Cell Host & Microbe, Volume 18

Supplemental Information

Strain-Specific V3 and CD4 Binding Site Autologous HIV-1 Neutralizing Antibodies Select Neutralization-Resistant Viruses M. Anthony Moody, Feng Gao, Thaddeus C. Gurley, Joshua D. Amos, Amit Kumar, Bhavna Hora, Dawn J. Marshall, John F. Whitesides, Shi-Mao Xia, Robert Parks, Krissey E. Lloyd, Kwan-Ki Hwang, Xiaozhi Lu, Mattia Bonsignori, Andrés Finzi, Nathan A. Vandergrift, S. Munir Alam, Guido Ferrari, Xiaoying Shen, Georgia D. Tomaras, Gift Kamanga, Myron S. Cohen, Noel E. Sam, Saidi Kapiga, Elin S. Gray, Nancy L. Tumba, Lynn Morris, Susan Zolla-Pazner, Miroslaw K. Gorny, John R. Mascola, Beatrice H. Hahn, George M. Shaw, Joseph G. Sodroski, Hua-Xin Liao, David C. Montefiori, Peter T. Hraber, Bette T. Korber, and Barton F. Haynes  

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Abbreviations:

ADCC, antibody-dependent cellular cytotoxicity; APC, allophycocyanin; bnAb, broadly

neutralizing antibody; C3-V4, third constant-variable loop 4; CHAVI, Center for HIV/AIDS

Vaccine Immunology; CD4bs, CD4 binding site; ELISA, enzyme-linked immunosorbant assay;

Env, envelope; Ig, immunoglobulin; mAb, monoclonal antibody; MTCT, mother-to-child

transmission; nAb, neutralizing antibody; PBMC, peripheral blood mononuclear cells; PE,

phycoerythrin; RT, reverse transcription; SGA, single genome amplication; SHIV, simian-human

immunodeficiency virus; T/F, transmitted/founder; V1V2, first and second variable region; V3,

third variable region; VH/κ/λ, variable region heavy chain / kappa chain / lambda chain; vRNA,

viral RNA.

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Experimental Procedures:

Ethics statement. The clinical material used for the present study was obtained as a part of the

CHAVI 001 observational study. The participants studied here were identified during the

screening of CHAVI 001 and CHAVI 008 subjects for the presence of neutralization breadth

(Tomaras et al., 2011). The present work was performed under a protocol approved by the Duke

University Health System Institutional Review Board for Clinical Investigations. These original

studies with human subjects from which we obtained the clinical material herein studied were

approved by the Kilimanjaro Christian Medical Centre Research Ethics Committee, the Tanzania

National Institutes for Medical Research Ethics Coordinating Committee, and the Institutional

Review Boards of the London School of Hygiene and Tropical Medicine and Duke University as

well as by the NIH Human Subject Review Committee.

Clinical material. The participants in this study (CH0457 and CH505) were recruited in 2008 in

Tanzania and Malawi, respectively. At the time of recruitment, CH0457 had been chronically

infected with a subtype C virus for an unknown period. This participant did not receive

antiretroviral drug therapy during the study period. Peripheral blood collections were performed

at weeks 0, 2, 4, 8, 12, 16, 24, 48, 72, and 96 of observation. Blood was processed for peripheral

blood mononuclear cells (PBMC), plasma, and serum, all of which were cryopreserved for

transport to the research laboratories. Participant CH505 was recruited 4 weeks following HIV-1

infection and has been described previously (Liao et al., 2013).

Flow cytometry panel antibodies, recombinant proteins, and assay control antibodies. The

gp120ConC core protein was produced as described (Gray et al., 2011a) and labeled with Pacific

Blue and Alexa Fluor (AF) 647 using fluorochrome labeling kits (Invitrogen, Carlsbad, CA). The

protein batches were confirmed to bind to CD4 expressed on the surface of the H9 T cell line as

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a quality control after conjugation. Setup for flow cytometry was performed as described

(Moody et al., 2012b). Sorting was performed using antibodies reactive with surface IgM

(FITC), surface IgD (phycoerythrin [PE]), CD3 (PE-Cy5), CD16 (PE-Cy5), CD235a (PE-Cy5),

and CD19 (allophycocyanin [APC]-Cy7) (BD Biosciences, San Jose, CA); CD14 (PE-Cy5)

(Invitrogen, Carlsbad, CA); CD27 (PE-Cy7) and CD38 (APC-Alexa Fluor 700) (Beckman

Coulter, Brea, CA).

Hyperimmune HIV-1 globulin subtype C (HIVIG-C) is a mixture of purified IgG from 5

subtype C HIV-1-infected plasma donors in South Africa (Johannesburg blood bank). (Morris et

al., 2011). Genetic subtype was confirmed by SGA sequencing of the plasma Envs. The 5 IgG

samples included in HIVIG-C were selected among 35 IgG samples for having the greatest

magnitude and breadth of neutralizing activity against a panel of 6 tier 2 viruses. Palivizumab, a

humanized monoclonal antibody against the F protein of respiratory syncytial virus, was

purchased from MedImmune, LLC (Gaithersburg, MD). Negative control CH65 is a mAb

directed against the sialic acid binding site of hemagglutinin (Moody et al., 2011; Whittle et al.,

2011). Positive control CH31 is a bnAb directed against the CD4bs (Bonsignori et al., 2012; Wu

et al., 2011), as is positive control CH106 (Liao et al., 2013). Positive control CD4bs-directed

BNAb HJ16 (Corti et al., 2010) was provided as a generous donation from Davide Corti

(Institute for Research in Biomedicine, Bellinzona, Switzerland).

Antibody reactivity by binding antibody multiplex assay, enzyme-linked immunosorbent assay

(ELISA), and peptide microarray. Expressed mAbs were studied for reactivity to HIV-1 antigens

using a standardized custom binding antibody multiplex assay using Luminex (Tomaras et al.,

2008). All assays were run under conditions compliant with Good Clinical Laboratory Practice,

including tracking of positive controls by Levy-Jennings charts. FDA-compliant software, Bio-

Plex Manager, version 5.0 (Bio-Rad, Hercules, CA), was utilized for the analysis of specimens.

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Screening by binding antibody multiplex assays was performed against a panel of HIV-1

antigens (gp140ConC, gp120ConC full length, gp140ConB, gp140ConG, gp140JR.FL); mAbs that had a

blank-bead-subtracted value greater than 2000 units and greater than 1000 times the mAb IgG

concentration in µg/mL were evaluated further. Binding of all mAbs was confirmed by

subsequent assays on mAbs prepared from transfected cells at large scales.

ELISA testing of mAbs was performed as described (Alam et al., 2008); testing was

considered positive if the optical density reading at 405 nm was above 0.3 units and greater than

4-fold over background. Epitope mapping by cross-clade peptide microarray was performed as

described (Tomaras et al., 2011).

Flow cytometric analysis and single-cell sorting. We previously reported that CH0457 had broad

neutralizing activity in plasma that could be absorbed by a subtype C consensus (ConC) gp120

protein that lacked V1V2 and V3 loops (gp120ConC core) (Tomaras et al., 2011). To isolate

neutralizing antibody-producing memory B cells, we used antigen-specific sorting.

Fluorescently-labeled gp120ConC core protein was used to isolate Env-reactive memory B cells

using a dual-color technique (Gray et al., 2011b; Moody et al., 2012a). We sorted samples from

the week 8 and week 12 time points, and in both cases we isolated antigen-specific B cells from

which immunoglobulin (Ig) genes were recovered (Fig. S1 online). In total, we isolated 19 heavy

chains with paired light chains and found that when expressed as mAbs, 12/19 (63%) were

reactive with one or more consensus Env proteins from clades A, B, C, G and CRF01_AE; 11 of

these mAbs were carried forward for further study (Table S1).

Single-cell sorting was performed using a BD FACSAria II (BD Biosciences, San Jose,

CA) and the flow cytometry data were analyzed using FlowJo (Treestar, Ashland, OR). Antigen-

specific memory B cells were identified by using gp120ConC core labeled with Alexa Fluor 647

and Pacific Blue; cells were gated on CD3− CD14− CD16− CD235a− CD19+ surface IgD−

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gp120ConC core+/+. Single cells were directly sorted into 96-well plates containing 20 µL per

well of reverse transcription (RT) reaction buffer (5 µL of 5′ first-strand cDNA buffer, 0.5 µL of

RNaseOUT [Invitrogen, Carlsbad, CA], 1.25 µL of dithiothreitol, 0.0625 µL Igepal CA-630

[Sigma, St. Louis, MO], 13.25 µL of distilled H2O [dH2O; Invitrogen, Carlsbad, CA]); plates

were stored at −80°C until use and after sorting were again stored at −80°C until PCR was

performed.

Memory B Cell Cultures. IgG+ memory cells were isolated from PBMCs from subject CH505

collected 41 or 176 weeks after HIV-1 transmission and cultured as described (Bonsignori et al.,

2011; Gao et al., 2014). Briefly, cells were resuspended in complete medium containing 2.5

µg/mL CpG ODN2006 (tlrl-2006; InvivoGen, San Diego, CA), 5 µM CHK2 kinase inhibitor

(Calbiochem/EMD Chemicals, Billerica, MA), and Epstein-Barr virus (200 µL supernatant of

B95-8 cells/104 memory B cells); and incubated in bulk overnight at 37°C in 5% CO2. After

overnight incubation, viable IgG+ memory B cells were transferred at a cell density of 2

cells/well in 96-well round-bottom tissue culture plates containing ODN2006, CHK2 kinase

inhibitor, and 5,000 irradiated (7,500 cGy) CD40 ligand–expressing L cells per well (Gao et al.,

2014). DH151 and DH228 were isolated from cultures that displayed binding to the CH505

transmitted/founder gp140 Env after 14 days of in vitro stimulation. RNA from positive cultures

was extracted by using standard procedures (RNeasy minikit; QIAGEN, Valencia, CA), and the

genes encoding (Ig) VH, Vκ, and Vλ genes were isolated as described below.

