Upload
donguyet
View
215
Download
1
Embed Size (px)
Citation preview
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
2 of 29
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.
3 of 29
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
4 of 29
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.
5 of 29
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−
6 of 29
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
7 of 29
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′-
8 of 29
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
9 of 29
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
10 of 29
(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
11 of 29
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
12 of 29
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
13 of 29
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.
14 of 29
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%
15 of 29
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.
16 of 29
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.
p 17 of 29
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
References
Alam, S.M., Scearce, R.M., Parks, R.J., Plonk, K., Plonk, S.G., Sutherland, L.L., Gorny, M.K., Zolla-Pazner, S., VanLeeuwen, S., Moody, M.A., et al. (2008). Human immunodeficiency virus type 1 gp41 antibodies that mask membrane proximal region epitopes: antibody binding kinetics, induction, and potential for regulation in acute infection. J Virol 82, 115–125.
Balla-Jhagjhoorsingh, S.S., Corti, D., Heyndrickx, L., Willems, E., Vereecken, K., Davis, D., and Vanham, G. (2013). The N276 glycosylation site is required for HIV-1 neutralization by the CD4 binding site specific HJ16 monoclonal antibody. PLoS ONE 8, e68863.
Balla-Jhagjhoorsingh, S.S., Willems, B., Heyndrickx, L., Heyndrickx, L., Vereecken, K., Janssens, W., Seaman, M.S., Corti, D., Lanzavecchia, A., Davis, D., et al. (2011). Characterization of neutralizing profiles in HIV-1 infected patients from whom the HJ16, HGN194 and HK20 mAbs were obtained. PLoS ONE 6, e25488.
Bonsignori, M., Hwang, K.-K., Chen, X., Tsao, C.-Y., Morris, L., Gray, E., Marshall, D.J., Crump, J.A., Kapiga, S.H., Sam, N.E., et al. (2011). Analysis of a clonal lineage of HIV-1 envelope V2/V3 conformational epitope-specific broadly neutralizing antibodies and their inferred unmutated common ancestors. J Virol 85, 9998–10009.
Bonsignori, M., Montefiori, D.C., Wu, X., Chen, X., Hwang, K.-K., Tsao, C.-Y., Kozink, D.M., Parks, R.J., Tomaras, G.D., Crump, J.A., et al. (2012). Two distinct broadly neutralizing antibody specificities of different clonal lineages in a single HIV-1-infected donor: implications for vaccine design. J Virol 86, 4688–4692.
Bonsignori, M., Wiehe, K., Grimm, S.K., Lynch, R., Yang, G., Kozink, D.M., Perrin, F., Cooper, A.J., Hwang, K.-K., Chen, X., et al. (2014). An autoreactive antibody from an SLE/HIV-1 individual broadly neutralizes HIV-1. J Clin Invest 124, 1835–1843.
Corti, D., Langedijk, J.P.M., Hinz, A., Seaman, M.S., Vanzetta, F., Fernandez-Rodriguez, B.M., Silacci, C., Pinna, D., Jarrossay, D., Balla-Jhagjhoorsingh, S., et al. (2010). Analysis of Memory B Cell Responses and Isolation of Novel Monoclonal Antibodies with Neutralizing Breadth from HIV-1-Infected Individuals. PLoS ONE 5, e8805.
Désormeaux, A., Coutu, M., Medjahed, H., Pacheco, B., Herschhorn, A., Gu, C., Xiang, S.-H., Mao, Y., Sodroski, J., and Finzi, A. (2013). The highly conserved layer-3 component of the HIV-1 gp120 inner domain is critical for CD4-required conformational transitions. J Virol 87, 2549–2562.
Ewing, B., and Green, P. (1998). Base-calling of automated sequencer traces using phred. II. Error probabilities. Genome Res. 8, 186–194.
Ewing, B., Hillier, L., Wendl, M.C., and Green, P. (1998). Base-calling of automated
p 26 of 29
sequencer traces using phred. I. Accuracy assessment. Genome Res. 8, 175–185.
