www.sciencemag.org/cgi/content/full/science.aah3945/DC1

Supplementary Materials for

Priming HIV-1 broadly neutralizing antibody precursors in human Ig loci transgenic mice

Devin Sok, Bryan Briney, Joseph G. Jardine, Daniel W. Kulp, Sergey Menis, Matthias Pauthner,

Andrew Wood, E-Chiang Lee, Khoa M. Le, Meaghan Jones, Alejandra Ramos, Oleksandr Kalyuzhniy, Yumiko Adachi, Michael Kubitz, Skye MacPherson, Allan Bradley, Glenn A.

Friedrich, William R. Schief,* Dennis R. Burton*

*Corresponding author. Email: burton@scripps.edu (D.R.B.); schief@scripps.edu (W.R.S)

Published 8 September 2016 on Science First Release DOI: 10.1126/science.aah3945

This PDF file includes:

Materials and Methods Figs. S1 to S11 Tables S1 to S6 References

Materials and Methods

Protein production and purification eOD monomers and 60mers were produced and purified as described previously (17), with eOD 60mers produced in the presence of 14 mM kifunensine in HEK293F or HEK293H cells (Invitrogen), and eOD monomers produced without kifunensine in HEK293F or HEK293H cells. BG505 SOSIP trimers were produced as previously described in HEK293F cells (26). All immunogens had endotoxin levels < 10 EU/mg.

Serum binding titers by ELISA eOD ELISAs were performed as described previously with minor modifications

(26). Microlon 96-well plates (Corning) were coated overnight with antigen (eOD-GT8, eOD-GT8 KO) at 2 μg/mL in PBS (25 μl/well). Plates were washed with PBS + 0.2% Tween20 and then blocked with 1% FBS + 5% skim milk + 0.2% Tween20 for 1 h at RT. Serially diluted sera in PBS + 1% FBS + 0.2% Tween20 were then added for 2 h at RT. Plates were then washed and alkaline phosphatase-labeled goat-anti-mouse immunoglobulin G (IgG) (Jackson Immunoresearch, Suffolk, England) was added for 1 h at a 1:2000 dilution in PBS + 1% FBS + 0.2% Tween20 at RT. After washing, absorption was measured at 450 nm.

Single-cell sorting by flow cytometry Mice spleen and lymph node samples were processed for single B cell sorting based

on previously described methods (6, 46, 47). In brief, mouse spleens and lymph nodes were stained with primary fluorophore-conjugated antibodies to murine CD4, CD8, F4/80, CD11c, Gr-1, CD19, B220, IgD, IgM, CD38, and GL7 markers. Memory B cells were selected for the phenotype CD19+, B220+, CD4-, CD8-, F4/80-, CD11c-, Gr-1-, IgM-, IgD-, while CD38 and GL7 markers were monitored to measure germinal center B cell frequencies (CD38-, GL7+). Naïve B cells were selected for the phenotype CD19+, B220+, CD4-, CD8-, F4/80-, CD11c-, Gr-1-, IgM+, IgD+. For antigen-specific staining, 50 nM of biotinylated AviTag eOD-GT8 monomer and its CD4bs KO varaint (eOD-GT8 KO) (26) were coupled to Streptavidin-AF488 and Streptavidin-PE (Life Technologoies) in equimolar ratios, respectively. B cells of interest were single-cell sorted into 96 well plates containing lysis buffer on a BD FACSAria Fusion sorter and immediately stored at -80°C (6, 46, 47). For sorting of B cells from unimmunized mice, a total of 1 million events were recorded to determine frequencies of antigen-specific memory B cells, and the remainder of the sample was single cell sorted for antigen-specific naïve B cells. For sorting of B cells from immunized mice, a total of 1 million events were recorded to determine frequencies of antigen-specific memory B cells and the remainder of the sample was single cell sorted for antigen-specific memory B cells.

Single B-cell RT-PCR, gene amplification, and sequencing Reverse transcription and subsequent PCR amplification of heavy and light chain

variable genes were performed using SuperScript III (Life Technologies) according to published protocols (6, 46, 47). All PCR reactions were performed in 25 µl volume with 2.5 µl of cDNA transcript using HotStar Taq DNA polymerase master mix (Qiagen) and mixtures of previously described primers (46). Second round nested-PCR reactions were

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performed using Phusion proof reading polymerase (NEB). A third round of PCR was performed using primers with barcodes specific to the plate number and well location as well as adapters appropriate for sequencing on an Illumina MiSeq. This reaction was performed in a 25-μL volume with HotStar Taq DNA polymerase master mix (Qiagen). Amplified IgG heavy- and light-chain variable regions were sequenced on an Illumina MiSeq (600-base v3 reagent kit; Illumina) and reads corresponding to the same plate/well location were combined into consensus sequences. Germline assignment and sequence annotation of the consensus sequences was performed with AbStar (www.github.com/briney/abstar). Rapid amplification of cDNA ends (RACE) next-generation sequencing

