Dose response curve slope helps predict therapeutic potency and breadth of HIV broadly neutralizing antibodies

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1 Received 8 Mr 5 Accepted Aug 5 Pulished 9 Sep 5 Dose response curve slope helps predict therpeutic potency nd redth of HIV rodly neutrlizing ntiodies Nichols E. We, Dvid C. Montefiori & Benhur Lee,3 DOI:.38/ncomms9443 OPEN A new genertion of HIV rodly neutrlizing ntiodies (nas) with remrkle potency, redth nd epitope diversity hs rejuvented interest in immunotherpeutic strtegies. Potencies defined y in vitro IC 5 nd IC 8 vlues (5 nd 8% inhiitory concentrtions) figure prominently into the selection of clinicl cndidtes; however, much higher therpeutic levels will e required to reduce multiple logs of virus nd impede escpe. Here we predict na potency t therpeutic levels y nlysing dose response curve slopes, nd show tht slope is independent of IC 5 /IC 8 nd specificlly reltes to na epitope clss. With few exceptions, CD4-inding site nd V3- nas exhiit slopes 4, indictive of higher expected therpeutic effectiveness, wheres V-, gp4 memrne-proximl externl region (MPER) nd gp gp4 nas exhiit less fvourle slopes o. Our results indicte tht slope is one mjor predictor of oth potency nd redth for nas t cliniclly relevnt concentrtions, nd my etter coordinte the reltionship etween na epitope structure nd therpeutic expecttions. Deprtment of Microiology, Immunology nd Moleculr Genetics, University of Cliforni, Los Angeles, Cliforni 94, USA. Deprtment of Surgery, Duke University Medicl Center, Durhm, North Crolin 77, USA. 3 Deprtment of Microiology, Ichn School of Medicine t Mount Sini, New York, New York 9, USA. Correspondence nd requests for mterils should e ddressed to D.C.M. (emil: dvid.montefiori@duke.edu) or to B.L. (emil: enhur.lee@mssm.edu). NATURE COMMUNICATIONS 6:8443 DOI:.38/ncomms & 5 Mcmilln Pulishers Limited. All rights reserved.

2 NATURE COMMUNICATIONS DOI:.38/ncomms9443 Severl regions of the HIV- envelope glycoprotein spike re vulnerle to rodly neutrlizing ntiodies (nas); these regions include the CD4-inding site () of gp (refs 4), -dependent epitopes in the second nd third vrile regions (V nd V3) of gp (refs 5 8), liner epitopes in the memrne-proximl externl region (MPER) of gp4 (refs 9 ) nd -dependent epitopes tht ridge gp nd gp4 (refs 5). This ssortment cretes opportunities for comintions of nas to trget multiple epitopes in n effort to chieve optiml coverge nd impede escpe 6. Indeed, the identifiction nd chrcteriztion of these nas hs generted renewed optimism tht novel vccines cn e designed to elicit similr types of ntiodies 7,8. The extrordinry redth nd potency of some of the newer nas lso fford promising opportunities for immunotherpy of estlished infection. Recent proof-of-concept studies with pssively delivered nas in HIV-infected humnized mice nd simin humn immunodeficiency virus (SHIV)-infected mcques hve generted encourging therpeutic results, especilly when comintions of nas were used 9 3. Moreover, single infusion with the na, 3BNC7, ws recently shown to reduce plsm virl lod y.8.5 log in chroniclly infected humns 4. These therpeutic enefits might e improved in the presence of stndrd ntiretrovirl drugs nd host utologous neutrlizing ntiodies. Mesurements of na potency nd redth re trditionlly determined y the concentrtion of ntiody tht inhiits either 5% (IC 5 ) or 8% (IC 8 ) of fixed virus inoculum in dose response single-cycle infection ssy in vitro. While these neutrliztion thresholds might e sufficient in prophylctic vccine setting, where the multiplicity of infection during trnsmission is reltively low 5 7 they fll fr elow the effective therpeutic dose rnge tht will e required to inhiit multiple logs of virus nd impede escpe in n infected individul. Another cliniclly relevnt dimension of dose response curves is slope, which my e more ccurte mesure of potency t therpeuticlly relevnt inhiition levels. Studies with ntiretrovirl drugs hve shown tht the slope cn e used to more relily predict clinicl outcome thn IC 5 lone. Instntneous inhiitory potentil (IIP) is n dditionl phrmcodynmic metric tht goes further y incorporting oth slope nd IC 5 to predict the numer of logs of infection reduced t ny given concentrtion of drug in single-round infection ssy 8,9. Together, IC 5 nd slope determine the full rnge of ctivity for given ntiretrovirl gent, nd IIP puts these prmeters into more clinicl context. For ntiretrovirl drugs, the slope of the sigmoidl dose response curve is relted to specific inhiitory mechnisms defined y the coopertive rectivity of inhiitors nd their trgets 8,3 3. Here we show tht the neutrliztion slopes of nas ply n importnt role in forming therpeutic expecttions from in vitro neutrliztion curves nd cn complement nd extend trditionl IC 5 /IC 8 -sed nlyses. We lso find tht slope is more strongly ssocited with neutrliztion redth thn IC 5. With some exceptions, na slopes generlly segregte y epitope clss suggesting tht like HIV inhiitors, na slopes re lso relted to specific mechnisms of neutrliztion, thus, this prmeter might id in the development of novel, highly effective immunotherpies. While oth slope nd IC 5 re fundmentl properties of na ctivity in vitro, na slopes re rrely considered when predicting therpeutic potency. Our results show tht this mechnistic prmeter hs significnt impct on predicted therpeutic potency nd dds new dimension to the development of novel immunotherpeutics. Results Impct of slope on predicted therpeutic potencies of nas. IC 5 nd IC 8 re common metrics used to estlish clinicl expecttions of na ctivity from experimentl results in vitro nd to identify nas with high potentil for dvncement into clinicl trils. While useful, these prmeters lone offer only limited description of neutrliztion ctivity. An dditionl nd often neglected prmeter, the dose response slope, ws strongly ssocited with clinicl outcome in the context of smll-molecule HIV inhiitors, which exhiited wide rnge of clss-specific nd mechnism-specific slopes 9 3. To our knowledge, only one previous study exmined in ny detil the slopes of HIV- na dose response curves, nd this ws mostly done in the context of ssessing the effects of comintions with erlier nas:, G nd F5 (ref. 33). Here we otined dose response curve slopes for 4 nas nd solule CD4 () ssyed in TZM-l cells ginst glol pnel of moleculrly cloned HIV Env-pseudotyped reference viruses 34 (Supplementry Tle ). To cquire dditionl positive neutrliztion results, suset of nas ws ssyed ginst five dditionl Env-pseudotyped reference viruses 35 (Supplementry Tle ). The nas represented six epitope clsses including the nas VRC (refs,4), 3BNC7 (ref. 3), CH3 (ref. 4) nd HJ6 (ref. ); the V- nas PG9, PG6 (ref. 5) nd CH (ref. 8); the V3- nas PGT8 (ref. 6), -74 (ref. 7) nd PGT (ref. 6); the high mnnose cluster () na G (ref. 36); the gp4 MPER nas F5, 4E (refs,) nd E8 (ref. 9); nd the gp/gp4 na PGT5 (ref. 4). Dose response neutrliztion curves for PG6 (V ) nd CH3 () ssyed ginst four Envs re shown in Fig. s exmples of some of the most mrked slope differences oserved. Regrdless of differences in IC 5 (Fig., top), PG6 exhiited shllow dose-dependent rise in neutrliztion reltive to the steeper rise seen with CH3 (Fig. ), which is indicted y the lower dose response curve slope for PG6 (Fig., ottom; compre lue with ornge rs). These results were trnsformed using the medin-effect eqution 37 (eqution (), Supplementry Fig., where f is percent neutrliztion, D is ntiody concentrtion, D m is IC 5 nd m is slope), to give the liner dose responses shown in Fig. c. This form revels tht for ny given Env, the higher slope of CH3 reltive to PG6 cuses the corresponding neutrliztion curves to converge towrds n intersection point nd then diverge s concentrtion continues to increse. This intersection defines the concentrtion (D i ; eqution (), where D m,, m nd D m,, m re the IC 5 s nd slopes for PG6 nd CH3, respectively) nd inhiition level (f i ; eqution (3), where D m nd m re the IC 5 nd slope of either PG6 or CH3, respectively) t which oth PG6 nd CH3 were eqully effective ginst the sme Env. f log ¼ m logðdþ mlogðd m Þ ðþ f " D i ¼ D m # m; m D m; m m ðþ f i ¼ m ð3þ þ D i D m The impct of these intersections on potency is illustrted in Fig. d, where the 5% inhiitory concentrtion of PG6 ws 5- nd,5-fold lower thn CH3 for Envs 57 nd Ce76, respectively. The potency of CH3 progressively pproched tht of PG6 for 8 nd 9% inhiition, where eventully CH3 ws,- nd 5-fold more potent thn PG6 NATURE COMMUNICATIONS 6:8443 DOI:.38/ncomms & 5 Mcmilln Pulishers Limited. All rights reserved.

3 NATURE COMMUNICATIONS DOI:.38/ncomms9443 ARTICLE Neutrliztion c log(f /f u ) % 8% 6% 4% % %.5.5 PG6 CH3 57 Ce76 CH9 CNE8 % 3 log(na (µg ml )) 99% 97% 9% 76% 5%.5 4% % 3 log(na (µg ml )) Neutrliztion IC 5 (µg ml ) d na (µg ml ) ,,.... PG6 CH3 57 Ce76 CH9 CNE8 57 Ce76 CH9 CNE8 Env 57 Env Ce76 IC 5 IC 8 IC 9 IC 99 IC 5 IC 8 IC 9 IC 99 Figure Effect of the slope on neutrliztion nd potency. () Hill plots of neutrliztion curves for PG6 (lue) nd CH3 (ornge) ginst four representtive Envs from our pnel. ()IC 5 (top) nd slope (ottom) vlues determined y medin-effect fitting (Methods). (c) Liner medin-effect plots of neutrliztion for the sme dt in, where IC 5 flls t the x intercept nd slope descries the ngle of ech curve reltive to the x xis. Intersections (crosses) indicte where oth PG6 nd CH3 gve the sme neutrliztion t the sme concentrtion for ech Env. (d) Potencies of PG6 nd CH3 ginst two Envs with the gretest difference in IC 5. Dt shown in () nd (c) re the verge of two replictes nd error rs indicte s.d. for 99% inhiition, reflecting the convergence, intersection nd divergence of the curves. Medin-effect extrpoltions to Z99% inhiition were experimentlly verified using titre-reduction ssy, where CH3 produced 3-log reduction in virl titre t times its IC 8 concentrtion (4 mgml ) compred with PG6, which produced only.9-log reduction in virl titre t times its IC 8 (.44 mgml ) ginst Env Ce76 (Supplementry Fig. ). Thus, chnges in reltive potency t therpeuticlly relevnt na concentrtions re the direct result of the slope s differentil effect on neutrliztion. Importntly, these sme slope-driven fetures were strongly ssocited with the clinicl ctivity of HIV inhiitors, while IC 5 lone ws not correlted to the historicl clinicl properties of HIV inhiitors 8,3. BnA clsses hve chrcteristic slopes. The IC 5 nd slope vlues for ech na ssyed ginst our entire pnel of Envs (Supplementry Tle ) re shown in Fig., (see lso Supplementry Tle ), illustrting the full rnge of vlues oserved in the complete dt set. Virus/nA comintions tht did not rech t lest 5% neutrliztion t the highest na concentrtions tested were excluded due to wek or non-detectle ctivity. We lso note tht some neutrliztion curves exceeded 5% ut plteued elow % (Supplementry Fig. 3), indicting tht portion of the virus ws refrctory to the na. Consistent with previous reports 5,3,5,38,39 we mostly oserved such incomplete neutrliztion for -trgeting nas (CH, PG6, PG9, G nd PGT5). Incomplete neutrliztion of geneticlly clonl Env-pseudovirions is likely mnifesttion of lterntive post-trnsltionl modifictions giving rise to heterogenous popultion of Env spikes, resulting in n epigenetic mixture of sensitive nd resistnt virions. Exmples re post-trnsltionl vriility in sequon occupncy 4 nd composition 38,4, oth of which could profoundly ffect nas tht either require s prt of their epitope, or re suject to shielding. BnAs tht re etter le to tolerte this epigenetic vriility re more likely to chieve % neutrliztion in the ssy. We excluded na/env comintions tht exhiited incomplete neutrliztion (tht is, curves tht plteu elow 95%) ecuse their full neutrliztion potentil fell within the mesurle rnge of the ssy (o-log reduction in infectivity). To compenste for minor ssy vrince, 95% ws used s the upper threshold for plteus tht were considered truly indictive of incomplete neutrliztion. CH exhiited plteus elow 95% neutrliztion ginst every Env in our pnel, while such plteus for PG6, PG9, G nd PGT5 were only oserved mong minor suset of Envs (Supplementry Tle ). Collectively, few sttisticlly significnt differences in slope were oserved within ech na epitope clss for those na/ Env comintions chieving complete neutrliztion within our detection limits, suggesting tht slope is primrily feture of the trget epitope. One exception to this generl rule ws, which gve slopes significntly lower thn those of the na clss (.95±.3 for nd.37±.3 for nas comined, Po., Student s t-test). Although the slopes of PG9 were generlly higher thn those of PG6, this difference did not rech sttisticl significnce (.9±. for PG9 nd.6±.4 for PG6, P ¼.8). No significnt correltion etween slope nd IC 5 ws oserved for ny na clss, reflecting the fundmentl independence of these two prmeters. However, ech clss of nas clustered differentilly in the lndscpe of IC 5 nd slope vlues (Fig. c). Tht (high slope/moderte IC 5, excluding ), V- (low slope/dispersed IC 5 ), MPER (low slope/high IC 5 ) nd V3- (high slope/low IC 5 ) nas clustered into distinct qudrnts suggest tht nas in ech prticulr clss occupy different phenotypic lndscpe defined y oth IC 5 nd slope. The E8 MPER na represents nother interesting exception s it exhiited significntly lower IC 5 s thn 4E nd F5 despite hving similr slopes (geometric men IC 5 NATURE COMMUNICATIONS 6:8443 DOI:.38/ncomms & 5 Mcmilln Pulishers Limited. All rights reserved.

