Virus glycoprotein nanodisc platform for vaccine analytics

Domain: neuroscience
Published: 2026-02-10
Editorial outcome: major_revision

Abstract
Transmembrane glycoproteins of enveloped viruses are targets of neutralizing antibodies and essential vaccine antigens. mRNA-LNP technology allows in vivo expression of transmembrane glycoproteins, but in vitro biophysical characterization of transmembrane antigens and analysis of post-immunization antibody responses typically rely on soluble proteins. Here, we present a platform for assembling transmembrane glycoprotein vaccine candidates into lipid nanodiscs. We demonstrate the utility of nanodiscs in HIV membrane proximal external region (MPER)-targeting vaccine development by binding assays using surface plasmon resonance (SPR), ex vivo B cell sorting with fluorescence-activated cell sorting (FACS), and by determining the structure of a prototypical HIV MPER-targeting immunogen nanodisc in complex with three broadly neutralizing antibodies (bnAbs), including MPER bnAb 10E8, to 3.5 Å by cryogenic electron microscopy (cryo-EM), providing a template for structure-based immunogen design. To demonstrate general applicability we characterize Ebola virus glycoprotein nanodiscs. Overall, the platform offers a tool for accelerating development of next-generation vaccines.

Introduction
In the past two decades, nanodiscs have emerged as a means to scaffold protein. Since the first apolipoprotein-derived membrane encapsulate transmembrane proteins into a stable and native-like scaffold proteins (MSPs) were introduced, several variations and environment. In most cases, proteins are extracted from the mem- alternativescaffoldshavebeendescribed,allowingforavarietyofdisc branebydetergentsolubilizationandthenassembledintoananodisc sizes and versatile experimentalapproaches1–5. Structural studies of byremovalofthedetergentinthepresenceoflipidmoleculesanda membraneproteinshaveperhapsbenefittedthemostfromnanodiscs, resultinginimportantadvancesinunderstandingofmembranepro- teinstructure-functionrelationship6,7. Theyhavealsobeenusedsuc- cessfullyinavarietyoffunctionalandbiophysicalapproaches,suchas surfaceplasmonresonance(SPR)8–10.

Viralglycoproteinsofenvelopedvirusesaremembraneproteins and essentialtargets for vaccine development. Structure-based vac- cinedesignwasanintegralpartofthesuccessfulCOVID-19vaccine development11–13,and asignificantportionofthe groundworkstem- med from decades of HIV vaccine development14. Structure-based methods, combined with rapidly advancing computational protein design approaches and mRNA lipid nanoparticle (mRNA-LNP) tech- nology, are now revolutionizing iterative vaccine development15,16. Immunogen delivery by mRNA-LNP expands the available immuno- genstomorenative-likeconstructsthatmaycontainthemembrane- proximal epitopes, such as the recently developed cleavage- independentNFLtrimersexpressingHIVEnv17andtheMD39.3gp151 Env trimer used in the HVTN302 clinical trial (NCT05217641), but recombinanttransmembraneglycoproteinproductionforbiophysical characterization of the immunogens remains challenging. In most studies,membraneproteinsaretruncatedbeforethetransmembrane domaintoobtainsolubleectodomainswithhigherexpressionlevels andeasierhandlingandapplicabilityindownstreamanalyticalmeth- ods.Theregionsexcludedtypicallyincludethemembrane-proximal externalregion (MPER), transmembrane domain (TM), and intracel- lularC-terminaldomain(CT).IninfluenzaHA,theanchorregionana- logoustoHIVMPERjoinstheectodomainasflexiblelinkerstothree transmembranehelicesandallowsthetrimertotiltinrelationtothe bilayer surface18. Both Env MPER and HA anchor have highly conserved epitopes for neutralizing antibodies (nAbs), making them attractive targets for vaccine development19–23. Significant progress in MPER- targeted HIV vaccine development has been made recently with peptide-liposome formulations showing induction of heterologous neutralizing antibody B cell lineages in humans, and with mRNA deliveredgermline-targeting(GT)epitopescaffoldefficientlyinducing antibody precursors in non-human primates (NHPs) and mice24,25. Analysis of antibodies elicited by these vaccines, as well as further immunogen development, would benefit from inclusion of the full epitope in recombinantly expressed proteins. For example, recombi- nantproteinsmatchingthetransmembraneimmunogensdeliveredas mRNAarerequiredforaccurateimmunogenassessmentiniterative vaccinedesignmethods,suchasSPRandstructuralstudies.Impor- tantly,transmembraneversionsofEnvmayalsoberequiredformore accurate representations of the glycan shield, as many neutralizing antibodiesbindspecificallytoglycans,andglycoproteinsexpressedas transmembrane proteins have been shown to have glycan shield compositionsclosertothenativeglycanshield26–28.

Some uses of nanodiscs to support vaccine development have beendemonstrated,particularlyineffortstocharacterizethestruc- tures of HIV MPER-targeting HIV antibody epitopes and transmem- branedomainsofvirusglycoproteinsintheirnativemembranebilayer environment29–33. The nanodisc assembly platform presented here providesthereproducibility,scalability,andaccuratereplicationofthe vaccine candidate properties that enable routine use of transmem- brane viral glycoproteins in key methods employed in iterative, rational vaccine design. We demonstrate the use of nanodiscs to measureantibodyaffinitiesbySPR,asFACSprobestosortantigen- specificBcellsfrommouseandnon-humanprimate(NHP)immuni- zationexperiments,andfordeterminingthecryo-EMstructureofa prototypeHIVboostvaccinecandidateincomplexwiththreebroadly neutralizingantibodies(bnAbs)thattargetdistinctsitesofvulner- ability on the HIV Env surface. We highlight the benefits of the approachbyreportingthestructureoftheentireproteinepitopeofan HIVMPER-targetingbnAb10E8.Finally,weshowtheadaptabilityofthe platformtootherglycoproteinsbyassemblingengineeredEbolavirus glycoproteininnanodiscsusingidenticalconditionstoHIVEnv.

Results
Glycoproteinnanodiscassemblyandanalysisworkflow
Weestablishedaworkflowthatprovidesmaterialsuitableformethods commonly used in rational, iterative vaccine design (Fig. 1). The workflowwassetupusingtheengineeredandhighlyexpressingHIV Env construct BG505 MD39.3 gp15134,35 that was recently tested in nonhuman primates and a phase 1 clinical trial HVTN 302 (NCT05217641)36,37.Alinker,followedbyanHRV3Cproteasecleavage siteandastreptag,wasaddedtotheintracellularC-terminus,leading toananodiscassemblyconstructbasethatwillhereonbereferredto asEnvgp151ND(Fig.2a,b).Thisconstructwasthenusedtooptimize thethroughputandreproducibilityofnanodiscproduction,leadingto astandardizedworkflow(Fig.2c).Inshort,transmembraneglycopro- teins(GPs)wereexpressedonthesurfaceofhumanFreeStyle293-F cells, followed by detergent solubilization with TX-100-containing buffer.Asthetransmembranedomainofviralglycoproteinsiscom- posedofonlythreehelices—oneperprotomer—detergentsolubiliza- tionbyTX-100wasnotexpectedtodisturbthequaternarystructureof the engineered GP trimer,asindicated by the binding of structure- specific antibodies to extracted GPs27,28. The detergent-solubilized glycoprotein was then assembled into nanodiscs using the MSP1D1scaffoldwithorwithoutabiotintag.Whileavarietyofscaffold proteinsareavailable,weoptedtoreducevariablesandfocusedon usingthestandardMSP1D1scaffoldforallpurposes.Thedeveloped “batch assembly” approach, resembling previously described “on- column”38 and “on-bead”39 methods, facilitated reproducibility and efficient assembly while GPs remained bound to the matrix. Empty nanodiscsforcontrolexperimentswereproducedbyomittingGPin theassemblystepandusingthescaffoldproteinHis-tagwithNiNTA affinity purification prior to SEC. Utilizing this workflow, up to 12 samples wereroutinely processed simultaneously in 5 days from transfectiontopurifiednanodisc. Oneliterofcellsyieldedapproxi- mately100to700µgofpureGPnanodiscs,whichwassufficientfor onetothreeendpointassays.Forexample,asingleproductionbatch producedEBOVGPnanodiscs,anHIVEnvnanodisc-Fabcomplexfor cryo-EMstructuralstudies,andeightFACSprobesforBcellsorting. FinalGPnanodiscpreparationswerestableforatleastthreemonthsat +4°C based on absorbance at 280nm, presence of trimeric GP in nanodiscinnegativestainEM(ns-EM)2Dclassaverages,andactivityin SPRanalysis(Fig.3b).

Theworkflowwasthen pilotedinanimmunogen development projectaimingtoincreasetheaffinityoftheMPER-targetingantibody 10E8andthereforeguideimmuneresponsestotheMPERdomain.In thenativemembranecontext,theC-terminalMPERhelixthatistar- getedbytheheavy chain complementarity-determining region 3 (HCDR3)of10E8isstericallyoccluded(Fig.2b),andoneofthedesign goalswastoimprovetheMPERaccess.Tothisend,N-linkedglycosy- lationsitesatpositionsN88,N618,andN625(HxB2numbering)adja- cent to the epitope were removed at the base of the trimer. An additionalR696Smutationthatimproved10E8bindingwhilemain- taining trimeric Env conformation was discovered by mammalian surfacedisplaylibraryscreening.Earlierstructuralanalysisindicated that R696 resides at the intersection of the three transmembrane helices29,40; therefore, this mutation was predicted to disrupt helix interactionsandfurtheropenthetrimerbase.Theresultingconstruct willbereferredtoasEnvgp151MPERND.AnegativecontrolforMPER antibody binding and a KO FACS probe for epitope-specific B cell sorting, designated as Env gp151 MPER-KO ND, was also generated (Fig. 2a). This construct included MPER mutations W672A, F673R, T676R,N677I,L679M,W680E,Y681W,and K683D,which werepre- viouslyreportedtocollectivelypreventthebindingofMPER-targeting antibodieswhilepreservingtheoveralltrimericstructureofEnvand other bnAb epitopes24, or screened for this study to improve the expressionoftheconstruct.TheprototypicalEbolaGPimmunogen EBOVGPMayingaNDwasbasedonaconstructwithtrimerstabilizing mutations T577P and K588F41, to which the native EBOV GP trans- membrane(TM)domainandthesameintracellularelementsasinthe HIVEnvconstructswereadded.Sequencesofgeneratedconstructsare given in Supplementary Table 1. Assembled nanodiscs generally exhibitedbroadpeaksinsizeexclusionchromatography(SEC)spec- tra, indicative of extensive glycosylation and variation in hydro- dynamicradiusthatmayresultfrompositionalheterogeneityofEnvin thedisc,and>95%purityandbandsoftheexpectedmolecularweight asexaminedbySDS-PAGE(SupplementaryFig.1a,b).Massspectro- metricglycananalysisusingtheDeGlyPHERmethod42wasdoneprior toandafternanodiscassemblytoascertainsite-specificglycanoccu- pancyandprocessivityofEnvconstructs.Thisanalysisconfirmedthe presenceofallexpectedglycansaswellasahighproportionofcomplex glycans in transmembrane constructs characteristic of native, transmembraneHIVEnv(SupplementaryFig.1c)26–28.Inadditiontothe MPER-targetingimmunogenconstructsandEBOVGPMayingaND,a germline-targetingHIVvaccinecandidateN332-GT5wasproducedasa germline-targetingHIVvaccinecandidateN332-GT5wasproducedasa gp151transmembraneconstructandassembledintonanodiscsusing the standardized workflow to test general applicability (Fig. 2 and SupplementaryFig.2a)35–37,43,44.Germline-targetingimmunogenN332- GT5 gp140, a soluble trimer, showed proportions of complex-type glycans matching closely those of the transmembrane version. All transmembraneEnvsshowedhigheroverallglycanoccupancyinthe gp41subunitcomparedtosolubleEnvgp140.