PCR isolation and analysis of immunoglobulin (Ig) VH, Vκ, and Vλ genes. Single-cell PCR was

performed as described (Liao et al., 2009; Moody et al., 2012b; Wrammert et al., 2008). PCR

amplicons were sequenced in forward and reverse directions using a BigDye sequencing kit on

an ABI 3730XL (Applied Biosystems, Foster City, CA). Sequence base calling was performed

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using Phred (Ewing and Green, 1998; Ewing et al., 1998), forward and reverse strands were

assembled using an algorithm based on the quality scores at each position (Kepler et al., 2010).

Local alignment with known sequences was used to determine Ig isotype (Smith and Waterman,

1981); V, D, and J region genes, complementarity-determining region 3 (CDR3) lengths, and

mutation frequencies were determined using SoDA (Volpe et al., 2006). Clonal lineages of

antibodies were determined as described (Moody et al., 2011; 2012a) and were confirmed by

alignment of complete V(D)J sequences. Maximum-likelihood trees for clonal lineages were

generated using V(D)J regions (excluding constant region sequences); trees were constructed

(dnaml), reorganized (retree), and plotted (drawgram) with the PHYLIP package, version 3.69

(Felsenstein, 2009).

Expression of VH and Vκ/λ as full-length IgG1 mAbs. PCR was used to assemble isolated Ig VH

and Vκ/λ gene pairs into linear full-length Ig heavy- and light-chain gene expression cassettes as

described (Liao et al., 2009). Human embryonic kidney cell line 293T (ATCC, Manassas, VA)

was grown to near confluence in six-well tissue culture plates (Becton Dickinson, Franklin

Lakes, NJ) and transfected with 2 µg per well of both IgH and Igκ/λ purified PCR-produced

cassettes using Effectene (Qiagen, Valencia, CA). Culture supernatants were harvested 3 days

after transfection and concentrated 4-fold using centrifugal concentrators; expressed IgG was

quantitated by ELISA (Gray et al., 2009); tested mAbs were expressed at 10 µg/mL up to 20

mg/mL. Larger-scale production of mAbs was performed using synthesized linear IgH and Igκ/λ

gene constructs (GeneScript, Piscataway, NJ).

Amplification of full-length env genes. Viral RNA (vRNA) was prepared from plasma samples

(400 µL) using the EZ1Virus Mini Kit V2.0 on BIO ROBOT EZ1 (Qiagen; Valencia, CA).

Reverse transcription was performed with 20 µL of vRNA and 80 pmol primer 1.R3.B3R (5′-

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ACTACTTGAAGCACTCAAGGCAAGCTTTATTG-3′) in 50 µL using Superscript III

(Invitrogen; Carlsbad, CA). The 3′ half genomes were amplified by single genome amplication

(SGA) as previous described (Jiang et al., 2011; Salazar-Gonzalez et al., 2009), using 07For7

(5′CAAATTAYAAAAATTCAAAATTTTCGGGTTTATTACAG-3′) and 2.R3.B6R (5′-

TGAAGCACTCAAGGCAAGCTTTATTGAGGC-3′) as first round primers, and VIF1 (5′-

GGGTTTATTACAGGGACAGCAGAG-3′) and Low2c (5′-

TGAGGCTTAAGCAGTGGGTTCC-3′) as the second round primers. The PCR products were

purified with the QiaQuick PCR Purification kit (Qiagen; Valencia, CA). The env gene

sequences were obtained by cycle-sequencing and dye terminator methods with an ABI 3730XL

genetic analyzer (Applied Biosystems; Foster City, CA). Individual sequence contigs from each

env SGA were assembled and edited using the Sequencher program 4.7 (Gene Codes; Ann

Arbor, MI).

Amplification of HIV-1 env genes from PBMCs by SGA. Proviral DNA was extracted from

3×106 PBMCs at the enrollment (week 0) time point using the QIAamp DNA Blood and Tissue

kit (Qiagen; Valencia, CA). The HIV-1 rev/env cassette was amplified from the genomic DNA

using the single genome amplification (SGA) method. The PCR primers and conditions were the

same as those used for viral RNA templates extracted from plasma.

Generation of pseudoviruses. The CMV promoter was added to the 5′ end of each env gene

amplified by SGA using the promoter addition PCR (pPCR) method as described (Kirchherr et

al., 2007). The pPCR product was used for generation of pseudoviruses by cotransfecting with

the env-deficient HIV-1 backbone pSG3Δenv into 293T cells in a 6-well tissue culture plate

using FuGENE6 transfection reagent (Roche Diagnostics; Indianapolis, IN) according to

manufacturer instructions. Transfected cells were maintained in DMEM with 10% FBS at 37°C

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with 5% CO2. Forty-eight hours after transfection, supernatants were harvested and stored in

20% FBS medium at −80°C.

Neutralization assay in TZM-bl cells. Neutralizing antibody assays in TZM-bl cells were

performed as described (Montefiori, 2005). Antibodies were tested at concentrations up to 50

µg/mL using eight serial 3-fold dilutions. Control antibodies include HJ16 which was generously

provided by D. Corti (Institute for Research in Biomedicine, Università della Svizzera Italiana,

Bellinzona, Switzerland). Env-pseudotyped viruses were added to the antibody dilutions at a

predetermined titer to produce measurable infection and incubated for 1 h. TZM-bl cells were

added and incubated for 48 h. Firefly luciferase (Luc) activity was measured as a function of

relative luminescence units (RLU) using a Britelite Luminescence Reporter Gene Assay System

as described by the supplier (Perkin-Elmer Life Sciences, Waltham, MA). Neutralization was

calculated as the reduction in RLU in test wells compared with control wells after subtraction of

background RLU in cell control wells and reported as mAb 50% inhibitory concentration (IC50)

in µg/mL. Env-pseudotyped viruses were prepared in 293T cells and titrated in TZM-bl cells as

described (Montefiori, 2005).

Neutralization data that appear in Figure 2 includes published reports (Balla-

Jhagjhoorsingh et al., 2011; Corti et al., 2010) and data generated as described above.

Mapping of mAb specificities by neutralization. Single amino acid substitutions were introduced

into the consensus C (ConC) or B.RHPA Env by oligonucleotide-directed PCR mutagenesis

using the QuickChange site-directed mutagenesis kit (Stratagene, La Jolla, California). Alanine

or conserved mutations were introduced in C1 (L125A), V1 (R132A/T), C2 (S256A, N289K),

C3 (T372V, T373M, S375M), C5 (G471E), the β23 sheet of C4 (R456W), as well as the CD4bs

(D-loop: N276A/Q, T278A, N279D; α5: D474A, M475A; and gp120 inner domain layer 3

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(Désormeaux et al., 2013): R476A). The ability of antibodies to neutralize pseudoviruses

containing Env point mutations was assessed and compared to the wild-type pseudovirus

neutralization. A fifteen-fold or higher increase in IC50 titer from the wild-type to the mutant was

considered positive.

Molecular structure. The deposited crystal structure for gp120C.YU2 (PDB accession code 1G9N)

(Kwong et al., 2000) was used to display identified amino acid residues that were determined to

be critical for binding. The structure was rendered in PyMOL with CD4 in light gray, gp120 in

light blue, and mAb 17b not shown). The critical residues were highlighted and side chains

shown for those residues (Fig. 1C).

Statistical analysis. Graphs of the data were created using GraphPad Prism (GraphPad Software,

La Jolla, CA) with layout in Illustrator CS5 (Adobe, San Jose, CA). Statistical tests were

performed in SAS, version 9.2 (SAS Institute, Cary, NC) or in R, version 2.15.2 (R Foundation

for Statistical Computing, Vienna, Austria). The statistical test used is noted when p values are

presented. Env sequence phylogenies were inferred using PhyML (Guindon and Gascuel, 2003)

with the HIVw substitution model (Nickle et al., 2007). Compartmentalization was tested using

the Slatkin-Madison test (Zárate et al., 2007).

Supplemental Results:

Isolation of nAbs. Antibodies from CH0457 were isolated by antigen-specific B cell sorting

using a clade C consensus Env protein. Following PCR of sorted cells, we isolated 11 mAbs

from the week 8 sample of which 5 were HIV-1 Env reactive; from the week 12 sample we

isolated 8 mAbs of which 6 were HIV-1 Env reactive. Analysis of these mAbs showed that they

consisted of two clonal lineages that spanned the two time points and two additional mAbs that

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were not related. Clonal lineage CH13 consisted of six monoclonal antibodies (mAbs) of IgG1

isotype (CH13, CH16, CH17, CH18, CH45, CH46) that used VH1~69*01 / JH3*02 and

VK1~39*01 / JK4*01 genes. Epitope mapping with binding and neutralization assays

demonstrated that the CH13 lineage antibody bound to the CD4bs and were sensitive to

mutations at D386, E370, I371, S375, and K421 (Fig. 1c; Tables S2 and S3). Two additional

mAbs, CH14 and CH48, were not clonally related to any other mAbs isolated nor to each other,

and both mAbs mapped in Env peptide binding assays to the HIV-1 Env third variable (V3) loop

(Fig. 1d; Table S4). Like clonal lineage CH13, mAbs CH14 and CH48 neutralized only tier 1

but not tier 2 heterologous HIV-1 strains (Fig. 2).

The second group of mAbs, clonal lineage CH27 (Fig. 1b), consisted of three mAbs that

used VH3~66*02 / JH2*01 and VK3~20*01 / JK1*01 (CH27, CH28, CH44). Two members of this

clonal lineage (CH27 and CH28) were found to be isotype IgA2 while the third was IgG1 (Table

S1). All were expressed as IgG1 mAbs. Testing of this group of mAbs using HIV-1 strain

B.RHPA mutants demonstrated that they were sensitive to changes at N276 and T278,

suggesting that the CH27 lineage consisted of HJ16-like CD4bs-directed bnAbs (Balla-

Jhagjhoorsingh et al., 2013) (Table S3). Surface plasmon resonance studies of mAbs from the

CH27 lineage and HJ16 showed that they cross-blocked each other for binding to HIV-1 Env

(Fig. S1).

