Materi Covid-19
Materi Covid-19
Materi Covid-19
Correspondence
mhoffmann@dpz.eu (M.H.),
spoehlmann@dpz.eu (S.P.)
In brief
The SARS-CoV-2 Omicron variant is
rapidly spreading worldwide and a public
health concern. Experiments show that
this variant is resistant against several
therapeutic antibodies for COVID-19 and
efficiently evades antibodies induced
upon infection or double BNT162b2
vaccination, but not triple BNT162b2 or
ChAdOx1/BNT162b2 vaccination.
Highlights
d Omicron uses human and animal ACE2 for host cell entry
Article
The Omicron variant is highly resistant
against antibody-mediated neutralization:
Implications for control of the COVID-19 pandemic
Markus Hoffmann,1,2,7,* Nadine Krüger,1,7 Sebastian Schulz,3 Anne Cossmann,4 Cheila Rocha,1 Amy Kempf,1,2
Inga Nehlmeier,1 Luise Graichen,1,2 Anna-Sophie Moldenhauer,1 Martin S. Winkler,5 Martin Lier,5
Alexandra Dopfer-Jablonka,4,6 Hans-Martin Jäck,3,7 Georg M.N. Behrens,4,6,7 and Stefan Pöhlmann1,2,7,8,*
1Infection Biology Unit, German Primate Center, Kellnerweg 4, 37077 Göttingen, Germany
2Faculty of Biology and Psychology, Georg-August-University Göttingen, Wilhelmsplatz 1, 37073 Göttingen, Germany
3Division of Molecular Immunology, Department of Internal Medicine 3, Friedrich-Alexander University of Erlangen-Nürnberg, Glückstraße 6,
SUMMARY
The rapid spread of the SARS-CoV-2 Omicron variant suggests that the virus might become globally domi-
nant. Further, the high number of mutations in the viral spike protein raised concerns that the virus might
evade antibodies induced by infection or vaccination. Here, we report that the Omicron spike was resistant
against most therapeutic antibodies but remained susceptible to inhibition by sotrovimab. Similarly, the Om-
icron spike evaded neutralization by antibodies from convalescent patients or individuals vaccinated with the
BioNTech-Pfizer vaccine (BNT162b2) with 12- to 44-fold higher efficiency than the spike of the Delta variant.
Neutralization of the Omicron spike by antibodies induced upon heterologous ChAdOx1 (Astra Zeneca-Ox-
ford)/BNT162b2 vaccination or vaccination with three doses of BNT162b2 was more efficient, but the Omi-
cron spike still evaded neutralization more efficiently than the Delta spike. These findings indicate that
most therapeutic antibodies will be ineffective against the Omicron variant and that double immunization
with BNT162b2 might not adequately protect against severe disease induced by this variant.
study suggested that the Omicron variant is more adept at infect- hibits 37 mutations as compared with the Wuhan-Hu-1 spike (Fig-
ing convalescent individuals as compared with previously circu- ures 1C and 1D). Thirteen of these changes are unique, while the
lating variants (Abdullah, 2021; Pulliam et al., 2021). Thus, the remaining changes are known from variants of interest or concern
Omicron variant constitutes a rapidly emerging threat to public (Haseltine, 2021). Specifically, 11 mutations, including 6 deletions
health and might undermine global efforts to control the and 1 insertion, are located in the N-terminal domain (NTD) and
COVID-19 pandemic. However, the susceptibility of the Omicron mutations N211D and ins214EPE are unique. Some of the dele-
variant to antibody-mediated neutralization remains to be tions are found in other VOCs and might reduce antibody binding
analyzed. or increase spike expression (Harvey et al., 2021b; Li et al., 2021b;
Here, we report that the Omicron S protein evades antibodies Liu et al., 2021; Meng et al., 2021; Wang et al., 2021; Wheatley
with up to 44-fold higher efficiency than the spike of the Delta et al., 2021). Another 15 mutations are found in the receptor-bind-
variant, rendering therapeutic antibodies ineffective and likely ing domain (RBD) (Figures 1C–1E), of which G339D, S371L,
compromising protection by antibodies induced upon infection S373P, and S375F are unique, and several were previously shown
or vaccination with two doses of BNT162b2 (BNT). to modulate ACE2 binding and/or antibody evasion (Harvey et al.,
2021b; Li et al., 2021b; Liu et al., 2021; Wang et al., 2021; Wheatley
RESULTS et al., 2021). Further, five mutations reside between the RBD and
the S1/S2 site, including the unique mutation T547K and mutation
The NTD and RBD of the Omicron spike are highly P681H, which may modulate cleavage at the S1/S2 site by the
mutated host protease furin (Hoffmann et al., 2020a; Zhang et al., 2021a)
The first sequences of the Omicron variant were deposited into (Figures 1C and 1D). Finally, five mutations are present in the S2
the GISAID (Global Initiative on Sharing All Influenza Data) data- subunit, all of them unique (Haseltine, 2021).
base on November 22 and 23, 2021 (Figure 1A). These se-
quences were obtained from patients in Botswana and South Af- Omicron spike binds human ACE2 and mediates
rica as well as from a traveler returning to Hong Kong from South efficient entry into cell lines
Africa. Subsequently, the number of deposited Omicron se- For analysis of host-cell entry, we employed vesicular stomatitis
quences rapidly increased (Figure 1A) and the virus was also de- virus (VSV) particles pseudotyped with SARS-CoV-2 S proteins.
tected in Europe, Asia, and the United States due to infected air These pseudotyped particles adequately mimic key features of
travelers (Figure 1B). Analysis of the Omicron genomic sequence SARS-CoV-2 entry into target cells, including receptor and pro-
revealed striking differences as compared with other known tease choice and neutralization by antibodies (Hoffmann et al.,
SARS-CoV-2 variants, suggesting extensive, independent evo- 2020b; Riepler et al., 2020; Schmidt et al., 2020).
lution, potentially in an isolated human population, immunocom- We first asked whether the Omicron spike differs from the
promised patients, or an unknown animal species (Kupfersch- spike of other VOCs regarding target cell choice and entry effi-
midt, 2021). ciency. The spike from SARS-CoV-2 B.1 (which is identical to
The Omicron spike (isolate hCoV-19/Botswana/R40B58_BHP_ the S protein of the Wuhan-Hu-1 isolate, except for the presence
3321001245/2021; GISAID Accession ID: EPI_ISL_6640919) ex- of mutation D614G) was analyzed in parallel, since this virus
Figure 1. The Omicron spike mediates entry into cell lines with different efficiency as compared with B.1 and Delta spike, binds human ACE2
efficiently, and utilizes a broad range of animal ACE2 orthologues as receptor
(A) Epidemiology of SARS-CoV-2 Omicron variant. Purple bars indicate the number of newly reported isolates per day while the blue line shows the cumulative
number of isolates as of November 17, 2021. (Based on data deposited in the GISAID database).