GPnanodiscbindingkineticstoantibodiesmeasuredbySPR
SPRisanessentialmethodiniterativevaccinedesignthatmeasures antibodyaffinitiestoengineeredimmunogens,assessingtheeffectof the designed features as well as affinities of antibodies induced by vaccination (Fig. 1). We set to establish scalable and reproducible conditions for measuring antibody binding kinetics against trans- membraneGPsinnanodiscsinthreedistinctSPRmodalities(Fig.3a). ModalityAutilizedananti-affinitytagcapturestrategyinwhichanti- strep-tag antibody was covalently immobilized on the chip surface, glycoproteinnanodiscswerecapturedusingtheintracellularstreptag, and Fab analytes were employed to study monovalent interactions. ModalityBemployed alowsurfacedensityIgGcapturestrategy,in whichanti-humanIgGcaptureantibodywasfirstcovalentlyimmobi- lized,andglycoprotein-specificIgGligandswerecapturedatreduced capture time to limitthe ligand density and thereby reduce avidity effects. Glycoprotein nanodiscs were then injected as analytes. In modality C, nanodisc capture was followed by IgG flow as analyte insteadofFabs,allowingasemi-quantitativehigh-throughputscouting approachwithlownanodiscsampleconsumptionandhighsensitivity. EstablishingthesethreemodalitiesfornanodiscGPsallowedflexibility inexperimentaldesignbasedondataqualityrequirementsandsample availability.

Next,weusedtheGPnanodiscSPRmodalitiestoestablishbase- linekineticsforneutralizingandnon-neutralizingmonoclonalHIVEnv antibodies spanning diverse epitopes (Fig. 3b–d, Supplementary Fig.2b–d).ModalityAmeasuredaffinitiesof100to250nMfor10E8to Env gp151 ND design base construct (Fig. 3b) and slightly higher apparentaffinityof36to42nMusingmodalityB.UsingmodalityA,we observeda70-foldincreasein10E8affinity(250nMvs3.6nM)toEnv gp151MPERNDasaresultofremovaloftheMPERepitope-proximal glycans and the mutation R696S in TM (Fig. 3c). Incorporation of MPER-KO mutations completely abrogated 10E8 binding. Kinetic analysisindicatedthattheimprovedaffinityintheengineeredimmu- nogenwasprimarilyattributedtoadecreasedoff-rate(8.1×10-3s-1for Env gp151 ND versus 3.5×10-4s-1 for Env gp151 MPER ND (Fig. 3d). AdditionalbnAbstargetingtheMPERandotherepitopesonthesur- faceofEnvwerealsotestedformorecomprehensiveantigenicpro- filing. This confirmed binding of MPER-targeting antibodies to Env gp151ND(SupplementaryFig.2b),andshowedasexpectedthatnon- MPERbnAbshavesimilaraffinitiesforsolubleEnvgp140andEnvgp151 ND(SupplementaryFig.2c).Non-neutralizingantibodies(non-nAbs) RM19RandRM20A3exhibitedreducedornobindingtoEnvgp151ND ascomparedtoEnvgp140(SupplementaryFig.2d).Thesetwonon- nAbs target the baseof the trimer, which is exposed inEnv gp140, whereasinEnvgp151NDaccessispresumablyrestrictedbyMPERand TMdomainsaswellasthelipidsurfaceofthenanodisc.Finally,we employedmodalityCtoconfirmthebindingoftwoEbolaGPglycan cap-specific antibodies EBOV-296 and 13C6 to EBOV GP nanodiscs, followed by monovalent affinity determination using modality A (Fig.3e).Asexpected,antibodiesEBOV-296and13C6boundwithhigh affinity(46nMand16nM,respectively)toEBOVGPnanodiscsbutdid not interactwith the HIV Env gp151 ND. Conversely, HIV MPER bnAb PGZL1 bound toEnv gp151 NDbutdid not interactwith the EBOV GP ND. Collectively,thesedatademonstratethatGPnanodiscsandSPRcanbe used to characterize the antigenic landscape of the designed trans- membraneimmunogen,informimmunogenselectionforinvivostu- dies,identifysuitablecomplexesforstructuralstudiesbycryo-EM,and supportGPnanodiscuseasBcellsortingprobesinFACS(Fig.1).

FACSsortingofBcellsfromimmunizedanimalmodelsusing nanodiscGPprobes
SingleBcellsortingandsequencingareroutinelyusedtoanalyzeB cellresponsesinducedinvivo.Typically,biotin-taggedvariantsof theimmunogenareusedwithstreptavidin.Thisallowsconjugation ofthesameprobetomultiplefluorochromestoincreaseselection specificityandtopreventisolationoffluorochrome-specificBcells. By including a matching sorting probe with KO mutations in the targetedepitope,epitope-specificresponsescanbeseparatedfrom off-targetresponsesagainstunrelatedepitopes.SolubleGPprotein constructssolelyconsistingoftheectodomainaremostoftenused as antigenic baits. For immunization studies where membrane- proximal epitopes are of interest, such as the HIV MPER, soluble probeswouldnotbeabletoextractallappropriateresponsestothe targeted epitope due to several challenges which include (1) the hydrophobicityoftheepitoperegionswhichmakebaitproduction challenging; (2) non-native or incomplete structural conformation oftheepitopeinshorterpeptiderepresentationsoftheantigen;and (3)missinglipidmembranecomponentsthatconstituteaportionof thenativeepitope.Here,weusedabiotinylatedMSP1D1scaffoldto generate nanodisc GP FACS probes. We first tested Env gp151 ND probe binding to FACS compensation beads conjugated to 10E8, DH511, or BG18 bnAbs (Fig. 4a). The background signal was mea- sured as binding to either MSP1D1 scaffold protein alone, empty nanodiscassembledwithDOPClipids,ortoemptynanodiscwitha mixtureofneutralandchargedlipids.Envgp151NDexhibitedsub- stantiallyhighersignalcomparedtocontrols,confirmingthatade- quate separation from background signal can be detected. We detectednobindingwithanyofthetestedprobestounimmunized C57BL/6(B6)mousesplenocytes,andastrongsignalagainstengi- neeredRamoscellsexpressingthebnAbVRC01asitsBcellreceptor (BCR;Fig.4a)45.ThisdataindicatedthatEnvnanodiscprobesweresuitableforthedetectionofBcellsfrommicebyflowcytometrythat specificallytargetEnv.

We then proceeded with a pilot immunization experiment in themicetoexploreifvaccine-elicitedantigen-specificBcellscouldbe identifiedusingEnvnanodiscsprobes.Weutilizedtransgenicmice expressingthehumanD3-3andJ 6genesthatallowmicetoexpress longHCDR3s,includingHCDR3precursorsofHIVMPERbnAbs10E8, DH511,andLN0124.Micewereintramuscularlyinjectedwith10µgof mRNA-LNPs encoding HIV Env gp151 MPER ND construct. Animals were sacrificed, and spleens and lymph nodes were harvested 6 weeks post-immunization. B cells were stained with immunogen- matched(WT)nanodiscprobesontwodistinctfluorophoresandEnv gp151MPER-KONDnanodiscprobewithKOmutationsintheMPER to identify MPER-specific cells (Fig. 4b and Supplementary Fig. 3). Among class-switched memory B cells (defined as CD19+IgD-IgM-), 0.7% of cellswere determined to beantigen-specific,comparedto 0.15%withintheIgD+IgM+naïveBcellpopulation(Fig.4c).This~4.7- foldincreasewasstatisticallysignificant(p=0.0177),indicatingthat the mRNA immunization elicited HIV Env gp151 MPER-specific responsesthatcouldbedetectedbyEnvnanodiscprobes.Thepro- portionofWTEnvnanodisc-bindingBcellsthatselectivelyboundto the 10E8 epitope (%KO- of WT++ B cells) was indistinguishable betweenthenaïveandmemoryBcellcompartments,suggestingthat theimmunogendidnotelicitMPERepitope-specificresponses,and that further engineering or inclusion of a prior germline-targeting primingimmunogenintheimmunizationregimenwouldberequired toachievethatcapacity24,46.

Antigen-specific(WT++)mousememoryBcellsweresortedand theirBCRssequencedtoassesstherobustnessofnanodiscprobesin the selection of antigen-specific BCRs. Eight antibodies from each animalwererandomlyselectedformonoclonalantibodyproduction. Anadditional12antibodieswithlong(≥19aminoacid(aa))HCDR3s wereselected,andduplicateantibodies(containingidenticalheavy- andlightchains)wereremoved,resultingin54totalantibodies.Genes encodingtheseantibodiesweresynthesized,and49of54antibodies weresuccessfullyexpressed,purifiedandusedinSPRmodalityBto measure affinities against Env nanodiscs (Fig. 4d). At 1μM analyte concentration, no non-specific responses to empty nanodiscs were detected.Outofthetestedantibodies,47%boundtosolublegp140 matchingtheimmunogenectodomain,and65%boundtoEnvgp151 MPER ND or Env gp151 MPER-KO ND. Considerable binding to the ectodomain,similarbindingtotheMPER-targetingimmunogenandits MPERKOversion,andoverallsimilargeometricmeanaffinitytothe testedEnvnanodiscsprovidedevidencethattheelicitedantibodies werenottargetingtheMPERpeptidebutboundtotheexposedbase epitopeorotheroff-targetepitopes.Nevertheless,usingthenanodiscs asFACSsortingprobesandinSPR,wewereabletosortandmeasure affinitiesofseveralantibodiesthatwerespecifictothetransmembrane Envgp151version(e.g.,Ab_38,Ab_48,Ab_79;SupplementaryFig.3b).

Non-human primates (NHPs) are an important model for late- stage pre-clinical vaccine development and are often used as final validation beforeclinicaltrials. Rhesusmacaques,likehumans,have extensive genetic diversity within the immunoglobulin loci and are alsocapableofmakinglongHCDR3s47–49.Totestwhethernanodisc- basedFACStetramerprobescouldbesuccessfullyusedtoassessRMB cellresponses,experimentswerecarriedoutusingPBMCsfromRMs immunizedpreviouslywithsolubleEnvMD3950.PBMCswerestained withthenanodiscprobestodeterminewhethertherewassubstantial backgroundbindingtothelipidcomponentofthenanodiscprobes. Pre-immunization PBMCs showed low background binding of IgD- memoryBcellstonanodisc,whilePBMCsfrom2-weekspostanMD39 boosterimmunizationshowedhighbindingtothenanodiscprobes (Fig.5a).Next,thenanodiscprobeswerecompareddirectlytosoluble Envgp140proteinprobestocharacterizeBcellsfromRMsthathad beenimmunizedwithMD39.3gp151mRNA(matchingEnvgp151ND sequence)4-weeksprior36.Acrosstwoanimals,therewerenodiffer- encesinthefrequencyoftetramer++cellsbetweentheEnvgp151ND nanodisc and Env gp140 soluble protein probes (Fig. 5b). Taken together,thesedatashowthatnanodiscsbearingstabilizedHIVEnv constructscanbedeployedastetramerprobesfortheevaluationof pre-clinicalHIVBcellresponsesinNHPs.

