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https://doi.org/10.1038/s41591-022-01792-5
Serum neutralization of SARS-CoV-2 Omicron
sublineages BA.1 and BA.2 in patients receiving
monoclonal antibodies
Timothée Bruel 1,2,16 ✉
, Jérôme Hadjadj3,16, Piet Maes 4,16, Delphine Planas1,2, Aymeric Seve5,
Isabelle Staropoli1, Florence Guivel-Benhassine1, Françoise Porrot 1, William-Henry Bolland1,6,
Yann Nguyen 3, Marion Casadevall3, Caroline Charre7,8,9, Hélène Péré10,11, David Veyer10,11,
Matthieu Prot12, Artem Baidaliuk12, Lize Cuypers13, Cyril Planchais14, Hugo Mouquet14, Guy Baele 4,
Luc Mouthon3, Laurent Hocqueloux 5, Etienne Simon-Loriere 12,17, Emmanuel André13,15,17,
Benjamin Terrier3,17, Thierry Prazuck5,17 and Olivier Schwartz 1,2,17 ✉
The severe acute respiratory syndrome coronavirus 2 Omicron BA.1 sublineage has been supplanted in many countries by the
BA.2 sublineage. BA.2 differs from BA.1 by about 21 mutations in its spike. In this study, we first compared the sensitivity of
BA.1 and BA.2 to neutralization by nine therapeutic monoclonal antibodies (mAbs). In contrast to BA.1, BA.2 was sensitive to
cilgavimab, partly inhibited by imdevimab and resistant to adintrevimab and sotrovimab. We then analyzed sera from 29 immu-
nocompromised individuals up to 1 month after administration of Ronapreve (casirivimab and imdevimab) and/or Evusheld
(cilgavimab and tixagevimab) antibody cocktails. All treated individuals displayed elevated antibody levels in their sera, which
efficiently neutralized the Delta variant. Sera from Ronapreve recipients did not neutralize BA.1 and weakly inhibited BA.2.
Neutralization of BA.1 and BA.2 was detected in 19 and 29 out of 29 Evusheld recipients, respectively. As compared to the Delta
variant, neutralizing titers were more markedly decreased against BA.1 (344-fold) than BA.2 (nine-fold). We further report
four breakthrough Omicron infections among the 29 individuals, indicating that antibody treatment did not fully prevent infec-
tion. Collectively, BA.1 and BA.2 exhibit noticeable differences in their sensitivity to therapeutic mAbs. Anti-Omicron neutral-
izing activity of Ronapreve and, to a lesser extent, that of Evusheld is reduced in patients’ sera.
The severe acute respiratory syndrome coronavirus 2 strain and prior variants of concern (VOCs), to an extent similar to
(SARS-CoV-2) Omicron variant comprises three main sublin- BA.1 (r
efs. 11–14). BA.2 also displays a marked decreased sensitivity to
eages, termed BA.1, BA.2 and BA.3 (ref
. 1). The original BA.1 many neutralizing mAbs when compared to previous VOCs
11,12,14–16.
sublineage (also termed B.1.1.529) was identified in November 2021
Neutralizing mAbs targeting the receptor-binding domain
and became dominant worldwide in about 2 months. BA.1 dem- (RBD) of the SARS-CoV-2 spike protein have been isolated from
onstrated considerable escape from neutralization by mAbs and COVID-19 convalescent individuals and demonstrated efficacy in
sera from vaccinated individuals
2–10. BA.2 cases have now sharply preventing or treating disease in human
s17,18. Some mAbs are used
increased, suggesting that it is more transmissible and possesses in combination, such as Ronapreve (imdevimab and casirivimab)
a selective advantage over BA.1. As of March 2022, BA.2 was the from Regeneron and Evusheld (cilgavimab and tixagevimab) from
dominant sublineage in many countries, including Denmark, the AstraZeneca. Evusheld mAbs are modified in their Fc regions to
Philippines, South Africa, France and Belgium. BA.1 and BA.2 have improve half-life and decrease Fc effector function
s8. Post-exposure
many mutations in common, but about 21 mutations in the spike administration of Ronapreve prevented 84% of infections in a ran-
protein differentiate the two sublineages (Fig.
1a). Neutralizing domized clinical trial, which was conducted before Omicron cir-
activity of sera from Coronavirus Disease 2019 (COVID-19) vac- culation
19. In a preclinical model, Evusheld protected macaques
cine recipients is reduced against BA.2 relative to the ancestral from infection with ancestral SARS-CoV-2 (ref
. 20). A press release
1Institut Pasteur, Université Paris Cité, CNRS UMR3569, Virus and Immunity Unit, Paris, France. 2Vaccine Research Institute, Créteil, France. 3Department
of Internal Medicine, National Reference Center for Rare Systemic Autoimmune Diseases, AP-HP, APHP.CUP, Hôpital Cochin, Paris, France. 4KU Leuven,
Department of Microbiology, Immunology and Transplantation, Laboratory of Clinical and Epidemiological Virology, Leuven, Belgium. 5CHR d’Orléans,
Service de Maladies Infectieuses, Orléans, France. 6Université Paris Cité, École doctorale BioSPC 562, Paris, France. 7Université Paris Cité, Faculté de
Médecine, Paris, France. 8INSERM U1016, CNRS UMR8104, Institut Cochin, Paris, France. 9AP-HP, Laboratoire de Virologie, CHU Cochin, Paris, France.
