JVI Accepts, published online ahead of print on 5 November 2014 J. Virol. doi:10.1128/JVI.02905-14 Copyright © 2014, American Society for Microbiology. All Rights Reserved.
1
Two classes of broadly neutralizing antibodies within a single lineage directed to
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the high-mannose patch of HIV Envelope
3 4
Katie J. Doores,1 Leopold Kong,3,4,5,6 Stefanie A. Krumm,1 Khoa M. Le,2,3,4 Devin
5
Sok,2,3,4 Uri Laserson,7 Fernando Garces,3,4,5,6 Pascal Poignard, 2,3,4 Ian A. Wilson,3,4,5,6
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Dennis R. Burton,2,3,4
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1
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Medicine, Guy's Hospital, London, United Kingdom. 2Department of Immunology and
Department of Infectious Diseases, King's College London Faculty of Life Sciences and
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Microbial Science, 3IAVI Neutralizing Antibody Center, The Scripps Research Institute,
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4
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Integrative Structural and Computational Biology and 6Skaggs Institute for Chemical
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Biology, The Scripps Research Institute, La Jolla, California, United States of America.
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7
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Health Sciences and Technology, Cambridge, MA.
Center for HIV/AIDS Vaccine Immunology and Immunogen Discovery, 5Department of
Department of Genetics, Harvard Medical School, Boston, MA. 8Harvard-MIT Division of
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Running title: HIV bnAb evolution in donor 36
18 19
Address correspondence to Katie J. Doores,
[email protected], and Dennis R.
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Burton,
[email protected].
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*Present address: Cloudera, 433 California St. Ste 1100, San Francisco, CA
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Abstract length: 247
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Document length: 6252
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27
Abstract
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The high-mannose patch of HIV envelope (Env) elicits broadly neutralizing antibodies
29
(bnAbs) during natural infection relatively frequently and consequently this region has
30
become a major target of vaccine design. However, it has also become clear that
31
antibody recognition of the region is complex due to the quaternary nature of the bnAb
32
epitopes and variability in neighboring loops and glycans critical to the epitopes. BnAbs
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against this region have some shared and some distinguishing features that are crucial
34
to understand in order to design optimal immunogens that can induce different classes
35
of bnAbs against this region. Here, we compare two branches of a single antibody
36
lineage, in which all members recognize the high-mannose patch. One branch
37
(prototype bnAb PGT128) has a 6-amino acid insertion in CDRH2 that is crucial for
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broad neutralization. Antibodies in this branch appear to favor a glycan site at N332 on
39
gp120 and somatic hypermutation is required to accommodate the neighboring V1 loop
40
glycans and glycan heterogeneity. The other branch (prototype bnAb PGT130) lacks the
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CDRH2 insertion. Antibodies in this branch are noticeably effective at neutralizing
42
viruses with an alternate N334 glycan site but are less able to accommodate glycan
43
heterogeneity. We identify a new somatic variant within this branch that is predominantly
44
dependent on N334. The crystal structure of PGT130 offers some insight into these
45
differences compared to PGT128.
46
required to elicit bnAbs that have the optimal characteristics of the two branches of the
47
lineage described.
We conclude that different immunogens may be
48 49
Importance
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Development of an HIV vaccine is of vital importance for control of new infections and it
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is thought that elicitation of HIV bnAbs will be an important component of an effective
52
vaccine. Increasingly, bnAbs that bind to the cluster of high-mannose glycans on the HIV 2
53
envelope glycoprotein, gp120, are being highlighted as important templates for vaccine
54
design. In particular, bnAbs from donor 36 (PGTs125-131) have been shown to be
55
extremely broad and potent. Combination of these bnAbs enhanced neutralization
56
breadth considerably suggesting that an optimal immunogen should elicit several
57
antibodies from this family. Here we study the evolution of this antibody family to inform
58
immunogen design. We identify two classes of bnAb that differ in their recognition of the
59
high-mannose patch and show that different immunogens may be required to elicit these
60
different classes.
61
3
62
Introduction
63 64
It is becoming increasingly apparent that the “high-mannose patch” on the outer-domain
65
of the HIV envelope glycoprotein, gp120, is a site of great vulnerability to HIV-1 broadly
66
neutralizing antibodies (bnAbs) (1-7). The high-mannose patch is a region rich in high-
67
mannose glycans centered around the N332 glycan in many isolates but replaced by an
68
N334 glycan in other isolates (5). In still other isolates, both N332 and N334 are lacking
69
but nevertheless the high-mannose region still persists (8-12). A prototype bnAb to the
70
high-mannose patch, PGT121, protects against SHIV challenge in macaques at low
71
serum concentrations (13) and is highly effective at reducing viral load in established
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SHIV infection (14). Serum neutralization from many HIV ‘elite neutralizers’ can be
73
mapped to this high-mannose patch (4, 6, 7) and several families of bnAbs have been
74
isolated from a number of different donors of which the prototypes include PGT121,
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PGT124, 10-1074, PGT128 and PGT135 (1-3, 15-17). These antibodies recognize the
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high-mannose patch using distinct but overlapping modes of binding, each interacting
77
with different combinations of N-linked glycans and protein components in addition to the
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N332 glycan (1-3, 15-17).
79 80
We have recently shown that bnAbs targeting the high-mannose patch can be
81
promiscuous in their N-glycan site recognition and can, in certain cases, utilize the
82
glycans at positions N136/7, N295 or N334 in the absence of the N332 glycan (18) and,
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in some cases, bind to both high-mannose and complex sugars (2, 16). This ability to
84
utilize other N-linked glycan sites may help counter neutralization escape mediated by
85
shifting or elimination of glycosylation sites. We observed that not only were there
86
differences in the extent of promiscuity of high-mannose patch recognition between
4
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donors but also within antibody families from individual donors. This was particularly
88
apparent for bnAbs isolated from IAVI Protocol G donor 36 (PGTs125-131) (3, 4) (18).