GPnanodiscsincryo-EMstructuralstudies
To demonstrate how structuralstudies of nanodiscGPs can inform vaccine design, we set out to determine the structure of prototype MPERvaccinecandidateEnvgp151MPERNDincomplexwiththe10E8 Fab by cryo-EM. We included two other HIV bnAbs: VRC01, which recognizes the CD4 binding site, and BG18, which targets the N332 glycan supersite. This antibody cocktail complex serves three pur- poses:First,theaddedFabsimprovetheorientationdistributionof particlesoncryo-EMgrids.Second,theymayreducethechancesof freeze-denaturationbypreventingparticlesfromadheringtotheair- water interface during grid preparation. Third, determining the structureofamembrane-embeddedtrimerincomplexwithmultiple bnAbs targeting different sites would assist the development of a protectiveHIVvaccinethatwilllikelyrequiretheelicitationofbnAbs targeting several sites to elicit sufficient neutralization breadth and potencytocounterglobalEnvdiversity.Atrimerboostwouldbenefit frombeingabletoengagethreeormoresites(e.g.,N332supersite/V3- glycan,CD4-bindingsite,andMPER,towhichApex/V2-glycanand/or fusion peptide couldalsobe included) in a single immunogen. The high-resolution structure of the 10E8 epitope has thus far been resolvedonlywithx-raycrystallographyandincomplexwithMPER peptidealone51–54.Afterconfirmingfeaturestypicalofnanodiscswith trimericGPwithorwithoutFabsinns-EM2Dclassaverages,wepro- ceededtocryo-EManalysis(SupplementaryFig.4a,b).Fromourcryo- EMdata,weproducedtworeconstructions:thecomplexwithtwo10E8 Fabsat4.3Å,andthecomplexwithasingle10E8Fabat3.5Åresolu- tion,whichenabledatomic-levelinterpretation.Wenotedthatwhile 10E8, VRC01, and BG18 were all added at the same step after the nanodisc batch assembly at ~10x molar excess, all VRC01 and BG18 sites were occupied, but the 10E8 binding site was partially underoccupiedinallreconstructions,suggestingsterichindranceof theMPERepitopewithincreasing10E8occupancy(Fig.6a–c).Inall reconstructions,10E8iswedgedbetweentheEnvectodomainandthe nanodisclipidbilayer.TheMPERantibody-inducedEnvtiltanglewas evaluated by aligning the low-pass filtered maps to the nanodisc representing the bilayer plane. A single 10E8 Fab appears to be accommodated with minimal Env tilting while binding of a second 10E8FabpushesEnvintoamoretiltedposition(Fig.6b).Unlikeinour previousWTEnvAMC011nanodisccryo-EMreconstruction,density fortheTMdomainremainedundetected.Thismayhavebeendueto theR696Smutationdisruptingthestabilizingconnectionpointofthe threehelicesinthemiddleofthebilayer29.Additionally,truncationof theCTmayhaveallowedtheTMhelicestoadoptlessdefinedpositions withinthebilayer.Theclasswithasingle10E8Fabboundwassub- jectedto3Dvariabilityanalysis(3DVA),whichrevealeddistinctcon- formations of the MPER helix with ~30° lateral twist of the Fab in conjunction with an apparent dislocation of the adjacent protomer (SupplementaryFig.4c,d;Supplementaryvideo1).

UsingthemodelofEnvgp151MPERNDcomplex,wewereable toresolvenovel structuraldetailsof the 10E8 epitope,including a binding pocket for 10E8 antibodyheavychain frameworkregion3 (HFR3),formedbyEnvgp120andgp41subunits(Fig.6d,inset5).We identified14aain10E8contactingtheCterminusofgp120,helices α6 to α8 of gp41 in protomer 1, and L660 of gp41 in protomer 2. These contacts are either in the binding pocket or in the helices formingacollararoundtheterminiofgp120.Strikingly,thisbinding pocketappearedtobetargetedbytheHCDR3ofanotherHIVbnAb, 3BC315,thathasbeenshowntoacceleratetrimerdissociation55.To confirm this similarity in engaging the binding pocket, we deter- minedthecryo-EMstructureofEnvgp140(BG505MD39.3)incom- plexwithBG18,VRC01,and3BC315Fabsto3.1Åresolution(Fig.6f; SupplementaryFig.4f,g).3BC315engagesthebindingpocketmainly withH100A,G100BandY100EatthetipoftheHCDR3.In10E8,the interactionisdrivenbyI75,S74andN76ofHFR3(Fig.6d,inset5). Theobservedsimilarityofthebindingpocketengagementledusto proposethattheseantibodiesmayshareacommonmechanismfor trimerdestabilization.Aspartofthebindingpocket,bothantibodies contactW623 ingp41(Fig. 6d,inset5;and Fig.6f), which is>99% conservedacross>5,000diverseHIVstrainsfromtheLANLdatabase (http://www.hiv.lanl.gov),andhaspreviouslybeenshowntobepart ofa tryptophanclaspcontributingtoastabilizingfour-helixcollar aroundtheterminiofgp12056.Engagingthebindingpocketleadsto wideningofthegp41protomer-protomergap(Fig.6g),whichwould eventuallyleadtodestabilizationofthecontactsinthecollararound theterminiofgp120andprematuretriggeringofthefusionpeptide. This would normally occur during virus entry-associated Env rear- rangementsthatfollowCD4receptorbinding.

We examinedthefunctionalimportanceofthecontactsidentified fromthecryo-EMstructurebyconductingpseudovirusneutralization assays(Fig.7a).Reversionsofmature10E8HFR3paratoperesiduesto theirpredictedgermlinesequencesallreducedneutralizationactivity, withF77Tresultinginapproximately8-foldreduction,suggestingthat the identified contacts contribute to 10E8 neutralization potency (Fig.7b).Mutationswithinthe10E8epitopewereintroducedtothe BG505T332NEnvsequencecommonlyusedasacontrolinHIVneu- tralizationpanels.MutatingthekeyMPERresidues(N671A,W672Aand K683A) engaged by the 10E8 HCDR3 completely abrogated neu- tralization,asexpected57.Mutating10E8contactresiduesvisualizedin thenewstructurealsoshowedsubstantialeffectson10E8neutraliza- tion.MutationsR500A,T536A,L619A,S620A,D624AandN625Aeach ledtomodest(1.5-to2.7-fold)decreasesinneutralizationsensitivityto 10E8(Fig.7c).T499GandM545Aresultedinmodest(2-fold)increases insensitivityto10E8.Notably,mutationL660Aresultedina7.5-fold increase in 10E8 neutralization sensitivity (IC from 0.15 to 0.02). 50 Thus, the new complex structure identified multiple residues that impact10E8neutralizationpotency.

Another novel structural feature of the 10E8 antibody was the dynamicremodelingoftheN-terminalMPERhelix,mostlikelydriven byinteractionswithHCDR1(D30,N31andW33)andHCDR2(G52C, G53, G54, W55, Fig. 6d, e; Supplementary video 1). The root mean square deviation (RMSD) of this HCDR1 and HCDR2 10E8 paratope regionis1.52Åcomparedtoapublishedx-raystructureof10E8Fabin complexwithagp41peptide(PDB4G6F).Incontrast,theRMSDof HFR3 between the nanodisc cryo-EM complex and peptide-bound crystalstructureis0.46Å.Althoughthe10E8crystalstructurefitwell intothecryo-EMmap(SupplementaryFig.5a),wecalculatedanRMSD of9.59ÅoftheentireMPERdomainbetweenthetwostructures.The difference between the MPER in the two structures stems predominantlyfromtheN-terminalportionoftheMPERthatiscon- nected to the rest of the ectodomain in the nanodisc Env cryo-EM structure.Furthercomparisonofthesestructuresshowedthatwhen aligned to the MPER peptide, 10E8 in the crystal structure adopts a~25°rotatedposition(SupplementaryFig.5).Althoughtheyengage the same residues within the MPER helix, only 14 of 29 contacting residuessintheparatopeand15of26intheepitopewereobservedin thecrystalstructureascomparedtothecryo-EMstructure.Compar- isonofthecryo-EMcomplexstructurewithcrystalstructuresofother MPERbnAbsboundtoMPERpeptidesshowedthatotherMPERbnAbs can accommodate the same approximate space between the lipid bilayer and ectodomain as 10E8 but adopt different binding poses. ThissuggeststhatalthoughtheMPERpeptidecontactsremaincon- served, variations in interactions are likely outside the core MPER epitope,andMPERbnAbscanadoptawiderangeofbindingmodes, which is alsoevident in our cryo-EM data (Supplementaryvideo 1). Taken together, the comparison to previous MPER peptide-bnAb crystalstructuressuggeststhatthenativemembraneenvironmentof EnvisrequiredforcompletestructuralcharacterizationofMPERtar- getingbnAbs.Ourobservationsofthenative,dynamicbindingmode ofthe10E8bnAbandstructuralinformationoftheentiresidechain contactnetworkwillinformthedevelopmentofnext-generationMPER targetingvaccines.

Methods
Ethicalstatement
This work complies with all relevant ethical regulations. All animal proceduresandexperimentswereperformedundertheapprovalof ScrippsResearchIACUC20-0001-2.

Proteinexpressionandnanodiscassembly.Thismethoddescription aimstoprovideacompleteplatformthatenablesroutineuseofthe nanodisc approach for iterative vaccine design using viral particlelipidcompositionwasusedcontaining45%DOPC,7%DOPS glycoproteins.Whileretainingtheversatilityofamodularapproach, (1,2-dioleoyl-sn-glycero-3-phospho-L-serine),7%DOPA(1-palmitoyl-2- eachstep is simplified and validated formaximizing reproducibility oleoyl-sn-glycero-3-phosphate),7%DOPE(1-palmitoyl-2-oleoyl-sn-gly- andscalability.Allglycoproteinandantibodyconstructswerecodon cero-3-phosphoethanolamine), 7% DOPG (1,2-dioleoyl-sn-glycero-3- optimizedforexpressioninhumancellsandsynthesizedandcloned phospho-(1’-rac-glycerol)),5%sphingomyelin,2%PI(4,5)P2(L-α-phos- intoexpressionvectorspHLSec(glycoproteins)oritsvariantspCW- phatidylinositol-4,5-bisphosphate),and20%CHS.Scaffoldproteinwas CHIg-hG1,pFabCW,pCW-CLig-hL2orpCW-CLig-hk(IgGheavychains, eitherproducedin-houseusingstandardE.coliexpressionmethodor Fabheavychains,lambdalightchainsandkappalightchains,respec- purchased from Sigma-Aldrich (membrane scaffold protein 1D1 tively)byGenScriptBiotech.Antibodieswereexpressedandpurified #M6574orbiotinylatedmembranescaffoldprotein1D1BTN#MSP13). from HEK293F (Thermo Fisher Scientific, #R79007) Ramos cells The engineering of strep-tagged construct and batch assembly according to the manufacturer’s instructions using rProtein A insteadofnanodiscassemblyafterGPelutionfromtheaffinitymatrix SepharoseFastFlowresin(Cytiva,#17127903)asdescribedearlier24,47. wasconsideredacriticalpoint,leadingtoimprovednanodiscassem- Antibodies10E8,EBOV-296,EBOV-13C6,PGZL1,DH511,BG18,VRC01, blyascomparedtoourearlierwork.Withbatchassembly,theentire 4E10, PGZL1 H4K3, PGZL1 gVmDmJ, VRC42.01, VRC43.01, 35022, workflow had better reproducibility, and other conditions, such as PGT128,PGT145,10E8UCA,3074,B6,F105,RM19RandRM20A3were lipid and scaffold proportions, had less effect on yield and sample expressedinhouse.

