Vaccine-elicited primate antibodies use a distinctapproach to the HIV-1 primary receptor bindingsite informing vaccine redesignKaren Trana,1, Christian Poulsena,1, Javier Guenagaa,1, Natalia de Valb, Richard Wilsona, Christopher Sundlingc,Yuxing Lia,d, Robyn L. Stanfieldb, Ian A. Wilsona,b,e, Andrew B. Warda,b,e, Gunilla B. Karlsson Hedestamc,and Richard T. Wyatta,d,2

aInternational AIDS Vaccine Initiative Neutralizing Antibody Center, Departments of bIntegrative Structural and Computational Biology and dImmunologyand Microbial Science, and eCenter for HIV/AIDS Vaccine Immunology and Immunogen Discovery, The Scripps Research Institute, La Jolla, CA 92037;and cDepartment of Microbiology, Tumor and Cell Biology, Karolinska Institutet, 171 77 Stockholm, Sweden

Edited* by Michel C. Nussenzweig, The Rockefeller University, New York, NY, and approved January 7, 2014 (received for review October 16, 2013)

HIV-1 neutralization requires Ab accessibility to the functionalenvelope glycoprotein (Env) spike. We recently reported the isolationof previously unidentified vaccine-elicited, CD4 binding site (CD4bs)-directed mAbs from rhesus macaques immunized with soluble Envtrimers, indicating that this region is immunogenic in the context ofsubunit vaccination. To elucidate the interaction of the trimer-elicited mAbs with gp120 and their insufficient interaction withthe HIV-1 primary isolate spike, we crystallized the Fab frag-ments of two mAbs, GE136 and GE148. Alanine scanning of theircomplementarity-determining regions, coupled with epitope scan-ning of their epitopes on gp120, revealed putative contact residuesat the Ab/gp120 interface. Docking of the GE136 and GE148 Fabsto gp120, coupled with EM reconstructions of these nonbroadlyneutralizing mAbs (non-bNAbs) binding to gp120 monomers andEM modeling to well-ordered trimers, suggested Ab approach tothe CD4bs by a vertical angle of access relative to the more lateralmode of interaction used by the CD4bs-directed bNAbs VRC01 andPGV04. Fitting the structures into the available cryo-EM native spikedensity indicated clashes between these two vaccine-elicited mAbsand the topside variable region spike cap, whereas the bNAbsduck under this quaternary shield to access the CD4bs effectivelyon primary HIV isolates. These results provide a structural basisfor the limited neutralizing breadth observed by current vaccine-induced, CD4bs-directed Abs and highlight the need for better orderedtrimer immunogens. The analysis presented here therefore providesvaluable information to guide HIV-1 vaccine immunogen redesign.

NHP | neutralizing antibodies | mode of recognition

Entry of HIV-1 into susceptible primate CD4+ chemokinereceptor 5 (CCR5)+ target cells is mediated by the trimeric

surface envelope glycoproteins (Envs). The exterior Env, gp120,binds to the primary receptor, CD4, and to the coreceptor,CCR5. The conserved CD4 binding site (CD4bs) on gp120 issurrounded by highly variable regions and glycan shielding (1).The CD4bs is a major target for neutralizing Abs (2), and aplethora of potent and broadly neutralizing Abs (bNAbs) to thisregion, elicited during chronic HIV-1 infection, were isolatedrecently (3–7). In addition to the obvious rationale that theprimary virus receptor binding site is a desired region to targetfor vaccine-induced B-cell responses, the identification of potentCD4bs-directed bNAbs, such as VRC01, strengthens the conceptthat targeting the conserved CD4bs is a desirable approach toaccomplish broad neutralization (8–10). Although the use ofEnv-based vaccines to elicit bNAbs to the CD4bs has been un-successful so far, CD4bs-directed Abs of more limited neutrali-zation breadth have been elicited (11–13). Recently, a panel ofsuch Abs was isolated from single-cell–sorted memory B cellsfrom nonhuman primates (NHPs) inoculated with soluble HIV-1Env trimers (gp140-F, foldon trimers) (11, 14). Alanine (Ala)scanning of gp120 and subsequent binding studies revealed that

the epitopes of these vaccine-elicited mAbs partially overlap withthe CD4bs-directed bNAb VRC01 but more closely align with thehuman infection-elicited non-bNAb, F105 (15).To elucidate better the limitations of current vaccine-elicited,

CD4bs-directed mAbs to access the functional Env spike, wecrystallized the Fabs of two members of this class, GE148 andGE136. Using the high-resolution structures of the unligandedmAbs, we defined properties of epitope recognition using a sys-tematic analysis of the Ab–antigen interaction by paratope Alascans and mapping of their putative gp120-interactive regions byboth binding and virus neutralization assays. Using this in-formation and available gp120 core structures in concert withClusPro (16) docking, we obtained relatively high-resolutionmodels of the NHP mAb/gp120 complexes. To validate thisanalysis, we examined additional substitutions at predicted contactresidues and observed increased neutralization potencies for bothmAbs. The results indicated that the vaccine-elicited mAbs pri-marily used their heavy chain (HC) complementarity-determiningregion 3 (HCDR3) and light chain (LC) complementarity-determining region 1 (LCDR1) to interact with their cognate

Significance

The development of broadly neutralizing antibodies (bNAbs) toHIV-1 is often thought to be a key component of a successfulvaccine. A common target of bNAbs is the conserved CD4binding site (CD4bs) on the HIV envelope glycoprotein (Env)trimeric spike. Although CD4bs-directed bNAbs have beenisolated from infected individuals, elicitation of such bNAbs byEnv vaccination has proven difficult. To help understand thelimitations of current immunogens, we structurally character-ized two vaccine-elicited, CD4bs-directed non-bNAbs from pri-mates. We demonstrate that these vaccine-elicited Abs attempta vertical approach to the CD4bs, thereby clashing with thevariable region of the trimeric spike cap, whereas CD4bs-directed bNAbs adopt angles of approach that avoid suchclashes. This analysis can inform future vaccine redesign.

Author contributions: K.T., C.P., J.G., N.d.V., I.A.W., A.B.W., G.B.K.H., and R.T.W. designedresearch; K.T., C.P., J.G., N.d.V., R.W., and R.L.S. performed research; R.W., C.S., and Y.L.contributed new reagents/analytic tools; K.T., C.P., J.G., N.d.V., C.S., Y.L., R.L.S., I.A.W.,A.B.W., G.B.K.H., and R.T.W. analyzed data; and K.T., C.P., J.G., N.d.V., I.A.W., A.B.W.,G.B.K.H., and R.T.W. wrote the paper.

The authors declare no conflict of interest.

*This Direct Submission article had a prearranged editor.

Data deposition: The structure factors have been deposited in the Protein Data Bank,www.pdb.org (PDB ID codes 4KTD and 4KTE).1K.T., C.P., and J.G. contributed equally to this work.2To whom correspondence should be addressed. E-mail: wyatt@scripps.edu.

This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10.1073/pnas.1319512111/-/DCSupplemental.

E738–E747 | PNAS | Published online February 3, 2014 www.pnas.org/cgi/doi/10.1073/pnas.1319512111

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epitopes in the gp120 CD4bs. In silico docking and subsequentsuperimposition to available gp120 trimer models from electrontomography data of the native spike (17) revealed that the Ablikely approaches at a vertical angle to gain access to the CD4bs.This mode of approach was confirmed by negative-stain EMreconstructions of GE136 and GE148 in complex with gp120,and subsequent fitting to Env trimer reconstructions showed thataccess to the CD4bs in properly folded Env trimers that mimicthe native Env spike was not achievable.Thus, the results presented here indicate that these two vac-

cine-elicited, CD4bs-directed mAbs cannot access the functionalspike of primary HIV-1 isolates from a vertical angle of approach,where accessibility to the CD4bs is limited by steric clashes withthe overlying variable (V1/V2/V3) “loop” region cap (V-regioncap) on the membrane-distal region of the native spike. Thismode of binding is in contrast to the more lateral angles of ap-proach used by bNAbs, such as VRC01 (4, 18). These resultsprovide a structural explanation in the context of the functionalHIV-1 spike for the limited neutralization breadth displayed byCD4bs-directed Abs elicited by the current foldon trimers (11).EM studies of the foldon trimers confirmed that they have anopen configuration compared with native Env spikes, likelyallowing exposure of immunogenic CD4bs elements that are notexposed in the functional spike. These insights are invaluable forinformed second-generation iterative immunogen redesign toenhance vaccine-induced B-cell responses against circulatingHIV-1 variants.

