SARS-CoV-2 variant B.1.617 is resistant to Bamlanivimab and evades antibodies induced by infection and vaccination

TL;DR: In this article, the authors analyzed whether B.1.617 is more adept in entering cells and/or evades antibody responses, and revealed that antibody evasion may contribute to the rapid spread of this variant.

Abstract: The emergence of SARS-CoV-2 variants threatens efforts to contain the COVID-19 pandemic. The number of COVID-19 cases and deaths in India has risen steeply in recent weeks and a novel SARS-CoV-2 variant, B.1.617, is believed to be responsible for many of these cases. The spike protein of B.1.617 harbors two mutations in the receptor binding domain, which interacts with the ACE2 receptor and constitutes the main target of neutralizing antibodies. Therefore, we analyzed whether B.1.617 is more adept in entering cells and/or evades antibody responses. B.1.617 entered two out of eight cell lines tested with slightly increased efficiency and was blocked by entry inhibitors. In contrast, B.1.617 was resistant against Bamlanivimab, an antibody used for COVID-19 treatment. Finally, B.1.617 evaded antibodies induced by infection or vaccination, although with moderate efficiency. Collectively, our study reveals that antibody evasion of B.1.617 may contribute to the rapid spread of this variant.

Summary

INTRODUCTION

• The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is the causative agent of the devastating coronavirus disease 2019 (COVID-19) pandemic, which more than one year after its emergence is associated with record numbers in cases and deaths (W.H.O., 2021) .
• Strategies to fight the COVID-19 pandemic either by vaccines or non-pharmaceutical interventions have been threatened by the emergence of SARS-CoV-2 variants of concern (VOC).
• These variants harbor mutations that confer increased transmissibility or immune evasion (Plante et al., 2021) .
• The S protein is incorporated into the viral membrane and facilitates viral entry into target cells.
• It is believed that many cases are due to infection with a novel variant, B.1.617, that harbors eight mutations in the S protein, including mutations L452R and E484Q within the RBD, which introduce changes at amino acid positions known to modulate antibody-mediated neutralization (Li et al., 2021; Li et al., 2020) .

The S protein of variant B.1.617 mediates increased entry into certain human intestinal and lung cell lines

• These pseudotyped particles faithfully mimic cell entry of SARS-CoV-2 and have been previously used to identify host factors required for SARS-CoV-2 cell entry and to study neutralization of SARS-CoV-2 by antibodies (Hoffmann et al., 2021a; Hoffmann et al., 2020; Riepler et al., 2020; Schmidt et al., 2020) .
• The only exceptions were Caco-2 and Calu-3 cells (and 293T cells in case of B.1.351), which are derived from human intestine and lung, respectively, and for which the S protein of B.1.617 (and B.1.351 ) mediated entry with moderately increased efficiency .
• In addition, the authors investigated S protein-driven cell entry into BHK-21 cells, which were transfected either with empty plasmid or ACE2 expression plasmid.

Soluble ACE2 and Camostat inhibit cell entry driven by the S protein of variant B.1.617

• The authors next examined whether entry of B.1.617 can be blocked by inhibitors targeting the RBD (soluble ACE2) and proteolytic activation of the S protein.
• Soluble ACE2 binds to the RBD and blocks subsequent engagement of membrane bound ACE2.
• The authors analyzed whether these antibodies were able to inhibit host cell entry driven by the S protein of variant B.1.617.
• These results suggest that Casirivimab and particularly Bamlanivimab monotherapy may not be suitable for treatment of patients infected with variant B1.617.

SARS-CoV-2 infection induces the generation of neutralizing antibodies in most infected patients

• And it is believed that these antibody responses are important for protection from re-infection (Rodda et al., 2021; Wajnberg et al., 2020) .
• Therefore, the authors determined whether variant B.1.617 evades inhibition by antibodies, which might contribute to its increasing transmission dynamics.
• For this, the authors analyzed antibody-mediated neutralization using plasma samples obtained from 15 COVID-19 patients at the intensive care unit of Göttingen University Hospital (Table S1 ).
• These plasma samples were prescreened for neutralizing activity and tested for their ability to block host cell entry driven by WT S protein and the S protein of variant B.1.617.
• The S protein of variant B.1.351 served as control since this S protein efficiently evades antibody-mediated neutralization (Hoffmann et al., 2021a) .