10INSERM, Functional Genomics of Solid Tumors (FunGeST), Centre de Recherche des Cordeliers, Université de Paris and Sorbonne Université, Paris,
France. 11Laboratoire de Virologie, Service de Microbiologie, Hôpital Européen Georges Pompidou, Assistance Publique des Hôpitaux de Paris, Paris, France.
12G5 Evolutionary Genomics of RNA Viruses, Institut Pasteur, Université Paris Cité, Paris, France. 13University Hospitals Leuven, Department of Laboratory
Medicine, National Reference Centre for Respiratory Pathogens, Leuven, Belgium. 14Humoral Immunology Laboratory, Institut Pasteur, Université Paris
Cité, INSERM U1222, Paris, France. 15KU Leuven, Department of Microbiology, Immunology and Transplantation, Laboratory of Clinical Microbiology,
Leuven, Belgium. 16These authors contributed equally: Timothée Bruel, Jérôme Hadjadj, Piet Maes. 17These authors jointly supervised this work: Etienne
Simon-Loriere, Emmanuel André, Benjamin Terrier, Thierry Prazuck, Olivier Schwartz. ✉
e-mail: xxxxxxxx.xxxxx@xxxxxxx.xx;
xxxxxxx.xxxxxxxx@xxxxxxx.xx
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a
1
100
180
260
340
420
500
580
660
795
965
1,135
1,273
Spike
S1/S2
ORF1ab
NTD
RBD
RBM
SD1
SD2
N
19
24 25 26 27
67 69 70
95 142 143 144 145 156 157 158 211 212 213 214 339 371 373 375 376 405 408 417 440 446 452 477 478 484 493 496 498 501 505 547 614 655 679 681 764 796 856 950 954 969 981
Ancestral
Delta
BA.1
BA.2
Common mutations
Unique mutations
b
Bamlanivimab
Etesivimab
Casirivimab
Imdevimab
Adintrevimab
120
120
120
120
120
100
100
100
100
100
80
80
80
80
80
60
60
60
60
60
40
40
40
40
40
20
20
20
20
20
0
0
0
0
0
–20
–20
–20
–20
–20
–3
–2
–1
0
1
2
3
4
–3
–2
–1
0
1
2
3
4
–3
–2
–1
0
1
2
3
4
–3
–2
–1
0
1
2
3
4
–3
–2
–1
0
1
2
3
4
10
10
10
10
10
10
10
10
10
10
10
10
10
10
10
10
10
10
10
10
10
10
10
10
10
10
10
10
10
10
10
10
10
10
10
10
10
10
10
10
Sotrovimab
Regdanvimab
Tixagevimab
Cilgavimab
Evusheld
Neutralization (%)
120
120
120
120
120
100
100
100
100
100
80
80
80
80
80
60
60
60
60
60
40
40
40
40
40
20
20
20
20
20
0
0
0
0
0
–20
–20
–20
–20
–20
–3
–2
–1
0
1
2
3
4
–3
–2
–1
0
1
2
3
4
–3
–2
–1
0
1
2
3
4
–3
–2
–1
0
1
2
3
4
–3
–2
–1
0
1
2
3
4
10
10
10
10
10
10
10
10
10
10
10
10
10
10
10
10
10
10
10
10
10
10
10
10
10
10
10
10
10
10
10
10
10
10
10
10
10
10
10
10
Concentration (ng ml–1)
Delta
BA.1
BA.2
Fig. 1 | Sensitivity of Omicron BA.1 and BA.2 to therapeutic mAbs. a, Mutational landscape of Omicron BA.1 and BA.2 spike proteins. Domains of the
protein are color-coded: NTD, N-Terminal Domain; RBD, Receptor-Binding Domain; RBM, Receptor-Binding Motif; SD1, subdomain 1; SD2, subdomain 2, S1/
S2, region proximal to the furin cleavage site. Mutations in the amino acid sequence are indicated in comparison to the ancestral Wuhan-Hu-1 sequence
(GenBank:
NC_045512). Light orange boxes indicate mutations shared by BA.1 and BA.2, and orange boxes indicate mutations unique to BA.1 and BA.2.
b, Neutralization curves of mAbs. Dose–response analysis of the neutralization by the indicated antibodies and by Evusheld, a combination of cilgavimab and
tixagevimab. Data are mean ± s.d. of 2–8 independent experiments. The IC50 values for each antibody are presented in T
able 1. NTD, N-terminal domain.
from AstraZeneca indicated that intra-muscular administration the neutralizing activity of the antibodies in sera from immunocom-
of Evusheld (300 mg) reduced symptomatic disease by 83%
21. The promised individuals who had received Ronapreve and/or Evusheld.
efficacy of Evusheld in preventing virus infection is not known.
