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LETTER doi:10.1038/nature14264

AAV-expressed eCD4-Ig provides durable protectionfrom multiple SHIV challengesMatthew R. Gardner1*, Lisa M. Kattenhorn2*, Hema R. Kondur1, Markus von Schaewen3, Tatyana Dorfman1, Jessica J. Chiang2,Kevin G. Haworth4, Julie M. Decker5, Michael D. Alpert2,6, Charles C. Bailey1, Ernest S. Neale Jr2, Christoph H. Fellinger1,Vinita R. Joshi1, SebastianP. Fuchs7, JoseM. Martinez-Navio7, Brian D. Quinlan1,AnnieY.Yao2, Hugo Mouquet8,9, Jason Gorman10,Baoshan Zhang10, Pascal Poignard11, Michel C. Nussenzweig8,12, Dennis R. Burton11,13, Peter D. Kwong10, Michael Piatak Jr14,Jeffrey D. Lifson14, Guangping Gao15, Ronald C. Desrosiers2,7, David T. Evans16, Beatrice H. Hahn5, Alexander Ploss3,Paula M. Cannon4, Michael S. Seaman17 & Michael Farzan1

Long-term in vivoexpression of a broad and potent entry inhibitorcould circumvent the need for a conventional vaccine for HIV-1.Adeno-associated virus (AAV) vectors can stably express HIV-1broadly neutralizing antibodies (bNAbs)1,2. However, even the bestbNAbs neutralize 1050% of HIV-1 isolates inefficiently (80% in-

hibitoryconcentration (IC80).5mg ml2

1), suggesting that high con-centrationsof these antibodies would be necessary to achieve generalprotection36. Here we show that eCD4-Ig, a fusion of CD4-Ig with asmall CCR5-mimetic sulfopeptide, binds avidly andcooperatively tothe HIV-1 envelope glycoprotein (Env) and is more potent than thebestbNAbs(geometric mean half-maximuminhibitory concentration(IC50),0.05mg ml

21). BecauseeCD4-Ig bindsonlyconservedregionsofEnv,it isalsomuchbroader thanany bNAb. For example, eCD4-Ig efficiently neutralized 100% of a diverse panel of neutralization-resistant HIV-1,HIV-2 and simianimmunodeficiencyvirusisolates,including a comprehensiveset of isolatesresistantto theCD4-bindingsite bNAbs VRC01, NIH45-46and 3BNC117. Rhesus macaques inoc-ulated with an AAV vector stably expressed 1777 mg ml21 of fullyfunctional rhesuseCD4-Ig formorethan 40 weeks,and thesemacaques

were protected from several infectious challenges with SHIV-AD8.Rhesus eCD4-Ig was also markedly less immunogenic than rhesusforms of fourwell-characterizedbNAbs. Ourdata suggest thatAAV-delivered eCD4-Ig can function like an effective HIV-1 vaccine.

Rhesus macaques inoculated with an AAV-based gene-therapy vec-tor express antibody-like immunoadhesins for years, and these immu-noadhesins afforded partial protection from a neutralization-sensitivesimian immunodeficiency virus(SIV)2, suggesting that long-term steril-izing protectionfromHIV-1 mightbe achievable without a conventional

vaccine. Full-length AAV-expressed bNAbs also protected humanizedmice from an HIV-1 challenge1,7. However, a large fraction of HIV-1isolatesremainpartiallyor wholly resistant to eventhe bestbNAbs,withIC80values greater than 5 mg ml

21 measured under optimal in vitroconditions36 (ExtendedData Table 1). Higher concentrations willpro-

bably be necessary for broad-based protection in vivo, but primate stud-ies suggest that these concentrations will be difficult to establish inhumans2,8. Aneffective AAV-basedvaccinemay thereforerequirebroaderand more potent inhibitors of HIV-1 entry.