Soluble Env GP was purified as described earlier61. The trans- quality29.Nanodiscbatchassemblywasdoneasfollows.Strep-tactin membraneglycoproteinpurificationmethodwasmodifiedfromthe matrix with detergent-solubilized GP bound was drained by gravity earlierprotocol29.Foroneliteroftransfectedcells,1mgofDNAwas flow,afterwhichthegravityflowcolumnwascapped,and400µlof mixedwith25mlofOPTI-MEMmedium(ThermoFisherScientific,# wash buffer 3wasadded tothe matrix.50% matrix slurrywas then 31985062)andsterilefiltratedthrough0.22µmfilterunit.3mgofPEI transferred to a 5ml test tube. Lipids were added at approx. 350× MAX(Polysciences,#24765)wasseparatelymixedwith25mlofOPTI- molarexcessandscaffoldat6×molarexcessinrelationtotheglyco- MEM,filteredandthencombinedwiththeDNA.CombinedDNAand protein.Glycoproteinamountboundtothematrixwasestimatedwith PEIMAXwerethenaddedtoHEK293Fcellsat1million/mldensity,and aseparateinitialexperimentwheretheproteinwaselutedafterthelast cellswereincubatedat37°C,8%CO ,and80%humidityundershaking washstep.Minorvariationsweremadetoglycoprotein:scaffoldand 2 at180rpmfor3days.Cellswereharvestedbycentrifugationat1500 glycoprotein:lipid ratios without a significant difference in the final rcfin4°Cfor15min,washedwith~300mlcoldPBS,andcentrifuged yield.Matrixwithglycoprotein,lipidsandscaffoldwasincubatedfor againat1500rcfin4°Cfor15min.Pelletedcellswerelysedwith~5ml 1hat4°CpriortotheadditionofSM-2bio-beads(Bio-Rad,#1523920). of lysis buffer per gramof cells (50mMTris-HCl (pH 7.4), 300mM Bio-beadswereactivatedaccordingtothemanufacturer’sinstructions NaCl,0.5%TX-100).Priortouse,approximately1proteaseinhibitor inmethanol,followedbyextensivewashingwithdeionizedwater.Bio- cocktail tablet per 50ml of lysis buffer, and 2ml of BioLock (IBA beads were added to approximately 50% v/v with the nanodisc Lifesciences, # 2-0205-050) per liter of cells was added to the lysis assemblymixture,i.e.,ifthetotalvolumeoftheassemblyreactionwas buffer.Cellswerelysedfor1hat4°Cinanoverheadrotatingmixer. 1.5ml,thefinalvolumeafterbio-beadadditioninthe5mltesttubewas Lysedcellswerecentrifugedfor1hat25,000rcf,followedbyfiltration ~3ml.Detergentwasthenremovedbyincubationinarotatingshaker ofthesupernatantthrougha0.22µmbottletopfiltrationunit.This overnightat4°C.Thefollowingday,anadditional~20%ofthetotal resultedinaclearedlysatethatwaspassedthroughStrep-tactinXT reactionvolumeoffreshbio-beadswasadded,followedbyanaddi- 4Flowmatrix(IBALifesciences,#2-5010-025)inagravityflowcolumn. tional1hincubationatroomtemperaturetoensurecompleteremoval Approx.400µlofdrainedmatrixwasusedperliterofcells.Matrixwas ofdetergent.Next,thecontentsofthetesttubeweretransferredtoa thenwashedthreetimeswith2mlofwashbuffer1(50mMTris-HCl newgravityflowcolumnwithapipettetipwiththefirst~2mmcutoff (pH7.4),300mMNaCl,0.1%(w/v)CHAPS,0.03mg/mLdeoxycholate), toallowpipettingbiobeadsandmatrix.Mixturewaswashedincolumn threetimeswith2mlofwashbuffer2(50mMTris/HClpH7.4,500mM twicewith4mlofTBSandelutedwithStrep-tactinelutionbufferBXT NaCl, 1mM EDTA, 0.1% n-dodecyl-ß-D-maltoside (DDM), 0.01% cho- in 500µl fractions. A280 of eluted fractions were monitored, and lesteryl hemisuccinate (CHS), 0.03mg/ml deoxycholate) and three fractionscontainingproteinwerepooledandconcentratedto<500µl times2mlofwash buffer3(50mMTris/HCl,pH7.4,150mMNaCl, forSECperformedusingaSuperose6Increase10/300column(Cytiva) 0.02%DDM,0.002%CHS,0.03mg/mldeoxycholate).Afterthis,the at 0.75ml/min in TBS. SDS-PAGE and ns-EM analysis were used to matrixwithdetergent-solubilizedGPwastransferredtoa5mltesttube confirmthepresenceofglycoproteinandscaffoldprotein,atypical forthenanodiscassemblystep. disc appearance in ns-EM 2D class averages and glycoprotein on Lipidstockswerepreparedpriortodiscassemblyin1mMfinal thedisc concentrations.Premade10%DDM/0.1%CHSstock(Anatrace,#D310- For structural biology purposes, Fabs were added to the GP CH210)wasusedthroughoutthestudy.Alllipidsinthisstudywere nanodiscsaftertheassembly,whilethenanodiscwasstillboundto prepared from chloroform stocks (Avanti Polar Lipids). Chloroform strep-tactinmatrix.Afterthesecondadditionofbio-beads,eachFab wasfirstevaporatedunderagentlenitrogenstreamuntilatransparent wasaddedat~10×molarexcess,andthemixturewasincubatedforan filmwasformedaroundtheglassvial.Lipidfilmwasthenrehydrated additional2hatroomtemperature.Complexelutionandthefollowing for1hatroomtemperatureinlipidrehydrationbuffer(50mMTris/ stepswereidenticaltothosedescribedaboveforunligandedglyco- HCl,pH7.4,150mMNaCl,0.1%DDM,0.01%CHS)inavolumebringing proteinnanodiscs.FractionswerepooledafterSEC,andthesample thelipidstockconcentrationto5mM.Afterthoroughmixingbyvor- wasconcentratedeitherto1mg/mlforFACS,SPRandotherpurposes, texing,thisstockwasdilutedfurthertoafinallipidstocksolutionat orupto11mg/mlforcryo-EMsamplepreparation.

IdentificationofR696SmutationonHIVEnvbymammalian directedevolution
A250aminoacidlongsite-saturationmutagenesis(NNK)scanofgp41 wassynthesizedatSGI-DNA.AfragmentofthelibrarycontainingNNKs from positions T606 toF699 (94positions)wasclonedintoBG505 MD39.2gp151.Thislibrarywasstablyintegratedinto293T-rtTA3Gcell line65usingthelentivirus-basedmammalian displayprotocoldescri- bedpreviously34,35,61.Afterinducinglibraryexpressionovernight,the cellswerestainedwith10E8IgGandPGT145Fabandthosewiththe highestbindingto10E8thatwerealsopositiveforPGT145weresorted. After three rounds of sorting, the library DNA was extracted and sequencedbySangersequencing.8of19sequenceshadamutationat position696,whichwasmutatedfromRtoS,A,V,IandL.Themost frequentmutation,R696S,wasselectedforfurthercharacterization.

Massspectrometry
ProteinaseKtreatmentanddeglycosylation.HIVEnvglycoprotein (when membrane-bound, denatured in6M urea)was exchanged to water using Microcon Ultracel PL-10 centrifugal filter. Glycoprotein wasreducedwith5mMtris(2-carboxyethyl)phosphinehydrochloride (TCEP-HCl)andalkylatedwith10mM2-Chloroacetamidein100mM ammoniumacetatefor20minatroomtemperature(RT,24°C).Initial protein-leveldeglycosylationwasperformedusing250UofEndoHfor 5µgtrimer,for1hat37°C.Glycoproteinwasdigestedwith1:25Pro- teinaseK(PK)for30minat37°C.PKwasdenaturedbyincubatingat 90°C for 15min, then cooled to RT. Peptides were deglycosylated againwith250UEndoHfor1hat37°C,thenfrozenat–80°Cand lyophilized. 100 U PNGase F was lyophilized, resuspended in 20µl 100mMammoniumbicarbonatepreparedinH 18O,andaddedtothe 2 lyophilizedpeptides.Reactionswerethenincubatedfor1hat37°C, subsequentlyanalyzedbyLC-MS/MS.

LC-MS/MS.SampleswereanalyzedonaQExactiveHF-Xmassspec- trometer. Samples were injected directly onto a 25cm, 100 μm ID columnpackedwithBEH1.7μmC18resin.Sampleswereseparatedata flowrateof300nL/minonanEASY-nLC1200UHPLC.BuffersAandB were 0.1% formicacidin 5% and 80% acetonitrile, respectively. The followinggradientwasused:1–25%Bover160min,anincreaseto40% Bover40min,anincreaseto90%Boveranother10minand30minat 90%Bforatotalruntimeof240min.Thecolumnwasre-equilibrated with solution A prior to the injection of the sample. Peptides were elutedfromthetipofthecolumnandnanosprayeddirectlyintothe massspectrometerbyapplicationof2.8kVatthebackofthecolumn. Themassspectrometerwasoperatedinadata-dependentmode.Full MS1scanswerecollectedintheOrbitrapat120,000resolution.The tenmostabundantionsperscanwereselectedforHCDMS/MSat25 NCE.Dynamicexclusion wasenabled with anexclusionduration of 10s,andsinglychargedionswereexcluded.

Dataprocessing.Proteinandpeptideidentificationweredonewith theIntegratedProteomicsPipeline(IP2).Tandemmassspectrawere extracted from raw files using RawConverter66 and searched with ProLuCID67 against a database comprising UniProt reviewed (Swiss- Prot) proteome for Homo sapiens (UP000005640), UniProt amino acidsequencesforEndoH(P04067),PNGaseF(Q9XBM8),andPro- teinaseK(P06873),aminoacidsequencesfortheexaminedproteins, andalistofgeneralproteincontaminants.Thesearchspaceincluded no cleavage-specificity. Carbamidomethylation (+57.02146C) was consideredastaticmodification.DeamidationinthepresenceofH 18O 2 (+2.988261N), GlcNAc (+203.079373N), oxidation (+15.994915M) and N-terminal pyroglutamate formation (–17.026549 Q) were con- sidered differential modifications. Data was searched with 50 ppm precursoriontoleranceand50ppmfragmentiontolerance.Identified proteinswerefilteredusingDTASelect268andutilizingatarget-decoy databasesearchstrategytolimitthefalsediscoveryrateto1%,atthe spectrumlevel69.Aminimumof1peptideperproteinandnotryptic endperpeptidewererequired,andprecursordeltamasscut-offwas fixed at 15 ppm. Statistical models for peptide mass modification (modstat)wereapplied.Census270label-freeanalysiswasperformed basedontheprecursorpeakarea,witha15ppmprecursormasstol- erance and 0.1min retention time tolerance. “Match between runs” wasusedtofindmissingpeptidesbetweenruns.Dataanalysisusing GlycoMSQuant42 was implemented to automate the analysis. Gly- coMSQuantsummedprecursorpeakareasacrossreplicates,discarded peptides without NGS, and discarded misidentified peptides when N-glycan remnant-mass modifications were localized to non-NGS asparagines and corrected/fixed N-glycan mislocalization where appropriate.

SPR
Thekineticsandbindingaffinitiesofantibody-antigeninteractionswere analyzedusingeithertheProteOnXPR36system(Bio-Rad)orCarterra LSA.TBSpH7.4(20mMTris,150mMNaCl)supplementedwithBSAat 1mg/mlwithoutdetergentswasusedasrunningbufferinallexperi- ments.InProteOnXPR36,HC30MXanTecsensorchipswereutilized.In 1mg/mlwithoutdetergentswasusedasrunningbufferinallexperi- ments.InProteOnXPR36,HC30MXanTecsensorchipswereutilized.In modalitiesBandC,anti-humanIgG(Fc)antibody(GE,#BR-1008-39) wasusedforcapturingIgG(ligand)atlowdensitieswithnanodiscGPas theanalyte.InmodalityA,anti-Strep-tagantibody(pAb,rabbit,Gen- Script, #A00875) was used for capturing Strep-tagged nanodiscs GP (ligand) followed by GP specific Fab as the analyte. Approximately 6000-8000 response units (RU) of the capture antibody were cova- lentlyattachedtothesensorsurfaceusingEDC-NHSchemistry.ForIgG- antigeninteractionstudies,approximately50to100RUsofIgGsata concentrationof0.3μg/mlwereimmobilizedoneachflowcell.ForFab- antigeninteractionstudies,approximately300to400RUsofantigenat 10μg/mlwerecapturedoneachflowcell.Analyteswereintroducedto theflowcellatarateof30μl/minfor3min,followedbyadissociation phaseof5min.Regenerationwasperformedwith1.7%or0.85%phos- phoricacid,eachwithacontacttimeof60–180s,repeatedfourtimes percycle.DataanalysiswasconductedusingProteOnManagersoftware (Bio-Rad),includingrawsensogramprocessing,interspotreferencing, andcolumndoublereferencing.Equilibriumor kineticfitswere per- formedusingtheLangmuirmodelasneeded.

