1 Two classes of broadly neutralizing antibodies ... - Journal of Virology

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Nov 5, 2014 - Two classes of broadly neutralizing antibodies within a single lineage ..... 4 μL 5x FS buffer, 1 μL 10 μM dNTP mix, 1 μL 0.1 M DTT, 1 μL RNase inhibitor ... RNA/DNA hybrid was removed by adding 4 μL E. coli RNase H (Enzymatics). ...... 636. PGT130 CDRH2 and G32C permitting disulfide formation). 637.
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.

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Two classes of broadly neutralizing antibodies within a single lineage directed to

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the high-mannose patch of HIV Envelope

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Katie J. Doores,1 Leopold Kong,3,4,5,6 Stefanie A. Krumm,1 Khoa M. Le,2,3,4 Devin

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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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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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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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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

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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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Abstract

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The high-mannose patch of HIV envelope (Env) elicits broadly neutralizing antibodies

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(bnAbs) during natural infection relatively frequently and consequently this region has

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become a major target of vaccine design. However, it has also become clear that

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antibody recognition of the region is complex due to the quaternary nature of the bnAb

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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

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to understand in order to design optimal immunogens that can induce different classes

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of bnAbs against this region. Here, we compare two branches of a single antibody

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lineage, in which all members recognize the high-mannose patch. One branch

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(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

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gp120 and somatic hypermutation is required to accommodate the neighboring V1 loop

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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

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viruses with an alternate N334 glycan site but are less able to accommodate glycan

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heterogeneity. We identify a new somatic variant within this branch that is predominantly

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dependent on N334. The crystal structure of PGT130 offers some insight into these

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differences compared to PGT128.

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required to elicit bnAbs that have the optimal characteristics of the two branches of the

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lineage described.

We conclude that different immunogens may be

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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

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vaccine. Increasingly, bnAbs that bind to the cluster of high-mannose glycans on the HIV 2

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envelope glycoprotein, gp120, are being highlighted as important templates for vaccine

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design. In particular, bnAbs from donor 36 (PGTs125-131) have been shown to be

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extremely broad and potent. Combination of these bnAbs enhanced neutralization

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breadth considerably suggesting that an optimal immunogen should elicit several

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antibodies from this family. Here we study the evolution of this antibody family to inform

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immunogen design. We identify two classes of bnAb that differ in their recognition of the

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high-mannose patch and show that different immunogens may be required to elicit these

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different classes.

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Introduction

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It is becoming increasingly apparent that the “high-mannose patch” on the outer-domain

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of the HIV envelope glycoprotein, gp120, is a site of great vulnerability to HIV-1 broadly

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neutralizing antibodies (bnAbs) (1-7). The high-mannose patch is a region rich in high-

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mannose glycans centered around the N332 glycan in many isolates but replaced by an

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N334 glycan in other isolates (5). In still other isolates, both N332 and N334 are lacking

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but nevertheless the high-mannose region still persists (8-12). A prototype bnAb to the

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high-mannose patch, PGT121, protects against SHIV challenge in macaques at low

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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

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mapped to this high-mannose patch (4, 6, 7) and several families of bnAbs have been

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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

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with different combinations of N-linked glycans and protein components in addition to the

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N332 glycan (1-3, 15-17).

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We have recently shown that bnAbs targeting the high-mannose patch can be

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promiscuous in their N-glycan site recognition and can, in certain cases, utilize the

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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

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utilize other N-linked glycan sites may help counter neutralization escape mediated by

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shifting or elimination of glycosylation sites. We observed that not only were there

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differences in the extent of promiscuity of high-mannose patch recognition between

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donors but also within antibody families from individual donors. This was particularly

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apparent for bnAbs isolated from IAVI Protocol G donor 36 (PGTs125-131) (3, 4) (18).

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Surprisingly, PGT130 could neutralize 68% of viruses naturally displaying an N334

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glycan compared to PGT128 that could only neutralize 39%. Further, PGT130 was able

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to neutralize 45% of a panel of 80 N332A/N334A mutant pseudoviruses unlike PGT128

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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).

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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).

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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

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diverse and promiscuous neutralizing antibody response similar to the serum of elite

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neutralizer donor 36 (18).