The breadth of an antibody depends on the conservation of its epi-tope. The two most conserved epitopes of HIV-1 Env are its CD4- and

coreceptor-binding sites911. The immunoadhesin form of CD4, CD4-Ig,has been extensively studied asa therapeutic.It neutralizes most iso-lates, irreversibly inactivates Env, and is demonstrated safe for use inhumans1215. However, itsaffinityfor Envis lower than thoseof bNAbs16,andits potency is furthercompromisedby its parallelability to promote

infection17. Mimetics of the primary HIV-1 coreceptor CCR5, in par-ticularpeptides based on its tyrosine-sulfated amino terminus, havealsobeen characterized18,19. These sulfopeptidesbind Envspecificallybutwithlow affinity in the absenceof CD4, in part because they include hydro-phobic residues and O-linked glycosylation that impede their associa-tion with Env18,20. CCR5mim1, a 15-amino-acid sulfopeptide derivedfrom the HIV-1 neutralizing antibody E51 (ref. 21), lacks these interfer-ingelements(Fig.1a)and binds Envwith higher affinity than CCR5-basedpeptides20,22. Reflecting the conservation of the sulfotyrosine-bindingpocketsof Env9,10, CCR5mim1binds bothCCR5-andCXCR4-dependentEnv proteins from all HIV-1 clades20,22.

We reasoned that a fusion of CD4-Ig and CCR5mim1 would bindEnv cooperatively and with higher avidity than either molecule alone.Accordingly, three fusion proteins were generated (sequences in Ex-

tendedData Fig. 1).CCR5mim1was inserted at eitherthe CD4-Igaminoterminus (fusion 1), between the CD4 and Fc domain (fusion 2), or atthe CD4-Ig carboxy terminus (fusion 3, renamed eCD4-Ig). All threeCD4-Ig variants neutralized CCR5- and CXCR4-dependent isolatesmore efficiently than did CD4-Ig, with eCD4-Ig consistently the mostpotent (Extended Data Fig. 2a, b). eCD4-Ig neutralized a wider panelof HIV-1 isolatesand SIVmac316with 10-to 100-fold lower IC50valuesthan CD4-Ig (Fig. 1b). Improved neutralization of SIVmac316 is con-sistentwith conservation of the sulfotyrosine-binding pockets of Env9,10,and a first indication of the exceptional breadth of eCD4-Ig.

To understand betterthe markedly greater potency of eCD4-Ig rela-tiveto CD4-Ig, we compared theirabilities to bindcell-surface-expressedEnv trimers (Fig. 1c). At low concentrations, eCD4-Ig bound thesetrimers more efficiently than did CD4-Ig. Surprisingly, eCD4-Ig satu-

rated trimer-expressing cells with approximately one-third less boundproteinthan CD4-Ig,suggesting that thesulfopeptidesof eCD4-IgmadesomeCD4-bindingsites inaccessible. eCD4-Ig alsoless efficientlypro-moted HIV-1 infection of CCR5-positive, CD4-negative cells thanCD4-Ig (Fig. 1d), presumably because its sulfopeptides blocked virionaccess to cell-surface CCR5. Heterodimers of CD4-Ig and eCD4-Ig23

*These authors contributed equally to this work.

1Department of InfectiousDiseases, TheScrippsResearchInstitute, Jupiter,Florida33458, USA.2Departmentof ComparativePathology, HarvardMedicalSchool,New EnglandPrimateResearchCenter,

Southborough,Massachusetts 01772,USA. 3Department of Molecular Biology, Princeton University, Princeton, New Jersey 08544, USA. 4Department of Molecular Microbiology and Immunology, Keck

School of Medicine of the University of Southern California, Los Angeles, California 90033, USA. 5Departments of Medicine and Microbiology, Perelman School of Medicine, University of Pennsylvania,

Philadelphia,Pennsylvania 19104, USA.6Immunathon Inc.,Cambridge,Massachusetts 02141, USA.7Departmentof Pathology, Universityof Miami MillerSchoolof Medicine,Miami, Florida33136, USA.8Laboratory of Molecular Immunology, The Rockefeller University, New York, New York 10065, USA. 9Department of Immunology, Institut Pasteur, Paris, 75015, France. 10Vaccine Research Center,

NationalInstitutes of Health, Bethesda,Maryland 20892, USA.11Department ofImmunology andMicrobial Science, IAVINeutralizingAntibodyCenter,and Center forHIV/AIDSVaccineImmunology and

Immunogen Discovery,The Scripps Research Institute, La Jolla, California92037, USA. 12Howard Hughes Medical Institute, New York, NewYork 10065,USA. 13Ragon Instituteof MGH,MIT and Harvard,

Cambridge, Massachusetts 02139, USA. 14AIDS and Cancer Virus Program, Leidos Biomedical Research, Incorporated, Frederick National Laboratory for Cancer Research, Frederick, Maryland 21702,

USA. 15GeneTherapyCenter,University of MassachusettsMedicalSchool,Worcester,Massachusetts 01655, USA.16Department of Pathology andLaboratoryMedicine,University of Wisconsin,Madison,

Wisconsin 53711, USA. 17Beth Israel Deaconess Medical Center, Boston, Massachusetts 02215, USA.