Total RNA from splenocytes was extracted from each naïve mouse (RNeasy Maxi Kit, Qiagen). 2.75ul of total RNA was reverse transcribed in 10ul total of total reaction volume using 1ul of oligo-dT and 1ul of template-switching primer (Table S6) using SuperScript II (Thermo Fisher, USA) and using the following cycling program: 42C for 90 minutes, 70C for 15 minutes. 2.5ul of reverse transcription product was used in each of two round 1 PCR reactions, one using 10pmol of each gene-specific primer for heavy chains (IgG and IgM, see Table S6 and the other using 10pmol of each primer for light chains (IgK and IgL, see Table S6. Each reaction also contained 2.5ul of 10x Universal Primer Mix (Zhu et al., 2001 Biotechniques) and 12.5 uL HotStarTaq Plus 2x MasterMax (Qiagen) in a total reaction volume of 25ul. Amplification was performed using the following cycling parameters: 95C for 5 minutes; 5 cycles of: 95C for 30 seconds, 72C for 2 minutes; 5 cycles of: 95C for 30 seconds, 70C for 30 seconds, 72C for 2 minutes; 25 cycles of: 95C for 30 seconds, 68C for 30 seconds, 72C for 2 minutes; 72C for 10 minutes. Indexing PCR was performed in a 25uL total reaction volume (HotStarTaq Plus, Qiagen) using 10pmol of each indexing primer (Table S6) and using the following cycling program: 95C for 5 minutes; 15 cycles of: 95C for 30 seconds, 58C for 30 seconds, 72C for 2 minutes; 72C for 10 minutes. Indexed PCR products were purified using 0.75 volumes of SPRIselect beads (Beckman Coulter Genomics) and eluted in 50uL of water. Samples from each donor were quantified using fluorometry (Qubit; Life Technologies), pooled at approximately equimolar concentrations and each sample pool was requantified before sequencing on an Illumina MiSeq (MiSeq Reagent Kit v3, 600-cycle).

Processing of NGS sequence data

Using the AbStar analysis pipeline (www.github.com/briney/abstar), raw sequencing reads were quality trimmed with Sickle (www.github.com/najoshi/sickle), adapters were removed with cutadapt (48), and paired reads were merged with PANDAseq (49). Germline gene assignment and sequence annotation was performed with AbStar and output was deposited into a MongoDB database. For NGS analysis of single-cell sorted B cells, all reads from the same plate/well location were binned using dual indexes added during amplification. In order to prevent primer-dimers and rare contaminant sequences from causing errors in consensus sequence calculation, the reads from each well were clustered at 97.5% identity, only the largest cluster of sequences was used to compute the consensus sequence for each well, and consensus sequences were only reported for wells in which the largest cluster contained a majority of sequences in the well. Although the

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number of reads per well varied substantially due to amplification efficiency and variability between sequencing runs, wells from which consensus sequences were computed typically contained 200-2000 sequencing reads. When identifying expanded non-VRC01-class lineages, lineage assignments were determined using Clonify (50).

Selection of antibody sequences for protein production

The 21 VRC01-class pairs from experiment #1 that were unique at the amino acid level were synthesized for production and purification, and of those, 20 were successfully purified and then evaluated by SPR. Antibody production

Heavy- and light-chain plasmids were cotransfected (1:1 ratio) in 293 FreeStyle cells using 293fectin (Invitrogen). Transfections were performed according to the manufacturer’s protocol, and antibody supernatants were harvested 4-5 days after transfection. Antibody supernatants were purified over Protein A Sepharose 4 Fast Flow (GE healthcare) columns, eluted with 0.1M citric acid (pH 3.0), and dialyzed against phosphate-buffered saline.

Surface plasmon resonance (SPR)

We measured kinetics and affinities of antibody-antigen interactions on a ProteOn XPR36 (Bio-Rad) using GLC Sensor Chip (Bio-Rad) and 1x HBS-EP+ pH 7.4

running buffer (20x stock from Teknova, Cat. No H8022) supplemented with BSA at 1mg/ml. We followed the Human Antibody Capture Kit instructions (Cat. No BR-1008-39 from GE) to prepare chip surfaces for ligand capture. In a typical experiment, about 6000 RU of capture antibody was amine-coupled in all 6 flow cells of the GLC Chip. Regeneration was accomplished using 3M Magnesium Chloride with 180 seconds contact time and injected four times per each cycle. Raw sensograms were analyzed using ProteOn Manager software (Bio-Rad), including interspot and column double referencing, and either Equilibrium fits or Kinetic fits with Langmuir model, or both, were employed when applicable. Analyte concentrations were measured on a NanoDrop 2000c Spectrophotometer using Absorption signal at 280 nm.

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Fig. S1. VH1-2 allele usage in humans. (A) VH gene frequencies measured from 99,678 heavy chain sequences from eight healthy human donors, aggregating alleles for each gene. The overall VH1-2 frequency was 3.1% and mean±standard deviation among the eight donors was 2.9±1.3%. (B) Frequencies of VH1-2 alleles among the 3110 VH1-2 genes in this dataset. The mean±standard deviation of the combined frequency of alleles *02, *03 and *04 was 84.2±21.3% among the eight donors.

