/funder. It is made available under a
The copyright holder for this preprintthis version posted August 16, 2020. . https://doi.org/10.1101/2020.08.15.252320doi: bioRxiv preprint

Frequently asked questions

Q1. What contributions have the authors mentioned in the paper "A sars-cov-2 neutralizing antibody protects from lung pathology in a covid-19 hamster model" ?

Kreye et al. this paper proposed a neutralizing mAbs targeting the receptor-binding domain ( RBD ) of SARS-CoV-2 to prevent viral entry and subsequent replication. 

Q2. What have the authors stated for future works in "A sars-cov-2 neutralizing antibody protects from lung pathology in a covid-19 hamster model" ?

Future research will need to clarify if additional mechanisms like triggering conformational changes in the spike protein upon antibody binding contribute to virus neutralization, as reported for SARS-CoV ( Walls et al., 2019 ). Whether self-reactive antibodies could contribute to extra-pulmonary symptoms in COVID-19, awaits further studies and should be closely monitored also in vaccination trials. This finding suggests that the self-reactivity observed in this study may not be limited to mAbs of the early humoral immune response in SARS-CoV-2 infections. 

Q3. What is the role of mAbs in the development of COVID-19?

Systemic application of CV07-209 in a hamstermodel of SARS-CoV-2 infection led to profound reduction of clinical, paraclinical and histopathologicalCOVID-19 pathology, thereby reflecting its potential for translational application in patients withCOVID-19. 

Q4. What is the role of self-reactive antibodies in COVID-19?

Whether self-reactiveantibodies could contribute to extra-pulmonary symptoms in COVID-19, awaits further studies andshould be closely monitored also in vaccination trials. 

Q5. How much weight did the hamsters lose after receiving mAb?

Hamsters under control mAb treatment lost 5.5±4.4% (mean±SD) of body weight, whereas those thatreceived mAb CV07-209 as a therapeutic or prophylactic single dose gained 2.2±3.4% or 4.8±3.4%weight after 5 days post-infection (dpi), respectively. 

Q6. How long did the animals have to acclimate to the conditions?

The animals had ad libitum access to food andwater and were allowed to acclimate to these conditions for seven days prior to prophylactictreatment and infection. 

Q7. How many antibodies were screened for binding to RBD?

86 were defined as strongly binding to RBD (defined as detectable binding at 10ng/ml in an RBD ELISA) and then assessed for neutralization of authentic SARS-CoV-2 at 25 and250 ng/ml using mAb-containing cell culture supernatants. 

Q8. How was the mixture incubated for 15 minutes?

one volume of biotinylated mcAbsat 100 ng/ml was added and the mixture incubated for additional 15 minutes, followed by detectionusing HRP-conjugated streptavidin (Roche Diagnostics) and 1-step Ultra TMB. 

Q9. How many plaque forming units were diluted in OptiPro?

For each dilution step, mAbs were diluted inOptiPro and mixed 1:1 with 200 μl virus (Munich isolate 984) (Wolfel et al., 2020) solution containing100 plaque forming units. 

Q10. What is the hk CV38-177 s1-mbc Ig?

1 1-44 1 AAWDDSLNGYV 0 0.15 HL CV38-173 S1-MBC IgG3 3-30-3 4 ARDYGGYNYN 4 3-1 2 QAWDSSTVV 0 9.92 - n.t. HK CV38-177 38-3 S1-MBC IgG1 3-9 4 AKDMVVVAIFGVGPFDY 

Q11. What is the way to prevent reactivity with host antigens?

Such reactivity with host antigens should ideally be prevented byimmunological tolerance mechanisms, but complete exclusion of such antibodies would generate“holes” in the antibody repertoire. 

Q12. How was the goat anti-human IgG-Alexa Fluor 488 treated?

After three PBS washing steps, goat anti-human IgG-Alexa Fluor 488 (Dianova, 109-545-003) diluted in blocking solution was applied for 2hours at room temperature before additional three washes and mounting using DAPI-containingFluoroshield. 

Q13. How did the authors select the mAbs from the COVID-19 patients?

the authors systematically selected 18 strongly neutralizing mAbs out of 598 antibodies from 10 COVID-19 patients by characterization of their biophysical properties, authentic SARS-CoV-2 neutralization,and exclusion of off-target binding to murine tissue. 

Q14. What is the role of mAbs in neutralizing SARS-CoV-2?

Futureresearch will need to clarify if additional mechanisms like triggering conformational changes in thespike protein upon antibody binding contribute to virus neutralization, as reported for SARS-CoV(Walls et al., 2019). 

Q15. HL CV01-227 ASC IgA1 3-30 4 AKPGGE?

2 3-1 1 QAWDSSTACV 3 n.exp. HL CV01-227 ASC IgA1 3-30 4 AKGSPLLGFGGVDY 0 5-39 3 AIWYSSSLV 1 n.exp. HK CV01-228 ASC IgA1 3-7 4 ARVGASDYDYVWGTRTLDS 

Q16. What was the Fab binding domain of the SARS-CoV-2 spike?

The receptor binding domain (RBD; residues 319-541) of the SARS-CoV-2 spike (S) protein wasexpressed in High Five cells and purified using affinity and size exclusion chromatography asdescribed previously (Yuan et al., 2020b).