Kineticsandaffinityofantibody-antigeninteractionsonCarterra LSAusingCMDPSensorChip(Carterra)forcaptureIgG–flownano- disc(modalityB)weredoneasfollows.Chipsurfaceswereprepared according to the Carterra software instructions. In a typical experi- ment, approximately 500–700 RU of capture antibody (South- ernBioTech,#2047-01)in10mMSodiumAcetate,pH4.5,wasamine coupledontheCMDPchip,takingspecialcarewiththeconcentration rangeoftheaminecouplingreagents.WeusedN-Hydroxysuccinimide (NHS) and 1-Ethyl-3-(3-dimethylaminopropyl) carbodiimide hydro- chloride (EDC) from the Amine Coupling Kit (GE, #BR-1000-50). Highestcouplinglevelsofcaptureantibodywereachievedbyusing10 timesdilutedNHSandEDCduringsurfacepreparationrunsascom- paredtothekitinstructions(10mlofwatereachtogive11.5mg/mland 75mg/ml, respectively, according to the kit instructions). Thus, the concentrationsofNHSandEDCwere1.15mg/mland7.5mg/ml,and theactivationtimewasreducedto1min.Theconcentratedstocksof NHSandEDCwerestoredfrozenin−20°Cforupto2monthswithout noticeablelossofactivity.Thecaptureantibodywasusedatacon- centrationof25µg/mlwith10mincontacttime.PhosphoricAcid1.7% wasusedasaregenerationsolutionwith60scontacttimeandinjected threetimespercycle.Theconcentrationofligandswasapproximately 1µg/ml,andthecontacttimewas5min.Rawsensogramswereana- lyzed using Kinetics software (Carterra) with interspot and blank doublereferencing,andtheLangmuirmodel.Analyteconcentrations werequantifiedonaNanoDrop2000cspectrophotometerusingthe absorptionsignalat280nm.Analyteswerebufferexchangedintothe runningbufferusingdialysis.Inatypicalrun,wecoveredabroadrange ofaffinitiesandusedtworeferencingpracticesdependingontheoff- rate of the ligand. For fast off-rate (faster than 9×10-3 1/s), we use automatedbatchreferencingthatincludesoverlayy-alineandhigher analyteconcentrations.Forslowoff-rates(9×10-31/sorless),weusea manual process referencing that includes serial y-align and lower analyte concentrations. After automated data analysis by Kinetics software,weperformedadditionalfilteringtoremovedatasetswith thehighestresponsesignalssmallerthansignalsfromnegativecon- trolsusinganRscript.

FACS
ValidationofEnv-Nanodiscproteinsasfluorescentbaits.Strepta- vidin(SA)conjugated-antigenbaitswerepreparedbycombiningbio- tinylatedEnv-nanodiscoremptynanodiscwithfluorescentSAatroom temperatureforatleast1hinthedarkatabait:SAmolarratioof2:1. ControlbeadsforFACSweregeneratedbyconjugatingvariousbnAbs to compensation beads. Mouse anti-human IgG (BD Biosciences, # 555784)wasfirstcapturedontoanti-mouseIgKcompensationbeads (BDBiosciences,#552843),followedbyawashstepandasecondary captureofbnAbsofinterest.VRC01-expressingRamoscells45,C57BL/ 6splenocytes(TheJacksonLaboratory,RRID:IMSR_JAX:000664),and thepreparedbnAb-conjugatedbeadswereincubatedwiththenano- discGP:SAbaitsatabaitconcentrationof10-50nMinthefinalstaining volumefor30minat4°Cinthedark.DatawereacquiredonaBDFACS MelodyandanalyzedinFlowJov10(FlowJo,LLC).

Mouse immunization studies. hD3-3/JH6 mice were immunized as previouslydescribedat2–4monthsofage24.Miceweremaintainedon a12-hwhitelightcycle,withlightsonfrom06:00AMto07:00PM.The room temperature was kept between 20°C and 26°C, and the humiditywasmaintainedbetween30%and70%.Allsixmiceweremale based on availability. Briefly, homozygous hD3-3/JH6 mice were injected with 10µg (50µl total volume) of Moderna mRNA LNPs encodingEnvgp151MPERI.M.underanesthesia(5%isofluraneinduc- tion)intheleftquadricepsmuscle.Aftersixweeks,micewereeutha- nized with compressed CO (100%) in a clear chamber to allow for 2 visualizationofrespirationandsubsequentdeathviarespiratoryces- sation.Bloodwascollectedfromthechestcavitypriortotheremoval ofthespleenandlymphnodes(mesenteric,inguinal,andpopliteal). Tissueswereplacedin3mlFACSbuffer(1×PBSCa/Mg++free,1mM EDTA,25mMHEPES,pH7.0,1%heat-inactivatedFBS)onice,andtis- sues were disassociated using the rough ends of two sandblasted microscope slides, followed by centrifugation (460 rcf for 5min at 4°C). Red blood cell lysis was performed using 1ml of ACK buffer (QualityBiological)for2minonicebeforelysiswashaltedbyadding 14mlFACSbufferpersample.Postlysisandcentrifugation(460×gfor 5min),cells wereresuspended in3mlBambankerfreezingmedium (BulldogBio)priortofiltrationthroughacotton-plugged,borosilicate pasteur pipette into a borosilicate glass test tube. 1ml filtered-cell solutionwassubsequentlydividedintothreecryovials/mouse,which wereprecooledinastyrofoamrackondryice.Cellswerestoredat −80°Cfor2–7dayspriortolong-termstorageinliquidnitrogen.All work followed IACUC guidelines associated with animal protocol number20-0001-2.

MousesamplepreparationandBcellsorting.Mousefrozensple- nocytes and lymphocytes were thawed and stained as previously described24,59. Briefly, after thawing and counting, totalB cells were isolated by negative selection using the EasySep Mouse Pan-B Cell Isolation Kit (StemCell, #19844) according to the manufacturer’s instructions.SA-conjugatedbaitswerepreparedasdescribedabove. Wildtype antigen baits were conjugated to BV421-SA (BioLegend, #405225)andAlexaFluor647-SA(Invitrogen,#S21374),whileepitope knockout(KO)baitswereconjugatedtohashtaggedTotalSeq-CPESA (BioLegend,#405261).PurifiedBcellswerestainedat4°Cindark,with appropriate baits and antibody master-mix consisting of FITC anti- CD19 (BioLegend, #152404), BV786 anti-IgM (BD Biosciences, #743328), PerCP-Cy5.5 anti-IgD (BD Biosciences, #564273), APC-Cy7 anti-F4/80(BioLegend,#123118),APC-Cy7anti-CD11c(BDBiosciences, #561241),APC-Cy7anti-Ly6C(BDBiosciences,#557661),APC-H7anti- CD8a (BD Biosciences, #560182), and APC-H7 anti-CD4 (BD Bios- ciences, #560181), all used at 1:100 dilution and prepared in FACS buffer (1% v/v heat inactivated FBS, 1mM EDTA, 1mM HEPES in 1× DPBS).TheKObaitwasfirstaddedtocellswiththeantibodymaster mixfor15min,followedbytheadditionofWTbaitsforanadditional 30min.Eachbaitwasusedatafinalbaitconcentrationof100nMinthe staining volume or 0.5µg per sample. During the addition of the antibody master mix, a unique TotalSeq-C anti-mousehashtag anti- body (BioLegend) was added to each sample at a concentration of 2.5µl/upto20millioncells.Followingantibodystaining,1mlof1:300 live/deadstaindilutedinFACSbuffer(Invitrogen,#L34966)wasadded toeachsampleforanadditional15minat4°C,thenwashedwith10ml of FACS buffer. Cells were resuspended in a final volume of 500µl FACSbufferandsortedonaBDFACSMelody.Cellswerebulksorted intoa4°CchilledPCRplatewellcontaining20µlof0.2µmfilteredFBS, andprocessedforBCRsequencingviathe10×GenomicsSingleCell ImmuneProfilingaccordingtokitprotocols,withminormodifications outlinedpreviously71.PooledlibrariesweresequencedonanIllumina NextSeq2000usinga100-cycleP3reagentkit(Illumina,#20040559).

BCR sequence analysis. Sequence analysis Raw sequencing data were demultiplexed, processed into assembled VDJ contigs and counts matrix files, and assigned to specific animal IDs based on TotalSeq-C antibody hashtag counts using Cell Ranger (v6.1) and scab as previously described71. Gene assignment, annotation, and formatting into Adaptive Immune Receptor Repertoire (AIRR) format72forpairedheavyandlightchainantibodysequenceswere performed using Sequencing Analysis and Data library for Immu- noinformaticsExploration(SADIE)withapreviouslydescribedcus- tom germline reference database that included all known mouse antibodygenesplusthehumanIGHD3-3andIGHJ6genesthatwere knockedintohD3-3/JH6mice24. Eightantibodiesfromeach animal wererandomlyselectedforinvitroanalysis,exceptforoneanimal from which only two total heavy/light pairs were recovered. In addition,12antibodieswithlongHCDR3s(>19aa)wereselectedfor invitrocharacterization.

NHP flow cytometry. Frozen NHP PBMC samples were thawed in RPMImediawith10%FBS,1%GlutaMAX,and1%Penicillin/Strepto- mycin(R10).NHPsamplesusedinthisstudywerefromanimalsthat werereportedinpreviousstudies36,50.Therecoveredcellswerethen stained with the appropriate antibody panel. Fluorescent antigen probes were constructed by combining fluorophore-conjugated streptavidin and biotinylated nanodisc GP or soluble Env probes withanappropriatevolumeofPBSinsmallincrementsover45minat roomtemperature(RT).Thawedcellswereincubatedwithprobesfor 30minat4°C.Amastermixofsurfacestainingantibodieswasthen added and incubated for 30min at 4°C. Cells were washed and preparedforacquisition onCytekAurora (CytekBiosciences). The followingreagentswereusedinthePBMCstainingpanel:AlexaFluor 647 Streptavidin (BioLegend #405237), BV421 Streptavidin (BioLe- gend #405225), PE Streptavidin (BioLegend #405245), PE-Cy7 Streptavidin (BioLegend #405206), BV711 Streptavidin (BioLegend #405241), BUV615 Streptavidin (BioLegend #613013), Viability eFluor780 (1:2000,Invitrogen #65-0865-14), CD3BV510 (1:100, BD Biosciences #569488), CD14 BV510 (1:100, BioLegend #301842), CD8a BV510 (1:100, BD Biosciences #563256), CD16 BV510 (1:100, BioLegend #302048), CD20 (1:100, BUV395 BD Biosciences #563782), IgG BV605 (1:100, BD Biosciences #563246), and IgD AF488(1:50,SouthernBiotech#2030-30).

EM. Negative stain EM was used for quality control of nanodisc assemblyandforinitialassessmentofantibodycomplexesforcryo- EM.Glycoproteinnanodiscornanodiscwiththreefoldmolarexcessof Fabwasappliedtoa400meshsizeCugridat0.04-0.06mg/mlcon- centration, blotted off with filter paper and stained with 2% uranyl formatefor60s.NsEMdatawerecollectedonaTecnaiSpiritmicro- scopeoperatingat120keV,usingaTietz4k×4kTemCam-F416CMOS cameraandLeginonautomatedimagecollectionsoftware73.Datawas processedusingtheRelion3.0imageprocessingpipeline66.

