Bonsignori - CH01-CH04 antibodies paper - SOM post ...jvi.asm.org/content/suppl/2011/09/02/85.19.9998.DC1/Supplemental...10 100 1000 10000 >40000 [IgG] (ng/ml) CH03 CH04 CH05 CH01

Supplemental Material

Analysis of a Clonal Lineage of HIV-1 Envelope V2/V3 Conformational Epitope-Specific Broadly Neutralizing Antibodies and Their Inferred

Unmutated Common Ancestors

Mattia Bonsignori, Kwan-Ki Hwang, Xi Chen, Chun-Yen Tsao, Lynn Morris, Elin Gray, Dawn J. Marshall, John A. Crump, Saidi H. Kapiga, Noel E. Sam, Faruk Sinangil, Marie Pancera,

Yang Yongping, Baoshan Zhang, Jiang Zhu, Peter D. Kwong, Sijy O’Dell, John R. Mascola, Lan Wu, Gary J. Nabel, Sanjay Phogat, Michael S. Seaman, John F. Whitesides, M. Anthony

Moody, Garnett Kelsoe, Xinzhen Yang, Joseph Sodroski, George M. Shaw, David Montefiori, Thomas B. Kepler, Georgia D. Tomaras, S. Munir Alam, Hua-Xin Liao, and Barton F. Haynes

10

100

1000

10000

>40000

[Ig

G]

(ng

/ml)

CH03

CH04CH05

CH01

CH02

Figure S1 – Total IgG concentration from culture supernatants of IgG+ memory B cells cultured for 14 days at clonal or near clonal dilution. IgG+ memory B cells from a Tanzanian female subject chronically infected with a clade A HIV-1 were cultured at 8 cells/well for 14 days. The total IgG concentration at the end of the 14-day stimulation for each of the ~3600 cultures is shown on the y‐axis. Each culture is represented by a dot. The red line indicates the median IgG concentration, which is 705 ng/ml. The cultures containing the CH01, CH02, CH03 and CH04 mAbs are shown with red dots.

Table S1 – Registry of viruses used in the neutralization assay for screening the B memory B cell cultures after in vitro stimulation.

Isolate Accession

no. Clade Origin Contributor(s)

MN B USA David C. Montefiori

SF162 EU123924 B USA Leonidas Stamatatos

BaL DQ318211 B USA John Mascola

Bx08 GQ855765 B France David C. Montefiori

6535 AY835438 B USA David C. Montefiori

QH0692 AY835439 B Trinidad David C. Montefiori

SC42 AY835441 B Trinidad Beatrice Hahn

PVO AY835444 B Italy David C. Montefiori

TRO AY835445 B Italy David C. Montefiori

AC10 AY835446 B USA David C. Montefiori

RHPA AY835447 B USA Beatrice Hahn

THRO AY835448 B USA Beatrice Hahn

REJO AY835449 B USA Beatrice Hahn

TRJO AY835450 B USA Beatrice Hahn

WITO AY835451 B USA Beatrice Hahn

CAAN AY835452 B USA Beatrice Hahn

WEAU EU289202 B USA George Shaw

1006 EU289183 B USA George Shaw







SC05 EU289200 B Trinidad George Shaw

MW965 U08455 C Malawi Beatrice Hahn

Du156 DQ411852 C S. Africa Carolyn Williamson / Lynn Morris

Isolate Accession




ZM197M DQ388515 C Zambia Beatrice Hahn




ZM53M AY423984 C Zambia Beatrice Hahn

ZM109F AY424138 C Zambia Beatrice Hahn

ZM135M AY424079 C Zambia Beatrice Hahn

CAP45 DQ435682 C S. Africa Lynn Morris

CAP210 DQ435683 C S. Africa Lynn Morris

Ce1086 FJ444395, FJ444410

C Malawi Ronald Swanstrom

Ce0393 FJ444215, FJ444208


Ce1176 FJ444437, FJ444444


Ce2010 FJ444561, FJ444553


Ce0682 FJ444325 C Malawi Ronald Swanstrom




Ce704809221 FJ444103 C S. Africa Ronald Swanstrom

Q461 AF407156 A/D Kenya Julie Overbaugh

BF1266 C Malawi Ronald Swanstrom

246F FJ496194 C Zambia Beatrice Hahn

249M EU166866, EU166872

C Zambia Beatrice Hahn

ZM247 FJ496204 C Zambia Beatrice Hahn

7030 FJ443999 C Malawi Beatrice Hahn

1394C9G1 FJ444529 C Malawi Beatrice Hahn

Isolate Accession


Q23.17 AF004885 A Kenya Julie Overbaugh

Q769 AF407158 A Kenya Julie Overbaugh



191955 A Uganda George Shaw / Beatrice Hahn

191084 A1 Uganda George Shaw / Beatrice Hahn

9004SS A Uganda Beatrice Hahn

21020 A Kenya George Shaw / Beatrice Hahn

R18553 A Rwanda Beatrice Hahn

851891 A Kenya Jerome Kim

257 EU513185 CRF02_AG Cameroon Dennis Ellenberger/ Sal Butera

928 EU513199 CRF02_AG Cote d’Ivoire

Dennis Ellenberger/ Sal Butera








NP03 CRF01_AE

92TH023 CRF01_AE

CM244 CRF01_AE

C1080 CRF01_AE Thailand Francine McCutchan

R2184 CRF01_AE Thailand Francine McCutchan






620345 CRF01_AE Thailand Jerome Kim

Isolate Accession


703357 CRF01_AE Thailand Jerome Kim

X1193 EU885761 GIB Spain Michael Thomson

P0402 EU885759 GIB Portugal Michael Thomson

X1254 EU885762 GIB Spain Michael Thomson

X2088 EU885764 GIB Ghana Michael Thomson

X2131 FJ817368 GIB Spain Michael Thomson

P1981 FJ817369 GIB Spain Michael Thomson

X1632 FJ817370 GIB Spain Michael Thomson

3016 D Tanzania Francine McCutchan

A07412M1.vrc12 HM215357 D Uganda Francine McCutchan/John Mascola

231965.c01 D Uganda Jerome Kim

231965.c02 D Uganda Jerome Kim

A.