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Fig. S2. Frequency analysis of VRC01-class light chain features in humans and unimmunized HK mice among all light chains or those with ≤ 5 nt mutations in VL. (A) L-CDR3 length (amino acid) frequency distribution. Distributions of all light chains are shown as solid lines, and distributions of light chain sequences with ≤ 5 nt mutations in VL are shown as dotted lines. (B) Frequency of 5-amino acid L-CDR3s. (C) Frequency of Vκ genes used by known VRC01-class antibodies (Vκ1-33, Vκ3-15, and Vκ3-20). (D) Frequency of light chains with 5-amino acid L-CDR3s and Vκ genes used by known VRC01-class antibodies. In (B)-(D) points represent frequencies for individual mice, and bars represent mean ± SD. Each mouse was sequenced once. Light chains with ≤ 5 nt mutations in VL comprised 59±3% of HK mouse light chains and 27±11% of human light chains.

Fig. S3. ELISA binding of VRC01 and germline-reverted VRC01 to eOD-GT8, eOD-GT8 KO and eOD-GT8 KO2. (A) VRC01 binding to eOD-GT8 and eOD-GT8 KO. (B) Germline-reverted VRC01 (VRC01 GL) binding to eOD-GT8 and eOD-GT8 KO. (C) VRC01 binding to eOD-GT8 and eOD-GT8 KO2. (D) Germline-reverted VRC01 binding to eOD-GT8 and eOD-GT8 KO2. The mutations in eOD-GT8 KO2 are, in HxB2 numbering, N280R, S365L and F371R. These mutations are different than the mutations in eOD-GT8 KO, which are D368R, N279A, D276N, and R287T (26). Data are plotted as mean ± SD with each dilution point measured in duplicate. Data are representative of two experiments.

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Fig. S4. Serum binding ELISA results Serum samples from day 0, 14, 28, and 42 post prime were collected and evaluated for antigen binding to eOD-GT8 and eOD-GT8 KO monomers by ELISA. Both serum IC50 and endpoint titers are shown in graphs on the left, and area above KO graphs are shown on the right. Area above KO is the difference between the areas under the eOD-GT8 and eOD-GT8 KO ELISA titrations. Data are plotted as mean ± SD.

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Fig. S5. Design and increased low-pH stability of eOD-GT8 d41m3 60mer. (A) Alignment of eOD-GT8 60mer and eOD-GT8 d41m3-60mer with 4 additional stabilizing mutations (P54C, I82C, K131C, S142C) and 3 active site mutations (F22A, H88S, R127A) (B) Denaturing and reducing SDS-PAGE. Lane 1: Novex Sharp Molecular Weight Marker, Lane 2: eOD-GT8 60mer, Lane 3: eOD-GT8 d41m3-60mer (C) SEC trace for eOD-GT8 60mer and eOD-GT8 d41m3-60mer at pH 7 in black. Molecular weight calculated across the peak in blue. (D) SEC trace for eOD-GT8 60mer and eOD-GT8 d41m3-60mer at pH 4 in black. Molecular weight calculated across the peak in blue. eOD-GT8 60mer partially dissociates into pentamers at low pH. eOD-GT8 d41m3-60mer remains fully intact.

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Fig. S6. Immunogenicity of eOD-GT8 d41m3 60mer in Kymab mice. (A) Epitope-specific (eOD-GT8+/eOD-GT8-KO-) memory B cell frequencies from HK (squares) and HKL (circles) mice immunized with eOD-GT8 60mer (red) or eOD-GT8 d41m3 60mer (blue). Points represent frequencies for individual mice, and bars show mean ± SD. (B) Number of animals that produced VRC01-class pairs in response to immunization with eOD-GT8 60mer or eOD-GT8 d41m3 60mer, with HK and HKL data combined. (C) Number of animals that produced VRC01-class pairs in response to immunization with eOD-GT8 60mer or eOD-GT8 d41m3 60mer, with HK and HKL data separated. (D) Number of animals that produced 5 aa L-CDR3s in in response to immunization with eOD-GT8 60mer or eOD-GT8 d41m3 60mer, with HK and HKL data combined. (E) Number of animals that elicited 5 aa L-CDR3s in response to immunization with eOD-GT8 60mer or eOD-GT8 d41m3 60mer, with HK and HKL data separated.

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A

B

Fig. S7. Amino acid sequences of 26 amino-acid-sequence unique VRC01-class antibodies elicited by eOD-GT8 60mer in Kymab mice. (A) Heavy chain sequences (B) Light chain sequences. Two other VRC01-class antibodies were unique at the nucleotide sequence level in the heavy and light chains but were identical the amino acid level. Amino-acid-identical antibodies always came from the same animal.