Both Ronapreve and Evushled received emergency use approval for
Results
pre-exposure prophylaxis (PrEP) in many countries. However, in We isolated a BA.2 variant from a nasopharyngeal swab that was
cell culture systems, BA.1 is resistant to casirivimab and imdevimab initial y sequenced at the National Reference Center of UZ/KU
and partial y evades cilgavimab and tixagevima
b3,4,6,
9. Different stud- Leuven (Belgium). The virus was amplified by two passages on Vero
ies have reported an 11–183-fold increase in the 50% inhibitory E6 cel s and re-sequenced (Pango lineage BA.2, 21L (Omicron),
concentration (IC50) of Evusheld against BA.1 relative to ancestral according to Nextstrain, GISAID accession ID: EPI_ISL_10654979)
strains
22. As BA.1 was becoming predominant, these results moti- (Fig.
1a). When compared to the Delta variant (B.1.617), the BA.2
vated the switch of emergency use from Ronapreve to Evusheld for spike protein contained 30 changes, with 18 modifications that
PreP in immunocompromised individuals. Besides Ronapreve and are shared with BA.1 (Fig.
1a). The modifications are dispersed
Evusheld, other mAbs are in clinical use. For instance, sotrovimab, throughout the spike but display a preferential accumulation in the
a pan-coronavirus antibody, is indicated for treatment of infected N-terminal domain and the RBD (Fig.
1a). Viral stocks were titrated
individuals at risk for severe disease
23. The relative capacity of mAbs using S-Fuse cel s. These reporter cel s become GFP+ upon infec-
to neutralize Omicron BA.1 and BA.2 sublineages is poorly charac- tion, allowing rapid measurement of viral infectivity and neutral-
terized, with discordant preliminary results regarding mAbs such as izing antibody activit
y24,25. Syncytia were observed in BA.2-infected
sotrovimab and imdevimab. The clinical significance of the reduced S-Fuse cel s, with a size similar to those induced by BA.1 (Extended
sensitivity of Omicron BA.1 and BA.2 to neutralizing antibodies Data Fig. 1). As previously reported
4, Delta-infected cel s formed
in cell culture remains unknown. To address this question, we first large syncytia, bigger than BA.1-infected or BA.2-infected cel s
evaluated the sensitivity of infectious BA.1 and BA.2 isolates to nine (Extended Data Fig. 1). This suggests that BA.1 and BA.2 may
therapeutic mAbs in a cell culture system. We then directly measured behave similarly in terms of fusogenicity and fitness.
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Table 1 | IC
Table 2 | Characteristics of patients
50 of therapeutic mAbs against Delta and against
Omicron BA.1 and BA.2
‘Orléans’
‘Cochin’
Total (%)
Delta
BA1
BA2
cohort
cohort
Bamlanivimab
>9,000
>9,000
>9,000
Patient characteristics
Etesivimab
3.8
>9,000
>9,000
n
8
21
29
Casirivimab
0.58
>9,000
>9,000
Age
58 (42–78)
62 (31–92) 61 (31–92)
Imdevimab
1.2
>9,000
693
Female
6
8
14 (64)
Adintrevimab
4.5
198
>9,000
Male
2
13
15 (52)
Regdavimab
23
>9,000
>9,000
Obesity
3
2
5 (17)
Sotrovimab
280
1,508
>9,000
Diseases
Tixagevimab
3.2
>9,000
>9,000
Rheumatoid arthritis
5
2
7 (24)
Cilgavimab
8.5
1,988
9.3
Kidney graft
2
0
2 (7)
Evusheld
2.6
715
23
Ayelodysplasia
1
0
1 (3)
IC
ANCA-associtaed vasculitis
0
17
17 (60)
50 (ng ml−1).
Polychondritis
0
1
1 (3)
Lupus
0
1
1 (3)
We first measured the sensitivity of BA.2 to a panel of nine mAbs
Medications
that were, or are currently, in clinical use
8,
26–31. These mAbs belong Rituximab (anti-CD20)
5
17
22 (76)
to the four main classes of anti-RBD antibodies, which are defined Infliximab (anti-TNF)
1
1 (3)
by their binding sit
e18,
32,33. In addition to the antibodies present in
Ronapreve (casirivimab and imdevimab) and Evusheld (cilgavimab
Prednisone
4
10
14 (48)
and tixagevimab), we tested the following antibodies: bamlanivimab
Mycofenolate mofetil
2
1
3 (10)
and etesevimab (class 2 and class 1, respectively) were initial y Methotrexate
0
3
3 (10)
mixed in the Eli Lil y cocktail and are no longer in clinical use; reg-
5-azacytidine
1
0
1 (3)
danvimab (Regkirona) (Cel trion) is a class 1 antibody; sotrovimab
(Xevudy) by GlaxoSmithKline and Vir Biotechnology is a class 3 Tacrolimus
1
0
1 (3)
antibody that targets an epitope outside of the receptor-binding Cyclosporin
1
0
1 (3)
motif (RBM); and adintrevimab (ADG20, Adagio) binds to an epi-
Vaccines
tope located in between class 1 and class 4 sites. We compared the 1st doses
activity of these nine mAbs against the Delta variant and against the
Omicron BA.1 and BA.2 sublineages (Fig
. 1b).
Pfizer
8
20
28 (97)
Seven antibodies (bamlanivimab, etesevimab, casirivimab, AstraZeneca
0
1
1 (3)
sotrovimab, adintrevimab, regdanvimab and tixagevimab) were 2nd doses
inactive against BA.2. The two other antibodies (imdevimab and
cilgavimab) displayed an IC
Pfizer
8
20
28 (97)
50 of 693 ng ml−1 and 9 ng ml−1 against
BA.2, respectively (Fig
. 1b and Tab
le 1), indicating that they were AstraZeneca
1
1 (3)
more active against BA.2 than BA.1. The addition of tixagevimab 3rd doses
to cilgavimab in the Evusheld cocktail was not more efficient than Pfizer
8
20
28 (97)
cilgavimab alone (Fig.