10 20 30 40 50 60 70....|....| ....|....| ....|....| ....|....| ....|....| ....|....| ....|....|

0219RUA-1 ATGCCGATGG GCTCCCTGCA GCCCCTGGCC ACCCTGTACC TGCTGGGCAT GCTGGTGGCC TCCGTGCTGG0219RUA-2 .......... .......... .......... .......... .......... .......... ..........CH01 ...GA..CA. A.A.A..C.T ..TATG..-- ----.ACTG. ....CT.GG. T.CA.--.TT C.AC..G..ACH02 ...GA..CA. A.A.A..C.T ..TATG..-- ----.ACTG. ....CT.GG. T.CA.--.TT C.AC..G..ACH03 ...GA..CA. A.A.A..C.T ..TATG..-- ----.ACTG. ....CT.GG. T.CA.--.TT C.AC..G..ACH04 ...GA..CA. A.A.A..C.T ..TATG..-- ----.ACTG. ....CT.GG. T.CA.--.TT C.AC..G..ACH05 ...GA..CA. A.A.A..C.T ..TATG..-- ----.ACTG. ....CT.GG. T.CA.--.TT C.AC..G..A

80 90 100 110 120 130 140....|....| ....|....| ....|....| ....|....| ....|....| ....|....| ....|....|

0219RUA-1 CCGAGGTGCA GCTGGTGGAG TCTGGGGGAG GTGTGGTACG GCCTGGGGGG TCCCTGAGAC TCTCCTGTGC0219RUA-2 .......... .......... .......... .......... .......... .......... ..........CH01 .-.....T.. .......... .......C.A A...T..... ...G...... .......... ........AACH02 .-.....T.. .......... .........A .......G.. ...G...... .......... ........AGCH03 .-.....T.. .......... .......... .......G.. ...G...... .......... ..........CH04 .-.....T.. .......... .......... ..C.CA.... ...G...... .......... ........AACH05 .-.....T.. .......... .......... ..C.CA.... ...G...... .......... ........AA

150 160 170 180 190 200 210....|....| ....|....| ....|....| ....|....| ....|....| ....|....| ....|....|

0219RUA-1 AGCCTCTGGA TTCACCTTTG ATGATTATGG CATGAGCTGG GTCCGCCAAG CTCCAGGGAA GGGGCTGGAG0219RUA-2 .......... .......... .......... .......... .......... .......... ..........CH01 ...G..C... ....T..... .AA...T... TT.T..T... ........G. .......... ......TC..CH02 ...G..C... ....T..... .GA.C..... .C...CT... .......... T......... A.....AC.TCH03 ...G..C... ....TT.... .GA.C..C.. .T...CT... .......... T......... A......C.TCH04 ..G...C..T ....T..... .GA...T... .T.CG..... .......... G......... ..........CH05 ..G...C..T ....T..... .GA...T... .T.CG..... .......... G......... ..........

220 230 240 250 260 270 280....|....| ....|....| ....|....| ....|....| ....|....| ....|....| ....|....|

0219RUA-1 TGGGTCTCTG GTATTAATTG GAATGGTGGT AGCACAGGTT ATGCAGACTC TGTGAAGGGC CGATTCACCA0219RUA-2 .......... .......... .......... .......... .......... .......... ..........CH01 ......G... ..C....... .......... GA....C... .......... .......... .......GA.CH02 ........C. .G..G..... .......... GA...GC... .......... .....G.... .....T.G..CH03 ........C. ....G..... .......... GA...GC... .......... .....G.... .......G..CH04 .....G.... .C.C...... ......A... GA.T..C... ...G...... .......... ........A.CH05 .....G.... .C.C...... ......A... GA.T..C... ...G...... .......... ........A.

290 300 310 320 330 340 350....|....| ....|....| ....|....| ....|....| ....|....| ....|....| ....|....|

0219RUA-1 TCTCCAGAGA CAACGCCAAG AACTCCCTGT ATCTGCAAAT GAACAGTCTG AGAGCCGAGG ACACGGCCTT0219RUA-2 .......... .......... .......... .......... .......... .......... ..........CH01 .G........ ....T...G. ..T.TTG... ..T..G.C.. .G.T.AAG.. G...T...C. ..........CH02 .G........ ....AG...C ...AT.GCA. .......... ...T.A.... ....TG.... ..........CH03 .G........ ....AG...T ..TAT.GCA. .......... ...A.A.... ....T...C. ..........CH04 .......... ....AG...C ..T.T.G.C. .C........ .......... ...C...... ........A.CH05 .......... ....AG...C ..T.T.G.C. .C........ .......... ...C...... ........A.

360 370 380 390 400 410 420....|....| ....|....| ....|....| ....|....| ....|....| ....|....| ....|....|

0219RUA-1 GTATTACTGT GCGAGAGGGA CCGATTACAC TATTGACGAC CAGGGGATCC GTTATCAAGG TTCGGGGACC0219RUA-2 .......... .......... .......... .......... .......... T......... ..........CH01 C......... .......... .......... .......... GC........ A...C..... ..........CH02 .........C .......... .......... G..A...... .....A.GAT T......... A.........CH03 .......... .......... .......... G..A...... .....A..TT T....A.... ..........CH04 A.....T... .......... .......... .........T .......... .......... .........TCH05 A.....T... .......... .......... .........T .......... .......... .........T

430 440 450 460 470 480 490....|....| ....|....| ....|....| ....|....| ....|....| ....|....| ....|....|

0219RUA-1 TTCTGGTACT TCGATCTCTG GGGCCGTGGC ACCCTGGTCA CTGTCTCCTC AGCGTCGACC AAGGGCCCAT0219RUA-2 .......... .......... .......... .......... .......... .......... ..........CH01 .......... .......... .......... .......... G......T.. ...C..C... ..........CH02 .......... .....T.T.. .......... ..A....... .......T.. ...C..C... ..........CH03 .......... .......... .......... .......... .......T.. ...C..C... ..........CH04 .......... .....G.... ......C... .......... .G........ ...C..C... ..........CH05 .......... .....G.... ......C... .......... .G........ ...C..C... ..........