Felsenstein, J. (2009). PHYLIP (Phylogeny Inference Package).
Gao, F., Bonsignori, M., Kumar, A., Xia, S.-M., Lu, X., Cai, F., Hwang, K.-K., Song, H., Zhou, T., Lynch, R.M., et al. (2014). Cooperation of B Cell Lineages in Induction of HIV-1-Broadly Neutralizing Antibodies. Cell 158, 481–491.
Gorny, M.K., VanCott, T.C., Hioe, C., Israel, Z.R., Michael, N.L., Conley, A.J., Williams, C., Kessler, J.A., Chigurupati, P., Burda, S., et al. (1997). Human monoclonal antibodies to the V3 loop of HIV-1 with intra- and interclade cross-reactivity. 159, 5114–5122.
Gorny, M.K., Wang, X.-H., Williams, C., Volsky, B., Revesz, K., Witover, B., Burda, S., Urbanski, M., Nyambi, P., Krachmarov, C., et al. (2009). Preferential use of the VH5-51 gene segment by the human immune response to code for antibodies against the V3 domain of HIV-1. Mol Immunol 46, 917–926.
Gray, E.S., Madiga, M.C., Hermanus, T., Moore, P.L., Wibmer, C.K., Tumba, N.L., Werner, L., Mlisana, K., Sibeko, S., Williamson, C., et al. (2011a). The Neutralization Breadth of HIV-1 Develops Incrementally over Four Years and Is Associated with CD4+ T Cell Decline and High Viral Load during Acute Infection. J Virol 85, 4828–4840.
Gray, E.S., Moody, M.A., Wibmer, C.K., Chen, X., Marshall, D., Amos, J., Moore, P.L., Foulger, A., Yu, J.S., Lambson, B., et al. (2011b). Isolation of a monoclonal antibody that targets the alpha-2 helix of gp120 and represents the initial autologous neutralizing-antibody response in an HIV-1 subtype C-infected individual. J Virol 85, 7719–7729.
Gray, E.S., Taylor, N., Wycuff, D., Moore, P.L., Tomaras, G.D., Wibmer, C.K., Puren, A., DeCamp, A., Gilbert, P.B., Wood, B., et al. (2009). Antibody specificities associated with neutralization breadth in plasma from human immunodeficiency virus type 1 subtype C-infected blood donors. J Virol 83, 8925–8937.
Guindon, S., and Gascuel, O. (2003). A simple, fast, and accurate algorithm to estimate large phylogenies by maximum likelihood. Syst. Biol. 52, 696–704.
Harris, R.S., and Liddament, M.T. (2004). Retroviral restriction by APOBEC proteins. Nat Rev Immunol 4, 868–877.
Jeffs, S.A., Gorny, M.K., Williams, C., Revesz, K., Volsky, B., Burda, S., Wang, X.H., Bandres, J., Zolla-Pazner, S., and Holmes, H. (2001). Characterization of human monoclonal antibodies selected with a hypervariable loop-deleted recombinant HIV-1(IIIB) gp120. Immunol. Lett. 79, 209–213.
Jiang, C., Parrish, N.F., Wilen, C.B., Li, H., Chen, Y., Pavlicek, J.W., Berg, A., Lu, X., Song, H., Tilton, J.C., et al. (2011). Primary infection by a human immunodeficiency virus with atypical coreceptor tropism. J Virol 85, 10669–10681.
p 27 of 29
Kepler, T.B., Sample, C., Hudak, K., Roach, J., Haines, A., Walsh, A., and Ramsburg, E.A. (2010). Chiropteran types I and II interferon genes inferred from genome sequencing traces by a statistical gene-family assembler. BMC Genomics 11, 444.
Kirchherr, J.L., Lu, X., Kasongo, W., Chalwe, V., Mwananyanda, L., Musonda, R.M., Xia, S.-M., Scearce, R.M., Montefiori, D.C., Haynes, B.F., et al. (2007). High throughput functional analysis of HIV-1 env genes without cloning. Journal of Virological Methods 143, 104–111.
