Supplementary Information for. Heavy chain-only IgG2b-llama antibody effects near-pan HIV-1 neutralization by

Size: px

Start display at page:

Download "Supplementary Information for. Heavy chain-only IgG2b-llama antibody effects near-pan HIV-1 neutralization by"

Jocelyn Dixon
5 years ago
Views:

1 Supplementary Information for Heavy chain-only IgG2b-llama antibody effects near-pan HIV-1 neutralization by recognizing a CD4-induced epitope that includes elements of co-receptor- and CD4-binding sites Priyamvada Acharya 1, Timothy Luongo 1, Ivelin Georgiev 1, Julie Matz 2, Stephen D. Schmidt 1, Mark K. Louder 1, Pascal Kessler 3, Yongping Yang 1, Krisha McKee 1, Sijy O Dell 1, Lei Chen 1, Daniel Baty 2, Patrick Chames 2, Loïc Martin 3, John R. Mascola 1 and Peter D. Kwong 1* 1 Vaccine Research Center, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, Maryland, USA. 2 Inserm, U1068, CRCM, Marseille, France. 3 CEA, ibitecs, Service d Ingénierie Moléculaire des Protéines, Gif-sur-Yvette, F-91191, France.

2 Table of contents Supplementary Figures... 1 Figure S1. Binding of CD4i antibodies 17b and 48d to YU2 gp120 core min in absence and presence of soluble, 2-domain CD Figure S2. Effect of glycan at position 386 on sensitivity of viruses to neutralization by JM4- IgG2b Figure S3. Comparison of JM4 sequence with its putative germline precursor and with other broadly neutralizing antibodies Supplementary Tables... 4 Table S1. Crystallographic data collection and refinement statistics... 4 Table S2. Interactions at the interface between JM4 and the gp120 YU2 core e polypeptide... 5 Table S3. Interactions at the interface between JM4 and the sugar at Asn 386 of gp120 YU2 core e... 6 Table S4. Hydrogen bonds, salt bridges and water mediated hydrogen bond formed at the JM4-gp120 YU2 core e interface Table S5. Neutralization IC 50 values for VRC01, JM4, JM4-IgG2b and JM4-IgG3 against a panel of 30 viruses including 12 that are known to be resistant to the CD4-binding site directed broadly neutralizing antibody VRC Table S6. Neutralization IC 50 values for CD4i antibodies 17b, 48d and 412d, their Fab fragments, and VHH-only antibody JM4 against a diversified panel of 21 viruses Table S7. Neutralization IC50 values for JM4-IgG2b against a diversified panel of 195 viruses....10

3 K D = 3.17 nm k a = 8.48(2)e 5 M 1 s 1 k d = (3) s 1 K D= 23.5 nm k a= 1.272(5)e 6 M 1 s 1 k = 0.298(1) s 1 d Figure S1. Binding of CD4i antibodies 17b and 48d to YU2 gp120 core min in absence and presence of soluble, 2-domain CD4. 17b (top pan els) and 48d ( bottom panels) IgGs were immobilized on a CM5 chip. YU2 core min gp120 was injected in absence (left panels) and in presence (right panels) of soluble CD4 (scd4). The binding affinities and kinetic parameters of each interaction are listed with the numbers in parenthesis indicating the error in the last significant digit. CD4i antibodies 17b and 48d show weak binding to YU2 core min gp120 in absence of CD4. Binding is enhanced ~10-fold in the presence of scd4. CD4 stabilizes the CD4- bound conformation of YU2 core min gp120 that allows binding of CD4i antibodies to the CD4-induced epitope of co-receptor binding on gp120. 1

4 p= IC50 (μg/ml) Isolates with a glycan at position 386 N=178 Isolates lacking glycan at position 386 N=17 Figure S2. Effect of glycan at position 386 on sensitivity of viruses to neutralization by JM4-IgG2b. HIV-1 isolates were grouped based on presence or absence of glycan at position 386. Each square represents neutralization IC 50 of a virus. Solid squares represent viruses that have a glycan at position 386 and empty squares represent viruses that lack this glycan. IC 50 >50 µg/ml were assigned a value of 50 µg/ml and are surrounded by a dashed rectangle in the graph. Neutralization IC 50 values for the two groups were compared using non-parametric Mann-Whitney test. No statistically significant difference was observed between the two groups. 2