500 510 520 530 540 550 560....|....| ....|....| ....|....| ....|....| ....|....| ....|....| ....|....|

0219RUA-1 CGGTCTTCCC CCTGGCACCC TCCTCCAAGA GCACCTCTGG GGGCACAGCG GCCCTGGGCT GCCTGGTCAA0219RUA-2 .......... .......... .......... .......... .......... .......... ..........CH01 .......... .......... .......G.. .......... .......... .......... ..........CH02 .......... .......... .......... .......... .......... .......... ..........CH03 .......... .......... .......... .......... .......... .......... ..........CH04 .......... .......... .......... .......... .......... .......... ..........CH05 .......... .......... .......... .......... .......... .......... ..........

570 580 590 600 610 620 630....|....| ....|....| ....|....| ....|....| ....|....| ....|....| ....|....|

0219RUA-1 GGACTACTTC CCCGAACCGG TGACGGTGTC GTGGAACTCA GGCGCCCTGA CCAGCGGCGT GCACACCTTC0219RUA-2 .......... .......... .......... .......... .......... .......... ..........CH01 .......... .......... .......... .......... .......... .......... ..........CH02 .......... .......... .......... .........G .......... .......... ..........CH03 .......... .......... .......... .......... .......... .......... ..........CH04 .......... .......... .......... .......... .......... .......... ..........CH05 .......... .......... .......... .......... .......... .......... ..........

640 650 660 670 680 690 700....|....| ....|....| ....|....| ....|....| ....|....| ....|....| ....|....|

0219RUA-1 CCGGCTGTCC TACAGTCCTC AGGACTCTAC TCCCTCAGCA GCGTGGTGAC CGTGCCCTCC AGCAGCTTGG0219RUA-2 .......... .......... .......... .......... .......... .......... ..........CH01 .......... .......... .......... .......... .......... .......... ..........CH02 .......... .......... .......... .......... .......... .......... ..........CH03 .......... .......... .......... .......... .......... .......... ..........CH04 .......... .......... .......... .......... .......... .......... ..........CH05 .......... .......... .......... .......... .......... .......... ..........

710 720 730 740 750 760 770....|....| ....|....| ....|....| ....|....| ....|....| ....|....| ....|....|

0219RUA-1 GTACCCAGAC CTACATCTGC AACGTGAATC ACAAGCCCAG CAACACCAAG GTGGACAAGA GAGTTGAGCC0219RUA-2 .......... .......... .......... .......... .......... .......... ..........CH01 .......... .......... .......... .......... .......... .......... ..........CH02 .......... .......... .......... .......... .......... .......... ..........CH03 .......... .......... .......... .......... .......... .......... ..........CH04 .......... .......... .......... .......... .......... .......... ..........CH05 .......... .......... .......... .......... .......... .......... ..........

780 790 800 810 820 830 840....|....| ....|....| ....|....| ....|....| ....|....| ....|....| ....|....|

0219RUA-1 CAAATCTTGT GACAAAACTC ACACATGCCC ACCGTGCCCA GCACCTGAAC TCCTGGGGGG ACCGTCAGTC0219RUA-2 .......... .......... .......... .......... .......... .......... ..........CH01 .......... .......... .......... .......... .......... .......... ..........CH02 .......... .......... .......... .......... .......... .......... ..........CH03 .......... .......... .......... .......... .......... .......... ..........CH04 .......... .......... .......... .......... .......... .......... ..........CH05 .......... .......... .......... .......... .......... .......... ..........

B.

10 20 30 40 50 60 70 80....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|

0219RUA-1 MPMGSLQPLATLYLLGMLVASVLAEVQLVESGGGVVRPGGSLRLSCAASGFTFDDYGMSWVRQAPGKGLEWVSGINWNGG0219RUA-2 ................................................................................CH01 .ET---DT.LLWV..LWVPG.TGD........AN............K....I.ENF.F...........Q..A.L.....CH02 .ET---DT.LLWV..LWVPG.TGD.........S............R....I.EN..LT....V.....H....M.....CH03 .ET---DT.LLWV..LWVPG.TGD...........................I.EN..LT....V.....H....M.....CH04 .ET---DT.LLWV..LWVPG.TGD..........LI..........KG...I.ENF.FG....G..........T.....CH05 .ET---DT.LLWV..LWVPG.TGD..........LI..........KG...I.ENF.FG....G..........T.....

90 100 110 120 130 140 150 160....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|

0219RUA-1 STGYADSVKGRFTISRDNAKNSLYLQMNSLRAEDTALYYCARGTDYTIDDQGIRYQGSGTFWYFDLWGRGTLVTVSSAST0219RUA-2 .....................................................L..........................CH01 D.R.........RM....SR.FV..D.DKVGVD...F.............A..H...................S......CH02 D.R.....R...SM....SN.IA.....N..V....................RF...........F..............CH03 D.R.....R...SM....SN.IA....KN..VD....................F.K........................CH04 DSR.G.............SN.FV........P....I............................V..............CH05 DSR.G.............SN.FV........P....I............................V..............

170 180 190 200 210 220 230 240....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|

0219RUA-1 KGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYIC0219RUA-2 ................................................................................CH01 ............R...................................................................CH02 ................................................................................CH03 ................................................................................CH04 ................................................................................CH05 ................................................................................