Kwong, P.D., Wyatt, R., Majeed, S., Robinson, J., Sweet, R.W., Sodroski, J., and Hendrickson, W.A. (2000). Structures of HIV-1 gp120 envelope glycoproteins from laboratory-adapted and primary isolates. Structure 8, 1329–1339.
Liao, H.-X., Levesque, M.C., Nagel, A., Dixon, A., Zhang, R., Walter, E., Parks, R., Whitesides, J., Marshall, D.J., Hwang, K.-K., et al. (2009). High-throughput isolation of immunoglobulin genes from single human B cells and expression as monoclonal antibodies. Journal of Virological Methods 158, 171–179.
Liao, H.-X., Lynch, R., Zhou, T., Gao, F., Alam, S.M., Boyd, S.D., Fire, A.Z., Roskin, K.M., Schramm, C.A., Zhang, Z., et al. (2013). Co-evolution of a broadly neutralizing HIV-1 antibody and founder virus. Nature 496, 469–476.
Maljkovic Berry, I., Athreya, G., Kothari, M., Daniels, M., Bruno, W.J., Korber, B., Kuiken, C., Ribeiro, R.M., and Leitner, T. (2009). The evolutionary rate dynamically tracks changes in HIV-1 epidemics: application of a simple method for optimizing the evolutionary rate in phylogenetic trees with longitudinal data. Epidemics 1, 230–239.
Maljkovic Berry, I., Ribeiro, R., Kothari, M., Athreya, G., Daniels, M., Lee, H.Y., Bruno, W., and Leitner, T. (2007). Unequal evolutionary rates in the human immunodeficiency virus type 1 (HIV-1) pandemic: the evolutionary rate of HIV-1 slows down when the epidemic rate increases. J Virol 81, 10625–10635.
Montefiori, D.C. (2005). Evaluating neutralizing antibodies against HIV, SIV, and SHIV in luciferase reporter gene assays. Curr Protoc Immunol Chapter 12, Unit12.11.
Montefiori, D.C., Karnasuta, C., Huang, Y., Ahmed, H., Gilbert, P., de Souza, M.S., mclinden, R., Tovanabutra, S., Laurence-Chenine, A., Sanders-Buell, E., et al. (2012). Magnitude and breadth of the neutralizing antibody response in the RV144 and Vax003 HIV-1 vaccine efficacy trials. Journal of Infectious Diseases 206, 431–441.
Moody, M.A., Trama, A.M., Bonsignori, M., Tsao, C., Drinker, M.S., Gurley, T.C., Amos, J.D., Eudailey, J.A., Armand, L.C., Parks, R., et al. (2012a). Antibody lineages with evidence of somatic hypermutation persisting for >4 years in a South African subject with broad neutralizing activity. Retrovirology 9, P85.
Moody, M.A., Yates, N.L., Amos, J.D., Drinker, M.S., Eudailey, J.A., Gurley, T.C., Marshall, D.J., Whitesides, J.F., Chen, X., Foulger, A., et al. (2012b). HIV-1 gp120 vaccine induces affinity maturation in both new and persistent antibody clonal lineages. J
p 28 of 29
Virol 86, 7496–7507.
Moody, M.A., Zhang, R., Walter, E.B., Woods, C.W., Ginsburg, G.S., McClain, M.T., Denny, T.N., Chen, X., Munshaw, S., Marshall, D.J., et al. (2011). H3N2 influenza infection elicits more cross-reactive and less clonally expanded anti-hemagglutinin antibodies than influenza vaccination. PLoS ONE 6, e25797.
Moore, J.P., Sattentau, Q.J., Wyatt, R., and Sodroski, J. (1994). Probing the structure of the human immunodeficiency virus surface glycoprotein gp120 with a panel of monoclonal antibodies. J Virol 68, 469–484.
Morris, L., Chen, X., Alam, M., Tomaras, G., Zhang, R., Marshall, D.J., Chen, B., Parks, R., Foulger, A., Jaeger, F., et al. (2011). Isolation of a human anti-HIV gp41 membrane proximal region neutralizing antibody by antigen-specific single B cell sorting. PLoS ONE 6, e23532.