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Fig. S8. Number of mice that produced VRC01-class pairs or 5-amino acid L-CDR3s in response to eOD-GT8 60mer immunization, according to regimen. (A) Number of mice that produced VRC01-class pairs 14 day post prime. (B) Number of mice that produced 5-amino acid L-CDR3s 14 days post prime. (C) Number of mice that produced VRC01-class pairs 42 days post prime. (D) Number of mice that produced 5-amino acid L-CDR3s 42 days post prime.

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Fig. S9. Surface plasmon resonance (SPR)-determined dissociation constants for 20 eOD-GT8 60mer-induced VRC01-class antibodies binding to eOD-GT8 and two epitope mutants. Bars show geometric mean (GM) and GM ×/÷ geometric standard deviation (GSD).

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Fig. S10. ELISA binding of eOD-GT8 60mer-induced VRC01-class mAbs to eOD-GT8 monomer and BG505 SOSIP native-like trimer. PGT145, a trimer-specific bnAb that should bind only to BG505 SOSIP, and 12A12, a VRC01-class bnAb that should bind to both eOD-GT8 and BG505 SOSIP, are included as positive controls. Starting concentration for all antibodies was 50 µg/ml.

10-4 10-2 100 1020

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Antibody Concentration (μg/mL)

OD405

BG505 SOSIP.664

Ky_001

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12A12

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eOD-GT8 monomer

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Fig. S10 cont'd

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Ky_0025

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Fig. S11. Results of TZM-bl neutralization assays testing eOD-GT8 60mer-induced VRC01-class mAbs for their ability to neutralize the HXB2 lab-adapted strain of HIV. Starting concentration for all antibodies was 10 µg/ml. bnAb 12A12 was included as a positive control. Data are plotted as mean ± SD, with each dilution point measured in duplicate.

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Table S1. Frequencies of VRC01-class heavy and light chain features in one HL and two HKL mice. Kappa (κ) frequencies are among all kappa chains; lambda (λ) frequencies are among all lambda chains.

mouse frequencies VH1-2*04 5-aa L-CDR3 κ 5-aa L-CDR3

λ 5-aa L-CDR3

VRC01-class κ 5-aa L-CDR3

VRC01-class λ HL (N=1) 2.05% --- 0.033% --- 0.0030%

HKL (N=2) 0.990% 0.0442% 0.053% 0.0032% 0.0011%

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Table S2.

Antibody gene analysis by mouse for experiment 1 at day 14 post prime.

Animal(s) Group HCs LCs Pairs VH1-2 5aa L-CDR3

VH1-2 pairs

5aa L-

CDR3 pairs

VRC01-class pairs

HK 30.2F eOD17_20ug_ISCO 77 123 69 0 0 0 0 0 HK 35.2E eOD17_20ug_ISCO 11 73 9 0 0 0 0 0 HL 89.4A eOD17_20ug_ISCO 12 2 2 0 0 0 0 0 HL 89.4B eOD17_20ug_ISCO 23 10 7 3 0 0 0 0

TOTAL 123 208 87 3 0 0 0 0

HK 25.2A GT8_20ug_ISCO 72 89 62 5 3 5 3 3 HK 25.2B GT8_20ug_ISCO 58 60 44 3 0 3 0 0 HL 81.5A GT8_20ug_ISCO 46 32 23 1 0 0 0 0 HL 81.5B GT8_20ug_ISCO 0 0 0 0 0 0 0 0

TOTAL 176 181 129 9 3 8 3 3

HK 30.2B GT8_20ug_RIBI 107 69 61 6 1 5 1 1 HK 30.2C GT8_20ug_RIBI 105 85 72 1 0 0 0 0 HL 98.1B GT8_20ug_RIBI 84 26 19 4 0 1 0 0 HL 98.1C GT8_20ug_RIBI 44 53 29 1 0 0 0 0

TOTAL 340 233 181 12 1 6 1 1

HK 30.2A GT8_4ug_ISCO 57 61 53 4 2 4 2 2 HK 33.2A GT8_4ug_ISCO 69 96 50 1 0 1 0 0 HL 97.1A GT8_4ug_ISCO 44 53 26 3 1 1 0 0 HL 97.1B GT8_4ug_ISCO 87 17 12 7 0 0 0 0

TOTAL 257 227 141 15 3 6 2 2

HK 31.2C GT8_4ug_RIBI 84 41 33 0 0 0 0 0 HK 31.2D GT8_4ug_RIBI 11 35 7 0 1 0 0 0 HL 104.1B GT8_4ug_RIBI 38 36 21 6 2 3 0 0 HL 104.1C GT8_4ug_RIBI 38 36 29 8 1 8 1 1

TOTAL 171 148 90 14 4 11 1 1

HK 19.5A unimmunized 0 0 0 0 0 0 0 0 HK 19.5B unimmunized 0 0 0 0 0 0 0 0 HK 19.5C unimmunized 19 42 16 1 0 0 0 0 HL 95.5A unimmunized 66 20 14 4 0 1 0 0 HL 95.5C unimmunized 51 40 29 2 0 1 0 0 HL 95.5B unimmunized 0 0 0 0 0 0 0 0

TOTAL 136 102 59 7 0 2 0 0

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Table S3 Antibody gene analysis by mouse for experiment 1 at day 42 post prime.