250 260 270 280 290 300 310 320....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|

0219RUA-1 NVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVD0219RUA-2 ................................................................................CH01 ................................................................................CH02 ................................................................................CH03 ................................................................................CH04 ................................................................................CH05 ................................................................................

330 340 350 360 370 380 390 400....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|

0219RUA-1 GVEVHNAKTKPREEQYNATYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIAATISKAKGQPREPQVYTLPPSREEMTK0219RUA-2 ................................................................................CH01 ................................................................................CH02 ................................................................................CH03 ................................................................................CH04 ................................................................................CH05 ................................................................................

410 420 430 440 450 460 470 480....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|

0219RUA-1 NQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKS0219RUA-2 ................................................................................CH01 ................................................................................CH02 ................................................................................CH03 ................................................................................CH04 ................................................................................CH05 ................................................................................

V

V D J

C.

10 20 30 40 50 60 70....|....| ....|....| ....|....| ....|....| ....|....| ....|....| ....|....|

0219RUA-1 ATGGAGACAG ACACACTCCT GCTATGGGTA CTGCTGCTCT GGGTTCCAGG TTCCACTGGT GACGAAATTG0219RUA-2 .......... .......... .......... .......... .......... .......... ..........CH01 .......... .......... .......... .......... .......... .......... ..........CH02 .......... .......... .......... .......... .......... .......... ..........CH03 .......... .......... .......... .......... .......... .......... ..........CH04 .......... .......... .......... .......... .......... .......... ..........CH05 .......... .......... .......... .......... .......... .......... .....C..CC

80 90 100 110 120 130 140....|....| ....|....| ....|....| ....|....| ....|....| ....|....| ....|....|

0219RUA-1 TGTTGACGCA GTCTCCAGGC ACCCTGTCTT TGTCTCCAGG GGAAAGAGCC ACCCTCTCCT GCAGGGCCAG0219RUA-2 .......... .......... .......... .......... .......... .......... ..........CH01 .....G.... .......... .......... .......... .......... .......... ..........CH02 .......... ........C. .........G .......G.. ...G...... .......... ..........CH03 .......... ........C. .......... .......G.. .......... .......... ..........CH04 .......A.. ........A. .......... ....C..... ...G...... ........A. ..........CH05 A......C.. .......TC. T........G CA...GTG.. A..C....T. ...A..A.T. ..C....A..

150 160 170 180 190 200 210....|....| ....|....| ....|....| ....|....| ....|....| ....|....| ....|....|

0219RUA-1 TCAGAGTGTT AGCAGCAGCT ACTTAGCCTG GTACCAGCAG AAACCTGGCC AGGCTCCCAG GCTCCTCATC0219RUA-2 .......... .......... .......... .......... .......... .......... ..........CH01 ...C.A...C CA.CC..AA. .T..C..... .......... ..G....... ..T.....C. A.........CH02 .....A...C CA.CC...A. .T..C..... ...T..A..A ....G..... ..T....... ..........CH03 .........C CA.CC..AA. .T..C..... .......... .......... ..T....... ..........CH04 .......... CA......A. ....T..... .........T .......... ..C....... A.........CH05 ....G.CA.. ---..A.ATG .T....G... ...T...... ..G..A..TA .A..C.AT.A ..........

220 230 240 250 260 270 280....|....| ....|....| ....|....| ....|....| ....|....| ....|....| ....|....|

0219RUA-1 TATGGTGCAT CCAGCAGGGC CACTGGCATC CCAGACAGGT TCAGTGGCAG TGGGTCTGGG ACAGACTTCA0219RUA-2 .......... .......... .......... .......... .......... .......... ..........CH01 .......GG. ...C...... .G.......T ....G..A.. ....C..... .......... ..C.......CH02 C..A...G.. ...C...... .G........ G......... ........G. .........A .TGC......CH03 ...A...G.. ...CT..... .G........ G......... ........G. .........A .T.C......CH04 .......GG. ...C...... .......... ..TA.T..A. ......C.G. C......... ...C.G....CH05 ....C..... .T..TTTACA A.G...GG.. ...TCAC... ....C..... .........C .....T....

290 300 310 320 330 340 350....|....| ....|....| ....|....| ....|....| ....|....| ....|....| ....|....|

0219RUA-1 CTCTCACCAT CAGCAGACTG GAGCCTGAAG ATTTTGCAGT GTATTACTGT CAGCAGTATG GTAGCTCCCC0219RUA-2 .......... .......... .......... .......... .......... .......... ..........CH01 .......... ...TC..G.. ..C....... .......... T......... .......... ..G.......CH02 .......... ..C....G.. .......... .......... C....T.... ..A..A..C. ..G.T..T..CH03 .......... ..C....G.. .......... .......... .....T...C ..A..A..C. ..G.T.....CH04 ........G. ..A....... ...G...... .......G.. A......... .......... ..C.......CH05 .......... ......C... C......... ....C...AC T......... .TA..AG..T AC..T.A...

360 370 380 390 400 410 420....|....| ....|....| ....|....| ....|....| ....|....| ....|....| ....|....|

0219RUA-1 GTACACGTTC GGCCAAGGGA CCAAGGTGGA AATCAAACGA ACTGTGGCTG CACCATCTGT CTTCATCTTC0219RUA-2 .......... .......... .......... .......... .......... .......... ..........CH01 .......... .......... .......... .......... .......... .......... ..........CH02 C......... .....G.... ...G...... .C...G.... .......... .......... ..........CH03 C......... .....G.... .......... .C...GG... .......... .......... ..........CH04 .......... .......... .......... G....G.... .......... .......... ..........CH05 ...T..T..T .....G.... ....CC.... G.....G... .......... .......... ..........

