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1
Resilience of S309 and AZD7442 monoclonal antibody treatments against
2
infection by SARS-CoV-2 Omicron lineage strains
3
4
James Brett Case1, Samantha Mackin1,2, John Errico2, Zhenlu Chong1, Emily A. Madden1,
5
Barbara Guarino3, Michael A. Schmid3, Kim Rosenthal4, Kuishu Ren4, Ana Jung2, Lindsay
6
Droit5, Scott A. Handley2, Peter J. Halfmann6, Yoshihiro Kawaoka6,7,8, James E. Crowe, Jr.9,10,11,
7
Daved H. Fremont2,5,12, Herbert W. Virgin2,13,14, Yueh-Ming Loo4, Mark T. Esser4, Lisa A.
8
Purcell13, Davide Corti3, and Michael S. Diamond1,2,5,15,16†
9
1
10
Department of Medicine, Washington University School of Medicine, St. Louis, MO.
2
11
Department of Pathology & Immunology, Washington University School of Medicine, St.
12
Louis, MO.
3
13
Humabs BioMed SA, a subsidiary of Vir Biotechnology, Bellinzona, Switzerland.
4
14
Vaccines and Immune Therapies, BioPharmaceuticals R&D, AstraZeneca, Gaithersburg, MD.
5
15
Department of Molecular Microbiology, Washington University School of Medicine, St. Louis,
16
MO.
6
17
Influenza Research Institute, Department of Pathobiological Sciences, School of Veterinary
18
Medicine, University of Wisconsin-Madison, Madison, WI.
7
19
Division of Virology, Institute of Medical Science, University of Tokyo, Tokyo, Japan.
8
20
The Research Center for Global Viral Diseases, National Center for Global Health and
21
Medicine
22
Research Institute, Tokyo, Japan.
9
23
Vanderbilt Vaccine Center, Vanderbilt University Medical Center, Nashville, TN.
10
24
Department of Pediatrics, Vanderbilt University Medical Center, Nashville, TN.
11
25
Department of Pathology, Microbiology, and Immunology, Vanderbilt University Medical
26
Center, Nashville, TN.
12
27
Department of Biochemistry & Molecular Biophysics, Washington University School of
28
Medicine, St. Louis, MO.
1
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13
29
Vir Biotechnology, San Francisco, CA.
14
30
University of Texas Southwestern Medical Center, Dallas, TX.
15
31
Andrew M. and Jane M. Bursky Center for Human Immunology and Immunotherapy
32
Programs, Washington University School of Medicine, Saint Louis, MO.
16
33
Center for Vaccines and Immunity to Microbial Pathogens, Washington University School of
34
Medicine, Saint Louis, MO.
35
36
†
Corresponding author:
2
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37
ABSTRACT
38
Omicron variant strains encode large numbers of changes in the spike protein
39
compared to historical SARS-CoV-2 isolates. Although in vitro studies have suggested that
several monoclonal antibody therapies lose neutralizing activity against Omicron variants1-
40
4
41
, the effects in vivo remain largely unknown. Here, we report on the protective efficacy
42
against three SARS-CoV-2 Omicron lineage strains (BA.1, BA.1.1, and BA.2) of two
43
monoclonal antibody therapeutics (S309 [Vir Biotechnology] monotherapy and AZD7442
44
[AstraZeneca] combination), which correspond to ones used to treat or prevent SARS-
45
CoV-2 infections in humans. Despite losses in neutralization potency in cell culture, S309 or
46
AZD7442 treatments reduced BA.1, BA.1.1, and BA.2 lung infection in susceptible mice
47
that express human ACE2 (K18-hACE2). Correlation analyses between in vitro
48
neutralizing activity and reductions in viral burden in K18-hACE2 or human Fcγ
R
49
transgenic mice suggest that S309 and AZD7442 have different mechanisms of protection
50
against Omicron variants, with S309 utilizing Fc effector function interactions and
51
AZD7442 acting principally by direct neutralization. Our data in mice demonstrate the
52
resilience of S309 and AZD7442 mAbs against emerging SARS-CoV-2 variant strains and
53
provide insight into the relationship between loss of antibody neutralization potency and
54
retained protection in vivo.
55
3
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56
MAIN TEXT
57
Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) variant strains continue
58
to emerge and spread globally despite currently employed countermeasures and public health
59
mandates. Since late 2020, variants of concern (VOC) and interest (VOI) have arisen due to
60
continued SARS-CoV-2 evolution. Many variants contain substitutions in the N-terminal domain
61
(NTD) and the receptor binding motif (RBM) of the receptor binding domain (RBD). Omicron
62
lineage variants containing the largest numbers of spike protein changes described so far have
63
emerged, spread globally, and become dominant. Moreover, cell-based studies suggest that the
64
neutralizing activity of many monoclonal antibodies (mAbs) with Emergency Use Authorization
65
(EUA) status or in advanced clinical development is diminished or abolished against Omicron
66
lineage strains (BA.1, BA.1.1, and BA.2)1,2,5,6. However, the effect of mutations that compromise
67
antibody neutralization on their efficacy
in vivo against SARS-CoV-2 remains less clear. Indeed,
68
for some classes of broadly neutralizing mAbs against influenza7,8 and Ebola9,10 viruses, there is
69
no strict correlation between neutralizing activity
in vitro and protection in animal models. Here,
70
using mAbs that are currently in use to prevent or treat SARS-CoV-2 infection, we evaluated
71
how the antigenic shift in Omicron variants affects neutralization in cells and protection in mice.
72
73
MAb neutralization against Omicron lineage viruses
74
We analyzed the substitutions in the RBDs of BA.1 (B.1.1.529), BA.1.1 (B.1.1.529
75
R346K), and BA.2 strains
(Fig. 1a, Extended Data Fig. 1) in the context of the structurally-
76
defined binding epitopes of S309, a cross-reactive SARS-CoV mAb and the parent of
77
Sotrovimab [VIR-7831], and AZD8895 and AZD1061, two mAbs that together (AZD7442) form
78
the clinically-used Evusheld combination treatment (
Fig. 1b-e, Extended Data Fig. 1). S309
4
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79
binds a conserved epitope on the RBD that is spatially distinct from the RBM11 and the
80
AZD8895 and AZD1061 antibodies bind non-overlapping RBM epitopes12. Across Omicron
81
lineage strains, substitutions at several antibody contact residues have occurred (
S309: G339D,
82
R346K, N440K;
AZD8895: K417N, S477N, T478K, E484A, Q493R;
AZD1061: R346K,
83
N440K, E484A, Q493R).
