Staged induction of HIV-1 glycan dependent broadly · George M. Shaw,3 Beatrice H. Hahn,3 Stephen C. Harrison,4 Bette T. Korber,11† Barton F. Haynes1,2† A preventive HIV-1 vaccine

SC I ENCE TRANS LAT IONAL MED I C I N E | R E S EARCH ART I C L E

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1Department of Medicine, Duke University School of Medicine, Duke University MedicalCenter, Durham, NC 27710, USA. 2DukeHumanVaccine Institute, Durham, NC 27710, USA.3Departments of Medicine and Microbiology, Perelman School of Medicine, University ofPennsylvania, Philadelphia, PA19104,USA. 4LaboratoryofMolecularMedicine, BostonChil-dren’s Hospital, HarvardMedical School, Boston,MA02115, USA. 5Department of ChemicalBiology, Memorial Sloan Kettering Cancer Center, New York, NY 10065, USA. 6Departmentof Surgery, Duke University School of Medicine, Duke University Medical Center, Durham,NC 27710, USA. 7Vaccine Research Center, National Institute of Allergy and Infectious Dis-eases, National Institutes of Health, Bethesda, MD 20892, USA. 8Department of Immunol-ogy, Duke University School of Medicine, Duke University Medical Center, Durham, NC27710, USA. 9National Institute for Communicable Diseases, Johannesburg 2131, SouthAfrica. 10Department of Medicine, University of Alabama at Birmingham, Birmingham,AL35294,USA. 11LosAlamosNational Laboratory, LosAlamos,NM87545,USA. 12Universityof North Carolina Project, Kamuzu Central Hospital, Lilongwe, Malawi. 13Departments ofMedicine, Epidemiology, andMicrobiology and Immunology, University of North Carolina,Chapel Hill, NC 27599, USA. 14Department ofMicrobiology andDepartment ofMathemat-ics and Statistics, Boston University, Boston, MA 02215, USA. 15Department of Pediatrics,Duke University School of Medicine, Duke University Medical Center, Durham, NC 27710,USA.*Corresponding author. Email: [email protected] (M.B.); [email protected] (B.T.K.); [email protected] (B.F.H.)†These authors contributed equally to this work.‡Present address: FHI 360, Lilongwe, Malawi.

Bonsignori et al., Sci. Transl. Med. 9, eaai7514 (2017) 15 March 2017

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Staged induction of HIV-1 glycan–dependent broadlyneutralizing antibodiesMattia Bonsignori,1,2*† Edward F. Kreider,3† Daniela Fera,4† R. Ryan Meyerhoff,1,2† Todd Bradley,1,2†

Kevin Wiehe,1,2 S. Munir Alam,1,2 Baptiste Aussedat,5 William E. Walkowicz,5 Kwan-Ki Hwang,2

KevinO. Saunders,2,6 Ruijun Zhang,2MorganA.Gladden,2 AnthonyMonroe,2 Amit Kumar,2 Shi-MaoXia,2

Melissa Cooper,2 Mark K. Louder,7 Krisha McKee,7 Robert T. Bailer,7 Brendan W. Pier,4 Claudia A. Jette,4

Garnett Kelsoe,2,8 Wilton B. Williams,1,2 Lynn Morris,9 John Kappes,10 Kshitij Wagh,11 Gift Kamanga,12‡

Myron S. Cohen,13 Peter T. Hraber,11 David C. Montefiori,2,6 Ashley Trama,2 Hua-Xin Liao,1,2

Thomas B. Kepler,14 M. Anthony Moody,2,8,15 Feng Gao,1,2 Samuel J. Danishefsky,5 John R. Mascola,7

George M. Shaw,3 Beatrice H. Hahn,3 Stephen C. Harrison,4 Bette T. Korber,11*† Barton F. Haynes1,2*†

A preventive HIV-1 vaccine should induce HIV-1–specific broadly neutralizing antibodies (bnAbs). However, bnAbsgenerally require high levels of somatic hypermutation (SHM) to acquire breadth, and current vaccine strategies havenot been successful in inducing bnAbs. Because bnAbs directed against a glycosylated site adjacent to the third var-iable loop (V3) of theHIV-1 envelopeprotein require limited SHM, the V3-glycan epitope is an attractive vaccine target.By studying the cooperation among multiple V3-glycan B cell lineages and their coevolution with autologous virusthroughout 5 years of infection, we identify key events in the ontogeny of a V3-glycan bnAb. Two autologous neu-tralizing antibody lineages selected for virus escape mutations and consequently allowed initiation and affinity mat-uration of a V3-glycan bnAb lineage. The nucleotide substitution required to initiate the bnAb lineage occurred at alow-probability site for activation-induced cytidine deaminase activity. Cooperation of B cell lineages and an im-probable mutation critical for bnAb activity defined the necessary events leading to breadth in this V3-glycan bnAblineage. These findings may, in part, explain why initiation of V3-glycan bnAbs is rare, and suggest an immunizationstrategy for inducing similar V3-glycan bnAbs.

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INTRODUCTIONA vaccine to prevent HIV-1 infection should include immunogens thatcan induce broadly neutralizing antibodies (bnAbs) (1, 2). Of the fivemajor targets for bnAbs, the glycan-rich apex of the HIV-1 envelope(Env) trimer and the base of the third variable loop (V3) are distin-guished by the potency of antibodies directed against them (3–8). Al-though these antibodies have less breadth than those directed againstthe CD4-binding site (CD4bs) or the gp41 membrane-proximal exter-nal region (MPER), one current goal of vaccine development is to elicitthem in combination with other bnAb specificities to achieve broadcoverage of transmitted/founder (TF) viruses to prevent HIV-1 integra-tion upon exposure (1, 2).

Mapping the coevolution of virus and antibody lineages over timeinforms vaccine design by defining the succession of HIV-1 Env var-iants that evolve in vivo during the course of bnAb development (9–11).Antibody lineages with overlapping specificities can influence eachother’s affinity maturation by selecting for synergistic or antagonisticescape mutations: an example of such “cooperating” lineages is providedby two CD4bs-directed bnAbs that we characterized previously (11, 12).Thus, cooperating antibody lineages and their viral escape mutants al-low identification of the specific Envs, among the diverse repertoire ofmutated Envs that develop within the autologous quasi-species in theinfected individual, that stimulate bnAb development and that we wishto mimic in a vaccine.