Plasma samples from CH0457 taken from weeks 8 and 96 were tested against the same

panel of heterologous viruses (Fig. 2). Neutralization titers against heterologous viruses were

similar at the two chronic infection time points, despite the fact that the samples were collected

nearly two years apart. Plasma antibodies neutralized all tier 1 isolates, consistent with the clonal

lineage CH13 mAbs and V3 mAbs CH14 and CH48 neutralization patterns. Of the 10

heterologous HIV isolates neutralized by plasma at >1:1000 dilution, nine viruses were

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neutralized by lineage CH27 mAbs at <2µg/mL (Fig. 2). Thus, the isolated mAbs accounted for

the majority of CH0457 plasma heterologous virus neutralization.

We isolated restricted V3 neutralizing antibodies from a second HIV-1-infected African

individual, CH505, by clonal memory B cell culture performed on samples taken at 41 weeks

and 176 weeks after transmission (Liao et al., 2013). This individual eventually developed a

CD4bs clonal lineage (termed CH103) at 136 weeks after transmission (Liao et al., 2013). At

each time point, memory B cells were cultured as described (Bonsignori et al., 2014): from week

41 we cultured 27,950 memory B cells yielding 17 mAbs that were reactive with Env; from

week 176 we cultured 35,100 memory B cells yielding 8 mAbs reactive with Env. Screening of

all 25 mAbs identified one mAb from each time point that mapped to the V3 loop.

Validation of CH0457 sequence integrity. To determine if there was any evidence for multiple

infection or contamination, particularly given that there were two distinctive clades in the

CH0457 sample, we did the following tests using the tools at the Los Alamos HIV database

(www.hiv.lanl.gov). First we made a DNA consensus of the sequences from the persistent minor

clade and the major lineage in CH0457. We then then used HIV-BLAST to these compare the

two consensus sequences against the HIV database. Both consensus sequences are closest to

natural sequences from CH0457 already in GenBank, supporting that they came from the same

quasispecies, and same individual. At the DNA level, the consensus from the persistent minor

clade shared between 94 and 97% identity in Blast searches with other CH0457 sequences from

the cominant clade. In contrast, the next closest match shared only 87%; it was a C clade isolate

from Malawi. We then extracted all full length Env sequences from Tanzania; there were 388 of

them. We combined these with the HIV subtyping reference set, and the consensus sequences

from CH0457, and made a neighbor joining phylogeny based on these 435 reference and

Tanzanian sequences. The two consensus sequences from the 2 distinctive within-subject

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CH0457 lineages always clustered together, among natural sequences from CH0457, forming a

monophyletic group with high bootstrap support in a neighbor joining tree (data not shown, as

this was a quality control check). This again indicates that the unusual clade is not a recurrent

contamination, or independent infecting strain, and that both lineages evolved from a single

infecting strain within CH0457, and had diverged prior to the first sample taken during chronic

infection.

This view was further supported by the addition of the PBMC proviral DNA sequences

from the enrollment time point, that were considered to be biologically “archived” in the host

represening virus that had been replicating prior to the time of enrollment. These sequences

revealed intermediate steps between the two distinctive lineages found in the CH0457 SGA

sequences (Fig. S2). Among the proviral sequences, there were 6 that were highly significantly

enriched for G-to-A hypermutated in Apobec motifs (Hypermut, www.hiv.lanl.gov) (Harris and

Liddament, 2004; Rose and Korber, 2000) giving rise to multiple stop codons in Env resulting in

clearly inactive virus. These are evident as a hypermutated cluster in the fully phylogenetic tree

shown in Fig. S2 (w0.41c, w0.40c, w0.19c, w0.c, w0.13c, w0.48c; highlighted by an asterisk).

There were no other significantly hypermutated sequences in the proviral set, and none among

the SGA viral sequences.

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Table S1, related to Figure 1: HIV-1 Env-reactive antibodies isolated from CH0457. heavy chain light chain week isotype VH JH CDR3

length mutation frequency

V J CDR3 length

mutation frequency

non-lineage CH14 12 IgG1 1~69*04 3*02 17 14.8% κ 4~1*01 3*01 9 8.2% CH48 12 IgG1 4~30-4*01 4-02 19 9.5% λ 2~14*03 3*02 9 6.2% Lineage CH13 CH13 8 IgG1 1~69*01 4*01 17 9.1% κ 1~39*01 4*01 9 4.0% CH16 12 IgG1 1~69*01 4*01 17 12.9% κ 1~39*01 4*01 9 9.0% CH17 12 IgG1 1~69*01 4*01 17 9.9% κ 1~39*01 4*01 9 5.3% CH18 12 IgG1 1~69*01 4*01 17 9.4% κ 1~39*01 4*01 9 4.3% CH45 8 IgG1 1~69*01 4*01 17 8.3% κ 1~39*01 4*01 9 9.6% CH46 8 IgG1 1~69*01 4*01 17 9.1% κ 1~39*01 4*01 9 8.7% average 9.8% 6.8% Lineage CH27 CH27 8 IgA2 3~66*02 2*01 10 15.3% κ 3~20*01 1*01 11 15.6% CH28 12 IgA2 3~66*02 2*01 10 14.0% κ 3~20*01 1*01 11 15.6% CH44 8 IgG1 3~66*02 2*01 10 17.7% κ 3~20*01 1*01 11 16.5% average 15.7% 15.9%

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Table S2, related to Figure 1: Mapping of mAbs by binding to gp120 mutants. mAb binding assay to gp120* B.HXBc2† B.YU2 E370K K421A R476A D477A D368A E370A I371A S375W Lineage CH13 CH13 0.04 0.31 0.79 1.08 0.18 0.23 0.31 0.29 CH16 0.27 0.73 1.34 1.10 0.79 0.48 0.71 0.41 CH17 0.07 0.68 0.91 1.23 0.78 0.46 0.60 0.37 * Data normalized vs. binding to wild type gp120 protein. † Additional mutants tested for which no binding change was observed: B.HXBc2 K429E, D474V, M475S; B.YU2 G473A, M475A, ∆V1/V2/V3. Lineage members CH18, CH45, and CH46 not tested.

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Table S3, related to Figure 1: Mapping of mAbs by neutralization. Neutralization* B.RHPA clade C consensus† N160K N276A T278A T278A

R456W R132A R132T T372V

T373M S375M D474A

Lineage CH13 CH13 –§ – – – >100 1.8 >50 >100 16 CH16 – – – – 0.5 0.5 7.3 >32 1.3 CH17 – – – – 91 >55 19 >100 10 CH18 – – – – 0.4 >15 >9 >15 2 CH45 – – – – >20 >20 9 >36 8.1 CH46 – – – – – – – – – Lineage CH27 CH27 0.1 7.6 7.1 7 0.7 1 2.1 2.3 0.4 CH28 0.3 >333 >333 >307 0.8 0.9 2.8 1.7 0.8 CH44 0.2 >106 >106 >1000 1.5 3.2 2.5 2.6 0.6 Control mAb HJ16 0.5 >10 >10 >1000 – – – – – * Data shown is fold increase in concentration required to produce 50% neutralization (increase in IC50 in µg/mL of mAb). † Other mutants of clade C consensus tested that did not show changes >20 fold for any tested mAb: L125A, S256A, N289K, G471E, M475A, R476A. § – = not tested.

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Table S4, related to Figures 1 and 3: Mapping of V3-directed mAbs by ELISA. EC50* V3 loop peptides scaffolded V3 loop antigens Env constructs ConB† ConC ConS gp70

B.MN3 gp70

AE.92TH023 gp70

ConAG gp70 ConC

RSC3 ∆RSC3

CH0457 mAbs non-lineage CH14 0.05 0.03 0.02 NB‡ 0.004 –§ – NB NB CH48 0.05 0.03 0.005 1.0 6.1 – – NB NB CH505 mAbs non-lineage DH151 0.15 0.009 0.008 NB‡ 0.003 0.002 0.002 NB NB DH228 NB NB 0.008 NB NB 1.50 2.52 NB NB * Data shown is half maximal effective concentration (EC50) in µg/mL of mAb. † ConB = clade B consensus; ConC = clade C consensus; ConS = group M consensus. ‡ NB = no binding observed. § – = not tested.

p 18 of 29

Supporting Figures

Figure S1, related to Figures 1 and 2. Cross blocking of HJ16 and lineage CH27 mAbs.

Antibodies from lineage CH27 were tested for cross-blocking against HJ16.

Taken together, the data suggest that the binding sites for the lineage CH27 mAbs and

HJ16 overlap but are not identical.

A. HJ16 was immobilized on a surface plasmon resonance chip and antibody-

Env mixtures were flowed over the chip to determine if the antibody-Env

complex bound to HJ16. Control mAb palivizumab was the control; non-

neutralizing anti-HIV-1 mAb 16H3 did not significantly block binding to

HJ16. In contrast, HJ16 blocked to 96% as expected, while CH27 and CH44

blocked about 1/3 of binding to HJ16.

B. CH27 immoblized on a chip was able to bind to Env mixed with palivizumab

or 16H3, but binding was partially blocked when Env was mixed with CH27,

CH44, or HJ16.

C. CH44 immoblized on a chip was able to bind to Env mixed with palivizumab

or 16H3, but binding was blocked when Env was mixed with CH27, CH44, or

HJ16.