89
Surprisingly, PGT130 could neutralize 68% of viruses naturally displaying an N334
90
glycan compared to PGT128 that could only neutralize 39%. Further, PGT130 was able
91
to neutralize 45% of a panel of 80 N332A/N334A mutant pseudoviruses unlike PGT128
92
that could only neutralize 23%. When combined, this family of bnAbs is able to neutralize
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an additional 14 viruses (12%) from a 120-pseudovirus panel compared to PGT128
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alone again suggesting complementary differences in epitope recognition (18).
95
Phylogenetic analysis of PGT125-131 sequences has shown that these bnAbs cluster
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into two distinct classes, PGT125-128 and PGT130-131 (19). PGT128 in the first class
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has been the most studied due to its superior potency and slightly higher breadth (3, 15).
98
However, little has been reported on the epitope recognition by the second class,
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PGT130 and PGT131, which are more N334-dependent. Further characterization of the
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PGT130 epitope and comparison of the evolution of the two classes of high-mannose
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patch binding bnAbs in this individual may help in the design of immunogens that elicit a
102
diverse and promiscuous neutralizing antibody response similar to the serum of elite
103
neutralizer donor 36 (18).
104 105
Here, we use next generation sequencing (NGS) and a recently reported phylogenic
106
method (ImmuniTree) to model lineage evolution and diversity of the bnAb response in
107
donor 36 (20). We show that, whilst the two branches described originated from the
108
same recombination event, the distinct structural features that evolved are critical for
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their N332 and/or N334 binding modes. A heavy chain insertion in the CDRH2 of the
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PGT128 branch appears necessary to avoid V1 loop glycosylation but this appears to
111
limit the ability to bind to N334. We also show that N334-dependence of the PGT130
112
branch arose at an early time point and identify a new somatic variant that is 5
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predominantly N334-dependent. High levels of somatic hypermutation mutation (SHM) in
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both branches are required to accommodate the glycan heterogeneity present on HIV
115
envelope and to avoid the V1 loop glycans in specific HIV strains. However, PGT130 is
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most sensitive to Env glycan heterogeneity. A crystal structure of unliganded PGT130
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Fab compared to PGT128 Fab shows that the CDRH2 insertion and CDRL1 deletion are
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likely in close proximity to glycans in the high-mannose patch when PGT130 binds to its
119
epitope in support of the above conclusions. Our data suggest that in order to elicit a
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diverse bnAb response similar to that in donor 36 different immunogens may be required
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to elicit these two antibody classes.
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6
124
Methods
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Ethics statement.
126
Peripheral blood mononuclear cells (PBMCs) were obtained from donor 36, an HIV-1
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infected donor from the IAVI Protocol G cohort (21). All human samples were collected
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with written informed consent under clinical protocols approved by the Republic of
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Rwanda National Ethics Committee, the Emory University Institutional Review Board,
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the University of Zambia Research Ethics Committee, the Charing Cross Research
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Ethics Committee, the UVRI Science and Ethics Committee, the University of New South
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Wales Research Ethics Committee. St. Vincent’s Hospital and Eastern Sydney Area
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Health Service, Kenyatta National Hospital Ethics and Research Committee, University
134
of Cape Town Research Ethics Committee, the International Institutional Review Board,
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the Mahidol University Ethics Committee, the Walter Reed Army Institute of Research
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(WRAIR) Institutional Review Board, and the Ivory Coast Comite ́ National d’Ethique des
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Sciences de la Vie et de la Sante ́ (CNESVS).
138 139
Cell sorting and RNA extraction.
140
Frozen vials of 107 PBMCs were thawed and washed before staining with Pacific Blue
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labeled anti-CD3 (UCHT1), Pacific Blue labeled anti-CD14 (M5E2), FITC labeled anti-
142
CD19 (HIB19), PE- Cy5 labeled anti-CD10 (HI10a), PE labeled anti-CD27 (M-T271), and
143
APC labeled anti-CD21 (B-ly4), all from BD Biosciences. Sorts were performed on a high
144
speed BD FACSAria into miRVana lysis buffer (Ambion). Immature B cells, exhausted
145
tissue-like memory, activated mature B cells, resting memory B cells, and short-lived
146
peripheral plasmablasts were stained using previously described markers (22). Total
147
RNA from the pooled sorted cells was then extracted using the mirVana RNA extraction
7
148
kit (Ambion) according to manufacturer’s instructions and quantitated on a 2100
149
Bioanalyzer (Agilent).
150 151
454 sequencing library preparation (20).
152
Reverse transcription was performed with 10 L total RNA and 2 L RT primer mix (50
153
M oligo-dT and 25 M random hexamer). The mixture was heated at 95oC for 1 min,
154
65oC for 5 min, then cooled on ice for 1 min. For each reaction, a mix was prepared with
155
4 L 5x FS buffer, 1 L 10 M dNTP mix, 1 L 0.1 M DTT, 1 L RNase inhibitor
156
(Enzymatics), and 1 L SuperScript III RT (Invitrogen). This mix was added to the
157
reaction and incubated at 25 oC for 10 min, 35 oC for 5 min, 55 oC for 45 min, and 85 oC
158
for 5 min. RNA/DNA hybrid was removed by adding 4 L E. coli RNase H (Enzymatics).
159
PCR reactions were assembled using 13.75 L water, 5 L cDNA, 5 L 5x HF buffer, 0.5
160
L 10 mM dNTP, 0.25 L of each 100 M primer stock, and 0.25 L Phusion Hot Start.
161
The reaction was cycled at 98 oC (60 s), 24 cycles of 98 oC (10 s), 62 oC (20 s), and 72 oC
162
(20 s), with a final extension at 72 oC (5 min). Samples were purified on a QIAquick
163
column and run on a 2% agarose E-gel. The desired bands were purified using the
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Qiagen MinElute gel extraction kit, eluted twice with 10 mL EB buffer, and quantitated on
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a 2100 Bioanalyzer. Samples were sent to SeqWright for 454 sequencing, which was
166
performed per manufacturer’s instructions.