For cryo-EM, nanodisc with lipid mix was complexed with 10× molar excess of VRC01, BG18 and 10E8 Fabs after batch nanodisc assembly,whileboundtotheaffinitymatrix.Theelutedcomplexwas thenSECpurified(Superose6Increasecolumn,Cytiva),concentrated to 8mg/mland frozen on graphene oxide grids (GO onQuantifoils R1.2/1.3,Cu,400mesh,ElectronMicroscopySciences)witha30swait time,blotforceof0,andblottimeof2.5-3s.FluorinatedFos-Choline-8 (Anatrace # F300F) was added to the sample immediately prior to freezingtoafinalconcentrationof3mM.GOgrids,highsamplecon- centration and fluorinated Fos-Choline-8 were necessary to prevent freeze-denaturation,ensurehighparticlecountpermicrographand prevent orientationbias. 11,871 micrographs werecollected atpixel size 0.718Å using a Thermo Fisher Scientific Glacios 2 microscope operating at 200kV and equipped with a Thermo Fisher Scientific Falcon4icamerausingatotaldoseof44.89e-/Å2.Automateddata collection was performed using the EPU software (Thermo Fisher Scientific),andimageswerewrittenintheEERframeformat.Micro- graphswerepreprocessedusingcryoSPARCLive74,includingmotion andCTFcorrection.Briefly,particleswerepickedwithTopaz75after trainingtheneuralnetworkwithasubsetofparticlesfirstpickedwith blobpicker.Pickedparticlesweresubjectedto3roundsof2Dclassi- ficationtoobtainafinalparticlestackof317,792particles.Thesewere thenextractedfrommicrographsusingaboxsizeof540pix.Maps containingoneortwocopiesof10E8Fabwereseparatedusinghet- erogeneous reconstruction and 3D classification tasks. A class with 7,964 particles containing two copies of 10E8 was refined to 4.3Å resolution.Aclasswith111,002particlescontainingasingle10E8was examinedwith3Dvariabilityanalysis76.Furtherclassificationresulted inafinal,cleanedparticlestackcontaining63,366particles,whichwas thensubjectedtoNon-UniformRefinement77,leadingtoa3.5Åreso- lutionmap,whichwasthenusedformodelbuilding.Weattributethe higher resolution in comparison to our earlier approaches29 to improvements in nanodisc incorporation efficiency resulting from batch assembly, which in turn allowed the use of Fluorinated Fos- Choline-8 in combination with high protein concentration and gra- pheneoxidegrids.Theseledtoanabundanceofparticleswithside- view orientation that are required for high-resolution structure determination.Additionally,improvementsinmicroscopeandcamera hardware, as well as data processing software, enabled faster grid conditionscreeningandlargerdatasets.Datacollectionandproces- sing statistics are summarized in Supplementary Table 2. Model buildingwasinitiatedbydockinginavailablestructuresof10E8Fab (PDB 4G6F), BG18 Fab (PDB 6DFG),VRC01 Fab (PDB 3NGB) and an AlphaFold378-generated model of the trimer into the map using ChimeraX79.Manual adjustmentwasdoneusing Coot80,and further refinement was done using Phenix Real Space Refine81 and Rosetta Relax82. Final model statistics are summarized in Supplementary Table2.

Thesolublegp140(MD39.3)cryo-EMstructurewasdoneasfol- lows:VRC01,BG18and3BC315Fabswereaddedat4.3×molarexcessto purifiedEnvglycoprotein.ThecomplexwaspurifiedusingaHiLoad Superdex20016/600pgcolumn(Cytiva)andconcentratedto10mg/ ml.SampleswerevitrifiedusingaThermoFisherScientificVitrobot Mark IV operating at 100% humidity, 4°C and 3–6s blot times, on UltrAuFoil1.2/1.3-300holeygoldfilmgrids(ElectronMicroscopySci- ences). Prior to grid application, lauryl maltose neopentyl glycol (LMNG)wasaddedtoafinalconcentrationof0.005mM.Imagingwas performedusingthesamemicroscopeandconditionsasforEnvgp151 MPERNDcomplex.AfterinitialcryoSPARCparticlepickingusingBlob Picker,templates were selected from 2D classification and used for TemplatePicker.Atotalof886,907particleswereextractedusinga boxsizeof576,downsampledto144.Afteradditionalroundsof2D classification,anAbInitioReconstructionwasperformed,followedby Non-Uniform refinement. A single 3BC315 Fab was visible in the C1 reconstruction. A mask was created over 3BC315, and a 3D classificationwasperformedtoremovetrimerswithno3BC315parti- cles.Afinalstackof243,503wasre-extractedanddownsampledtoa boxsizeof400(resultinginapixelsizeof1.034Å)andsubjectedto Non-Uniform Refinement, which resulted in a 3.1Å reconstruction. Modelbuildingwasperformedasabove,andfinalmodelstatisticsare summarizedinSupplementaryTable2.

Maps and models have been deposited to the EMDB and PDB, respectively, under the accession codes listed in Supplementary Table2.

TZM-blPseudovirus(PSV)Neutralizationassay.PSVwereproduced in human embryonic kidney 293T cells (RRID:CVCL 0063) co- transfected using FuGENE 6 (Promega t#E2691) with pseudovirus Env-expressing plasmid and Env-deficient backbone plasmid (PSG3DEnv). PSVs were harvested 72h post-transfection and kept frozen at −80°C until use. On the experimental day, viruses were prepared by diluting with D10 media (Dulbecco’s Modified Eagle Medium (Gibco #10313-021), 10% FBS (Omega Scientific #FB-02),1× PenStrep(Gibco#15070-063),and1×GlutaMAX(Gibco#35050-061) accordingtopreviouslydefinedtiters.DEAE-Dextran(SpectrumChe- mical #DE-132) (5ug/ml final concentration) was added to all PSV excepttoMLVcontrolduetoitspotency.Equalvolumesofserially dilutedmAbsandcontrolsatastartingconcentrationof25μg/ml(Test mAbs and Den3 negative control), 10μg/ml (VRC01) and 5μg/ml (BG18)wereincubatedwithHIVPSVinaround-bottom96-wellplate (Costar#3788)at37°Cfor1h.TZM-blcells(RRID:CVCL_B478)were seeded24hpriorat100,000cells/mlin50μl/wellofhalf-area96-well plates(Costar#3688).Theplateswerethenremovedfromthe37°C incubator,culturemediacarefullyaspirated,and25μlofdilutedmAb +PSVaddedtoeachwellbeforefurtherincubationat37°Cfor24hina humidifiedatmosphereof5%CO2.After24hincubation,75μlofcul- ture media was added, and the plates were incubated in the same conditionsforanadditional48h.After48h(total72h),culturemedia wereremoved,andcellswerelysedwith45μl/well1×LuciferaseCul- tureLysisbuffer(Promega#E1531)for20minatRT.Neutralizationwas measured by adding 30μl luciferase reagent (substrate) per well (Promega#E1501)andmeasuringluminescence.IC wascalculated 50 usingaone-siteFitLogIC nonlinearregressioncurvefitconstrained 50 from0-100%inGraphPadPrism10.6.1.

Statistics and reproducibility. Details of statistical analyses can be found in the Figure legends. Statistical analysis using the indicated testsandplottingofalldatawereperformedusingGraphpadPrism 10.6.1.Datacollectionandanalysiswerenotperformedblindtothe conditions of the experiments.Formouse experiments, the sample sizeofn=6perindependentexperimentwasdeterminedbyanimal availability.