Here, we describe the coevolution of an HIV-1 Env quasi-speciesand a memory B cell lineage of gp120 V3-glycan–directed bnAbs inan acutely infected individual followed over time as broadly neutraliz-ing plasma activity developed. To follow virus evolution, we sequenced~1200 HIV-1 env genes sampled over a 5-year period; to follow theantibody response, we identified natural heavy- and light-chain pairsof six antibodies from a bnAb lineage, designated DH270, andaugmented this lineage by next-generation sequencing (NGS). Struc-tural studies defined the position of the DH270 Fab on gp140 Env. Wealso found two B cell lineages (DH272 and DH475) with neutraliza-tion patterns that likely selected for observed viral escape variants,which, in turn, stimulated the DH270 lineage to potent neutralizationbreadth. We found a mutation in the DH270 heavy chain thatoccurred early in affinity maturation at a disfavored activation-inducedcytidine deaminase (AID) site and that was necessary for bnAb lineageinitiation. This improbable mutation can explain the long period ofantigenic stimulation needed for initial expansion of the bnAb B celllineage in this individual.

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RESULTSThree N332 V3-glycan–dependent antibody lineagesWe studied an African male from Malawi (CH848) followed from thetime of infection up to 5 years after transmission. He was infected witha clade C virus, developed plasma neutralization breadth 3.5 years af-ter transmission, and did not receive antiretroviral therapy during thistime as per country treatment guidelines. Reduced plasma neutraliza-tion of N332A Env–mutated HIV-1 pseudoviruses and plasma neu-

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tralization fingerprinting demonstratedthe presence of N332-sensitive bnAbs(table S1) (13). To identify these anti-bodies, we studied memory B cells fromweeks 205, 232, and 234 after infectionusing memory B cell cultures (14) andantigen-specific sorting (15, 16) andfound three N332-sensitive lineages, de-signated DH270, DH272, and DH475.Their genealogy was augmented by theNGS of memory B cell complementaryDNA from seven time points from week11 to week 240 after transmission.

DH270 antibodies were recoveredfrommemory B cells at all three samplingtimes (weeks 205, 232, and 234), and ex-pansion of the clone did not occur untilweek 186 (Fig. 1A and table S2). Clonalexpansion was concurrent with the devel-opment of plasma neutralization breadth(tableS3), andmembers of theDH270 line-age also displayed neutralization breadth(Fig. 1B and data set S1). Themost potentDH270 lineage bnAb (DH270.6) was iso-lated using a fluorophore-labeled Man9-V3glycopeptide that isamimicof theV3-glycanbnAb epitope (16), which comprises a dis-continuous 30–amino acid residue peptidesegmentwithin gp120V3 and is represen-tative of the PGT128-boundminimal ep-itope described by Pejchal et al. (17). Thesynthetic Man9-V3 glycopeptide includeshigh-mannose glycan residues (Man9)each at N301 and N332 and was synthe-sized using a chemical process similar tothat described previously (18, 19). TheV3-glycan bnAb PGT128 affinity for theMan9-V3 glycopeptide was similar to thatof PGT128 for the BG505 SOSIP trimer,and Man9-V3 glycopeptide was thereforean effective affinity bait for the isolation ofV3-glycan bnAbs (16). The lineage derivedfrom a VH1-2*02 rearrangement thatproduced a CDRH3 [complementarity-determiningregion(CDR)H3]of20–aminoacid residues paired with a light chain en-coded by Vl2-23 (fig. S1). Neutralizationassays and competitionwith theV3-glycanbnAbs PGT125 and PGT128 confirmed de-pendenceof lineageon thepotentialN-linkedglycosylation (PNG) site at N332 (fig. S2).


The DH475 mAb was recovered from memory B cells at week 232after transmission by antigen-specific sorting using the fluorophore-labeled Man9-V3 glycopeptide (16). The earliest DH475 lineageVH(D)JH rearrangements were identified with the use of NGS atweek 64 after transmission (fig. S3A and table S2). Its heavy chaincame from VH3-23*01 [VH (variable gene of immunoglobulin heavychain) mutation frequency, 10.1%] paired with a Vl4-69*02 lightchain (fig. S3B).

Fig. 1. DH270 lineage with time of appearance and neutralization by selected members. (A) Phylogenetic relation-ship of six monoclonal antibodies (mAbs) and 93 NGS VH(D)JH sequence reads in the DH270 clonal lineage. External nodes(filled circles) represent VH(D)JH nucleotide sequences of either antibodies retrieved from cultured and sortedmemoryB cells (labeled) or a curated data set of NGS VH(D)JH rearrangement reads (unlabeled). Coloring is by time of isolation.Samples from weeks 11, 19, 64, 111, 160, 186, and 240 were tested, and time points from which no NGS reads withinthe lineage were retrieved are reported in table S2. Internal nodes (open circles) represent inferred ancestral inter-mediate sequences. Units for branch-length estimates are nucleotide substitution per site. (B) Neutralization dendro-grams display single mAb neutralization of a genetically diverse panel of 207 HIV-1 isolates. Coloring is by medianinhibitory concentration (IC50). See also data set S1.

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The DH272 mAb came from cultured memory B cells obtained atweek 205 after transmission. DH272 lineage VH(D)JH rearrangementswere detected as early as 19 weeks after transmission by NGS (fig. S3Aand table S2). The DH272 heavy chain used VH1-2*02, as did DH270,but it paired with a Vk2-30 light chain. Its CDR H3 was 17 aminoacids long; VH mutation was 14.9%. DH272, an immunoglobulin A(IgA) isotype, had a six-nucleotide deletion in FR H3 (fig. S3B).

For both DH272 and DH475 lineages, binding to CH848 TF Envgp120 depended on the N332 PNG site (fig. S3C). DH272 bindingalso depended on the N301 PNG site (fig. S3C). Neither lineagehad neutralization breadth (fig. S3D).

Evolution of the CH848 virus quasi-speciesWe sequenced 1223 HIV-1 3′ half single genomes from the virus inplasma collected at 26 time points over 246 weeks. Analysis of se-quences from the earliest plasma sample indicated that CH848 hadbeen infected with a single, subtype clade C founder virus, ~ 17 (con-fidence interval, 14 to 19) days before screening (figs. S4 and S5). Byweek 51 after infection, 91% of the sequences had acquired an iden-tical 10-residue deletion in variable loop 1 (V1), a region that includesthe PGT128-proximal residues 133 to 135 and 141 (figs. S6 and S7).Further changes accrued during the ensuing 4 years, including addi-tional insertions and deletions in V1, mutations in the 324GDIR327 mo-tif within the V3 loop, deletion or shifting of N-linked glycosylationsites at positions 301 and 322, and mutations at PGT128-proximalpositions in V1, V3, and C4; however, none of these escape variantswent to fixation during the 4.5 years of follow-up (figs. S6 to S9).