4 NATURE COMMUNICATIONS DOI:.38/ncomms9443 V MPER V3 gp/gp4. V MPER V3 gp/gp4 IC 5 (µg ml ) VRC CH3 3BNC7 HJ6 PG6 PG9 4E F5 E8 74 PGT8 PGT G PGT5 na VRC CH3 3BNC7 HJ6 PG6 PG9 4E F5 E8 74 PGT8 PGT G PGT5 na c VRC 3BNC7 CH3 HJ6 PG6 V PG9 d... 4E MPER F5 E8.... IC 5 (µg ml ) 74 PGT8 PGT V V MPER V3 Epitope clss gp/gp4 Figure Slope nd IC 5 chrcteristics of na epitope clsses. ()IC 5 nd () slope vlues of nas nd (ottom ctegories) ginst ech Env in our pnel (circles). Brs indicte geometric men IC 5 s with 95% confidence intervl (CI) or men slopes with 95% CI. Antiodies re grouped y epitope clss (top ctegories). (c) Lndscpe of slope nd IC 5 vlues for (top left), V- (top right), MPER (ottom left) nd V3- (ottom right) na clsses. Dshed lines indicte qudrnts of high/low IC 5 nd high/low slope. (d) Slopes of ech na epitope clss ginst ech Env in our pnel. Brs indicte men nd 95% CI. All dt shown re from na/env comintions tht chieved complete neutrliztion nd re derived from medin-effect fits of two-replicte verges. for E8 ¼.6 mgml versus 3.5 nd 3.6 mgml for 4E nd F5, respectively; Po., one-wy nlysis of vrince (ANOVA); Fig.,). The slopes of ech na epitope clss could e further ctegorized into the three groups (Fig. d) s those hving slopes 4 (, V3 ), those hving slopes B () nd those hving slopes o (V, gp/gp4, MPER) with high sttisticl significnce (Po., one-wy ANOVA). Notly, nd ech na exhiited rnge of slope vlues mong the viruses in our pnel, indicting tht the slope is lso Env dependent. This will e n importnt considertion when interpreting clinicl enefits mong ptient popultion receiving pssive na therpy. CD4-sed immunodhesins. In ddition to on fide nas, immunodhesins consisting of effector domins fused to the IgG Fc region represent novel clss of rtionlly designed ntivirl therpeutics. For exmple, CD4-Ig consists of the CD4 D nd D domins fused to the Fc domin of IgG (IgG-Fc). Very recently, n enhnced version (ecd4-ig) ws descried in which mimetic peptide derived from the N terminus of the mjor HIV coreceptor, CCR5, ws fused to the C terminus of the CD4-Ig Fc domin 4. ecd4-ig ws le to neutrlize n exceptionlly rod rry of HIV- Envs with n incresed potency reltive to CD4-Ig. We nlysed the dose response curves of ecd4-ig nd CD4-Ig to determine if differences in slope my ccount for the enhnced potency of ecd4-ig nd compred these with the slopes nd IC 5 s of the nas nd. First-order pproximtions of slopes cn e otined from ville IC 5 nd IC 8 concentrtions y using the liner medin-effect form (see eqution (6) in Methods), thus, medin effect reduces the complex curvture of the stndrd sigmoidl Hill curve into liner form (Fig. c nd Supplementry Fig. ) tht simplifies mthemticl nlysis nd llows one to pproximte slopes from limited set of ville dt 37. Both IC 5 nd IC 8 vlues re pulicly ville for the CD4-sed immunodhesins. Figure 3, shows the pproximted slopes nd pulished IC 5 vlues for CD4-Ig nd ecd4-ig. Interestingly, Fig. 3 shows tht ecd4-ig does not hve consistently higher slope compred with CD4-Ig cross the pnel of Envs nlysed (.78±. nd.87±. for CD4-Ig nd ecd4-ig, respectively). Indeed, oth the CD4 immunodhesins nd ll hve similr slopes (.95±.7 for ). However, ecd4-ig consistently exhiited.4 log lower IC 5 compred with CD4-Ig (Po., Student s t-test) (Fig. 3). Thus, the enhnced potency of ecd4-ig reltive to CD4-Ig cn e ttriuted to its lower IC 5. Tht the slope does not differ etween, CD4-Ig nd ecd4-ig my lso indicte tht these mechnisms of inhiition re the sme nd re predominted y the initil CD4-inding event. Conversely, 4 NATURE COMMUNICATIONS 6:8443 DOI:.38/ncomms & 5 Mcmilln Pulishers Limited. All rights reserved.