(B) Global distribution of SARS-CoV-2 Omicron variant. Sequences retrieved from the GISAID database were grouped based on the continent where they were
detected (as of November 17, 2021).
(C) Schematic overview and domain organization of the S proteins of SARS-CoV-2 Omicron variant. Mutations compared with the SARS-CoV-2 Wuhan-Hu-1
isolate are highlighted in red. (NTD, N-terminal domain; RBD, receptor-binding domain; TD, transmembrane domain).
(D) Location of Omicron-specific mutations in the context of the trimeric spike protein. (S1 subunit, light blue; RBD, dark blue; S2 subunit, gray; S1/S2 and S20
cleavage sites; mutated amino acid residues, red (compared with the S protein of the SARS-CoV-2 Wuhan-Hu-1 isolate).
(E) Locations of Omicron-specific mutations in the context of the RBD. (RBD, gray; RBD residues that interact with ACE2, green; ACE2-interacting RBD residues
that are mutated in Omicron spike, red: non-ACE2-interacting RBD residues that are mutated in Omicron spike, orange).
(F) Particles bearing the indicated S proteins were inoculated onto different cell lines, and S-protein-driven cell entry was analyzed at 16–18 h postinoculation by
measuring the activity of virus-encoded firefly luciferase in cell lysates. Presented are the mean from 3 to 12 biological replicates (each conducted with four
technical replicates) in which S-protein-driven cell entry was normalized against B.1 (set as 1). (Error bars: SEM).
(G) Binding of soluble ACE2 to B.1 or Omicron spike proteins. 293T cells expressing the indicated S protein (or no S protein) following transfection were
sequentially incubated with soluble ACE2 harboring a C-terminal Fc-tag derived from human IgG and AlexaFlour-488-conjugated antihuman antibody. Finally,
ACE2 binding was analyzed by flow cytometry. Cells incubated with only secondary antibody served as controls. Presented is the mean geometric mean channel
fluorescence from six biological replicates (each conducted with single samples). (Error bars: standard deviation).
(H) Particles bearing the indicated S proteins or VSV-G were inoculated on BHK-21 cells that were previously transfected to express the indicated ACE2 or-
thologues or empty vector. S protein-driven cell entry was analyzed as described in (F). Presented are the mean data from three biological replicates (each
conducted with four technical replicates) in which signals obtained from particles bearing no viral glycoprotein (indicated by dashed line) were used for
normalization (set as 1). (Error bars: SEM).
(F–H): statistical significance of differences between individual groups was assessed by two-tailed Students t test: p > 0.05, not significant (ns); *p < 0.05; **p <
0.01; ***p < 0.001.
circulated early in the pandemic and does not harbor mutations In addition, the antibody sotrovimab was shown to inhibit
found in the S proteins of VOCs. For the analysis of cell tropism, SARS-CoV-2 and related viruses and was found to protect pa-
we employed the cell lines Vero (African green monkey, kidney), tients from COVID-19 (Gupta et al., 2021). Since the Omicron
293T (human kidney), and A549 (human lung), which were engi- spike harbors several mutations within the structures that are
neered to express high levels of ACE2 (A549-ACE2), Huh-7 (hu- recognized by these antibodies (Figure 2B), we investigated
man liver), Caco-2 (human colon) and Calu-3 (human lung) cells. whether the antibodies were still able to neutralize the Omicron
All cell lines were highly susceptible to entry driven by VSV-G and spike. All antibodies inhibited entry driven by the B.1 spike in a
SARS-CoV-2 B.1 spike (Figures 1F and S1). Further, all VOC S robust and concentration-dependent manner, while a control
proteins facilitated robust entry into the cell lines analyzed, but immunoglobulin was inactive (Figure 2C). In contrast, entry
subtle differences were noted. Thus, the Delta spike mediated driven by the Omicron spike was fully resistant against bamlani-
increased entry into Calu-3 and Caco-2 cells, in keeping with vimab, etesevimab, and imdevimab and largely resistant against
published data (Arora et al., 2021b), while the Omicron spike casirivimab. In agreement with these findings, a cocktail of bam-
facilitated increased entry into Vero, Huh-7, and particularly lanivimab and etesevimab failed to inhibit entry mediated by the
293T cells (Figure 1F). Further, B.1 and Omicron spike mediated Omicron spike, while inhibition by a cocktail of casirivimab and
comparable entry into A549-ACE2 cells while entry driven by the imdevimab was inefficient (Figure 2C). In contrast, sotrovimab
other S proteins was less efficient (Figure 1F). Finally, the Omi- was active against Omicron spike, although inhibition was
cron spike bound efficiently to human ACE2 (Figure 1G) and slightly less efficient than that measured for B.1 spike (Figure 2C).
used ACE2 for host-cell entry (see below), indicating that the mu- In sum, the Omicron spike is resistant against several antibodies
tations in the RBD do not compromise ACE2 interactions. In used for COVID-19 treatment.