1b and Table
1). These results are in line with
recent repor
ts11,
12,14 and highlight substantial differences in the neu-
Moderna
1
1 (3)
tralization profiles of BA.1 and BA.2.
4th doses
We next measured antibody levels and neutralization activity in
Pfizer
3
3
6 (21)
the sera of 29 immunocompromised individuals before and after Previous COVID-19
0
1
1 (3)
administration of Evusheld (Tab
le 2). Some individuals (
n = 18 of
29) were previously treated with Ronapreve 10−49 days (mean, 35 PrEP
days) before Evusheld administration. The first group of patients was
Ronapreve
3
15
18 (62)
a cohort of eight individuals (six females and two males) from the Evusheld
8
21
29 (100)
Centre Hospitalier Regional of Orléans, France, with pre-existing
conditions, including rheumatoid arthritis (RA,
n = 5), kidney
transplantation (
n = 2) and myelodysplasia (
n = 1). Most patients doses of ChadOX-1 (AstraZeneca) and one dose of mRNA-1273
were receiving anti-CD20 (rituximab) (
n = 5) and prednisone (Moderna). Three patients received a 4th dose of BNT162b2, and
(
n = 4). These treatments were maintained before and after vacci- another had a history of COVID-19. They were mostly treated with
nation and at the time of administration of the anti-SARS-CoV-2 rituximab (
n = 17). Fifteen of the 21 individuals were already receiv-
mAbs. The patients were previously vaccinated with three doses ing Ronapreve. None of the 29 individuals elicited antibodies above
of BNT162b2 (Pfzier/BioNTech), and three had a 4th dose. Three 264 binding antibody units per milliliter (BAU ml−1) after vaccina-
patients received Ronapreve as PrEP 4−7 weeks before Evusheld. tion and were, thus, eligible to receive Evusheld PreP, according to
The second group of twenty-one patients (thirteen females and French health authority guidelin
es34.
eight males) came from Hôpital Cochin in Paris. They were suf-
We first analyzed the eight individuals from the Orléans cohort,
fering from autoimmune diseases, including RA (
n = 2), vasculitis as longitudinal samples were available (Fig
. 2a). We used the S-Flow
(
n = 17), polychondritis (
n = 1) and lupus (
n = 1). They were vacci- assay to quantify anti-spike IgGs in sera collected at days 0, 3, 15 and
nated with three doses of BNT162b2, except one who received two 30 after Evusheld administration. Day 30 sampling was available
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a
Evusheld
Ronapreve + Evusheld
#1
#2
#3
#5
#7
#4
#6
#8
104
)–1 103
102
101
Anti-S IgGs (BAU ml
100
106
)
105
50
(ED
104
tion
103
traliza
102
Neu
101
0
3
15 30
0
3
15 30
0
3
15 30
0
3
15 30
0
3
15 30
0
3
15 30
0
3
15 30
0
3
15 30
Days after Evusheld administration
b
c
Evusheld +
****
Naive
Ronapreve
Evusheld
Ronapreve
105
107
)
****
**
**
*
–1
104
) 106
****
****
50
****
*
105
103
(ED
**
tion 104
102
103
traliza
**
101
Anti-S IgGs (BAU ml
102
Neu
100
101
Delta BA.1 BA.2
Delta BA.1 BA.2
Delta BA.1 BA.2
Delta BA.1 BA.2
Naive
Ronapreve Evusheld
Delta
BA.1
BA.2
Ronapreve +
Evusheld
Fig. 2 | Neutralization of Delta and Omicron BA.1 and BA.2 by sera of immunocompromised individuals receiving Ronapreve and/or Evusheld as a
pre-exposure prophylaxis. a, Eight individuals from the Orléans cohort were followed longitudinally, before and after Evusheld administration. Anti-S IgGs
were measured using the flow cytometry-based S-Flow assay (top panel). Neutralization of Delta and Omicron BA.1 and BA.2 was measured with the
S-Fuse assay (bottom panel). The dotted lines indicate the limit of detection of the assays. Three individuals received first Ronapreve and then Evusheld.
b, Anti-S IgG levels in sera of individuals before PrEP (naive;
n = 11), treated with Ronapreve (
n = 18), treated with Evusheld (
n = 11) or treated with both
Ronapreve and Evusheld (
n = 18). Two-sided Kruskall–Wallis test with Dunn’s multiple comparison correction. Naive versus Ronapreve (
P < 0.0001), naive
versus Ronapreve+Evusheld (
P < 0.0001), Evusheld versus Ronapreve+Evusheld (
P = 0.024).
c, Sero-neutralization of Delta and Omicron BA.1 and BA.2
in the same individuals as in
b. Two-sided Friedman tests with Dunn’s multiple comparison correction were performed to compare the different groups.