Peer review rounds
[{"round": 1, "reviewer_comments": {"reviewer_1": "The manuscript by Schief and colleagues describes the use of nanodiscs to generate soluble membrane anchored viral glycoproteins such as HIV-1 Env as tools for biophysical characterization of vaccine antigens that have been formulated as mRNA LNPs to produce membrane anchored immunogens. Furthermore, nanodisc-Env can be used to analyze antibody responses after vaccination. The authors employ the system to further analyze Env complex formation with the MPER-targeting bnAb 10E8.\nMajor findings are:\nEnv gp151 MPER ND with three gp41 glycosylation sites removed and a R696S mutation binds to 10E8 with nanomolar affinity. Removing the glycans helps to increase the affinity from 200 nM to 3.6 nM.\nGlycosylation of Env gp151 ND is close to native Env glycosylation.\nBiotinylated nanodisc-Env was used as a bait to sort B cells from mice immunized with Env gp151 MPER ND mRNA-LNPs. No MPER-specific responses were detected. Abs were cloned and confirmed no binding to MPER.\nNanodisc-Env can be used as tetramer probes to evaluate B cell responses in NHPs.\nCryoEM structures of nanodisc-Env in complex with 10E8 Fabs showed 3:1 and 3:2 complexes, but no 3:3 complexes. Novel features of the 10E8 epitope include 10E8 contacts with the C-terminus of gp120 and with gp41 helices 6 to 8.\nIn summary, the described workflow to characterize membrane-anchored viral glycoproteins such as Env, is important to characterize mRNA-LNP formulated antigens and their immune response. The application of the workflow should be of general interest to the vaccine field.\nThe following points need to be addressed to strengthen he conclusions of the manuscript:\nFig. 3: The SPR graphs should show the KD values. 3e shows gp151 ND binding to PGZL1; What is its affinity for gp151 MPER ND? Supp. Fig. 2 The KD measurements seem to be the result of on eexperiment? Could the authors provide triplicates for the MPER antibodies?\nThe authors describe three different SPR setups to test binding. Which ones were used for the data presented in 3c?\nWhat were the affinities of the Ebola Gp antibodies?\nThe cryoEM structures are interesting as they define the MPER epitope to be more complex. The previously reported affinities of 10E8 to MPER peptides including partial or complete TM is approximately 10x higher. How do the authors explain this discrepancy? In fact, I would expect that the additional contacts of 10E8 with gp120 and gp41 would have increased the 10E8 binding affinity.\nDoes the removal of the glycosylation sites correlate with the additional 10E8 binding to gp120 and gp41 and thereby explain the increased 10E8 binding to gp151 MPER ND as compared to gp151 ND? The difference is 55-fold? In contrast, PGZL1 binding does not seem to be affected by the glycans? The angle of approach of different MPER bnAbs varies by up to approximately 34°; could all known MPER bnAbs bind to the structure of gp151 MPER ND? Can this be modelled?\nMode of binding: The authors should show a comparison of 10E8 binding to peptides and to the MPER epitope within the new structure. Are the MPER epitope contacts all the same?\nFinally, are the additional contacts of 10E8 important for its function? Do mutations thereof affect neutralization?", "reviewer_2": "In this paper the authors describe an improved method for presenting glycoproteins in lipid nanodiscs; demonstrate three uses of such discs, for proteins from two different pathogens; and present beautiful structural data for an important HIV neutralizing antibody 10E8. This paper is a significant advance over the main authors’ previous publication (Rantalainen, K. et al. HIV-1 Envelope and MPER Antibody Structures in Lipid 915 Assemblies. Cell Rep 31, 107583 (2020)). The advances are: the standardization and higher throughput of the improved method, which should make it easier for other labs to adopt; and the higher resolution structural data.\nThis reviewer is enthusiastic about the paper. The method should be of great interest in structural biology and the 10E8 structure will be of interest in the HIV vaccine and antibody field. While this reviewer does not have expertise in some of the methods here, they seem quite complete. The figures are clear and visually appealing.\nMajor comments:\n- The description of the comparison of the new 10E8 structure with the published peptide-only structure (PDB 4G6F), was a little hard to understand. A main or supplementary figure would help the reader visualize the comparison. This could include the full epitope-paratope regions of the paratope and epitope overlays separately.\n- Authors should say something about the method optimization. What parameters did they test? Key for other labs that might use this method, what parameters were most important?\n- What enabled the better EM structure? Are the discs substantially different from the ones described in the 2020 paper?\n- Does the structure validate the predicted effects of the new mutations that the authors put into Env gp151 MPER ND? This should be analyzed.\nMinor comments:\n- These are sticky probes – hi background on naïve B cell population within splenocytes, which may limit utility. The authors see higher Ag+ in memory cells in 5/6 mice but do not show unvax mice, so formally the discs could be sticking nonspecifically to memory. It would be helpful to test on unvaccinated mice. This is what they did for NHP in fig 5. This reviewer suggests but does not require this experiment.\n- Watch the order of supp figures – callout to S4 comes early\n- Line 168 “higher” than what?\n- Fig 4A legend: need to say what gray is\n- Fig 4D legend: the mRNA did not encode nanodiscs, did it?\n- Mention why you do flow using one probe in 2 colors\n- line 342, “they will stabilize the trimer and prevent protomers from dissociating.” While you may expect that for an antibody like PGT145, this is unlikely to be an effect of VRC01 and BG18.\n- Line 357 “Unlike in our previous WT Env AMC011 nanodisc 357 cryo-EM reconstruction, density for the TM domain remained undetected, presumably due to the R696S mutation disrupting the stabilizing connection point of the three helices in the middle of the bilayer.” Other hypotheses? Don’t presume.\n- Make colors match in 6a and 6b (use 6b scheme in 6a)\n- Line 389 “indicating that epitope presentation in a more native context recapitulates interactions more faithfully than epitope peptide alone” - we can’t know that, your new structure could have unknown artifacts. This reviewer would leave out that clause, and say “suggests” when making the same point in line 395.", "reviewer_3": "The manuscript by Rantalainen et al. reports a study seemingly proposing nanodisc-solubilized virus glycoprotein for vaccine candidate design. Although the title reads like that the authors are treating nanodisc approach as a vaccine platform, the paper itself only studies HIV envelope glycoprotein (Env), by mixing data from in vitro characterization with structural studies, but with little or no direct data to support that the nanodisc-solubilized Env would provide safe, effective vaccination in any primate animal model. The authors did present preliminary FACS data from mouse model and some data for using the nanodisc for sorting non-human primate cells. But unfortunately, these data insufficiently justify that the nanodisc-derived method would be a viable vaccine formula or platform. Using nanodisc to solubilize membrane proteins, including the HIV Env, have been extensively studied and documented in the literature. Without introducing any novel concept, this paper attempts to “rebrand” the nanodisc-based method as a vaccine design platform. Although this idea is hypothetically feasible, the current manuscript falls short in proving this idea. The major concerns are in the following.\n(1) What is the antigenicity and immunogenicity of the nanodisc itself in both mouse and primate models? Is it safe to use the present composition of the nanodisc to form an Env vaccine? What side effects are expected for these nanodiscs as a vaccine?\n(2) What is the difference of Env-solubilizing nanodisc particle as compared to mRNA-LNP in terms of lipid composition and immunogenicity?\n(3) Is the Env nanodisc designed to only elicit the MPER-targeting bnAbs? Can other epitopes of bnAbs be incorporated in the same design? Is the nanodisc necessary for presenting MPER-derived epitopes?\n(4) What is the advantage or disadvantage of the nanodisc Env as a vaccine candidate over those engineered soluble gp140 Envs?\n(5) Are there any cryo-EM densities of the nanodiscs in the antibody-bound Env? Are there any cryo-EM structural evidence that the nanodiscs better preserve the bnAbs-eliciting MPER or any other epitopes?\n(6) Are there any direct structural evidence that the Env nanodiscs are better vaccine design than gp140 Env mimetics?\n(7) How is the MPER-targeting bnAbs modifying or causing the rearrangement of MPER structure? Would the presence of nanodisc make the MPER epitope less accessible for bnAbs? Have the nanodisc design been optimized to maximally expose the MPER epitopes? If so, how was it done?\n(8) As the safety and side effects of mRNA-LNP still remains a concern for certain patient groups and demands in-depth investigation with more evidence, how do the authors recommend on the necessary steps for nanodisc to become successful as part of a vaccine design?"}, "author_response": "We thank the reviewers for valuable comments. Please find point-by-point responses below\nReviewer #1 (Remarks to the Author):\nThe manuscript by Schief and colleagues describes the use of nanodiscs to generate soluble\nmembrane anchored viral glycoproteins such as HIV-1 Env as tools for biophysical\ncharacterization of vaccine antigens that have been formulated as mRNA LNPs to produce\nmembrane anchored immunogens. Furthermore, nanodisc-Env can be used to analyze antibody\nresponses after vaccination. The authors employ the system to further analyze Env complex\nformation with the MPER-targeting bnAb 10E8.\nMajor findings are:\nEnv gp151 MPER ND with three gp41 glycosylation sites removed and a R696S mutation binds\nto 10E8 with nanomolar affinity. Removing the glycans helps to increase the affinity from 200 nM\nto 3.6 nM.\nGlycosylation of Env gp151 ND is close to native Env glycosylation.\nBiotinylated nanodisc-Env was used as a bait to sort B cells from mice immunized with Env gp151\nMPER ND mRNA-LNPs. No MPER-specific responses were detected. Abs were cloned and\nconfirmed no binding to MPER.\nNanodisc-Env can be used as tetramer probes to evaluate B cell responses in NHPs.\nCryoEM structures of nanodisc-Env in complex with 10E8 Fabs showed 3:1 and 3:2 complexes,\nbut no 3:3 complexes. Novel features of the 10E8 epitope include 10E8 contacts with the C-\nterminus of gp120 and with gp41 helices 6 to 8.\nIn summary, the described workflow to characterize membrane-anchored viral glycoproteins such\nas Env, is important to characterize mRNA-LNP formulated antigens and their immune response.\nThe application of the workflow should be of general interest to the vaccine field.\nResponse: We thank the reviewer for the careful review and valuable comments\nThe following points need to be addressed to strengthen he conclusions of the manuscript:\nFig. 3: The SPR graphs should show the KD values. 3e shows gp151 ND binding to PGZL1; What\nis its affinity for gp151 MPER ND? Supp. Fig. 2 The KD measurements seem to be the result of\non eexperiment? Could the authors provide triplicates for the MPER antibodies?\nResponse: We thank the reviewer for this suggestion. We added KD values to Figure 3. The\nprevious Figure 3e exemplified a measurement with SPR modality C that, although highly\nsensitive, yields semi-quantitative binding results rather than monovalent K s and therefore is an\nD\nalternative approach for confirming antibody binding with high sensitivity and low sample\nconsumption. To strengthen the conclusions and to report K s, we performed additional SPR\nD\nexperiments with HIV and Ebola antibodies as Fabs (Modality A). These are now added to revised\nFig. 3e and in Supp. Fig . 2, including a selection of MPER antibody affinities measured with\nmodality A in triplicates. We also added additional text describing the differences between\nmodalities.\nUpon addressing this point, we noted a missing detail in the experiment description – all SPR\nexperiments related to this point were done with nanodiscs assembled with DOPC lipids only. The\npurpose was to simplify the baseline experiments by excluding the possible lipid headgroup\ncharge effect to MPER antibody binding. We have corrected the materials and methods section\nand figure legends accordingly.\nThe authors describe three different SPR setups to test binding. Which ones were used for the\ndata presented in 3c?\nResponse: Figure 3c uses modality A (nanodisc capture – Flow Fab). This is now indicated in the\nfigure legend and we have indicated this also in the figure next to SPR data\nWhat were the affinities of the Ebola Gp antibodies?\nResponse: We have now expressed Ebola antibodies EBOV-296 and EBOV-13C6 as Fabs and\nmeasured the affinities to EBOV GP Mayinga ND (46 nM and 16 nM, respectively). These are\nnow given in Fig. 3 e.\nThe cryoEM structures are interesting as they define the MPER epitope to be more complex. The\npreviously reported affinities of 10E8 to MPER peptides including partial or complete TM is\napproximately 10x higher. How do the authors explain this discrepancy? In fact, I would expect\nthat the additional contacts of 10E8 with gp120 and gp41 would have increased the 10E8 binding\naffinity.\nDoes the removal of the glycosylation sites correlate with the additional 10E8 binding to gp120\nand gp41 and thereby explain the increased 10E8 binding to gp151 MPER ND as compared to\ngp151 ND? The difference is 55-fold? In contrast, PGZL1 binding does not seem to be affected\nby the glycans?\nResponse: We thank the reviewer for the insightful comment. We propose that the previously\nreported higher affinity to partial MPER peptide is stemming from the absence of steric restrictions\nimposed by additional gp120 and gp41 glycans and lipid surface contacts. This Is supported by\nincreased affinity of 10E8 to Env gp151 MPER ND where three glycans around the MPER epitope\nare deleted. We have now included this in the discussion with relevant references.\nThe angle of approach of different MPER bnAbs varies by up to approximately 34°; could all\nknown MPER bnAbs bind to the structure of gp151 MPER ND? Can this be modelled?\nMode of binding: The authors should show a comparison of 10E8 binding to peptides and to the\nMPER epitope within the new structure. Are the MPER epitope contacts all the same?\nResponse: We have added an additional supplementary figure 5 to illustrate these important\ncomparisons. Aligning published crystal structures with MPER peptide in our cryo-EM structure\nallows visualizing how the binding pose in crystal structures would be accommodated in the\ncontext of the entire Env and the lipid bilayer. This analysis shows that these crystal structures\nmay represent a binding mode adopted in the absence of complete epitope and that MPER\nantibodies can adopt a wide range of binding modes. We also noted that only 14 out of 29\ncontacting residues in the paratope and 15 out of 26 contacting residues in the epitope were\nobserved in the 10E8 crystal structure compared to the cryo-EM structure. We have included\nthese observations in results and discussion.\nFinally, are the additional contacts of 10E8 important for its function? Do mutations thereof affect\nneutralization?\nResponse: We thank the reviewer for this important question. We have now conducted\nneutralization assays with key point mutations discovered from the new structure. The results are\npresented in Fig 7. These data show that reverting paratope residues of 10E8 FRH3 to their\ngermline sequences results in up to eightfold loss in neutralization efficiency with 10E8. Modest\nloss in neutralization efficiency was observed with several epitope mutations with notable\nexception of L660A resulting in approx. sevenfold increase. These observations are now included\nin the last paragraph of results section and in discussion. Together they show that newly identified\ncontacts can influence 10E8 neutralization and be used to improve vaccine designs.\nReviewer #2 (Remarks to the Author):\nIn this paper the authors describe an improved method for presenting glycoproteins in lipid\nnanodiscs; demonstrate three uses of such discs, for proteins from two different pathogens; and\npresent beautiful structural data for an important HIV neutralizing antibody 10E8. This paper is a\nsignificant advance over the main authors’ previous publication (Rantalainen, K. et al. HIV-1\nEnvelope and MPER Antibody Structures in Lipid Assemblies. Cell Rep 31, 107583 (2020)). The\nadvances are: the standardization and higher throughput of the improved method, which should\nmake it easier for other labs to adopt; and the higher resolution structural data.