sum, the Omicron spike binds to ACE2 and mediates robust en-
try into diverse ACE2-positive cell lines. The Omicron spike evades neutralization by antibodies
induced upon infection and BNT vaccination with high
The Omicron spike can efficiently use ACE2 orthologues efficiency
from different animal species for cell entry The resistance against several antibodies used for COVID-19
We next asked whether the Omicron spike can use human ACE2 therapy suggested that the Omicron spike might also evade an-
and ACE2 orthologues from different animal species, including tibodies induced upon infection and vaccination. Indeed, sera/
horseshoe bat, masked palm civet, raccoon dog, and pangolin, plasma collected within two months of convalescence from
for entry into target cells. Entry driven by the B.1 and Delta spikes mild or severe COVID-19 inhibited entry driven by the Omicron
as well as VSV-G served as controls. The expression of the ACE2 spike 80-fold less efficiently as compared with the B.1 spike
orthologues did not modulate entry driven by VSV-G but in most and 44-fold less efficiently as compared with the Delta spike,
cases allowed robust and roughly comparable entry driven by with 9 out of 17 sera tested being unable to neutralize particles
the B.1, Delta, and Omicron spikes (Figure 1H). Two exceptions bearing Omicron spike (Figures 3A and S2). The samples were
were noted. Murine ACE2 was used more efficiently by the Delta collected in Germany during the first COVID-19 wave (Table
spike as compared with the B.1 spike and supported entry driven S1), when neither the Alpha nor the Delta variant predominated,
by the Omicron spike with the highest efficiency (Figure 1H). suggesting the antibodies raised against the virus circulating at
Further, the B.1 spike was unable to use ACE2 from Pearson’s the beginning of the pandemic offer little to no protection against
horseshoe bat (Rhinolophus pearsonii) for entry while the Delta the Omicron variant.
and particularly the Omicron spike used this receptor with high The mRNA-based vaccine BNT efficiently protects against
efficiency (Figure 1H). Collectively, these data reveal broad us- COVID-19 (Polack et al., 2020) and is frequently used in Europe
age of ACE2 orthologues by Omicron spike for host-cell entry, and the United States. Sera collected within one to three months
which might hint toward high zoonotic potential. after the second dose of BNT (10 sera were collected within
1 month, one serum was collected within 3 months; Table S2) in-
Omicron spike is inhibited by soluble ACE2 but is hibited entry by the Omicron spike with 34-fold lower efficiency
resistant against several antibodies used for COVID-19 as compared with the B.1. spike and with 12-fold lower efficiency
treatment as compared with the Delta spike (Figures 3B and S2). These
We next asked whether the Omicron spike can be inhibited by results suggest that two immunizations with BNT, which can pro-
soluble ACE2, which binds to the RBD, blocks host-cell entry, vide more than 90% protection from severe disease upon infec-
and is currently being developed for COVID-19 therapy (Monteil tion with the Delta variant (Chemaitelly et al., 2021), might be
et al., 2020). Soluble ACE2 did not modulate entry driven by VSV- markedly less effective against the Omicron variant.
G but robustly and comparably inhibited entry driven by B.1,
Delta, and Omicron spikes (Figure 2A), indicating that soluble Evidence that heterologous and booster vaccination
ACE2 should be suitable for treatment of patients infected with may provide improved protection against the Omicron
the Omicron variant. variant
Several recombinant, neutralizing monoclonal antibodies We next explored whether strategies known to increase produc-
were identified that inhibit SARS-CoV-2 infection and cocktails tion of neutralizing antibodies relative to BNT/BNT immunization
of casirivimab and imdevimab (REGN-COV2, Regeneron) (Wein- might afford better protection against the Omicron variant. Het-
reich et al., 2021) and etesevimab and bamlanivimab (Eli Lilly) erologous vaccination with a first dose of ChAdO1-nCoV-19/
(Dougan et al., 2021) are currently used for COVID-19 therapy. AZD1222 (AZ) (Voysey et al., 2021) and a second dose of BNT
Figure 2. The Omicron spike evades neutralization by four out of five monoclonal antibodies but is efficiently inhibited by soluble ACE2
(A) Inhibition of S-protein-driven cell entry by soluble ACE2. Particles harboring the indicated S proteins were preincubated (30 min, 37 C) with different dilutions
of soluble ACE2 before being inoculated onto Vero cells. S-protein-driven cell entry was analyzed as described in Figure 1F. Presented are the mean data from
three biological relocates (each conducted with four technical replicates).
(B) Locations of Omicron-specific mutations in the context of the RBD epitopes targeted by casirivimab, imdevimab, bamlanivimab, etesevimab, and sotrovimab.
(RBD, gray; epitope targeted by the antibody, blue; Omicron-specific mutations within the epitope, red; Omicron-specific mutations outside the epitope, orange).
(C) Omicron spike is resistant against four out of five monoclonal antibodies used for treatment of COVID-19 patients. Particles harboring the indicated S proteins
were preincubated (30 min, 37 C) with different concentrations of recombinant monoclonal antibody before being inoculated onto Vero cells. S-protein-driven
cell entry was analyzed as described in Figure 1F. Presented are the mean data from three biological replicates (each conducted with four technical replicates).
(A and C): statistical significance of differences between individual groups was assessed by two-way analysis of variance with Dunnett’s (A) or Sidak’s (C) post
hoc tests: p > 0.05, not significant (ns); *p < 0.05; **p < 0.01; ***p < 0.001.
was shown to induce higher neutralizing antibody titers as collected within three months after BNT/BNT vaccination (Fig-
compared with the corresponding homologous vaccinations ure 3C). Inhibition of Omicron-spike-driven entry by sera from
(Barros-Martins et al., 2021; Behrens et al., 2021). Sera collected AZ/BNT-vaccinated individuals was 14-fold less efficient as
within one month after heterologous AZ/BNT vaccination (Table compared to B.1. spike but only 3-fold less efficient relative to
S2) exhibited higher neutralizing activity as compared with sera Delta spike (Figure 3C). Further, inhibition of the Omicron spike
DISCUSSION
(C) The experiment was performed as described in (A) but sera from AZ/BNT-
vaccinated individuals were analyzed (n = 10).
(D) The experiment was performed as described in (A) but sera from BNT/BNT/
BNT-vaccinated individuals were analyzed (n = 10).
(A–D) Patient identifiers are indicated on the x axes. The reciprocal serum/
Figure 3. The Omicron spike shows high resistance against anti- plasma dilution factors that caused a 50% reduction in S protein-driven cell
bodies elicited upon infection or vaccination entry (neutralization titer 50, NT50) are shown. Left panels show individual
(A) Particles bearing the indicated S proteins were preincubated (30 min, 37 C) NT50 values clustered per SARS-CoV-2 variant. Black lines and numerical
with different dilutions of convalescent sera/plasma (n = 17) before being values in brackets indicate median NT50 values, whereas right panels show
inoculated onto Vero cells. S-protein-driven cell entry was analyzed as serum/plasma-specific NT50 values ranked from highest to lowest based on
described in Figure 1F. Black triangles indicate patients with severe disease NT50 for B.1. Numerical values indicate the median fold change in neutrali-
that required admission to the intensive care unit; all other patients showed zation between individual SARS-CoV-2 variants. Dashed lines indicate the
mild disease. lowest serum dilution tested. Statistical significance of differences between
(B) The experiment was performed as described in (A) but sera from BNT/BNT- individual groups was assessed by Wilcoxon matched-pairs signed rank test:
vaccinated individuals were analyzed (n = 11). p > 0.05, not significant (ns); *p < 0.05; **p < 0.01; ***p < 0.001).