A

time (s)400 6002000-100

-10

100

80

60

80

40

0

resp

onse

uni

ts

Y Y YHJ16 on chip

mAb +gp120C.1086

Y

mAb % blocking of HJ16palivizumab control16H3 6.9CH27 38CH44 33HJ16 96

B

time (s)400 6002000-100

-16

160

128

96

64

32

0

resp

onse

uni

ts

Y Y YCH27 on chip

mAb +gp120C.1086

Y

mAb % blocking of CH27palivizumab control16H3 8.5CH27 70CH44 53HJ16 84

C

time (s)400 6002000-100

-30

300

240

180

120

60

0

resp

onse

uni

ts

Y Y YCH44 on chip

mAb +gp120C.1086

Y

mAb % blocking of CH44palivizumab control16H3 6.4CH27 48CH44 66HJ16 65

p 19 of 29

w0.27c w0.22c w0.5c w0.35c*** w2.11 w0.14c w0.29c*** w0.24c w2.7** w0.4c w2.20 w0.6 w0.55c w2.4** w0.3c w16.8** w12.30 w4.16 w2.15 w96.24 w72.2* w96.12 w72.21 w72.10 w48.9 w72.23 w72.18** w72.25 w96.37 w96.20 w0.53c w0.11c w24.10 w48.20 w24.11** w12.15* w0.54c w0.50c w0.15c w4.12 w0.2c w0.56c w0.45c w0.51c w0.18c w2.21 w0.39c w0.5 w0.27 w0.41c w0.40c w0.19c w0.1c w0.13c w0.48c w2.16** w0.17c w2.5 w2.17 w0.11 w2.19 w2.3 w4.13 w0.21 w4.18 w0.24 w0.2 w0.16c w4.7 w4.5 w0.57c w0.16 w0.12 w2.2 w2.1 w2.14 w4.15 w4.1 w0.22 w0.19 w0.18 w4.2 w0.7 w0.25 w4.8 w0.9 w2.12 w4.11 w2.10 w0.31c w4.14 w2.8 w0.23** w0.34c w0.10c w2.13 w0.44c w0.26 w4.19 w8.11 w0.20 w0.10 w4.6 w4.3 w0.17 w0.9c w0.38c w4.20 w4.17 w0.21c w0.42c w12.17* w12.7 w12.4** w24.14 w24.4** w24.2* w0.30c w8.15 w4.9** w12.6* w8.8 w8.24 w12.28 w8.20 w8.5 w12.8 w8.27 w8.13 w8.23 w8.10 w8.26 w8.9 w8.25 w8.18 w8.12 w8.22 w8.6 w12.14 w12.12* w8.4 w8.3* w0.28c w0.47c w8.14 w12.24** w16.22 w16.26 w12.2 w24.1 w16.21* w24.9 w24.3* w24.13 w16.9 w16.17* w24.12 w16.20 w12.22 w12.26 w12.10 w12.27 w16.18 w24.6 w16.16 w16.6 w16.13 w16.3 w16.15 w12.3 w12.1 w12.19 w12.21 w12.11 w12.29 w16.19 w16.2 w16.4 w12.13 w12.20 w12.16 w16.10 w12.32** w16.5 w16.14 w0.46c w0.8c w16.12 w16.24 w16.25 w16.11 w16.7 w0.25c w24.16 w48.17 w48.22 w48.13 w48.1 w48.15 w48.10 w48.3 w48.8 w48.16 w48.4 w48.5 w48.19 w48.2 w48.23 w72.22 w96.15 w72.8 w72.19 w72.7 w72.3 w96.13*** w72.27 w72.11 w72.26 w72.12 w72.1 w96.27 w96.34 w72.4 w72.20 w96.40 w72.15 w72.17 w96.11 w96.17 w96.19 w96.14 w96.29 w96.3 w96.1 w96.2 w96.21 w96.25 w96.32 w96.4 w96.38 w96.36 w96.39 w96.9 w96.23 w96.10 w96.30 w96.6 w96.7 w96.35 w96.33 w96.16 w96.5*** w96.28

w0 35cw0 29c

w2 7

w2 4w16 8

w72 2

w72 18

w24 11w12 15

w2 16

w0 23

w12 17w12 4w24 4w24 2

w4 9w12 6

w12 12w8 3

w12 24

w16 21w24 3

w16 17

w12 32

w96 13

w96 5

*

9110087

92 9697

67 9990 76

98

61

6670

99 99 62 97 82

9589

686169

837679

60

87

9966

8467648296

98

84

99

100

8188

CH14

CH14

↑

↓

CH16

CH16

↑

↓0.05 Env Variants

MatchMismatch

Sample TimepointProviruschronic enrollmentweek +2week +4week +8week +12week +16week +24week +48week +72week +96

Neut. TitermAbµg/ml 50 20 − 50 10 − 20 5 − 10 2 − 5 1 − 2 0.5 − 1 0.2 − 0.5 0.2≤

>>>>>>>>

Indel

V1 V2 LoopD

CD4Loop

V4β23

V5β24

V3

||||||| | ||||||||||||||||||||||||||| |||||||||||||||||||||||||||||||||||||||||||||| |||||||||||||||||||||||||||||||||||||||||||| |||||||||||||||||||||||||||||||||||||||||||||||||||||||| ||||||||||||||||||||||||||||||||||||||||||||||| CD4 contact residues

VirusTimepoint

↓

VirusTimepoint

↑

Env Position100 200 300 400 500 600 700 800

p 20 of 29

Figure S2, related to Figure 4. HIV-1 env gene evolution in participant CH0457.

Env phylogeny from CH0457 during chronic infection is shown. A pixel map

(left) depicts mutations where each site differs from the consensus of earliest plasma

Envs, whether mutations (red) or insertions/deletions (black). Each row in the tree and

the pixel map depicts a distinct Env isolated from longitudinal samples; i.e., week 0

(enrollment; red) through week 96 post-enrollment (purple). Env provirus sequenced

from PBMCs in the enrollment sample are also shown (grey). The phylogeny was

inferred from protein sequences by PhyML (Guindon and Gascuel, 2003) with the HIVw

substitution model (Nickle et al., 2007). Node labels indicate at least 60% bootstrap

support. Root placement was chosen to minimize the sum of variances among within-

timepoint distances (Maljkovic Berry et al., 2009; 2007). A group of six provirus-derived

Envs was enriched for APOBEC3G hypermutations (Rose and Korber, 2000), as

identified by a square bracket and asterisk. Neutralization titers (µg/mL) from two

representative mAbs (CH14, CH16) are shown in two columns between the pixel map

and the tree for the subset of Envs assayed. Locations of V1-V5 and other Env landmarks

are shown by (faint grey boxes) and sites that contact CD4 are shown near the top of the

pixel map (pink tic marks).

