bind to saturation. Graphs represent reference-
subtracted relative“analyte binding late”report
points (RUs), determined by aligning sensor-
grams to Y (RUs) = 0, beginning at the mAb
association stage using Biacore 8K Insights
Evaluation Software (Cytiva). All assays were
performed using a Biacore 8K+ (Cytiva) SPR
spectrometer.
ACE2-binding inhibition assay
ACE2-binding inhibition was completed, as
previously described ( 7 ), using 1:5 diluted BAL
and NS samples.
Lentiviral pseudovirus neutralization assay
Pseudotyped lentiviral reporter viruses were
produced by the cotransfection of plasmids
encoding SARS-CoV-2 proteins from multiple
variants, a luciferase reporter, lentivirus back-
bone, and human transmembrane protease
serine 2 (TMPRSS2) genes as previously de-
scribed ( 17 , 44 ). Reagent details are shown in
table S5. Sera were tested, in duplicate, for
neutralizing activity against the pseudovi-
ruses, and percent neutralization was cal-
culated in GraphPad Prism software version
9.0.2. The lower limit of quantification was
1:40 ID 50.
VSV pseudovirus neutralization assay
SARS-CoV-2 pseudotyped recombinant VSV-
DG-firefly luciferase viruses were made by co-
transfection of plasmid expressing full-length
S, and subsequent infection with VSVDG-
firefly-luciferase and neutralization assays
were completed on sera samples as previously
described ( 15 , 45 ); reagent details are described
in table S5. The lower limit of quantification
was 1:40 ID 50.
Focus reduction neutralization test
Focus reduction neutralization tests were per-
formed on sera samples, in duplicate, as pre-
viously described ( 40 ). Reagent details are
provided in table S5. For samples that did not
neutralize 50% of virus at the limit of detec-
tion,“ 5 ”was plotted and used for geometric
mean calculations.
B cell probe-binding assay
Cryopreserved peripheral blood mononuclear
cells (PBMCs) were thawed and washed in
wash buffer (4% heat-inactivated newborn calf
serum/0.02% NaN 3 /phenol-free RPMI). Cells
were incubated with streptavidin-BV605 (BD
Biosciences)–labeledbS-2P and streptavidin-
BUV661 (BD Biosciences)–labeled WA-1 S-2P
for30minat4°C,washedtwicein1×PBS,and
incubated with Aqua live/dead fixable dead
cell stain (Thermo Fisher Scientific) for 20 min
at room temperature. After two washes in
wash buffer, cells were incubated with primary
antibodies for 20 min at room temperature.
The following antibodies were used (monoclo-
nal unless indicated): IgD fluorescein isothio-
cyanate (FITC, goat polyclonal, Southern Biotech),
IgM PerCP-Cy5.5 (clone G20-127, BD Bio-
sciences), IgA Dylight 405 (goat polyclonal,
Jackson ImmunoResearch), CD20 BV570 (clone
2H7, BioLegend), CD27 BV650 (clone O323,
BioLegend),CD14BV785(cloneM5E2,BioLegend),
CD8 BUV395 (clone RPA-T8, BD Biosciences),
CD16 BUV496 (clone 3G8, BD Biosciences),
CD4 BUV737 (clone SK3, BD Biosciences), CD19
APC (clone J3-119, Beckman Coulter), IgG Alexa
Fluor 700 (clone G18-145, BD Biosciences), CD3
APC-Cy7 (clone SP34.2, BD Biosciences), CD38
PE (clone OKT10, Caprico Biotechnologies),
CD21 PE-Cy5 (clone B-ly4, BD Biosciences),
and CXCR5 PE-Cy7 (clone MU5UBEE, Thermo
Fisher Scientific). Cells were washed twice in
wash buffer, and residual red blood cells were
lysed using BD FACS Lysing Solution (BD Bio-
sciences) for 10 min at room temperature.
After two additional washes, cells were fixed
in 0.5% formaldehyde (Tousimis Research)
All antibodies were previously titrated to de-
termine the optimal concentration. Samples
were acquired on an BD FACSymphony flow
cytometer and analyzed using FlowJo soft-
ware version 10.7.2 (BD Biosciences).Intracellular cytokine staining
Cryopreserved PBMCs were thawed and rested
overnight in a 37°C, 5% CO 2 incubator. The next
morning, cells were stimulated with SARS-
CoV-2 S protein peptide pools (S1 and S2) that
were matched to the vaccine insert [composed
of 158 and 157 individual peptides, respec-
tively, as 15mers overlapping by 11 amino acids
in 100% dimethyl sulfoxide (DMSO), JPT Pep-
tides] at a final concentration of 2mg/ml in the
presence of 3 mM monensin for 6 hours.