The findings that the Omicron spike facilitated efficient entry pandemic indicate that this high level of protection might not
into several human cell lines and robustly bound human ACE2 apply to reinfection with the Omicron variant. Thus, neutralization
suggest that the Omicron variant readily infects human cells. of the Omicron spike was 80-fold less efficient as compared with
Nevertheless, some peculiarities of host cell entry driven by the controls and several sera did not exert neutralizing activity.
Omicron spike relative to the B.1 spike were noted. The Omicron Although neutralization by sera from patients infected with the
spike mediated slightly but significantly augmented entry into Delta variant remains to be examined, it is likely that convales-
cell lines which only allow for cathepsin L-(293T, Huh-7, A549- cent patients might not be adequately protected against symp-
ACE2) but not TMPRSS2-dependent entry, due to low or absent tomatic reinfection with the Omicron variant, in keeping with
TMPRSS2 expression (Hoffmann et al., 2020b). We previously recent data (Pulliam et al., 2021).
observed a similar phenotype for the S protein of another African Neutralizing antibody responses are also believed to be critical
SARS-CoV-2 lineage, A.30 (Arora et al., 2021a), and the muta- for protection against COVID-19 by BNT vaccination (Corbett
tions in the spike responsible for this phenotype remain to be et al., 2021; Feng et al., 2021; Gilbert et al., 2021). We found
determined. Further, the Omicron spike used ACE2 proteins that antibodies induced upon BNT/BNT immunization neutral-
from different animal species with high efficiency for entry. The ized the Omicron spike with 34-fold reduced efficiency as
efficient usage of murine ACE2 is in keeping with the presence compared with B.1. Therefore, two doses of BNT might not pro-
of the mutations K417N and N501Y, which can also emerge vide robust protection against severe disease induced by the
upon SARS-CoV-2 adaptation to experimentally infected mice Omicron variant and adaptation of the vaccine to the new variant
(Huang et al., 2021; Sun et al., 2021; Zhang et al., 2021b). In addi- seems required. In the meantime, heterologous AZ/BNT vacci-
tion, the Omicron spike harbors mutations Q493R and Q498R, nation or a BNT booster (BNT/BNT/BNT) might afford some pro-
which are related to exchanges Q493K and Q498H, which tection against the Omicron variant, since sera from individuals
were also detected in mouse-adapted SARS-CoV-2 (Huang who had received the respective vaccinations neutralized the
et al., 2021; Sun et al., 2021; Zhang et al., 2021b). However, Omicron spike with appreciable efficiency. However, it remains
some of these mutations are present in other VOCs and cannot to be determined whether this protection is transient or long
be taken as evidence that the Omicron variant may have evolved lasting.
in infected mice in the wild. In addition, the Omicron spike was
able to use ACE2 from the Pearson’s horseshoe bat (Rhinolo- Limitations of the study
phus pearsonii) with high efficiency. Again, this finding does Our study has several limitations, including the analysis of a
not indicate that the Omicron variant infects these animals limited number of sera/plasma collected within 3 months after
(which are found in Asia) in the wild but rather reflects the ability vaccination and use of pseudotyped virus instead of authentic
of the Omicron spike to use diverse ACE2 orthologues for entry. SARS-CoV-2 Omicron variant, which was not available to us.
Collectively, our results suggest that the mutations in the Omi- Further, T cell responses were not determined in our study. How-
cron spike are compatible with robust usage of diverse ACE2 or- ever, given the important role that antibodies play in immune pro-
thologues for entry and might thus have broadened the ability of tection against SARS-CoV-2, our results suggest that antibody
the Omicron variant to infect animal species. cocktails used for therapy and likely also vaccines must be
Cocktails of the antibodies casirivimab and imdevimab as well adapted for efficient protection against the Omicron variant.
as etesevimab and bamlanivimab are used for COVID-19 ther- While such adaptations are in progress, heterologous or booster
apy. Entry driven by the Omicron spike was fully or largely resis- immunizations could help to limit the impact of the Omicron
tant against each of these antibodies and against the antibody variant on public health, and conventional control measures
cocktails, most likely due to the mutations K417N, N440K, like face masks and social distancing should be maintained.
G446S, S477N, T478K, E484A, Q493R, G496S, Q498R,
N501Y, and Y505H, which are located within or close to the epi- STAR+METHODS
topes bound by these antibodies (Figure 2B). Thus, two
frequently used COVID-19 treatment options will not be available Detailed methods are provided in the online version of this paper
to combat the Omicron variant. In contrast, sotrovimab, a pan- and include the following:
sarbecovirus-neutralizing antibody (Gupta et al., 2021),
remained active against the Omicron spike, in keeping with so- d KEY RESOURCES TABLE
trovimab recognizing an epitope not substantially altered by mu- d RESOURCE AVAILABILITY
tations found in the Omicron spike (Figure 2B). Finally, soluble B Lead contact
ACE2 robustly blocked entry driven by the Omicron spike and B Materials availability
might be an option for treatment of patients infected with the B Data and code availability
Omicron variant. d EXPERIMENTAL MODEL AND SUBJECT DETAILS
Studies conducted before the emergence of the Omicron B Cell culture
variant indicated that convalescent COVID-19 patients are effi- d METHOD DETAILS
ciently protected against reinfection and antibody responses B Expression plasmids
likely play an important role in protection (Addetia et al., 2020; B Sequence analysis and protein models
Hall et al., 2021; Hansen et al., 2021; Harvey et al., 2021a; Kram- B Pseudotyping
mer, 2021; Lumley et al., 2021). Our findings with serum/plasma B Analysis of spike protein-mediated cell entry
samples collected in Germany during the first wave of the B Analysis of ACE2 binding
Abdullah, F. (2021). Tshwane District Omicron variant patient profile - early fea- Gilbert, P.B., Montefiori, D.C., McDermott, A.B., Fong, Y., Benkeser, D., Deng,
tures. https://www.samrc.ac.za/news/tshwane-district-omicron-variant-patient- W., Zhou, H., Houchens, C.R., Martins, K., Jayashankar, L., et al. (2021). Im-
profile-early-features. mune correlates analysis of the mRNA-1273 COVID-19 vaccine efficacy clin-
ical trial. Science, eab3435. https://doi.org/10.1126/science.abm3425.