84
Because of these sequence changes, we assessed the neutralizing activity of S309,
85
AZD8895, AZD1061, and AZD7442 against BA.1, BA.1.1, and BA.2 viruses in Vero-
86
TMPRSS2 cells. For these studies, we used mAbs that correspond to the products in clinical use
87
which have Fc modifications: S309-LS [M428L/N434S], AZD8895-YTE/TM
88
[M252Y/S254T/T256E and L234F/L235E/P331S], AZD1061-YTE/TM, and AZD7442-
89
YTE/TM. The LS and YTE Fc substitutions result in extended antibody half-life in humans, and
90
the TM changes reduce Fc effector functions13. Compared to the historical WA1/2020 D614G
91
strain (hereafter D614G), antibody incubation with BA.1 was associated with 2.5-fold (S309-
92
LS), 25-fold (AZD7442-YTE/TM), 118-fold (AZD8895-YTE/TM), and 206-fold (AZD1061-
93
YTE/TM) reductions in neutralization potency (
Fig. 1f-o), which agree with experiments with
94
authentic or pseudotyped SARS-CoV-21,2,5,6. Some differences were observed with BA.1.1:
95
whereas S309-LS and AZD8895-YTE/TM were only slightly less effective against BA.1.1
96
compared to BA.1, the neutralizing activity of AZD1061-YTE/TM was reduced by almost
97
1,700-fold. Despite the decrease in activity of the AZD1061-YTE/TM component, the
98
AZD7442-YTE/TM combination still showed inhibitory activity against BA.1.1 with a 176-fold
99
reduction compared to D614G. Whereas small (no change to 5-fold) reductions in neutralization
100
activity were observed with AZD1061-YTE/TM and AZD7442-YTE/TM against BA.2, larger
101
reductions (32- and 68-fold) were observed for S309-LS and AZD8895-YTE/TM compared to
5
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.
102
D614G. Overall, these data demonstrate that S309 retains potency against BA.1 and BA.1.1
103
strains but has less
in vitro neutralizing activity against BA.2, and the AZD7442 combination
104
shows reduced yet residual activity against strains from all three Omicron lineages.
105
106
MAb protection in vivo against Omicron viruses
107
Because S309 and AZD7442 mAbs might act
in vivo by a combination of mechanisms
108
that are not fully reflected by
in vitro neutralization potency assays, we evaluated the effects of
109
the mutations observed in BA.1, BA.1.1 and BA.2 on efficacy in animals. For these studies, we
110
used S309-LS and a different form of AZD7442, which contained only the TM substitutions and
111
not the YTE modification. Although the YTE modification promotes antibody recycling to
112
confer extended antibody half-life in humans and non-human primates, it accelerates antibody
113
elimination in rodents14. To assess the efficacy of S309-LS and AZD7442-TM
in vivo, we
114
administered a single 200 μg (~10 mg/kg total) mAb dose to K18-hACE2 transgenic mice by
115
intraperitoneal injection one day prior to intranasal inoculation with BA.1, BA.1.1, or BA.2
116
strains. Although Omicron lineage viruses are less pathogenic in mice, they still replicate to high
117
levels in the lungs of K18-hACE2 mice15. Nonetheless, as preliminary studies suggested slightly
118
different kinetics of replication and spread in mice, we harvested samples at 7 dpi for BA.1 and
119
BA.1.1 and 6 dpi for BA.216. In BA.1 and BA.1.1-infected mice, S309-LS mAb reduced viral
120
burden in the lung, nasal turbinates, and nasal washes at 7 dpi compared to isotype mAb-control
121
treated mice (
Fig. 2a-d). Nonetheless, control of infection, as judged by viral RNA levels, was
122
less efficient against BA.1 (182-fold reduction) and BA.1.1 (39-fold reduction) viruses than
123
against D614G (>500,000-fold reduction). Despite the diminished neutralizing activity against
124
BA.2
in vitro, S309-LS treatment reduced viral RNA levels in the lungs of BA.2-infected mice
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125
substantially (742-fold reduction) (
Fig 2a, b). Protection by S309-LS was not observed in the
126
nasal turbinates or nasal washes of mice challenged with BA.2 (
Fig. 2c, d), in part due to the low
127
and variable levels of infection with this variant. AZD7442-TM treatment differentially reduced
128
viral burden in the lungs of mice against D614G (>400,000-fold reduction in viral RNA), BA.1
129
(91-fold reduction in viral RNA), BA.1.1 (4-fold reduction in viral RNA), and BA.2 (>100,000-
130
fold reduction in viral RNA) (
Fig. 2e, f). Protection in the upper respiratory tract was less
131
consistent, as AZD7442-TM treatment lowered viral RNA levels in the nasal washes of D614G
132
and BA.1-infected mice but not in BA.1.1 or BA.2-infected mice and failed to reduce D614G,
133
BA.1, BA.1.1, or BA.2 infection in the nasal turbinates (
Fig. 2g, h).
134
As an independent metric of mAb protection, we measured cytokine and chemokine
135
levels in the lung homogenates of S309-LS and AZD7442-TM treated animals infected with
136
Omicron variant strains (
Fig 2i-j, and Extended Data Fig. 2, 3). All infected K18-hACE2 mice
137
receiving isotype control mAbs had increased expression levels of several pro-inflammatory
138
cytokines and chemokines such as G-CSF, GM-CSF, IFN-γ, IL-1β, IL-6, CXCL-10, CCL-2, and
139
CCL-4 when compared to uninfected mice. In contrast, mice treated with AZD7442-TM mAbs
140
and infected with BA.1 or BA.2 but not BA.1.1. showed reduced levels of pro-inflammatory
141
cytokines and chemokines, which is consistent with effects on viral burden (
Fig. 2e, f). In
142
comparison to the isotype controls, mice treated with S309-LS had lower levels of cytokines and
143
chemokines in lung homogenates after infection with all three Omicron variants, although the
144
protection against BA.2-induced inflammation was less than against BA.1. or BA.1.1. Overall,
145
these experiments suggest that despite losses in neutralizing potency in cell culture, S309-LS or
146
AZD7442-TM can limit inflammation in the lung caused by Omicron variants.