Simultaneous with the first detection of DH270 lineage antibodiesat week 186, four autologous virus clades emerged, which defined dis-tinct immunological resistance profiles of the CH848 autologous quasi-species (fig. S6). The first clade included viruses that shifted the PNGsite at N332 to N334 (fig. S6, open circles), and despite the fact that thismutation was associated with complete resistance to the DH270 line-age bnAbs, this clade was detected only transiently and at a relativelylow frequency (7 to 33% per sample), suggesting a balance where im-mune escape was countered by a cost in virological fitness. Conversely,viruses in the other three clades retained N332 and persisted through-out the 5 years of sampling. Viruses in the second clade resistedDH270 lineage neutralization and comprised gp120 Envs that werenot bound by the DH270 antibodies (fig. S6, triangles, and data setS2). The third and fourth clades defined autologous viruses whosegp120 Env was bound by DH270 lineage antibodies but that were eitheronly weakly neutralized by the most mature members of the DH270lineage (fig. S6, “X,” and data set S2) or completely neutralization-resistant (fig. S6, “+,” and data set S2), respectively. Persistence offour divergent clades in the CH848 Env, each with distinctive immu-nological resistance phenotypes, suggests that multiple distinctive immuneescape routes were explored and selected, allowing continuing Envescape mutations to accrue in distinct frameworks and exposing theantibody to Env diversity that may have been necessary to acquireneutralization breadth.

Ontogeny of DH270 lineage and acquisition ofneutralization breadthAs with other V3-glycan bnAbs, viral neutralization clade specificityand intraclade breadth of DH270 depended primarily on the frequen-cy of the N332 glycosylation site within the relevant clade (Fig. 2A).Only 1 of the 62 pseudoviruses tested that lacked the PNG site atN332, the B clade virus 5768.04, was sensitive to DH270.5 and


DH270.6 (data set S1). Across the full panel of M group HIV-1 virusisolates used in neutralization assays, the loss of the PNG N332 sitesaccounted for 70% of the observed neutralization resistance. The cir-culating recombinant form CRF01 rarely has this glycosylation site[3% of sequences in the Los Alamos database and 4% (1 of 23) inour test panel], and the DH270 lineage antibodies did not neutralizeCRF01 strains (Fig. 2A). As a consequence of the N332 PNG site re-quirement of V3-glycan bnAbs to neutralize, in vitro estimation ofneutralizing breadth was affected simply by the fraction of CRF01viruses included in the panel. Other V3-glycan bnAbs (10-1074,PGT121, and PGT128) shared this N332 glycan dependency, butPGT121 and PGT128 were not as restrictive (data set S1) (5, 6, 8).Antibody 10-1074 was similar to DH270.6, in that it more strictly re-quired the N332 PNG site, and its neutralization potency correlatedwith that of DH270.6 (Pearson’s r = 0.63, P = 8.0 × 10−13) (8).

Heterologous breadth and potency of the DH270 lineage antibo-dies increased with the accumulation of VH mutations, and althoughDH270.UCA did not neutralize heterologous HIV-1, five amino acidsubstitutions in DH270.IA4 (four in the heavy chain and one in thelight chain) were sufficient to initiate the bnAb lineage and conferheterologous neutralization (Fig. 2, B and C, and data set S3).

The capacity of the early DH270 lineage members to neutralizeheterologous viruses correlated with the presence of short V1 loops(Fig. 2D). As the lineage evolved, it gained capacity to neutralize viruseswith longer V1 loops, although with reduced potency (Fig. 2D and fig.S10, A to C). Neutralization of the same virus panel by the V3-glycanbnAbs 10-1074, PGT121, and PGT128 followed the same inverse cor-relation between potency and V1 length (fig. S10, D to F).

Mutations in the DH270 antibody lineage that initiatedheterologous neutralizationThe likelihood of AID-generated somatic mutation in immunoglobulingenes has strong nucleotide sequence dependence (20, 21). Moreover,we have recently shown for CD4bs bnAbs that VH sites of high intrinsicmutability determine many sites of somatic hypermutation (11).Like the VRC01-class CD4bs bnAbs, both DH270 and DH272 usedVH1-2*02, although unlike the CD4bs bnAbs, V3-glycan bnAbs ingeneral can use quite disparate VH gene segments (3, 17, 22–25).Antibodies in both lineages have mutations at the same amino acidpositions that correspond to sites of intrinsic mutability that we iden-tified in theVH1-2*02CD4bs bnAbs (fig. S11A) (11). InHIV-1–negativeindividuals, we identified 20 amino acids that frequentlymutate from theVH1-2*02 germline sequence (fig. S11A). Twelve of these 20 amino acidswere also frequentlymutated in theDH270 lineage antibodies, and 11 ofthese 12 amino acids mutated to 1 of the 2 most frequent amino acidsmutated in non–HIV-1 VH1-2*02 sequences (identity conformity).G57Rwas the lone exception. DH272mutated in 6 of these 12 positions,and CD4bs bnAb VRC01 mutated in 11 of 12 positions (fig. S11A).

The presence of the canonical VH1-2*02 allele in individual CH848was confirmed by genomic DNA sequencing (fig. S11B). Four nucle-otide changes in the DH270.UCA conferred heterologous neutraliza-tion activity on the next intermediate antibody (IA4). The G92A andG102A nucleotide mutations in DH270.IA4 (and in DH272) occurredat “canonical” AID hotspots (DGYW) and encoded amino acid sub-stitutions G31D and M34I, respectively (Fig. 3A). G164C (G164A forDH272) was in a “noncanonical” AID hotspot with a comparable levelof mutability (20) and encoded the S55T (N for DH272) substitution(Fig. 3A). In contrast, the G169C mutation in DH270.IA4, which en-coded the G57R amino acid mutation, occurred at a site with a very

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low predicted level of mutability (20), generated a canonical cold spot(GTC), and disrupted the overlapping AID hotspot at G170 within thesame codon, which was instead used by DH272 and resulted in theG57V substitution (Fig. 3A). Thus, although both the DH270 bnAband DH272 autologous neutralizing lineages had mutations at Gly57,the substitution in the DH270 lineage (G57R) was an improbable


event, whereas the substitution (G57V) in the DH272 lineage wasmuch more probable.