Addetia, A., Crawford, K.H.D., Dingens, A., Zhu, H., Roychoudhury, P., Huang,
M.-L., Jerome, K.R., Bloom, J.D., and Greninger, A.L. (2020). Neutralizing an- Graham, F. (2021). Daily briefing: Omicron was already spreading in Europe.
tibodies correlate with protection from SARS-CoV-2 in humans during a fishery Nature. https://doi.org/10.1038/d41586-021-03610-3.
vessel outbreak with a high attack rate. J. Clin. Microbiol. 58 https://doi.org/10. Gu, H., Krishnan, P., Ng, D.Y.M., Chang, L.D.J., Liu, G.Y.Z., Cheng, S.S.M.,
1128/JCM.02107-20. Hui, M.M.Y., Fan, M.C.Y., Wan, J.H.L., Lau, L.H.K., et al. (2021). Probable
Arora, P., Rocha, C., Kempf, A., Nehlmeier, I., Graichen, L., Winkler, M.S., Lier, transmission of SARS-CoV-2 omicron variant in quarantine hotel, Hong
M., Schulz, S., Jäck, H.-M., Cossmann, A., et al. (2021a). The spike protein of Kong, China, November 2021. Emerg. Infect. Dis. 28 https://doi.org/10.
SARS-CoV-2 variant A.30 is heavily mutated and evades vaccine-induced an- 3201/eid2802.212422.
tibodies with high efficiency. Cell. Mol. Immunol. 18, 2673–2675. https://doi. Gupta, A., Gonzalez-Rojas, Y., Juarez, E., Crespo Casal, M., Moya, J., Falci,
org/10.1038/s41423-021-00779-5. D.R., Sarkis, E., Solis, J., Zheng, H., Scott, N., et al. (2021). Early treatment
STAR+METHODS
Continued
REAGENT or RESOURCE SOURCE IDENTIFIER
Vaccinee serum (4797) Laboratory of Georg M.N. Behrens N/A
Vaccinee serum (6187) Laboratory of Georg M.N. Behrens N/A
Vaccinee serum (6227) Laboratory of Georg M.N. Behrens N/A
Vaccinee serum (6208) Laboratory of Georg M.N. Behrens N/A
Vaccinee serum (6043) Laboratory of Georg M.N. Behrens N/A
Vaccinee serum (6029) Laboratory of Georg M.N. Behrens N/A
Vaccinee serum (6025) Laboratory of Georg M.N. Behrens N/A
Vaccinee serum (6028) Laboratory of Georg M.N. Behrens N/A
Vaccinee serum (6023) Laboratory of Georg M.N. Behrens N/A
Vaccinee serum (6054) Laboratory of Georg M.N. Behrens N/A
Vaccinee serum (6003) Laboratory of Georg M.N. Behrens N/A
Vaccinee serum (7539) Laboratory of Georg M.N. Behrens N/A
Vaccinee serum (7533) Laboratory of Georg M.N. Behrens N/A
Vaccinee serum (7423) Laboratory of Georg M.N. Behrens N/A
Vaccinee serum (7426) Laboratory of Georg M.N. Behrens N/A
Vaccinee serum (7532) Laboratory of Georg M.N. Behrens N/A
Vaccinee serum (7580) Laboratory of Georg M.N. Behrens N/A
Vaccinee serum (7773) Laboratory of Georg M.N. Behrens N/A
Vaccinee serum (7562) Laboratory of Georg M.N. Behrens N/A
Vaccinee serum (7585) Laboratory of Georg M.N. Behrens N/A
Vaccinee serum (7595) Laboratory of Georg M.N. Behrens N/A
Chemicals, peptides, and recombinant proteins
Soluble human ACE2 (sol-hACE2-Fc) Laboratory of Stefan Pöhlmann N/A
Critical commercial assays
Beetle-Juice Kit PJK Cat# 102511
GeneArt Gibson Assembly HiFi Master Mix Thermo Fisher Scientific Cat# A46627
Experimental models: Cell lines
293T DSMZ Cat# ACC-635;
RRID: CVCL_0063
A549 (ACE2) Laboratory of Stefan Pöhlmann N/A
BHK-21 Laboratory of Georg Herrler ATCC Cat# CCL-10;
RRID:CVCL_1915
Caco-2 Laboratory of Stefan Pöhlmann ATCC Cat# HTB-37; RRID: CVCL_0025
Calu-3 Laboratory of Stephan Ludwig ATCC Cat# HTB-55;
RRID: CVCL_0609
Huh-7 Laboratory of Thomas Pietschmann JCRB Cat# JCRB0403;
RRID: CVCL_0336
Vero Laboratory of Andrea Maisner ATCC Cat# CRL-1586;
RRID: CVCL_0574
Oligonucleotides
Human ACE2 (NotI) F (TGATCC Sigma-Aldrich N/A
GCGGCCGCATGTCAAGCTCT
TCCTGGCTCC)
Human ACE2-cMYC (PacI) R (GATCCG Sigma-Aldrich N/A
TTAATTAACTACAGATCTTCTTCGCTA
ATCAGTTTCTGTTCAAAGGAGGTCTG
AACATCATCAG)
Masked Palm civet ACE2 (NotI) F Sigma-Aldrich N/A
(TGATCCGCGGCCGCATGTCA
GGCTCTTTCTGGCTCC)
(Continued on next page)
Continued
REAGENT or RESOURCE SOURCE IDENTIFIER
Masked Palm civet ACE2-cMYC (PacI) Sigma-Aldrich N/A
R (GATCCGTTAATTAACTACAGATCTT
CTTCGCTAATCAGTTTCTGTTCAAATG
AAGTCTGAACGTCATCAGC)
Raccoon dog ACE2 (NotI) F (TGATCC Sigma-Aldrich N/A
GCGGCCGCGCCACCATGTCAGGC
TCTTCCTGGCTCC)