7
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.
147
We next evaluated whether the differences in neutralizing activity of S309-LS and
148
AZD7442-YTE/TM correlated with lung viral burden after infection with the three Omicron
149
strains. The change in AZD7442-YTE/TM neutralizing activity associated directly with the
150
differences in lung viral burden of each Omicron variant (
Fig. 2k). This relationship is consistent
151
with its likely mechanism of action, virus neutralization and inhibition of entry17,18. The
152
AZD7442-TM version we used, like the clinical drug Evusheld, encodes for modifications in the
153
constant region of the mAb heavy chains that profoundly decrease binding to Fc-gamma
154
receptors (FcγRs) and complement components (19 and
Fig 3a). In comparison, for S309-LS, a
155
similar direct correlation between changes in neutralization potency
in vitro and reductions in
156
viral burden
in vivo was not observed (
Fig. 2l), indicating a possible additional protective
157
mechanism beyond virus neutralization.
158
159
S309-LS employs Fc effector functions to protect against SARS-CoV-2 variants
160
To evaluate a potential role for Fc effector functions in S309 mAb-mediated protection
161
against Omicron strains, we engineered loss-of-function GRLR mutations (G236R, L328R) into
162
the Fc domain of the human IgG1 heavy chain of S309; these substitutions eliminate antibody
163
binding to FcγRs13. Introduction of the GRLR mutations abrogated binding to hFcγRI and
164
hFcγRIIIa, as expected (
Fig. 3a) but did not affect neutralization of the SARS-CoV-2 strains
165
(
Extended Data Fig. 4). Next, we compared VIR-7831 (the clinical form of S309-LS) and
166
S309-GRLR in an
in vitro antibody-dependent cell cytotoxicity (ADCC) assay. When target cells
167
expressing similar levels of Wuhan-D614, BA.1, or BA.2 spike proteins on the cell surface (
Fig.
168
3b) were incubated with VIR-7831 mAb, we observed some reductions in binding to Omicron
169
spike proteins compared to mAb S2X324, an antibody that retains neutralizing activity against
8
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.
170
BA.1, BA.1.1, and BA.2 and engages a distinct epitope in the RBM4. Despite the differences in
171
binding, target cells expressing Wuhan-D614, BA.1, or BA.2 spike proteins were lysed
172
efficiently by primary natural killer (NK) cells (antibody-dependent cellular cytotoxicity,
173
ADCC) isolated from four donors by VIR-7831 but not by S309-GRLR (
Fig. 3c, d, Extended
174
Data Fig. 5). Similarly, primary CD14+ monocytes isolated from five donors mediated
175
comparable antibody-dependent cellular phagocytosis (ADCP) of target cells expressing Wuhan-
176
D614, BA.1, or BA.2 spike proteins by VIR-7831 but not by S309-GRLR (
Fig. 3e, f, Extended
177
Data Fig. 6, 7).
178
To evaluate the role of effector functions
in vivo in S309-LS mAb-mediated protection
179
against Omicron variant strains, we treated K18-hACE2 mice with a single 200 μg (~10 mg/kg
180
total) dose of S309-GRLR mAb by intraperitoneal injection one day prior to intranasal
181
inoculation with D614G, BA.1, or BA.2 strains. At 6 (BA.2) or 7 (D614G and BA.1) dpi, viral
182
RNA levels in the lungs, nasal turbinates, and nasal washes were measured (
Fig. 3g-i). Although
183
S309-GRLR treatment reduced viral burden in the lung and nasal turbinates of D614G-infected
184
mice, it did not limit infection by BA.1 and BA.2 strains in the tissues tested. To corroborate
185
these findings, we treated human FcγR (hFcγR) transgenic C57BL/6 mice20 with a single 3
186
mg/kg dose of S309-LS or S309-GRLR mAbs one day prior to inoculation with a SARS-CoV-2
187
Beta (B.1.351) isolate; we used the Beta isolate for these studies because Omicron strains
188
replicate poorly in conventional C57BL/6 mice lacking expression of hACE216. At 2 or 4 dpi,
189
S309-LS mAb-treated hFcγR mice showed markedly reduced levels of viral RNA (49 to 127-
190
fold) or infectious virus (56- to 538-fold) in the lung compared to the isotype control-treated
191
mice, whereas animals administered S309-GRLR showed smaller (2.3- to 13-fold) differences,
192
most of which did not attain statistical significance (
Fig. 3j, k). Collectively, these data suggest
9
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.
193
that the protection mediated by S309-LS mAb
in vivo is mediated at least in part by Fc effector
194
functions and engagement of FcγRs.
195
DISCUSSION
196
Due to the continued emergence of SARS-CoV-2 variants encoding an increasing
197
number of amino acid changes in the spike protein, antibody countermeasure efficacy requires
198
continued monitoring. When the BA.1 Omicron virus emerged in late 2021, five mAb therapies
199
were in late-stage clinical development or had acquired EUA status.
In vitro assays with
200
pseudoviruses5 and authentic viruses1 established that mAb therapies from Regeneron
201
(REGN10933 and REGN10987), Lilly (LY-CoV555 and LY-CoV016), and Celltrion (CT-P59)
202
showed a complete loss in neutralizing activity against BA.1. Subsequent experiments in K18-
203
hACE2 mice confirmed that the REGN-COV2 mAb cocktail completely lost its efficacy against
204
the BA.1 variant21. More recently, an additional antibody (LY-CoV1404, bebtelovimab), which
205
shows considerable neutralization activity against a range of SARS-CoV-2 strains, received EUA
206
status22, although protection data
in vivo against VOC, including Omicron, has not yet been
207
published.
208
We compared the
in vitro neutralizing activity and
in vivo efficacy of S309 (parent mAb
209
of Sotrovimab) and AZD7442 (Evusheld) that correspond to the clinically-used products. Our
210
study establishes the utility of S309 and AZD7442 mAbs against highly divergent SARS-CoV-2
211
variants. Despite losses in neutralization potency against BA.1, BA.1.1, and BA.2 strains, S309-
212
LS and AZD7442-TM reduced viral burden and pro-inflammatory cytokine levels in the lungs of
213
K18-hACE2 mice, albeit with some differences in activity and mechanisms of action. Although
214
AZD7442-TM had a limited protective effect on viral burden in the nasal washes and nasal
215
turbinates of infected mice, this was not entirely unexpected, as studies with the parental mAbs
10
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.