5 A FR CDR FR CDR2 - Germline: QVQLVESGGGLVQAGGSLRLSCAASGRTFSSYAMGWFRQAPGKEREFVAAISWSGGSTYY 60 JM4 : EVQLVESGGGLVQPGGSLRLSCAASGFTLDYYSIGWFRQAPGKEREGVSCISDSDGRTYY 60 :************.************ *:. *::************ *:.** *.* *** FR Germline: ADSVKGRFTISRDNAKNTVYLQMNSLKPEDTAVYYCAA 98 JM4 : ADSVKGRFTISRDNAKNTVYLQMNSLKPEDTAVYYCAT 98 *************************************: B FR CDR FR CDR2 - JM4 EVQLVESGGGLVQPGGSLRLSCAASGFTLDYYSIGWFRQAPGKEREGVSCISDSDGRTYY 60 J3 EVQLVESGGGLVQAGGFLRLSCELRGSIFNQYAMAWFRQAPGKEREFVAGMG---AVPHY 57 A12 AVQLVESGGGLVQAGGSLRLSCTASGRISSSYDMGWFRQAPGKEREFVAAISWSGGTTDY FR CDR3 JM4 ADSVKGRFTISRDNAKNTVYLQMNSLKPEDTAVYYCATDCT----VDPSLLYV-MDYY J3 GEFVKGRFTISRDNAKSTVYLQMSSLKPEDTAIYFCARSKS----TYISYNSNGYDYW A12 ADSVKGRFAISKDNAKNAVSLQMNSLKPEDTAVYYCAAKWRPL--RYSDYPSN-SDYYDW FR4---- JM4 GKGTQVTVSS- 123 J3 GRGTQVTVSS- 121 A12 GQGTQVTVSS- 127 Figure S3. Comparison of JM4 sequence with its putative germline precursor and with other broadly neutralizing antibodies. (A) Sequence analysis of mature JM4 with its putative germline antibody. (B) Sequence analysis of JM4 and other antibodies isolated from llama, J3 and A12. 3

6 Table S1. Crystallographic data collection and refinement statistics YU2gp120core e + M48U1 + JM4 Data collection Space group P2 1 Cell constants a, b, c (Å) α, β, γ ( ) 96.8, 86.2, , 102.9, 90.0 Wavelength (Å) 1.00 Resolution (Å) ( ) R sym 10.6 (46.0) I / σi 11.4 (1.2) Completeness (%) 77.2 (45.1) Redundancy 4.5 (2.3) Refinement Resolution (Å) ( ) Unique reflections 98838(1329) R work / R free (%) 0.192/0.240 No. atoms (non hydrogen) Protein Water 558 B-factors (Å 2 ) Overall Protein Water R.m.s. deviations Bond lengths (Å) Bond angles ( ) 1.2 Ramachandran Favored (%) 95 Outliers (%) 1 Values in parentheses are for highest-resolution shell. R sym = Σ I <I> /Σ<I>, where I is the observed intensity, and <I> is the average intensity of multiple observations of symmetry-related reflections. R = Σhkl Fobs Fcalc /Σhkl Fobs. R free is calculated from 5% of the reflections excluded from refinement. 4

7 Table S2. Interactions at the interface between JM4 and the gp120 YU2 core e polypeptide. Accessible Surface Area, Å² (ASA) and Buried Surface Area, Å² (BSA) were calculated using the EBI PISA server ( Residues contributing to the interface through H bonds or salt bridges are indicated by a blue letter H or a red letter S, respectively. The bars ( ) quantify buried area percentage, one bar per 10%. JM4 ASA BSA gp120 YU2 ASA BSA CDR H3 CDR H2 CDR H1 N-term GLU VAL GLY PHE TYR 31 H TYR SER SER ASP 53 HS ARG 57 H TYR 59 H THR ASP 99 H CYS THR ASP 103 H SER 105 H LEU TYR VAL 109 H MET ASP 111 HS TYR CYS VAL LEU VAL GLY 324 H ASP ILE ARG 327 HS GLN 328 H ASP PRO VAL THR TYR ASN 386 H PRO CYS ARG ILE LYS 421 HS GLN 422 H ILE 423 H ASN 425 H LYS 432 S MET TYR ALA PRO ARG