ResultsCrystal Structures of GE136 and GE148. To define the properties ofCD4bs-directed Abs elicited by YU2 gp140-F trimers in NHPs,we analyzed the interactions of GE136 and GE148 with severalforms of Env. A summary of the gene use, HCDR3 length, bind-ing, and neutralization capacity of GE136 and GE148 is shown inFig. 1A. Although these mAbs bind with high affinity to mono-meric gp120, their neutralization breadth is largely restricted toneutralization-sensitive tier 1 viruses, with limited activity againsttier 2 viruses in the TZM-bl neutralization assay (19). However,they neutralized two clade B tier 2 isolates, RHPA and SC22, inthe more sensitive A3R5 neutralization assay (20) (Fig. S1).To obtain a deeper understanding of the properties of vaccine-

induced, CD4bs-directed Abs, we crystallized the GE136 andGE148 Fab fragments using high-throughput crystallizationscreening and iterative optimization (21). We obtained crystalsof both Fabs that diffracted to a resolution of 2.0 Å and 1.8 Å,respectively. The structural data are summarized in Table 1. Asexpected, the overall structures of the GE136 [Protein DataBank (PDB) ID code 4KTD] and GE148 (PDB ID code 4KTE)Fabs reveal that the six CDRs of both Fabs form a discontinuoussurface that comprises their binding sites (Fig. 1B, Top). Whenexamined from a “head-on” perspective of the Ab binding sites(paratope), each mAb displayed relatively prominent and pro-truding centrally positioned HCDR3s (Fig. 1B, Middle). Analysiswith University of California, San Francisco Chimera softwarerevealed the hydrophobic character of the molecular surface ofeach mAb, demonstrating that the Ab HCDR3 loops were highlyhydrophobic at their surface-exposed “tips” (Fig. 1B, Bottom),

A

B C

-4.5 0 4.5

GE148 GE136

V

C 1

V

CC 1

V

C

VCDR1CDR2CDR3

F105 b13

b12 CH103

L

L

H

H

H

H

L

L

PGV04VRC01

D

mAb CDR3 Sequence Tier 1A Tier 1B Tier 2

GE136 4.11 14.3 CVREGIVLVNLAVKNWFDVW 18 + + -

GE148 4.57 13.0 CARVQNIVVVFTIKEFFELW 18 ++ + -

Infection-induced Human CD4bs non-bNAbs

F105 4–59 8.2 CARGPVPAVFYGDYRLDPW 17 + + -

b13 3–30 7.1 CARDIGLKGEHYDILTAYGPDY 20 + + -

b12 1–3 20.4 CARVGPYSWDDSPQDNYYMDVW 20 ++++ ++++ +++

Vaccine-induced NHP CD4bs non-bNAbs

CDR3 Length

VH Family

VH Mutation Frequency

(% aa)

Neutralization Profile

CH103 4–59 16.8 CASLPRGQLVNAYFRN 14 ++++ ++++ +++

VRC01 1–2 41.8 CTRGKNCDYNWDFEHW 14 ++++ ++++ ++++

PGV04 1–2 45.9 CARQKFYTGGQGWYFDL 15 ++++ ++++ ++++

Hydrophobicity

Infection-induced Human CD4bs bNAbs

-4.5 0 4.5Hydrophobicity

Fig. 1. Properties of CD4bs mAbs and crystalstructures of GE148 and GE136 are illustrated. (A)Key features of GE136 and GE148 are listed com-pared with examples of human infection-elicitedAbs. Neutralization potency ranges are qualitativelysummarized as nonneutralizing (−) to very potentlyneutralizing (++++). VH, HC variable region gene. (B)Ribbon and surface representations of GE148 (PDB IDcode 4KTE, Left) and GE136 (PDB ID code 4KTD, Right)with HCs (darker shade) and LCs (lighter shade)marked. CDR1 (navy), CDR2 (cyan), and CDR3 (gold)are marked (Top), and their distribution is shownbelow in the surface representations of the Ab par-atopes (Middle). (Bottom) Hydrophobic surface rep-resentations of GE148 and GE136 are shown, with thetips of the respective HCDR3s circled in red (hydro-philic, purple; intermediate, white; hydrophobic, tan;see scale). (C) Hydrophobicity of human infection-elicited CD4bs-directed non-bNAbs F105 (PDB ID code1U6A) and b13 (PDB ID code 3IDX); their HCDR3 tipregions are circled in red. (D) Hydrophobicity of hu-man infection-elicited CD4bs-directed bNAbs b12(PDB ID code 2NY7), CH103 (PDB ID code 4JAM),VRC01 (PDB ID code 3NGB), and PGV04 (PDB ID code3SE9); their HCDR3 tip regions are circled in red.

Tran et al. PNAS | Published online February 3, 2014 | E739

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indicating that hydrophobic interactions might participate sig-nificantly in epitope recognition. This finding was consistent withanalysis of additional vaccine-elicited, CD4bs-directed mAbs,which also displayed hydrophobic HCDR3s (Fig. S2A). Usingthe structure coordinates of the human CD4bs-directed mAbsF105 and b13 (PDB ID codes 1U6A and 3IDX) (22), we ana-lyzed the molecular surface of these non-bNAbs and found thatthey too possessed hydrophobic HCDR3 tips (Fig. 1C). In con-trast, b12 (PDB ID code 2NY7), which is structurally relatedto b13 but is markedly more broadly neutralizing (22), lackeda hydrophobic HCDR3, as did the recently described CD4bs-directed bNAb CH103 (PDB ID code 4JAM) (6), which uses itsHCDR3 to bind gp120 (Fig. 1D). The bNAbs VRC01 (PDB IDcode 3NGB) and PGV04 (PDB ID code 3SE9) (Fig. 1D) orVRC23 (PDB ID code 4J6R), VRC03 (PDB ID code 3SE8), andVRC06 (PDB ID code 4JB9) (Fig. S2B), which are less relevantfor this comparison because they do not use their HCDR3s forrecognition of the gp120 CD4bs, also possessed nonhydrophobicHCDR3s (4, 23). Electrostatic analyses of GE136 and GE148were consistent with the hydrophobic nature of their HCDR3s(Fig. S2C).

Ala Scanning of GE148 CDRs Reveals Affinity-Related Effects. Next,we sought to define the specific interactions of GE148 andGE136 with their epitopes in the gp120 CD4bs. We used selectedforms of Env, both in soluble formats and in the context of thefunctional virus spike, to determine the interactions of the vac-cine-elicited mAbs with Env. To begin, we used our availableunliganded Fab structures as the basis to interrogate interactions

at the CD4bs. An extensive Ala scan of the GE148 HCDRsand LCDRs was performed (Fig. 2) to decipher any specificand critical contacts of GE148 with gp120 Env. Delineationof the framework and CDR regions is shown in Fig. S2D. Apanel of the GE148 IgG HCDR Ala mutants was used initiallyto assess recognition of monomeric YU2 gp120 by ELISAcompared with the unmodified WT GE148 IgG (Fig. 2A, leftcolumns). Somewhat surprisingly, we found little impact ofmost Ala substitutions on mAb recognition except for a singleresidue on the C-terminal flank of the HCDR3 loop at posi-tion K100e (Kabat numbering). Because the immunogen thatelicited GE148 was a YU2-based trimer, we reasoned that perhapsthe relative affinity (avidity) to the homologous Env was too highto detect single-residue effects of Ala substitution on GE148 rec-ognition. Accordingly, we performed a similar scan on an alternateEnv target of a different strain, the HXBc2 core variant, V3S (24).Again, we detected reduced binding at residue K100e (Fig. 2A), aswell as a weaker effect at E100f. Because GE148 binds the cys-teine-stabilized HXBc2-based core, 2CC (24), but with 100-foldlower affinity compared with the isogenic nonstabilized core (11),V3S, we performed a “low-affinity” Ala scan and found severaladditional residues that reduced recognition of 2CC by the GE148Ala variants. The strongest effect again centered on residue K100e,but Ala substitutions of flanking residues F100g and E100f alsodisplayed greatly reduced binding. Several other residues in theHCDR3 and HCDR2 regions also affected binding. To confirmthe specificity of these effects in a format assessing direct affinityrather than ELISA-based avidity, we measured the bindingkinetics of the Abs to gp120 by biolayer light interferometry (BLI;