167 168
Primers:
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Heavy chain: 20111028-IGHV4 (CRGCTCCCAGATGGGTCCTGT) and 20080924-IGHG
170
(CSGATGGGCCCTTGGTGG).
171
Light chain: 20111028-IGLV2-8 (TCACTCAGGGCACAGGGTCCTG) and 20111028-
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IGLC (AGAGGAGGGYGGGAACAGAGTG).
8
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Raw data processing: VDJ alignment and clone definition.
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Raw sequencing data were processed using in-house tools written in python. Reads
176
were split into barcodes, size-filtered, and aligned to IMGT’s germline VDJ reference
177
database. The scores were kept low, as we were interested in sequences that were very
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highly mutated. The V region is aligned first, then removed, followed by J, then removed,
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followed by D. The IMGT-defined CDR3 sequence of each read was then extracted. The
180
CDR3 sequences were sorted by abundance and clustered with USEARCH5.1 with the
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options ‘‘–minlen 0 –global –usersort – iddef 1 –id 0.9’’. Finally, each CDR3 sequence
182
was aligned to the ‘‘target’’ antibody sequences of PGT125-131 to determine a
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‘‘divergence’’ value from these antibodies (20).
184 185
Antibody variant identification and analysis.
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The divergence-mutation plots are used as a tool to identify reads that are similar to the
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known PGT125-131 antibodies (20). High-identity clusters of sequences and clusters
188
that are above background are manually identified and used as input for a phylogeny
189
inference with ImmuniTree. ImmuniTree is a dedicated algorithm for building immune
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receptor sequence trees from high-throughput DNA sequencing. It implements a
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Bayesian model of somatic hypermutation of clones, including probabilistic models of
192
SHM and sequencing error and performs Markov chain Monte Carlo over the tree
193
structure, birth/death times of the subclones, birth/death, mutation, and sequencing error
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rates, subclone consensus sequences, and assignment of reads to nodes. The tree
195
structure is also used for multiple computations and to overlay different information. We
196
estimate the selection pressure that a given node has experienced using the BASELINe
197
algorithm. It performs a Bayesian estimation of selection pressure by comparing the
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observed number of replacement/silent mutations in the CDRs/FWRs of the node 9
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consensus sequence. For a thorough description of the ImmuniTree algorithm see
200
Reference 20.
201 202
Pseudovirus production and neutralization assays.
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To produce pseudoviruses, plasmids encoding Env were co-transfected with an Env-
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deficient genomic backbone plasmid (pSG3∆Env) in a 1:2 ratio with the transfection
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reagent PEI (1 mg/mL, 1:3 PEI:total DNA, Polysciences) into HEK 293T cells or GnT1-/-
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deficient 293S cells. Pseudoviruses were harvested 72 hours post transfection for use in
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neutralization assays. Glycosidase inhibitors were added at the time of transfection at
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the following concentrations as described in ref. (23): 25 M kifunensine and 20 M
209
swainsonine.
210 211
Neutralizing activity was assessed using a single round replication pseudovirus assay
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with TZM-bl target cells, as described previously (3). Briefly, TZM-bl cells were seeded in
213
a 96-well flat bottom plate at a concentration of 20,000 cells/well. The serially diluted
214
virus/antibody mixture, which was pre-incubated for 1 hr, was then added to the cells
215
and luminescence was quantified 72 hrs following infection via lysis and addition of
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Bright-GloTM Luciferase substrate (Promega). To determine IC50 values, serial dilutions
217
of mAbs were incubated with virus and the dose-response curves were fitted using
218
nonlinear regression.
219 220
Antibody expression and purification.
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Antibody sequences for predicted precursors were synthesized (IDT DNA, San Diego,
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CA) and cloned into previously described heavy and light chain vectors (24). Antibody
223
plasmids were co-transfected at a 1:1 ratio in 293 FreeStyle cells using 293fectin
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(Invitrogen). Transfections were performed according to the manufacturer’s protocol and 10
225
antibody supernatants were harvested four days following transfection. Antibody
226
supernatants were purified over a protein A column, eluted with 0.1M Glycine (pH 3.5),
227
and buffer exchanged into PBS.
228 229
To prepare the PGT130 Fab fragment for crystallization studies, the Fc CH2-3 domains
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in the expression vector containing the full length IgG were deleted and soluble Fab was
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produced by transient transfection of 293S cells with the recombinant DNA. The Fab
232
was purified using an anti-human lambda affinity column followed by passage through a
233
monoS 10/100 GL cation exchange column (GE Healthcare). The Fab was then treated
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with endoglycosidase H (N.E.B) to cleave the N-linked glycans and purified through size
235
exclusion using a Superdex 200 (GE Healthcare) column.
236 237
Antibody and envelope mutations.
238
Mutations in the PGT heavy chain, the HIV-1 envelope glycoprotein were introduced
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using QuikChange site-directed mutagenesis (Stratagene, La Jolla, CA). Mutations were
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verified by DNA sequencing (Eton Biosciences, La Jolla, CA and MWG Eurofins,
241
Germany).
242 243
Crystallization
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The PGT130 Fab was concentrated to 8-12 mg/mL using an Amicon centrifugal
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ultrafiltration unit (Millipore) with a 10 kDa molecular weight cutoff. Fab samples were
246
screened for crystallization using the 384 conditions of the JCSG Core Suite (Qiagen) at
247
both 277 and 293 K using the JCSG/IAVI robotic Crystalmation system (Rigaku).
248
Crystallization was performed using the nanodroplet vapor diffusion method (25) with
249
standard JCSG crystallization protocols (26). Sitting drops composed of 200 nl of protein
250
sample were mixed with 200 nl of crystallization reagent in a sitting drop format and 11
251
were equilibrated against a 50 µl reservoir of crystallization reagent. After approximately
252
3 days at 20oC, crystals of Fab 130 formed in 40% (w/v) PEG 400, 0.2 M NaCl and 0.1
253
M Na/K phosphate, pH 6.2 (JCSG Core Suite 2, well C11).