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Here, we use next generation sequencing (NGS) and a recently reported phylogenic

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method (ImmuniTree) to model lineage evolution and diversity of the bnAb response in

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donor 36 (20). We show that, whilst the two branches described originated from the

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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

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limit the ability to bind to N334. We also show that N334-dependence of the PGT130

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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

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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

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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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Methods

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Ethics statement.

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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

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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).

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Cell sorting and RNA extraction.

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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-

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CD19 (HIB19), PE- Cy5 labeled anti-CD10 (HI10a), PE labeled anti-CD27 (M-T271), and

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APC labeled anti-CD21 (B-ly4), all from BD Biosciences. Sorts were performed on a high

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speed BD FACSAria into miRVana lysis buffer (Ambion). Immature B cells, exhausted

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tissue-like memory, activated mature B cells, resting memory B cells, and short-lived

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peripheral plasmablasts were stained using previously described markers (22). Total

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RNA from the pooled sorted cells was then extracted using the mirVana RNA extraction

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kit (Ambion) according to manufacturer’s instructions and quantitated on a 2100

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Bioanalyzer (Agilent).

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454 sequencing library preparation (20).

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Reverse transcription was performed with 10 L total RNA and 2 L RT primer mix (50

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M oligo-dT and 25 M random hexamer). The mixture was heated at 95oC for 1 min,

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65oC for 5 min, then cooled on ice for 1 min. For each reaction, a mix was prepared with

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4 L 5x FS buffer, 1 L 10 M dNTP mix, 1 L 0.1 M DTT, 1 L RNase inhibitor

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(Enzymatics), and 1 L SuperScript III RT (Invitrogen). This mix was added to the

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reaction and incubated at 25 oC for 10 min, 35 oC for 5 min, 55 oC for 45 min, and 85 oC

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for 5 min. RNA/DNA hybrid was removed by adding 4 L E. coli RNase H (Enzymatics).

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PCR reactions were assembled using 13.75 L water, 5 L cDNA, 5 L 5x HF buffer, 0.5

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L 10 mM dNTP, 0.25 L of each 100 M primer stock, and 0.25 L Phusion Hot Start.

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The reaction was cycled at 98 oC (60 s), 24 cycles of 98 oC (10 s), 62 oC (20 s), and 72 oC

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(20 s), with a final extension at 72 oC (5 min). Samples were purified on a QIAquick

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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

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performed per manufacturer’s instructions.

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Primers:

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Heavy chain: 20111028-IGHV4 (CRGCTCCCAGATGGGTCCTGT) and 20080924-IGHG

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(CSGATGGGCCCTTGGTGG).

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Light chain: 20111028-IGLV2-8 (TCACTCAGGGCACAGGGTCCTG) and 20111028-

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IGLC (AGAGGAGGGYGGGAACAGAGTG).

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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

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were split into barcodes, size-filtered, and aligned to IMGT’s germline VDJ reference

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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

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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

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was aligned to the ‘‘target’’ antibody sequences of PGT125-131 to determine a

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‘‘divergence’’ value from these antibodies (20).

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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

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that are above background are manually identified and used as input for a phylogeny

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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

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SHM and sequencing error and performs Markov chain Monte Carlo over the tree

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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

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structure is also used for multiple computations and to overlay different information. We

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estimate the selection pressure that a given node has experienced using the BASELINe

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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

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Reference 20.

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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

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swainsonine.

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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

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a 96-well flat bottom plate at a concentration of 20,000 cells/well. The serially diluted

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virus/antibody mixture, which was pre-incubated for 1 hr, was then added to the cells

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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

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of mAbs were incubated with virus and the dose-response curves were fitted using

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nonlinear regression.

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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

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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

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antibody supernatants were harvested four days following transfection. Antibody

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supernatants were purified over a protein A column, eluted with 0.1M Glycine (pH 3.5),

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and buffer exchanged into PBS.

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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

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was purified using an anti-human lambda affinity column followed by passage through a

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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

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exclusion using a Superdex 200 (GE Healthcare) column.

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Antibody and envelope mutations.

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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,

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Germany).

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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

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screened for crystallization using the 384 conditions of the JCSG Core Suite (Qiagen) at

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both 277 and 293 K using the JCSG/IAVI robotic Crystalmation system (Rigaku).