Table 1. Crystallographic statistics

Crystal GE136 GE148

Data collectionWavelength, Å 1.033 1.033Space group P212121 P212121a, b, c; Å 44.92, 71.50, 154.24 42.71, 72.04, 175.74Resolution range, Å 40.0–2.0 (2.05–2.00) 20.0–1.8 (1.85–1.80)Measured reflections 176,417 369,218Unique reflections 33,654 (2,470) 51,192 (3,982)Rsym*, % 10.5 (54.0) 6.2 (49.7)Rmeas

†, % 11.7 (60.2) 6.6 (53.5)Completeness 97.6 (97.4) 99.8 (100)<I/σI> 12.3 (4.6) 21.1 (4.1)Multiplicity 5.2 (4.8) 7.2 (7.3)

RefinementNo. of reflections 33,611 51,191Wilson B, Å2 33.1 27.5Average B, Å2 (overall/main chain) 29.5/27.4 25.9/21.7Rcryst

‡, % 21.4 16.5Rfree

§, % 24.3 19.4rmsd from ideal

Bond length, Å 0.003 0.006Bond angles, ° 0.80 1.15

Dihedral angles, ° 11.6 12.9Ramachandran plot¶

Most favored region, % 98.6 97.9Additional allowed regions, % 1.4 2.1

I, integrated intensity; σI, estimated SD of that intensity.*Rsym = (ΣhklΣijIi(hkl) − <I(hkl)>)/ΣhklΣκi(hkl), where Ii(hkl) is the intensity of the ith measurement of reflection(hkl) and <I(hkl)> is the average intensity.†Rmeas = (Σhkl (sqrt(Nhkl/(Nhkl − 1))ΣijIi(hkl) − <I(hkl)>)/ΣhklΣki(hkl), where Ii(hkl) is the intensity of the ith mea-surement of reflection (hkl) and <I(hkl)> is the average intensity.‡Rcryst = (ΣhkljFo − Fcj/ΣhklFo), where Fo and Fc are the observed and calculated structure factors, respectively.§Rfree is calculated as for Rwork, but from a randomly selected subset of the data (5%) that were excluded fromthe refinement (49).¶Chen et al. (50).

E740 | www.pnas.org/cgi/doi/10.1073/pnas.1319512111 Tran et al.

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Fig. 2A). Here, we found effects with much larger magnitude thatwere again focused around the tip of HCDR3. The kineticsrevealed that the decreased affinity to gp120 was largely due tothe rapid off-rate of the mutant mAbs (Fig. 2B and Fig. S3).To assess the impact of these substitutions regarding neu-

tralizing capacity, we performed virus neutralization comparingWT GE148 with the HC Ala mutants (Fig. 2A, right columns).GE148 IgG neutralizes HXBc2 relatively potently (11) and, inthis instance, with an IC50 of 0.17 μg/mL. Similar to the broaderrange of residues affected in the low-affinity 2CC ELISA scan,neutralization of the HXBc2 virus was negatively influenced bymultiple Ala substitutions in HCDR3. We then performed anadditional scan using the SF162 virus, which GE148 neutralizes(11) but with a slightly lower IC50 potency of 0.62 μg/mL. Again,we detected several Ala substitution residues clustered at the tipof HCDR3 that decreased SF162 neutralizing capacity in a pat-tern similar to the 2CC or HXBc2 scan. Analysis in the context ofneutralization of the MN virus produced similar results. TheGE148 LCDRs (Fig. 2C) were similarly scanned, and, again, moreeffects with respect to the low-affinity 2CC scan were detectedcompared with full-length or core gp120 scan by ELISA, roughlyparalleling the affinity scan of gp120 by BLI. The binding kineticsconfirmed that the decreased affinity was largely due to morerapid off-rates (Fig. S3). Regarding neutralization, we saw moredisperse effects in the GE148 LC compared with the HC, andthese effects were of greatest magnitude on the C-terminal sideof LCDR1, at the base of LCDR2, and in the N-terminal regionof LCDR3. These data suggest that when non-bNAbs, such asGE148, are able to access their epitopes on the functional virusspike of tier 1 viruses, they achieve effective virus neutralizationand that such a productive interaction with the Env spike is largelydetermined by mAb off-rate.

Modeling of GE148 and gp120 Interactions. Further insight into thebinding interaction between GE148 and Env was gained throughcomputer modeling. The GE148 Fab crystal structure was dockedonto the gp120 structure derived from the F105-bound core gp120(PDB ID code 3HI1; hereafter referred to as “core gp120”)using ClusPro 2.0. The best-fit model was then obtained usingthe available data we had from the Ala scan of the GE148 par-atope (Fig. 2) and epitope on gp120 (11). In Fig. 3A (Left), theresidues in the GE148 CDRs that had the greatest impact onneutralization of the three viruses tested when substituted to Alaare highlighted in red. The structure of core gp120 is presentedin Fig. 3A (Right), with the CD4 loop highlighted in yellow,where the Ala substitutions that had the greatest effects onGE148 recognition were located (11), including residues D368and E370, which are critical recognition determinants of manyCD4bs mAbs. As demonstrated in Fig. 3B, the best-fit model showsa docking orientation that is consistent with proximal inter-actions of the GE148 CDRs with the gp120 CD4 loop. We alsoperformed a similar docking analysis of GE148 but used thegp120 derived from the b13-bound conformation (PDB ID code3IDX) instead, which resulted in a convergent solution that closelymatched the F105-bound model (Fig. S4).Interestingly, the model of GE148 docked to the F105-bound

conformation of gp120 (Fig. 3C, Left) predicts that the K100eresidue on the C-terminal side of the HCDR3 loop interacts withD368 and E370 of the gp120 CD4 loop, potentially involvinghydrogen bonding (Fig. 3C, Right). Such an interaction is notsurprising, because the published GE Abs were selected asCD4bs-directed B cells in the original flow cytometric single-cellsorting approach based upon a D368R substitution. In addition,the modeling suggests interaction of F100b with the hydrophobicphenylalanine (Phe)-43 cavity on the gp120 molecular surface.The fitting of both of these residues is reminiscent of the CD4interaction with gp120 defined by crystallography, in which the

(K , nM)BLI Affinity

YU2gp120

HXBc2 coreV3S

G26A 0.04 0.05 0.17 0.66 0.16 0.50 1.45G27A 0.04 0.06 0.23 0.97 0.08 0.34 0.14S28A 0.04 0.06 0.30 1.69 0.31 0.14 2.66I29A 0.04 0.06 0.21 1.71 3.00 0.39 3.72S30A 0.04 0.05 0.15 0.82 0.23 1.90 1.07G31A 0.05 0.06 0.20 0.11 0.04 0.15 0.16G32A 0.04 0.06 0.27 0.11 0.23 1.55 3.06Y33A 0.04 0.06 0.10 0.43 1.88 0.70 4.00G34A 0.04 0.06 0.15 0.17 0.30 0.32 0.66

I51A 0.05 0.08 0.69 0.74 0.49 0.01 0.17Y52A 0.05 0.10 1.90 1.24 0.59 0.31 36.88S52aA 0.05 0.06 0.26 0.43 0.67 0.68 1.94S53A 0.04 0.07 0.78 2.08 0.13 0.77 1.72S54A 0.05 0.07 0.48 0.18 0.09 0.07 0.45G55A 0.04 0.06 0.34 0.65 0.27 0.99 2.50S56A 0.04 0.08 0.59 0.77 0.29 1.77 4.00T57A 0.06 0.06 3.79 1.21 0.97 1.06 4.53

R94A 0.05 0.23 1.22 0.83 0.89 0.14 0.63V95A 0.05 0.09 1.23 4.10 >50 0.48 >50Q96A 0.04 0.06 0.31 0.67 0.30 0.25 7.02N97A 0.04 0.04 0.12 0.41 0.33 0.77 0.25I98A 0.04 0.08 3.55 1.51 0.27 1.83 >50V99A 0.05 0.07 0.98 0.25 0.48 1.96 4.15V100A 0.04 0.11 3.87 3.50 1.47 5.07 >50V100aA 0.05 0.08 0.73 0.63 0.31 0.43 7.50F100bA 0.03 0.20 2.9 25.90 >50 >50 >50T100cA 0.04 0.07 0.29 0.30 0.54 0.03 0.59I100dA 0.04 0.14 1.58 12.40 >50 >50 >50K100eA 0.71 1.98 7.37 133 >50 >50 >50E100fA 0.04 0.34 7.49 126 >50 >50 >50F100gA 0.03 0.12 >50 4.03 4.84 3.02 >50F100hA 0.05 0.13 1.10 5.34 0.03 5.50 >50E101A 0.04 0.07 2.72 4.74 0.19 0.71 0.23L102A 0.04 0.06 0.74 0.85 0.23 0.04 0.05