Raccoon dog ACE2-cMYC (PacI) R (GATC Sigma-Aldrich N/A
CGTTAATTAACTACAGATCTTCTTCGCTA
ATCAGTTTCTGTTCAAATGAAGTCTGA
GCATCATCCAC)
American mink ACE2 (NotI) F (TGATCC Sigma-Aldrich N/A
GCGGCCGCATGTTAGGCTCTTCCTG
GCTCC)
American mink ACE2-cMYC (PacI) R Sigma-Aldrich N/A
(GATCCGTTAATTAACTACAGATCTTCTT
CGCTAATCAGTTTCTGTTCAAATGACGT
CTGAACATCATCGAC)
Cat ACE2 (NotI) F (TGATCCGCGGCCGC Sigma-Aldrich N/A
ATGTCAGGCTCTTTCTGGCTCC)
Cat ACE2-cMYC (PacI) R (GATCCG Sigma-Aldrich N/A
TTAATTAACTACAGATCTTCTTCGC
TAATCAGTTTCTGTTCAAATGAAGT
CTGAACATCATCAGC)
Red fox ACE2 (NotI) F (TGATCC Sigma-Aldrich N/A
GCGGCCGCATGAGCGGCTCC
AGCTGGCTGC)
Red fox ACE2-cMYC (PacI) R (GATCCG Sigma-Aldrich N/A
TTAATTAACTACAGATCTTCTTCGCTAA
TCAGTTTCTGTTCGAAGCTTGTCTG
GGCGTCATCC)
Malayan pangolin ACE2 (NotI) F (TGATCC Sigma-Aldrich N/A
GCGGCCGCATGAGCGGCAGCAGCTG
GCTGC)
Malayan pangolin ACE2-cMYC (PacI) R Sigma-Aldrich N/A
(GATCCGTTAATTAACTACAGATCTTCT
TCGCTAATCAGTTTCTGTTCGAAGCT
GGTCTGCACGTCGTCC)
Pig ACE2 (NotI) F (TGATCC Sigma-Aldrich N/A
GCGGCCGCATGTCAGGC
TCTTTCTGGCTCC)
Pig ACE2-cMYC (PacI) R (GATCCG Sigma-Aldrich N/A
TTAATTAACTACAGATCTTCTTCG
CTAATCAGTTTCTGTTCAAACGA
AGTCTGAATGTCATCG)
Mouse ACE2 (NotI) F (TGATCC Sigma-Aldrich N/A
GCGGCCGCATGTCCAGCTCC
TCCTGGCTCC)
Mouse ACE2-cMYC (PacI) R (GATCCG Sigma-Aldrich N/A
TTAATTAACTACAGATCTTCTTCGCTA
ATCAGTTTCTGTTCAAAGGAAGTCTG
AGCATCATCAC)
Bat (Rhinolophus affinis) ACE2 (NotI) F Sigma-Aldrich N/A
(TGATCCGCGGCCGCATGTCAGGCT
CTTCCTGGCTCC)
(Continued on next page)
Continued
REAGENT or RESOURCE SOURCE IDENTIFIER
Bat (Rhinolophus affinis) ACE2-cMYC (PacI) Sigma-Aldrich N/A
R (GATCCGTTAATTAACTACAGATCTTC
TTCGCTAATCAGTTTCTGTTCAAAGGAG
GTCTGAACATCATCACC)
Bat (Rhinolophus landeri) ACE2 (NotI) F Sigma-Aldrich N/A
(TGATCCGCGGCCGCATGTCAGGCT
CTTCCTGGCTCTTTC)
Bat (Rhinolophus landeri) ACE2-cMYC Sigma-Aldrich N/A
(PacI) R (GATCCGTTAATTAACTACAG
ATCTTCTTCGCTAATCAGTTTCTGTTC
AAAGGAGGTCTGAACATCATCACC)
Bat (Rhinolophus pearsonii) ACE2 (NotI) Sigma-Aldrich N/A
F (TGATCCGCGGCCGCATGTCAGGCT
CTTTCTGGTTCC)
Bat (Rhinolophus pearsonii) ACE2-cMYC Sigma-Aldrich N/A
(PacI) R (GATCCGTTAATTAACTACAGA
TCTTCTTCGCTAATCAGTTTCTGTTC
AAAGGAGGTCTGAACATCATCACC)
Bat (Rhinolophus sinicus) ACE2 (NotI) F Sigma-Aldrich N/A
(TGATCCGCGGCCGCATGTCAGGCTC
TTCCTGGCTCC)
Bat (Rhinolophus sinicus) ACE2-cMYC Sigma-Aldrich N/A
(PacI) R (GATCCGTTAATTAACTACAG
ATCTTCTTCGCTAATCAGTTTCTGTTCA
AACGAAGTCTGAACATCATCACC)
SARS-2-S Seq-01 (CAAGATCTACAGCA Sigma-Aldrich N/A
AGCACACC)
SARS-2-S Seq-02 (GTCGGCGGCAACTA Sigma-Aldrich N/A
CAATTAC)
SARS-2-S Seq-03 (GCTGTCTGATCGGA Sigma-Aldrich N/A
GCCGAG)
SARS-2-S Seq-04 (TGAGATGATCGCCCA Sigma-Aldrich N/A
GTACAC)
SARS-2-S Seq-05 (GCCATCTGCCACGA Sigma-Aldrich N/A
CGGCAAAG)
pCG1 F (CCTGGGCAACGTGCTGGT) Sigma-Aldrich N/A
pCG1 R (GTCAGATGCTCAAGGGG Sigma-Aldrich N/A
CTTCA)
Recombinant DNA
SARS-2-SD18 (Omicron), Thermo Fisher Scientific N/A
codon-optimized, DNA strings
Plasmid: pCG1 Laboratory of Roberto Cattaneo N/A
Plasmid: pCAGGS-VSV-G Laboratory of Stefan Pöhlmann N/A
Plasmid: pCAGGS-DsRed Laboratory of Stefan Pöhlmann N/A
Plasmid: pCG1-SARS-2-SD18 (B.1), Laboratory of Stefan Pöhlmann N/A
codon-optimized
Plasmid: pCG1-SARS-2-SD18 (Alpha), Laboratory of Stefan Pöhlmann N/A
codon-optimized
Plasmid: pCG1-SARS-2-SD18 (Beta), Laboratory of Stefan Pöhlmann N/A
codon-optimized
Plasmid: pCG1-SARS-2-SD18 (Gamma), Laboratory of Stefan Pöhlmann N/A
codon-optimized
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Continued
REAGENT or RESOURCE SOURCE IDENTIFIER
Plasmid: pCG1-SARS-2-SD18 (Delta), Laboratory of Stefan Pöhlmann N/A
codon-optimized
Plasmid: pCG1-SARS-2-SD18 (Omicron), This study N/A
codon-optimized
Plasmid: pQCXIP_Human ACE2-cMYC This study N/A
Plasmid: pQCXIP_Masked palm civet This study N/A
ACE2-cMYC
Plasmid: pQCXIP_Raccoon dog This study N/A
ACE2-cMYC
Plasmid: pQCXIP_American Mink This study N/A
ACE2-cMYC
Plasmid: pQCXIP_Cat ACE2-cMYC This study N/A
Plasmid: pQCXIP_Red fox ACE2-cMYC This study N/A