Ronapreve: Delta versus BA.1 (
P ≤ 0.0001), Delta versus BA.2 (
P ≤ 0.0081), BA.1 versus BA.2 (
P ≤ 0.0081); Evusheld: Delta versus BA.1 (
P ≤ 0.0001), BA.1
versus BA.2 (
P ≤ 0.031); Evusheld+Ronapreve: Delta versus BA.1 (
P ≤ 0.0001), Delta versus BA.2 (
P ≤ 0.0081), BA.1 versus BA.2 (
P ≤ 0.0081).
for only four individuals. In the five Ronapreve-naive individuals, did not neutralize BA.1. After Evusheld treatment, seven of eight
administration of Evusheld led to a sharp increase of anti-spike IgGs individuals neutralized BA.1 at different time points between days
(from 5–57 BAU ml−1 before treatment to 195–1,290 BAU ml−1 after 3 and 30. Titers were, however, very low, ranging from 27 to 128 at
treatment) (Fig.
2a). As expected, the three individuals who initial y day 15. For most of the patients, we observed an increase of antibody
received Ronapreve had anti-spike antibodies (788-1,016 BAU ml−1) levels between days 3 and 15, reflecting the pharmacokinetics of the
at the time of Evusheld administration (day 0), with no detectable antibodies. The delayed and low neutralizing activity of the sera at
effect of Evusheld on antibody levels (Fig.
2a). In all patients, levels day 3 against BA.1 was likely due to the poor antiviral activity of
of anti-spike antibodies were stable or slightly increasing between the mAbs against this viral isolate. A low level of BA.2 neutralizing
days 3 and 30 (Fig
. 2a).
activity was detectable in the three Ronapreve-treated individuals,
We then measured the neutralizing activity of the sera against in line with the ability of imdevimab to neutralize BA.2 (Fig
. 1b).
Delta and Omicron BA.1 and BA.2 by calculating 50% effective dilu- Sera from the five Ronapreve-naive individuals did not neutralize
tion (ED50) titers with the S-Fuse assay (Fig
. 2a). None of the five BA.2 at day 0. Evusheld administration raised BA.2 neutralization in
Ronapreve-naive individuals had detectable neutralization activity all individuals, with titers reaching up to an ED50 of 3,534 at day 15
at day 0. Evusheld administration led to a sharp increase of neu- (Fig.
2b). Neutralization titers for the three viral lineages were stable
tralizing activity against Delta, with ED50s between 788 and 1,016. for six of eight individuals, consistent with Evusheld’s long half-lif
e20.
For the three individuals having previously received Ronapreve,
We extended this analysis to the 21 individuals of the second
Evusheld administration did not increase their levels of neutraliza- group, who were sampled at a single time point, 15–30 days after
tion against Delta. In line with in vitro experiments (Fig.
1b and refs. Evusheld administration. We combined the results obtained with
4,6), sera from Ronapreve-naive and Ronapreve-treated individuals the first group of eight individuals at day 15 to collectively analyze
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Table 3 | Summary of breakthrough cases
Case
Diagnostic
Strain
Days after Evusheld Anti-S (BAu ml−
1)
Neutralization BA.1 (ED50)
COVID-19
1
PCR+ screening
Omicron
15
9,630
351
Mild
2
PCR+ screening
Omicron
12
5,736
7,5
Mild
3
PCR+ screening
Omicron
21
1,786
36
Mild
4
PCR+ sequencing
BA.1
23
4,536
31
Severe
29 individuals. The nine Ronapreve-naive individuals had low levels Omicron sublineages with regard to their sensitivity to therapeu-
of anti-spike antibodies (below 264 BAU ml−1), reflecting the inef- tic mAbs. Considering that these variants have sequential y domi-
ficacy of the vaccination (Fig.
2b). Ronapreve or Evusheld therapy nated the pandemic in the last few months, and the vulnerability
strongly and similarly increased anti-spike IgGs in the sera (median of immunocompromised individuals to both Omicron infection
of 3,263 BAU ml−1 and 1,321 BAU ml−1) (Fig.
2b). These levels were and severe disease, our results support the importance of genomic
not higher in individuals who successively received the two treat- surveil ance. Rapid genotyping or sequencing will need to be intro-
ments (Fig.
2b).
duced in clinical practice to better inform treatment of patients
We next measured neutralization titers in the 29 sera (Fig. with COVID-19. For pre-exposure prophylaxis, which was the
2c). The untreated individuals did not neutralize any of the three application studied here, it will be important to use mAbs that
strains. Ronapreve-treated individuals efficiently neutralized Delta, cover both BA.1 and BA.2 (for example, bebtelovimab)
14,35, espe-
were inactive against BA.1 and poorly neutralized BA.2. Sera from cial y in regions where both sublineages are prevalent. Our results
Evusheld-treated and Ronapreve+Evusheld-treated individuals also show that measuring antibody levels with standard serology
were efficient against Delta (ED50 of 15,109 and 71,324, respec- assays that currently use an ancestral spike antigen does not inform
tively), barely neutralized BA.1 (ED50 of 44 and 42, respectively) and on protection. Future work will help determine whether adapted,
quite efficiently neutralized BA.2 (ED50 of 1,673 and 1,882, repre- lineage-specific, serological or neutralization assays can be used as
senting a nine-fold and 38-fold decrease, respectively, compared to a marker of clinical efficacy.