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neutralized less potently than eCD4-Ig (Fig. 1e and Extended DataFig. 2ce), indicating that both eCD4-Ig sulfopeptides engage the Envtrimer, consistent with a model of eCD4-Ig bound to Env (ExtendedData Fig. 3) and previous studies of CCR5mim1 (ref. 24). Thus, themarkedly greater potency of eCD4-Ig relative to CD4-Ig is due in partto the higher avidity with which it binds Env and to its decreased abil-

ity to promote infection.

We nextassessedeCD4-Ig under more physiologicalconditions. Weobserved thateCD4-Ig,but not CD4-Ig, halted replication of infectious

viruses in human peripheral blood mononuclear cells (PBMC) at con-centrationsaslowas125ngml21 (Extended DataFig. 1f,g).Weadmin-istered sufficient eCD4-Ig to humanized NOD/SCID/Il2rg2/2 (NSG)mice to maintain serum concentrations of 24 mg ml21 at the time ofchallenge. Five eCD4-Ig-treated mice and six control mice were chal-lenged intravenouslywith 53 104 infectious units of HIV-1NL4-3. Five

outof sixcontrol mice, butno eCD4-Ig-inoculatedmice, were infected(Fig. 1f and Extended Data Fig. 2h). Five weeks later, three eCD4-Ig-treated miceand theuninfectedcontrol mouse wererechallenged.Again,no eCD4-Ig-treated mouse was infected, whereas the control mousebecame infected.

We then characterized the ability of eCD4-Ig to neutralize a diversepanel of neutralization-resistant tier 2 and 3 viruses25 (Extended DataFigs 4a and 5a). In parallel, we assayed three additional eCD4-Ig var-iants. Inthefirst, eCD4-Igmim2, CCR5mim1 wasreplacedby CCR5mim2,which differs from CCR5mim1 by a single Ala to Tyr substitution 22.We also introduced a previously characterized Gln40 to Alamutationintothe CD4domain1 of eCD4-Ig (eCD4-IgQ40A)16. Bothmutationswerecombined in a final variant (eCD4-IgQ40A,mim2). eCD4-Ig and these

variantssubstantiallyoutperformed CD4-Igfor every virus in the panel,

typically improving neutralization potency by 20- to.

200-fold. Under-scoring its breadth, eCD4-Ig neutralized SIVmac251 33 times moreefficientlythan CD4-Ig. In general, the more neutralization-resistant a

virus, the better eCD4-Igand its variants performedrelative to CD4-Ig.In most cases, replacement of CCR5mim1 with CCR5mim2 modestlyimproved neutralization. Similarly, the Gln40Ala mutation also im-proved neutralization of most HIV-1 isolates, but not of SIVmac251.

We compared eCD4-Ig, eCD4-Igmim2 and eCD4-IgQ40A,mim2with apanel of 12 antibodies andinhibitors using three additional HIV-1iso-lates (Fig. 2a and Extended Data Fig. 6a, b). eCD4-Ig and its variantsneutralized the SG3and YU2 isolates more efficiently than anyof theseinhibitors. Five bNAbs neutralized JR-CSF more efficiently than anyeCD4-Ig variant, but four of these could not neutralize SG3. All eCD4-Igvariantsneutralized these isolateswith IC50valuesless than 0.3mg ml

21,which is more efficiently than CD4-Ig, the tetrameric CD4-Ig variantPRO-542 (refs 12,14),or theantibodies 2G12, 4E10 andVRC01.eCD4-Ig and its variants, but not three CD4-binding site bNAbs, neutralizedtheneutralization-resistantSIVmac239as wellas HIV-2 strain ST (Fig. 2bandExtended Data Fig.6c). As observed with SIVmac251, theGln40Ala

variant was less efficient at neutralizing SIVmac239 and HIV-2. Thepotency of these eCD4-Ig variants was also reflected in their abilitiesto mediate antibody-dependent cell-mediated cytotoxicity (ADCC).eCD4-Ig, eCD4-Igmim2 and eCD4-IgQ40A,mim2 each facilitated 3040times more killing of infected cells by CD161 natural killer cells26 thandidCD4-Ig or theantibody IgGb12(Fig. 2c). Thus theC-terminalmodi-fication of eCD4-Ig did not interfere with the ADCC effector functionof its Fc domain.