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Crystallization was performed using the nanodroplet vapor diffusion method (25) with

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standard JCSG crystallization protocols (26). Sitting drops composed of 200 nl of protein

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sample were mixed with 200 nl of crystallization reagent in a sitting drop format and 11

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were equilibrated against a 50 µl reservoir of crystallization reagent. After approximately

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3 days at 20oC, crystals of Fab 130 formed in 40% (w/v) PEG 400, 0.2 M NaCl and 0.1

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M Na/K phosphate, pH 6.2 (JCSG Core Suite 2, well C11).

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Data collection

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The PGT130 crystals diffracted to 2.75 Å at beamline 08ID-1 of the Canadian Light

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Source (CLS). Data were collected at 100 K to a completeness of 91.4% with an overall

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Rsym of 0.13 (0.67 in the high resolution shell) (Table S3). Data were indexed, processed

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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.

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Structure Refinement

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The structure was determined by the molecular replacement method using Phaser (28)

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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

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Rfree values are 24.5% and 26.9%, respectively.

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Results

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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.

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Six bnAbs have previously been isolated from donor 36 (PGTs125-131) and the binding

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of all to gp120 is strongly dependent on the N-linked glycans at positions N301 and

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N332 (3, 18). Analyses of the antibody sequences show that they share common germ

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line V- and J-genes (IGHV4-39, IGHJ5*02 and IGLV2-8, IGLJ3*02) as well as long

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stretches of identical N-nucleotides and common mutated residues. The antibody

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sequences cluster into two distant but clonally related branches that have evolved from

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the same VDJ recombination event (3, 19). The two branches differ by the presence of a

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6-residue insertion in CDRH2 and a 5-residue deletion in CDRL1 of bnAbs in the

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PGT125-128 branch (Table 1 and Figure S8). To determine the importance of these

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indels for divergence in high-mannose patch recognition, we constructed two sets of

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complementary mutant bnAbs; those in which the PGT128 CDRH2 insertion was

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removed or the PGT128 CDRL1 deletion restored and those in which the insertion or

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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).

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The CDRH2 insertion in PGT128 has previously been shown to be important for

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neutralization of JRFL (15). This insertion is shown here to be critical for neutralization

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breadth and potency across the cross-clade indicator panel (Figure 1C). However,

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restoration of the CDRL1 deletion in PGT128 had no effect on neutralization activity

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(Figure 1B). PGT128 is more potent than its sister clone PGT130 (3), but the increased

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potency is not simply due to the CDRH2 insertion and heavy chain disulfide between

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CDRH1 and CDRH2 in PGT128; when residues S50-A52c (insertion) and G32C

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mutation (permitting disulfide formation) were introduced into PGT130, no neutralization

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was observed (Figure 1F). This result suggests that PGT130 has evolved distinct

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somatic mutations to enable recognition of the high-mannose patch region in the

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absence of the heavy chain insertion.

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N332/N334 dependence arises early in the PGT130 branch

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To explore the evolution of the high-mannose patch bnAb response and the divergence

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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,

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longitudinal samples were not available from this individual and, therefore, only PBMCs

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from a single time point at which the bnAbs were isolated were sequenced and used to

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model the lineage evolution of the PGT128 family. To produce libraries for 454

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sequencing, gene-specific primers were used on the population of 78,000 sorted IgG+

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memory B cells to amplify the IGHV4-39 and IGLV2-08 gene families from which

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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

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(Figure S1a and S1b) were generated using a new Bayesian phylogeny algorithm,

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ImmuniTree, that models somatic hypermutation for the PGT125-131 family of bnAbs

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(see methods for full details). The trees consist of both inferred and observed nodes with

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the observed nodes being the most highly mutated. To address sequencing errors, the

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algorithm collapsed the 390,563 heavy chain sequences and 711,575 light chain

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sequences into 108 heavy chain nodes (named 1H to 108H) and 136 light chain nodes

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(named 1L to 136L). Of these nodes, 24 heavy chain and 32 light chain nodes were

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inferred statistically and did not have direct sequencing data. As we were interested in

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whether less mutated inferred intermediates had neutralizing activity, heavy and light

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chain nodes with varying levels of mutation at key branch points were selected and

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paired to generate a simplified tree (Figure 2A, Table 1 and Table S1).

