Antigen modification regulates competition of broad and narrow neutralizing HIV antibodies

to structural analysis,including other ferritins that could not be solved by x-ray crystallography. During the first4e–/Å2on gold substrates,1to 2Åof in-plane motion remain.These first few electrons are critical,as they potentially contain the most high-resolution information(26,27). Future work will focus on substrate design and image acquisition conditions to reduce the initial motion even more.Along with further improve-ments in electron detector efficiency,this will bring cryo-EM to the physical limits imposed by the homogeneity of the protein preparation, the electron scattering cross sections of the bio-logical specimen(4),and the counting statistics of detecting individual electrons.We anticipate that high-efficiency,stationary particle imaging will allow:(i)measurement and modeling of the progressive damage to the primary and second-ary structure of the molecules,(ii)improved re-finements using dose-fractionation that includes the use of tilt,and(iii)direct modeling and re-finement of molecular structure from particle images.This will enable routine structure deter-mination for many molecules and complexes that are currently too difficult to be practical. REFERENCES AND NOTES1. A.Amunts et al.,Science343,1485–1489(2014).2.M.Liao,E.Cao,D.Julius,Y.Cheng,Nature504,107–112(2013).3.M.Allegretti,ls,G.McMullan,W.Kühlbrandt,J.Vonck,eLife3,e01963–e01963(2014).4.R.Henderson,Q.Rev.Biophys.28,171–193(1995).5.P.B.Rosenthal,R.Henderson,J.Mol.Biol.333,721–745(2003).6.R.Henderson,G.McMullan,Microscopy62,43–50(2013).7. A.Faruqi,R.Henderson,M.Pryddetch,P.Allport,A.Evans,Nucl.Instrum.Methods546,170–175(2005).8.R.Henderson,R.M.Glaeser,Ultramicroscopy16,139–150(1985).9. E.R.Wright,C.V.Iancu,W.F.Tivol,G.J.Jensen,J.Struct.Biol.153,241–252(2006).10.R.M.Glaeser,G.McMullan,A.R.Faruqi,R.Henderson,Ultramicroscopy111,90–100(2011).11. A.F.Brilot et al.,J.Struct.Biol.177,630–637(2012).12.G.McMullan,S.Chen,R.Henderson,A.R.Faruqi,Ultramicroscopy109,1126–1143(2009).13.G.McMullan,A.R.Faruqi,D.Clare,R.Henderson,Ultramicroscopy147,156–163(2014).14.M.G.Campbell et al.,Structure20,1823–1828(2012).15.X.-C.Bai,I.S.Fernandez,G.McMullan,S.H.Scheres,eLife2,e00461(2013).16.X.Li et al.,Nat.Methods10,584–590(2013).17. C.J.Russo,L.A.Passmore,Nat.Methods11,649–652(2014).18.D.Rhinow,W.Kühlbrandt,Ultramicroscopy108,698–705(2008).19.C.Yoshioka,B.Carragher,C.S.Potter,Microsc.Microanal.16,43–53(2010).20.C.J.Russo,L.A.Passmore,J.Struct.Biol.187,112–118(2014).21.S.H.Banyard,D.K.Stammers,P.M.Harrison,Nature271,282–284(1978).22.R.R.Crichton,J.-P.Declercq,Biochim.Biophys.Acta1800,706–718(2010).23.W.H.Massover,Micron24,389–437(1993).24.S.H.W.Scheres,J.Struct.Biol.180,519–530(2012).25.N.de Val,J.-P.Declercq,C.K.Lim,R.R.Crichton,J.Inorg.Biochem.112,77–84(2012).26.W.Baumeister,M.Hahn,J.Seredynski,L.M.Herbertz,Ultramicroscopy1,377–382(1976).27.H.Stark,F.Zemlin,C.Boettcher,Ultramicroscopy63,75–79(1996).ACKNOWLEDGMENTSWe thank R.Henderson for guidance and advice throughout this project;ls and W.Kühlbrandt for the use of the Polara electron microscope at the Max Planck Institute of Biophysics, Frankfurt;I.S.Fernandez and the V.Ramakrishnan lab for the gift of ribosomes;G.McMullan,S.Chen,C.Savva,J.Grimmett,T.Darling,and M.Skehel for technical assistance;our colleagues at the Laboratory of Molecular Biology—especially S.Scheres, G.Murshudov,and R.A.Crowther—for many helpful discussions;and R.A.Crowther,V.Ramakrishnan,and E.Rajendra for a criticalreading of the manuscript.This work was supported by theEuropean Research Council(ERC)under the European Union’sSeventh Framework Programme(FP7/2007-2013)/ERC