254 255
Data collection
256
The PGT130 crystals diffracted to 2.75 Å at beamline 08ID-1 of the Canadian Light
257
Source (CLS). Data were collected at 100 K to a completeness of 91.4% with an overall
258
Rsym of 0.13 (0.67 in the high resolution shell) (Table S3). Data were indexed, processed
259
and scaled with HKL-2000 (27) in the orthorhombic space group P212121 with unit cell
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parameters a = 67.0Å, b = 69.1Å, c = 272.2Å.
261 262
Structure Refinement
263
The structure was determined by the molecular replacement method using Phaser (28)
264
with the PGT 128 Fab (PDBID: 3TV3) as an initial model. Model building was carried out
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using Coot-0.7 (29) and refinement was implemented with Phenix (30). Final Rcryst and
266
Rfree values are 24.5% and 26.9%, respectively.
267
12
268
Results
269 270
bnAbs in donor 36 arise from the same recombination event but differ by the
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presence of a heavy chain insertion and light chain deletion.
272
Six bnAbs have previously been isolated from donor 36 (PGTs125-131) and the binding
273
of all to gp120 is strongly dependent on the N-linked glycans at positions N301 and
274
N332 (3, 18). Analyses of the antibody sequences show that they share common germ
275
line V- and J-genes (IGHV4-39, IGHJ5*02 and IGLV2-8, IGLJ3*02) as well as long
276
stretches of identical N-nucleotides and common mutated residues. The antibody
277
sequences cluster into two distant but clonally related branches that have evolved from
278
the same VDJ recombination event (3, 19). The two branches differ by the presence of a
279
6-residue insertion in CDRH2 and a 5-residue deletion in CDRL1 of bnAbs in the
280
PGT125-128 branch (Table 1 and Figure S8). To determine the importance of these
281
indels for divergence in high-mannose patch recognition, we constructed two sets of
282
complementary mutant bnAbs; those in which the PGT128 CDRH2 insertion was
283
removed or the PGT128 CDRL1 deletion restored and those in which the insertion or
284
deletion was incorporated into PGT130. Neutralization breadth and potency of the
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mutant nAbs was assessed on an indicator panel of 6-pseudoviruses (21) (Figure 1).
286
The CDRH2 insertion in PGT128 has previously been shown to be important for
287
neutralization of JRFL (15). This insertion is shown here to be critical for neutralization
288
breadth and potency across the cross-clade indicator panel (Figure 1C). However,
289
restoration of the CDRL1 deletion in PGT128 had no effect on neutralization activity
290
(Figure 1B). PGT128 is more potent than its sister clone PGT130 (3), but the increased
291
potency is not simply due to the CDRH2 insertion and heavy chain disulfide between
292
CDRH1 and CDRH2 in PGT128; when residues S50-A52c (insertion) and G32C
13
293
mutation (permitting disulfide formation) were introduced into PGT130, no neutralization
294
was observed (Figure 1F). This result suggests that PGT130 has evolved distinct
295
somatic mutations to enable recognition of the high-mannose patch region in the
296
absence of the heavy chain insertion.
297 298
N332/N334 dependence arises early in the PGT130 branch
299
To explore the evolution of the high-mannose patch bnAb response and the divergence
300
in N332/N334 dependence in this donor, we used 454 deep sequencing and a recently
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published program, ImmuniTree, designed to model SHM (20). Unfortunately,
302
longitudinal samples were not available from this individual and, therefore, only PBMCs
303
from a single time point at which the bnAbs were isolated were sequenced and used to
304
model the lineage evolution of the PGT128 family. To produce libraries for 454
305
sequencing, gene-specific primers were used on the population of 78,000 sorted IgG+
306
memory B cells to amplify the IGHV4-39 and IGLV2-08 gene families from which
307
PGT128 and PGT130 were derived (19). 454 sequencing of the library yielded 390,563
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and 711,575 heavy and light chain reads respectively. Heavy and light chain trees
309
(Figure S1a and S1b) were generated using a new Bayesian phylogeny algorithm,
310
ImmuniTree, that models somatic hypermutation for the PGT125-131 family of bnAbs
311
(see methods for full details). The trees consist of both inferred and observed nodes with
312
the observed nodes being the most highly mutated. To address sequencing errors, the
313
algorithm collapsed the 390,563 heavy chain sequences and 711,575 light chain
314
sequences into 108 heavy chain nodes (named 1H to 108H) and 136 light chain nodes
315
(named 1L to 136L). Of these nodes, 24 heavy chain and 32 light chain nodes were
316
inferred statistically and did not have direct sequencing data. As we were interested in
317
whether less mutated inferred intermediates had neutralizing activity, heavy and light
14
318
chain nodes with varying levels of mutation at key branch points were selected and
319
paired to generate a simplified tree (Figure 2A, Table 1 and Table S1).
320 321
Breadth and potency of the purified antibodies were initially determined on the 6-
322
pseudovirus indicator panel (Figure 2B; neutralization data for a larger panel of chimeric
323
bnAbs can be found in Figure S2). As seen for other families of bnAbs, no HIV reactivity
324
for the predicted germ line antibody was detected on the 6-virus panel (Figure 2B) and
325
no binding to the corresponding recombinant gp120 glycoprotein was observed in ELISA
326
(data not shown) (31-35). Within the PGT128 branch, the least mutated predicted
327
intermediate 95H 71L (12% mutated in nucleotide sequence of the heavy and light V-
328
and J-gene segments) was able to neutralize all 6 viruses albeit at a slightly lower
329
potency than the fully mutated PGT128 (16%). In the PGT130 branch, intermediate 3H
330
3L (11%) showed no neutralization activity. This is in contrast to the PGT121 family of
331
bnAbs where 46% neutralization was observed with 6% SHM (20). However, chimeric
332
antibody 74H 3L (13%) showed very limited neutralization activity.