216
COV2-2196 and COV2-2130 showed less protection in nasal washes than lungs against multiple
217
SARS-CoV-2 VOC23. Moreover, studies in non-human primates with anti-SARS-CoV-2 human
218
mAbs showed the concentrations in nasopharyngeal washes are approximately 0.1% of those
219
found in the serum24, which likely explains their diminished benefit in this tissue compartment.
220
We also assessed whether the reductions in mAb neutralization potency against Omicron
221
variant strains correlated with the observed changes in viral burden. For AZD7442-TM, which
222
contains L234F/L235E/P331S modifications that abolish Fc receptor engagement13 and were
223
introduced to decrease the potential risk of antibody-dependent enhancement of disease18,
224
antibody-mediated reductions in viral titer corresponded directly with neutralization activity
225
against Omicron variant strains; thus, neutralization is likely a key protective mechanism for
226
these RBM-specific mAbs. For S309-LS, which only contains half-life extending M428L/N434S
227
modifications in the human IgG1 Fc domain, and exhibits Fc effector functions including ADCC
228
and ADCP11, changes in neutralization potency did not linearly relate to changes in lung viral
229
titer. S309-LS mAb treatment still conferred significant protection in the lungs of mice infected
230
with BA.2 despite a substantial loss in neutralizing activity. Because of these results, we
231
evaluated the contributions of Fc effector functions in protection in mice using S309-GRLR,
232
which has G236R/L328R mutations in the Fc domain that abrogate binding to FcγRs13. We
233
observed that intact S309-LS but not S309-GRLR mAb protected K18-hACE2 and hFcγR mice
234
against SARS-CoV-2 variant strains. These results are consistent with prior studies showing a
beneficial role of Fc-effector functions in antibody mediated protection in mice and hamsters25-
235
29
236
, and may explain why mAbs with markedly different
in vitro neutralization potencies against
237
SARS-CoV-2 strains show similar protective activity in animals
238
(https://opendata.ncats.nih.gov/covid19/animal). Furthermore, they also demonstrate that for
11
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.
239
some mAbs, Fc effector functions can compensate for losses in neutralization potency against
240
SARS-CoV-2 variants and act as a protective mechanism
in vivo. Thus, effector functions can
241
contribute to resilience of some mAbs against Omicron and other VOC30,31. We speculate that
242
the stoichiometric threshold and antibody occupancy requirements for Fc effector function
243
activity may be lower than for virion neutralization32; if so, this property might clarify how
244
antibodies with reduced neutralizing potency against VOC that still bind spike protein on the
245
virion or surface of infected cells retain protective activity
in vivo.
246
Limitations of study. We note several limitations of our study: (a) Female K18-hACE2
247
mice were used to allow for group caging. Follow-up experiments in male mice to confirm and
248
extend these results are needed. (b) The BA.1, BA.1.1., and BA.2 viruses are less pathogenic in
249
mice than the D614G virus16,33-35. This could lead to an overestimation of protection compared to
250
other more virulent strains in mice. (c) We only evaluated the efficacy of S309 or AZD7442 as
251
prophylaxis. Whereas AZD7442 is authorized only as preventive agent, post-exposure
252
therapeutic studies with both mAbs and Omicron variants may provide further insight as to
253
effects on potency. Moreover, the relationship between initial viral dosing and antibody
254
protection against Omicron variants was not explored. (d) Several experiments were performed
255
in transgenic mice that over-express human ACE2 receptors. High levels of cellular hACE2 can
256
diminish the neutralizing activity of mAbs that bind non-RBM sites of the SARS-CoV-2
257
spike36,37. Thus, studies in hACE2-transgenic mice could underestimate the efficacy of mAbs
258
binding outside of the RBM. Challenge studies in other animal models and ultimately humans
259
will be required for corroboration, including the contribution of Fc effector functions to mAb
260
efficacy.
12
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.
261
Collectively, our data expand on recent
in vitro findings with BA.1 strains by evaluating
262
the level of protection conferred by treatment with two EUA mAbs against the three currently
263
dominant Omicron variants. While S309-LS (and by extension Sotrovimab) and AZD7442-TM
264
(Evusheld) retained inhibitory activity against several Omicron lineage strains, the impact of
265
shifts in neutralization potency
in vitro may not directly predict dosing in the clinical setting.
266
Finally, our studies highlight the potential of both mAb neutralization and Fc effector function
267
mechanisms in protecting against SARS-CoV-2-mediated disease and suggest mechanisms of
268
action for withstanding mutations in variant strains that reduce but do not abrogate mAb binding
269
and neutralization.
270
271
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272
ACKNOWLEDGEMENTS
273
This study was supported by grants and contracts from the NIH (R01 AI157155, U01
274
AI151810, NIAID Centers of Excellence for Influenza Research and Response (CEIRR) contract
275
75N93019C00051) and the Defense Advanced Research Projects Agency (DARPA; HR0011-
276
18-2-0001). J.B.C. is supported by a Helen Hay Whitney Foundation postdoctoral fellowship.
277
E.A.M. is supported by a W.M. Keck postdoctoral fellowship from Washington University. We
278
thank
and
for technical support.
279
280
AUTHOR CONTRIBUTIONS
281
J.B.C. performed and analyzed neutralization assays. J.M.E. performed structural analyses
282
with guidance from D.H.F. J.B.C., S.M., Z.C., and E.A.M. performed mouse experiments and
283
viral burden analyses. J.B.C. propagated and validated SARS-CoV-2 viruses. B.G. and M.A.S.
284
designed, performed, and analyzed
in vitro Fc-mediated effector function studies. K. Rosenthal,
285
and K. Ren performed antibody analyses. A.J., L.D., and S.A.H. performed deep sequencing
286
analysis. L.A.P., D.C., Y-M.L., and M.T.E. provided mAbs. P.J.H. and Y.K. provided SARS-
287
CoV-2 strains. J.E.C. and H.W.V. provided key intellectual contributions to the design of the
288
study D.H.F. and M.S.D. obtained funding and supervised the research. J.B.C. and M.S.D. wrote
289
the initial draft, with the other authors providing editorial comments.