5 NATURE COMMUNICATIONS DOI:.38/ncomms9443 ARTICLE P<. P< P=NS IC 5 (µg ml )..... CD4-Ig ecd4-ig CD4-Ig ecd4-ig Figure 3 Slope nd IC 5 chrcteristics of CD4 immunodhesins. Neutrliztion slopes () nd IC 5 s() of the nas comined nd CD4 immunodhesin regents, CD4-Ig nd ecd4-ig. Slope nd IC 5 vlues for nas nd re from Fig. nd reproduced here for ese of comprison. Slopes for CD4-Ig nd ecd4-ig were estimted from pulished IC 5 nd IC 8 vlues 4 using eqution (6) (Methods). Thus, slopes were only estimted for suset of Envs (n ¼ 4) where discrete IC 5 nd IC 8 vlues were reported. Brs represent geometric men IC 5 with 95% CI or men slope with 95% CI nd P vlue (one-wy ANOVA) compres slopes of nas with, CD4-Ig nd ecd4-ig comined in pnel, ll other P vlues re Student s t-test. the higher slopes of nas reltive to, CD4-Ig nd ecd4-ig suggest the neutrlizing mechnisms of these nas might e distinct from those of the CD4 immunodhesins. trditionl metrics (IC 5 nd IC 8 ). IIP ¼ log þ D D m m ð4þ Neutrliztion redth is strongly ssocited with slope. The overll therpeutic potentil of nas will depend on the diversity of HIV isoltes tht re neutrlized within cliniclly relevnt rnge of concentrtion. Bredth is trditionlly defined s the percentge of isoltes for which na cn chieve 5 or 8% neutrliztion elow designted concentrtion, usully 5 mgml. Becuse slope defines the chnges in na concentrtion necessry to increse inhiition, we sought to determine how this property ffects neutrliztion redth t incresing therpeutic thresholds, rther thn simply using IC 5 nd IC 8 vlues t fixed na concentrtion. Figure 4 shows the redth of ech na t incresing thresholds of IC 5,IC 8, IC 9 nd IC 99 using medin-effect-fitted curves. Bredth scores cross these incresing thresholds chnged more mrkedly for nas with chrcteristiclly lower slopes (V, MPER nd gp/gp4) thn for nas with chrcteristiclly higher slopes ( nd V3 ). The improved nd nrrow distriution of potencies for E8 resulted in dely of this effect to higher neutrliztion thresholds, where the extrpolted IC 99 redth decreses from to 4%. For isoltes tht were sensitive to ech na (IC 5 o5 mgml ), redth t the more therpeuticlly relevnt IC 99 threshold (tht is, potency needed for -log inhiition) ws strongly ssocited with slope (Fig. 4). Trditionl mesures of potency showed moderte ssocition with neutrliztion redth (IC 8 ) or none t ll (IC 5 ) (Fig. 4c). The MPER nas 4E nd F5, with the exception of E8, were excluded from this ltter nlysis ecuse they exhiited zero redth t the IC 99 threshold. IIP defines clinicl expecttions using oth IC 5 nd slope. The neutrliztion potency of n ntiody cn e more completely descried when oth slope nd IC 5 re used to determine the IIP. IIP ws first used in n explntory frmework tht ccounted for the mrked differences in clinicl potency mong the extnt clsses of ntiretrovirl drugs 8,9, which could not e ccounted for y differences in IC 5 lone. IIP uses oth slope nd IC 5 to descrie the log decrese y which single-round infection is reduced y the ntivirl gent t given concentrtion (D) in vitro (see eqution (4), where slope is m nd IC 5 is D m, nd Supplementry Fig. 4). Thus, IIP serves s more precise therpeutic expecttion for ny given dose of na thn As n exponentil prmeter, smll differences in slope (m) cn led to lrge differences in IIP s D increses. As n illustrtive exmple, we clculted the IIP of four nas ginst single Env clone (57) t, 5 nd mgml (Fig. 5). These four nas from the indicted epitope clsses (, V nd gp/gp4) lso exhiit different m vlues with this Env. A mrked increse in IIP (on log-scle) is seen s the concentrtion of na increses, most notly for nas with higher slopes (CH3 nd 3BNC7). This pttern is even more pprent when exmining the dose response curves shown in Supplementry Fig. 4. Using eqution (4) nd the sme methodology, we clculted the IIP of the entire pnel of nas t 5 mgml, which is common threshold concentrtion used to ssess the therpeutic potentil of nas (Fig. 5). By incorporting oth slope nd IC 5, IIP revels some striking results. For exmple, the V3- nas with higher slopes (.5±.3) were predicted to reduce virl infectivity y 3 logs more thn the MPER nas with lower slopes (.8±.) (men IIP 4.9±.9 for V3- nas versus IIP.4±.6 for MPER nas; Po., Student s t-test). In generl, the IIP reflected the slopes (Fig. ) of ech na clss except the V- nas, where the wide rnge of IC 5 vlues (Fig. ) resulted in n eqully wide distriution of IIPs. Indeed, PG6 exhiited one of the highest IIPs (7.6) ginst Env 737, which reflected the chrcteristiclly low IC 5 (. mgml ) of PG6 when coupled to n unusully high slope (.69) for this na (medin PG6 slope.6±.4) ginst this prticulr Env. The IIPs of the CD4 immunodhesin regents (CD4Im) reflected their differences in IC 5, where ecd4-ig chieved.4 log greter reduction in infection thn CD4-Ig (IIP ¼.6±.7 for CD4-Ig nd 3.±. for ecd4-ig, Po.). Recll tht the IC 5 s of ecd4-ig were.4 logs lower thn those for CD4-Ig ut no significnt differences in slope were oserved (Fig. 3). The sme ws oserved for E8, which gve geometric men IC 5 s tht were.3 logs lower thn F5 nd 4E resulting in B-log increse in IIP. Altogether, these dt suggest tht the comintion of slope nd IC 5 vlues reflected in the IIP metric hs considerle explntory potentil tht cn complement nd inform evlution of the therpeutic efficcy of nas in the sme wy these three metrics illuminte our understnding of the clinicl potency of smll-molecule inhiitors. NATURE COMMUNICATIONS 6:8443 DOI:.38/ncomms & 5 Mcmilln Pulishers Limited. All rights reserved.

6 NATURE COMMUNICATIONS DOI:.38/ncomms9443 % Neutrliztion threshold 5% 8% 9% 99% CD4Im V MPER V3 gp/gp4 Isoltes neutrlized 8% 6% 4% %.5.5 % VRC CH3 3BNC7 r = % CI=.63 to.963 P<. HJ6 V MPER (E8) V3 PG6 c IC 8 (µg ml ) PG9. 4E na P=.49 gp/gp % Isoltes with % Isoltes with % Isoltes with IC 99 < 5 µg ml IC 99 < 5 µg ml IC 99 < 5 µg ml F5 E8 r = % CI=.847 to.5-74 IC 8 (µg ml ) PGT8.. PGT P =.6 G PGT5 r = % CI=.84 to.9 Figure 4 Effect of slope on neutrliztion redth. () Neutrliztion redths determined from dose response curves of ech na ginst our totl Env pnel t incresing neutrliztion thresholds. A na is considered non-neutrlizing for prticulr Env t given inhiitory threshold when the respective inhiitory concentrtion (IC 5,IC 8,IC 9 or IC 99 )is45 mgml. Antiodies re ordered y epitope clss. Correltions of slope (), IC 8 (c, left) nd IC 5 (c, right) to redth t 99% neutrliztion for ech na, grouped y epitope clss (symols), where redth excludes Envs giving no detectile neutrliztion within the prmeters of our ssy (Methods). Person correltions (r), 95% CI nd ssocited P vlues re indicted ove ech grph. IIP µg ml 5 µg ml µg ml na CH3 PG6 3BNC7 PGT5 Slope IIP (5 µg ml ) VRC CH3 3BNC7 HJ6 CD4Im V CD4-lg ecd4-lg PG6 PG9 na MPER 4E F5 E8 74 V3 PGT8 PGT gp/gp4 G PGT5 Figure 5 IIP incorportes oth slope nd IC 5. () The, 5 nd mgml IIPs for CH3, PG6, 3BNC7 nd PGT5 ginst Env 57 with slopes indicted. () 5mgml IIPs for ll nas ginst our Env pnel where complete neutrliztion ws oserved. Brs indicte men nd 95% CI. Clinicl implictions of na slopes. Two studies in humns demonstrted moderte trnsient reduction in plsm viremi when G, F5 nd 4E were co-dministered immeditely efore tretment interruption in sujects who egn stndrd ntiretrovirl therpy during cute infection 43,44. Results of detiled nlysis of escpe vrints in the treted sujects suggested tht G ws the only ntiody in the comintion tht exerted pressure on the virus. On the other hnd, results of n in-depth nlysis showing tht escpe in vitro my e more difficult for F5 nd 4E thn for G suggests tht perhps ll three nas were needed for the oserved trnsient effect on viremi 45. We oserved in our dt set moderte ut sttisticlly 6 NATURE COMMUNICATIONS 6:8443 DOI:.38/ncomms & 5 Mcmilln Pulishers Limited. All rights reserved.