333 334
To investigate how the differences in N332 and N334 dependency arose between the
335
two branches, we next tested intermediate and mature bnAbs on a 54-cross clade
336
pseudovirus panel (Table S2 and Figure 3A and 3B) that included 41 viruses with a
337
glycan at position N332, 12 viruses with a glycan at position N334 and one virus lacking
338
both N332/N334. 95% of clade CRF01_AE viruses have a glycan site at position N334
339
whereas the remaining subtypes have >75% of viruses with a glycan site at position
340
N332 (36). For both the PGT128 and PGT130 branches, an increased level of SHM was
341
seen to increase breath and potency of neutralization as illustrated in the cumulative
342
frequency distribution plot (Figure 3C). As shown previously, PGT130 neutralized more
343
N334 viruses (73%) than PGT128 (58%) (18) and PGT128 neutralized more N332 15
344
viruses (98%) than PGT130 (73%). The less mutated PGT130 branch bnAb 74H 3L,
345
although only able to neutralize 13% of viruses, was able to neutralize 42% of N334
346
viruses compared to 5% of N332 viruses (Figure 3A and 3B). This suggests that the
347
PGT130 branch may have initially evolved to be more N334 specific before developing
348
N332 reactivity. To further explore the N332/N334 dependency of the two bnAb classes,
349
we next tested them against two mutated forms of the 6-virus panel where the glycan at
350
N332 was either shifted to N334 or removed by an N332A mutation (Figure 3D) (18).
351
Notably, 74H 3L could now neutralize JRCSF N334 and 92RW020 N334, although not
352
the N332A versions, further supporting a preference for N334 (Figure 3D).
353 354
A somatic variant that is predominantly N334 dependent is observed in the
355
PGT130 branch
356
In addition to clonal variants that cluster into the PGT128 and PGT130 branches, new
357
heavy and light chain sequences were identified within the PGT130 branch of the heavy
358
and light chain trees. 9H (24% mutated) also contains no indels but is significantly
359
divergent from PGT130, and was paired with 46L and this new somatic variant 9H 46L
360
(19%) was seen to be predominantly N334-dependent. This dependency was further
361
confirmed on a small number of N334A variant viruses (Figure 3D and Figure S3A-B)
362
and the N334 shifted panel (Figure 3D). The data suggest that predominantly N334-
363
dependent somatic variants exist within the polyclonal response against the high-
364
mannose patch, although single B cell cloning would be required to confirm the correct
365
heavy and light chain pairing. Of note, although 9H 46L is more N334 dependent, the
366
epitope is still centered on the N301 glycan site as shown by its inability to neutralize a
367
JRCSF N334 variant lacking the N301 glycan (Figure S3C-D).
368 369
High levels of SHM are necessary to accommodate V1 loop glycans 16
370
Many of the N332-dependent bnAbs require binding to additional neighboring N-linked
371
glycans for neutralization (1, 15, 17). Sok et al. recently showed that putative precursors
372
of PGT121 are dependent on both N301 and N332 glycans and that the dependency on
373
the N301 glycan is reduced with increased SHM (20). To determine how SHM and indels
374
impact on the dependency of these two classes of bnAbs on N-linked glycans within the
375
high-mannose patch, we used a panel of JRCSF glycan mutant viruses and measured
376
impact on neutralization by PGT128, PGT130, 95H 71L, 3H 130L and PGT128-insert
377
(Figure S4 and Figure 4A). Glycan sites N135, N295 and N301 were chosen based on
378
the recent HIV envelope trimer crystal structure that revealed their close proximity to the
379
PGT128 epitope (17). Interestingly, 3H 130L and PGT128-insert showed enhanced
380
neutralization for the N135A mutant virus suggesting increased SHM and/or the heavy
381
chain insertion might allow better accommodation of this V1 loop glycan. To explore this
382
hypothesis further, we measured neutralization of a small panel of V1 loop glycan
383
mutant pseudoviruses (Figure 4B-H). The results were somewhat strain dependent; for
384
N332 viruses, removal of the N135/6/7 glycan enhanced neutralization whereas, for
385
N334 viruses, removal of the N141, N149 or N151 glycans enhanced neutralization. The
386
difference in V1 loop glycan shielding between N332 and N334 containing viruses likely
387
reflects the differing proximities of the glycans arising from the different protein
388
sequences. The PGT130 bnAb class, including 9H 46L, was much more sensitive to
389
removal of V1 loop glycans than the PGT128 class (Figure 4). Removal of a V1 loop
390
glycan allowed some viruses to become sensitive to neutralization by PGT128-insert
391
when the wild-type virus had been resistant. However the germ line version of the
392
antibody remained ineffective against all mutant viruses (data not shown). Overall, it
393
appears that the V1 loop glycans limit the putative precursor bnAbs from fully accessing
394
their epitopes but that this can be overcome by additional SHM and/or indels.
395 17
396
High levels of SHM are required to accommodate glycan heterogeneity on HIV
397
Envelope.
398
Analysis of the neutralization profiles of a number of inferred intermediate bnAbs, and on
399
occasion PGT130, showed viruses neutralized incompletely with a low neutralization
400
plateau and/or a shallow neutralization curve (Figure S5). We have previously shown
401
that incomplete neutralization can be a result, at least in part, of envelope glycan
402
heterogeneity giving rise to a population of viruses that are resistant to neutralization
403
(23). This might suggest that less mutated bnAbs and the PGT130 class of bnAbs are
404
more restricted in the type of glycan they can recognize at the key glycosylation sites
405
(N332/N334 and N301) and therefore are unable to neutralize the complete population
406
of heterogeneously glycosylated virus particles. We therefore measured the impact of
407
changes in envelope glycosylation on neutralization sensitivity for an antibody panel by
408
making BJOX015000.11.5 (Figure 5) and JRFL (Figure S6) in the presence of the
409
glycosidase inhibitors kifunensine and swainsonine and in the GnT1-/- deficient cell line,
410
293S (23). We observed different sensitivities towards changes in glycosylation profile
411
between the classes of bnAbs and the virus strain.