Negative controls received an equal concen-
tration of DMSO instead of peptides (final
concentration 0.5%). Intracellular cytokine
staining was performed as described previ-
ously ( 46 ). The following mAbs were used:
CD3 APC-Cy7 (clone SP34.2, BD Biosciences),
CD4 PE-Cy5.5 (clone S3.5, Invitrogen), CD8
BV570 (clone RPA-T8, BioLegend), CD45RA
PE-Cy5 (clone 5H9, BD Biosciences), CCR7
BV650 (clone G043H7, BioLegend), CXCR5 PE
(clone MU5UBEE, Thermo Fisher), CXCR3
BV711 (clone 1C6/CXCR3, BD Biosciences),
PD-1 BUV737 (clone EH12.1, BD Biosciences),
ICOS Pe-Cy7 (clone C398.4A, BioLegend),
CD69 ECD (cloneTP1.55.3, Beckman Coulter),
interferon-g(IFN-g) Ax700 (clone B27, BioLegend),
interleukin-2 (IL-2) BV750 (clone MQ1-17H12,
BD Biosciences), IL-4 BB700 (clone MP4-25D2,
BD Biosciences), tumor necrosis factor (TNF)–
FITC (clone Mab11, BD Biosciences), IL-13 BV421
(clone JES10-5A2, BD Biosciences), IL-17 BV605
(clone BL168, BioLegend), IL-21 Ax647 (clone
3A3-N2.1, BD Biosciences), and CD154 BV785
(clone 24-31, BioLegend). The Aqua live/dead
fixable dead cell stain kit (Thermo FisherScientific) was used to exclude dead cells. All
antibodies were previously titrated to deter-
mine the optimal concentration. Samples were
acquired on a BD FACSymphony flow cytom-
eter and analyzed using FlowJo software ver-
sion 9.9.6 (TreeStar). The gating schema is
represented in fig. S8.Statistical analysis
Graphs show data from individual animals,
with dotted lines indicating assay limits of
detection. Groups were compared for viral
load and immune responses using Welch’s
ttests; data were analyzed on the log 10 scale
for viral loads and appropriate immune as-
says. Changes over time were summarized
using the change on the log 10 scale, and sta-
tistical significance was determined using
pairedttests. When more than two variants
were compared, as in Fig. 1, A to F, a Holm’s
adjustment for multiple comparisons was
used on the set ofPvalues to determine sig-
nificance. There were no adjustments for mul-
tiple comparisons across different analyses
in this descriptive study. Comparisons drawn
between vaccination groups for serum anti-
body epitope analysis were performed using
unpairedttests, within each time point eval-
uated. Analyses were performed in R software
version 4.0.2 and GraphPad Prism software
versions 8.2 and 9.0.2.REFERENCESANDNOTES- F. P. Polacket al.,N. Engl. J. Med. 383 , 2603– 2615
(2020). - L. R. Badenet al.,N. Engl. J. Med. 384 , 403– 416
(2021). - M. Voyseyet al.,Lancet 397 , 99–111 (2021).
- J. Sadoffet al.,N. Engl. J. Med. 384 , 2187–2201 (2021).
- P. T. Heathet al.,N. Engl. J. Med. 385 , 1172– 1183
(2021). - C. Liuet al.,Cell 184 , 4220–4236.e13 (2021).
- A. Peguet al.,Science 373 , 1372–1377 (2021).
- Q. Liet al.,Cell 182 , 1284–1294.e9 (2020).
- H. Tegallyet al.,Nature 592 , 438–443 (2021).
- C. K. Wibmeret al.,Nat. Med. 27 , 622–625 (2021).
- E. Andreanocet al., bioRxiv 424451 [Preprint]
(2020). - M. Hoffmannet al.,Cell 184 , 2384–2393.e12 (2021).
- W. F. Garcia-Beltranet al.,Cell 184 , 2372–2383.e9
(2021). - N. Doria-Roseet al.,N. Engl. J. Med. 384 , 2259– 2261
(2021). - K. Wuet al.,N. Engl. J. Med. 384 , 1468–1470 (2021).
- T. Tadaet al., bioRxiv 452771 [Preprint] (2021).
- K. Wuet al., medRxiv 2021.2005.2005.21256716
[Preprint] (2021). - K. S. Corbettet al.,Science 373 , eabj0299 (2021).
- K. S. Corbettet al.,N. Engl. J. Med. 383 , 1544– 1555
(2020). - M. Guebre-Xabieret al.,Vaccine 38 , 7892– 7896
(2020). - N. B. Mercadoet al.,Nature 586 , 583–588 (2020).
- J. Yuet al.,Science 369 , 806–811 (2020).
- P. J. Klasse, D. F. Nixon, J. P. Moore,Sci. Adv. 7 , eabe8065
(2021). - N. van Doremalenet al.,Nature 586 , 578–582 (2020).
- E. S. Winkleret al.,Cell 184 , 1804–1820.e16 (2021).
- J. R. Francicaet al.,Sci. Transl. Med. 13 , eabi4547
(2021). - L. Wanget al.,Science 373 , eabh1766 (2021).
- G. D. Victora, M. C. Nussenzweig,Annu. Rev. Immunol. 30 ,
429 – 457 (2012). - J. S. Turneret al.,Nature 595 , 421–425 (2021).
1352 10 DECEMBER 2021•VOL 374 ISSUE 6573 science.orgSCIENCE
RESEARCH | RESEARCH ARTICLES