430 440 450 460 470 480 490....|....| ....|....| ....|....| ....|....| ....|....| ....|....| ....|....|

0219RUA-1 CCGCCATCTG ATGAGCAATT GAAATCTGGT ACCGCCTCTG TTGTGTGCCT GCTGAATAAC TTCTATCCCA0219RUA-2 .......... .......... .......... .......... .......... .......... ..........CH01 .......... .......G.. .........A ..T....... .......... .......... ..........CH02 .......... .......G.. .........A ..T....... .......... .......... ..........CH03 .......... .......G.. .........A ..T....... .......... .......... ..........CH04 .......... .......G.. .........A ..T....... .......... .......... ..........CH05 .......... .......G.. .........A ..T....... .......... .......... ..........

500 510 520 530 540 550 560....|....| ....|....| ....|....| ....|....| ....|....| ....|....| ....|....|

0219RUA-1 GAGAGGCCAA AGTACAGTGG AAGGTGGATA ACGCCCTCCA ATCGGGTAAC TCCCAGGAGA GTGTCACAGA0219RUA-2 .......... .......... .......... .......... .......... .......... ..........CH01 .......... .......... .......... .......... .......... .......... ..........CH02 .......... .......... .......... .......... .......... .......... ..........CH03 .......... .......... .......... .......... .......... .......... ..........CH04 .......... .......... .......... .......... .......... .......... ..........CH05 .......... .......... .......... .......... .......... .......... ..........

570 580 590 600 610 620 630....|....| ....|....| ....|....| ....|....| ....|....| ....|....| ....|....|

0219RUA-1 GCAGGACAGC AAGGACAGCA CCTACAGCCT CAGCAGCACC CTGACGCTGA GCAAAGCAGA CTACGAGAAA0219RUA-2 .......... .......... .......... .......... .......... .......... ..........CH01 .......... .......... .......... .......... .......... .......... ..........CH02 .......... .......... .......... .......... .......... .......... ..........CH03 .......... .......... .......... .......... .......... .......... ..........CH04 .......... .......... .......... .......... .......... .......... ..........CH05 .......... .......... .......... .......... .......... .......... ..........

640 650 660 670 680 690 700....|....| ....|....| ....|....| ....|....| ....|....| ....|....| ....|....|

0219RUA-1 CACAAAGTCT ACGCCTGCGA AGTCACCCAT CAGGGCCTGA GCTCGCCCGT CACAAAGAGC TTCAACAGGG0219RUA-2 .......... .......... .......... .......... .......... .......... ..........CH01 .......... .......... .......... .......... .......... .......... ..........CH02 .......... .......... .......... .......... .......... .......... ..........CH03 .......... .......... .......... .......... .......... .......... ..........CH04 .......... .......... .......... .......... .......... .......... ..........CH05 .......... .......... .......... .......... .......... .......... ..........

710....|....| .

0219RUA-1 GAGAGTGTTG A0219RUA-2 .......... .CH01 .......... .CH02 .......... .CH03 .......... .CH04 .......... .CH05 .......... .

D.

Figure S2 –Heavy and light chain alignments of nucleotide and amino acid sequences of clonal lineage monoclonal antibodies CH01, CH02, CH03 and CH04 and their putative reverted unmutated ancestor antibodies. All the sequences shown were aligned to 0219RUA-1. (A, B) heavy

chain nucleotide (A) and amino acid (B) sequences are shown. In B, the red box indicates the HCDR3 and the colored lines indicate the V (red line), D (orange line) and J (brown line) regions. The light chain alignments are shown in C for the nucleotide sequences and in D for the amino acid sequences.

10 20 30 40 50 60 70 80....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|

0219RUA-1 -ETDTLLLWVLLLWVPGSTGDEIVLTQSPGTLSLSPGERATLSCRASQSVSSSYLAWYQQKPGQAPRLLIYGASSRATGI0219RUA-2 -...............................................................................CH01 M........................A.....................HN.HPK.F.........S.......G.T..A..CH02 M............................A...V..............N.HPR.F......R..S.....HSG.T..A..CH03 M............................A....................HPK.F.........S......SG.T..A..CH04 M............................D....................H.R.F....H....P.......G.T.....CH05 M....................D.Q.....SS..A.V.D.V.IT.....GIR-ND.G.......K.HK....A...LQS.V

90 100 110 120 130 140 150 160....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|

0219RUA-1 PDRFSGSGSGTDFTLTISRLEPEDFAVYYCQQYGSSPYTFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNN0219RUA-2 ................................................................................CH01 .GK................VD.............G.............................................CH02 A.....G...MH.....T.V........F.....G.........R..LR...............................CH03 A.....G...IH.....T.V........F.....G............LR...............................CH04 .N...AG....Q....VN...A............R.............R...............................CH05 .S................S.Q.....T...L.DY.Y........NL..................................

170 180 190 200 210 220 230....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|.

0219RUA-1 FYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC0219RUA-2 ............................................................................CH01 ............................................................................CH02 ............................................................................CH03 ............................................................................CH04 ............................................................................CH05 ............................................................................

Figure S3 – Phylogenetic tree of the CH01-CH04 monoclonal antibodies. – The phylogenetic tree shows the evolutionary distance of the V(D)J nucleotide sequences of the CH01-CH04 monoclonal antibodies. The tree is rooted on the nucleotide sequence of the common putative reverted unmutated ancestor, inferred as described in the Materials and Methods section.

Figure S4 – Neutralization breadth of CH01-CH04 and PG9/PG16 monoclonal antibodies. PG9 and PG16 mAbs were tested for neutralizing activity against the same panel of 83 tier-2 isolates used for CH01-CH04 mAbs. CH01-CH04 mAbs neutralized a subset of the pseudoviruses neutralized by PG9 and PG16 and all the pseudoviruses neutralized by CH01-CH04 were also neutralized by PG9 and PG16. The table shows the neutralization breadth of CH01-CH04 and PG9/PG16 mAbs on a representative panel of 20 tier-2 pseudotyped lentiviruses. Results are expressed as IC50 (µg/ml) and color coded according to the legend.