The G31D and M31I substitutions that occurred in AID hotspotsbecame fixed in both lineages, and S55T eventually became prevalentalso in the DH272 lineage (Fig. 3B). By week 111 after transmission,all DH272 lineage VH(D)JH transcripts sequenced by NGS harbored a

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ig. 2. Heterologous breadth in the DH270neage. (A) Neutralizing activity of DH270.1H270.5, and DH270.6 bnAbs (columns) fo07 tier 2 heterologous viruses (rows). Coloringby neutralization IC50 (in mg/ml). The first colmn displays the presence of a PNG site aosition 332 (blue), at N334 (orange), or aeither one (black). The second column indiates the clade of each individual HIV-1 strainnd is color-coded as indicated: clade A, greenlade B, blue; clade C, yellow; clade D, purpleRF01, pink; clade G, cyan; others, gray. See datat S1. (B). Heterologous neutralization of alH270 lineage antibodies for a 24-virus paneolor coding for the presence of PNG siteslade, and IC50 is the same as in (A). See fig. S1nd data set S3. (C) Covariation between VH mutation frequencies (x axis), neutralization

breadth (y axis, top), and potency (y axis, bottom) of individual antibodies against viruses with a PNG site at position N332 from the larger (left) and smaller (right) pseudoviruspanels. (D) Correlation between viral V1 loop length and DH270 lineage antibody neutralization. Top: Neutralization of 17 viruses (with N332; sensitive to at least one DH270lineage antibody) by selected DH270 lineage antibodies from unmutated common ancestor (UCA) to mature bnAbs (x axis). Viruses are identified by their respective V1 looplengths (y axis); for each virus, neutralization sensitivity is indicated by an open circle, and resistance is indicated by a solid circle. The P value is a Wilcoxon rank sumcomparison of V1 length distributions between sensitive and resistant viruses. Bottom: Regression lines (IC50 for neutralization versus V1 loop length) for DH270.1 andDH270.6, with a P value based on Kendall’s t.




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mutation in the Gly57 codon, which resulted in the predominance ofan encoded aspartic acid (Fig. 3B). In contrast, only 6 of 758 (0.8%)DH270 lineage transcripts isolated 186 weeks after transmission hadVal57 or Asp57; 48 of 758 (6.3%) retained Gly57, whereas over two-thirds, 514 of 758 (67.8%), had G57R (Fig. 3B).

Because the rare G169C nucleotide mutation in DH270.IA4 intro-duced a cold spot and simultaneously disrupted the overlapping AID


hotspot, it had a high probability of beingmaintained once it occurred, and indeed,it was present in 523 of 758 (68%)DH270lineageVH sequences identifiedwithNGSat week 186 after transmission (Fig. 3C).

Reversion of Arg57 to Gly abrogatedDH270.IA4 neutralization of autologousand heterologous HIV-1 isolates (Fig. 3D).A DH270.IA4 R57V mutant (with thebase change that would have occurredhad the overlapping AID hotspot beenused) also greatly reduced DH270.IA4neutralization, confirming that Arg57,rather than the absence of Gly57, wasresponsible for the acquired neutraliz-ing activity (Fig. 3D). Finally, the DH270.UCA G57R mutant neutralized bothautologous and heterologous viruses,confirming that G57R alone could con-fer neutralizing activity on the DH270germline antibody (Fig. 3E). Thus, theimprobable G169C mutation conferredreactivity against autologous virus andinitiated acquisition of heterologousneutralization breadth in the DH270lineage.

A search for an Env that might selectfor the critical G57R mutation in DH270.UCA or IA4-like antibodies yielded Env10.17 at week 135 of infection (fig. S12,A and B), which derived from the onlyautologous virus Env that DH270.IA4could bind. DH270.IA4 binding to Env10.17 depended on the presence ofArg57, and reversion of Arg57 to Gly(R57G) was necessary and sufficientto abrogate binding (fig. S12A). Also,binding to Env 10.17 was acquired byDH270.UCA upon introduction of theG57R mutation (fig. S12B).

Autologous neutralizing antibodylineages that cooperatedwith DH270Evidence for functional interaction amongthe three N332-dependent lineages camefrom the respective neutralization profilesagainst a panel of 90 autologous virusesfrom TF to week 240 after transmission(Fig. 4A and data set S2). Both DH475andDH272 neutralized autologous virusesisolated during the first year of infection,

which were resistant to most DH270 lineage antibodies (only DH270.IA1 and DH270.4 neutralized weakly) (Fig. 4A). DH475 neutralizedviruses from week 15 to week 39, and DH272 neutralized the CH848TF and all viruses isolated up to week 51, when viruses that resistedDH475 and DH272 became strongly sensitive to the more mature anti-bodies in theDH270 lineage (VHnucleotidemutation frequency,≥5.6%)(Fig. 4A).

Fig. 3. A singledisfavoredmutationearlyduringDH270 clonal development conferredneutralizingactivity on theV3-glycan bnAbDH270precursor antibodies. (A) Nucleotide alignment of DH270.IA4 andDH272 to VH1-2*02 sequenceat the four VH positions that mutated from DH270.UCA to DH270.IA4. The mutated codons are highlighted in yellow. AIDhotspots are indicated by red lines (solid, canonical; dashed, noncanonical); AID cold spots are indicated by blue lines (solid,canonical; dashed, noncanonical) (20). At position 169, DH270.IA4 retained positional conformity with DH272 but not iden-tity conformity (red boxes). (B) Sequence logo plot of amino acids mutated from germline (top) in the NGS reads of theDH270 (middle) and DH272 (bottom) lineages at weeks 186 and 111 after transmission, respectively. Red asterisks indicateamino acids mutated in DH270.IA4. The black arrow indicates lack of identity conformity between the two lineages atamino acid position 57. (C) Sequence logo plot of nucleotide mutations (positions 165 to 173) in the DH270 and DH272lineages at weeks 186 and 111 after transmission, respectively. The arrow indicates position 169. (D) Effect of reversionmutations on DH270.IA4 neutralization. Coloring is by IC50. WT, wild type. (E) Effect of G57Rmutation on DH270.UCA auto-logous (top) and heterologous (bottom) neutralizing activity.

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The identification of specific mutations implicated in the switchof virus sensitivity was complicated by the high levels of mutationsaccumulated by virus Env over time (fig. S13 and data set S4). Weidentified virus signatures that defined the DH270.1 and DH272/DH475 immunotypes and introduced four of them, in various com-binations, into the DH272/DH475-sensitive virus that was closest insequence to the DH270.1-sensitive immunotype: a 10–amino aciddeletion in V1 (D134-143); a D185N mutation in V2, which intro-duced an N-linked glycosylation site; an N413Y mutation in V4,which disrupted an N-linked glycosylation site; and a 2–amino aciddeletion (D463-464) in V5.


The large V1 deletion was critical for DH270.1 neutralization, withsmaller contributions from the other changes; the V1 deletionincreased virus resistance to DH475 (3.5-fold increase). V1 loop–mediated resistance to DH475 neutralization increased further whencombined with the D463-464 V5 deletion (5-fold increase) (Fig. 4B).

