donors (geometric mean titers = 1595, 727, and
854 for D614G, Beta, and Delta, respectively)
(Fig. 1, H to J). Furthermore, about 80% of
donor sera displayed low but detectable neu-
tralizing activity against Omicron, with 100%
neutralization titers ranging from <10 to
90 (Fig. 1K). Taken together, heterologous
ChAdOx1:mRNA-1273 prime-boost vaccina-
tion elicits superior binding and neutraliz-
ing antibody responses to SARS-CoV-2 and
VOCs relative to homologous ChAdOx1:ChAdOx1
vaccination.
Previous studies of other viral infections
have shown that booster vaccination typically
induces a rapid and robust antigen-specific
plasmablast response that peaks at approxi-
mately day 7 after immunization and accounts
for a relatively large proportion of peripheral
blood B cells ( 15 , 16 ). We therefore assessed the
frequency of plasmablasts in peripheral blood
7 to 10 days after homologous and heterolo-
gous booster vaccination (fig. S2A). Although
we observed modest plasmablast expansion in
a subset of ChAdOx1-boosted donors, the me-
dian frequency of plasmablasts across all do-
nors was not significantly elevated relative to
preboost or unpaired prepandemic healthy
donor samples (Fig. 2A). By contrast, heterol-
ogous booster immunization induced a robust
plasmablast response in almost all donors, in
which plasmablast frequencies averaged 3%
and accounted for up to 7% of all CD19+periph-
eral blood B cells (Fig. 2A).
We next investigated the magnitude and
specificities of the S-specific MBC response in-
duced after both prime and boost immunization.Because ChAdOx1 encodes WT S, which may
elicit B cell responses to both pre- and post-
fusion conformations of S, we evaluated B cell
responses to both prefusion-stabilized (S-2P)
and WT (unstabilized) forms of the S pro-
tein (Fig. 2B and fig. S2B). SDS–polyacrylamide
gel electrophoresis (SDS-PAGE) analysis re-
vealed that the recombinant WT S prepara-
tion used for B cell staining contained both
uncleaved and cleaved forms of S, the latter
likely representing a mixture of prefusion, post-
fusion, and non-native or dissociated forms of
S (fig. S3). The frequency of total S (WT + S-2P)–
specific IgG+B cells ranged from 0 to 3.7%
after ChAdOx1 prime immunization, and homol-
ogous booster immunization did not signifi-
cantly enhance this response in most donors
(Fig. 2C). By contrast, mRNA-1273 boosterSCIENCEscience.org 4 MARCH 2022•VOL 375 ISSUE 6584 1045
A BCDE FChAdOx1:
ChAdOx1
ChAdOx1:
mRNA-1273ChAdOx1:ChAdOx1
No. of VOCs bound
(KD< 50nM)ChAdOx1
mRNA-1273ChAdOx1
mRNA-1273ChAdOx1
mRNA-1273ChAdOx1
mRNA-1273ChAdOx1
mRNA-1273ChAdOx1
mRNA-1273020406080100% of anti-RBD antibodies<22-1010-100>100Fold reduction
in KDBooster
type:Beta Gamma Delta Kappa LambdaOmicronRBD K(nM)D45
1
2
3
4076Beta
Gamma
Delta
Kappa
Lambda
Omicron<22-1010-100>100mAb class undefined11/222/334AntibodyNeut. IC (^50) class
<0.02
0.02-0.2
0.2-2
2-10
10
Fold
reduction
in KD
Wuhan-1 neut.
IC 50 (μg/ml)
ChAdOx1:mRNA-1273
ChAdOx1
mRNA-1273
ChAdOx1
mRNA-1273
ChAdOx1
mRNA-1273
ChAdOx1
mRNA-1273
0
20
40
60
80
100
% of anti-NTD antibodies
Beta Gamma Delta Omicron
<2
2-10
10-100
100
Fold reduction
in KD
Booster
type:
ChAdOx1:
ChAdOx1
ChAdOx1:
mRNA-1273
NTD K
(nM)D
Neutralizing Non-neutralizing
Wuhan-1
Beta
GammaDeltaKappaLambdaOmicron
0.1
1
10
100
0.082 0.052
Wuhan-1
Beta
Gamma
Delta
Omicron
0.01
0.1
1
10
100
Fig. 4. Breadth of antibody binding to SARS-CoV-2 variants.(A) Proportion
of RBD-reactive antibodies with the indicated fold reduction in Fab binding affinity to
RBDs incorporating mutations present in the Beta, Gamma, Delta, Kappa, Lambda,
and Omicron variants relative to the Wuhan-1 RBD, as determined with a bead-based
flow cytometric assay. (B) Fab binding affinities of RBD-directed antibodies to
Wuhan-1 and variant RBDs. Antibodies that did not reach half-maximal saturation at
the highest concentration tested (100 nM) are shown asKd>100 nM. Black bars
denote medians. (C) Heatmap showing the fold reduction in affinity to variant RBDs
compared with the Wuhan-1 RBD among neutralizing and non-neutralizing antibodies
to RBD. The top bar indicates the neutralization inhibitory concentration (IC 50 )
of each mAb against MLV-SARS-CoV-2 Wuhan-1. Antibodies that did not reach
50% neutralization at 10mg/ml were classified as non-neutralizing. The bottom bar
denotes the class of each antibody. (D) Proportion of NTD-directed antibodies
with the indicated fold reductions in binding activity to variant NTDs relative to the
Wuhan-1 NTD. (E) Fab binding affinities of NTD-directed antibodies to Wuhan-1
and variant NTDs. Antibodies that did not reach half-maximal saturation at 100 nM
are shown asKd>100 nM. Black bars denote medians. (F) Combined proportions
of antibodies to RBD and NTD that bound the indicated number of variants of
concern (Beta, Gamma, Delta, and Omicron) withKd≤50 nM. The number in
the center of the pie indicates the total number of antibodies tested. Statistical
significance was determined by [(B) and (E)] two-sided Mann-WhitneyUtests or (F)
FisherÕs exact test.KD, equilibrium dissociation constant; *P< 0.05.
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