7 NATURE COMMUNICATIONS DOI:.38/ncomms9443 ARTICLE log(f /f u ) 3 G MPER (F5, 4E) log(na (µg ml )) 99.9% 99% 9% 5% % 4 Neutrliztion na (µg ml ),,,. IC 8 IC 9 IC 99 F5 4E G F5 4E G F5 4E G c IIP na (µg ml ) F5 4E G Figure 6 Sensitivity of therpeuticlly relevnt potencies to smll differences in slope. () Medin-effect plot of men neutrliztion for G (red solid line) nd MPER nas (4E nd F5 comined, green solid line) determined from the comined medin-effect curves of these nas ginst ech Env on our pnel tht ws neutrlized. Shded res indicte the corresponding s.d. () IC 8,IC 9 nd IC 99 potencies of F5, 4E nd G ginst ech Env in our pnel (symols). Brs indicte geometric men nd 95% CI. Dotted oxes illustrte the rnge of pek/trough plsm concentrtions for ech na reported in humn trils 38,39.(c) IIPs of F5, 4E nd G ginst our Env pnel t verge pek serum concentrtions reported in humn trils 43,44. significnt differences in slope etween G nd the MPER nas F5 nd 4E (G slope ¼.±.; comined F5 nd 4E slopes ¼.83±., P ¼., Student s t-test). As shown in Fig. 6, this moderte difference is compounded t higher neutrliztion thresholds, such tht G chieves 99% neutrliztion t n verge of 75 mgml, wheres F5 nd 4E required Zmgml. Figure 6 shows the rnge of F5, 4E nd G pek/trough plsm concentrtions estimted from humn trils 43,44. While the IC 8 s nd IC 9 s of F5 nd 4E fll within or elow this rnge, only G remined predominntly within or elow this rnge t IC 99. The IIP of these nas ginst our Env pnel t verge pek serum concentrtions 43,44 provide more clinicl description of expected efficcy, where F5 nd 4E chieved nrrow distriution of moderte IIP (.64±.44 nd.68±.56 for F5 nd 4E, respectively) nd G chieved n IIP 43 for over hlf the Envs on our pnel tht were sensitive to this na (Fig. 6c, men IIP 3.3±.5). These results illuminte potentil mechnisms for the exclusive G escpe oserved with this triple therpy in humns, in ddition to the reltive ese of G escpe in vitro 46 nd the distinct phrmcokinetic properties of these three nas, where ccumultion of G results in greter concentrtions in vivo 43,44. The higher verge IIP of G ginst our Env pnel suggests this na would likely exert greter neutrlizing ctivity nd selective pressure thn F5 or 4E; however, the rod distriution of G IIP reltive to the nrrow distriution of MPER IIP lso suggest roder lndscpe of potentil resistnce muttions for G, represented y our Env pnel. Overll, our results suggest tht even sutle differences in slope cn give rise to importnt differences in IIP nd clinicl outcome. Three new nas hve een evluted in pssive immunotherpy experiments in mcques, ech of which exhiited chrcteristic slopes 4 in our study. As monotherpy, PGT (V3 ) ws profoundly effective ginst estlished SHIV- SF6P3 infection 3, wheres 3BNC7 () nd -74 (V3 ) were profoundly effective ginst estlished SHIV- AD8EO infection, resulting in up to 3-log reductions in plsm viremi in ech cse. Using ville pulished dose response dt, we estimted the slope for PGT ginst the SHIV- SF63P3 chllenge stock to e B, nd the slopes for 3BNC7 nd -74 ginst SHIV-AD8EO chllenge stock to e.59 nd.9, respectively. Finlly, s mentioned erlier, single infusion with 3BNC7 ws recently shown to reduce plsm virl lod in chroniclly infected humns s long s therpeutic levels were present 4. While these results further indicte tht nas with slopes 4 re ssocited with positive clinicl outcomes, pucity of pssive immunotherpy dt with nas tht exhiit lower slopes precludes quntittive verifiction of their potentil clinicl enefits t this time. Discussion Next-genertion nas re currently eing considered for immunotherpy due to their enhnced potency nd redth of neutrliztion. In ddition to these fctors, other considertions such s scle-up mnufcturility, sfety, phrmcokinetics, immunogenicity nd ese of escpe, just to nme few, will determine the clinicl success of nas. Furthermore, neither redth nor potency (or ny in vitro test for tht mtter) cn esily predict the ese of escpe nd fitness of escpe muttions to ny prticulr na in vivo. Indeed, it is unlikely tht monotherpy with ny one na, no mtter how potent or rod, will succeed, especilly since ll extnt nas hve known resistnce muttions. Nonetheless, redth nd potency, imperfect surrogte mesures s they re of therpeutic efficcy, re criticl components of the evlution of na cndidtes (or na comintions) for in vivo efficcy trils. Importntly, redth nd potency re trditionlly defined y in vitro IC 5 nd IC 8 vlues tht re well elow the therpeutic threshold nd these metrics only offer limited, fixed description of na ctivity. Here we show tht the dose response curve slope is more relile indictor of na redth nd potency t more therpeuticlly relevnt doses. The current stte-of-the-rt does not consider the slope prmeter when prioritizing which na or comintion of nas to dvnce to humn trils. We elieve our nlysis cn complement these incresingly sophisticted efforts 6 nd enhnce the cliniclly predictive power of in vitro surrogte ssys for na potency. More importntly, inclusion of the estlished IIP metric, which incorportes oth IC 5 nd slope, dds nother cliniclly relevnt dimension to our nlysis (Fig. 5). The IIP metric hs proven utility in predicting the clinicl potency of ntiretrovirl drugs nd drug comintions 8,9,3. It is derived from phrmcodynmic model tht predicts the log decrese in virus infection when the ntivirl gent, in this cse the na, is extrpolted to given cliniclly relevnt concentrtion. Predicted IIPs or IIP ve ( more sophisticted metric tht includes dditionl phrmcokinetic prmeters such s hlf-life of the na, ut criticlly still includes the IC 5 nd slope prmeters) cn help guide the determintion of effective dosing rnges nd intervls. NATURE COMMUNICATIONS 6:8443 DOI:.38/ncomms & 5 Mcmilln Pulishers Limited. All rights reserved.