The V1 loop of the TF virus (34 residues) was longer than the av-erage V1 length of 28 residues (range, 11 to 64) of HIV-1 Env se-quences found in the Los Alamos Sequence Database (26). Asobserved for heterologous neutralization, DH270 lineage antibodiesacquired the ability to neutralize larger fractions of autologous virusesas maturation progressed by gaining activity for viruses with longerV1 loops, although at the expense of lower potency (fig. S14, A toC). This correlation was less clear for gp120 binding (fig. S14, D toF), however, suggesting that the V1 loop length dependency of V3-glycan bnAb neutralization has a conformational component. Thus,DH475 cooperated with the DH270 bnAb lineage by selecting viralescape mutants sensitive to bnAb lineage members.

For DH272, the viral variants that we made did not implicate aspecific cooperating escape mutation. The D134-143 (V1 deletion)mutated virus remained sensitive to DH272 neutralization; both com-binations of the V1 deletion in our panel that were resistant to DH272and sensitive to DH270.1 included D185N, which also caused DH272resistance on its own but did not lead to DH270.1 sensitivity (Fig. 4C).Thus, we have suggestive, but not definitive, evidence that DH272 alsoparticipated in selecting escape mutants for the DH270 bnAb lineage.

Structure of DH270 lineage membersWe determined crystal structures for the single-chain variable frag-ment of DH270.1 and the Fabs of DH270.UCA3, DH270.3,DH270.5, and DH270.6, as well as DH272 (table S4). Because of un-certainty in the inferred sequence of the germline precursor (fig. S15,A and B), we also determined the structure of DH270.UCA1, whichhas a somewhat differently configured CDR H3 loop (fig. S15C); re-configuration of this loop during early affinity maturation could ac-count for the observed increase with respect to the UCA inheterologous neutralization by several intermediates. The variable do-mains of the DH270 antibodies superposed well, indicating that affin-ity maturation modulated the antibody-antigen interface withoutsubstantially changing the antibody conformation (Fig. 5A). Muta-tions accumulated at different positions for DH270 lineage bnAbsin distinct branches (fig. S16), possibly accounting for their distinctneutralization properties. DH272 had a CDR H3 configured different-ly from that of DH270 lineage members and a significantly longerCDR L1 (Fig. 5B), compatible with their distinct neutralization profiles.

We also compared the structures of DH270 lineage memberswith those of other N332-dependent bnAbs. All appear to haveone long CDR loop that can extend through the network of glycanson the surface of the gp120 subunit and contact the “shielded” pro-tein surface. The lateral surfaces of the Fab variable module canthen interact with the reconfigured or displaced glycans to eitherside. PGT128 has a long CDR H2 (Fig. 5C), in which a six-residueinsertion is critical for neutralization breadth and potency (5, 17).PGT124 has a shorter and differently configured CDR H2 loop, buta long CDR H3 instead (Fig. 5D) (27).

Structure of the DH270–HIV-1 Env complexWe determined a three-dimensional image reconstruction, fromnegative-stain electron microscopy (EM), of the DH270.1 Fabbound with a gp140 trimer (92Br SOSIP.664) (Fig. 5, E and F,

Fig. 4. Cooperation among DH270, DH272, and DH475 N332-dependentV3-glycan neutralizing antibody lineages. (A) Neutralizing activity of DH272,DH475, and DH270 lineage antibodies (columns) against 90 autologous virusesisolated from CH848 over time (rows). Neutralization potency (IC50) is shown asindicated in the bar. For each pseudovirus, presence of a PNG site at N332 or N334,and V1 loop length are indicated in the right columns. See also data set S2. (B and C)Susceptibility of autologous viruses bearing selected immunotype-specific mutationsto DH270.1 and to DH475 (B) or DH272 (C).

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and fig. S17). The threeDH270.1 Fabs proj-ect laterally,with their axesnearlynormal tothe threefold of gp140, in a distinctly more“horizontal” orientation than seen forPGT124, PGT135, and PGT128 (Fig. 5, Gand H, and fig. S18). This orientationaldifference is consistent with differences be-tween DH270 and PGT124 or PGT128 inthe lengths and configurations of theirCDRloops,which required an alternativeDH270bnAb position when docked onto the sur-face of the Env trimer. We docked theBG505 SOSIP coordinates (28) and theFab into the EM reconstruction and furtherconstrained the EM reconstruction imageby the observed effects of BG505 SOSIPmutations in the gp140 surface image (figs.S17 and S19). Asp325 was essential forbinding DH270.1 because it is a potentialpartner for Arg57 on the Fab. MutatingAsp321 led to a modest loss in affinity;R327A had no effect (fig. S20). These datafurther distinguish DH270 from PGT124and PGT128. Individually mutating W101,Y105, D107, D115, Y116, or W117 inDH270.1 to alanine substantially reducedbinding to the SOSIP trimer, as did pair-wise mutation to the alanines of S106 andS109. The effects of these mutations illus-trate the critical role of the CDR H3 loopin binding with HIV-1 Env (fig. S20).

DH270.UCA bindingThe DH270.UCA did not bind to any of the 120 CH848 autologousgp120 Env glycoproteins isolated from time of infection to week245 after infection, including the TF Env (Fig. 6A). DH270.UCA,as well as maturation intermediate antibodies, also did not recognizefree glycans or cell surface membrane–expressed gp160 trimers (Fig.


6B). Conversely, the DH270.UCA bound to theMan9-V3 synthetic gly-copeptide mimic of the V3-glycan bnAb gp120 epitope (fig. S21A) andalso bound to the aglycone form of the same peptide (fig. S21B). Simi-larly, the early intermediate antibodies (IA4, IA3, and IA2) each boundto both the Man9-V3 glycopeptide and its aglycone form, and their

Fig. 5. Fab/scFv crystal structures and three-dimensional reconstruction of DH270.1 boundwith the 92BR SOSIP.664 trimer. Superpositionof backbone ribbon diagrams for the DH270 lineagemembers: UCA1 (gray), DH270.1 (green), andDH270.6 (blue)alone (A),with theDH272cooperatingantibody (red) (B), with PGT128 (magenta) (C), andwith PGT124 (orange) (D). Arrows indicate major dif-ferences inCDR regions. Top (E) andside (F) viewsof afit of the DH270.1 Fab (green) and the BG505 SOSIPtrimer (gray)ontoamapobtainedfromnegative-stainEM. Top (G) and side (H) views of the BG505 trimer[Protein Data Bank (PDB) ID: 5ACO] (28) (gray, withV1-V2 and V3 loops highlighted in red and blue, re-spectively) bound to PGT124 (PDB ID: 4R2G) (27)(orange), PGT128 (PDB ID: 3TYG) (17) (magenta),PGT135 (PDB ID: 4JM2) (22) (cyan), and DH270.1(green), superposed. The arrows indicate the direc-tion of the principal axis of each of the bnAb Fabs;the color of each arrow matches that of thecorresponding bnAb. See also fig. S18.