Delta) (Fig
. 2c). After Evusheld administration, eight of 11 individ-
Our study has limitations. The relatively low number of indi-
uals, who did not previously receive Ronapreve, had neutralization viduals analyzed did not allow us to evaluate the clinical efficacy of
activity against BA.1 in their sera, and all neutralized BA.2. This Evusheld against BA.2. We did not have access to nasopharyngeal
confirmed that Evusheld is more active against BA.2 than BA.1. samples of the individuals. Measuring antibody levels and neutraliz-
There was no major difference in the neutralization titers in indi- ing activity in these types of samples could provide insights into the
viduals having received only Evusheld or the successive combina- capacity of mAbs to neutralize Omicron sublineages at the infec-
tion of Ronapreve and Evusheld (Fig.
2c). The neutralizing activity tion site. We also did not test neutralization of the BA.1.1 and BA.3
against Delta correlated to anti-spike IgG levels, whereas this was sublineages of Omicron. Future experiments with these sublineages
not the case for BA.1 and BA.2 (Extended Data Fig. 2). This reflects are needed to determine the antiviral activity of mAbs against the
an uncoupling of the capacity of the antibodies to bind to the spike full landscape of the Omicron clade, which we recently proposed
from the ancestral Wuhan strain and to neutralize Omicron BA.1 to be considered as a distinct SARS-CoV-2 serotype from ancestral
and BA.2 strains. Altogether, these data show that administration strains and previous varian
ts36. We observed that syncytia induced
of Evusheld in immunocompromised individuals elicits poor sera by BA.1 and BA.2 are of similar size and smaller than those formed
neutralizing activity against BA.1 and better activity against BA.2.
by Delta-infected cel s. Future experiments are warranted to deter-
In agreement with the decreased sero-neutralization activity mine affinity to ACE2 and other characteristics of the BA.2 spike. It
of Evusheld-treated individuals against BA.1, we observed four will also be informative to study the binding of the sera to BA.1 and
breakthrough infections among the 29 participants. A summary BA.2 spike proteins to confirm the neutralization results obtained
of the cases is provided in Tab
le 3 and Extended Data Fig. 3, along with infectious viral strains.
with the serology and neutralization data of the closest sampling
Although clinical trials that can provide a complete evaluation
point. The four cases came from the second cohort of patients. of the effect of BA.2 on the treatment efficacy of mAbs have yet to
Polymerase chain reaction (PCR) screening confirmed Omicron be completed, based on our observation of breakthrough infections
infection for the four cases but did not allow for distinction we expect more frequent treatment failures. It is also possible that
between BA.1 and BA.2. However, in France, at the time of the the progressive accumulation of further mutations will increase the
sampling, BA.1 represented 90% of sequenced cases, whereas BA.2 level of resistance of BA.1 or BA.2 to mAbs during prolonged infec-
was detected in less than 10% of cases. Sequencing was performed tion. The low or intermediate sensitivity to Ronapreve and Evusheld,
only for case 4 and confirmed BA.1 infection in this individual. when used as a pre-exposure prophylaxis in immunocompromised
Three of the four individuals received sotrovimab after diagno- individuals at risk for severe disease, is of potential concern. One
sis, according to French guidelines. Three cases were classified as can speculate that the risk that further escape mutations will arise
mild disease, whereas case 4 was classified as severe and required in these individuals is higher compared to Delta. We, therefore, rec-
hospitalization. Despite detection of high levels of anti-spike anti- ommend a close follow-up of these individuals, particularly in case
bodies in the sera, the neutralization titers against BA.1 were low of prolonged infection despite treatment.
and ranged between <7.5 and 351 for the four individuals (Tab
le 3
and Extended Data Fig. 3). These four cases indicate that Evusheld
Online content
neither protects against Omicron infection nor ful y prevents Any methods, additional references, Nature Research report-
severe disease.
ing summaries, source data, extended data, supplementary infor-
mation, acknowledgements, peer review information; details of
Discussion
author contributions and competing interests; and statements of
We highlight here substantial differences not only between the data and code availability are available a
t https://doi.org/10.1038/
Delta and Omicron variants but also between BA.1 and BA.2
s41591-022-01792-5.
NATuRE MEDICINE | www.nature.com/naturemedicine
Articles
Nature MediciNe
Received: 6 March 2022; Accepted: 22 March 2022;
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Articles
Methods
using 4% PFA. A standard curve with serial dilutions of a human anti-spike
No statistical methods were used to predetermine sample size. The experiments
monoclonal antibody (mAb48) was acquired in each assay to standardize the
were not randomized, and the investigators were not blinded to allocation during
results as a binding Unit (BU). Data were acquired on an Attune NxT instrument
experiments and outcome assessment. Our research complies with all relevant
using Attune NxT software version 3.2.2 (Life Technologies) and analyzed with
ethical regulation, as detailed in the ‘Cohorts’ section.
FlowJo version 10.7.1 software (see Extended Data Fig. 4 for gating strategy).
