Nature - USA (2020-08-20)

Nature | Vol 584 | 20 August 2020 | 453

antibodies were slightly less potent, with IC 50 values ranging from 0.013
to 0.767 μg ml−1. A common feature of the NTD mAbs was the plateauing
of virus neutralization at levels short of 100%. Two antibodies, catego-
rized as ‘other’, neutralized with IC 50 values of 0.071 and 0.652 μg ml−1.
Antibody neutralization of the authentic or live SARS-CoV-2 (strain
USA-WA1/2020) was carried out using Vero cells inoculated with a mul-
tiplicity of infection of 0.1. As shown in the bottom portion of Fig. 2b,
the RBD-directed antibodies again neutralized the virus but with IC 50
values of 0.0007 to 0.209 μg ml−1; the NTD-directed antibodies showed
similar potency, with IC 50 values ranging from 0.007 to 0.109 μg ml−1.
Here, the plateauing effect seen in the pseudovirus neutralization assay
was less apparent. Antibodies 2-43 and 2-51 neutralized the live virus
with IC 50 values of 0.003 and 0.007 μg ml−1, respectively. Overall, nine
mAbs exhibited high potency in neutralizing authentic SARS-CoV-2
in vitro with IC 50 values of 0.009 μg ml−1 or less, including four against

RBD (2-15, 2-7, 1-57, and 1-20), three against NTD (2-17, 5-24, and 4-8),

and two against undetermined regions on the S trimer (2-43 and 2-51).

Patient 2 alone contributed five of the top nine SARS-CoV-2 neutralizing

mAbs. A correlation of the results of the two virus-neutralizing assays

is shown in Extended Data Fig. 4.

Epitope mapping

All 19 potent neutralizing mAbs (Fig.  2 ) were further studied in antibody

competition experiments to gain insight into their epitopes. We also

chose 12 mAbs that bound the S trimer strongly during the initial super-

natant screen, including 5 that bound RBD (1-97, 2-26, 4-13, 4-24, and

4-29) and 7 that did not bind RBD (1-21, 2-29, 4-15, 4-32, 4-33, 4-41, and

5-45). Four of these mAbs were weak in neutralizing SARS-CoV-2 pseu-

dovirus, and the remaining eight were non-neutralizing (Extended Data

AUC

Competitor antibodies

AUC

Competitor antibodies

1-875-241-68 4-82-512-174-184-195-7 4-324-332-295-454-414-151-21 2-15 2-41-572-382-302-7 1-204-202-362-26CR30 22 1-974-294-244-13

Biotin

ylated

antibodie

s

1-87 382244 387 500402 762 590 829417 630 589 1,1581,0531,0291,0841,072

Biotinylated antibodies

2-15 104 256142 613671 408190 2481 ,0161,07 81 ,0631,0468 97 1,0669 75

5-24 350179 491 448508 654 503 869522 750 692 1,0321,003 866 1,1061,041 2-45 4 86 76 337 458568 79 1511 ,1821,12 41 ,0171,1799 71 1,1881,099

1-68 149131 190 322239 531 389 718274 385 458 1,304 994 1,0671,138 985 1-57 92 244 124627673 336128 2231 ,123 953961 905968 950957

4-8 319271 442 247449 503 441 727325 549 950 1,5731,3951,3461,2451,015 2-38 114101 89 137 236122 88 1081 ,326 754753 6191 ,4741,8391,596

2-51 224158 275 314220 577 382 705352 575 614 1,2881,3211,3671,4181,156 2-30 116170 111399 190 1,0969 7141 819585 939169 1,0791,1341,144

2-17 299192 409 550557 478 431 826324 539 622 894 1,059 871 1,0101,086 2-76 5598 72 407 1,002 87 76 1,3199 36 1,25 6966 1,1726 95 909950

4-18 552594 673 808724 571 478 996652 798 915 988 1,0011,0741,013 957 1-20 215321 242846 541816 178 318320 5981 ,220 983932 938914

4-19 361320 423 657660 539 651 638844 957 1,076 813 1,2831,155 973 961 4-20 127208 151468 272834 134191 157519 1,3279 51 811958 798

5-7 615376 691 598788 937 739 1,196 379 1,4381,391 799691 1,016 8751 ,053 2-36 855773 857776 965952 179244 221 576529 906794 942902

4-32 420339 565 429488 627 640 1,0691,325 325 356 570644 982 1,0621,066 2-261,6871,5431,0809 05 805981 236320 271 308325742 441675 396

4-33 400281 491 483516 701 542 1,0581,320 245 293 385675 793 1,0291,000 CR30 22 986945 1,1407 18 988912 1,1988 47 147 332205 552 444 667173

2-291,2681,0351,1051,7611,4041,050 969 854309 415 384 226833 766 1,633 991 1-97 9761 ,039 974975 8731 ,072 950 9651 ,006 656803 169 359269 162

5-451,0181,0441,0041,1301,0971,1421,0541,026 621 989 1,0391,031 320 409 9261 ,006 4-29 870995 1,0241,051 873906 665880724 518651179 213 236102

4-411,0091,0031,073 969 1,121 915 1,020 987739 1,035 979 893373 320 855 1,0054-249 83 1,1499 42 9411 ,001 847 742767892 551723108 191 150 96

4-151,0821,1601,1131,0191,1191,0341,0401,066 689 984 6651 ,367 590 514 498 1,6814-139 78 1,0439 96 978907 960972 969981 903855 465584 532298

1-211,1671,0781,090 990999 1,056 983 1,0361,0261,1411,0361,0461,046 997 1,126 408 ACE 2 161325 219840 584145 101101 136518 983843 905871 1,076

NTD ++++– +++++––+ ––– RBDmut ––++++–– +++++++

Non-RBD binders RBD binders

A

B

C D E H

F

G

a No competitionCompetition

b

Potent neutralizing antibody

Binding knocked out by L455R, A475R, and G502R

Non-neutralizing antibody

Competition by cell-surface staining

2-38 2-30

1-57

2-4*

1-20*

4-20*

2-7

2-15*

2-43

1-97

4-29 4-24

4-13

2-26

CR3022

2-36

5-45 4-41

4-15

1-21

5-7

5-24

2-29

4-32

4-33

1-68

4-19

4-8

2-17

4-18

1-87

2-51

NTD RBD

Strong competition with ACE2 Binding to NTD *

Neutralizing antibody

A

B

D

E

F G

H

C

Fig. 3 | Epitope mapping of select neutralizing and non-neutralizing mAbs.
a, Competition results of non-RBD binders (left) and RBD binders (right) in
blocking binding of ACE2 or biotinylated mAb to the S trimer. In addition, the
ability of each mAb to bind NTD and RBDmut is shown. The numbers in each box
show the area under each competition curve (AUC) as tested by ELISA. Plus and

minus signs indicate binding and no binding, respectively, of the mAb to the

protein. The letters A to H at the bottom denote clusters of antibody epitopes

defined by the competition experiments. b, Venn diagram interpretation of

results from a and Extended Data Fig. 6b.