grantagreement no.261151and MRC grant U105192715.C.J.R.and L.A.P.are inventors on a patent application filed by theMRC on the gold substrates.The coordinates of the refinedapoferritin model are deposited in the Protein Data Bank underaccession code4v1w and the EM density map is depositedSUPPLEMENTARY MATERIALS/content/346/6215/1377/suppl/DC1Materials and MethodsFigs.S1to S10Table S1References(28–40)Movie S14August2014;accepted13November2014 glycoprotein(Env)elicits a polyclonal an-tibody response that targets diverse epi-topes(1,2).Antibodies that display narrowbreadth of neutralization(narrow neutral-izing antibodies;nNAbs)develop during the firstmonths of infection whereas those capable of neu-tralizing heterologous viruses(broadly neutralizingantibodies;bNAbs)develop several years later in~10to30%of HIV-1–positive individuals(3).bNAbsisolated from HIV-1–infected patients are more pro-tective than nNAbs in experimental HIV-1/SHIVinfection(4)and will likely be a key component ofan effective HIV-1vaccine.Even though nNAbs andbNAbs target the same regions of Env(2,5–7),re-combinant Env(rEnv)immunogens are poorly rec-ognized by germline-reverted(gl)bNAbs(glbNAbs)and their corresponding B cell receptors(BCRs)(5,8–21),suggesting that the lack of bNAb gen-eration during immunization may be due toinefficient stimulation of naïve bNAb BCR pro-genitors(17,20).In contrast,little is known aboutgenitors of nNAbs.Understanding why B cellresponses against nNAb epitopes dominate overthose targeted by bNAbs in the context of rEnvimmunization will inform on basic immunolog-ical mechanisms of epitope competition and pro-vide new information relevant to the developmentof an effective HIV-1vaccine.Here we investigated whether glnNAbs fromdistinct clonal lineages that targeted the CD4-binding site(BS)and V3regions of Env(2)alsodisplay minimal rEnv recognition.Amino acid dif-ferences between the mutated and gl sequences ofnNAbs range from2.4to7.3%for the heavy chainsand2.7to5.6%for the light chains for the nNAbCD4-BS antibodies(table S1and fig.S1).In con-trast,prototypic CD4-BS bNAbs,VRC01(33.9%heavy,23%light),NIH45-46(a clonal relative ofVRC01;39.8%heavy,26.1%light),b12(21%heavy,19%light),8ANC131(33%heavy,24%light),andCH103(12.7%heavy,10%light)are more mutated(5,8,16,22).The anti-V3nNAbs are more mutated(11.6to21.6%heavy,9.7to13.8%light)than theanti-CD4-BS nNAbs.In contrast to the anti-CD4-BS glbNAbs,whichdo not bind rEnv(5,8,16,17,20)(table S2),glnNAbsdisplayed broad Env recognition(from51to100%)(table S2).The binding affinities of the glnNAbswere generally weaker than those of the corre-sponding mutated antibodies,owing to increasedoff rates in most cases(fig.S2).Whereas the glVRC01class bNAbs were unable to neutralize any of the 1Seattle Biomedical Research Institute,Seattle,WA98109,USA.2Laboratory of Molecular Immunology,The RockefellerUniversity,New York,NY10065,USA.3Laboratory of HumoralResponse to Pathogens,Department of Immunology,InstitutPasteur and CNRS-URA1961,75015Paris,France.4Departmentof Global Health,University of Washington,Seattle,WA98109,USA.*These authors contributed equally to this work.†Present address:Fred Hutchinson Cancer Research Center,Seattle,WA98109,USA.‡Present address:GlycoVaxyn,Grabenstrasse3,CH-8952Schlieren,Switzerland.