412 413
Neutralization by PGT128 remained largely unaffected by changes in viral glycosylation
414
profile although some increase in neutralization sensitivity was observed for viruses
415
grown in the presence of kifunensine, which leads to homogeneous Man9GlcNAc2
416
glycans. Within the PGT128 branch, the less mutated 95H 71L showed increased
417
potency against BJOX015000.11.5 when glycan heterogeneity was decreased by any of
418
three conditions (Figure 5). The increase brought the inferred precursor up to the level of
419
PGT128 in terms of isolate neutralization potency suggesting that, for this isolate,
420
PGT128 was better able to cope with glycan heterogeneity than the precursor antibody.
421
The effects were less pronounced for the JRFL isolate (Figure S6), which might already 18
422
have a more homogeneous or optimal glycan profile. In contrast, the PGT130 branch
423
antibodies were more sensitive to changes in glycosylation. PGT130 had a reduced
424
potency against both the kifunensine and 293S prepared JRFL pseudoviruses but an
425
increased potency against the swainsonine virus (Figure S6). Very little effect was
426
observed for the BJOX015000.11.5 viruses (Figure 5). Less mutated 74H 3L and new
427
somatic variant 9H 46L were unable to neutralize either the virus prepared with
428
kifunensine or made in 293S cells and virus made in the presence of swainsonine was
429
neutralized with a slightly increased potency. This suggests that, for some donor 36
430
bnAbs, either certain glycan structures cannot be accommodated within the antibody-
431
binding site resulting in a greater sensitivity to glycan heterogeneity or alternatively, the
432
larger Man9GlcNAc2 structure present in the V1 loop might prevent engagement of the
433
antibody with its epitope. For example, in a similar manner to PG9 (23, 37), it is possible
434
that PGT130 or 74H 3L might not be able to accommodate a Man9GlcNAc2 glycan at
435
position N301 and/or binding affinity is enhanced if N301 is a hybrid glycan for some
436
viruses. Similarly, members of the PGT121 family have shown differing abilities to
437
recognize both complex and high-mannose sugars (2, 16). Overall, SHM affects the
438
ability of bnAbs to accommodate glycan heterogeneity on HIV Envelope and this may
439
impact protective efficacy in vivo.
440 441
A crystal structure of PGT130 Fab highlights differing modes of binding by the
442
antibody classes
443
To better understand how the different structural features of the PGT128 and PGT130
444
branches of the bnAb family might impact the antibody combining site and subsequent
445
recognition of the high-mannose patch and N332/N334 dependency, we next
446
determined the crystal structure of PGT130 Fab to 2.75Å resolution (Figure 6A). In the
447
unliganded state, the CDR loops of PGT130 appear highly flexible, resulting in poorly 19
448
defined electron density, especially for the CDR H3 loop, which is largely disordered. Of
449
note, electron density for single GlcNAc moieties are defined at the N-linked
450
glycosylation sites N24LC and N82bHC. The antigen combining sites of the unliganded
451
PGT130 Fab and PGT128 structures overlay very well except at the 6-amino acid heavy
452
chain insertion and the 5-amino acid light chain deletion in PGT128 (Figure 6B). The
453
CDRL1 of PGT130 adopts a more helical conformation consistent with Chothia
454
canonical L1 loop class 6 compared to the L1 loop of PGT128, which is not consistent
455
with any Chothia canonical class, presumably due to the 5-amino acid deletion (38). A
456
model of the PGT128 Fab epitope on the BG505 SOSIP trimer crystal structure ((17)
457
and Figure 6C) reveals the close proximity of the CDRL1 loop to the N137 V1 loop
458
glycan and the tight association of the CDRH2 loop insertion with the N332 glycan on
459
the gp120 core. Thus, the CDRH2 loop may constrain the PGT128 interaction towards
460
the V3 loop and away from the V1 loop and the 5-amino acid deletion in the PGT128
461
CDRL1 may allow better accommodation of V1 loop glycosylation (Figure 6D). This is
462
corroborated by PGT128 being less tolerant of the N334 glycan shift compared to
463
PGT130, which does not include the indels. Additionally, neutralization for the V1 loop
464
glycan mutant viruses by PGT128 lacking the heavy chain insertion supports its function
465
to avoid the V1 loop glycans (Figure 4). However, reintroducing the CDRL1 deletion into
466
the fully mutated PGT128 does not have a significant impact on neutralization potency
467
for the fully mutated bnAb or the PGT128 variant lacking the heavy chain insertion
468
(Figure 1). Thus, these structural differences indicate diverging strategies for recognition
469
of the high-mannose patch: PGT128 focuses on the more conserved elements of the
470
high-mannose patch and V3 loop region at the cost of lacking tolerance for variation at
471
V3 glycans while PGT130 is more sensitive to V1 glycans at the cost of greater breadth
472
and potency in general.
473 20
474
Antibody residues that contact the N332 glycan are conserved across all somatic
475
variants and predicted precursor bnAbs.
476
To map the key interactions between the PGT130 combining site and gp120, we
477
performed paratope mapping of PGT130. Residues shown to be critical for PGT128
478
binding to high-mannose and eODmV3 protein (15) were mutated to alanine in PGT130
479
and their impact on neutralization of JR-CSF and JR-FL pseudovirus (Figure 6E) and on
480
gp120 binding (Figure S7) measured. Residues shown to be critical for PGT128
481
interaction with Man8/9GlcNAc2 at position N332 (HCDR3 residues W100eA, K100gA,
482
and LCDR3 W95A and D95aA) were also found to be critical for PGT130 neutralization
483
and gp120 binding suggesting similar modes of recognition for this high-mannose glycan.