Diminished neutralization by plasma from Comirnaty/BNT162b2 vaccinated patients

• Vaccination with Comirnaty/BNT162b2 has been shown to be safe and to protect against COVID-19 with high efficiency (Polack et al., 2020) .
• The vaccine induces antibody and T cell responses (Grifoni et al., 2020; Peng et al., 2020) and the neutralizing antibodies triggered by vaccination are believed to be important for vaccine-induced protection against SARS-CoV-2 infection.
• Therefore, the authors analyzed whether cell entry driven by the S protein of variant B.1.617 can be efficiently inhibited by plasma from Comirnaty/BNT162b2 vaccinated individuals (Table S2 ).
• To address this question, the authors analyzed neutralization by 15 plasma samples obtained from vaccinees two to three weeks after they had received the second vaccine dose.
• Thus, variant B.1.617 can partially evade control by antibodies induced by vaccination with Comirnaty/BNT162b2.

DISCUSSION

• The recent surge in COVID-19 cases and deaths in India is paralleled by the spread of the novel SARS-CoV-2 variant B.1.617.
• The B.1.617 S protein facilitated moderately increased entry into the human lung and intestinal cell lines Calu-3 and Caco-2, respectively, and this effect was much less pronounced when ACE2 was overexpressed in Calu-3 cells.
• Another explanation for the increased spread of variant B.1.617 in India might be immune evasion, i.e. the ability to spread in a population in which a substantial portion of individuals has preexisting immune responses against SARS-CoV-2.
• The RBD of the B.1.617 S protein harbors two mutations associated with (L452R) or suspected (E484Q) of antibody evasion.

Cell

• Authentication of cell lines was performed by STR-typing, amplification and sequencing of a fragment of the cytochrome c oxidase gene, microscopic examination and/or according to their growth characteristics.
• In addition, cell lines were routinely tested for contamination by mycoplasma.

Expression plasmids

• In order to generate the expression vector for the S protein of SARS-CoV-2 variant B.1.617, the required mutations were inserted into the WT SARS-CoV-2 S sequence by overlap extension PCR.
• The resulting open reading frame was further inserted into the pCG1 plasmid (kindly provided by Roberto Cattaneo, Mayo Clinic College of Medicine, Rochester, MN, USA), making use of the unique BamHI and XbaI restriction sites.
• Sequence integrity was verified by sequencing using a commercial sequencing service (Microsynth SeqLab).
• Transfection of 293T cells was carried out by the calcium-phosphate precipitation method, while BHK-21 cells were transfected using Lipofectamine LTX (Thermo Fisher Scientific).

Preparation of vesicular stomatitis virus pseudotypes

• For this study, the authors employed rhabdoviral pseudotype particles that are based on a replicationdeficient VSV vector that lacks the genetic information for VSV-G and instead codes for two reporter proteins, enhanced green fluorescent protein and firefly luciferase (FLuc), VSV * ΔG-FLuc (kindly provided by Gert Zimmer, Institute of Virology and Immunology, Mittelhäusern, Switzerland) (Berger Rentsch and Zimmer, 2011) .
• Thereafter, cells received culture medium containing anti-VSV-G antibody (culture supernatant from I1-hybridoma cells; ATCC no.
• CRL-2700; except for cells expressing VSV-G, which received only medium) and incubated for 16-18 h.
• Finally, the clarified supernatant was aliquoted and stored at -80 °C.