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Fig. 6. DH270 lineage antibodybinding to autologous CH848Env components. (A) Binding ofDH270 lineage antibodies (col-umn) to 120 CH848 autologousgp120 Env glycoproteins (rows)grouped on the basis of time ofisolation (w, week; d, day; blackand white blocks). The last threerows show the neutralizationprofile of the three autologousviruses that lost the PNG at posi-tion N332 (blue blocks). V1 aminoacid length of each virus is color-coded as indicated. Antibodybinding is measured by enzyme-linked immunosorbent assay,expressed as log area under thecurve (LogAUC), and color-codedon the basis of the categoriesshown in the histogram. The his-togram shows the distribution ofthe measured values in eachcategory. The black arrow indi-cated Env 10.17. Viruses isolatedat and after week 186, whichis the time of first evidence ofDH270 lineage presence, arehighlighted in different colorsaccording to week of isolation.(B) Left: Binding toCH848 TFmu-tants with disrupted N301 and/orN332 glycan sites. Results areexpressed as LogAUC. VH muta-tion frequency is shown in paren-theses for each antibody (see alsofig. S1A). Middle: Binding toCH848 Env trimer expressed onthe cell surface of Chinese ham-ster ovary cells. Results are ex-pressed as maximum percentageof binding and are representativeof duplicate experiments. DH270antibodies are in red. Palivizumabis the negative control (gray area).The curves indicate binding to thesurface antigen on a scale of 0 to100 (y axis); the highest peak be-tween the test antibody and thenegative control sets the value of100. Right: Binding to free glycansmeasured on a microarray. Resultsare the average of background-subtracted triplicate measure-ments and are expressed in relativeunits (RU).

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binding was stronger to the aglycone V3 peptide than to the Man9-V3glycopeptide (fig. S21B). Overall, binding of the DH270.UCA andearly intermediate antibodies to the Man9-V3 glycopeptide was low(>10 mM) (fig. S21A). DH270.1 (VH nucleotide mutation frequency,5.6%) bound the glycopeptide with higher affinity than did the aglycone(Kd,glycopeptide = 331 nM) (fig. S21, A and B), and as mutations accumu-lated, binding of theMan9-V3 glycopeptide also increased, culminatingin a Kd (apparent dissociation constant) of 188 nM in the most potentbnAb, DH270.6, which did not bind to the aglycone V3 peptide (fig.S21, A and B). Thus, both the Man9-V3 glycopeptide and the aglyconeV3 peptide bound to the DH270.UCA, and antibody binding wasindependent of glycans until the DH270 lineage had acquired a nucle-otide mutation frequency of ~6%.


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DISCUSSIONWe can reconstruct from the data presented here a plausible series ofevents during the development of a V3-glycan bnAb in a natural in-fection. The DH272 and DH475 lineages neutralized the autologousTF and early viruses, and the resulting escape viruses were neutralizedby the DH270 lineage. In particular, V1 deletions were necessary forneutralization of all but the most mature DH270 lineage antibodies.DH475 (and possibly DH272) escape variants stimulated DH270 af-finity maturation, including both somatic mutations at sites of intrin-sic mutability (11) and a crucial, improbable mutation at an AID coldspot within CDR H2 (G57R). The G57R mutation initiated expansionof the DH270 bnAb lineage. The low probability of this heterologousneutralization-conferring mutation and the complex lineage interac-tions that occurred is one explanation for why it took 4.5 years forthe DH270 lineage to expand.

The CH848 viral population underwent a transition from a longV1 loop in the TF (34 residues) to short loops (16 to 17 residues)when escaping DH272/DH475 and facilitating expansion of DH270,to restoration of longer V1 loops later in infection as resistance toDH270 intermediates developed. Later DH270 antibodies adapted toviruses with longer V1 loops, allowing recognition of a broaderspectrum of Envs and enhancing breadth. DH270.6 could neutralizeheterologous viruses regardless of V1 loop length, but viruses withlong loops tended to be less sensitive to it. Association of long V1 loopswith reduced sensitivity was evident for three other V3-glycan bnAbsisolated from other individuals and may be a general feature of this class.

The V1 loop deletions in CH848 autologous virus removed thePNG site at position 137. Although the hypervariable nature of theV1 loop (which evolves by insertion and deletion, resulting in extremelength heterogeneity, as well as extreme variation in the number ofPNG sites) complicates the interpretation of direct comparisons amongunrelated HIV-1 strains, it is worth noting that a PNG site in thisregion specified as N137 was shown to be important for regulating af-finity maturation of the PGT121 V3-glycan bnAb family, with somemembers of the lineage (PGT121 to PGT123) evolving to bind andothers (PGT124) to accommodate or avoid this glycan (29).

Because we cannot foresee the susceptibility of each specificpotential TF virus to a particular bnAb lineage it will be importantfor a vaccine to induce bnAbs against multiple epitopes on theHIV-1 Env to minimize TF virus escape (30, 31). In particular, induc-tion of bnAb specificities beyond the HIV-1 V3-glycan epitope is crit-ical for use in Asian populations where CRF01 strains (which, for themost part, lack the N332 PNG site required for efficient neutralizationby V3-glycan bnAbs) are frequently observed.


Regardingwhatmight have stimulated theUCAof theDH270 bnAblineage the absence of detectable binding to the CH848 TF Env raised atleast two possibilities. One is that the lineage arose at the end of year 1,either from a primary response to viruses present at that time (for ex-ample, with deletions in V1-V2) or from a subversion of an antibodylineage initially elicited by some other antigens. The other is that somealtered form of the CH848 TF Env protein (for example, shed gp120, ora fragment of it) exposed theV3 loop and theN301 andN332 glycans ina way that bound and stimulated the germline B cell receptor (BCR),although the native CH848 TF Env did not. Our findings suggest thata denatured, fragmented, or otherwise modified form of Env may haveinitiated the DH270 lineage. We cannot exclude that the DH270.UCAcould not bind to autologous Env as an IgG but could potentially betriggered as an IgM BCR on a cell surface.

It will be important to define how often an improbable mutationsuch as G57R determines the time it takes for a bnAb lineage in anHIV-1–infected individual to develop and how many of the accompa-nying mutations are necessary for potency or breadth, rather than be-ing nonessential mutations at AID mutational hotspots (11, 32).Mutations of the latter type might condition the outcome or modulatethe impact of a key, improbable mutation, without contributing direct-ly to affinity. Should the occurrence of an unlikely mutation be rate-limiting for breadth or potency in many other cases, a program ofrational immunogen design will need to focus on modified Envs mostlikely to select very strongly for improbable yet critical antibody nu-cleotide changes.