290
291
COMPETING FINANCIAL INTERESTS
292
M.S.D. is a consultant for Inbios, Vir Biotechnology, Senda Biosciences, and Carnival
293
Corporation, and on the Scientific Advisory Boards of Moderna and Immunome. The Diamond
294
laboratory has received funding support in sponsored research agreements from Moderna, Vir
14
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.
295
Biotechnology, and Emergent BioSolutions. J.E.C. has served as a consultant for Luna
296
Innovations, Merck, and GlaxoSmithKline, is a member of the Scientific Advisory Board of
297
Meissa Vaccines and is founder of IDBiologics. The Crowe laboratory has received sponsored
298
research agreements from AstraZeneca, Takeda, and IDBiologics during the conduct of the
299
study. Vanderbilt University has applied for patents for some of the antibodies in this paper, for
300
which J.E.C. is an inventor. B.G., M.A.S, H.W.V., D.C., and L.A.P. are employees of Vir
301
Biotechnology and may hold equity in Vir Biotechnology. L.A.P. is a former employee and may
302
hold equity in Regeneron Pharmaceuticals. H.W.V. is a founder and holds shares in PierianDx
303
and Casma Therapeutics. Neither company provided resources to this study. Y.K. has received
304
unrelated funding support from Daiichi Sankyo Pharmaceutical, Toyama Chemical, Tauns
305
Laboratories, Inc., Shionogi & Co. LTD, Otsuka Pharmaceutical, KM Biologics, Kyoritsu
306
Seiyaku, Shinya Corporation, and Fuji Rebio. K. Rosenthal, K. Ren, Y-M.L. and M.T.E. are
307
employees of AstraZeneca and may hold stock in AstraZeneca. All other authors declare no
308
competing financial interests.
309
310
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311
FIGURE LEGENDS
312
Figure 1. Neutralization of Omicron lineage strains by mAbs. a, One protomer of the
313
SARS-CoV-2 spike trimer (PDB: 7C2L) is depicted with BA.2 variant amino acid substitutions
314
labelled and shown as red spheres. The N-terminal domain (NTD), RBD, RBM, and S2 are
315
colored in yellow, green, magenta, and blue, respectively. All mutated residues in the BA.2 RBD
316
relative to WA1/2020 are indicated in
b, and the BA.2 RBD bound by mAbs S309 (orange,
317
PDB: 6WPS) (
b), AZD8895 (green, PDB: 7L7D) (
c), and AZD1061 (purple, PDB:7L7E) (
d) are
318
shown. BA.2 mutations in the respective epitopes of each mAb are shaded red, whereas those
319
outside the epitope are shaded green.
e, Multiple sequence alignment showing the epitope
320
footprints of each mAb on the SARS-CoV-2 RBD (orange, S309; green, AZD8895; purple,
321
AZD1061). The WA1/2020 RBD is shown in the last row with relative variant sequence changes
322
indicated. Red circles below the sequence alignment indicate hACE2 contact residues on the
323
SARS-CoV-2 RBD38. Structural analysis and depictions were generated using UCSF
324
ChimeraX39.
f-i, Neutralization curves in Vero-TMPRSS2 cells with the indicated SARS-CoV-2
325
strain and mAb. The average of three to four experiments performed in technical duplicate are
326
shown.
j-m, Comparison of EC50 values for the indicated mAb against D614G, BA.1, BA.1.1,
327
and BA.2 viruses. Data are the average of three experiments, error bars indicate standard error of
328
the mean (SEM), and the dashed line indicates the upper limit of detection (one-way ANOVA
329
with Dunnett’s test; ns, not significant, *
P < 0.05, **
P < 0.01, ***
P < 0.001, ****
P <
330
0.0001).
n, Summary of the EC50 values for each mAb against the indicated SARS-CoV-2 strain.
331
o, Summary of the fold-change in EC50 values for each mAb against the indicated Omicron strain
332
relative to SARS-CoV-2 D614G.
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333
Figure 2. Antibody protection against Omicron variants in K18-hACE2 mice. a-j,
334
Eight-week-old female K18-hACE2 mice received 200 μg (about 10 mg/kg) of the indicated
335
mAb treatment by intraperitoneal injection one day before intranasal inoculation with 103 FFU of
336
the indicated SARS-CoV-2 strain. Tissues were collected at six (BA.2) or seven days (all other
337
strains) after inoculation. Viral RNA levels in the lungs (
a,
e)
, nasal turbinates (
c,
g), and nasal
338
washes (
d, h) were determined by RT-qPCR, and infectious virus in the lungs (
b, f) was assayed
339
by plaque assay (lines indicate median ± SEM.; n = 6-8 mice per group, two experiments; Mann-
340
Whitney test between isotype and mAb treatment; ns, not significant; *
P < 0.05, **
P < 0.01,
341
***,
P < 0.001).
i-j, Heat map of cytokine and chemokine protein expression levels in lung
342
homogenates from the indicated groups. Data are presented as log2-transformed fold-change over
343
naive mice. Blue, reduction; red, increase.
k-l, Correlation analysis. The fold-change in EC50
344
value of AZD7442-YTE/TM (
k) and S309-LS (
l) for D614G and each Omicron variant strain are
345
plotted on the x-axis. The fold-change in lung viral RNA titer between the respective isotype or
346
mAb-treated groups against each Omicron variant strain are plotted on the y-axis. Best-fit lines
347
were calculated using a simple linear regression. Two-tailed Pearson correlation was used to
348
calculate the R2 and P values indicated within each panel.
349
Figure 3. Role of Fc-effector functions in S309 mAb-mediated protection. a, Binding
350
of AZD7442-TM, S309-LS, or S309-GRLR mAbs to hFcγRI or hFcγRIIIa (two experiments;
351
dotted lines indicate the limit of detection).
b, ExpiCHO-S cells were transiently transfected with
352
plasmids encoding the indicated SARS-CoV-2
spike protein. 24 to 48 h later, cells were
353
harvested, washed, and stained with the indicated concentrations of VIR-7831 or S2X324 mAbs
354
to assess binding to the cell surface. Representative histograms from two-three experiments are
355
shown.
c, ExpiCHO-S cells transiently transfected with Wuhan-1 D614, BA.1, or BA.2 spike
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.