Production of soluble ACE2

• For the production of soluble ACE2 fused to the Fc portion of human immunoglobulin G (IgG), sol-ACE2, 293T cells were grown in a T-75 flask and transfected with 20 µg of sol-ACE2 expression plasmid.
• Then, the culture supernatant was collected and the cells received fresh medium and were further incubated.
• After an additional 24 h, the culture supernatant was harvested and centrifuged as described before.
• Next, the clarified supernatants from both harvests were combined, loaded onto Vivaspin protein concentrator columns with a molecular weight cutoff of 30 kDa and centrifuged at 4,000 x g at 4 °C until a concentration factor of 20 was achieved.
• Finally, the concentrated sol-ACE2 was aliquoted and stored at -80 °C.

Collection of serum and plasma samples

• All plasma samples were heat-inactivated (56 °C, 30 min) before analysis.
• Further, all plasma were pre-screened for the presence of neutralizing activity against WT SARS-CoV-2 S using a pseudotype neutralization test.
• Convalescent plasma samples were collected from COVID-19 patients treated at the intensive care unit of the University Medicine Göttingen under approval given by the ethic committee of the University Medicine Göttingen (SeptImmun Study 25/4/19 Ü).
• Plasma from individuals vaccinated with BioNTech/Pfizer vaccine BNT162b2/ Comirnaty were obtained 24-31 days after the second dose.
• The study was approved by the Institutional Review Board of Hannover Medical School (8973_BO_K_2020).

Transduction of target cells

• All transduction experiments were carried out in 96-well format at a cell confluency of 50-80%.
• For experiments addressing cell tropism and entry efficiency, Vero, Caco-2, Calu-3, Calu-3 (ACE2), 293T, A549 (ACE2), A549 (ACE2+TMPRSS2) and Huh-7 target cells were inoculated with identical volumes of pseudotype preparations.
• For this, the culture supernatant was aspirated.

Pseudotype particle neutralization test

• In all cases, particles incubated only with medium served as control.
• Transduction efficiency was determined at 16-18 h postinoculation as described above.

Data analysis

• The results presented in this study represent average (mean) data obtained from three biological Data normalization was performed in the following fashion: (i) For comparison of entry efficiency by the different S proteins, transduction was normalized against WT SARS-CoV-2 S (set as 100%).
• Alternatively, transduction was normalized against the background signal (luminescence measured for cells inoculated with particles bearing no viral glycoprotein; set as 1).
• Evidence of escape of SARS-CoV-2 variant B.1.351 from natural and vaccine-induced sera.
• A pneumonia outbreak associated with a new coronavirus of probable bat origin.

Full text

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SARS-CoV-2 variant B.1.617 is resistant to Bamlanivimab and evades
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antibodies induced by infection and vaccination
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Markus Hoffmann,
1,2,6,*
Heike Hofmann-Winkler,
1,6
Nadine Krüger,
1,6
Amy Kempf,
1,2
4
Inga Nehlmeier,
1
Luise Graichen,
1,2
Anzhalika Sidarovich,
1,2
Anna-Sophie Moldenhauer,
1
5
Martin S. Winkler,
3
Sebastian Schulz,
4
Hans-Martin Jäck,
4
Metodi V. Stankov,
5
6
Georg M. N. Behrens,
5
Stefan Pöhlmann
1,2,*
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8
1
Infection Biology Unit, German Primate Center, Kellnerweg 4, 37077 Göttingen, Germany
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2
Faculty of Biology and Psychology, Georg-August-University Göttingen, Wilhelmsplatz 1,
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37073 Göttingen, Germany
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3
Department of Anaesthesiology, University of Göttingen Medical Center, Göttingen, Georg-
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August University of Göttingen, Robert-Koch-Straße 40, 37075 Göttingen, Germany
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4
Division of Molecular Immunology, Department of Internal Medicine 3, Friedrich-Alexander
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University of Erlangen-Nürnberg, Glückstraße 6, 91054 Erlangen, Germany
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5
Department for Rheumatology and Immunology, Hannover Medical School, Carl-Neuberg-Str.
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1, 30625 Hannover, Germany
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6
These authors contributed equally
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Lead contact
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*Correspondence: mhoffmann@dpz.eu (M.H.), spoehlmann@dpz.eu (S.P.)
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.CC-BY-NC-ND 4.0 International licenseavailable under a
was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made
The copyright holder for this preprint (whichthis version posted May 5, 2021. ; https://doi.org/10.1101/2021.05.04.442663doi: bioRxiv preprint