The following proposal for a strategy for inducing V3-glycanbnAbs recreates the events that led to bnAb induction in CH848:starting by priming with a ligand that binds the bnAb UCA, suchas the synthetic glycopeptide mimic of the V3-glycan bnAb gp120 ep-itope, and then boosting with an Env that can select G57R CDR H2mutants, followed by Envs with progressive V1 lengths (fig. S22). Wehypothesize that more direct targeting of V3-glycan UCAs andintermediate antibodies can accelerate the time of V3-glycan bnAb de-velopment in the setting of vaccination.

A limitation of this approach is that the selection of immunogenswas based on the analysis of a single lineage from a single individual,and how frequently DH270-like lineages are present in the generalpopulation is unknown. Finally, our study describes a generalstrategy for the design of vaccine immunogens that can select specif-ic antibody mutations, thereby directing antibody lineage maturationpathways.

MATERIAL AND METHODSStudy designThe CH848 donor, from which the DH270, DH272, and DH475 an-tibody lineages were isolated, is an African male enrolled in theCHAVI (Center for HIV/AIDS Vaccine Immunology) 001 acuteHIV-1 infection cohort (33) and followed for 5 years, after whichhe started antiretroviral therapy. During this time, viral load rangedfrom 8927 to 442,749 copies/ml (median, 61,064 copies/ml), andCD4 counts ranged from 288 to 624 cells/mm3 (median, 350 cells/mm3). The time of infection was estimated by analyzing the sequencediversity in the first available sample using the Poisson-Fitter tool, asdescribed in (10). Results were consistent with a single founder virusestablishing the infection (34).

The mAbs DH270.1 and DH270.3 were isolated from culturedmemory B cells isolated 205 weeks after transmission (14). The mAbs

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DH270.6 and DH475 were isolated from Man9-V3 glycopeptide–specific memory B cells collected 232 and 234 weeks after transmis-sion, respectively, using direct sorting. The mAbs DH270.2, DH270.4,and DH270.5 were isolated from memory B cells collected 232 weeksafter transmission that bound to Consensus C gp120 Env but not toConsensus C N332A gp120 Env, using direct sorting.

Experimental methodsDetailed description of experimental methods is provided in Supple-mentary Materials and Methods.

Statistical analysesStatistical analysis was performed using R. The specific tests used todetermine significance are reported for each instance in the text.


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SUPPLEMENTARY MATERIALSwww.sciencetranslationalmedicine.org/cgi/content/full/9/381/eaai7514/DC1Materials and MethodsFig. S1. Characteristics of DH270 lineage mAbs.Fig. S2. DH270 lineage displays an N332-dependent V3-glycan bnAb functional profile.Fig. S3. DH475 and DH272 are strain-specific, N332-glycan–dependent antibodies.Fig. S4. CH848 was infected by a single TF virus.Fig. S5. CH848 was infected by a subtype C virus.Fig. S6. Coevolution of CH848 autologous virus and the N332-dependent V3-glycan antibodylineages DH272, DH475, and DH270.Fig. S7. Mutations in CH848 Env over time.Fig. S8. Accumulation of amino acid mutations in CH848 virus over time.Fig. S9. CH848 virus lineage maximum likelihood phylogenetic tree rooted on the TFsequence.Fig. S10. Inverse correlation between the potency of V3-glycan bnAbs and V1 length shown forthe full panel of 207 viruses.Fig. S11. Role of VH1-2*02 intrinsic mutability in determining DH270 lineage antibody somatichypermutation.Fig. S12. Effect of the G57R mutation on DH270.IA4 and DH270.UCA binding to Env 10.17gp120.Fig. S13. Virus signature analysis.Fig. S14. Autologous Env V1 length associations with DH270 lineage neutralization and gp120binding.Fig. S15. Sequence and structural comparison of DH270.UCA1 and DH270.UCA3.Fig. S16. Accumulation of mutation in DH270 lineage antibodies.Fig. S17. Negative-stain EM of DH270 Fab in complex with the 92BR SOSIP.664 trimer.Fig. S18. DH270.1 and other N332 bnAbs bound to the 92BR SOSIP.664 trimer.Fig. S19. DH270.1 binding kinetics to 92BR SOSIP.664 trimers with mutated PNG sites.Fig. S20. DH270.1 binding kinetics to 92BR SOSIP.664 trimer with additional mutations.Fig. S21. Man9-V3 glycopeptide binding of DH270 lineage antibodies.Fig. S22. Example of an immunization regimen derived from studies of virus-bnAb coevolutionin CH848.Table S1. N332-dependent CH848 plasma neutralization.Table S2. NGS longitudinal sampling of VH(D)JH rearrangements assigned to the DH270,DH272, and DH475 lineages from memory B cell mRNA.Table S3. CH848 plasma neutralization breadth over time.Table S4. Data collection and refinement statistics.Data set S1. DH270 lineage heterologous neutralization (207-virus panel).Data set S2. Autologous binding and neutralization of DH270, DH272, and DH475 lineages.Data set S3. Heterologous neutralization on a 24-virus panel.Data set S4. Virus signatures.References (36–52)

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Acknowledgments: We thank the following for technical assistance: M. C. Mangiapani,R. J. Parks, C. Vivian, K. Lloyd, D. M. Kozink, F. Perrin, A. J. Cooper, A. Foulger, G. Hernandez,J. Pritchett, S. Arora, T. Gurley, L. Armand, A. Eaton, A. Wang, and the Duke Human VaccineInstitute (DHVI) Flow Core Facility from DHVI; G. H. Learn, T. G. Decker, and Y. Li from theUniversity of Pennsylvania; N. Tumba from the National Institute for Communicable Diseases(Johannesburg); the beamline staff at 24-ID-C and 24-ID-E of the Advanced Photon Source,and the Harvard Medical School EM staff. We also thank K. Soderberg (DHVI) for projectmanagement and the HIV Vaccine Trials Network for the continued use of NGSprevaccination samples from the HVTN082 and HVTN204 trials (35). Funding: E.F.K. andR.R.M. are supported by the F30 Ruth L. Kirschstein National Research Service Award(AI112426 and AI122982-0, respectively). D.F. is supported by an F32 fellowship(1F32AI116355-01) from the NIH. R.R.M. is supported by a Medical Scientist Training Programgrant (T32GM007171). K.O.S. is supported by a National Institute of Allergy and InfectiousDiseases (NIAID) grant (R01-AI120801). This work was funded by UM1 AI100645 from theDuke CHAVI–Immunogen Discovery, Division of AIDS, NIAID, NIH (to B.F.H.). Authorcontributions: M.B., B.H.H., S.C.H., B.T.K., and B.F.H. coordinated and designed the study,analyzed and evaluated the data, and wrote and edited the manuscript and figures; E.F.K.,