356
proteins were incubated with the indicated concentrations of VIR-7831 or S309-GRLR mAb and
357
mixed with purified NK cells isolated from healthy donors at a ratio of 1:9 (target:effector). Cell
358
lysis was determined by a lactate dehydrogenase release assay. The error bars indicate standard
359
deviations (SD).
d, Area under the curve (AUC) analyses from four NK donors
(
Extended Data
360
Fig. 5).
e, ExpiCHO-S cells transiently transfected with Wuhan-1 D614, BA.1, or BA.2 spike
361
proteins and fluorescently labelled with PKH67 were incubated with the indicated concentrations
362
of VIR-7831 or S309-GRLR mAb and mixed with PBMCs labelled with CellTrace Violet from
363
healthy donors at a ratio of 1:20 (target:PBMCs). Association of CD14+ monocytes with spike-
364
expressing target cells (ADCP) was determined by flow cytometry. The error bars indicate SD.
f,
365
AUC analyses of VIR-7831 and S309-GRLR for each Omicron variant for four donors. (
g-i)
366
Eight-week-old female K18-hACE2 mice or (
j-k) 12-week-old male hFcγR mice received a
367
single 10
mg/kg or 3 mg/kg dose respectively, of S309-LS or S309-GRLR mAb by
368
intraperitoneal injection one day before intranasal inoculation with 103 FFU of D614G, BA.1, or
369
BA.2 (
g-i) or 105 FFU of Beta (B.1.351) (
j-k). Tissues were collected at 2 (B.1.351), 4 (B.1.351),
370
6 (BA.2), or 7 (D614G and BA.1) dpi. Viral RNA levels in the lungs (
g, j-k),
nasal turbinates
371
(
h), and nasal washes (
i) were determined by RT-qPCR, and infectious virus in the lungs (
j-k)
372
was measured by plaque assay (
g-k; lines indicate median ± SEM.;
g-i and
j-k; n = 8 and
373
10 mice per group, respectively; two experiments;
g-i; (Mann-Whitney test between isotype and
374
mAb treatment; ns, not significant; **
P < 0.01, ***,
P < 0.001);
j-k; one-way ANOVA with
375
Tukey’s multiple comparisons test; ns, not significant; *
P < 0.05, **
P < 0.01, ***,
P < 0.001,
376
****,
P < 0.0001).
377
378
EXTENDED DATA FIGURES
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379
Extended Data Figure 1. BA.1.1 spike protein substitutions and mAb epitopes.
380
Mutated residues in the BA.1.1 RBD relative to WA1/2020 are indicated in green in all three
381
panels. The BA.1.1 RBD bound by mAbs S309 (orange, PDB: 6WPS) (
a), AZD8895 (pale
382
green, PDB: 7L7D) (
b), and AZD1061 (purple, PDB:7L7E) (
c) are shown. BA.1.1 substitutions
383
in the respective epitopes of each mAb are shaded red, whereas those outside the epitope are
384
shaded green. Structural analysis and depictions were generated using UCSF ChimeraX39.
385
Extended Data Figure 2. Cytokine and chemokine induction after S309-LS
386
treatment and SARS-CoV-2 infection. Individual graphs of cytokine and chemokine protein
387
levels in the lungs of S309-LS mAb-treated K18-hACE2 mice at 6 (BA.2) or 7 dpi (all other
388
strains) with the indicated SARS-CoV-2 strain (line indicates median; n = 3 naive, n = 6-8 for all
389
other groups (Mann-Whitney test with comparison between the isotype control and mAb: *,
P <
390
0.05, **,
P < 0.01, ***,
P < 0.001).
391
Extended Data Figure 3. Cytokine and chemokine induction after AZD7442-TM
392
treatment and SARS-CoV-2 infection. Individual graphs of cytokine and chemokine protein
393
levels in the lungs of AZD7442-TM mAb-treated K18-hACE2 mice at 6 (BA.2) or 7 dpi (all
394
other strains) with the indicated SARS-CoV-2 strain (line indicates median; n = 3 naive, n = 8
395
for all other groups (Mann-Whitney test with comparison between the isotype control and mAb:
396
*,
P < 0.05, **,
P < 0.01, ***,
P < 0.001).
397
Extended Data Figure 4. Neutralization of SARS-CoV-2 variants by S309-LS and
398
S309-GRLR mAbs. Neutralization curves in Vero-TMPRSS2 cells comparing infection of the
399
indicated SARS-CoV-2 strain in the presence of each mAb. The average of two experiments
400
performed in technical duplicate are shown. For D614G, BA.1, and BA.2 strains, the S309-LS
401
neutralization data from
Fig. 1f are shown for comparison.
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402
Extended Data Figure 5. VIR-7831-mediated antibody-dependent cell cytotoxicity
403
with NK cells. ExpiCHO-S cells transiently transfected with expression plasmids encoding
404
Wuhan D614, BA.1, or BA.2 spike proteins were incubated with the indicated concentrations of
405
VIR-7831 or S309-GRLR and mixed with NK cells isolated from healthy donors at a ratio of 1:9
406
(target:effector). Target cell lysis was determined by a lactate dehydrogenase release assay. The
407
error bars indicate SDs. Each panel is an individual donor. Donors 1 and 3 are heterozygous for
408
F158 and V158 FcγRIIIa, whereas donors 2 and 4 are homozygous for V158.
409
Extended Data Figure 6. VIR-7831-mediated antibody-dependent cell phagocytosis
410
with monocytes. ExpiCHO-S cells transiently transfected with Wuhan-1 D614, BA.1, or BA.2
411
spike proteins and fluorescently labelled with PKH67 were incubated with the indicated
412
concentrations of VIR-7831 or S309-GRLR mAb and mixed with PBMCs labelled with
413
CellTrace Violet from healthy donors carrying different FcγRIIA and IIIA genotypes at a ratio of
414
1:20 (target:PBMCs). Association of CD14+ monocytes with spike-expressing target cells
415
(ADCP) was determined by flow cytometry. The error bars indicate SD. Each panel is an
416
individual donor.
417
Extended Data Figure 7. Gating strategy for CD14+ monocytes used for antibody-
418
dependent cell phagocytosis assays. From PBMCs, monocytes were gated as CD3– CD19–
419
CD14+ cells. For ADCP, % FITC+ CellTrace Violet+ CD14+ monocytes were gated as indicated.