8 Table S3. Interactions at the interface between JM4 and the sugar at Asn 386 of gp120 YU2 core e. Accessible Surface Area, Å² (ASA) and Buried Surface Area, Å² (BSA) were calculated using the EBI PISA server ( Residues contributing to the interface through H bonds are indicated by a blue letter H. The bars ( ) quantify buried area percentage, one bar per 10%. JM4 ASA BSA NAG 386 ASA BSA CDR H2 CDR H1 GLU PRO SER LEU LEU TYR 108 H VAL NAG

9 Table S4. Hydrogen bonds, salt bridges and water mediated hydrogen bond formed at the JM4-gp120 YU2 core e interface. JM4 Hydrogen bonds Distance (Å) gp120 YU2 CDR H1 CDR H2 CDR H3 CDR H2 CDR H3 TYR 31 [ O ] 2.02 GLN 422 [HE22] TYR 59 [ HH ] 2.05 GLY 324 [ O ] ASP 53 [ OD1] 2.4 ARG 327 [HH12] ASP 53 [ OD1] 2.25 ARG 327 [HH22] ASP 99 [ OD1] 2.18 GLN 422 [ H ] ASP 99 [ OD2] 2.01 ILE 423 [ H ] ASP 103 [ OD2] 2.28 GLN 328 [HE22] SER 105 [ O ] 1.65 ASN 386 [HD21] TYR 108 [H ] 2.44 NAG 886 [ O7 ] VAL 109 [ O ] 2.13 LYS 421 [ HZ3] ASP 111 [ OD2] 2.06 ASN 425 [HD22] Salt bridges JM4 Distance (Å) gp120 YU2 ASP 53 [ OD1] 3.15 ARG 327 [ NH1] ASP 53 [ OD1] 3.04 ARG 327 [ NH2] ASP 53 [ OD2] 3.28 ARG 327 [ NH1] ASP 111 [ OD1] 3.95 LYS 432 [ NZ ] ASP 111 [ OD1] 3.33 LYS 421 [ NZ ] ASP 111 [ OD2] 3.09 LYS 432 [ NZ ] Water mediated hydrogen bond JM4 Water gp120 YU2 CDR H3 ASP 111 W1 and W2 Bridging sheet ILE 423 7

10 Table S5. Neutralization IC 50 values for VRC01, JM4, JM4-IgG2b and JM4-IgG3 against a panel of 30 viruses including 12 that are known to be resistant to the CD4-binding site directed broadly neutralizing antibody VRC01. IC 50 values are color-coded: IC 50 < 1.0 μg/ml, red; IC 50 between 1.0 and 50 μg/ml, yellow; IC 50 > 50 μg/ml, white. Clade Virus VRC01 JM4 JM4-IgG2b JM4- IgG3 A KER KER >50 >50 Q >50 >50 >50 Q842.d >50 >50 >50 UG >50 >50 >50 AC 6540.v4.c1 >50 >50 >50 >50 AG >50 >50 >50 >50 B C > >50 >50 BG >50 >50 BL01 > > CAAN.A >50 >50 JR-CSF JR-FL >50 >50 PVO >50 >50 TRO >50 >50 YU >50 > V3.C10 >50 >50 >50 >50 CAP210. E8 >50 >50 >50 >50 Du >50 >50 Du >50 >50 >50 >50 Du > >50 >50 SO >50 >50 TV1.29 >50 >50 >50 >50 TZA > >50 >50 ZM >50 >50 ZM >50 ZM >50 >50 D vrc15 > G X2088_c9 > >50 >50 Non- HIV SVA-MLV >50 >50 >50 >50 SIVmac >50 >50 >50 >50 8