Animal(s) Group HCs LCs Pairs VH1-2 5aa L-CDR3s

VH1-2 pairs

5aa L-CDR3 pairs

VRC01-class pairs

HK W35-2F eOD17_20ug_ISCO 28 34 22 0 0 0 0 0 HK W35.2G eOD17_20ug_ISCO 0 0 0 0 0 0 0 0 HK W30.1A eOD17_20ug_ISCO 0 0 0 0 0 0 0 0 HL E90-4A eOD17_20ug_ISCO 204 76 64 6 0 0 0 0 HL E90-4B eOD17_20ug_ISCO 15 15 11 0 0 0 0 0 HL E94.6A eOD17_20ug_ISCO 10 8 5 1 0 1 0 0

TOTAL 257 133 102 7 0 1 0 0

HK W25.2E GT8_20ug_ISCO 75 119 47 0 0 0 0 0 HK W25.2D GT8_20ug_ISCO 173 151 119 2 1 2 1 1 HK W25.2C GT8_20ug_ISCO 116 121 62 1 1 1 1 1 HL E84.6B GT8_20ug_ISCO 111 153 90 25 13 22 6 6 HL E84.6A GT8_20ug_ISCO 178 160 120 9 2 6 1 1 HL E83.7A GT8_20ug_ISCO 150 139 104 18 1 13 1 0

TOTAL 803 843 542 55 18 44 10 9

HK W31.2B GT8_20ug_RIBI 111 126 94 2 3 2 2 2 HK W31.2A GT8_20ug_RIBI 153 148 123 1 0 1 0 0 HK W30.2D GT8_20ug_RIBI 101 60 50 0 0 0 0 0 HL E102.1B GT8_20ug_RIBI 69 44 35 10 0 6 0 0 HL E102.1A GT8_20ug_RIBI 31 15 4 1 0 1 0 0 HL E104.1A GT8_20ug_RIBI 0 0 0 0 0 0 0 0

TOTAL 465 393 306 14 3 10 2 2

HK W34.1B GT8_4ug_ISCO 53 97 49 1 1 0 0 0 HK W34.1A GT8_4ug_ISCO 89 80 46 3 4 2 2 1 HK W33.1C GT8_4ug_ISCO 74 82 70 0 0 0 0 0 HL E98.1A GT8_4ug_ISCO 125 186 109 7 0 7 0 0 HL E97.1D GT8_4ug_ISCO 90 68 51 6 2 5 2 2 HL E97.1C GT8_4ug_ISCO 47 74 39 1 0 1 0 0

TOTAL 478 587 364 18 7 15 4 3

HK W35.2A GT8_4ug_RIBI 108 85 75 0 0 0 0 0 HK W31.2F GT8_4ug_RIBI 134 131 123 1 1 1 0 0 HK W31.2E GT8_4ug_RIBI 89 94 79 2 2 2 2 2 HL E103.1B GT8_4ug_RIBI 61 28 23 7 0 3 0 0 HL E103.1A GT8_4ug_RIBI 83 48 41 12 0 10 0 0 HL E102.1C GT8_4ug_RIBI 33 41 29 2 2 2 1 1

TOTAL 508 427 370 24 5 18 3 3

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Table S4. Antibody gene analysis by mouse for experiment 2 at day 42 post prime.

Animal(s) Group HCs LCs Pairs VH1-2 5aa L-CDR3

VH1-2 pairs

5aa L-CDR3 pairs

VRC01-class pairs

HKL FC27-1D BG505-SOSIP-D664 25 13 8 1 0 1 0 0 HKL FC26-1B BG505-SOSIP-D664 15 17 11 0 0 0 0 0 HKL FC25-1F BG505-SOSIP-D664 16 39 11 1 0 1 0 0 HKL FC25-1E BG505-SOSIP-D664 22 31 20 0 0 0 0 0 HKL FC25-1D BG505-SOSIP-D664 11 13 7 0 0 0 0 0 HK DK37-1C BG505-SOSIP-D664 3 26 2 1 0 0 0 0 HK DK29-3D BG505-SOSIP-D664 18 30 14 0 0 0 0 0 HK DK29-3C BG505-SOSIP-D664 52 101 36 0 0 0 0 0 HK DK29-3B BG505-SOSIP-D664 35 103 23 1 0 0 0 0 HK DK29-3A BG505-SOSIP-D664 12 15 4 0 0 0 0 0 TOTAL 209 388 136 4 0 2 0 0