420
The gate of positive cells was set based on the no mAb control.
421
422
SUPPLEMENTAL TABLE TITLES
423
Supplementary Table 1. Omicron variant strain mutations as determined by next-
424
generation sequencing.
20
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425
21
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426
METHODS
427
Cells.
Vero-TMPRSS240 and Vero-hACE2-TMPRRS241 cells were cultured at 37°C in
428
Dulbecco’s Modified Eagle medium (DMEM) supplemented with 10% fetal bovine serum
429
(FBS), 10 mM HEPES pH 7.3, 1 mM sodium pyruvate, 1× non-essential amino acids, and
430
100 U/ml of penicillin–streptomycin. Vero-TMPRSS2 cells were supplemented with 5 μg/mL
431
of blasticidin. Vero-hACE2-TMPRSS2 cells were supplemented with 10 µg/mL of puromycin.
432
ExpiCHO-S cells were obtained from Thermo Fisher Scientific. All cells routinely tested
433
negative for mycoplasma using a PCR-based assay.
434
Viruses. The Beta (B.1.351) and Omicron (BA.1 (R346), BA.1.1 (R346K), and BA.2)
435
strains were obtained from nasopharyngeal isolates. All virus stocks were generated in Vero-
436
TMPRSS2 cells and subjected to next-generation sequencing as described previously41 to
437
confirm the presence and stability of expected substitutions (see
Supplementary Table 1). All
438
virus experiments were performed in an approved biosafety level 3 (BSL-3) facility.
439
Monoclonal antibody purification. The mAbs studied in this paper, S309, AZD8895,
440
AZD1061, and the AZD7442 cocktail have been described previously4,11,18.
441
S309-LS and S309-GRLR were produced in ExpiCHO-S cells and affinity-purified using
442
HiTrap Protein A columns (GE Healthcare, HiTrap mAb select Xtra #28-4082-61) followed by
443
buffer exchange to histidine buffer (20 mM histidine, 8% sucrose, pH 6.0) using HiPrep 26/10
444
desalting columns. The final products were sterilized after passage through 0.22 μm filters and
445
stored at 4°C. VIR-7831 (clinical lead variant of S309-LS) was produced at WuXi Biologics.
446
AZD8895 and AZD1061 mAbs were cloned into mammalian expression vectors and
447
expressed as IgG1 constructs with the TM (L234F/L235E/P331S) Fc modification with or
448
without a second YTE (M252Y/S254T/T256E) modification to extend half-life in humans.
22
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.
449
MAbs were expressed in 293F cells after transfection with 293fectin (Thermo Fisher Scientific)
450
and isolated from supernatants by affinity chromatography using Protein A or Protein G columns
451
(GE Healthcare). MAbs were eluted with 0.1 M glycine at low pH and dialyzed into PBS.
452
Mouse experiments. Animal studies were carried out in accordance with the
453
recommendations in the Guide for the Care and Use of Laboratory Animals of the National
454
Institutes of Health. The protocols were approved by the Institutional Animal Care and Use
455
Committee at the Washington University School of Medicine (assurance number A3381–01).
456
Virus inoculations were performed under anesthesia that was induced and maintained with
457
ketamine hydrochloride and xylazine, and all efforts were made to minimize animal suffering.
458
Heterozygous K18-hACE2 C57BL/6J mice (strain: 2B6.Cg-Tg(K18-ACE2)2Prlmn/J)
459
and wild-type C57BL/6J (strain: 000664) mice were obtained from The Jackson Laboratory.
Human FcγR transgenic mice20 (FcγRα-/-
460
461
/hFcγRI+/hFcγRIIAR131+/hFcγRIIB+/hFcγRIIIAF158+/hFcγRIIIB+) were a generous gift (J.
462
Ravetch, Rockefeller University) and bred at Washington University. All animals were housed in
463
groups and fed standard chow diets. For experiments with K18-hACE2 mice, eight- to ten-week-
464
old female mice were administered the indicated doses of the respective SARS-CoV-2 strains
465
(see Figure legends) by intranasal administration. For hFcγR mouse experiments, 12-week-old
466
male mice were administered 105 FFU of a Beta (B.1.351) isolate by intranasal administration.
In
467
vivo studies were not blinded, and mice were randomly assigned to treatment groups. No sample-
468
size calculations were performed to power each study. Instead, sample sizes were determined
469
based on prior
in vivo virus challenge experiments. Mice were administered the indicated mAb
470
dose by intraperitoneal injection one day before intranasal inoculation with the indicated SARS-
23
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.
471
CoV-2 strain. AZD7442-TM (lacking the YTE modification that accelerates antibody
472
elimination in rodents) was used in mouse studies.
473
Focus reduction neutralization test. Serial dilutions of mAbs were incubated with 102
474
focus-forming units (FFU) of different strains or variants of SARS-CoV-2 for 1 h at 37°C.
475
Antibody-virus complexes were added to Vero-TMPRSS2 cell monolayers in 96-well plates and
476
incubated at 37°C for 1 h. Subsequently, cells were overlaid with 1% (w/v) methylcellulose in
477
MEM. Plates were harvested 48-72 h later by removing overlays and fixing with 4% PFA in PBS
478
for 20 min at room temperature. Plates were washed and incubated with an oligoclonal pool of
479
SARS2-2, SARS2-11, SARS2-16, SARS2-31, SARS2-38, SARS2-57, and SARS2-7142. Plates
480
with Omicron variant strains were additionally incubated with CR3022 and a pool of anti-
481
SARS-CoV-2 mAbs that cross-react with SARS-CoV43. Subsequently, samples were incubated
482
with HRP-conjugated goat anti-mouse IgG (Sigma, 12-349) and HRP-conjugated goat anti-
483
human IgG (Sigma, A6029) in PBS supplemented with 0.1% saponin and 0.1% bovine serum
484
albumin. SARS-CoV-2-infected cell foci were visualized using TrueBlue peroxidase substrate
485
(KPL) and quantitated on an ImmunoSpot microanalyzer (Cellular Technologies).