The sensitivity is 99.2% with a 95% confidence interval of 97.69–99.78%, and
Cohorts. Immunocompromised individuals receiving Evusheld were
the specificity i
s 100% (98.5–100%)40. To determine BAU ml−1, we analyzed a
recruited in two centers (CHR d’Orléans and Hôpital Cochin) in the French
series of vaccinated (
n = 144), convalescent (
n = 59) samples and World Health
cities of Orléans and Paris. The ‘Orléans’ cohort is an ongoing prospective,
Organization international reference sera (20/136 and 20/130) on S-Flow and
monocentric, longitudinal, observational cohort clinical study aiming to
on two commercial y available ELISAs (Abbott 147 and Beckmann 56). Using
describe the kinetics of neutralizing antibodies after SARS-CoV-2 infection
this dataset, we performed a Passing–Pablok regression, which shows that the
or vaccination (ClinicalTrials.gov identifier:
NCT04750720). This study was
relationship between BU and BAU ml−1 is linear, allowing calculation of BAU ml−1
approved by the Est II (Besançon) ethical committee. At enrollment, written
using S-Flow dat
a41.
informed consent was collected, and participants completed a questionnaire
that covered sociodemographic characteristics, clinical information and data
Statistical analysis. Flow cytometry data were analyzed with FlowJo version 10
related to anti-SARS-CoV-2 vaccination. Blood sampling was performed on the
software. Calculations were performed using Excel 365 (Microsoft). Figures were
day of Evusheld infusion and after 3 days, 15 days and 1 month. The ‘Cochin’
drawn on Prism 9 (GraphPad Software). Statistical analysis was conducted using
cohort is a prospective, monocentric, longitudinal, observational clinical study
GraphPad Prism 9. Statistical significance between different groups was calculated
(NCT04870411) enrolling immunocompromised individuals with rheumatic
using Kruskall–Wallis test with Dunn’s multiple comparions, Friedman tests with
diseases, aiming at describing immunological responses to COVID-19
Dunn’s multiple comparison correction and Spearman non-parametric correlation
vaccine in patients with autoimmune and inflammatory diseases treated with
test. All tests were two-sided.
immunosuppressants and/or biologics. Ethics approval was obtained from the
Comite de Protection des Personnes Nord-Ouest II. Leftover sera from usual
Reporting Summary. Further information on research design is available in the
care were used from these individuals in the setting of the local biological
Nature Research Reporting Summary linked to this article.
samples collection (RAPIDEM). A written informed consent was collected for al
participants. None of the study participants received compensation.
Data availability
All data supporting the findings of this study are available within the article or
Viral strains. The Delta strain was isolated from a nasopharyngeal swab of a
from the corresponding authors upon reasonable request without any restrictions.
hospitalized patient returning from India
37. The swab was provided and sequenced
Source data are provided for Figs. 1 and 2 and Extended Data Fig. 2. Source data are
by the virology laboratory of Hopital Européen Georges Pompidou (Assistance
provided with this paper.
Publique–Hopitaux de Paris). The Omicron strain was supplied and sequenced
by the NRC UZ/KU Leuven (Belgi
um)4. The BA.2 strain was isolated from a
nasopharyngeal swab sampled on 4 January 2022 from a 10-year-old male patient.
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d
Acknowledgements
4.
We thank the European Health Emergency Preparedness and Response Authority
(HERA) for supporting the work being done at Institut Pasteur and UK Leuven.
S-Fuse neutralization assay. U2OS-ACE2 GFP1-10 or GFP11 cel s, also
We thank J. Buchrieser, J. Puech and F. Gambaro for their help with the sequencing
termed S-Fuse cel s, become GFP+ when they are productively infected by
data analysis. We thank the patients who participated to this study. We thank
SARS-CoV-2. Cel s tested negative for mycoplasma. Cel s were mixed (ratio 1:1)
Y. Yazdanpanah, members of the Virus and Immunity Unit and other teams for
and plated at 8 × 103 per well in a μClear 96-well plate (Greiner Bio-One). The
discussions and help, N. Aulner and the UtechS Photonic BioImaging (UPBI) core
indicated SARS-CoV-2 strains were incubated with serial y diluted mAb or sera
facility (Institut Pasteur), a member of the France BioImaging network, for image
for 15 minutes at room temperature and added to S-Fuse cel s. The sera were
acquisition and analysis. The Opera system was co-funded by Institut Pasteur and the
heat-inactivated for 30 minutes at 56 °C before use. Eighteen hours later, cel s were
Région Ȋle-de-France (DIM1Health). We thank the KU Leuven University authorities
fixed with 2% paraformaldehyde (PFA), washed and stained with Hoechst (dilution
and J. Arnout, B. Lambrecht, C. Van Geet and L. Sels for their support. We thank F.