HKL FC28-1C eOD-17-c1-g28b-GS-60mer 34 84 25 1 0 0 0 0 HKL FC28-1B eOD-17-c1-g28b-GS-60mer 40 66 35 0 0 0 0 0 HKL FC21-2F eOD-17-c1-g28b-GS-60mer 45 73 37 0 0 0 0 0 HKL FC21-2E eOD-17-c1-g28b-GS-60mer 66 90 36 0 0 0 0 0 HKL FC21-2D eOD-17-c1-g28b-GS-60mer 36 75 24 0 0 0 0 0 HK DK29-3F eOD-17-c1-g28b-GS-60mer 9 27 0 0 0 0 0 0 HK DK29-3E eOD-17-c1-g28b-GS-60mer 0 18 0 0 0 0 0 0 HK DK26-2E eOD-17-c1-g28b-GS-60mer 17 27 7 0 0 0 0 0 HK DK20-3H eOD-17-c1-g28b-GS-60mer 41 53 12 3 0 1 0 0 HK DK20-3G eOD-17-c1-g28b-GS-60mer 53 77 36 3 0 1 0 0 TOTAL 341 590 212 7 0 2 0 0

HKL FC23-1D eOD-GT8-60mer 31 77 27 6 2 3 1 1 HKL FC20-1G eOD-GT8-60mer 37 76 25 0 0 0 0 0 HKL FC20-1F eOD-GT8-60mer 41 59 19 4 0 3 0 0 HKL FC20-1E eOD-GT8-60mer 74 94 57 3 3 2 2 2 HKL FC19-4D eOD-GT8-60mer 78 57 21 4 1 1 1 0 HK DK29-2K eOD-GT8-60mer 29 70 21 0 0 0 0 0 HK DK29-2J eOD-GT8-60mer 22 64 13 1 0 1 0 0 HK DK29-2I eOD-GT8-60mer 29 72 22 2 0 2 0 0 HK DK17-4H eOD-GT8-60mer 45 47 14 0 0 0 0 0 HK DK17-4E eOD-GT8-60mer 12 52 8 0 0 0 0 0 TOTAL 398 668 227 20 6 12 4 3

HKL FC26-1A eOD-GT8-d41-m3-60mer 56 81 27 4 5 3 1 1 HKL FC24-1B eOD-GT8-d41-m3-60mer 52 80 27 0 1 0 0 0 HKL FC24-1A eOD-GT8-d41-m3-60mer 32 21 6 0 0 0 0 0 HKL FC21-1F eOD-GT8-d41-m3-60mer 64 68 22 3 0 0 0 0 HK DK37-1B eOD-GT8-d41-m3-60mer 55 83 17 1 2 1 0 0 HK DK37-1A eOD-GT8-d41-m3-60mer 46 53 20 0 0 0 0 0 HK DK34-1F eOD-GT8-d41-m3-60mer 68 51 21 0 2 0 0 0 HK DK34-1E eOD-GT8-d41-m3-60mer 49 76 15 0 1 0 1 0 HK DK34-1D eOD-GT8-d41-m3-60mer 25 66 21 3 1 2 0 0

TOTAL 579 176 11 12 6 2 1

21

Table S5. Properties of VRC01-class antibodies elicited by eOD-GT8 60mer in Kymab mice. KD values are for monovalent interaction with eOD-GT8. ND, not determined.

V Gene J Gene D Gene CDR3 %

mut (nt)

% mut (aa)

KD

(nM)