p 21 of 29

B C DA

w0.2w0.5w0.7w0.9w0.10w0.20w0.23w0.25

CD4bs-directed mAbsgp120 V3 mAbs

mAbs without activity against Tier 2 viruses

w0.26w0.27

wee

k 0

w2.3w2.4w2.5w2.7w2.10w2.11w2.13w2.16w2.17w2.20

w4.8w4.9w4.11w4.12w4.13w4.19

w4.2

wee

k 2

wee

k 4

w8.3w8.4

w8.11w8.13w8.15w8.27

w8.10

wee

k 8

w16.7w16.8w16.11w16.14w16.17w16.21w16.25

wee

k 16

w12.4w12.6w12.12w12.15w12.17w12.24w12.28w12.32

wee

k 12

w24.2

w24.4w24.3

w24.6w24.9w24.11w24.14w24.16

wee

k 24

w48.22w48.23

wee

k 48

w96.13w96.12

wee

k 96

w72.25

w72.2w72.4

w48.20w48.17

w48.9w48.5

w48.13w48.15

w48.1

w48.4w48.2

w96.20

w96.37w96.30

w96.11w96.5w96.4

w72.18

w72.8w72.10

w72.21

w72.11

8.3

19

5.9

47

25

4.1

26

20

28

>50

CH48

20

>50

>50

>50

>50

29

23

>50

>50

45

>50

>50

>50

14

>50

>50

14

27

>50

17

>50

>50

5.4

13

6.8

3.3

>50

17

5.0

40

4.2

0.7

11

17

21

>50

1.7

0.6

1.5

45

37

>50

10

>50

>50

>50

>50

>50

>50

1.4

26

7.1

>50

>50

>50

>50

>50

>50

>50

>50

>50

>50

>50

>50

>50

40

5.1

46

>50

>50

>50

7.1

6.7

7.6

20

18

18

20

16

28

32

CH14

>50

18

19

31

29

48

0.7

17

36

>50

24

>50

43

13

13

>50

18

4.4

5.6

28

6.2

>50

13

3.3

13

3.3

50

29

7.7

>50

6.0

2.7

2.0

3.2

1.8

37

0.6

0.7

41

46

>50

9.3

>50

31

40

>50

8.4

38

>50

>50

>50

26

>50

21

>50

>50

>50

45

>50

18

22

31

40

>50

4.6

18

27

39

>50

35

>50

>50

>50

>50

>50

49

>50

>50

>50

>50

*F105

>50

>50

>50

>50

>50

>50

>50

>50

>50

>50

>50

>50

>50

>50

>50

>50

>50

48

>50

>50

>50

>50

>50

>50

>50

>50

>50

>50

>50

>50

>50

>50

>50

>50

>50

>50

>50

>50

>50

>50

>50

>50

>50

>50

NT

>50

>50

NT

NT

>50

>50

>50

>50

NT

>50

>50

NT

>50

NT

>50

>50

>50

>50

>50

NT

>50

>50

NT

>50

NT

>50

NT

NT

NT

wee

k 72

Lineage CH13

nAbs

Lineage CH27

11

14

16

23

11

18

25

16

26

8.5

6.1

>50

5.9

>50

8.9

14

7.8

41

20

6.5

31

7.7

21

18

15

3.8

29

17

20

12

16

18

16

1.8

3.3

1.9

9.5

28

13

10

9.8

17

2.2

7.3

6.9

12

13

1.8

18

11

2.5

3.7

5.2

0.8

1.1

9.3

3.4

0.9

>50

>50

0.7

>50

6.0

>50

2.5

1.9

2.3

1.9

31

4.8

>50

>50

5.2

1.2

0.7

3.7

>50

>50

>50

>50

>50

>50

>50 >50 >50 >50 >50

>50

>50

>50 >50 >50 >50

>50

>50

>50

>50

>50

>50

>50 >50

CH13

32

CH16

14

CH17

27

CH18

22 24 47

28 31 >50

>50 14 50

35 49 40

31

CH45

>50

46

>50

43

>50

46 45 >50 >50

32 >50

>50

>50 >50 38 >50 >50

>50 >50 >50 >50 >50

>50 >50 >50 >50 >50

>50 >50 >50 >50 >50

41 23 24 29 27

>50 >50 28 >50 >50

15 2.5 2.0 2.1 2.1

>50 32 >50 >50

>50 >50 >50

>50

>50 >50

>50 >50 >50 >50 >50

>50 45 >50 >50 >50

>50 >50 >50 >50 >50

>50 >50 >50 >50 >50

>50 >50 >50 >50 >50

>50 >50 >50 >50 >50

>50 >50 >50 >50 >50

>50 >50 >50 >50 >50

>50 9.7 28 >50

>50 >50 33

3.4

>50 >50

>50 >50 >50 >50 >50

>50 39 44 >50 >50

>50 >50 >50 >50 >50

>50 >50 >50 >50 >50

38 15 9.6 16 17

>50 15 >50 49

>50 5.1 7.0

15

17 20

37 4.6 14 13 14

>50 >50 >50 >50 >50

>50 >50 >50 >50 >50

27 6.7 7.6 12 16

>50 >50 >50 >50 >50

>50 8.9 28 >50

4.2 0.8 1.4

18

18 14

>50 7.8 7.3 29 >50

16 3.0 13 38 44

>50 >50 26 >50 >50

42 10 2.4 >50 >50

>50 >50 >50 >50 >50

15 6.1 9.5 8.02.3

>50 14 6.8 8.8 6.7

21 2.7 1.1 14 6.0

21 9.9 12 35 8.2

>50 >50 >50 >50 >50

>50 33 >50 >50 >50

>50 >50 >50 >50 >50

>50 >50 >50 >50 >50

>50 >50 >50 >50 >50

42 >50 >50 >50>50

32 >50 48 >50>50

>50>50

>50 >50 >50

29 >50 37

>50>50

23 9.1 28 >50>50

11 4.4 5.1 3.930

27 >50 22 >50>50

32 26 29 >50>50

>50 >50 >50 >50>50

>50 >50 >50 >50>50

36 >50 47 >50>50

>50 >50 >50 >50>50

>50 >50 >50 >50>50

29 >50 41 >50>50

20 >50 42 >50>50

29 >50 43 >50>50

>50>50 >50 >50 >50

50 >50 >50 >50>50

>50>50 40 >50 >50

19 36 30 >50>50

>50>50

22 >50 35

26 >50 36

>50>50

5.9 3.2 18 1249

24 >50 30 >50>50

36 >50 >50 >50>50

>50 >50 >50 >50>50

19 31 24 44>50

>50

CH27

>50

CH28

>50

>50 >50 >50

>50 >50 >50

>50 >50 >50

>50 >50 >50

>50 >50 >50

>50 >50 >50

>50 >50 >50

>50 >50 >50

>50 >50 >50

CH44

>50 >50 >50

>50 >50

>50 >50

>50 >50

>50 >50

>50 >50

>50 >50

>50 >50

>50 >50

>50 >50 NT

NT

NT

NT

NT

NT

NT

>50 >50

>50 >50

>50 >50

>50 >50

>50 >50

>50 >50

>50 >50

>50

>50

>50

>50

>50

>50

>50

NT

NT

>50 >50

>50 >50

>50 >50

>50 >50

>50 >50

>50 >50

>50 >50

>50

>50

>50

>50

>50

>50

>50

44 >50

>50 >50

>50 >50

>50 >50

>50 >50

>50 >50

>50 >50

>50

>50

>50

>50

>50

>50

>50

>50 >50

>50 >50

>50 >50

>50 >50

>50 >50

>50 >50

>50 >50

>50

>50

>50

>50

>50

>50

>50

>50 >50 >50

>50 >50

>50 >50

>50 >50

>50 >50

>50 >50

>50 >50

>50 >50

>50

>50

>50

>50

>50

>50

>50

>50 >50 >50

>50 32

>50 >50

>50

>50

>50 >50

>50 >50

>50

50

>50 >50

>50 >50

>50 >50 >50

>50 >50

>50 >50

>50

>50

>50 >50 >50

>50 >50 >50

>50 >50 >50

>50 >50 50

>50 >50

>50 >50

>50 >50

>50

>50

>50

>50 >50 >50

>50 >50

>50 >50

>50

>50

>50 >50

>50 >50

>50 >50

>50

>50

>50

>50 >50 >50

>50 >50 >50

>50 >50 >50

>50 >50 >50

>50

>50 >50 >50

50

>50

>50

>50

>50

>50

>50

>50

>50

>50

>50

>50

>50

>50

>50

>50

>50

>50

>50

>50

>50

>50

>50

>50

>50

>50

>50

>50

>50

>50

>50

>50

>50

>50

>50

>50

>50

>50

>50

>50

>50

>50

>50

>50

>50

>50

>50

>50

>50

>50

>50

>50

>50

>50

NT

>50

>50

NT

NT

>50

>50

>50

NT

>50

>50

NT

>50

NT

>50

>50

>50

>50

0.4

0.3

0.3

0.5

0.3

0.2

0.4

0.2

0.2

NT

0.17

0.06

<0.02

0.05

0.08

0.09

0.060.13

NT

>50

NT

>50

NT

NT

NT

*HJ16 *CH31 *CH106 *CH65

1.1

1.3

1.4

2.1

3.6

0.6

1.0

1.3

1.9

1.4

NT

NT

NT

NT

NT

NT

NT

NT

7.1

3.0

4.4

>50

9.5

5.0

5.9

NT

NT

1.8

3.6

3.0

1.9

1.4

6.5

6.9

22

>50

8.8

2.4

3.9

7.5

46

7.5

4.0

5.7

>50

2.8

3.3

2.3

8.3

2.5

3.1

2.6

1.0

7.9

NT

3.4

7.2

NT

NT

7.4

3.1

0.7

>50

NT

>50

7.8

NT

3.6

NT

22

4.9

>50

>50

3.5

NT

0.7

1.2

NT

0.7

NT

3.3

NT

NT

NT

>50

>50

>50

>50

>50

>50

>50

>50

>50

>50

>50

>50

>50

>50

>50

>50

>50

>50

>50

>50

>50

>50

>50

>50

>50

>50

>50

>50

>50

>50

>50

>50

>50

>50

>50

>50

>50

>50

>50

>50

>50

>50

>50

>50

>50

>50

>50

>50

>50

>50

>50

>50

>50

>50

>50

>50

>50

>50

>50

>50

>50

>50

>50

36

>50

>50

>50

>50

>50

>50

>50

>50

>50

>50

>50

>50

>50

>50

>50

>50

>50

>50

>50

>50

w0.57c

CD4bs-directed mAbsgp120 V3 mAbs

Tier 1 mAbs

>50

CH48

23

CH14

Lineage CH13

nAbs

Lineage CH27

>50

CH13

>50

CH16

>50

CH17

>50

CH18

>50

CH45

>50

CH27

>50

CH28

>50

CH44 HIVIG-C

96

w0.16c >5028 >50 >50 31 >50 >50 >50 >50 >50 103

w0.39c 279.4 >50 >50 >50 >50 >50 >50 >50 >50 104

w0.47c 4116 >50 >50 >50 >50 >50 >50 >50 >50 63

w0.46c >50>50 >50 >50 >50 >50 >50 >50 >50 >50 146

w0.8c >5040 >50 >50 >50 >50 >50 >50 >50 >50 85

w0.28c 1817 >50 >50 >50 >50 >50 >50 >50 >50 58

w0.34c 2712 >50 >50 >50 >50 >50 >50 >50 >50 70

w0.10c 154.4 45 >50 >50 25 26 >50 >50 >50 38

w0.31c >5016 >50 >50 >50 >50 >50 >50 >50 >50 83

w0.25c 3742 >50 >50 >50 >50 >50 >50 >50 >50 66

w0.17c 3418 >50 >50 >50 >50 >50 >50 >50 >50 40w0.42c >50>50 >50 >50 >50 >50 >50 >50 >50 >50 104

w0.9c 3217 >50 >50 >50 >50 >50 >50 >50 >50 81

w0.51c 187.1 >50 >50 46 >50 >50 >50 >50 >50 63

w0.45c 4915 >50 >50 >50 >50 >50 >50 >50 >50 157

w0.18c 1215 >50 >50 >50 >50 >50 >50 >50 >50 68

w0.27c 2814 >50 >50 >50 >50 >50 >50 >50 >50 73

w0.22c 2121 >50 >50 >50 >50 >50 >50 >50 >50 41

w0.5c 7.77.1 >50 >50 >50 >50 >50 >50 >50 >50 24

w0.4c 3845 >50 >50 >50 >50 >50 >50 >50 >50 43

w0.11c 107.2 28 21 20 16 11 >50 >50 >50 81

w0.53c 252.8 >50 >50 28 49 >50 >50 >50 >50 131

w0.15c 3512 >50 21 30 17 >50 >50 >50 >50 32

w0.56c 1412 37 39 10 >50 >50 28 >50 >50 43

w0.2c 1311 40 13 19 10 13 8.6 >50 49 48

w0.54c 2511 48 >50 >50 >50 >50 >50 >50 >50 81

w0.50c NTNT >50 >50 >50 >50 >50 >50 >50 >50 59

w0.55c >50>50 >50 >50 >50 >50 >50 >50 >50 >50

>50>50 >50 >50 >50 >50 >50 >50 >50 >50

142

w0.3c 80

w0.14c 2839 >50 >50 44 >50 42 >50 35 24 110

w0.29c >5036 >50 >50 >50 >50 >50 >50 0.8 0.5 42

w0.35c >50>50 >50 >50 >50 >50 >50 2.0 0.1 0.2 31

w0.24c 2410 26 18 3.7 22 38 >50 >50 >50 48

mAbIC50

(µg/mL)