Nickle, D.C., Heath, L., Jensen, M.A., Gilbert, P.B., Mullins, J.I., and Kosakovsky Pond, S.L. (2007). HIV-specific probabilistic models of protein evolution. PLoS ONE 2, e503.
Pantophlet, R., Wrin, T., Cavacini, L.A., Robinson, J.E., and Burton, D.R. (2008). Neutralizing activity of antibodies to the V3 loop region of HIV-1 gp120 relative to their epitope fine specificity. Virology 381, 251–260.
Posner, M.R., Hideshima, T., Cannon, T., Mukherjee, M., Mayer, K.H., and Byrn, R.A. (1991). An IgG human monoclonal antibody that reacts with HIV-1/GP120, inhibits virus binding to cells, and neutralizes infection. 146, 4325–4332.
Rose, P.P., and Korber, B.T. (2000). Detecting hypermutations in viral sequences with an emphasis on G --> A hypermutation. Bioinformatics 16, 400–401.
Salazar-Gonzalez, J.F., Salazar, M.G., Keele, B.F., Learn, G.H., Giorgi, E.E., Li, H., Decker, J.M., Wang, S., Baalwa, J., Kraus, M.H., et al. (2009). Genetic identity, biological phenotype, and evolutionary pathways of transmitted/founder viruses in acute and early HIV-1 infection. J Exp Med 206, 1273–1289.
Smith, T.F., and Waterman, M.S. (1981). Identification of common molecular subsequences. J Mol Biol 147, 195–197.
Swetnam, J., Shmelkov, E., Zolla-Pazner, S., and Cardozo, T. (2010). Comparative magnitude of cross-strain conservation of HIV variable loop neutralization epitopes. PLoS ONE 5, e15994.
Tomaras, G.D., Binley, J.M., Gray, E.S., Crooks, E.T., Osawa, K., Moore, P.L., Tumba, N., Tong, T., Shen, X., Yates, N.L., et al. (2011). Polyclonal B cell responses to conserved neutralization epitopes in a subset of HIV-1-infected individuals. J Virol 85, 11502–11519.
Tomaras, G.D., Yates, N.L., Liu, P., Qin, L., Fouda, G.G., Chavez, L.L., Decamp, A.C.,
p 29 of 29
Parks, R.J., Ashley, V.C., Lucas, J.T., et al. (2008). Initial B-cell responses to transmitted human immunodeficiency virus type 1: virion-binding immunoglobulin M (IgM) and IgG antibodies followed by plasma anti-gp41 antibodies with ineffective control of initial viremia. J Virol 82, 12449–12463.
Volpe, J.M., Cowell, L.G., and Kepler, T.B. (2006). SoDA: implementation of a 3D alignment algorithm for inference of antigen receptor recombinations. Bioinformatics 22, 438–444.
Whittle, J.R.R., Zhang, R., Khurana, S., King, L.R., Manischewitz, J., Golding, H., Dormitzer, P.R., Haynes, B.F., Walter, E.B., Moody, M.A., et al. (2011). Broadly neutralizing human antibody that recognizes the receptor-binding pocket of influenza virus hemagglutinin. Proceedings of the National Academy of Sciences 108, 14216–14221.
Wrammert, J., Smith, K., Miller, J., Langley, W.A., Kokko, K., Larsen, C., Zheng, N.-Y., Mays, I., Garman, L., Helms, C., et al. (2008). Rapid cloning of high-affinity human monoclonal antibodies against influenza virus. Nature 453, 667–671.
Wu, X., Zhou, T., Zhu, J., Zhang, B., Georgiev, I., Wang, C., Chen, X., Longo, N.S., Louder, M., McKee, K., et al. (2011). Focused evolution of HIV-1 neutralizing antibodies revealed by structures and deep sequencing. Science 333, 1593–1602.
Zárate, S., Pond, S.L.K., Shapshak, P., and Frost, S.D.W. (2007). Comparative study of methods for detecting sequence compartmentalization in human immunodeficiency virus type 1. J Virol 81, 6643–6651.