Ky_001_VRC01c

IGHV1-2 IGHJ3 IGHD3-10 CARDQGFAMVRGVTFDIW 0 0

910 IGKV3-15 IGKJ1 CQQYNNF 0.7 0

Ky_004_VRC01c

IGHV1-2 IGHJ4 IGHD1-26 CARGESGSYFDYW 0 0 ND

IGKV3-20 IGKJ3

CQQFETF 0 0

Ky_005-VRC01c

IGHV1-2 IGHJ4 IGHD3-10 CARAYYGSGSYFFDYW 1.4 2.1 150

IGKV1D-33 IGKJ5 CQQYVTF 1.1 2.3

Ky_006_VRC01c

IGHV1-2 IGHJ4 IGHD6-13 CARRYSSSWYFDYW 3.4 4.2 110

IGKV3-20 IGKJ4 CQQYGSF 1.1 1.1

Ky_007_VRC01c

IGHV1-2 IGHJ4 IGHD6-19 CARRGGYSSGWSYFDYW 0 0 1600

IGKV1-5 IGKJ5

CQQYNTF 0.7 0

Ky_008_VRC01c

IGHV1-2 IGHJ3 IGHD6-13 CARNIATTGDAFDIW 2.4 2.1 0.14

IGLV3-1 IGLJ3 CQAWDMF 0 0

Ky_009_VRC01c

IGHV1-2 IGHJ1 IGHD5-12 CARGGYGGYGWDFQHW 1.7 4.2 32

IGLV2-14 IGLJ3 CSSYTVF 0.7 1.1

Ky_010_VRC01c

IGHV1-2 IGHJ5 IGHD5-12 CARRGYSGYDWFDPW 0 0 1700

IGLV2-14 IGLJ1

CSSYTSF 2 0

Ky_012_VRC01c

IGHV1-2 IGHJ3 IGHD1-26 CARRGRHAFDIW 1.4 4.2 10

IGLV2-14 IGLJ1 CSSYTRF 2.4 0

Ky_014_VRC01c

IGHV1-2 IGHJ4 IGHD3-10 CARFYGSGSFFDYW 0 0 140

IGLV2-23 IGLJ3 CCSYVVF 2.9 5.6

Ky_015_VRC01c

IGHV1-2 IGHJ3 IGHD3-9 CARAVHYDILTGFGFDIW 1 3.1 46

IGLV2-14 IGLJ1

CSSYTSF 2.4 1.1

Ky_016_VRC01c

IGHV1-2 IGHJ6 IGHD6-19 CARKAVAGYYGMDVW 0.7 1 190

IGLV2-14 IGLJ3 CSSYTVF 0.7 0

Ky_018_VRC01c

IGHV1-2 IGHJ5 IGHD1-1 CARSGTTGTTGWFDPW 0 0 1000

IGLV2-14 IGLJ1 CSSYTSF 2 1.1

Ky_019_VRC01c

IGHV1-2 IGHJ4 IGHD3-16 CARGGGITTIFDYW 0 0 ND

IGLV2-14 IGLJ1

CTSYTTF 4.1 1.1

Ky_020_VRC01c

IGHV1-2 IGHJ4 IGHD1-26 CARGESGSYFDYW 0 0 ND

IGLV3-1 IGLJ3 CQAWDIF 0.7 1.1

Ky_021_VRC01c

IGHV1-2 IGHJ4 IGHD1-26 CARVSGSYFDYW 0 0 300

IGLV3-1 IGLJ3 CQAWDIF 1.1 1.2

22

Table S5. cont’d

Ky_022_VRC01c

IGHV1-2 IGHJ4 IGHD6-13 CARRYSSSWYFDYW 3.1 3.1 470

IGKV3-15 IGKJ3 CQQYNTF 0.7 1.1

Ky_023_VRC01c

IGHV1-2 IGHJ4 IGHD6-13 CARDRIAAAGTPFDYW 0 0 1800

IGKV4-1 IGKJ5 CQQYYTF 0.7 0

Ky_024_VRC01c

IGHV1-2 IGHJ4 IGHD3-10 CARVFYGSGSYWYFDYW 0 0 ND

IGKV1-5 IGKJ3 CQQYNSF 0.7 0

Ky_025_VRC01c

IGHV1-2 IGHJ4 IGHD3-22 CYYDSSGYYYW 0 0 350

IGKV3-15 IGKJ1 CQQYNTF 1.8 4.5

Ky_026_VRC01c

IGHV1-2 IGHJ4 IGHD3-10 CARGGYYYGSGGPYFDYW 1.4 4.2

190 IGKV3-15 IGKJ4 CQQYNTF 0.7 0

Ky_028_VRC01c

IGHV1-2 IGHJ4 IGHD1-26 CARGESGSYFDYW 1.4 3.1 6

IGKV1-5 IGKJ1 CQQYNTF 2.2 2.3

Ky_029_VRC01c

IGHV1-2 IGHJ4 IGHD6-13 CARPSYSSSWYFDYW 0 0 ND

IGKV3-15 IGKJ4 CQQYNTF 1.1 0

Ky_030_VRC01c

IGHV1-2 IGHJ5 IGHD5-18 CARGEYSSWFDPW 0 0 ND

IGVK1-5 IGKJ1 CQQYNTF 0.4 0

Ky_031_VRC01c

IGHV1-2 IGHJ4 IGHD1-26 CAREGSGSYDYW 0 0 ND

IGVK1-5 IGHJ4 CQQYNSF 0.7 1.1

Ky_032_VRC01c

IGHV1-2 IGHJ4 IGHD6-13 CAAGYSSSWYFDYW 0 0 ND

IGKV 3-20 IGKJ4 CQQYGSF 0 0

23

Table S6. Primers used for RACE next-generation sequencing. Nucleotides in bold are ribonucleotides.

Primer Name Primer Sequence

Oligo dT TTTTTTTTTTTTTTTTTTTTTTTTTVN TS-short_ill AAGCAGTGGTATCAACGCAGAGTAGACGTGTGCTCTTCCGATCTGGGGG TS_heel-carrier GTAATACGACTCACTATAGGGCAAGCAGTGGTATCAACGCAGAGT