A. IC50 (ug/ml)

CH01 CH02 CH03 CH04 PG9 PG16

Q23.17 wt 0.02 0.02 <0.02 0.02 <0.02 <0.02

N160K >50 >50 >50 >50 >50 >50

JRCSF wt 0.13 0.16 <0.02 <0.02 <0.02 <0.02

N160K >50 >50 >50 >50 >50 >50

JRFL

wt >50 n/a n/a n/a >50 >50

E168K 0.04 n/a n/a n/a <0.02 <0.02

+ N160K >50 n/a n/a n/a >50 >50

B.

PG16

0.001 0.01 0.1 1 10 100 10000

200

400

600

800wild-type

heterotrimer

MAb conc (g/ml)

MF

I

CH01

0.1 1 10 100 10000

500

1000

1500

MAb conc (g/ml)

MF

I

CH04

0.1 1 10 100 10000

200

400

600

800

1000

MAb conc (g/ml)

MF

I

CH02

0.1 1 10 100 10000

200

400

600

800

MAb conc (g/ml)

MF

I

IgG1b12

0.1 1 10 100 10000

50

100

150

200

250

MAb conc (g/ml)

MF

I

C.

Figure S5 – CH01-CH04 monoclonal antibodies have functional characteristics of PG9/PG16-like V2/V3 quaternary epitope-specific antibodies. (A) The N160K point mutation of the HIV-1 gp120 envelope abrogates neutralization mediated by the CH01-CH04 mAbs. (B) The N160Q mutation of both DU422 and ZM433 gp145 envelopes expressed on the cell surface of 293T cells inhibits the CH01-CH04 mAbs (blue line) to bind. The red line shows the Human IgG negative control. Results are shown as percentage of maximum binding. (C) The binding of CH01, CH02, CH04 mAbs to conC heterotrimers containing both wild-type and N160A-mutated conC protomers (red line) is 44 to 49% (CH01 = 44%, CH02 = 47%, CH04 = 44%) relative to binding to conC wild-type homotrimers (black line). PG16 (positive control) binding to conC heterotrimers is 49% compared to conC wild-type homotrimers whereas IgG1b2 (negative control) bound to the homo- and heterotrimers equally well. These data are in line with previously reported findings (Walker LM et al., Science; 326(5950):285-9, 2009). The x-axis shows the antibody concentration and the y-axis shows the mean fluorescence intensity. (D) CH01, CH02, CH03 and CH04 mAb neutralization of CM244 and WITO pseudotyped lentiviruses tested in TZM-bl cells is abrogated by treatment of 293T cells in which the viruses are grown with 50 µM of mannosidase I inhibitor kifunensine. Reported values are the concentrations at which the fluorescent units were reduced 50% relative to virus control wells. (E) PG9 and the CH01-CH04 bNabs block each other binding to the E.A244 gp120 envelope glycoprotein. The plot on the left shows the percentage of CH01-CH04 blocking of PG9 binding measured by SPR analysis and the plot on the right shows the percentage of PG9 blocking of CH01-CH04 binding. All the antibodies were tested at a concentration of 100 µg/ml.

D. ID50 (g/ml) in TZM-bl cells1

CM244.ec1/293T WITO.IMC.LucR/293T untreated + Kifunensine untreated + Kifunensine CH01 <0.02 >50 0.07 >50 CH02 0.03 >50 0.1 >50 CH03 <0.02 >50 0.1 >50 CH04 <0.02 >50 0.2 >50 PG9 <0.02 1.1 <0.02 12 PG16 <0.02 >50 <0.02 >50 HIVIG-C 113 205 119 410

24.9 27.122.2

18.1

0

10

20

30

40

50

60

70

80

90

100

CH01 CH02 CH03 CH04mAb blocked by PG9

CH01‐CH04 Binding

61.6

39.4

52.6

70.5

0

10

20

30

40

50

60

70

80

90

100

CH01 CH02 CH03 CH04

Blocking mAb

PG9 BindingE.

Table S2 – Neutralization of CH01/PG16 (A: IC50; B: IC80), CH02/PG16 and CH03/PG16 (C: IC50; D: IC80) chimeras, and CH01, CH02 and CH03 chimeric antibodies with permutations of the heavy and light chains (E: IC50; F: IC80).

A.

PG16 CH01PG16H+C

H01LCH01H+P

G16LA.Q842 <0.02 0.3 >50 >50

A.Q23.17 <0.02 <0.02 >50 >50

B.AC10 <0.02 0.1 >50 >50

B.REJO <0.02 0.06 >50 >50

B.WITO <0.02 <0.02 >50 >50

B.RHPA 0.8 15 >50 >50

C.DU156 <0.02 0.4 >50 >50

C.CAP45 <0.02 0.04 >50 >50

C.DU172 0.03 23 >50 >50

C.ZM233 <0.02 0.03 >50 >50

C.CAP210 <0.02 14 >50 >50

AG.278 0.4 1 >50 >50

SIVmac251 >50 >50 >50 >50

MuLV >50 >50 >50 >50

IC50 ug/ml B.

PG16 CH01PG16H+C

H01LCH01H+P

G16L

A.Q842 <0.02 >50 >50 >50

A.Q23.17 <0.02 0.03 >50 >50

B.AC10 <0.02 >50 >50 >50

B.REJO 0.1 3.1 >50 >50

B.WITO <0.02 0.03 >50 >50

B.RHPA 41 >50 >50 >50

C.DU156 0.02 12 >50 >50

C.CAP45 <0.02 0.4 >50 >50

C.DU172 0.2 >50 >50 >50

C.ZM233 <0.02 0.09 >50 >50

C.CAP210 0.09 >50 >50 >50

AG.278 42 9.8 >50 >50

SIVmac251 >50 >50 >50 >50

MuLV >50 >50 >50 >50

IC80 ug/ml

C.