11 Table S6. Neutralization IC 50 values for CD4i antibodies 17b, 48d and 412d, their Fab fragments, and VHH-only antibody JM4 against a diversified panel of 21 viruses. IC 50 values are color-coded: IC 50 < 1.0 μg/ml, red; IC 50 between 1.0 and 50 μg/ml, yellow; IC 50 > 50 μg/ml, white. IC 50 values clade virus 17b IgG 48D IgG 412d IgG 17b Fab 48d Fab 412d Fab JM4 VHH B BaL >50 >50 >50 >50 >50 > B JR-FL >50 >50 >50 >50 >50 > B JR-CSF >50 >50 > >50 > B YU2 >50 >50 >50 >50 >50 > B CAAN >50 >50 >50 >50 >50 > B PVO.04 >50 >50 >50 >50 >50 > B THRO.18 >50 >50 >50 >50 >50 > B TRO.11 >50 >50 >50 >50 >50 > B BG >50 >50 >50 >50 >50 > B >50 >50 >50 >50 >50 > C ZA >50 >50 >50 >50 >50 > C DU >50 >50 >50 >50 >50 > C ZM106.9 >50 >50 >50 >50 >50 >50 >50 A RW020.2 >50 >50 >50 >50 >50 > A Q23.17 >50 >50 >50 >50 >50 >50 >50 A Q168.a2 >50 >50 >50 >50 >50 >50 >50 A Q769.h5 >50 >50 >50 >50 >50 >50 >50 A KER >50 >50 >50 >50 >50 > B TRJO.58 >50 >50 >50 >50 >50 > C DU >50 >50 >50 >50 >50 >50 31 C ZM55.28 >50 >50 >50 >50 >50 >

12 Table S7. Neutralization IC 50 values for JM4-IgG2b against a diversified panel of 195 viruses. IC 50 values are color-coded: IC 50 < 1.0 μg/ml, red; IC 50 between 1.0 and 50 μg/ml, yellow; IC 50 > 50 μg/ml, white. Clade Virus IC 50 (μg/ml) glycan at 386? A 0260.v5.c36 >50 Y A 0330.v4.c3 >50 Y A 0439.v5.c1 >50 Y A 3365.v2.c20 >50 Y A 3415.v1.c1 >50 Y A 3718.v3.c11 >50 N A 398-F1_F6_20 >50 N A BB201.B42 >50 Y A BB539.2B13 >50 Y A BI369.9A >50 N A BS208.B1 >50 Y A KER Y A KER >50 Y A KNH >50 N A MB201.A1 >50 Y A MB539.2B7 >50 Y A MI369.A5 >50 N A MS208.A1 >50 Y A Q23.17 >50 Y A Q >50 N A Q769.d22 >50 Y A Q842.d12 >50 Y A QH209.14M.A2 >50 Y A RW020.2 >50 N A UG037.8 >50 Y AC 3301.V1.C24 >50 Y AC 3589.V1.C4 >50 Y AC 6540.v4.c1 >50 Y AC 6545.V4.C1 >50 Y ACD 0815.V3.C3 >50 Y ACD 6095.V1.C10 >50 Y AD 3468.V1.C12 >50 Y AD Q168.a2 >50 Y AD Q461.e2 >50 Y AE c1 >50 N AE BJOX >50 Y AE BJOX >50 Y AE BJOX >50 Y AE BJOX >50 Y AE C1080.c3 >50 Y AE C2101.c1 >50 Y AE C3347.c11 >50 Y AE C Y AE CM244.ec1 >50 Y AE CNE3 >50 Y AE CNE5 >50 Y AE CNE55 >50 Y AE CNE Y AE CNE59 >50 Y AE CNE8 >50 Y AE M02138 >50 Y 10