8 NATURE COMMUNICATIONS DOI:.38/ncomms9443 Mechnistic explntions for the different na curve slopes will require dditionl studies. We hypothesize tht for the geneticlly cloned Env-pseudovirions used here, slope is t lest prtilly determined y epigenetic heterogeneity within the Env glycoprotein spikes tht decorte the virus surfce. Exmples re vriility in sequon occupncy nd composition s mentioned ove. Trget heterogeneity hs een invoked to explin the slopes of other lignd-effector units 47 nd ws suggested to impct the slope of HIV inhiitors, including nas such s, G nd F5 (ref. 33). BnAs tht re etter le to tolerte post-trnsltionl Env heterogeneity, or whose epitopes re not ffected y this, would neutrlize ll virus prticles eqully well, resulting in slope of B. BnAs with lower threshold of tolernce would exhiit vrile neutrliztion efficiencies cross the heterogeneous virus popultion, resulting in slopes o. Here dequte na concentrtions my e cple of neutrlizing ll virions in the popultion; however, it is lso possile tht minor frction of virions would completely resist neutrliztion. Becuse we excluded ll neutrliztion curves tht exhiit incomplete neutrliztion in our ssy, the expected plteu representing the minor frction of resistnt virions would reside outside the rnge of the ssy (for exmple, plteu t 99.9% neutrliztion). Neutrliztion ssys with wider rnge of detection will e needed to ssess this ltter possiility. BnA slopes might lso e determined in prt y mechnisms of neutrliztion, such s n ility to ct t one or multiple stges of the fusion process 48,49. BnAs tht re le to inhiit t multiple stges might cooperte to explin in prt dose response curve slopes 4. Collectively our dt revel n ssocition etween na epitopes nd dose response slopes tht ridge the fields of structurl iology nd clinicl evlution, nd my help to guide the rtionl design nd testing of therpeuticlly effective ntiodies for HIV nd other pthogens. Methods Virus stocks. Virus stocks were prepred y trnsfection in 93T cells nd titrted in TZM-l cells s descried 5. A complete list of our HIV Env pnel is provided in Supplementry Tle. Neutrliztion ssy. The neutrlizing ctivity of nas ws mesured s function of reductions in luciferse (Luc) reporter gene expression fter single round of infection in TZM-l cells 5. TZM-l cells (lso clled JC57BL-3) were otined from the NIH AIDS Reserch nd Reference Regent Progrm, s contriuted y John Kppes nd Xioyun Wu. This is HeL cell clone tht ws engineered to express CD4 nd CCR5 (ref. 5) nd to contin integrted reporter genes for firefly luciferse nd Escherichi coli et-glctosidse under control of n HIV- long terminl repet 5. Briefly, pre-titrted dose of virus ws incuted with seril threefold dilutions of test smple in duplicte in totl volume of 5 ml for h t 37 C in 96-well flt-ottom culture pltes. Freshly trypsinized cells (, cells in ml of growth medium contining 75 mgml diethylminoethyl dextrn) were dded to ech well. One set of eight control wells received cells þ virus (virus control) nd nother set received cells only (ckground control). After 48 h of incution, ml of cells ws trnsferred to 96-well lck solid plte (Costr) for mesurements of luminescence using the Britelite Luminescence Reporter Gene Assy System (PerkinElmer Life Sciences). Assy stocks of moleculrly cloned Env-pseudotyped viruses were prepred y trnsfection in 93T/7 cells (Americn Type Culture Collection) nd titrted in TZM-l cells s descried 5. This ssy hs een formlly optimized nd vlidted 53 nd ws performed in complince with good clinicl lortory prctices, including prticiption in forml proficiency testing progrmme 54. Additionl informtion on the ssy nd ll supporting protocols my e found t: BnAs. 3BNC7 (ref. 3) nd -74 (ref. 7) were otined from Michel Nussenzweig (Rockefeller University). VRC (refs,4) ws otined from John Mscol (Vccine Reserch Center, NIAID, NIH). PG9 (ref. 5), PG6 (ref. 5), PGT8 (ref. 6) nd PGT5 (ref. 4) were otined from Dennis Burton (Scripps Reserch Institute). CH (ref. 8) nd CH3 (ref. 4) were otined from Brton Hynes (Duke University Medicl Center). HJ6 (ref. ) ws otined from Dvide Corti nd Antonio Lnzvecchi (Institute for Reserch in Biomedicine, USI Switzerlnd). G (ref. 36), F5 (refs,) nd 4E (refs,) were purchsed from PolyMun Scientific (Germny). Medin-effect nlysis. Slope nd IC 5 vlues were determined using the medin-effect method 37. This method involves liner trnsformtion of the stndrd Hill plot (Supplementry Fig. ), where neutrliztion is represented y log effect rtio (eqution () nd Supplementry Fig. ). Liner regression ws used to determine the slope (m)ndic 5 (D m ) vlues corresponding to the liner slope nd x intercept, respectively, of ech curve (Supplementry Fig. ). In ll cses, medin-effect fits were determined from the verge of two experimentl replictes for ech neutrliztion curve. Envs tht did not rech minimum 5% neutrliztion within the rnge of ntiody concentrtions used in ech neutrliztion ssy were considered non-neutrlized s well s Envs with IC 5 vlues ove 5 mgml (Supplementry Tle ). Neutrliztion intersection concentrtions (D i ) were determined using eqution (), which ws derived from eqution (). In cses where neutrliztion reched mximum plteu o95% (Supplementry Tle nd Supplementry Fig. 3) stepwise itertor (perl v5..4) ws used to fit mximum neutrliztion (N) to eqution (5) using the method of lest squres, where f is neutrliztion s percentge of mximum neutrliztion, N is mximum neutrliztion, D is na concentrtion (mgml ), m is slope nd D m is the concentrtion giving hlf mximum neutrliztion. f log ¼ m logðdþ m logðd m Þ ð5þ N f In ll cses, the IC 5 vlues reported re the concentrtions giving 5% mximum neutrliztion. IIP nlysis. IIPs were clculted using eqution (4) s previously descried 9, using fitted slope (m) ndic 5 (D m ) vlues. See Supplementry Fig. 4 for n illustrtive description of IIP. Slope estimtes. Eqution (6) ws used to estimte slope vlues from puliclly ville IC 5 nd IC 8 vlues, where m is slope nd log(4) is the chnge in the log effect rtio (logð f Þ, eqution ()) etween 5 nd 8% neutrliztion. f Eqution (6) ws derived from the liner medin-effect form descried y eqution (). logð4þ m ¼ logðic 8 Þ logðic 5 Þ Comprison of epitope clsses. Sttisticl differences in men slope etween the, V, V3, MPER, or gp/gp4 clsses were performed using one-wy ANOVA in GrphPd Prism 6. The vrince of slopes mong these clsses were equl nd followed norml distriution. Experimentl vlidtion of extrpolted potencies. Extrpoltion of inhiitory concentrtions using medin-effect-fitted slope nd IC 5 vlues ws investigted experimentlly using modifiction of the TZM-l ssy descried ove. Briefly, undiluted stocks of Env-pseudotyped viruses were incuted in the presence nd sence of the indicted concentrtions of nas for h t 37 C. Ech mixture ws then diluted serilly fourfold in qudruplicte for totl of dilutions in 96- well culture pltes. TZM-l cells were dded nd incuted t 37 C for 48 h. Infectious virl titre ws defined y the fold dilution of Ce76 virus stock, virus stock þ CH3 or virus stock þ PG6 mixtures giving, reltive light units (RLU) luciferse ctivity. The dynmic rnge of this ssy ws greter thn the stndrd TZM-l neutrliztion ssy, where we oserved mximum 3.-log reduction in virus titre (B99.9% neutrliztion). The chnge in virus titre reduction etween nd IC 8 concentrtions for PG6 nd CH3 were proportionte to their respective slopes, where the log titre reduction ws.9-fold higher for CH3 (etween nd IC 8 ) nd the log titre reduction ws.3- fold for PG6 (etween nd IC 8 ) (Supplementry Fig. ). Neutrliztion redth nd redth correltions. Neutrliztion redth ws defined s the percentge of Envs on our pnel tht gve 5, 8, 9 or 99% neutrliztion t concentrtions elow 5 mgml ccording to medin-effect fits. This clcultion includes Envs for which no detectile neutrliztion could e experimentlly oserved. To ccurtely represent the correltions of slope, IC 5 nd IC 8 to 99% neutrliztion redth, redths were re-clculted to exclude Envs tht gve no detectile neutrliztion within the concentrtion rnge used in our ssy. Sttisticl nlysis. Slope nd IC 5 vlues were determined from liner regression of medin-effect-trnsformed neutrliztion dt using Microsoft Excel. Person correltions, confidence intervls, t-tests nd one-wy ANOVA nlyses were conducted using GrphPd Prism 6. ð6þ 8 NATURE COMMUNICATIONS 6:8443 DOI:.38/ncomms & 5 Mcmilln Pulishers Limited. All rights reserved.