21-50

neg

11-20

5.1-10

2.1-5.0

1.1-2.0

0.6-1.0

0.2-0.5

<0.2

<20

21-50

51-100

101-200

201-500

501-1000

1001-2000

2001-5000

>5000

SerumID50

(reciprocaldilution)

>50T/F

w14.8w14.10w14.12w14.21

w20.3w20.4w20.7w20.8w20.9w20.11

w20.2

wee

k 14

wee

k 20

w30.5w30.6

w30.9w30.10w30.12

w30.8

wee

k 30

w53.3w53.6w53.8w53.9

wee

k 53

w20.14w20.15w20.19w20.21w20.22w20.23

w20.13

w20.24w20.25w20.26w20.27

w78.1w78.3w78.4w78.5

wee

k 78

w78.6w78.7w78.8w78.9w78.10w78.14w78.15w78.16w78.17w78.25w78.33w78.38

0.7

CH106

0.9

0.6

0.6

0.8

>50

3.8

0.6

14

3.1

6.4

w30.13w30.15

w30.18w30.19w30.20

w30.17 5.1

9.1

0.7

0.8

5.3

0.6

w30.21w30.23

w30.25w30.26w30.27

w30.24 6.5

23

12

10

9.1

0.7

w30.28w30.31

w30.34w30.36w30.37

w30.32 0.9

>50

9.6

35

10

0.7

8.2

16

14

w53.10w53.11w53.13 8.2

20

15

w53.14w53.15w53.16 15

12

8.7

w53.17w53.19w53.22 19

2.4

12

w53.25w53.27w53.28 12

10

13

w53.29w53.31w53.32 14

0.9

18

6.5

2.2

1.0

3.2

2.9

1.2

3.7

0.6

>50

14

1.4

1.9

6.6

8.3

9.2

15

>50

0.6

7.2

3.7

8.1

9.9

8.5

11

w100.A2w100.A3w100.A4w100.A5

wee

k 10

0

w100.A6w100.A10w100.A11w100.A12w100.A13w100.B3w100.B4w100.B6w100.B7w100.B8

3.2

7.0

4.0

3.9

9.0

6.1

46

4.5

2.4

0.7

2.7

3.7

49

3.3

>50

19

>50

>50

>50

>50

27

1.2

>50

>50

>50

26

>50

>50

>50

>50

>50

>50

>50

>50

44

6.4

>50

3.0

5.2

>50

42

>50

>50

15

>50

17

9.9

>50

1.7

24

>50

41

6.4

0.7

>50

35

>50

36

>50

>50

>50

>50

>50

2.4

>50

>50

8.2

49

2.3

12

>50

>50

>50

>50

23

>50

21

>50

>50

>50

>50

>50

>50

>50

>50

>50

>50

>50

12

>50

0.4

0.30.3

0.20.4

0.3

0.40.5

0.4

0.3

0.4 0.53.7

>50

>50

47

3.9

1.1

>50

>50

>50

8.4

>50

36

>50

3.7

32

13

>50

>50

>50

>50

>50

>50

3.3

>50

>50

>50

>50

>50

3.6

>50

>50

44

41

>50

50

>50

>50

>50

>50

>50

>50

33

>50

11

>50

>50

1.8

46

>50

19

>50

>50

2.5

11

>50

43

19

>50

>50

>50

>50

42

>50

>50

>50

>50

>50

>50

>50

>50

>50

>50

45

>50

>50

46

>50

>50

17

>50

>50

39

>50

43

>50

44

28

>50

>50

>50

>50

36

20

26

38

>50

>50

22

48

>50

>50

>50

27

19

21

0.1

0.04 0.03

0.11 0.14

37

>50

34

>50

DH151 DH228

>278

64

268

12

207

99

29

28

90

>278

158

102

209

271

258

51

556

276

>278

105

119

63

34

82

57

225

62

101

147

129

97

84

125

163

138

101

45

127

>278

126

107

107

132

186

187

65

170

175

113

124

70

60

100

98

147

237

114

134

91

40

44

263

56

230

92

130

108

122

155

141

105

137

159

140

122

115

87

83

266

86

222

93

>278

196

111

186

149

183

213

93

54

70

52

180

161

HIVIG-C

1110

5

172

8

891

305

180

82

132

>1111

81

285

666

>1111

513

42

1083

209

>1111

266

262

84

15

337

232

636

166

99

228

104

87

76

191

658

144

89

25

156

157

267

132

104

193

247

87

63

370

120

328

587

248

244

248

301

>1111

333

472

455

362

199

89

365

39

286

325

341

290

264

280

404

111

185

178

153

122

168

56

59

532

227

415

308

261

558

484

106

142

337

324

426

82

188

31

441

323

SA-C8

118

37

92

17

127

73

72

64

77

240

44

50

102

251

77

129

191

64

202

72

37

63

13

73

275

410

50

88

>1111

440

386

440

721

962

>1111

786

84

1051

1016

466

267

904

794

>1111

>1111

75

268

>1111

254

131

166

24

121

405

256

159

92

87

101

161

55

153

79

128

63

464

397

645

369

722

547

522

702

358

695

308

350

156

>1111

251

492

844

>1111

1022

>1111

>1111

736

631

>1111

593

220

444

109

643

969

SA-C36

790

149

583

34

614

340

182

138

195

950

365

254

678

623

453

216

740

427

817

240

241

137

74

372

342

417

242

242

353

451

371

422

376

>1111

710

189

113

374

313

476

278

261

288

549

792

147

346

650

265

450

289

200

175

510

490

512

497

278

218

221

96

564

340

510

311

342

271

198

465

373

767

164

252

299

>1111

230

>1111

199

507

302

865

591

>1111

569

749

896

487

685

667

796

207

377

132

530

625

SA-C82

>1111

371

>1111

8

>1111

906

197

198

417

>1111

870

784

548

>1111

>1111

307

>1111

>1111

>1111

361

>1111

363

50

414

463

>1111

70 199 208 323 255

261

575

180

945

757

>1111

868

>1111

507

22

111

>1111

>1111

886

957

250

>1111

57

430

657

>1111

628

585

554

656

329

583

611

>1111

>1111

835

1109

636

394

289

>1111

140

>1111

466

>1111

>1111

>1111

>1111

>1111

>1111

>1111

>1111

919

>1111

>1111

>1111

422

>1111

439

>1111

>1111

838

>1111

>1111

410

987

>1111

508

761

534

>1111

269

1103

>1111

SA-C102

2

1A

1B

2

1B

2

1B

1B

1B

2

2

1A

2

2

2

2

2

2

2

2

2

1B

2

2

1B

2

2

2

1B

2

2

2

1B

2

2

2

2

2

2

2

2

2

2

1B

2

1B

2

2

1B

2

2

2

2

2

2

2

2

2

2

2

1B

2

2

2

2

2

2

2

2

2

2

2

2

1B

2

2

2

1B

2

2

1B

2

2

2

2

2

2

2

2

2

2

2

2

2

2

2

TierClassification

mAbs withoutactivity againstTier 2 isolates nAbs

gp120 V3mAbs

Control Plasma fromClade C Infected Persons

169

206

186

168 279 181 223 223

142

220

189 294 213 196

183

165

197

225

275

161

155 249

wk 0

210

wk 2

143

wk4

235

wk8

246

wk16

437

wk24

1210

330 384 1279

261 292 1147

253 183 895

245 292 961

373 355 1895

235 237 1079

281 233 858

343 268 1314

411 399 834

wk48

w0.2w0.5w0.7w0.9w0.10w0.20w0.23w0.25

280 183 306

274 179 284

202 146 158

418 275 510

313

wk12

575

489

296

877

372

0457 serum Heterologous serum

HIVIG-C J520b

205 112 241 328w0.26 224 306w0.27

93

172

T322

66

S423

275

P436

86

C650c

52

290 194 557 216 72

292 145 372 121 64

316 55 441 89 65

670 729 1659 739 425

399 53 269 NT 62

342 153 471 196 123

H133644

NT

286

NT 179 NT NT 25 83

NT 55 NT NT 94 61

88 49 210 NT 46 105

NT

71 <20 92 NT <20 133

NT

199 81 141 NT 23 141

NT

151 44 89 NT <20 107

NT

NT

584 256 1221 NT 184 294

92

160

107

159

182

257

160

152

198484

wee

k 0

wee

k 0

PBM

C

174 293 305

244 265

216 286

179 308

156 222

374 332

214 231

240 237

w4.2w4.8w4.9w4.11w4.12w4.13w4.19

141

144

168

184

140

240

333583

149 130 79

294

149 211

191 310 201 255 469

147 254 157 238 224

89 113 94 127 190

178 249 206 252 281

163 150 113 148 218 1150

1268

1534

1258

456

1105

1290

wee

k 2

wee

k 4

209 161 301

360 437

152 404

427 364

162 203

324 434

302 556

283 355

w8.3w8.4w8.10w8.11w8.13w8.15w8.27

476

218

220

98

324

130

258363

191 154 158

225

258 271

318 253 276 222 357

92 66 75 116 130

305 475 169 260 553

151 252 128 214 328

362 444 502 492 699 1563

1555

1281

804

1601

1448

1432

wee

k 8

218 178 347

120 186

447 468

213 199

144 219

229 235

354 413

215 294

w16.7w16.8w16.11w16.14w16.17w16.21w16.25

214

205

373

434

409

446

135509

243 287 369

166

442 546

298 237 233 283 399

104 96 139 112 160

110 100 86 96 128

286 286 309 504 1235

81 106 102 121 389 918

684

854

1065

1207

1935