484
These residues are conserved between all family members and precursor antibodies
485
indicating that these mutations arose before divergence of the two branches (Figure S8).
486
This may suggest that the interaction with the N332 glycan was important for initial
487
selection of this bnAb family. Residues H59 and K64 in PGT128 that also contact the
488
N332 glycan are conserved in PGT130, but these had little effect on neutralization when
489
mutated in PGT128 (15). Residues within the CDRH2 of PGT130 (Ile51-Thr55) were also
490
identified as important for neutralization by PGT130. Based on the PGT130 Fab
491
structure, His50 and Ile51 likely play a structural role in maintaining the antibody-
492
combining site whereas Tyr53 and Thr54 are more likely forming key contacts with the
493
target epitope. Unlike PGT128, PGT130 has N-linked glycans in the variable regions of
494
its heavy and light chains. When these glycans were removed using site-directed
495
mutagenesis no significant effect on either breadth or potency was observed (Figure 6E)
496
suggesting these glycans are not important for the difference in epitope recognition by
497
the two classes of bnAbs.
498 499 21
500
Discussion
501 502
Insight into the development of broadly neutralizing antibodies (bnAbs) in HIV-infected
503
individuals is a major goal in HIV vaccine research (39-43). bnAbs targeting the glycan
504
shield must either bind the N-linked glycans within their binding site for increased affinity
505
or evolve to accommodate or avoid obstructing glycans. The bnAb response against the
506
high-mannose patch in Protocol G donor 36 uses a combination of these strategies.
507
Unlike less mutated forms of PGT121 that use additional N-linked glycans for
508
neutralization (17, 20), this family of bnAbs evolves to better accommodate or avoid the
509
V1 loop glycans (Figure 4) whilst gaining affinity from binding to both the
510
N332/N334/N295 and N301 glycans. From the small number of putative germ line
511
precursor bnAbs tested in this study, it can be deduced that increased SHM and indels
512
in this family of bnAbs have several effects: i) an increased breadth and potency of
513
neutralization (Figure 3C), ii) an ability to better accommodate changes in glycan
514
heterogeneity within the antibody combining site (Figure S5), iii) an ability to better
515
accommodate glycans within the V1 loop for some virus strains (Figure 4), and iv) an
516
increased ability to utilize other N-linked glycans within the high-mannose patch in the
517
absence of the N332 glycan (Figure 3D). Here we describe two related classes of bnAbs
518
against the high-mannose patch arising from a single recombination event in donor 36
519
that achieve these advantageous features differently. The most potent class, PGT128,
520
has a critical insertion in the CDRH2. The second more mutated class, PGT130,
521
although less potent is more able to tolerate different glycan patterns in the high-
522
mannose patch (Figure 3 and (18)).
523
22
524
We first consider the PGT128 class of bnAb. An important difference between the
525
PGT128 and PGT130 classes of bnAbs from donor 36 is the CDRH2 insertion and
526
CDRL1 deletion in the PGT128 branch that appears to occur at a relatively early time
527
point (Figure 1, Table 1, Figure S1). We have shown that a PGT128 variant lacking the
528
CDRH2 insert has very limited neutralization activity but neutralization can be increased
529
by removal of certain V1 loop glycans. These data, along with docking of PGT128 on the
530
BG505 SOSIP trimer (17), suggest the PGT128 CDRH2 insertion is necessary to
531
accommodate V1 loop glycosylation (Figure 6C and 6D). However, the tight association
532
between the CDRH2 loop insertion and the N332 glycan, although increasing
533
neutralization potency, appears to constrain the PGT128 interaction towards the V3 loop
534
thus reducing the ability of PGT128 to use alternate glycans in the vicinity of the high-
535
mannose patch in the absence of the N332 glycan (Figure 3 and (18)). SHM also plays
536
an important role in epitope recognition in the PGT128 bnAb class. Antibody 95H 71L,
537
which also has both the heavy chain insertion and light chain deletion but a lower level of
538
SHM, was found to be more sensitive to changes in glycan heterogeneity, removal of V1
539
loop glycosylation and had a reduced ability to neutralize N334 and N332A/N334A
540
mutant viruses.
541 542
The PGT130 class has no indels so relies solely on SHM for breadth, potency and use
543
of alternate glycans in binding to the high-mannose patch. A common feature of many
544
HIV bnAbs is the presence of long insertions and deletions that appear critical for HIV
545
reactivity (1, 20, 44-46) and it is a challenge for immunogen design to develop strategies
546
capable of eliciting antibodies with these unusual features (47). As demonstrated here
547
PGT130 is able to reach a similar level of neutralization breadth as PGT128 without
548
indels, albeit with a slightly higher degree of somatic mutation and a slightly lower
549
potency. The decreased potency of PGT130 may reflect its reduced ability to 23
550
accommodate V1 loop glycosylation near the CDRL1 loop within the binding site and its
551
greater sensitivity to changes in glycan heterogeneity (Figure 4 and 5 and Figure S6).
552
However, the subsequent benefit of PGT130 lacking the PGT128 indels is its noticeably
553
increased ability to neutralize N334 viruses. Here we show that the putative precursor of
554
PGT130, Ab 74H 3L, is able to neutralize 42% of N334 viruses compared to 5% of N332
555
viruses. This may suggest the PGT130 branch initially evolved to be predominantly
556
N334 dependent and upon increased SHM was able to bind more different
557
configurations of the high-mannose patch and neutralize N332 containing viruses. In
558
support of this hypothesis, we also identified an additional sub-branch (9H 46L) that
559
neutralized 50% of N334 viruses (with a median IC50 of 0.08 g/mL) compared to only
560
12% of N332 viruses.