9 NATURE COMMUNICATIONS DOI:.38/ncomms9443 ARTICLE Code vilility. In cses where n Env/nA comintion chieved mximum plteu in neutrliztion within our detection limit, lest squres itertive lgorithm ws used to fit mximum neutrliztion (N) ccording to eqution (5) using perl v5..4. This script is ville s Supplementry Dt. References. Wu, X. et l. Rtionl design of envelope identifies rodly neutrlizing humn monoclonl ntiodies to HIV-. Science 39, ().. Bll-Jhgjhoorsingh, S. S. et l. The N76 glycosyltion site is required for HIV- neutrliztion y the CD4 inding site specific HJ6 monoclonl ntiody. PLoS ONE 8, e68863 (3). 3. Scheid, J. F. et l. Sequence nd structurl convergence of rod nd potent HIV ntiodies tht mimic CD4 inding. Science 333, (). 4. Wu, X. et l. Focused evolution of HIV- neutrlizing ntiodies reveled y structures nd deep sequencing. Science 333, (). 5. Wlker, L. M. et l. Brod nd potent neutrlizing ntiodies from n Africn donor revel new HIV- vccine trget. Science 36, (9). 6. Wlker, L. M. et l. Brod neutrliztion coverge of HIV y multiple highly potent ntiodies. Nture 477, (). 7. Mouquet, H. et l. Complex-type N- recognition y potent rodly neutrlizing HIV ntiodies. Proc. Ntl Acd. Sci. USA 9, E368 E377 (). 8. Bonsignori, M. et l. Anlysis of clonl linege of HIV- envelope V/V3 conformtionl epitope-specific rodly neutrlizing ntiodies nd their inferred unmutted common ncestors. J. Virol. 85, (). 9. Hung, J. et l. Brod nd potent neutrliztion of HIV- y gp4-specific humn ntiody. Nture 49, 46 4 ().. Muster, T. et l. A conserved neutrlizing epitope on gp4 of humn immunodeficiency virus type. J. Virol. 67, (993).. Zwick, M. B. et l. Brodly neutrlizing ntiodies trgeted to the memrneproximl externl region of humn immunodeficiency virus type glycoprotein gp4. J. Virol. 75, ().. Schrf, L. et l. Antiody 8ANC95 revels site of rod vulnerility on the HIV- envelope spike. Cell Rep. 7, (4). 3. Flkowsk, E. et l. Brodly neutrlizing HIV ntiodies define dependent epitope on the prefusion conformtion of gp4 on cleved envelope trimers. Immunity 4, (4). 4. Blttner, C. et l. Structurl delinetion of quternry, clevge-dependent epitope t the gp4-gp interfce on intct HIV- Env trimers. Immunity 4, (4). 5. Hung, J. et l. Brod nd potent HIV- neutrliztion y humn ntiody tht inds the gp4-gp interfce. Nture 55, 38 4 (4). 6. Kong, R. et l. improving neutrliztion potency nd redth y comining rodly rective HIV- ntiodies trgeting mjor neutrliztion epitopes. J. Virol. 89, (5). 7. Burton, D. R. et l. A lueprint for HIV vccine discovery. Cell Host Microe, (). 8. Mscol, J. R. & Hynes, B. F. HIV- neutrlizing ntiodies: understnding nture s pthwys. Immunol. Rev. 54, 5 44 (3). 9. Klein, F. et l. HIV therpy y comintion of rodly neutrlizing ntiodies in humnized mice. Nture 49, 8 ().. Horwitz, J. A. et l. HIV- suppression nd durle control y comining single rodly neutrlizing ntiodies nd ntiretrovirl drugs in humnized mice. Proc. Ntl Acd. Sci. USA, (3).. Klein, F. et l. Enhnced HIV- immunotherpy y commonly rising ntiodies tht trget virus escpe vrints. J. Exp. Med., (4).. Shingi, M. et l. Antiody-medited immunotherpy of mcques chroniclly infected with SHIV suppresses viremi. Nture 53, 77 8 (3). 3. Brouch, D. H. et l. Therpeutic efficcy of potent neutrlizing HIV--specific monoclonl ntiodies in SHIV-infected rhesus monkeys. Nture 53, 4 8 (3). 4. Cskey, M. et l. Viremi suppressed in HIV--infected humns y rodly neutrlizing ntiody 3BNC7. Nture 5, (5). 5. Hlnd, R. E. et l. Inflmmtory genitl infections mitigte severe genetic ottleneck in heterosexul trnsmission of sutype A nd C HIV-. PLoS Pthog. 5, e74 (9). 6. Arhms, M.-R. et l. Quntitting the multiplicity of infection with humn immunodeficiency virus type sutype C revels non-poisson distriution of trnsmitted vrints. J. Virol. 83, (9). 7. Li, H. et l. High multiplicity infection y HIV- in men who hve sex with men. PLoS Pthog. 6, e89 (). 8. Shen, L. et l. Dose-response curve slope sets clss-specific limits on inhiitory potentil of nti-hiv drugs. Nt. Med. 4, (8). 9. Shen, L., Ri, S. A. & Silicino, R. F. A novel method for determining the inhiitory potentil of nti-hiv drugs. Trends Phrmcol. Sci. 3, 6 66 (9). 3. Smph, M. E. S., Shen, L., Jilek, B. L. & Silicino, R. F. Dose-response curve slope is missing dimension in the nlysis of HIV- drug resistnce. Proc. Ntl Acd. Sci. USA 8, (). 3. Jilek, B. L. et l. A quntittive sis for ntiretrovirl therpy for HIV- infection. Nt. Med. 8, (). 3. Lskey, S. B. & Silicino, R. F. A mechnistic theory to explin the efficcy of ntiretrovirl therpy. Nt. Rev. Microiol., (4). 33. Kets, T. J., Holuigue, S., Mtthews, K., Moore, J. P. & Klsse, P. J. Envglycoprotein heterogeneity s source of pprent synergy nd enhnced coopertivity in inhiition of HIV- infection y neutrlizing ntiodies nd entry inhiitors. Virology 4, 36 (). 34. decmp, A. et l. Glol pnel of HIV- Env reference strins for stndrdized ssessments of vccine-elicited neutrlizing ntiodies. J. Virol. 88, (4). 35. Li, M. et l. Humn immunodeficiency virus type env clones from cute nd erly sutype B infections for stndrdized ssessments of vccine-elicited neutrlizing ntiodies. J. Virol. 