§Corresponding author.E-mail:lstamata@viruses tested,three of the five glnNAbs exhibited neutralizing activity against tier1viruses(table S3). Overall,we conclude that the glnNAbs and glbNAbs recognize the CD4-BS on soluble and virion-associated Env differently(23,24).Two of the three anti-V3glnNAbs displayed neutralizing activity against several tier1viruses(table S3).We next investigated whether B cells stably expressing glnNAb and glVRC01-class BCRs(fig. S3)could become activated by(Fig.1A)and in-and C.As previously reported,none of the rEnvstested activated glVRC01-class B cells(17,20);however,they did activate glnNAb B cells tar-geting either the CD4-BS or V3(Fig.1A).Sim-ilarly,glnNAb B cells readily internalized diverserEnvs,whereas glVRC01class B cells did not(Fig.1B).Combined,the above results indicate thatrEnv immunogens can activate naïve nNAb Bcells but not naïve VRC01-class B cells.We recently reported that the disruption ofN460D,and N463D—on the clade C426c rEnv(herein called426c.NLGS.TM)confers binding toand activation of glNIH45-46and glVRC01B cells(17).In contrast,wild-type(WT)426c Env is rec-ognized by only the glnNAbs(table S2).gl1-154,gl1-695,gl1-732,and gl4-341nNAbs inhibitedbinding of glNIH45-46to426c.NLGS.TM(fig.S5A),an indication that the epitopes of the anti-CD4-BS glnNAbs used here overlap those of theVRC01-class glbNAbs.The anti-V3gl2-59mono-germline reverted antibodies to trimeric426c Env gp140variants measured by BLI.NA:binding was undetectable.426c NLGS.TM trimeric gp140426c NLGS.TM trimeric D V1D2D3 Apparent K A(1/M)k on(1/Ms)K off(1/s)Apparent K A(1/M)K on(1/Ms)K off(1/s)2.85×108 2.96×104 1.04×10-4 1.77×108 1.57×1048.85×10–56.30×1087.48×104 1.19×10–4 2.65×1089.71×103 3.66×10–54.12×107 1.61×104 3.91×10–4 3.31×1067.73×1035.88×10–46.77×1079.89×103 1.46×10–4 2.24×107 4.66×103 2.08×10–4NA NA NA NA NA NA4.04×1079.87×103 2.44×10–4 3.52×108 1.40×104 3.98×10–52.67×108 5.51×103 2.06×10–4 2.64×1089.07×1033.43×10–5NA NA NA NA NA NA1.19×1011 1.85×105 1.55×10–6NA NA NANA NA NA NA NA NAFig.2.Activation by and internalization of Envby glnNAb and glVRC01-class B cells in responseto modified Env immunogens.(A)glNIH45-46or glVRC01B cells were mixed with the indicatedglnNAb B cells.The pooled B cells were then chal-lenged with WT426c(top row),426c.NLGS.TM(mid-dle row),or426c.NLGS.TM.D V1-3(bottom row)at a1m M final concentration.Calcium flux was monitoredconcurrently in both B cell lines with the strategydepicted in fig.S6.Black arrow indicates time of Envaddition.(B)Area under the calcium flux curve de-termined from duplicate experiments in(A).Errorbars represent SD from the mean of10(glVRC01-class)or4replicates(other B cell lines).Green as-terisks and blue stars over the bars indicatesignificant differences(P<0.05)from glNIH45-46and glVRC01B cells,respectively,for each proteinusing a two-tailed Student’s t test.(C)Quantifica-tion of internalization of the indicated Envs byglVRC01class and glnNAb B cells.Error bars rep-resent SD from the mean of two independent ex-periments each performed in duplicate(n=4).Green astrices and blue stars represent significantdifferences(P<0.05)from glNIH45-46and glVRC01,respectively,for each protein using a two-tailedStudent’s t test.Fig.3.B cell activation and antigen uptake of Env by glVRC01-class B cellsin the presence of soluble anti-HIVantibodies.(A)Calcium flux was monitored in glNIH45-46(solid bars)or glVRC01(patterned bars)B cells incubated with 426c.NLGS.TM(white bars)or with426c.NLGS.TM.D V1-3(gray bars)in the presence of equimolar concentrations of the indicated antibodies.Bars repre-sent mean and SD of the area under the calcium flux response curve relative to that of Env in the absence of soluble mAb from three independent experiments. Asterisks represent a significant difference(P<0.05)in the calcium flux re-sponse between426c.NLGS.TM and426c.NLGS.TM.D V1-3using a two-tailed Student’s t test.Env internalization by glNIH45-46(B)or glVRC01(C)B cells incubated with426c.NLGS.TM(white bars)or426c.NLGS.TM.D V1-3(gray bars) in the absence or presence of