1435

wee

k 16

490 356 764

299 353

178 426

325 489

301 363

259 244

331 435

500 662

w12.4w12.6w12.12w12.15w12.17w12.24w12.28

150

286

280

210

199

446

2781350

260 248 266

894

286 647

194 225 253 291 748

117 116 118 216 214

246 252 181 301 424

342 261 209 325 385

197 205 238 255 393 787

1299

1746

546

1431

22892084

184 239 217 231 363w12.32 268262190 1276

wee

k 12

247 221

471 293

224 245

193 152

145 148

207 205

324 244

w24.2w24.3w24.4w24.6w24.9w24.11w24.14

227

238

474

190

121

135

252

326 323 239 370 547

512 563 411 516 429

273 293 267 404 787

202 251 201 228 316

116 215 137 266 240

131 128 95 189 235

232 294 210 426 832

185 254 160 158 406

122 94 154 271

731

3863547

999

497

253

783

202 219w24.16 1931469

wee

k 24

83 64

101 72

70 54

w48.1w48.2w48.4

123

127

112

94

76 47 101 7565

85 50 102 9993

95

100

65

wee

k 48

91 37 91 95 81 <20

102 29

85 24

w96.4w96.5w96.11

116

131

134

90

16993

38 32 55

76 58 105

7470

<20

40

<20

wee

k 96

1404 639 4343 723 726

127 28

1160 961

w72.2w72.4

w72.18w72.21w72.25

256

208

722

1116

103 109 166 221129

473 262 399 586529

2637

72 42 49 100 97 60 12497 74

185 96 99 164 214 124 603152 131

39

417

1249

1829

1736

1158

1908

825

1819

20801993

1632

wk72

1350

1936

1935

1746

334

1084

1629

1269

20241788

1463

1550

22211610

26351236

710

478

1124

1634

3559

1542

23722045540

1653

299743211385

658

3120553

548

271

451

2643877

260

402

217

91

110

75

938

72

160

156

599

1815

2712

29442754

2324 1318 1696 2512 1859 25803230 2087 1704

w0.29cw0.35c 2086 1945 3062

1321

1554

NT

NT

NT

1447

NT

2377

21691263

1723

930

444031672436

wk96

1096

1800

1805

1841

1534

1620

1652

376 339 413

435 914

1327 621

259 352

821 894

180 186

272 269

259 244

471 481

w2.4w2.5w2.7w2.10w2.11w2.13w2.16w2.17w2.20

116

181

276

171

193

343

193

156

292262 331 226 481 727710

140 197 160 382 1431289

228 324 271 1267610

177 199 204 280 1323249

211 217 207 680 899751

235 254 324 273 1389234

413 701 1120 1262 28751443

212 287 305 461 3144542

230 294 307 499 403 285

295

1653

1614

1856

1605

1658

566

3747354

137 167 170 213 242 296 1514w2.3 196208 1075 942

1140

1666

1938

1433

1637

1401

728

3048

37241426

1973

40931278

21972374631

1066

1589

1490

1560

3881987

1666

1339

927

34331183

506

284

571

1305

3562

26322272

85 50 88 126 61 41w48.5 163101 61 271

92 61

131 103

w48.17w48.20

105

159

82 62 88 8876

115 65 147 21290

87

108

156

277

109 73 125 115 101 77w48.13 85100 111 237 1101

105 77 121 143 161 121w48.9 76109 139 219 887

205 52w48.15 14572 54 60 7962 135 288

399

104 70 142 180 117 75

76 67

w48.22w48.23

87

99

104

86 75 148 25380

147

90

1228

755

3227

9174

8505

53456216

6359

5559

71877357

16,409

8227

5757

16,444

9368

21

46

33

42 34 31 51 90 87

97 63

70 109

w96.12

w96.30w96.37

195

101

324

58

12759

109 52 71

53 45 89

11799

83

69

103

74

98

95

128

85

86 51w96.20 69367102 88 52 53 71 55 86

90 53w96.13 1079896 81 43 83 65 114 108

151

4318

71

352

1334

136 31w72.8 182136 68 127 132151 41 187 471

207 91w72.10 24093 60 46 10190 62 60 81

119 22w72.11 273149 76 180 293121 29 180 511

1699

wee

k 72

334

NT <20 123 NT <20 50

NT 30 NT NT <20 69

68

144

NT 43 NT NT <20

NT 125 NT NT <20

NT 53 NT NT 28 52

p 22 of 29

Figure S3, related to Figures 3 and 4. Neutralization of autologous viruses from CH0457 and CH505.

A: Antibodies were tested against a panel of 84 pseudoviruses amplified from plasma from participant

CH0457 that spanned the study period. Antibodies from lineage CH13 neutralized 52/84 (62%) of isolates

tested and mAbs from this lineage were active against at least one isolate from each of the time points tested.

For mAbs from lineage CH13, neutralization titers ranged from 0.8-50 µg/mL. In contrast, mAbs from lineage

CH27 neutralized only 5/84 (6%) of isolates; neutralization titers ranged from 44-50 µg/mL. Control mAbs are

shown with asterisks above their names; narrow neutralizing CD4bs mAb F105 (Posner et al., 1991) weakly

neutralized 2/72 (2.8%) while bnAb HJ16 (Corti et al., 2010) potently neutralized 5/72 (6.9%) of pseudoviruses.

Anti-HIV-1 bnAbs CH31 (Wu et al., 2011) and CH106 (Liao et al., 2013) neutralized 73/84 (87%) and 55/62

(89%) respectively with titers ranging from < 0.02 to 46 µg/mL, while anti-influenza bnAb CH65 (Whittle et

al., 2011) weakly neutralized a single isolate (w72.4). Testing of the autologous viruses by these and additional

samples (Fig. S4A) was used to classify the viruses for neutralization sensitivity (Tier Classification).

B: HIV-1 Env sequences were amplified by single genome amplification from week 0 PBMC. Env

sequences from plasma are indicated by a “p”; cell derived sequences are indicated by a “c”. Pseudoviruses

made from these Env sequences were tested against the panel of mAbs isolated from CH0457. Of the 34

pseudoviruses tested, 28/34 (82%) were sensitive to the V3 mAbs CH14 and CH48 and 11/34 (32%) were

sensitive to the CD4bs-directed lineage CH13 mAbs. Only 5/34 (15%) of pseudoviruses were sensitive to the

nAb lineage CH27 mAbs; of these, the two Envs most distant in the phylogenetic tree from the week 0 plasma

Envs, w0.29c and w0.35c, were the most sensitive to neutralization (IC50 range 0.1-2.0 µg/mL).

C: Antibodies DH151 and DH228 were tested against a panel of 96 autologous pseudoviruses from

participant CH505. Tier 1 V3 mAbs neutralized 45/96 (47%, range 50-0.03µg/mL) of the autologous viruses.

Like CH0457 tier 1 V3 abs, mAbs DH151 and DH228 neutralized 7/96 (7.3 %) viruses at ≤ 2µg/mL. Testing of

the autologous viruses against HIVIG-C and a panel of well characterized sera from clade C infected

participants (SA-C8, SA-C36, SA-C82, SA-C102) (Fig. S4C) was used to classify the viruses for neutralization

sensitivity (Tier Classificiation).

D: Serum from participant CH0457 spanning the study period was tested against 84 autologous virus

isolates from the same time period and two autologous viruses isolated from PBMC. Control HIVIG-C pooled

antibodies are shown on the right. Serum antibodies from CH0457 neutralized autologous viruses from all early

time points, and serum from weeks 48, 72, and 96 showed greater potency against autologous viruses. Virus

isolates from week 96 were resistant to plasma from all time points, suggesting that a new escape event may

have occurred during the later study period. Six viruses were tested for sensitivity to a panel of five well

characterized serum samples; these viruses demonstrated an intermediate sensitivity to these sera, consistent

with an intermediate phenotype (tier 1b). Companion data for these sera against other HIV-1 strains is shown in

Fig. S4B.

p 23 of 29

A

mAbIC50

(µg/mL)

21-50

neg

11-20

5.1-10

2.1-5.0

1.1-2.0

0.6-1.0

0.2-0.5

<0.2

<20

21-50

51-100

101-200

201-500

501-1000

1001-2000

2001-5000

>5000

SerumID50

(reciprocaldilution)