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Breadth and potency of the purified antibodies were initially determined on the 6-

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pseudovirus indicator panel (Figure 2B; neutralization data for a larger panel of chimeric

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bnAbs can be found in Figure S2). As seen for other families of bnAbs, no HIV reactivity

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for the predicted germ line antibody was detected on the 6-virus panel (Figure 2B) and

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no binding to the corresponding recombinant gp120 glycoprotein was observed in ELISA

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(data not shown) (31-35). Within the PGT128 branch, the least mutated predicted

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intermediate 95H 71L (12% mutated in nucleotide sequence of the heavy and light V-

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and J-gene segments) was able to neutralize all 6 viruses albeit at a slightly lower

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potency than the fully mutated PGT128 (16%). In the PGT130 branch, intermediate 3H

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3L (11%) showed no neutralization activity. This is in contrast to the PGT121 family of

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bnAbs where 46% neutralization was observed with 6% SHM (20). However, chimeric

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antibody 74H 3L (13%) showed very limited neutralization activity.

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To investigate how the differences in N332 and N334 dependency arose between the

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two branches, we next tested intermediate and mature bnAbs on a 54-cross clade

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pseudovirus panel (Table S2 and Figure 3A and 3B) that included 41 viruses with a

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glycan at position N332, 12 viruses with a glycan at position N334 and one virus lacking

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both N332/N334. 95% of clade CRF01_AE viruses have a glycan site at position N334

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whereas the remaining subtypes have >75% of viruses with a glycan site at position

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N332 (36). For both the PGT128 and PGT130 branches, an increased level of SHM was

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seen to increase breath and potency of neutralization as illustrated in the cumulative

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frequency distribution plot (Figure 3C). As shown previously, PGT130 neutralized more

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N334 viruses (73%) than PGT128 (58%) (18) and PGT128 neutralized more N332 15

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viruses (98%) than PGT130 (73%). The less mutated PGT130 branch bnAb 74H 3L,

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although only able to neutralize 13% of viruses, was able to neutralize 42% of N334

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viruses compared to 5% of N332 viruses (Figure 3A and 3B). This suggests that the

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PGT130 branch may have initially evolved to be more N334 specific before developing

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N332 reactivity. To further explore the N332/N334 dependency of the two bnAb classes,

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we next tested them against two mutated forms of the 6-virus panel where the glycan at

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N332 was either shifted to N334 or removed by an N332A mutation (Figure 3D) (18).

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Notably, 74H 3L could now neutralize JRCSF N334 and 92RW020 N334, although not

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the N332A versions, further supporting a preference for N334 (Figure 3D).

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A somatic variant that is predominantly N334 dependent is observed in the

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PGT130 branch

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In addition to clonal variants that cluster into the PGT128 and PGT130 branches, new

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heavy and light chain sequences were identified within the PGT130 branch of the heavy

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and light chain trees. 9H (24% mutated) also contains no indels but is significantly

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divergent from PGT130, and was paired with 46L and this new somatic variant 9H 46L

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(19%) was seen to be predominantly N334-dependent. This dependency was further

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confirmed on a small number of N334A variant viruses (Figure 3D and Figure S3A-B)

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and the N334 shifted panel (Figure 3D). The data suggest that predominantly N334-

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dependent somatic variants exist within the polyclonal response against the high-

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mannose patch, although single B cell cloning would be required to confirm the correct

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heavy and light chain pairing. Of note, although 9H 46L is more N334 dependent, the

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epitope is still centered on the N301 glycan site as shown by its inability to neutralize a

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JRCSF N334 variant lacking the N301 glycan (Figure S3C-D).

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High levels of SHM are necessary to accommodate V1 loop glycans 16

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Many of the N332-dependent bnAbs require binding to additional neighboring N-linked

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glycans for neutralization (1, 15, 17). Sok et al. recently showed that putative precursors

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of PGT121 are dependent on both N301 and N332 glycans and that the dependency on

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the N301 glycan is reduced with increased SHM (20). To determine how SHM and indels

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impact on the dependency of these two classes of bnAbs on N-linked glycans within the

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high-mannose patch, we used a panel of JRCSF glycan mutant viruses and measured

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impact on neutralization by PGT128, PGT130, 95H 71L, 3H 130L and PGT128-insert

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(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