equimolar concentrations of the indicated nNAbs. Bars represent the mean of two independent experiments.Asterisk denotes significant difference(P<0.05)in fluorescence compared to cells incubated with Env alone using a two-tailed Student’s t test in(B)and(C).Dotted line corresponds to mean426c.NLGS.TM internalization by B cells in the absence of Ab in(B)and(C).in the binding of glNIH45-46(fig.S5A).The gl1-676,gl1-79,and gl2-1261antibodies,which do not bind426c.NLGS.TM(Table1),had no effect on the binding of glNIH45-46(fig.S5A).In germinal centers(GCs),B cells expressing higher-affinity BCRs selectively expand,whereas lower-affinity B cells are eliminated(25).The anti-CD4-BS gl1-154,gl1-695,gl1-732,and gl4-341,and the anti-V3gl2-59bound426c.NLGS.TM with a higher apparent affinity than glNIH45-46(Table1).glVRC01 bound more strongly to426c.NLGS.TM than gl1-154 and gl1-695,but had a slightly lower apparent affinity than the gl4-341and gl1-732mAbs.The gl2-59V3–directed mAb bound more strongly to 426c.NLGS.TM than any of the glCD4-BS Abs,con-sistent with the higher propensity of gl2-59B cells to be activated and take up rEnv(Fig.1).We antic-ipate that upon immunization with the426c.NLGS. TM,naïve2-59–like B cells,or B cells that target immunodominant epitopes on the variable regions of Env(V1,V2and V3),would have an advantage over naïve VRC01-class B cells in becoming acti-vated,taking up antigen,and obtaining T cell help. To investigate the effect that differences in binding affinity have on the relative activation of B cells expressing different anti-Env BCRs,we de-veloped an assay that allows for the concurrent monitoring of B cell activation in distinct B cell populations(fig.S6).As expected,WT426c ac-tivated glnNAb B cells(Fig.2A top row,and Fig. 2B),but not glVRC01-class B cells(Fig.2A top row, and Fig.2B).426c.NLGS.TM activated gl VRC01-class B cells,but with the exception of gl1-154,it activated glnNAb B cells more strongly(Fig.2A, middle row).426c.NLGS.TM activated gl1-695more strongly than glNIH45-46and comparably to glVRC01(Fig.2A,middle row,and Fig.2B). Because426c.NLGS.TM showed the strongest activation of gl2-59B cells,we aimed to abolish ac-tivation of B cells targeting the V1,V2,and V3re-gions of426c.NLGS.TM,by deleting these regions to create426c.NLGS.TM.D V1-3.As expected,426c. NLGS.TM.D V1-3did not bind to gl2-59mAb(Table1) and did not activate gl2-59B cells(Fig.2,A and B).With the exception of glNIH45-46,deletion of the variable loops reduced the apparent binding affinities of the anti-CD4-BS antibodies tested (Table1).Consistent with these changes in bind-ing,426c.NLGS.TM.D V1-3activated glNIH45-46B cells more robustly than426c.NLGS.TM(Fig.2,A and B).The relative activation of all but one anti-CD4-BS glnNAb B cell(gl4-341)by426c.NLGS. TM.D V1-3was less than that observed for glVRC01-class B cells(Fig.2,A and B).gl4-341B cells were activated comparably to glVRC01class B cells, although the activation was reduced relative to that observed with WT426c and426c.NLGS.TM (Fig.2,A and B).At lower concentrations of426c. NLGS.TM.D V1-3,only glVRC01-class and gl4-341 B cells were activated(fig.S7).To represent a more physiological B cell repertoire,we challenged glVRC01-class B cells mixed with an excess of pooled glnNAb,and non-Env reactive B cells (fig.S8).WT426c only activated nNAb B cells, and although426c.NLGS.TM could activate the minority glVRC01-class B cells,a preferential ac-tivation of the nNAb B cell pool was observed.In contrast,426c.NLGS.TM.D V1-3activated glVRC01-class B cells preferentially at higher concentrationsand exclusively at lower concentrations(fig.S8).When the internalization of rEnv by B cells