WT 0.04 0.06 0.23 1.35 0.17 0.62 3.84

HC CDR3

ELISA Binding (EC , µg/ml)

Neutralization (IC ,

HC CDR1

HC CDR2

BLI Affinity(K , nM)

YU2gp120

HXBc2 coreV3S 2CC

YU2gp120 HXBc2 SF162 MN

S27A 0.02 0.06 0.7 0.75 0.59 0.28 0.15G27aA 0.02 0.05 0.56 0.70 0.22 2.43 7.00I27bA 0.02 0.04 0.41 0.28 0.11 0.59 0.26N27cA 0.02 0.06 1.09 1.86 1.62 1.02 0.12V28A 0.02 0.05 0.65 2.15 0.51 1.20 0.57G29A 0.02 0.06 0.75 1.66 0.32 3.24 5.08S30A 0.02 0.05 0.67 1.71 0.28 0.75 0.86Y31A 0.03 0.2 12 144 >50 >50 >50S32A 0.02 0.13 >50 49.80 >50 >50 >50

Y50A 0.02 0.09 >50 9.40 >50 10.81 >50Y51A 0.02 0.05 >50 3.18 2.02 3.39 >50S52A 0.02 0.07 0.94 1.10 2.51 7.12 0.03D53A 0.02 0.05 0.46 1.99 1.31 4.96 0.82S54A 0.02 0.05 0.59 0.34 0.49 6.35 5.58S54aA 0.02 0.08 0.42 10.40 >50 8.90 >50K54bA 0.02 0.06 1.34 1.66 3.11 0.70 0.18

I90A 0.04 0.07 3.58 2.53 0.01 6.75 >50W91A 0.04 0.07 >50 2.67 0.19 7.22 >50H92A 0.04 0.07 >50 3.60 3.60 1.75 1.78N93A 0.04 0.07 0.29 0.98 0.54 5.39 0.23F94A 0.04 0.11 >50 19.40 >50 >50 >50C96A 0.04 0.08 0.31 1.75 0.28 1.44 2.03V97A 0.04 0.09 1.3 1.65 2.88 1.82 >50WT 0.04 0.06 0.23 1.35 0.17 0.62 3.84

LC CDR1

LC CDR2

LC CDR3

Neutralization (IC , µg/ml)

ELISA Binding (EC , µg/ml)A C

B

0.00.10.20.30.40.5

n m

0 120 240Time (sec)

WT

0.00.10.20.30.40.5

n m

0 120 240Time (sec)

HCDR3 E100fA

50 50 50 50 D D

2CC HXBc2 SF162 MN YU2gp120

500 nM

250 nM

125 nM

62.5 nM 500 nM

GE148 GE148

µg/ml)

Fig. 2. Ala scan of the GE148 paratope. (A) Effectsof Ala substitution in the HCDRs on binding to Envvariants (YU2 gp120, HXBc2 V3S core, and HXBc2stable core 2CC) by ELISA or BLI and on neutraliza-tion capacity compared with WT GE148 (WT, blue).Residues are numbered based on the Kabat num-bering system. EC50 (Ab concentration for half-maximal binding), Kd (binding affinity), and IC50 (Abconcentration for 50% neutralization) values areshown. Color-coding reflects the fold decrease inactivity compared with WT (four- to 10-fold, beige;10- to 20-fold, yellow; >20-fold or >50 μg/mL, red).(B) Binding kinetics for WT and the Ala mutantHCDR3 E100fA at different ligand concentrations(62.5–500 nM) are shown as representative exam-ples. The decreased binding affinity of GE148 Alamutants to gp120 is largely due to an increased off-rate (koff; on-rate, kon) compared with WT (WT:KD = 1.4 nM, kon = 1.2e5 M−1·s−1, koff = 1.6e4 s−1;HCDR3 E100fA: KD = 126 nM, kon = 1.2e5 M−1·s−1,koff = 1.5e2 s−1). (C) Effect of Ala substitution in theGE148 LCDRs on binding to Env and neutralizationcapacity as in A.

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R59 of CD4 interacts with D368 and the F43 of CD4 interactswith the so-named “Phe-43 cavity” (25). Similar structuralinteractions to that of CD4 have been described in previousstudies for several CD4bs-directed mAbs (22).To confirm the accuracy of the model, we performed site-

directed mutagenesis of residues predicted to interact betweenthe interface of the paratope of GE148 and the epitope of gp120.We substituted K100eE in the GE148 HCDR3 and observedthat this nonconservative change completely eliminated neu-tralization of HIV-1, consistent with the modeling (Fig. 3D).Similarly, altering F100bW, which is predicted to create a clashin the Phe-43 cavity of gp120, also reduced GE148 neutralizationcapacity. Next, we attempted to increase neutralization potencyby model-guided “gain-of-function” substitutions to increase hy-drophobic interactions between GE148 HCDR2 and HCDR3with hydrophobic elements of gp120. As predicted by the model,enhanced neutralization capacity was observed for changes atS53I/W, V99I, and T100cW (Fig. 3D). These data strongly suggest

that the GE148/gp120 complex model provides structural informa-tion at a relatively high level of resolution that can then be exploitedto both decrease and enhance the functional capacity of the mAb.

Ala Scan, Modeling of GE136, and Validation with F105. We nextperformed a similar modeling analysis using the Ala scan data ofGE136 (Fig. 4A) in conjunction with the published epitope scanof the footprint of this CD4bs-directed mAb on gp120 (11) todock GE136 with core gp120 (Fig. 4B). A convergent solutionwas also seen when the alternative gp120 core (PDB ID code3IDX) was used (Fig. S4). The model revealed potential inter-actions of GE136 K100e with D368 of gp120, a hydrogen bondbetween GE136 V100d and E370 of gp120, and a hydrophobicinteraction of GE136 L100b with the gp120 Phe-43 cavity (Fig.4C). Consistent with the modeling, substitution of these residuesto Ala (Fig. 4A) and K100eE (Fig. 4D) resulted in a loss of neu-tralization capacity. To confirm the accuracy of the model further,we designed substitutions to increase hydrophobic interactionsbetween the GE136 HCDR2 and HCDR3 with gp120 at residuesS54, L99, and L100b as predicted by the model, most of whichdid result in gains in GE136 neutralization potency (Fig. 4D).Although substitutions with Phe at L99 and L100b increasedGE136 neutralization potency, substitutions with tryptophan(Trp) did not. This may be due to the bulkiness of the Trp indolegroup compared with Phe benzyl group, which may not fit in thegp120 hydrophobic pocket due to steric hindrance.To validate the modeling methodology using an existing

structure of an Ab/Env ligand complex, we independently per-formed the docking and modeling by the same methods andconstraints with the human CD4bs-directed mAb F105, using theAla scan of the paratope (Fig. S5A) and epitope on gp120 (11) toobtain the best-fit model, which is remarkably close to the crystalstructure in terms of the contacts between the Ab CDRs andgp120 (Fig. S5B). We also compared the ClusPro model ofthe human CD4bs mAb b13 in complex with gp120 to the b13/gp120 crystal structure, with similar results (Fig. S5C).