2
SUMMARY
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The emergence of SARS-CoV-2 variants threatens efforts to contain the COVID-19
26
pandemic. The number of COVID-19 cases and deaths in India has risen steeply in recent
27
weeks and a novel SARS-CoV-2 variant, B.1.617, is believed to be responsible for many of
28
these cases. The spike protein of B.1.617 harbors two mutations in the receptor binding
29
domain, which interacts with the ACE2 receptor and constitutes the main target of
30
neutralizing antibodies. Therefore, we analyzed whether B.1.617 is more adept in entering
31
cells and/or evades antibody responses. B.1.617 entered two out of eight cell lines tested
32
with slightly increased efficiency and was blocked by entry inhibitors. In contrast, B.1.617
33
was resistant against Bamlanivimab, an antibody used for COVID-19 treatment. Finally,
34
B.1.617 evaded antibodies induced by infection or vaccination, although with moderate
35
efficiency. Collectively, our study reveals that antibody evasion of B.1.617 may contribute to
36
the rapid spread of this variant.
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.CC-BY-NC-ND 4.0 International licenseavailable under a
was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made
The copyright holder for this preprint (whichthis version posted May 5, 2021. ; https://doi.org/10.1101/2021.05.04.442663doi: bioRxiv preprint

3
INTRODUCTION
49
The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is the causative agent of the
50
devastating coronavirus disease 2019 (COVID-19) pandemic, which more than one year after its
51
emergence is associated with record numbers in cases and deaths (W.H.O., 2021). Effective
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antivirals are largely lacking, although recombinant antibodies targeting the viral spike protein
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(S) can significantly reduce viral load und have received emergency use authorization (EUA)
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(Chen et al., 2021a; Gottlieb et al., 2021). Drugs that target the dysregulated cytokine responses
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characteristic for COVID-19 are available but their clinical benefit is oversee-able (Tomazini et
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al., 2020; W.H.O. REACT Working Group et al., 2020). While progress in treatment
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development is moderate, mRNA- and vector-based vaccines are available that provide efficient
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protection against disease (Baden et al., 2021; Polack et al., 2020). As a consequence, vaccination
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is viewed as the key instrument in combating and ultimately ending the COVID-19 pandemic.
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Strategies to fight the COVID-19 pandemic either by vaccines or non-pharmaceutical
61
interventions have been threatened by the emergence of SARS-CoV-2 variants of concern
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(VOC). These variants harbor mutations that confer increased transmissibility or immune evasion
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(Plante et al., 2021). Studies of mutations present in VOC have mainly focused on the viral S
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protein. The S protein is incorporated into the viral membrane and facilitates viral entry into
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target cells. For this, the surface unit, S1, of the S protein first binds to the cellular receptor ACE2
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(Hoffmann et al., 2020; Zhou et al., 2020) via its receptor binding domain (RBD). Subsequently,
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the S protein is activated by TMPRSS2 or related cellular proteases (Hoffmann et al., 2021b;
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Hoffmann et al., 2020) and the transmembrane unit, S2, of the S protein facilitates fusion of the
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viral and a cellular membrane, allowing delivery of the viral genome into the host cell. These
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processes are essential for SARS-CoV-2 infection and are targeted by drugs and neutralizing
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antibodies.
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.CC-BY-NC-ND 4.0 International licenseavailable under a
was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made
The copyright holder for this preprint (whichthis version posted May 5, 2021. ; https://doi.org/10.1101/2021.05.04.442663doi: bioRxiv preprint