486
Measurement of viral RNA levels. Tissues were weighed and homogenized with
487
zirconia beads in a MagNA Lyser instrument (Roche Life Science) in 1 mL of DMEM medium
488
supplemented with 2% heat-inactivated FBS. Tissue homogenates were clarified by
489
centrifugation at 10,000 rpm for 5 min and stored at −80°C. RNA was extracted using the
490
MagMax mirVana Total RNA isolation kit (Thermo Fisher Scientific) on the Kingfisher Flex
491
extraction robot (Thermo Fisher Scientific). RNA was reverse transcribed and amplified using
492
the TaqMan RNA-to-CT 1-Step Kit (Thermo Fisher Scientific). Reverse transcription was
493
carried out at 48°C for 15 min followed by 2 min at 95°C. Amplification was accomplished over
24
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.
494
50 cycles as follows: 95°C for 15 s and 60°C for 1 min. Copies of SARS-CoV-2
N gene RNA in
495
samples were determined using a previously published assay44. Briefly, a TaqMan assay was
496
designed to target a highly conserved region of the
N gene (Forward primer:
497
ATGCTGCAATCGTGCTACAA; Reverse primer: GACTGCCGCCTCTGCTC; Probe: /56-
498
FAM/TCAAGGAAC/ZEN/AACATTGCCAA/3IABkFQ/). This region was included in an RNA
499
standard to allow for copy number determination down to 10 copies per reaction. The reaction
500
mixture contained final concentrations of primers and probe of 500 and 100 nM, respectively.
501
Viral plaque assay. Vero-TMPRSS2-hACE2 cells were seeded at a density of 1×105
502
cells per well in 24-well tissue culture plates. The following day, medium was removed and
503
replaced with 200 μL of material to be titrated diluted serially in DMEM supplemented with 2%
504
FBS. One hour later, 1 mL of methylcellulose overlay was added. Plates were incubated for 72 h,
505
then fixed with 4% paraformaldehyde (final concentration) in PBS for 20 min. Plates were
506
stained with 0.05% (w/v) crystal violet in 20% methanol and washed twice with distilled,
507
deionized water.
508
Transient expression of recombinant SARS-CoV-2 protein and flow cytometry.
509
ExpiCHO-S cells were seeded at 6 x 106 cells/mL in a volume of 5 mL in a 50 mL bioreactor.
510
The following day, cells were transfected with SARS-CoV-2 spike glycoprotein-encoding
511
pcDNA3.1(+) plasmids (BetaCoV/Wuhan-Hu-1/2019, accession number MN908947, Wuhan
512
D614; Omicron BA.1 and BA.2 generated by overlap PCR mutagenesis of the Wuhan D614
513
plasmid) harboring the Δ19 C-terminal truncation26. Spike encoding plasmids were diluted in
514
cold OptiPRO SFM (Life Technologies, 12309-050), mixed with ExpiFectamine CHO Reagent
515
(Life Technologies, A29130) and added to cells. Transfected cells were then incubated at 37˚C
516
with 8% CO2 with an orbital shaking speed of 250 RPM (orbital diameter of 25 mm) for 24 to 48
25
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(which 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
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available under a
.
517
h. Transiently transfected ExpiCHO-S cells were harvested and washed twice in wash buffer
518
(PBS 2% FBS, 2 mM EDTA). Cells were counted and distributed into round bottom 96-well
519
plates (Corning, 3799) and incubated with serial dilutions of mAb starting at 10 μg/mL. Alexa
520
Fluor647-labelled Goat Anti-human IgG secondary Ab (Jackson Immunoresearch, 109-606-098)
521
was prepared at 2 μg/mL and added onto cells after two washing steps. Cells were then washed
522
twice and resuspended in wash buffer for data acquisition at ZE5 cytometer (BioRad).
523
Fc-mediated effector functions. Primary cells were collected from healthy human
524
donors with informed consent and authorization via the
Comitato Etico Canton Ticino
525
(Switzerland). ADCC assays were performed using ExpiCHO-S cells transiently transfected with
526
SARS-CoV-2 spike glycoproteins (Wuhan D614, BA.1 or BA.2) as targets. NK cells were
527
isolated from fresh blood of healthy donors using the MACSxpress NK Isolation Kit (Miltenyi
528
Biotec, cat. no. 130-098-185). Target cells were incubated with titrated concentrations of mAbs
529
for 10 min and then with primary human NK cells at an effector:target ratio of 9:1. ADCC was
530
measured using LDH release assay (Cytotoxicity Detection Kit (LDH) (Roche; cat. no.
531
11644793001) after 4 h incubation at 37˚C.
532
ADCP assays were performed using ExpiCHO-S cells transiently transfected with SARS-
533
CoV-2 spike glycoproteins (Wuhan D614, BA.1, or BA.2) and labelled with PKH67 (Sigma
534
Aldrich) as targets. PMBCs from healthy donors were labelled with CellTrace Violet
535
(Invitrogen) and used as source of phagocytic effector cells. Target cells (10,00 per well) were
536
incubated with titrated concentrations of mAbs for 10 min and then mixed with PBMCs (200,000
537
per well). The next day, cells were stained with APC-labelled anti-CD14 mAb (BD Pharmingen),
538
BV605-labelled anti-CD16 mAb (Biolegend), BV711-labelled anti-CD19 mAb (Biolegend),
539
PerCP/Cy5.5-labelled anti-CD3 mAb (Biolegend), APC/Cy7-labelled anti-CD56 mAb
26
https://doi.org/10.1101/2022.03.17.484787
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(which 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
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available under a
.
540
(Biolegend) for the identification of CD14+ monocytes. After 20 min, cells were washed and
541
fixed with 4% paraformaldehyde before acquisition on a ZE5 Cell Analyzer (Biorad). Data were
542
analyzed using FlowJo software. The % ADCP was calculated as % of monocytes (CD3- CD19-
543
CD14+ cells) positive for PKH67.
544
Data availability. All data supporting the findings of this study are available within the
545
paper and are available from the corresponding author upon request.
546
Statistical analysis. All statistical tests were performed as described in the indicated
547
figure legends using Prism 8.0. Statistical significance was determined using a one-way ANOVA
548
when comparing three or more groups. When comparing two groups, a Mann-Whitney test was
549
performed. The number of independent experiments performed are indicated in the relevant
550
figure legends. For correlation analyses, best-fit lines were calculated using a simple linear
551
regression. Two-tailed Pearson correlation was used to calculate the R2 and P values indicated
552
within each panel.
553
27
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.
554
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