EM Analysis of GE136 and GE148 with gp120. To assess the inter-actions of GE148 and GE136 with gp120 by direct structuralanalysis further, we performed EM studies of these Abs interactingwith gp120. Initially, we generated equimolar mixtures of bothGE136 and GE148 Fabs in complex with monomeric full-lengthgp120. Following negative staining, EM images were generatedand analyzed. Reference-free 2D classification images were se-lected from the primary dataset to generate 3D reconstructions ofthese Fabs in complex with gp120 (Fig. S6 A and B). Fitting of theFab crystal structures of GE136 or GE148 with the core gp120was performed, which generated good fits with the density. Fittingof the GE136/gp120 and GE148/gp120 into these densities wasconsistent with the ClusPro modeling of the Abs docked to gp120.To constrain the fits within the density further and to derive an

orientation of the Abs and core gp120 in the context of the tri-meric spike, we added 2G12 to the analysis to provide a distinctlandmark on gp120. As a glycan-reactive bNAb, 2G12 binds to atleast three high-mannose glycans on the gp120 outer domainsurface, and the 2G12 crystal structure in complex with glycan isavailable (PDB ID code 1OM3) (26, 27). The 2G12 Ab is un-usual in that it binds via domain exchange, resulting in an IgGwith the two Fab regions tightly cross-linked. This domain swapcreates a unique multivalent surface compared with a conven-tional bivalent IgG, limiting 2G12 IgG binding to only one gp120monomer. Negative-stain, reference-free 2D class averages of 2G12generated a distinctive double-lobed pattern relative to the GE136Fab (Fig. 5A, Upper Left), aiding in orientation by providing a dis-tinctive feature for the EM analysis. Using this landmark and thefitting of the mAbs with gp120, we were able to assign individualelements of the 2D classification images of the complex to 2G12,core gp120, and GE136 (Fig. 5A, Upper Right). Manual docking

A

B

GE148 gp120

K100eF100b

E370 D368β20-21 V5

Loop D

V1

V

V

H

L

V

V

H

L

CCH

L

C

D

F100b

D368

K100e

S53W V99I

T100cW

E370

K100eE > -50F100bW > -50

S53I +4S53W +31V99I +19V99F +2

T100cW +4

Fig. 3. Docking models of GE148 onto gp120 and confirmatory sub-stitutions. (A, Left) Variable regions of GE148 HC (VH, dark green) and LC(VL, light green) with key residues of the paratope shown in red (i.e., resi-dues when mutated to Ala severely decreased Ab function), with their sidechains shown as a stick representation. (A, Right) Ribbon representation ofcore gp120 (PDB ID code 3HI1) with key features labeled. The amino acidswithin the gp120 CD4 loop that resulted in decreased Ab binding when al-tered in the gp120 Ala scan (11) are shown in yellow. (B) ClusPro model ofthe GE148 and core gp120 complex showing convergence of the Ab para-tope CDRs and the gp120 CD4 loop. (C) ClusPro model in the ribbon repre-sentation (Left) and a close-up view at the Ab interaction with the CD4bsloop (Right). HCDR3 residue K100e (red) is predicted to form hydrogen bonds(black dashed lines) with D368 and E370 on gp120 (yellow). F100b at the tip ofthe HCDR3 is positioned in proximity to the gp120 Phe-43 pocket in theCD4bs, resulting in a putative hydrophobic interaction. (D) Confirmatory Abmutations based on the ClusPro docking model were assayed for neutraliza-tion potency against the HIV-1 isolate SF162. K100e was predicted to abrogateAb neutralization when mutated to E, whereas S53W, V99I, and T100cW werepredicted to interact with hydrophobic elements of gp120 (shown in tan onthe molecular surface of core gp120). Fold changes compared with WT withenhanced activity (>+threefold) are highlighted in green, and those withdecreased activity (>−threefold) are highlighted in red.

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of the crystal structures of these proteins (PDB ID codes 1OM3,3HI1, and 4KTD, respectively) to form a complex was consistentwith the EM images and assignments (Fig. 5A, Lower). Using the2D-classified images, we generated a 3D density volume anddocked GE136, core gp120, and 2G12 into this density of thecomplex (Fig. 5B, Upper). The fitting of the 2G12 Fab and gp120coordinates into the density was consistent with an interaction ofthis mAb with the gp120 outer domain glycans that comprise itsepitope (Fig. 5B; highlighted in red on gp120). Similarly, theGE136 Fab was positioned to interact with its cognate epitope onthe CD4bs (Fig. 5B; highlighted in yellow). We then positionedthe 2G12/gp120/GE136 tricomplex above the published cryo-EMdensity of the functional spike [Electron Microscopy Data Bank(EMDB) ID 5019] (17), using the core gp120 molecular surface,the visible 2G12 epitope N-glycan, and the CD4 loop as land-marks (Fig. 5B). This analysis provides approximate angles ofapproach for the spike by both 2G12 and GE136 (Fig. 5B, arrows)and suggests that GE136 would have to approach the CD4bs fromthe top of the spike, which we examine in greater detail below.Weperformed a similar EM analysis on GE148 with core gp120 and2G12 with an analogous outcome (Fig. S6C). The EM 3D recon-structions shownwere generated at a resolution of∼20Å. Projectionmatching and Fourier shell correlation for 2G12/gp120/GE136tricomplex are shown in Fig. S7A.

Angle of Ab Approach to the CD4bs of the Functional Spike. To de-fine the orientations of the vaccine-induced mAbs with the gp120CD4bs in the context of the functional spike further, we performed

a similar analysis as that described above but now using the N332-directed bNAb PGT122 (28) Fab instead of the 2G12 IgG intricomplex with gp120 and the GE136 or GE148 Fab. Becausethe interaction of PGT122 with the soluble BG505 SOSIP.664gp140-cleaved trimeric spike mimetic was previously assessed byEM (EMDB ID 5624) (29), and although the non-bNAbs GE136and GE148 cannot bind to the functional spike of primaryisolates such as BG505, we nevertheless were able to approxi-mate the theoretical positions of the PGT122/gp120/GE136 andPGT122/gp120/GE148 tricomplexes in the context of the BG505SOSIP.664 using the PGT122 Fab density (derived from EMDBID 5624) as a feature in common (Fig. 5C, Left and Center).Similarly, we docked PGT122/gp120/PGV04 onto the BG505 tri-meric spike (Fig. 5C, Right) as a control and for comparison.Because the EM densities of both PGT122/BG505 SOSIP.664and PGV04/KNH1144 SOSIP.664 (EMDB ID 5706) are available(29, 30), we could confirm the accuracy of the tricomplex dockingon the trimeric spike. Projection matching and Fourier shell cor-relations for the tricomplexes are presented in Fig. S7 B–D. Thisanalysis revealed that GE136 and GE148 attempt to access theirrespective CD4bs epitopes from “the top of the spike” relative tothe lateral, “side approach” of PGV04 (Fig. 5C, side view and Fig.S8A, top view), which may be why GE136 and GE148 cannot bindthe SOSIP trimers or cannot neutralize primary HIV-1 isolates.The analyses described above indicated that access from the

top might be a general property of nonbroadly neutralizingCD4bs-directed mAbs to tier 1 isolates. To examine if bindingorientation to the functional Env spike discriminates between

A BNeutralization (IC , µg/ml)

ELISA Binding (EC , µg/ml)

HXBc2 SF162 MN YU2gp120

HXBc2 core

I51A 0.03 0.11 1.28 5.01 1.90S52A 0.02 0.08 0.15 0.55 0.57G52aA 0.05 0.19 1.63 4.16 2.16S53A 0.03 0.11 1.02 4.15 1.89S54A 0.03 0.11 0.38 3.38 2.47G55A 0.03 0.12 0.75 0.58 2.27D56A 0.03 0.17 0.43 3.16 18.29T57A 0.03 0.13 1.11 5.15 2.28

V93A 0.04 0.18 0.77 3.11 4.15R94A 0.04 0.13 0.05 0.21 0.89E95A 0.04 0.29 >50 >50 >50G96A 0.05 0.29 3.34 12.30 6.04I97A 0.05 0.09 10.75 0.16 8.86V98A 0.05 0.22 1.16 >50 3.31L99A 0.06 0.56 >50 >50 >50V100A 0.09 0.91 >50 >50 >50N100aA 0.02 0.06 1.59 14.66 2.97L100bA 0.05 0.47 >50 >50 >50V100dA 0.03 0.19 >50 6.04 18.90K100eA 0.16 1.90 >50 >50 >50N100fA 0.06 0.34 6.83 27.14 9.86W100gA 0.15 1.99 >50 >50 >50F100hA 0.05 0.33 >50 >50 16.71D101A 0.03 0.12 0.11 5.50 2.25V102A 0.03 0.13 0.90 8.65 1.89