1:1,000, Invitrogen). Images were acquired with an Opera Phenix high-content
Peira, V. Legros and L. Courtellemont for their help with the cohorts. UZ Leuven, as
confocal microscope (PerkinElmer). The GFP area and the number of nuclei were
national reference center for respiratory pathogens, is supported by Sciensano, which
quantified using Harmony software version 4.9 (PerkinElmer). The percentage
is grateful y acknowledged. Work in the O.S. lab is funded by Institut Pasteur, Urgence
of neutralization was calculated using the number of syncytia as value with the
COVID-19 Fundraising Campaign of Institut Pasteur, Fondation pour la Recherche
following formula: 100 × (1 − (value with serum − value in ‘non-infected’) / (value
Médicale (FRM), ANRS, the Vaccine Research Institute (ANR-10-LABX-77), Labex
in ‘no serum’ − value in ‘non-infected’)). Neutralizing activity of each serum was
IBEID (ANR-10-LABX-62-IBEID), ANR/FRM Flash Covid PROTEO-SARS-CoV-2,
expressed as the ED50. ED50 values (in μg ml−1 for mAbs and in dilution values
ANR Coronamito and IDISCOVR. Work in the UPBI facility is funded by grant ANR-
for sera) were calculated with a reconstructed curve using the percentage of the
10-INSB-04-01 and the Région Ȋle-de-France program DIM1Health. D.P. is supported
neutralization at the different concentrations. We previously reported correlations
by the Vaccine Research Institute. G.B. acknowledges support from the Internal
between neutralization titers obtained with the S-Fuse assay and both pseudovirus
Funds KU Leuven under grant agreement C14/18/094 and the Research Foundation–
neutralization and microneutralization assays
38,39.
Flanders (Fonds voor Wetenschappelijk Onderzoek–Vlaanderen, G0E1420N and
G098321N). P.M. acknowledges support from a COVID-19 research grant of ‘Fonds
Anti-spike serology. The S-Flow assay uses 293T cel s stably expressing the
Wetenschappelijk Onderzoek’/Research Foundation Flanders (grant G0H4420N). P.M.
spike protein (293T spike cel s) and 293T control cel s as control to detect
acknowledges support of a COVID-19 research grant from ‘Fonds Wetenschappelijk
anti-spike antibodies by flow cytometr
y40. In brief, the cel s were incubated at
Onderzoek’/Research Foundation Flanders (grant G0H4420N) and ‘Internal Funds
4 °C for 30 minutes with sera (1:300 dilution) in PBS containing 0.5% BSA and
KU Leuven’ (grant 3M170314). E.S.L. acknowledges funding from the INCEPTION
2 mM EDTA. Cel s were then washed with PBS and stained with an anti-human
program (Investissements d’Avenir grant ANR-16-CONV-0005). The funders of this
IgG Fc Alexa Fluor 647 antibody (109-605-170, Jackson Immuno Research).
study had no role in study design, data collection, analysis and interpretation or writing
After 30 minutes at 4 °C, cel s were washed with PBS and fixed for 10 minutes
of the article.
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Author contributions
Additional information
Experimental strategy and design: T.B., D.P., B.T., T.P. and O.S. Laboratory experiments:
Extended data is available for this paper at
https://doi.org/10.1038/s41591-022-01792-5.
T.B., D.P., I.S., F.G.B., F.P., W.H.B., C.C., H.P., D.V., M.P., A.B. and C.P. Cohort
Supplementary information The online version contains supplementary material
management and clinical research: T.B., J.H., D.P., A.S., I.S., Y.N., M.C., C.C., H.P., D.V.,
available a
t https://doi.org/10.1038/s41591-022-01792-5.
L.M., L.H., B.T., T.P. and O.S. Viral strains: P.M., F.G.B., H.P., D.V., L.C., G.B. and E.A.
Manuscript writing: T.B. and O.S. Manuscript editing: T.B., J.H., P.M., D.P., G.B., L.H.,
Correspondence and requests for materials should be addressed to Timothée Bruel
E.S.L., E.A., B.T. and O.S.
or Olivier Schwartz.
Peer review information Nature Medicine thanks Ailong Huang, Joshua Tan and the
Competing interests
other, anonymous, reviewer(s) for their contribution to the peer review of this work.
T.B., C.P., H.M. and O.S. have a pending patent application for an anti-RBD mAb
Saheli Sadanand was the primary editor on this article and managed its editorial process
not used in this study (PCT/FR2021/070522). All other authors declare no conflicts
and peer review in col aboration with the rest of the editorial team.
of interest.
Reprints and permissions information is available at
www.nature.com/reprints.
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Extended Data Fig. 1 | SARS-CoV-2 variants Delta, BA.1 and BA.2 induce syncytia in S-Fuse cells. S-Fuse cells that become GFP + upon cell-cell fusion
were exposed to the indicated SARS-CoV-2 strains. After 20 h, cells were stained with Hoechst to visualize nuclei. Syncytia (green) and nuclei (blue) are
shown. Representative images from three independent experiments are shown. Scale bar, 500 μm.
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Extended Data Fig. 2 | Correlation of neutralization capacity and anti-S antibody levels in individuals having received Ronapreve and/or Evusheld. Two-sided Spearman non-parametric correlations of neutralizing antibody titers against Delta, Omicron BA.1 and BA.2 and the level of anti-S IgG. R and
p-values are indicated.
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Extended Data Fig. 3 | Report of four Omicron breakthrough infections in Evusheld treated patients. A timeline indicates the key events for each of the 4
Omicron breakthrough cases. Patients’ characteristics and antibody measurement of the closest sampling point are indicated.
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Extended Data Fig. 4 | Gating strategy of the S-Flow assay. 293T cells stably expressing the Wuhan Spike were incubated with sera from patient treated
with monoclonal antibodies (dilution 1:300), stained with an anti-human secondary antibody and analyzed by flow-cytometry.
a. One representative
example of the gating strategy is shown. Gates are set on cells transfected with a control plasmid not encoding a spike.
b. An example of the signal
obtained by a reactive serum on spike expressing cells is shown.
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