561 562
The findings described here have implications for vaccine design. This study and our
563
previous work (18) suggest a spectrum of N332 and N334 dependency exists for bnAbs
564
that bind the high-mannose patch. At one extreme, from a single donor, donor 36,
565
PGT127 shows strong dependence on N332 (3, 18) and at the other extreme, 9H 46L
566
shows strong dependence on N334 (Figure 3B). PGT130 occupies the middle ground
567
and neutralizes both N332 and N334 viruses efficiently. A combination of bnAbs with
568
differing N332/N334 dependence will likely be most effective at limiting viral escape and
569
providing protection against all subtypes. Without longitudinal samples, it is not possible
570
to determine in detail how the two antibody classes diverged. However, despite the
571
differences described above, the crystal structures of PGT128 and PGT130 Fab are
572
remarkably similar except for the PGT128 indels. Paratope mapping (Figure 6E) and
573
sequence alignment (Figure S8) has shown conservation of residues critical for
574
N332/N334 binding across both bnAb classes and predicted precursor nAbs suggesting
575
a common pathway of maturation occurred before divergence and introduction of the 24
576
PGT128 indels. As shown by Moore et al., the differing N332/N334 specificity may have
577
arisen from the altering selective pressure on the virus from the two antibody branches
578
within this donor that drove the shifting of the N332/N334 glycan site (5). Therefore, in
579
terms of immunogen design, both immunogens with a glycan at position N332 and
580
immunogens with a glycan at position N334 may prove essential for eliciting a bnAb
581
response similar to that in donor 36.
582 583
Further, these findings suggest a series of immunogens that first lack key V1 loop glycan
584
sites (N137 or N149/N151) and then inclusion of these glycan sites might favor elicitation
585
of bnAbs that can better accommodate V1 loop glycosylation. The complexity of the
586
epitope of the donor 36 bnAbs, and similarly other high-mannose patch binding bnAbs (1,
587
16, 18, 20), indicates that immunogens focused only on the key contacts with the mature
588
bnAbs will likely elicit bnAbs with limited breadth and potency. Finally, the ability to
589
accommodate the natural glycan heterogeneity of virion-associated envelope may prove
590
essential for in vivo protection against 100% of virus particles. Therefore, although some
591
bnAbs have a higher binding affinity for certain glycoforms, immunogens designed to re-
592
elicit this family of bnAbs may need to incorporate glycan heterogeneity similar to that on
593
viral gp120 such that elicited bnAbs bind all possible envelope glycoforms and achieve
594
100% neutralization.
595 596
In summary, somatic hypermutation and indels in a family of bnAbs to the high-mannose
597
patch of HIV gp120 increases breadth and potency through more complete recognition
598
of glycan heterogeneity, accommodation of V1 loop glycans and the ability to utilize
599
alternate glycans. The level of SHM required for HIV reactivity is higher than for donor
600
17 bnAbs (20). Two classes of bnAbs within a single lineage directed to the high-
601
mannose patch exist in donor 36. The PGT128 class is superior in breadth and potency; 25
602
however, PGT130 would likely be more effective at countering potential escape through
603
mutation of glycan sites. Therefore, a vaccine capable of eliciting both classes of bnAb
604
would be highly advantageous (18). In terms of immunogen design, a combination of
605
immunogens displaying either the N332 or N334 glycan sites, with carefully positioned
606
V1 loop glycans and glycan heterogeneity which resembles that of the virus may be
607
needed to maximize the potential of eliciting antibodies of this specificity and a high
608
degree of neutralization breadth and potency.
609 610
Accession codes. Coordinates and structure factors for Fab PGT 130 has been
611
deposited in the Protein Data Bank under accession code 4RNR.
612 613
Acknowledgements:
614
We thank Christina Corbaci for assistance with graphics. This work was supported by
615
the International AIDS Vaccine Initiative through the Neutralizing Antibody Consortium
616
and Bill and Melinda Gates Center for Vaccine Discovery (D.R.B., I.A.W., P.P.), NIH R01
617
AI033292 (D.R.B.), AI84817 (I.A.W.) and 1U19AI090970 (P.P.), Center for HIV/AIDS
618
Vaccine Immunology and Immunogen Discovery Grant UM1AI100663 (D.R.B. and
619
I.A.W.) and an MRC Career Development Fellowship MR/K024426/1 (K.J.D).
620
26
621
Figure legends:
622 623
Table 1. Sequence characteristics of selected heavy and light chain bnAbs from
624
donor 36. Mutation frequency was calculated over the V-gene and J-gene as
625
nucleotides or amino acids differing from the putative germline sequence for (A) heavy
626
chain sequences and (B) light chain sequences. The CDR3 regions and insertions and
627
deletions were excluded from the analysis. CDR3 lengths were determined according to
628
the IMGT definition.
629 630
Figure 1. Importance of indels for neutralization breadth and potency by bnAbs
631
from donor 36. Neutralization breadth and potency of mutated and chimeric antibodies
632
were determined on a 6-pseudovirus panel previously shown to be predicative of
633
breadth and potency on a larger panel (21). A) PGT128, B) PGT+del (5 residue CDRL1
634
deletion restored), C) PGT128-insert (6 residue CDRH2 insertion removed), D) PGT128-
635
insert+del (6 residue CDRH2 insert removed and 5 residue CDRL1 deletion restored), E)
636
PGT130, and F) PGT130+insert (PGT128 6 residue CDRH2 insert introduced into
637
PGT130 CDRH2 and G32C permitting disulfide formation).
638 639
Figure 2. Breadth and potency of PGT125-131 variants identified by deep
640
sequencing and phylogenetic analysis. A) A simplified antibody tree representing
641
analysis of deep sequencing data using ImmuniTree (for full analysis see Figures S1a
642
and S1b). Mutation level is listed for heavy and light chains individually and based on
643
nucleotide changes from germ line over V and J segments, B) % breadth and potency
644
on the 6-virus panel of predicted precursor bnAbs of PGT128 and PGT130, and a new
645
somatic variant (9H 46L). Values are reported as IC50 (antibody concentrations required
27
646
to inhibit HIV activity by 50%) and measured in g/mL. The panels are coloured as
647
follows; red