79, 8 5 (5). 36. Scnln, C. N. et l. The rodly neutrlizing nti-humn immunodeficiency virus type ntiody G recognizes cluster of lph 4 mnnose residues on the outer fce of gp. J. Virol. 76, (). 37. Chou, T. C. & Tlly, P. Quntittive nlysis of dose-effect reltionships: the comined effects of multiple drugs or enzyme inhiitors. Adv. Enzyme Regul., 7 55 (984). 38. Doores, K. J. & Burton, D. R. Vrile loop dependency of the rod nd potent HIV--neutrlizing ntiodies PG9 nd PG6. J. Virol. 84, 5 5 (). 39. Sok, D. et l. Promiscuous site recognition y ntiodies to the highmnnose ptch of gp rodens neutrliztion of HIV. Sci. Trnsl. Med. 6, 36r63 (4). 4. Go, E. P. et l. Glycosyltion site-specific nlysis of HIV envelope proteins (JR-FL nd CON-S) revels mjor differences in glycosyltion site occupncy, glycoform profiles, nd ntigenic epitopes ccessiility. J. Proteome Res. 7, (8). 4. Pritchrd, L. K. et l. Glycn microheterogeneity t the PGT35 ntiody recognition site on HIV- gp revels moleculr mechnism for neutrliztion resistnce. J. Virol. 89, (5). 4. Grdner, M. R. et l. AAV-expressed ecd4-ig provides durle protection from multiple SHIV chllenges. Nture 59, 87 9 (5). 43. Trkol, A. et l. Dely of HIV- reound fter cesstion of ntiretrovirl therpy through pssive trnsfer of humn neutrlizing ntiodies. Nt. Med., 65 6 (5). 44. Mehndru, S. et l. Adjunctive pssive immunotherpy in humn immunodeficiency virus type -infected individuls treted with ntivirl therpy during cute nd erly infection. J. Virol. 8, 6 3 (7). 45. Mnrique, A. et l. In vivo nd in vitro escpe from neutrlizing ntiodies G, F5, nd 4E. J. Virol. 8, (7). 46. Trkol, A. et l. In vivo efficcy of humn immunodeficiency virus neutrlizing ntiodies: estimtes for protective titers. J. Virol. 8, (8). 47. Hoffmn, A. & Golderg, A. The reltionship etween receptor-effector unit heterogeneity nd the shpe of the concentrtion-effect profile: phrmcodynmic implictions. J. Phrmcokinet. Biophrm., (994). 48. Crooks, E. T. et l. Chrcterizing nti-hiv monoclonl ntiodies nd immune ser y defining the mechnism of neutrliztion. Hum. Antiodies 4, 3 (5). 49. Binley, J. M. et l. Redox-triggered infection y disulfide-shckled humn immunodeficiency virus type pseudovirions. J. Virol. 77, (3). 5. Montefiori, D. C. Mesuring HIV neutrliztion in luciferse reporter gene ssy. Methods Mol. Biol. 485, (9). 5. Pltt, E. J., Wehrly, K., Kuhmnn, S. E., Chesero, B. & Kt, D. Effects of CCR5 nd CD4 cell surfce concentrtions on infections y mcrophgetropic isoltes of humn immunodeficiency virus type. J. Virol. 7, (998). 5. Wei, X. et l. Emergence of resistnt humn immunodeficiency virus type in ptients receiving fusion inhiitor (T-) monotherpy. Antimicro. Agents Chemother. 46, (). 53. Srzotti-Kelsoe, M. et l. Optimiztion nd vlidtion of the TZM-l ssy for stndrdized ssessments of neutrlizing ntiodies ginst HIV-. J. Immunol. Methods 49, 3 46 (4). 54. Todd, C. A. et l. Development nd implementtion of n interntionl proficiency testing progrm for neutrlizing ntiody ssy for HIV- in TZM-l cells. J. Immunol. Methods 375, (). Acknowledgements We thnk Miroslw Bilsk, Amy Peterson, Jennifer Mrtin nd Elizeth Ycovelli for technicl ssistnce. We lso grtefully cknowledge Michel Nussenzweig, John Mscol, Dennis Burton, Brton Hynes, Dvide Corti nd Antonio Lnzvecchi for generously providing nas. Finlly, we thnk Bette Korer nd Kshitij Wgh for helpful NATURE COMMUNICATIONS 6:8443 DOI:.38/ncomms & 5 Mcmilln Pulishers Limited. All rights reserved.

10 NATURE COMMUNICATIONS DOI:.38/ncomms9443 discussions. This work ws supported y grnts from the US Ntionl Institutes of Helth (AI98, B.L.; T3 GM785; N.E.W.) nd from the Bill & Melind Gtes Foundtion Collortion for AIDS Vccine Discovery (#344, D.C.M.). B.L. lso cknowledges the Wrd-Colemn estte for endowing the Wrd-Colemn Chirs t the Ichn School of Medicine t Mount Sini. Author contriutions All uthors designed the experiments nd contriuted to mnuscript writing; neutrliztion ssys were performed in the D.C.M. lortory; N.E.W. performed the dt nd sttisticl nlyses. Additionl informtion Supplementry Informtion ccompnies this pper t nturecommunictions Competing finncil interests: The uthors declre no competing finncil interests. Reprints nd permission informtion is ville online t reprintsndpermissions/ How to cite this rticle: We, N. E. et l. Dose response curve slope helps predict therpeutic potency nd redth of HIV rodly neutrlizing ntiodies. Nt. Commun. 6:8443 doi:.38/ncomms9443 (5). This work is licensed under Cretive Commons Attriution 4. Interntionl License. The imges or other third prty mteril in this rticle re included in the rticle s Cretive Commons license, unless indicted otherwise in the credit line; if the mteril is not included under the Cretive Commons license, users will need to otin permission from the license holder to reproduce the mteril. To view copy of this license, visit NATURE COMMUNICATIONS 6:8443 DOI:.38/ncomms & 5 Mcmilln Pulishers Limited. All rights reserved.