Science - USA - 03.12.2021

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