Plasmid: pQCXIP_Pangolin ACE2-cMYC This study N/A
Plasmid: pQCXIP_Pig ACE2-cMYC This study N/A
Plasmid: pQCXIP_Mouse ACE2-cMYC This study N/A
Plasmid: pQCXIP_Bat (Rhinolophus affinis) This study N/A
ACE2-cMYC
Plasmid: pQCXIP_Bat (Rhinolophus landeri) This study N/A
ACE2-cMYC
Plasmid: pQCXIP_Bat (Rhinolophus This study N/A
pearsonii) ACE2-cMYC
Plasmid: pQCXIP_Bat (Rhinolophus sinicus) This study N/A
ACE2-cMYC
Plasmid: pCG1-solACE2-Fc Laboratory of Stefan Pöhlmann N/A
Software and algorithms
Hidex Sense Microplate Reader Software Hidex Deutschland Vertrieb GmbH https://www.hidex.de
YASARA (version 19.1.27) YASARA Biosciences GmbH http://www.yasara.org
Adobe Photoshop CS5 Extended (version Adobe https://www.adobe.com/
12.0 x 32)
GraphPad Prism (version 8.3.0(538)) GraphPad Software https://www.graphpad.com/
SWISS-MODEL online tool Protein Structure Bioinformatics Group, https://swissmodel.expasy.org
Swiss Institute of Bioinformatics
Biozentrum, University of Basel
Flowing software (version 2.5.1) Turku Bioscience https://bioscience.fi/services/cell-imaging/
flowing-software/
Microsoft Office Standard 2010 (version Microsoft Corporation https://products.office.com/
14.0.7232.5000)
Other
Complex of SARS-CoV-2 receptor binding (Hansen et al., 2020) https://www.rcsb.org/structure/6XDG
domain with the Fab fragments of two
neutralizing antibodies (PDB: 6XDG)
SARS-CoV 2 Spike Protein bound to LY- (Jones et al., 2021) https://www.rcsb.org/structure/7L3N
CoV555 (PDB: 7L3N)
Molecular basis for a potent human (Shi et al., 2020) https://www.rcsb.org/structure/7C01
neutralizing antibody targeting SARS-CoV-
2 RBD (PDB: 7C01)
Structure of the SARS-CoV-2 spike (Pinto et al., 2020) https://www.rcsb.org/structure/6WPT
glycoprotein in complex with the S309
neutralizing antibody Fab fragment (open
state) (PDB: 6WPT)
Distinct conformational states of (Cai et al., 2020) https://www.rcsb.org/structure/6XR8
SARS-CoV-2 spike protein (PDB: 6XR8)
RESOURCE AVAILABILITY
Lead contact
Requests for material can be directed to Markus Hoffmann (mhoffmann@dpz.eu) and the lead contact, Stefan Pöhlmann
(spoehlmann@dpz.eu).
Materials availability
All materials and reagents will be made available upon installment of a material transfer agreement (MTA).
Cell culture
The following cells lines were used in the present study: 293T (human, female, kidney; ACC-635, DSMZ; RRID: CVCL_0063), A549-
ACE2 ((Huang et al., 2021); based on parental A549 cells, human, male, lung; CRM-CCL-185, ATCC), BHK-21 (Syrian hamster, male,
kidney; ATCC Cat# CCL-10; RRID: CVCL_1915, kindly provided by Georg Herrler, University of Veterinary Medicine Hannover, Ger-
many), Vero (African green monkey kidney, female, kidney; CRL-1586, ATCC; RRID: CVCL_0574, kindly provided by Andrea Mais-
ner), Huh-7 cells (human, male, liver; JCRB Cat# JCRB0403; RRID: CVCL_0336, kindly provided by Thomas Pietschmann), Calu-3
(human, male, lung; HTB-55, ATCC; RRID: CVCL_0609, kindly provided by Stephan Ludwig) and Caco-2 cells (human, male, colon;
HTB-37, ATCC, RRID: CVCL_0025). 293T, BHK-21, Vero and Huh-7 cells were maintained in Dulbecco’s modified Eagle medium
(DMEM, PAN-Biotech). Calu-3 and Caco-2 cells were cultured in minimum essential medium (GIBCO) while A549-ACE2 cells
were maintained in DMEM/F-12 medium (GIBCO). All media were supplemented with 10% fetal bovine serum (Biochrom) and
100 U/ml penicillin and 0.1 mg/ml streptomycin (PAA). Further, media for Calu-3 and Caco-2 cells were supplemented with 1x
non-essential amino acid solution (from 100x stock, PAA) and 1 mM sodium pyruvate (GIBCO). Cell lines were validated 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, all cell lines were regularly tested for mycoplasma contamination. For transfection of 293T
cells, the calcium-phosphate based method was applied, while BHK-21 cells were transfected using Lipofectamine 2000 (Thermo
Fisher Scientific).
