Science - USA (2021-12-03)

(Antfer) #1

trimer, potentially allowing for increased
recruitment and/or selection of specific clones
that can bind conserved regions of RBD ( 43 , 44 ).
By contrast, convalescent individuals were
primed against native, nonstabilized spike pro-
tein. Taken together, our data indicate robust
B cell memory to multiple components of the
spike protein as well as currently described
VOCs that continues to evolve and increase in
frequency over time.


Clonal evolution of variant-specific
memory B cells
We next asked what differences may underly
variant-binding versus nonbinding proper-
ties of memory B cells. Here, we focused on
the Beta B.1.351 variant RBD containing the
K417N, E484K, and N501Y mutations because
this variant resulted in the greatest loss of
binding relative to WT RBD (Fig. 3, E, G, and
H). We designed a sorting panel to identify

three populations of memory B cells with
different antigen-binding specificities: (i) mem-
ory B cells that bind full-length spike but not
RBD, (ii) memory B cells that bind full-length
spike and WT RBD but not B.1.351 variant RBD,
and (iii) memory B cells that bind full-length
spike and cross-bind both WT and B.1.351 var-
iant RBD (Fig. 4A and fig. S5A). Naïve B cells
were also sorted as a control. These populations
were isolated from eight SARS-CoV-2–naïve

Goelet al.,Science 374 , eabm0829 (2021) 3 December 2021 7of17


Naive Recovered

Naive Recovered

0.0

2.5

5.0

7.5

WT RBD+
RBD++

% mutated VH NT

Overlapping Clones

****

WT RBD+
18177

RBD++
576 4419

Clonal Overlap (50% mcf)

0

25

50

75

100

Pre−Im

mun

e
Naive
Reco

vere

d

% of WT RBD+

B.1.351+

Naive

B
Spike

+ RBD−WT RBD+RBD++Nai
ve B
Spik
e+ RBD−WT RBD+RBD++

1

10

100

D20 Score

Clonality

ns0.078
0.057

ns0.069
*

A


D


RBD+
7.24

RBD++
20.3

RBD-
72.1

Gated on Spike+ Memory B

RBD WT - APC

RBD B.1.351 - PE

Isolate B Cells Cell Sorting

Label SARS-CoV-2-Specific
Memory B Cells

N = 8 N = 4

3-4 Months Post-Vaccine

BCR Sequencing

IgH

RBD-WT RBD+RBD++

Spike+ Memory B

Naive

Recovere

d

Cross-BindingB.1.351

Prior COVID-19

BC

H

G

I

J

Overlapping Clones

N = 576

Higher SHM in:
WT RBD+
Equal
RBD++

: 23.6%
: 45%

: 31.4%

E
Naive Recovered

(^0510152005101520)
0.0
0.1
0.2
0.3
Density
SHM
Naiv
e B
Spike+ RBD−
WT RBD+RBD++Nai
ve B
Spik
e+ RBD−WT RBD+RBD++
0
4
8
12
% mutated VH NT
SHM
N=117,451
N=23,583
N=15,340
N=5,954
N=105,913
N=13,277
N=10,091
N=2,147
% mutated VH NT
F
Clonal Lineages with Binding Overlap
WT RBD+ RBD++
IGHV3-53
CARELGDFAFDIW
IGHV3-53
CARDLEVYGMDVW
IGHV3-33
CARDVNDTTMALGPLYFYGMDVW
IGHV3-53
CARDLDYYGMDVW
IGHV3-53
RARDYGDLYFDYW
IGHV4-59
CARNGGSGRIGLDKKWAFDIW
IGHV3−49
IGHV5−10−1
IGHV3−13
IGHV4−38−2
IGHV6−1
IGHV1−24
IGHV1−8
IGHV3−74
IGHV1−3
IGHV2−70
IGHV3−11
IGHV3−15
IGHV4−61
IGHV2−5
IGHV4−4
IGHV3−53
IGHV3−66
IGHV1−18
IGHV5−51
IGHV1−46
IGHV4−31
IGHV3−9
IGHV3−48
IGHV4−34
IGHV1−2
IGHV3−21
IGHV3−7
IGHV4−39
IGHV4−59
IGHV1−69
IGHV3−33
IGHV3−23|3−23D
IGHV3−30
Recovered
Binding
Binding
Spike+ RBD−
WT RBD+
RBD++
Prior COVID-19
No
Ye s
5
10
15
0
% of Clones (by Column)
GL GL GL GL GL GL








**



  • ns

    Fig. 4. Variant-binding memory B cell clones use distinct VH genes and
    evolve through somatic hypermutation.(A) Experimental design for sorting
    and sequencing SARS-CoV-2Ðspecific memory B cells. (B) Frequency of RBD++
    (B.1.351 variant cross-binding) memory B cells as a percentage of total RBD+
    cells. (C) Percentage of sequence copies occupied by the top 20 ranked
    clones (D20) across naïve B cells and different antigen-binding memory B cell
    populations. (D) Heatmap and hierarchical clustering of VH gene usage
    frequencies in memory B cell clones across different antigen-binding populations.
    Data are represented as the percentage of clones with the indicated VH gene
    per column. (EandF) Somatic hypermutation (SHM) density plots (E) and
    boxplots of individual clones across naïve B cells and different antigen-binding
    memory B cell populations (F). Data are represented as the percent of
    mutated VH nucleotides. Number of clones sampled for each population
    is indicated. For (C) to (F), data were filtered on clones with productive
    rearrangements and≥2 copies. (G) Venn diagram of clonal lineages that are
    shared between WT RBD and RBD cross-binding (RBD++) populations. Data were
    filtered on the basis of larger clones with≥50% mean copy number frequency
    (mcf) in each sequencing library. (H) Example lineage trees of clones with
    overlapping binding to WT and B.1.351 variant RBD. VH genes and CDR3
    sequences are indicated. Numbers refer to mutations compared with the
    preceding vertical node. Colors indicate binding specificity, black dots indicate
    inferred nodes, and size is proportional to sequence copy number. GL, germline
    sequence. (I) Classification of SHM within overlapping clones. Each clone
    was defined as having higher (or equal) SHM in WT RBD binders or RBD++
    cross-binders on the basis of average levels of SHM for all WT RBD versus RBD++
    sequence variant copies within each lineage. (J) SHM levels within overlapping
    clones. Data are represented as the percentage of mutated VH nucleotides
    for WT RBD and RBD++sequence copies. Statistics were calculated using
    unpaired nonparametric Wilcoxon test, with BH correction for multiple comparisons
    in (C) and (F). Notches on boxplots in (F) and (J) indicate a 95% confidence
    interval of the median. P< 0.05; P< 0.01; P< 0.001; ****P< 0.0001;
    ns, not significant.
    RESEARCH | RESEARCH ARTICLE



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