4
The prototypic VOC with increased fitness is variant B.1.1.7, which emerged in the
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United Kingdom and is now spreading in many countries. B.1.1.7 replicates to higher levels in
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patients and is more efficiently transmitted between humans as compared to the previously
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circulating viruses (Frampton et al., 2021; Graham et al., 2021; Leung et al., 2021). The increased
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transmissibility might be linked to mutation N501Y in the RBD that might increase binding to
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ACE2 (Ali et al., 2021; Luan et al., 2021). However, the exact mechanisms underlying more
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robust transmission of B.1.1.7 remain to be elucidated. In contrast, differences in antibody-
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mediated neutralization between previously circulating viruses and variant B.1.1.7 are minor,
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with B.1.1.7 being slightly less sensitive to neutralization (Chen et al., 2021b; Collier et al., 2021;
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Hoffmann et al., 2021a; Kuzmina et al., 2021; Muik et al., 2021; Planas et al., 2021; Shen et al.,
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2021; Supasa et al., 2021; Wang et al., 2021a; Xie et al., 2021). In sum, B.1.1.7 shows increased
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fitness and will outcompete previously circulating viruses in an immunologically naïve
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population.
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In populations with a high percentage of individuals with pre-existing immune responses
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against SARS-CoV-2, viral variants that can evade immune control have a selective advantage.
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Variant B.1.351 that became dominant in South Africa (Tegally et al., 2021) and variant P.1 that
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became dominant in Brazil (Faria et al., 2021) are such variants (Chen et al., 2021b; Dejnirattisai
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et al., 2021; Edara et al., 2021; Garcia-Beltran et al., 2021; Hoffmann et al., 2021a; Kuzmina et
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al., 2021; Planas et al., 2021; Wang et al., 2021a; Zhou et al., 2021). These variants harbor
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mutations in the S protein that reduce neutralization by antibodies, including E484K, which is
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located in the RBD and is present in both, B.1.351 and P1 (Li et al., 2021; Liu et al., 2021; Wang
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et al., 2021c). At present, evasion form antibodies is most prominent for variant B.1.351 but it is
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unclear whether variants can arise that exhibit increased or even complete neutralization
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resistance.
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.CC-BY-NC-ND 4.0 International licenseavailable under a
was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made
The copyright holder for this preprint (whichthis version posted May 5, 2021. ; https://doi.org/10.1101/2021.05.04.442663doi: bioRxiv preprint

5
India has seen a steep increase in COVID-19 cases and deaths in the recent weeks
97
(W.H.O., 2021). It is believed that many cases are due to infection with a novel variant, B.1.617,
98
that harbors eight mutations in the S protein, including mutations L452R and E484Q within the
99
RBD, which introduce changes at amino acid positions known to modulate antibody-mediated
100
neutralization (Li et al., 2021; Li et al., 2020). However, it is at present unknown whether
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B.1.617 evades antibody-mediated neutralization. Similarly, it is unknown whether the variant
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exhibits an altered dependence on host cell factors for entry, which may alter cell tropism, entry
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efficiency and sensitivity to entry inhibitors.
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Here, we report that the S protein of B.1.617 mediates moderately enhanced entry into the
105
human lung- and intestine-derived cell lines Calu-3 and Caco-2, respectively, and that entry is
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inhibited by soluble ACE2 and Camostat, the latter of which targets TMPRSS2. In contrast, entry
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driven by the B.1.617 S protein was fully resistant to neutralization by a monoclonal antibody
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with EUA for COVID-19 treatment (Bamlanivimab). Finally, B.1.617 S protein-driven entry was
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partially resistant against neutralization by antibodies elicited upon infection or vaccination with
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the Comirnaty/BNT162b2 vaccine.
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.CC-BY-NC-ND 4.0 International licenseavailable under a
was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made
The copyright holder for this preprint (whichthis version posted May 5, 2021. ; https://doi.org/10.1101/2021.05.04.442663doi: bioRxiv preprint