WT 0.05 0.17 2.27 10.15 3.50

HC CDR2

HC CDR3

L100b E370

K421

K100e

D368

D

C

S54W

L100bF

L99FVH

C 1H

VL

CL

GE136

50 50

gp120

K100eE > -50

S54I +3S54W +17L99F +4L99W -3

L100bF +3L100bW -14

GE136

Fig. 4. GE136 Ala scans, modeling, and confirmatory substitutions. (A) Binding and neutralization activity of Ala substitution mutants in GE136 HCDR2 andHCDR3 (Kabat numbering) are shown compared with WT (blue). Decreases in activity compared with WT are highlighted (four- to 10-fold, beige; 10- to 20-fold, yellow; >20-fold or >50 μg/mL, red). (B) ClusPro model of GE136 with HC (dark brown) and LC (light brown) in complex with core gp120 (colored as in Fig.3A). Specific HCDR mutations that decreased Ab function in the Ala scan are shown in red. (C) Magnified view of potential molecular interactions betweenGE136 and the gp120 CD4bs loop. Potential hydrogen-bonding interactions between residues in GE136 and gp120 are indicated with black dashed lines.GE136 L100b (red), at the tip of the HCDR3, is another important residue that may contribute to the binding energy of the complex by a hydrophobic in-teraction. (D) GE136 mutants with substitutions in HCDR2 or HCDR3 predicted to enhance or decrease GE136–gp120 interactions were assayed for neu-tralization potency against the HIV-1 SF162 isolate. Fold changes compared with WT are shown, with enhancements greater than threefold highlighted ingreen and decreases greater than threefold highlighted in red.

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nonbroadly and broadly neutralizing CD4bs-directed Abs, whetherelicited by chronic HIV-1 infection or Env vaccination, we usedthe coordinates of core gp120 fitted into the cryoelectron to-mography density of the functional spike (EMDB ID 5019) (17)and superimposed the ClusPro models of the GE136 and GE148Fab/gp120 dicomplexes onto the gp120 trimer (PDB ID code3DNN) fitted in the density (Fig. 6A). To assess the relativeangles of approach for other CD4bs-directed mAbs, we per-formed a similar analysis using available coordinates and struc-tures of CD4bs-directed mAbs in complex with the gp120 core(Fig. 6A and Fig. S8B). As can clearly be seen, the vaccine- orinfection-induced non-bNAbs approach their respective epitopesin the CD4bs from the top of the spike. The modeling suggeststhat the non-bNAbs require a deeper penetration toward thetrimer axis, which is evident in the top views of GE136, GE148,and F105, where the non-bNAbs protrude further into theV-region cap area toward the trimer axis, as outlined by the blackdashed line (Fig. 6A, Lower). This likely creates clashes (redstarbursts in Fig. 6 and Fig. S8) with the V-region cap of neu-tralization-resistant viruses, thereby preventing mAb binding tothe spike and subsequent neutralization of the virus. Probablycontributing to these clashes are the N-linked glycans on thesurface of the trimeric spike emanating from the V1/V2/V3loops, which are not visible in the density. Such a route of accessto the spike may be achievable on the more neutralization-sensitive viruses that likely possess less tightly packed trimeric spikesbut would be hampered against the more tightly packed spikespresent on primary isolates. In contrast, the broadly neutralizingCD4bs-directed mAbs approach their epitopes in the functionalspike via a more lateral “side access” (18), avoiding clashes withthe quaternary topside cap of the functional spike. To clarifysuch clashes better, interaction of GE148 with the functionalspike is compared with the bNAb, VRC01. In Fig. 6B, GE148(ClusPro model with gp120) and VRC01 are docked into thefunctional spike (EMDB ID 5019) in a similar fashion as in Fig.6A. On the right, the spike is rotated 45° from the previous

orientation on the y axis and reveals substantial clashes of theGE148 HC and LC with cap elements of the trimeric spike, ashighlighted. In contrast, VRC01 approaches its epitope in theCD4bs laterally, bisecting the wedges defined by two adjacentprotomer arms of the spike, thus avoiding clashes with eitherthe V-region cap or adjacent protomers. A similar analysis wasperformed with GE148 and CH103 with comparable results (Fig.S8B), indicating that although CH103 binds slightly more prox-imal to the V-region cap of the native spike, access is stillachievable as long as the bNAb “splits the wedge” by stacking theHC and LC in a vertical manner to minimize width and clashesas it approaches the CD4bs. Comparison of GE136 access rel-ative to VRC01 yielded an analogous result as GE148 (Fig. S8C).These results prompted us to investigate the structural con-

formation of the YU2 gp140-F trimers used to elicit the GE136and GE148 mAbs by EM. As seen in Fig. 6C, these trimers ap-pear relatively heterogeneous in composition, indicating thatthey are likely open in their conformation compared with morefaithful mimetics of the native spike, such as the BG505 SOSIPtrimers (31). Collectively, the modeling and EM studies suggesta distinct difference in terms of angle of approach to the CD4bsbetween bNAbs and the class of non-bNAbs typified by the twomAbs studied here and suggest a strategy to modify currentsoluble trimeric immunogens to restrict access of B-cell receptors(BCRs) toward a “side angle” approach to the CD4bs whileoccluding a vertical approach, perhaps by strengthening inter-actions in the gp120 trimer association domains. Such an im-munogen design strategy might better elicit neutralizing Abs tothis recessed and important functionally conserved neutralizingdeterminant on the HIV-1 spike.

DiscussionRecently, we reported the isolation of previously undescribedEnv trimer vaccine-elicited, CD4bs-directed mAbs from rhesusmacaques, including GE136 and GE148, which neutralize tier 1HIV-1 strains relatively potently (11). Similar to the majority of

A

GE136

gp120

2G12

GE136 GE148PGT122 PGT122 PGT122

PGV04

gp120 gp120 gp120

B

C

GE136

GE136

2G12

2G12

gp120

Fig. 5. EM analysis of GE136 and GE148. (A) Two-dimensional class average of the gp120/2G12/GE136complex by EM is shown (Upper Left) and with thecomponents colored (Upper Right) as follows: 2G12(blue), gp120 (cyan), and GE136 (brown). (Lower)Surface representation of the 2G12 crystal structure(PDB ID code 1OM3, blue) and the ClusPro model ofGE136 (brown) bound to core gp120 (cyan) modeledfollowing EM data is shown. The patch of red ingp120 depicts the N332 and N295 glycan sites im-portant for recognition by 2G12. (B) EM density ofthe gp120/2G12/GE136 complex filled with ribbonrepresentations. GE136 (brown), 2G12 (blue), andgp120 core (cyan) are shown with the CD4bs (yellow)and the glycan sites, N295 and N332 (red). Superim-position of the GE136 tricomplex with the unli-ganded trimeric gp120 from published electrontomography data of the native BaL HIV viral spike toapproximate the angle of Ab access (2G12, blue ar-row; GE136, brown arrow) is illustrated. (C) EMdensity data of the broadly neutralizing antiglycanAb PGT122 bound to the SOSIP soluble trimer (EMDBID 5624) are shown in light yellow with the theo-retical superimposition of the EM density (gray) ofgp120/PGT122/GE136 (Left), gp120/PGT122/GE148(Center; note that GE136 and GE148 do not bindto the SOSIP trimers; hence, these are theoreticalsuperimpositions), and gp120/PGT122/PGV04 (Right)using the common PGT122 density. A ribbon repre-sentation of PGT122 (PDB ID code 4JY5, red) GE136(brown), GE148 (green), and PGV04 (pink), with themolecular surface of gp120 core (cyan), is shownfilling the EM data for identification of density.