TS_heel-specific GTAATACGACTCACTATAGGGC

hum_IgG_RACE ACACTCTTTCCCTACACGACGCTCTTCCGATCTNNNNNNSGATGGGCCCTTGGTGGARGC

hum_IgA_RACE ACACTCTTTCCCTACACGACGCTCTTCCGATCTNNNNNNCTTGGGGCTGGTCGGGGATG

hum_IgM_RACE ACACTCTTTCCCTACACGACGCTCTTCCGATCTNNNNNNGGTTGGGGCGGATGCACTCC

hum_IgK_RACE ACACTCTTTCCCTACACGACGCTCTTCCGATCTNNNNNNAGATGGTGCAGCCACAGTTC

hum_IgL_RACE ACACTCTTTCCCTACACGACGCTCTTCCGATCTNNNNNNGAGGGYGGGAACAGAGTGAC

ill_r2_ui01 CAAGCAGAAGACGGCATACGAGAGATCGGTCTCGGCATTCCTGCTGAAGATNNNNACAATGATGTGACTGGAGTTCAGACGTGTGCTCTTCCGATC

ill_r2_ui02 CAAGCAGAAGACGGCATACGAGAGATCGGTCTCGGCATTCCTGCTGAAGATNNNNAGGGATTTGTGACTGGAGTTCAGACGTGTGCTCTTCCGATC

ill_r2_ui03 CAAGCAGAAGACGGCATACGAGAGATCGGTCTCGGCATTCCTGCTGAAGATNNNNCCTTATTGGTGACTGGAGTTCAGACGTGTGCTCTTCCGATC

ill_r2_ui04 CAAGCAGAAGACGGCATACGAGAGATCGGTCTCGGCATTCCTGCTGAAGATNNNNTGTTCGCCGTGACTGGAGTTCAGACGTGTGCTCTTCCGATC

ill_r2_ui05 CAAGCAGAAGACGGCATACGAGAGATCGGTCTCGGCATTCCTGCTGAAGATNNNNAGGTAAAAGTGACTGGAGTTCAGACGTGTGCTCTTCCGATC

ill_r2_ui06 CAAGCAGAAGACGGCATACGAGAGATCGGTCTCGGCATTCCTGCTGAAGATNNNNGATGGGGTGTGACTGGAGTTCAGACGTGTGCTCTTCCGATC

ill_r2_ui07 CAAGCAGAAGACGGCATACGAGAGATCGGTCTCGGCATTCCTGCTGAAGATNNNNCGTCACTTGTGACTGGAGTTCAGACGTGTGCTCTTCCGATC

ill_r2_ui08 CAAGCAGAAGACGGCATACGAGAGATCGGTCTCGGCATTCCTGCTGAAGATNNNNGGCGACAAGTGACTGGAGTTCAGACGTGTGCTCTTCCGATC

ill_r2_ui09 CAAGCAGAAGACGGCATACGAGAGATCGGTCTCGGCATTCCTGCTGAAGATNNNNACTGTCAGGTGACTGGAGTTCAGACGTGTGCTCTTCCGATC

ill_r2_ui10 CAAGCAGAAGACGGCATACGAGAGATCGGTCTCGGCATTCCTGCTGAAGATNNNNGACTAACAGTGACTGGAGTTCAGACGTGTGCTCTTCCGATC

ill_r2_ui11 CAAGCAGAAGACGGCATACGAGAGATCGGTCTCGGCATTCCTGCTGAAGATNNNNAAATCCGGGTGACTGGAGTTCAGACGTGTGCTCTTCCGATC

ill_r2_ui12 CAAGCAGAAGACGGCATACGAGAGATCGGTCTCGGCATTCCTGCTGAAGATNNNNGGTGCCGTGTGACTGGAGTTCAGACGTGTGCTCTTCCGATC

ill_r2_ui13 CAAGCAGAAGACGGCATACGAGAGATCGGTCTCGGCATTCCTGCTGAAGATNNNNAAAGCCAAGTGACTGGAGTTCAGACGTGTGCTCTTCCGATC

ill_r2_ui14 CAAGCAGAAGACGGCATACGAGAGATCGGTCTCGGCATTCCTGCTGAAGATNNNNTCCTTGCAGTGACTGGAGTTCAGACGTGTGCTCTTCCGATC

ill_r2_ui15 CAAGCAGAAGACGGCATACGAGAGATCGGTCTCGGCATTCCTGCTGAAGATNNNNAAAACGTAGTGACTGGAGTTCAGACGTGTGCTCTTCCGATC

ill_r2_ui16 CAAGCAGAAGACGGCATACGAGAGATCGGTCTCGGCATTCCTGCTGAAGATNNNNACGTTTTAGTGACTGGAGTTCAGACGTGTGCTCTTCCGATC

ill_r2_ui17 CAAGCAGAAGACGGCATACGAGAGATCGGTCTCGGCATTCCTGCTGAAGATNNNNACCTGGCCGTGACTGGAGTTCAGACGTGTGCTCTTCCGATC

ill_r2_ui18 CAAGCAGAAGACGGCATACGAGAGATCGGTCTCGGCATTCCTGCTGAAGATNNNNATATGACCGTGACTGGAGTTCAGACGTGTGCTCTTCCGATC

ill_r2_ui19 CAAGCAGAAGACGGCATACGAGAGATCGGTCTCGGCATTCCTGCTGAAGATNNNNGTAGGTCCGTGACTGGAGTTCAGACGTGTGCTCTTCCGATC

ill_r2_ui20 CAAGCAGAAGACGGCATACGAGAGATCGGTCTCGGCATTCCTGCTGAAGATNNNNACCCAAGCGTGACTGGAGTTCAGACGTGTGCTCTTCCGATC

UT_Rev AATGATACGGCGACCACCGAGATCTACACTCTTTCCCTACACGACGCTC

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