w0.23

CD4bs-directed mAbsgp120 V3 mAbs

mAbs without activity against Tier 2 viruses

w2.4w2.7w2.16

w4.9

w16.17

w12.4

w12.24w12.32

w24.4w24.11

week 24

week 0PBMC

w72.18

26

CH48

>50

>50

>50

3.3

>50

1.7

1.5

>50

5.1

20

CH14

19

0.7

43

>50

3.3

37

0.6

>50

40

>50

*F105

>50

>50

>50

>50

>50

>50

>50

>50

>50

NT

NT

*2219

21

>25

>25

>25

18

>25

*2557

14

>24

>24

7.8

11

>24

*3074

5.8

3.6

24

2.7

4.5

18

*3869

6.9

3.9

25

2.3

4.8

>25

*447-52D

>25

>25

>25

>25

>25

>25

*838-12D

>18

15

>18

>18

16

>18

*F39F

>50

>50

>50

>50

>50

>50

*19b

>50

>50

>50

>50

>50

>50

*CH22

>25

>25

>25

>25

>25

>25

*CH23 *654-30D

*1008-30D

*729-30D

*1570D

>50

>50 6.4 15 4.3 14

>25 >25 16 15 >25 NT NT NT NT NT

>25 >25 >19 >25 >25 >25 >50 >50 >25 >50

>25 >25 14 21 >25 24 >50 >50 >25 >50

>25 >25 >25 >25

>50>25 >25 >25 >25

>50>25 >25 >25 >25

>25 >25 21 >25 >25 NT NT NT NT NT >25 >25 >25 >25

>25 >25 >25 >25 >25 NT NT NT NT NT >25 >25 >25 >25

14 9.1 3.7 1.9 >25 NT NT NT NT NT 13 >25 13 13

>25 4.8 0.45 1.9 >25 11 >50 >50 >25 >50 >25 >25 7.4 >25

>25 >25 18 23 >25 NT NT NT NT NT >25 >25 23 22

>25 >25 >25 >25

>25 >25 >25 >25

>25 >25 22 >25

>25 >25 15 >25

>25 >25 >25 >25

>50

>50

>50

>50

week 72

week 16

week 4

week 0

week 12

week 2

Lineage CH13

nAbs

Lineage CH27 Heterologous Serum

18

>50

14

6.5

1.9

13

7.3

12

1.8

3.7

>50

>50 >50 >50 >50

CH13 CH16 CH17 CH18 CH45

>50

>50 >50 >50 >50 >50

15 2.5 2.0 2.1 2.1

>50 >50 >50 >50 >50

>50 >50 >50 >50 >50

37 4.6 14 13 14

4.2 0.8 1.4 18 14

>50 >50 >50 >50 >50

15 6.1 9.5 8.02.3

21 2.7 1.1 14 6.0

>50 >50 >50 >50 >50

5.9 3.2 18 1249

CH27 CH28

>50 >50 >50

CH44

>50 >50

>50 >50

>50 >50 NT

NT

>50 >50 >50

NT

>50 >50 >50

>50 >50

>50 >50

>50

>50

>50 >50 >50

>50 >50

>50 >50

>50

>50

>50 >50 >50

>50

>50

>50

>50

>50

>50

>50

>50

>50

>50

NT

0.4

0.3

0.3

0.17

0.05

NT

*HJ16 *CH31 *CH106 *CH65

1.0

4.0>50 >50

0.96>50 >50

NT

NT

3.0

NT

3.9

7.5

3.3

8.3

3.1

NT

NT

>50

>50

>50

>50

>50

>50

>50

>50

>50

>50

>50

>50

>50

>50

J520b T322 S423 P436 C650c

670 729 1659 739 425

H133644

NT

399 53 269 NT 62 286

NT 179 NT NT 25 83

NT 55 NT NT 64 61

88 49 210 NT 46 105

71 <20 92 NT <20 133

199 81 141 NT 23 141

NT 30 NT NT <20 69

584 256 1221 NT 184 294

NT

NT

NT

NT

NT

342 153 471 196 123

172 66 275 86 52

290 194 557 216 72

292 145 372 121 64

316 55 441 89 65

1B

2

2

2

1B

1B

1B

1B

1B

2

2

2

w16.8 5.07.7 >5016 12 6.7 5.0 >25 NT NT NT NT NT 7.9 14 6.1 15 1.827 6.7 7.6 12 16 >50 >50 >50 >50 >50 >50 151 44 89 NT <20 107

w96.13 >50>50 >5022 20 11 17 >25 >25 >50 >50 >25 >50 >25 >25 24 >25week 96

3.4>50 29 >50 37 >50 >50 >50 >50 >50 7.4 >50 NT 53 NT NT 28 52

w96.5 >5022 >50>25 23 8.7 20 >25 >25 >50 >50 >25 >50 >25 >25 >25 >25 0.7>50 26 >50 36 >50 >50 >50 >50 0.06 0.7 >50 NT NT NT NT NT NT

2

2

w24.2w24.3

0.6

0.30.06>50

>50

5.6

12

2.5

1.3

1.4

0.512.2

1.1

>25

>25

6.6

4.4

>50

>50

>50

>50

>25

>25

16 >25 11 22

24 >25 >25 >25

>50

>50

13

18

>50 14 6.8 8.8 6.7

21 9.9 12 35 8.2

>50 >50

>50 >50

>50

>50

>50

>50

0.7 2.5

2.6

>50

>50

68

144

NT 43 NT NT <20

NT 125 NT NT <20

2

2

2

2

w12.15 >5024 24 1.5 11 >25 21 >50 >50 >25 >50 17 21 10 19 1716 3.0 13 38 44 >50 >50 >50 >50112.0 >50 >50 NT <20 123 NT <20 50 2

TierClassification

w0.29c >5036 >50 >50 >50 >50 >50 >50 0.8 0.5

w0.35c >50>50 >50 >50 >50 >50 >50 2.0 0.1 0.2

6535.3B clade

Tier 1b

J520b

160

T322

176

S423

678

P436

362

20975MN.3B clade

Tier 1a

>43740 >43740 26862 14280

C650c

301

QH0692.42

PVO.4

SC422661.8

B clade

Tier 2

48

<20

50

36

<20

39

84

96

84

49

63

49

37

42

37

Heterologous SerumB C

w14.12

w20.3w20.11

w30.13w30.28

w53.6

w20.15w20.24

w78.1w78.3

week 78

week 20

week 30

week 53

week 14

w78.15w78.16

1.2

>50

>50

>50

1.8

3.3

>50

1.7

0.7

>50

2.4

>50

2.5

3.6

>50

>50

>50

0.4 0.53.7

1.1

>50

0.04 0.03

DH151 DH228

12

90

276

>278

119

130

98

97

138

101

107

107

HIVIG-C

8

132

209

>1111

262

341

301

87

144

89

132

104

SA-C8

17

77

64

202

37

464

405

386

>1111

786

267

904

SA-C36

34

195

427

817

241

342

510

371

710

189

278

261

SA-C82

8

417

>1111

>1111

>1111

>1111

611

757

507

22

957

250

SA-C102

2

1B

1A

2

2

2

2

1B

2

2

2

1B

TierClassification

CH106

0.8

>50

0.6

14

2.9

0.6

1.4

1.9

>50

0.6

0.4

0.7

nAbs

Control Plasma fromClade C Infected Persons (IC50)

CD4bs-directed mAbsgp120 V3 mAbs

mAbs without activity against Tier 2 viruses

135.2 42

*CH18*2219*CH48*CH14 *2557 *3074 *3869 *447-52D

*654-30D

*1008-30D

*729-30D

*1570D

>25 >25 19 >25 >25 >25 >25 >25 >25

Heterologous Serum (ID50)

J520b T322 S423 P436 C650c H133644

376 NT 663 406 10 NT

>5013 49>25 >25 >25 >25 >25 >25 >25 >25 >25 348 167 269 243 10 NT

>507.5 49>25 >25 13 >25 >25 >25 >25 >25 >25 494 10 537 223 26 NT

>50>50 >50>25 >25 >25 >25 >25 >25 >25 >25 >25 35 10 472 30 10 NT

<0.02<0.02 2.1>25 >25 <0.02 0.3 15 7.1 >25 2.7 2.6 6237 5840 5499 5367 1147 NT

1.50.03 40>25 >25 0.4 >25 >25 >25 >25 >25 >25 174 58 645 132 24 NT

>504.7 >50>25 >25 >25 >25 >25 >25 >25 >25 >25 561 40 464 111 21 NT

>504.7 44>25 >25 8.9 >25 25 >25 >25 >25 >25 291 43 506 187 60 NT

w100.A13week 100 0.11 0.14 52 31 109 132 269 23.70.450.17 6.5>25 >25 0.2 >25 >25 >25 >25 2.8 10 NT <20 546 NT <20 33

2.10.4 >50>25 >25 0.8 >25 >25 >25 >25 23 >25 NT 74 907 NT <20 71

334.6 >50>25 >25 9.4 >25 >25 >25 >25 >25 >25 NT <20 546 NT <20 33

w30.5w30.12 3.0

>50

2.3

>50

57

101

232

99

275

88

342

242

463

575 2

1B

6.4

0.3

>5012 >50>25 >25 20 >25 >25 >25 >25 22 >25 299 10 517 160 10 NT

6.70.4 28>25 >25 1.5 >25 >25 >25 >25 23 >25 446 257 3146 506 40 NT

>5011 47>25 >25 >25 >25 >25 >25 >25 >25 >25 209 24 1308 75 10 NT

240.7 26>25 >25 7.0 >25 >25 >25 >25 15 18 358 303 602 NT 51 315

p 24 of 29

Figure S4, related to Figures 2, 3, and 4. Neutralization of mAbs and serum against

autologous viruses from CH0457 and CH505, and heterologous viruses: extended panels.

A: Data shown here include some neutralization data shown in Fig. 4A and Fig.

S3. We selected viruses reflective of different portions of the entire virus phylogram (Fig.

4; Fig. S2), selecting ten that were relatively resistant to autologous mAbs CH14, CH48,

and those from the CH13 lineage; and ten additional viruses that were more sensitive to

those mAbs. These twenty viruses were tested against a panel of V3 and CD4bs mAbs

with restricted neutralization profiles (Gorny et al., 1997; 2009; Jeffs et al., 2001;

Montefiori et al., 2012; Moore et al., 1994; Pantophlet et al., 2008; Swetnam et al., 2010)

and a panel of well-characterized HIV-1-infected patient serum samples. Antibodies

indicated by asterisks are heterologous mAbs from the references listed above as well as

other bnAbs; mAb CH65 is a negative control mAb against influenza. These

neutralization profiles were used to classify the pseudoviruses for neutralization

sensitivity.

B: Five HIV-1 isolates were tested against five well characterized serum samples.

The canonical tier 1 virus MN.3 was very sensitive to the serum samples. The

intermediate sensitive virus 6535.3 was more resistant than MN.3 but not as resistant as

the three tier 2 viruses.

C: Data shown in Fig. 4C are here supplemented with additional neutralization

data. As for (A) above, we selected viruses that were reflective of different portions of

the virus phylogram, and selected that were relatively resistant to autologous mAbs

DH151 and DH228 as well as viruses that were more sensitive to those mAbs. These

fifteen pseudoviruses were tested against a panel of mAbs and well characterized HIV-1-

infected patient serum samples. Antibodies indicated by asterisks are heterologous mAbs

from the references listed in the legend to (A) above as well as other bnAbs. Isolates that

were sensitive to the autologous V3 mAbs DH151 and CH228 were also mostly sensitive

to heterologous mAbs. Sensitivity to the mAbs and sera were used to refine the tier

classification shown in the rightmost column.

p 25 of 29

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