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HIV-1 infection–induced CD4bs-directed mAbs, these vaccine-elicited mAbs do not neutralize the more resistant tier 2 HIV-1isolates. To investigate the structural basis for the limited neu-tralization by these mAbs sharing this CD4bs-directed, non-broadly neutralizing phenotype, we solved the high-resolutionstructures of the GE136 and GE148 Fabs and performed a de-tailed examination of the potential mode of interaction of thesemAbs with the Env spike, including Ab paratope and epitopemapping, in silico docking of the mAbs to gp120, and EMstructural analysis with gp120. These data provide a clear ex-planation for the shortcomings of the nonbroadly neutralizingCD4bs-directed Abs elicited by the foldon Env trimer designsanalyzed here and may provide structural insight for rational

immunogen modification to improve the elicitation of bNAbsagainst the conserved HIV-1 primary receptor binding site. Onelimitation of the current analysis is that it directly reveals theangle of approach used by CD4bs mAbs generated by the foldontrimers. However, given the shared inability of many CD4bs-directed Abs elicited by the foldon trimers or other forms ofEnv to neutralize primary isolates, this congruence suggests thatmany Env immunogens that do not elicit bNAbs also generateCD4bs-directed mAbs that take similar suboptimal angles ofapproach to this conserved neutralization determinant.The crystal structures of GE136 and GE148 revealed modestly

protruding HCDR3s with hydrophobic tips, a property that isshared by other nonbroad CD4bs-directed mAbs but not by

A

GE148 GE136 b12 PGV04F105 VRC01

GE148

VRC01

45º

B C

~62º ~54º ~52º ~30º ~25º ~22º

non-bNAbs bNAbs

YU2 gp140-F Trimers Native Trimer

V-region cap

V-region cap

Fig. 6. Trimer docking model of GE136 and GE148. (A, Left) Models of the NHP non-bNAbs GE148 and GE136 and the human non-bNAb F105 docked ontothe trimeric gp120 (PDB ID code 3DNN) and fitted into the electron tomography density of the unliganded native spike (EMDB ID 5019). (A, Right) bNAbs b12,PGV04, and VRC01 are similarly docked. Both side views (Upper) and top views (Lower) are shown for each mAb. The dashed red lines on the spike density inthe models on the far right indicate the approximate location of the V-region cap. The angle of approach to the CD4bs, approximated by the central axisof the mAb (dotted line through the Ab rigid body) is indicated in degrees relative to a fully horizontal approach, parallel to the viral membrane. As can beseen, the non-bNAbs attempt to access the CD4bs vertically relative to the viral membrane, whereas the bNAbs more closely approximate a horizontal path.(Upper) Red starburst indicates clashes with the primary isolate spike density V-region cap if a more vertical angle of approach is attempted. The black dashedoutline marks protrusion of the mAb into the V-region cap density. Note that in this rendering, docking of the bNAbs into the unliganded spike density, b12assumes a more vertical orientation relative to the b12-liganded orientation described by Tran et al. (18), which can be attributed to b12-induced confor-mational changes of the spike, altering the angle of binding relative to VRC01 and the viral membrane. (B, Left) VRC01 and GE148 docked into the spikedensity (EMDB ID 5019) in the same orientation as in A. (B, Right) VRC01 and GE148 docking rotated ∼45° to reveal substantial clashes of GE148 HC and LCwith protomer proximal V cap elements of the trimeric primary isolate spike, as indicated by the red starburst, but few clashes for the bNAb. (C, Left)Representative reference-free 2D class averages of the YU2 gp140-F antigens that were used to inoculate the NHPs. The irregular shapes suggest that thesefoldon trimers are relatively open in configuration. (C, Right) Although held together by gp41 determinants and the foldon motif, nonneutralizing epitopes(arrows) are exposed and available for Ab recognition. Conversely, a native trimer provides constraints on Ab approach.

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bNAbs. Ala scanning of GE136 and GE148 indicated that theirHCDR3s harbor critical contacts for binding and neutralization,which is quite different from the bNAbs, such as VRC01 andPGV04, for which the HCDR3s form markedly fewer contactswith gp120 (4, 23, 32) but more in HCDR2 and frameworkregions (33). Many more effects on binding of GE148 wererevealed by the lower affinity interaction of this mAb with thestabilized gp120 core protein, 2CC, suggesting that the effect ofsingle Ala substitutions is more apparent if the mAb–gp120 in-teraction is of lower affinity. Interestingly, the lower affinityrecognition of GE148 for 2CC, and the resulting Ala scan rec-ognition profile of 2CC by ELISA, more closely mapped to theresidues on GE148 affected by Ala substitutions in the context ofneutralization than did a similar scan on gp120. This result mayrelate to more inherent flexibility in monomeric gp120 than tothe stabilized core and gp120 in the context of the native Envtrimer. These data suggest that HIV-1 neutralization may bea “threshold” event requiring mAb access to its epitope in thecontext of the functional spike, and that, if achieved, the mAbmust maintain this interaction long enough to inhibit virus entry.This interpretation is consistent with kinetic binding data withGE148, where decreases in affinities of the Ala substitutionswere determined mainly by their off-rate to gp120. The data arealso consistent with the general model that if an Ab neutralizesHIV-1, it must be able to bind to the functional viral spike gly-coprotein with a reasonable efficiency and occupancy, as pre-viously suggested (34). For HIV-1 CD4bs-directed mAbs, suchinteractions appear to be “all or none”; that is, if an Ab cannotaccess its spike due to steric occlusion, quaternary packing, orconformational masking, there is no detectable on-rate and es-sentially no binding or neutralization. This is opposed to a modelin which the CD4bs-directed mAb can access its epitope on theprimary isolate spike with a detectable on-rate but cannot induceconformational changes required for high-affinity binding tomediate neutralization. Such a two-step mechanism of bindinghas been proposed for CD4 interaction with HIV Env (35).The interactions defined from a combination of mutagenesis,

crystallography, EM, and modeling suggest that the hydrophobicHCDR3s of these mAbs protrude into the Phe-43 cavity andinvolve interaction of hydrophobic residues on both mAbs withthis cavity. The lack of such hydrophobicity in the HCDR3s ofother broadly effective mAbs, b12 and CH103, indicates that thisproperty is associated with non-bNAbs, although their HCDR3sinteract in different ways. This analysis suggests that perhapsdeeper penetration by the HCDR3s of non-bNAbs into thispocket or neighboring regions not available on primary HIV-1isolates limits neutralization. The structural models also suggestthat a basic residue in the HCDR3s of each mAb interacts withD368 or E370 in the gp120 CD4 binding loop, reminiscent ofsimilar cationic + hydrophobic motifs in F105 and b13 (22). TheGE148 mAb LC Ala scan revealed that many putative interactionsoutside of the HCDR3 were involved in neutralization, suggestingthis charge-plus-hydrophobic motif may be necessary, but notsufficient, for broad CD4bs-directed HIV-1 neutralization.Recognition of the primary isolate functional spike by the

GE136 and GE148 appears limited, as reflected by their modestneutralization capacity. The structural analysis, coupled with theneutralization data, suggests that these vaccine-elicited, CD4bs-directed mAbs adopt a mode of recognition involving an approachto the spike from the trimer apex to attempt access of their re-spective epitopes in the CD4bs. This mode of binding, reminiscentof that of F105 (22), contrasts to that used by the bNAbs VRC01and PGV04, which use a side approach, parallel to the viralmembrane, to “duck under” the quaternary V-region cap effec-tively to access their epitopes in the CD4bs efficiently. CD4bs-directed bNAbs, such as b12 and CH103, which are more limitedin neutralization breadth relative to VRC01 or PGV04, adopt a

slightly more vertical mode of recognition, invoking clashes withthe V-region cap on viruses that they cannot neutralize (36).For both the vaccine- and infection-elicited non-bNAbs ana-

lyzed in this study, it appears that steric clashes with the V-regioncap limit their neutralizing capacity against the tight quaternarypacking of most primary isolates. That the CD4bs-directed non-bNAbs appear relatively commonly elicited by the soluble trimerimmunogens used here suggests that these spike mimetics areopen in conformation, permitting unfettered top access of Bcells recognizing the CD4bs, resulting in the generation of non-bNAbs. Efforts to redesign the soluble foldon trimers to improvethe structural integrity of the V-region cap are warranted.Trimers displaying improved quaternary packing, such as solubleSOSIP trimers, may better elicit Abs to the conserved primaryreceptor binding site by restricting BCR access to a side ap-proach to the recessed CD4bs. However, more open trimersmight still have a useful role in priming B-cell responses to theCD4bs that is recessed on more native spikes. In sum, thepresent studies offer important insight on how vaccine-induced,CD4bs-directed mAbs interact with Env, providing a clear ex-planation for a long-standing problem in the HIV-1 vaccine field(37–39) that can now be rationally addressed through improvedsoluble trimer redesign to accelerate the development of an ef-fective HIV-1 vaccine.

Materials and MethodsExperimental methods used in this study are briefly summarized here. Ad-ditional details are presented in SI Materials and Methods.

Fab and Core gp120 Expression and Purification. Similar procedures were usedas described in previous studies (11, 24), and additional details can be foundin SI Materials and Methods.