wasquantitated,426c NLGS.TM was preferentiallyinternalized by nNAb B cells,whereas426cNLGS.TM.D V1-3was preferentially internalizedby glVRC01-class and gl4-341B cells(Fig.2C).The differential impact of removing the variableEnv regions on the binding affinities,B cell activa-tion,and antigen internalization for the bNAbsversus nNAbs to the CD4-BS is likely due to differ-ences in the angle of approach to the CD4-BS bythese two types of antibodies(16,22,24,26–29).Additionally,the nNAbs may make contacts withportions of the variable loops.Antibodies can enter GCs and inhibit BCRbinding to an immunogen unless the affinity ofthe BCR for the immunogen is higher than forthe soluble antibody(30).Through this feedbackmechanism,higher-affinity antibodies hinderthe ability of low-affinity,antigen-specific B cellsto receive T cell help and thus select for higher-affinity B cell clones(30).Because the apparentaffinities of glVRC01-class bNAbs for426c.NLGS.TM D V1-3are higher than those for the glnNAbsstudied here(Table1),we expect that such afeedback mechanism will provide a selectiveadvantage to glVRC01-class B cell responses ifthis rEnv is used as an immunogen.Inversely,owing to the higher apparent affinities of theglnNAbs for the426c.NLGS.TM relative to theglVRC01class bNAbs(Table1),we would expectthis immunogen to drive the development ofnNAb responses through the same antibody-feedback mechanism.Indeed,the binding ofanti-CD4-BS nNAbs to426c.NLGS.TM reducedthe activation of glVRC01-class B cells by thatrEnv(Fig.3A and fig.S9).In contrast,activationof glVRC01class B cells by426c.NLGS.TM.D V1-3rEnv was observed even in the presence of thegl1-154,and gl1-695Abs(Fig.3A and fig.S9).Thus,removal of the V1,V2,and V3regions reduces theability of some(but not all)anti-CD4nNAbs toblock rEnv activation of glVRC01class B cells.Similar observations were made when theinternalization of these two Envs by glVRC01class B cells was determined in the absence andpresence of glnNAbs(Fig.3,B and C).glNIH45-46B cells more readily internalized426c.NLGS.TM.D V1-3compared to426c.NLGS.TM,even inthe presence of inhibitory antibodies,with theexception of the gl4-341mAb(Fig.3B,comparewhite and gray bars).Overall,we conclude thatthe presence of anti-CD4-BS glnNAbs can inhibitthe activation of,and the antigen internalizationby,glVRC01-class B cells in response to the426c.NLGS.TM,but that these effects are less pro-nounced when the variable regions V1,V2,andV3are removed from this rEnv.Thus,selectingwhich immunogen is used during the primingphase of immunization(e.g.,426c.NLGS.TM versus426c.NLGS.TM.D V1-3)will be critically importantto the eventual elicitation of anti-CD4-BS bNAbs.In sum,our study provides a mechanistic expla-nation as to why rEnv immunogens elicit narrowrather than broadly neutralizing antibody re-sponses to the CD4-BS of Env.This is primarily dueto the broad Env-recognition properties of glnNAbBCRs,which contrasts with the inefficiency by whichbNAb BCRs recognize rEnv.Notably,we demon-strate that rational immunogen modifications canreduce(and in certain cases eliminate)the activa-tion of naïve B cells that give rise to such nNAbs,while promoting the activation of naïve B cellsthat give rise to glVRC01-class bNAbs,even whenthe epitope of these antibodies substantially over-lap.As such,our results are relevant to efforts toelicit protective antibody responses against notonly HIV-1but potentially other pathogens as well.REFERENCES AND NOTES1.H.Mouquet et al.,PLOS ONE6,e24078(2011).2.J.F.Scheid et al.,Nature458,636–640(2009).3.L.Stamatatos,L.Morris,D.R.Burton,J.R.Mascola,Nat.Med.15,866–870(2009).4. 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