PG16 CH02 CH03PG16H+C

H02LCH02H+P

G16LPG16H+C

H03LCH03H+P

G16L

A.Q842 <0.02 0.3 0.2 >50 >50 >50 >50

A.Q23.17 <0.02 <0.02 <0.02 >50 >50 >50 >50

B.AC10 <0.02 0.08 0.1 >50 >50 >50 >50

B.REJO <0.02 0.09 0.4 >50 >50 >50 >50

B.WITO <0.02 <0.02 <0.02 >50 >50 >50 >50

B.RHPA 1 >50 33 >50 >50 >50 >50

C.DU156 <0.02 1.9 0.2 >50 >50 >50 >50

C.CAP45 <0.02 0.05 0.03 >50 >50 >50 >50

C.DU172 0.03 32 10 >50 >50 >50 >50

C.ZM233 <0.02 0.05 0.02 >50 >50 >50 >50

C.CAP210 <0.02 >50 >50 >50 >50 >50 >50

AG.278 0.4 6.4 2.3 >50 >50 >50 >50

SIVmac251 >50 >50 >50 >50 >50 >50 >50

MuLV >50 >50 >50 >50 >50 >50 >50

IC50 ug/ml

D.

PG16 CH02 CH03PG16H+C

H02LCH02H+P

G16LPG16H+C

H03LCH03H+P

G16L

A.Q842 <0.02 >50 >50 >50 >50 >50 >50

A.Q23.17 <0.02 0.1 0.03 >50 >50 >50 >50

B.AC10 <0.02 >50 >50 >50 >50 >50 >50

B.REJO 0.05 >50 >50 >50 >50 >50 >50

B.WITO <0.02 0.02 0.02 >50 >50 >50 >50

B.RHPA 35 >50 >50 >50 >50 >50 >50

C.DU156 0.02 >50 4.6 >50 >50 >50 >50

C.CAP45 <0.02 >50 >50 >50 >50 >50 >50

C.DU172 0.1 >50 >50 >50 >50 >50 >50

C.ZM233 <0.02 0.2 0.09 >50 >50 >50 >50

C.CAP210 0.06 >50 >50 >50 >50 >50 >50

AG.278 >50 >50 >50 >50 >50 >50 >50

SIVmac251 >50 >50 >50 >50 >50 >50 >50

MuLV >50 >50 >50 >50 >50 >50 >50

IC80 ug/ml

E.

CH01 CH02 CH03CH01H+C

H02LCH01H+C

H03LCH02H+C

H01LCH02H+C

H03LCH03H+C

H01LCH03H+C

H02L

A.Q842 0.3 0.7 0.4 0.5 0.3 0.8 1.2 0.2 0.1

A.Q23.17 <0.02 0.03 <0.02 <0.02 <0.02 0.03 0.03 <0.02 <0.02

B.AC10 0.1 0.1 0.4 0.2 0.1 0.2 0.2 0.1 0.07

B.REJO 0.09 0.2 3.2 0.09 0.06 0.09 0.3 0.5 0.2

B.WITO <0.02 <0.02 0.03 <0.02 <0.02 <0.02 <0.02 <0.02 <0.02

B.RHPA 15 >50 48 9.8 8.8 23 >50 6.7 5.6

C.DU156 0.3 1.5 0.2 0.2 0.2 0.9 0.5 0.2 0.1

C.CAP45 0.03 0.05 0.08 0.04 0.03 0.1 0.1 0.03 <0.02

C.DU172 30 >50 12 15 10 >50 50 8.8 4.6

C.ZM233 0.05 0.06 0.04 0.03 0.04 0.06 0.03 0.02 0.02

C.CAP210 >50 >50 >50 25 15 >50 >50 8.6 >50

AG.278 0.8 3.9 1.8 0.9 1 5.4 4.3 1.3 0.6

SIVmac251 >50 >50 >50 >50 >50 >50 >50 >50 >50

MuLV >50 >50 >50 >50 >50 >50 >50 >50 >50

IC50 ug/ml

F.

CH01 CH02 CH03CH01H+C

H02LCH01H+C

H03LCH02H+C

H01LCH02H+C

H03LCH03H+C

H01LCH03H+C

H02L

A.Q842 >50 >50 >50 >50 >50 >50 >50 >50 >50

A.Q23.17 0.04 0.2 0.06 0.04 0.04 0.2 0.2 0.03 0.02

B.AC10 >50 >50 >50 >50 >50 >50 >50 >50 >50

B.REJO >50 >50 >50 >50 >50 >50 >50 >50 >50

B.WITO 0.03 0.05 0.1 0.03 0.03 0.05 0.03 0.03 0.02

B.RHPA >50 >50 >50 >50 >50 >50 >50 >50 >50

C.DU156 4.1 >50 2.6 2.7 1.7 >50 >50 2.6 1.3

C.CAP45 0.3 >50 >50 0.4 0.2 >50 >50 0.6 0.05

C.DU172 >50 >50 >50 >50 >50 >50 >50 >50 >50

C.ZM233 0.1 0.2 0.2 0.1 0.1 0.3 0.1 0.1 0.07

C.CAP210 >50 >50 >50 >50 >50 >50 >50 >50 >50

AG.278 7.3 >50 >50 6.9 8.3 >50 >50 13 11

SIVmac251 >50 >50 >50 >50 >50 >50 >50 >50 >50

MuLV >50 >50 >50 >50 >50 >50 >50 >50 >50

IC80 ug/ml

A.

B.