Crystallization and Structural Determination of the GE136 and GE148 Fabs.Initial crystallization trials in 384 conditions [Joint Center for StructuralGenomics (JCSG) core I–IV] were set up using the automated IAVI/JCSG/TSRICrystalMation robotic system (Rigaku) at the JCSG (www.jcsg.org). Diffrac-tion-quality crystals of the GE136 Fab fragment could be grown in a solutioncomposed of 0.1 M citric acid (pH 3.5) and 1.6 M ammonium sulfate (pH 4.0).For the GE148 Fab fragment, diffraction-quality crystals were obtained in0.17 M ammonium sulfate, 25.5% (wt/vol) PEG 4000, and 15% (vol/vol)glycerol. Diffraction data for the GE136 and GE148 Fab fragment werecollected on beamline (BL) 23-ID-D at the Advanced Photon Source and onBL 11-1 at the Stanford Synchrotron Radiation Lightsource, respectively. Thediffraction data were integrated and scaled using the XDS program packagein space group P212121. The initial model of GE148 and GE136 was solvedand built by the automated crystal structure determination platform Auto-Rickshaw (40), using the Fab HC and LC from PDB ID code 3G6D in the case ofGE148 and the Fab structure of GE148 for GE136. Auto-Rickshaw returnedmodels having about 95% of the model built by ARP/wARP. Final refine-ments were made by using Phenix refine, applying the TLS (translation/li-bration/screw-motion) method (41), and using manual rebuilding with Coot(42). Refinement statistics can be found in Table 1.

Ala Scanning, Binding, and Neutralization Assays. Similar procedures were usedas described in previous studies (11, 19), and additional details can be foundin SI Materials and Methods.

Modeling of NHP Ab Structures in Complex with gp120 Core. The ClusPro server2.0 (16) was used in the Ab mode to produce structural models of the NHPAbs GE148 and GE136 in complex with previously determined crystal struc-tures of core gp120. Published structures of the two non-bNAbs F105 andb13 (PDB ID codes 3HI1 and 3IDX) were used based on the criteria that thesemAbs displayed a similar binding and neutralizing profile as the NHP Abs. Toinput the gp120 core structure in the ClusPro server, a separate PDB file wascreated for each core gp120 from published structures, removing the humanmAbs to expose the CD4bs on the core molecular surface. The high-resolu-tion models of the complexes resulting from pairing each NHP mAb and thetwo gp120 core structures were obtained based primarily upon the availableAla scan data that we had determined experimentally. To confirm the ac-curacy of the ClusPro analysis, models of GE148 and GE136 in complex withthe gp120 core were used to identify paratope positions that, upon specific

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substitution, would affect the mAb loss or gain of function, primarily de-termined by HIV-1 neutralization capacity (SI Materials and Methods).

EM Sample Preparation and Imaging. Purified core gp120 was incubated witha 3M excess of several Fab fragments at 4 °C for 30 min. Different complexeswere analyzed as follows: (i) JRFLgp120 with GE136, (ii) JRFLgp120 withGE148, (iii) JRFLgp120 with GE136 and PGT122, (iv) JRFLgp120 with GE148and PGT122, (v) JRFLgp120 with GE136 and 2G12, (vi) JRFLgp120 with GE148and 2G12, and (vii) JRFLgp120 with PGT122 and PGV04 as a control. Negativelystained grids were prepared by applying 3 μL at ∼0.01 mg/mL purified com-plexes to a freshly glow-discharged, carbon-coated 400-Cu mesh grid andstained with 2% (wt/vol) uranyl formate or nanoW (Nanoprobes). Grids wereviewed using an FEI Tecnai Spirit electron microscope operating at 120 kV atthe National Resource for Automated Molecular Microscopy at The ScrippsResearch Institute. Images were acquired at a magnification of 52,000× witha defocus of ∼1 μm on a Tietz 4,000 × 4,000 complementary metal oxidesemiconductor camera using the Leginon (43) package at tilt increments of 10°,up to 50°. The tilts provided additional particle orientations to improve the imagereconstructions. The pixel size of the CCD camera was calibrated at this magni-fication to be 2.05 Å using a 2D catalase crystal with known cell parameters.

Data Processing and Image Reconstruction. All particles were automaticallyselected from micrographs using DoG Picker (44) and put into a particle stackwithin the Appion software package (45). Particles were binned by three(64 × 64-sized) boxes, and initial reference-free 2D class averages were cal-culated using Xmipp Clustering 2D Alignment (46). Particles with bound Fabswere selected into a substack and further classified into reference-free 2Dclass averages using Xmipp Clustering 2D Alignment (46). Ten ab initiomodels were generated from the final reference-free 2D class averages usingthe EMAN2 package (47). One of those 10 ab initio models was refined againstthe raw particle stack using EMAN1 projection-matching refinement (48). Theresolutions of the final models were determined using a Fourier shell corre-lation cutoff of 0.5 (Fig. S7). Model fitting into EM densities is described in SIMaterials and Methods.

ACKNOWLEDGMENTS. This study was supported by the International AIDSVaccine Initiative, Scripps Center for HIV/AIDS Vaccine Immunology andImmunogen Discovery, National Institutes of Health, Swedish ResearchCouncil, and Swedish International Development Agency/Departmentof Research Cooperation. C.P. was supported by a fellowship from theCarlsberg Foundation.

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Corrections

EARTH, ATMOSPHERIC, AND PLANETARY SCIENCESCorrection for “High-precision timeline for Earth’s most severeextinction,” by Seth D. Burgess, Samuel Bowring, and Shu-zhongShen, which appeared in issue 9, March 4, 2014, of Proc NatlAcad Sci USA (111:3316–3321; first published February 10, 2014;10.1073/pnas.1317692111).The authors note that Fig. 3 and its corresponding legend ap-

peared incorrectly. The corrected figure and legend appear below.

www.pnas.org/cgi/doi/10.1073/pnas.1403228111

BIOPHYSICS AND COMPUTATIONAL BIOLOGYCorrection for “Trends in structural coverage of the proteinuniverse and the impact of the Protein Structure Initiative,” byKamil Khafizov, Carlos Madrid-Aliste, Steven C. Almo, and AndrasFiser, which appeared in issue 10, March 11, 2014, of Proc NatlAcad Sci USA (111:3733–3738; first published February 24, 2014;10.1073/pnas.1321614111).The authors note that the following statement should be

added as a new Acknowledgments section: “This work was sup-ported by grants from the National Institutes of Health (GM094665,GM094662, GM096041).”

www.pnas.org/cgi/doi/10.1073/pnas.1404196111

IMMUNOLOGYCorrection for “Vaccine-elicited primate antibodies use a dis-tinct approach to the HIV-1 primary receptor binding site in-forming vaccine redesign,” by Karen Tran, Christian Poulsen,Javier Guenaga, Natalia de Val Alda, Richard Wilson, ChristopherSundling, Yuxing Li, Robyn L. Stanfield, Ian A. Wilson, Andrew B.Ward, Gunilla B. Karlsson Hedestam, and Richard T. Wyatt,which appeared in issue 7, February 18, 2014, of Proc Natl AcadSci USA (111:E738–E747; first published February 3, 2014; 10.1073/pnas.1319512111).The authors note that the author name Natalia de Val Alda

should instead appear as Natalia de Val. The corrected authorline appears below. The online version has been corrected.

Karen Tran, Christian Poulsen, Javier Guenaga, Nataliade Val, Richard Wilson, Christopher Sundling, Yuxing Li,Robyn L. Stanfield, Ian A. Wilson, Andrew B. Ward,Gunilla B. Karlsson Hedestam, and Richard T. Wyatt

www.pnas.org/cgi/doi/10.1073/pnas.1403776111

251.941 ± 0.037

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251.495 ± 0.064

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Fig. 3. Generalized Changshingian to Anisian carbonate carbon isotopiccomposition from South China. Bed thickness and number, carbonate carbonisotopic composition, weighted mean 206Pb/238U dates, and extinction in-terval (gray) within Fig. 1, Inset from this study and Cao et al. (11). Re-mainder of carbonate carbon isotopic composition from Payne et al. (52) andgeochronology from Galfetti et al. (54). Permian and Triassic conodont zonesfrom Ogg et al. (63). Stage/substage names are global standard chro-nostratigraphic units used by the International Commission on Stratigraphy.

5060 | PNAS | April 1, 2014 | vol. 111 | no. 13 www.pnas.org