D.F., T.B., K. Wiehe, K.O.S., A.M., and S.M.A. performed experiments, analyzed the data, andproduced the figures; M.B. directed the memory B cell cultures and performed intrinsicmutability analysis; R.R.M. isolated the antibodies through antigen-specific single-cellsorting; D.F., B.W.P., and C.A.J. performed structural studies; B.A., W.E.W., and S.J.D.synthesized the Man9-V3 glycopeptide; K.-K.H., M.A.G., and A.M. executed the memoryB cell cultures and antibody functional screenings; R.Z., A.T., H.-X.L., A.K., and F.G. producedthe recombinant proteins and pseudoviruses; S.-M.X., M.C., M.K.L., K.M., R.T.B., J.R.M., andD.C.M. performed neutralization studies; G. Kelsoe, J.R.M., and G.M.S. provided intellectualcontributions; W.B.W., L.M., and J.K. contributed the NGS data, CH848 plasma profiling, andtransfected cell lines, respectively; K. Wagh, P.T.H., and B.T.K. performed virus signatureand virus evolution analyses; G. Kamanga and M.S.C. supervised the clinical sites; M.A.M.supported single-cell sorting; T.B.K. performed computational analysis; and B.F.H. conceivedthe study. All authors approved the paper. Competing interests: M.B., R.R.M., M.A.M., H.-X.L.,T.B., K.O.S., P.T.H., B.T.K., and B.F.H. have filed patent application(s) covering DH270lineage antibodies and immunogens to induce such antibodies. B.A., H.-X.L., B.F.H., andS.J.D. have filed patent application(s) covering the V3 glycopeptide and its synthesis and useas an immunogen. All other authors declare that they have no competing interests. Dataand materials availability: The V(D)J rearrangement sequences of DH272, DH475, and theDH270 lineage antibodies (DH270.UCA, DH270.IA1 to DH270.IA4, and DH270.1 to DH270.6)have been deposited in GenBank, with accession numbers KY354938 to KY354963. NGSsequence data for clones DH270, DH272, and DH475 have been deposited in GenBank,with accession numbers KY347498 to KY347701. Coordinates and structure factors for UCA1,UCA3, DH270.1, DH270.3, DH270.5, DH270.6, and DH272 have been deposited in thePDB, with accession code 5U0R, 5U15, 5U0U, 5TPL, 5TPP, 5TQA, and 5TRP, respectively.The EM map of the 92BR SOSIP.664 trimer in complex with DH270.1 has been deposited inthe EM Data Bank, with accession code EMD-8507.

Submitted 6 August 2016Resubmitted 18 August 2016Accepted 31 January 2017Published 15 March 201710.1126/scitranslmed.aai7514

Citation: M. Bonsignori, E. F. Kreider, D. Fera, R. R. Meyerhoff, T. Bradley, K. Wiehe, S. M. Alam,B. Aussedat, W. E. Walkowicz, K.-K. Hwang, K. O. Saunders, R. Zhang, M. A. Gladden, A. Monroe,A. Kumar, S.-M. Xia, M. Cooper, M. K. Louder, K. McKee, R. T. Bailer, B. W. Pier, C. A. Jette,G. Kelsoe, W. B. Williams, L. Morris, J. Kappes, K. Wagh, G. Kamanga, M. S. Cohen,P. T. Hraber, D. C. Montefiori, A. Trama, H.-X. Liao, T. B. Kepler, M. A. Moody, F. Gao,S. J. Danishefsky, J. R. Mascola, G. M. Shaw, B. H. Hahn, S. C. Harrison, B. T. Korber,B. F. Haynes, Staged induction of HIV-1 glycan–dependent broadly neutralizing antibodies.Sci. Transl. Med. 9, eaai7514 (2017).

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dependent broadly neutralizing antibodies−Staged induction of HIV-1 glycan

Bette T. Korber and Barton F. HaynesHarrison,Moody, Feng Gao, Samuel J. Danishefsky, John R. Mascola, George M. Shaw, Beatrice H. Hahn, Stephen C.

Myron S. Cohen, Peter T. Hraber, David C. Montefiori, Ashley Trama, Hua-Xin Liao, Thomas B. Kepler, M. AnthonyW. Pier, Claudia A. Jette, Garnett Kelsoe, Wilton B. Williams, Lynn Morris, John Kappes, Kshitij Wagh, Gift Kamanga, Anthony Monroe, Amit Kumar, Shi-Mao Xia, Melissa Cooper, Mark K. Louder, Krisha McKee, Robert T. Bailer, BrendanBaptiste Aussedat, William E. Walkowicz, Kwan-Ki Hwang, Kevin O. Saunders, Ruijun Zhang, Morgan A. Gladden, Mattia Bonsignori, Edward F. Kreider, Daniela Fera, R. Ryan Meyerhoff, Todd Bradley, Kevin Wiehe, S. Munir Alam,

DOI: 10.1126/scitranslmed.aai7514, eaai7514.9Sci Transl Med

that protects against diverse strains of HIV.these types of antibodies do not develop more often. These tools and findings could pave the way for a vaccine developed broadly neutralizing antibodies. They also examined viral evolution over time and found clues as to whyglycopeptide and other baits to study the V3-glycan antibody responses of an HIV-infected individual that

. used this syntheticet alidentify B cells targeting this epitope and also used it to immunize macaques. Bonsignori . designed a synthetic glycopeptide that canet alepitope are often not detected in people infected with HIV. Alam

thisvaccine design. One such site is a glycan near the V3 loop of the envelope protein, but antibodies recognizing Although it typically evades the immune system, HIV does have sites of vulnerability that can be targeted in

Guiding anti-glycan antibodies

ARTICLE TOOLS http://stm.sciencemag.org/content/9/381/eaai7514

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Staged induction of HIV-1 glycan dependent broadly · George M. Shaw,3 Beatrice H. Hahn,3 Stephen C. Harrison,4 Bette T. Korber,11*† Barton F. Haynes1,2*† A preventive HIV-1 vaccine

Staged induction of HIV-1 glycan dependent broadly · George M. Shaw,3 Beatrice H. Hahn,3 Stephen C. Harrison,4 Bette T. Korber,11† Barton F. Haynes1,2† A preventive HIV-1 vaccine