\nThis reviewer is enthusiastic about the paper. The method should be of great interest in structural\nbiology and the 10E8 structure will be of interest in the HIV vaccine and antibody field. While this\nreviewer does not have expertise in some of the methods here, they seem quite complete. The\nfigures are clear and visually appealing.\nResponse: We thank for the positive and appreciative review of the manuscript.\nMajor comments:\n- The description of the comparison of the new 10E8 structure with the published peptide-only\nstructure (PDB 4G6F), was a little hard to understand. A main or supplementary figure would help\nthe reader visualize the comparison. This could include the full epitope-paratope regions of the\nparatope and epitope overlays separately.\nWe removed Supp. Fig panel 4e and extended this analysis in Supplemental Fig 5 to address\nsimilar point from Reviewer #1. While the contacts within the MPER peptide remained the same\nbetween the two structures, the crystal structure included only 14 out of 29 contacting residues in\nthe paratope and 15 out of 26 contacting residues in the epitope when compared to the cryo-EM\nstructure.\n- Authors should say something about the method optimization. What parameters did they test?\nKey for other labs that might use this method, what parameters were most important?\nResponse: We agree that this is a critical point and have added a sentence in methods section\nemphasizing the importance of the batch assembly approach where glycoprotein is bound to\naffinity matrix during nanodisc assembly. While we initially tested a variety of GP:scaffold:lipid\nratios, they did not have a significant difference in the nanodisc assembly efficiency when batch\nassembly approach was used. Batch assembly also improved the reproducibility of the entire\nmethod which is critically important for scalable routine use of the approach.\n- What enabled the better EM structure? Are the discs substantially different from the ones\ndescribed in the 2020 paper?\nResponse: This very important point is now addressed in Methods section. We attribute the\nimproved resolution of the EM structure mainly to stable Env-Fab complexes and higher protein\nconcentration achieved through improved nanodisc assembly conditions. This allowed us to use\na fluorinated Fos-choline detergent in the EM grid preparation. This detergent is known to improve\norientation distribution of particles by blocking the air-water interface and does not disturb the lipid\nbilayer could only be used at high nanodisc protein concentrations. Additionally, rapid progress\nof cryo-EM instrumentation and data collection allowed us to test more conditions and collect\nlarger datasets.\n- Does the structure validate the predicted effects of the new mutations that the authors put into\nEnv gp151 MPER ND? This should be analyzed.\nResponse: Mutations intended to improve 10E8 binding were glycan knockouts (KOs). As\nexpected, we did not observe densities for these glycans. In these parts the absence of features\nin the EM structure confirmed the intended effect of the mutations together with the increased\naffinity in SPR measurements. Although we were unable to resolve the transmembrane domain,\nthe lack of density in the middle of the nanodisc indicates that the R696S mutation has disrupted\nthe stabilizing interaction between the three TM helices. In our earlier work using WT Env\nconstruct, we were able to observe strong density, albeit at low resolution connecting the three\nhelices in the middle of the bilayer (Rantalainen, K. et al. HIV-1 Envelope and MPER Antibody\nStructures in Lipid Assemblies. Cell Rep 31, 107583 (2020)).\nMinor comments:\n- These are sticky probes – hi background on naïve B cell population within splenocytes, which\nmay limit utility. The authors see higher Ag+ in memory cells in 5/6 mice but do not show unvax\nmice, so formally the discs could be sticking nonspecifically to memory. It would be helpful to test\non unvaccinated mice. This is what they did for NHP in fig 5. This reviewer suggests but does not\nrequire this experiment.\nResponse: We included this important control to our early method optimization where we detected\nno binding to probes using unimmunized C57BL/6 (B6) mouse splenocytes (Fig 4a). We agree\nthat this is an important control and should be included in future pre-clinical assessments. We can\nalso monitor interactions with lipid membranes by including an empty nanodisc control in SPR\nwhen measuring apparent affinities of monoclonal antibodies from sorted B cells to immunogens\nin nanodiscs (Supplementary figure 3b).\n- Watch the order of supp figures – callout to S4 comes early\nResponse: We thank the reviewer. We have moved the reference to the EM control experiment\nto a more appropriate location (Results: GP nanodiscs in cryo-EM structural studies) and revised\nfigure callouts.\n- Line 168 “higher” than what?\nResponse: Added “compared to soluble Env gp140”\n- Fig 4A legend: need to say what gray is\nResponse: Reference to MSP1D1 scaffold negative control was added\n- Fig 4D legend: the mRNA did not encode nanodiscs, did it?\nResponse: Nanodiscs with Env sequences matching the immunogen were used for SPR. We\nhave now corrected and clarified the figure legend.\n- Mention why you do flow using one probe in 2 colors\nResponse: Two colors were used to enrich for double-positive cells that are binding the antigen\nand not one of the fluorophores. This is now clarified in the first paragraph of FACS results.\n- line 342, “they will stabilize the trimer and prevent protomers from dissociating.” While you may\nexpect that for an antibody like PGT145, this is unlikely to be an effect of VRC01 and BG18.\nResponse: We agree and have rephrased the sentence. The stabilization may rather be a\nsecondary effect of antibodies preventing the trimer from adhering to the air-water interface, which\ncould prevent the trimer from falling apart due to freeze denaturation upon EM grid preparation.\n- Line 357 “Unlike in our previous WT Env AMC011 nanodisc 357 cryo-EM reconstruction, density\nfor the TM domain remained undetected, presumably due to the R696S mutation disrupting the\nstabilizing connection point of the three helices in the middle of the bilayer.” Other hypotheses?\nDon’t presume.\nResponse: We have rephrased this. It could also be possible that lack of intracellular C-terminal\ndomain could make the TM domain connection less stable.\n- Make colors match in 6a and 6b (use 6b scheme in 6a)\nResponse: We thank the reviewer for noting this and have matched the colors in 6a and b.\n- Line 389 “indicating that epitope presentation in a more native context recapitulates interactions\nmore faithfully than epitope peptide alone” - we can’t know that, your new structure could have\nunknown artifacts. This reviewer would leave out that clause, and say “suggests” when making\nthe same point in line 395.\nResponse: We have changed this to “suggest”. While the nanodisc is a considerably more native\ncontext for the MPER epitope compared to MPER peptide alone, as supported by the well-defined\nectodomain binding pocket, we may have introduced other unknown artifacts as the reviewer\ncorrectly pointed out.\nReviewer #3 (Remarks to the Author):\nThe manuscript by Rantalainen et al. reports a study seemingly proposing nanodisc-solubilized\nvirus glycoprotein for vaccine candidate design. Although the title reads like that the authors are\ntreating nanodisc approach as a vaccine platform, the paper itself only studies HIV envelope\nglycoprotein (Env), by mixing data from in vitro characterization with structural studies, but with\nlittle or no direct data to support that the nanodisc-solubilized Env would provide safe, effective\nvaccination in any primate animal model. The authors did present preliminary FACS data from\nmouse model and some data for using the nanodisc for sorting non-human primate cells. But\nunfortunately, these data insufficiently justify that the nanodisc-derived method would be a viable\nvaccine formula or platform. Using nanodisc to solubilize membrane proteins, including the HIV\nEnv, have been extensively studied and documented in the literature. Without introducing any\nnovel concept, this paper attempts to “rebrand” the nanodisc-based method as a vaccine design\nplatform. Although this idea is hypothetically feasible, the current manuscript falls short in proving\nthis idea. The major concerns are in the following.\nResponse: We would like to apologize for any confusion. The manuscript does not intend to\npropose to use nanodiscs as vaccines. Rather it provides an analytical platform to support vaccine\ndevelopment. We have changed the title to Virus glycoprotein nanodisc platform for vaccine\nanalytics to better reflect this important distinction. As we indicated in the abstract (line 34),\nmRNA-LNP can now deliver transmembrane glycoprotein vaccine candidates, but biophysical\nand structural characterization still relies on recombinant protein production and soluble mimics\nare often used in place of immunogen sequences that encode the transmembrane protein. We\nsee mRNA-LNP as a superior delivery mechanism for transmembrane immunogens (e.g. covid19\nvaccines or, for HIV applications, see Parks et al. SciTransMed 2025, Ramezani-Rad et al.\nSciTransMed 2025, Xie Z. et al. Science 2025, Melzi et al. Immunity 2022). Therefore we did not\nattempt to to produce recombinant protein/nanodisc-based immunogens. Some of the reasons\ninclude poor translatability of nanodisc assembly to clinical material production, including\nendotoxin free production of scaffold protein and generally low expression levels of\ntransmembrane recombinant proteins. Instead, we present a platform that allows the critically\nimportant in vitro characterization methods of the immunogens and analysis of immune responses\nwhich, to the best of our knowledge, have not been described previously. These include novel\nconcepts for 1) accurate affinity measurement with SPR at high throughput using transmembrane\nglycoproteins in nanodiscs and 2) sorting of B cells with nanodisc probes that contain identical\nsequences to mRNAs that were used for immunization. In addition we present the most complete\nand detailed epitope of an MPER targeting HIV antibody with a novel binding pocket targeted by\n10E8, facilitated by methodological advances in nanodisc preparation for cryoEM. This structure\nmay be critically important for MPER targeted vaccine development as the structural template for\nrational design of this epitope component has thus far been missing.\n(1) What is the antigenicity and immunogenicity of the nanodisc itself in both mouse and primate\nmodels? Is it safe to use the present composition of the nanodisc to form an Env vaccine?\nWhat side effects are expected for these nanodiscs as a vaccine?\nResponse: We believe mRNA-LNP is far better immunogen delivery platform compared to\nrecombinant protein nanodisc glycoproteins and did not intend to present nanodiscs as an\nimmunogen delivery modality.\n(2) What is the difference of Env-solubilizing nanodisc particle as compared to mRNA-LNP in\nterms of lipid composition and immunogenicity?\nResponse: Currently the lipid composition that is associated with in vivo-expressed Env from\nmRNA-LNPs is unknown but assumed to be related to the lipid composition of the cells that are\ntransfected in vivo. Deciphering this would be a major undertaking and is outside the scope of\nthis study.\n(3) Is the Env nanodisc designed to only elicit the MPER-targeting bnAbs? Can other epitopes of\nbnAbs be incorporated in the same design? Is the nanodisc necessary for presenting MPER-\nderived epitopes?\nOur structure (Fig 6) indicates that the N332 glycan supersite targeted by bnAbs like BG18, and\nthe CD4-binding site targeted by bnAbs like VRC01 and N6, can both be engaged simultaneously\nwith the MPER site. We see that assembling designs that integrate multiple engineered epitopes\ninto nanodiscs could allow testing the responses more broadly by biophysical and structural\nmeans, while the nanodiscs themselves may not be needed (and are not currently intended) as\nimmunogens. The MPER epitope may be presented alone as a peptide on liposomes or partially\nin soluble Env trimer designs, but our structural characterization of the complete epitope indicates\nthat these may not recapitulate the entire epitope and can thus lead to impaired immune\nresponses compared to transmembrane Env designs such as BG505 MD39.3 gp151 that has\nperformed well in NHPs (Ramezani-Rad et al. SciTransMed 2025) and humans (Parks et al.\nSciTransMed 2025).\n(4) What is the advantage or disadvantage of the nanodisc Env as a vaccine candidate over those\nengineered soluble gp140 Envs?\nAs noted in earlier responses, using nanodiscs as immunogens would bring significant\ndisadvantages as they would be challenging to produce as endotoxin free material in adequate\namounts and could lead to off-target responses to scaffold proteins. Engineered soluble gp140\nimmunogens are generally truncated before the MPER epitope and therefore do not generally\npresent the MPER. mRNA can deliver engineered transmembrane Env designs in a native-like\ncontext and we thus consider them as the most promising approach to present a complete set of\nbnAb epitopes including the MPER.\n(5) Are there any cryo-EM densities of the nanodiscs in the antibody-bound Env? Are there any\ncryo-EM structural evidence that the nanodiscs better preserve the bnAbs-eliciting MPER or any\nother epitopes?\nWe can visualize the nanodisc portion of the sample by low-pass filtering the cryo-EM data as\nindicated in Fig 6a. While only nanodiscs can preserve the entire MPER -targeting bnAb epitope\n(lipid surface, MPER peptide and the rest of the ectodomain), we don’t have evidence indicating\nthat other epitopes would be differently engaged in nanodisc assembled Env vs. soluble Envs.\n(6) Are there any direct structural evidence that the Env nanodiscs are better vaccine design than\ngp140 Env mimetics?\nEnv nanodiscs allow studying transmembrane Env designs using approaches that have\ntraditionally been available only for soluble mimetics such as gp140. We don’t propose using Env\nnanodiscs as vaccines. Nanodiscs do allow structural studies of the MPER epitope that is deleted\nin gp140 mimetics and therefore we consider the structure in Fig 6 to be evidence that nanodiscs\nare suitable for structural studies of MPER targeting antibodies, unlike gp140 mimetics.\n(7) How is the MPER-targeting bnAbs modifying or causing the rearrangement of MPER\nstructure? Would the presence of nanodisc make the MPER epitope less accessible for bnAbs?\nHave the nanodisc design been optimized to maximally expose the MPER epitopes? If so, how\nwas it done?\nWe designed the Env gp151 MPER ND construct to improve the MPER epitope accessibility by\nremoving glycans that could mask the epitope and by adding the point mutation R696S in the TM\ndomain. The point mutation was discovered in a directed evolution experiment as described in\nmethods section Identification of R696S mutation on HIV Env by mammalian directed evolution.\n(8) As the safety and side effects of mRNA-LNP still remains a concern for certain patient groups\nand demands in-depth investigation with more evidence, how do the authors recommend on the\nnecessary steps for nanodisc to become successful as part of a vaccine design?\nAlthough nanodiscs are not envisioned as a practical platform for preparation of clinical trial\nmaterial, they can provide valuable additional data points for mRNA-LNP investigations e.g. by\nexamining how antibodies from mRNA-LNP immunizations engage the transmembrane Env in\nthe membrane context. They could also be used in combination with mRNA formulations in early\npre-clinical experiments to assess transmembrane designs as protein administered vaccines vs.\nmRNA-LNPs, although we have not pursued this later avenue."}, {"round": 2, "reviewer_comments": {"reviewer_1": "The authors responded to the previous concerns and included additional data that strengthen the manuscript for publication in Nature Communications", "reviewer_3": "The authors did not fully address my major concerns in terms of novelty and significance. Instead, the authors now admitted the previous proposal might be a bad one, and now just revised the phrase to re-sell the same results, without providing any further data to support fundamentally important advancement expected for this journal. Using nanodisc to study membrane proteins including Env has been around for decades, I don't see any major novelty and significance here. I am sorry that I cannot recommend its publication in Nature Communications."}, "author_response": ""}]

Decision letter