METHOD DETAILS
Expression plasmids
Plasmids encoding DsRed, VSV-G (vesicular stomatitis virus glycoprotein), soluble ACE2, SARS-CoV-2 S B.1 (codon optimized, con-
tains C-terminal truncation of the last 18 amino acid) and SARS-CoV-2 S of VOCs Alpha (B.1.1.7), Beta (B.1.351), Gamma (P.1) and
Delta (B.1.617.2) have been previously reported (Brinkmann et al., 2017; Hoffmann et al., 2021a, 2021b). The expression vector for
the Omicron spike (based on isolate hCoV-19/Botswana/R40B58_BHP_3321001245/2021; GISAID Accession ID: EPI_ISL_6640919)
was generated by Gibson assembly using five overlapping DNA strings (Thermo Fisher Scientific, sequences available upon request),
linearized (BamHI/XbaI digest) pCG1 plasmid and GeneArt Gibson Assembly HiFi Master Mix (Thermo Fisher Scientific). Gibson
assembly was performed according to manufacturer’s instructions. The pCG1 vector was kindly provided by Roberto Cattaneo
(Mayo Clinic College of Medicine, Rochester, MN, USA). To generate expression plasmids for different ACE2 orthologues (harboring
a C-terminal cMYC-epitope tag) the respective sequences were amplified from existing plasmids (human, pangolin, cat, masked
palm civet and red fox, Rhinolophus landeri ACE2) (Hoffmann et al., 2021b; Krüger et al., 2021; Li et al., 2021a) or ACE2 sequences
were obtained from a commercial gene synthesis service (raccoon dog, American mink, Pig, Mouse, Rhinolophus affinis, Rhinolo-
phus sinicus, Rhinolophus pearsonii; GeneArt). Expression plasmids for pangolin, masked palm civet and red fox ACE2 used as tem-
plates for cloning were kindly provided by Zhaohui Qian (Chinese Academy of Medical Sciences and Peking Union Medical College,
Beijing, China). ACE2 open reading frames were inserted into the pQCXIP expression vector making use of NotI and PacI restriction
sites. The integrity of all expression plasmids was confirmed using a commercial sequencing service (Microsynth SeqLab).
(https://swissmodel.expasy.org). Alternatively, the following published crystal structures were employed; PDB: 6XDG (Hansen et al.,
2020), PDB: 7L3N (Jones et al., 2021), PDB: 7C01 (Shi et al., 2020) or PDB: 6WPT (Pinto et al., 2020).
Pseudotyping
Rhabdoviral pseudotypes harboring SARS-CoV-2 S protein were produced as described (Kleine-Weber et al., 2019). In brief, 293T
cells were transfected with expression plasmids for SARS-CoV-2 S protein, VSV-G or empty plasmid (control). At 24 h posttransfec-
tion, cells were infected with at an MOI of 3 with a replication-deficient reporter VSV, termed VSV*DG-FLuc (Berger Rentsch and Zim-
mer, 2011). This virus lacks the ORF for VSV-G and codes for enhanced green fluorescent protein and firefly luciferase (FLuc). After
1 h of incubation, the input virus was removed and cells were washed with phosphate-buffered saline (PBS). Thereafter, culture me-
dium containing anti-VSV-G antibody (culture supernatant from I1-hybridoma cells; ATCC no. CRL-2700) was added in order to
neutralize residual inpunt virus. For cells expressing VSV-G, culture medium without antibody was added. After 16-18 h, the culture
supernatant was harvested, separated from cellular debris by centrifugation for 10 min at 4,000 x g at room temperature (RT), and the
clarified supernatants were aliquoted and stored at -80 C.
Neutralization assay
For neutralization experiments, S protein bearing particles were pre-incubated for 30 min at 37 C with Casirivimab, Imdevimab,
Bamlanivimab, Etesevimab, Casirivimab + Imdevimab, Bamlanivimab + Etesevimab, Sotrovimab or unrelated control human IgG
(5, 0.5, 0.05, 0.005, 0.0005 mg/ml). Alternatively, pseudotyped particles were pre-incubated with different dilutions of convalescent
or vaccinated plasma/serum (dilution range: 1:25 to 1:12,800). After incubation, the mixtures were added to Vero cells. Particles
exposed to medium without antibodies served as control. Transduction efficiency was determined at 16-18 h postinoculation by
determining luciferase activities in cell lysates, as described above.
The results on S protein-driven cell entry represent average (mean) data acquired from three to twelve biological replicates, each
conducted with four technical replicates. The transduction was normalized against SARS-CoV-2 S B.1 (= 1). Alternatively, transduc-
tion was normalized against the background signal (luminescence measured for cells inoculated with particles bearing no viral glyco-
protein; set as 1). For ACE2 binding, presented are the average (mean) geometric mean channel fluorescence data from six biological
replicates, each conducted with single samples. The results on neutralization of S protein-driven cell entry by monoclonal antibodies
and control IgG represent average (mean) data from three biological replicates (each conducted with technical quadruplicates) for
which transduction was normalized against samples that did not contain any antibody (= 0% inhibition). The results on inhibition
of S protein-driven cell entry by soluble ACE2 represent average (mean) data from three biological replicates (each conducted
with technical quadruplicates) for which transduction was normalized against samples that did not contain soluble ACE2 (= 0% in-
hibition). The results on neutralization of spike protein-driven cell entry by convalescent plasma/serum or serum from vaccinated in-
dividuals are based on a single experiment, which was conducted with technical quadruplicates. For data normalization, the plasma/
serum dilution factor that leads to 50% reduction in S protein-driven cell entry (neutralizing titer 50, NT50) was calculated. In addition,
for each plasma/serum the fold change in NT50 between the indicated SARS-CoV-2 variants was calculated.
Error bars are defined as either standard deviation (SD) or standard error of the mean (SEM). Data were analyzed using Microsoft
Excel (as part of the Microsoft Office software package, version 2019, Microsoft Corporation) and GraphPad Prism 8 version 8.4.3
(GraphPad Software). Statistical significance was analyzed by two-tailed Student’s t-test with Welch correction (pseudotype entry,
ACE2 binding), two-way analysis of variance (ANOVA) with Dunnett’s or Sidak’s post-hoc test (soluble ACE2 inhibition, and mono-
clonal antibody neutralization) or Wilcoxon matched-pairs signed rank test (serum/plasma neutralization). Only p-values of 0.05 or
lower were considered statistically significant (p > 0.05, not significant [ns]; p % 0.05, *; p % 0.01, **; p % 0.001, ***). Details on
the statistical test and the error bars can be found in the figure legends.
Supplemental figures
Figure S1. Cell entry driven by the spike proteins of VOC (related to Figure 1F)
Pseudotype entry data presented in Figure 1F normalized against the assay background. Pseudotype entry was normalized against signals obtained from cells
inoculated with particles bearing no viral glycoprotein (background, set as 1), and data obtained for particles bearing VSV-G were included. Error bars indicate
the SEM.
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Article