13 Clade Virus IC 50 (μg/ml) glycan at 386? AE R1166.c1 >50 Y AE R2184.c4 >50 Y AE R3265.c6 >50 Y AE TH023.6 >50 Y AE TH966.8 >50 Y AE TH >50 N AG >50 Y AG >50 Y AG >50 Y AG >50 Y AG >50 Y AG >50 Y AG DJ263.8 >50 Y AG T250-4 >50 Y AG T >50 Y AG T >50 Y AG T >50 Y AG T >50 N AG T >50 N AG T Y AG T280-5 >50 Y AG T33-7 >50 Y B >50 Y B >50 Y B >50 Y B >50 N B >50 Y B 45_01dG5 >50 Y B 89.6.DG 3.94 Y B AC10.29 >50 Y B ADA.DG Y B Bal Y B BaL Y B BG >50 Y B BL01.DG >50 Y B BR07.DG 6.35 Y B BX Y B CAAN.A2 >50 Y B CNE10 >50 Y B CNE Y B CNE Y B CNE Y B CNE Y B HO Y B HT Y B HXB2.DG Y B JRCSF.JB 17.2 Y B JRFL.JB >50 Y B MN Y B PVO.04 >50 Y B QH >50 Y B QH >50 Y B REJO Y B RHPA.7 >50 Y B SC422.8 >50 Y B SF162.LS Y B SS Y 11

14 Clade Virus IC 50 (μg/ml) glycan at 386? B THRO.18 >50 Y B TRJO.58 >50 Y B TRO.11 >50 Y B WITO.33 >50 Y B YU2.DG >50 Y BC CH >50 Y BC CH070.1 >50 Y BC CH117.4 >50 Y BC CH >50 Y BC CNE15 >50 Y BC CNE Y BC CNE20 >50 Y BC CNE21 >50 Y BC CNE40 >50 Y BC CNE7 >50 Y C >50 Y C >50 Y C Y C >50 Y C 0077_V1.C16 >50 Y C Y C 0921.V2.C14 >50 Y C >50 Y C >50 Y C >50 Y C Y C >50 Y C >50 Y C >50 Y C 3168.V4.C10 >50 Y C 3637.V5.C3 >50 Y C 3873.V1.C24 >50 Y C 6322.V4.C1 >50 Y C 6471.V1.C16 >50 Y C 6631.V3.C10 >50 Y C 6644.V2.C Y C 6785.V5.C14 >50 Y C 6838.V1.C35 >50 Y C 96ZM >50 Y C BR025.9 >50 Y C CAP210.E8 >50 Y C CAP244.D3 >50 Y C CAP45.G3 >50 Y C CNE30 >50 Y C CNE31 >50 Y C CNE53 >50 Y C CNE58 >50 Y C DU N C DU >50 Y C DU >50 Y C DU >50 N C DU >50 N C MW Y C SO18.18 >50 Y C TV1.29 >50 Y C TZA >50 Y C TZBD.02 >50 Y C ZA >50 Y 12

15 Clade Virus IC 50 (μg/ml) glycan at 386? C ZM106.9 >50 Y C ZM Y C ZM135.10a >50 Y C ZM >50 Y C ZM197.7 >50 Y C ZM >50 Y C ZM215.8 >50 Y C ZM249.1 >50 N C ZM53.12 >50 Y C ZM55.28a >50 Y CD 3326.V4.C3 >50 Y CD 3337.V2.C6 >50 Y CD 3817.v2.c59 >50 Y D E Y D c1 >50 N D >50 Y D 3016.v5.c45 >50 Y D vrc Y D 6405.v4.c34 >50 Y D A03349M1.vrc4a >50 Y D A07412M1.vrc12 >50 Y D NKU3006.ec1 >50 Y D UG Y G P0402.c2.11 >50 Y G P1981.C5.3 >50 Y G X1193.c1 >50 Y G X1254.c3 >50 Y G X1632.S2.B10 >50 Y G X2088.c9 >50 Y NA SIVmac SG3 >50 NA NA SVA.MLV >50 NA 13

P G K R P E W M G W L K P R G G A V N Y A R P L Q G R V T M T R D V Y S D T A F

P G K R P E W M G W L K P R G G A V N Y A R P L Q G R V T M T R D V Y S D T A F Supplementary Figure 1 VRC01 45-08-110497H 45-08-212510H 45-08-511533H 45-08-541880H Q V Q L V Q S G G Q M K K P G E S M R I S C R A S G Y E F I D C T L N W I R L A CAGGTGCAGCTGGTGCAGTCTGGGGGTCAGATGAAGAAGCCTGGCGAGTCGATGAGAATTTCTTGTCGGGCTTCTGGATATGAATTTATTGATTGTACGCTAAATTGGATTCGTCTGGCC