Figure S6 – Heavy chain sequence analyses of the CH01-CH04 mAbs predict that CH01-CH04 mAbs have structural characteristics similar to those of PG9 and PG16 V2/V3 quaternary epitope-specific monoclonal antibodies. We threaded the CH01 heavy chain sequence onto the PG16 heavy chain Fab structure. The PG16 HCDR3 has been previously reported and described as axe-shaped, with a “butt” towered by a long “blade”. Since the HCDR3 of CH01 differs in length and amino acid sequence from that of PG16, we used two different CH01 heavy chain alignments: (A) alignment 1 preserved the alignment of the heavy chain of CH01 to the blade of the PG16 HCDR3 structure whereas alignment 2 preserved the alignment to the butt of the PG16 HCDR3 structure. (B) Based on threading and DFIRE score, alignment 2 better predicts the Fab structure of the CH01-CH04 mAbs. (C) When the amino acid sequences of the CH01-CH04 mAbs was threaded onto Fab structures of monoclonal antibodies belonging to different groups (V2/V3 quaternary epitope-specific antibodies PG9 and PG16 and other CD4i monoclonal antibodies), the normalized DFIRE score predicted that the CH01-CH04 sequence fits better the structure of the V2/V3 quaternary epitope-specific antibodies. The values shown are normalized by dividing the DFIRE score obtained after threading onto the structure by the DFIRE score of sequence threaded onto the matched structure (i.e. PG16 sequence onto the PG16 structure). Values ranging from 1 to 1.4 (in green) are probably correct. Values from 1.5 to 1.9 are intermediate and are shown in orange, while values of 2.0 or above (in red) are unlikely to be correct. The HCDR3 of 47e, 412d and e51 monoclonal antibodies are disordered in crystal structure and modeled for threading purpose.

PG16 47e 412d 17b 48d X5 e51 CH01 CH02 CH03 CH04 PG9

PG16 1.0 1.6 1.9 2.0 1.6 2.2 1.8 1.2 1.2 1.2 1.1 1.0

47e 1.6 1.0 1.6 2.0 1.0 1.5 1.4 1.5 1.4 1.5 1.7 3.8

412d 3.6 1.9 1.0 1.5 1.3 2.8 2.4 2.5 1.9 3.2 3.1 3.5

17b 5.0 2.0 1.6 1.0 2.5 3.0 3.3 2.6 2.6 2.5 2.4 3.2

48d 4.4 2.9 4.0 2.7 1.0 3.0 4.5 4.6 4.3 4.0 4.5 3.2

X5 4.1 2.7 1.8 2.5 1.9 1.0 3.5 1.7 2.4 2.5 2.2 4.0

e51 3.1 1.8 2.0 2.1 1.4 2.1 1.0 2.3 2.6 3.3 2.3 4.0

Sequences

Structures

C.

Table S3: CH01-CH04 mAb epitope mapping on V2/V3 single point mutants of consensus C (conC) virus.

IC50 (µg/ml)

CH01 CH02 CH04 PG9 PG16

conC wild type 0.7 0.4 0.2 0.02 <0.002

fold effect of point mutations on IC50

V127A >100 >70 >150 27 57

S158A 2 4 2 1 1

F159A >100 >70 >150 11 266

N160A >100 >70 >150 >1000 >1000

L165A 1 2 1 2 2

R166A 1 16 5 1 1

D167N 1 1 1 1 1

K168A 13 22 15 1 1

K169E >100 >70 >150 >1000 >1000

K171A >100 >70 >150 6 24

V172E 1 1 1 1 1

L175A 2 3 1 1 1

I181A >100 >70 >150 13 17

K305A 1 1 1 2 2

I307A 2 3 3 4 5

R308A 1 3 1 1 1

I309A 1 1 0 3 2

Q315R 1 1 1 1 1

F317A 1 1 1 2 2

Y318A 1 1 1 2 1

A.

B.

HCV E2 Anaerobic Gut Flora

Aerobic Gut Flora

Influenza HA (Wisconsin)

CH01 <30 197 <30 <30

CH02 333 259 <30 <30

CH03 2910 287 98 <30

CH04 <30 <30 <30 <30

PG9 <30 <30 <30 <30

PG16 <30 <30 <30 <30

4E10 (pos ctrl) 89 83 69 <30

Synagis (neg ctrl) <30 <30 <30 <30

Figure S7 – Autoreactivity and polyreactivity of the CH01-CH04, PG9 and PG16 monoclonal antibodies. (A) Autoantigen reactivity of the CH01-CH04, PG9 and PG16 monoclonal antibodies was measured in a Luminex® AtheNA® Multi-Lyte ANA assay (Wampole Laboratories, Princeton, NJ). Serial dilutions of antibodies (x-axis) were tested against extractable nuclear antigens Sjogren’s syndrome antigen A (SSA) and B (SSB), Smith antigen (Sm), ribonucleoprotein (RNP), centromere B (Cent B), histone, scleroderma 70 (Scl

C.

70) and Jo-1 proteins and double-strand DNA (dsDNA). Binding is shown on the y-axis and is expressed in relative luminescence units (RLU). The dotted line shows the cut-off for positivity. 4E10 mAb is shown as positive control. (B) CH01-CH04 mAb reactivity to HIV-1 negative human epithelial HEp-2 cells was determined by indirect immunofluorescence on HEp-2 slides using FITC-conjugated goat anti-human Ig. Slides were photographed on an Olympus AX70 with SpotFlex FX1520 CCD with a UPlanFL 40x 0.75 NA objective at 25°C in the FITC channel using SPOT Software. All images were acquired for 12s except CH04 (10s) and 2F5 (6s). Image layout and scaling was performed in Adobe Photoshop without image manipulation. The size bar shown is 25 µm long. 2F5 and 17b mAbs are shown as positive and negative controls, respectively. CH03 mAb is weakly reactive. (C) Polyreactivity of the CH01-CH04 mAbs was measured using a Luminex® platform. Results are expressed as background-subtracted Relative Luminescence Units (RLU) using an antibody concentration of 50 µg/ml.

Documents

Bonsignori - CH01-CH04 antibodies paper - SOM post ...jvi.asm.org/content/suppl/2011/09/02/85.19.9998.DC1/Supplemental...10 100 1000 10000 >40000 [IgG] (ng/ml) CH03 CH04 CH05 CH01