We furtherevaluated eCD4-Ig,eCD4-Igmim2,eCD4-IgQ40A,mim2 andthe bNAb NIH45-46 using nearly every isolate reported to be resistantto either of the CD4bs antibodies NIH45-46 or 3BNC117 (ExtendedData Figs 4b and 5b). Both eCD4-Ig variants efficiently neutralized all38 resistant isolates assayed with IC50values ranging from ,0.001to1.453mg ml21. By contrast,26 isolatesin thispanel wereconfirmedto beresistant to NIH45-46. Previous reports found 29 and 18 isolates to beresistant to 3BCN117 and VRC01, respectively4,6. Figure3 andExtendedDataFig. 7 summarizethe neutralizationstudiescompiledfrom theex-periments in Figs 1 and 2 and Extended Data Figs 46, and from pre-

vious studies of VRC01 and 3BNC117 against the same isolates4. Theyshow that the geometric mean IC50 and IC80values of eCD4-Ig and its

variants are less than 0.05mg ml21 (500 pM) and 0.2mg ml21 (2 nM),respectively, roughly 34times lower than those of VRC01,NIH45-46or 3BNC117. Importantly, our lead eCD4-Ig variant, eCD4-Igmim2,neutralized 100% of the isolates assayed at concentrations (IC50,

1.5mg ml21; IC80,5.2mg ml21) thatare probablysustainablein humans.

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Figure 1| Functional characterization of eCD4-Ig. a, CD4-Ig iscomprisedofCD4 domains 1 and 2 (blue) fused to the human IgG1 Fc domain (grey). IneCD4-Ig, thesulfopeptide CCR5mim1 (red) is fused to theC terminus of CD4-Ig. The sequence of the CCR5 N terminus is provided for comparison.Common residues, including four CCR5 sulfotyrosines, are shown in red.CCR5mim1 Ala 4 (blue) is substituted with Tyr in CCR5mim2, describedbelow.b, HIV-1 pseudotyped with the Env proteins of the indicated HIV-1or SIV isolates was incubated with GHOST-CCR5 cells and varyingconcentrations of CD4-Ig (red) or eCD4-Ig (blue). Infection was measured asgreen fluorescent protein (GFP)-expression by flow cytometry. Errors ofreplicates are less than 20% of indicated values but not indicated for clarity.c, 293T cells transfected to express 89.6 or ADA Env proteins were incubatedwith the indicated concentrations of CD4-Ig (red), eCD4-Ig (blue) or IgG(grey) and analysed by flow cytometry.d, HIV-1 expressing luciferase andpseudotyped with the Env proteins of the indicated isolates was incubatedwith Cf2Th-CCR5 cells in the presence of varying concentrations of CD4-Ig(red) or eCD4-Ig (blue). Experiment was controlled with HIV-1 pseudotypedwith the VSV-G protein (grey). Infection normalized to the maximumvalue observed for each pseudovirus.e, HIV-1 pseudotyped with the 89.6 Envwas incubated with TZM-bl cells and varying concentration of CD4-Ig(red), eCD4-Ig (blue) or a CD4-Ig/eCD4-Ig heterodimer (green). Similarexperiments using additional Env proteinsare shown in Extended Data Fig. 2c,d.f, Infection curves of humanized NSG mice with 24 mg ml21 of serum

eCD4-Ig at time of HIV-1NL4-3challenges (blue line,n5

5), or mock treated(redline, n5 6) areshown. Three uninfected eCD4-Ig treated mice andthe soleuninfected mock treated mouse were rechallenged 5 weeks after the firstchallenge. Significant protection (P5 0.002; MantelCox test) was observedin the eCD4-Ig-treated group. Viral load measurements are shown inExtended Data Fig. 2h. Experiments inbewere performed at least twicewith each indicated isolate with similar results. Errors bars denote one s.e.m.of duplicates.

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indicating thatthe eCD4-Igwasefficientlysulfatedand fullyactive invivo.We alsocompared macaquehumoralresponses to expressed rh-eCD4-Ig and to four AAV-expressed bNAbs inoculated for a separate study.3BNC117, NIH45-45, 10-1074 and PGT121, each bearing rhesus IgG2and light-chain constant domains, elicited markedly higher endogen-ous antibodyresponsesthan did rh-eCD4-Ig,consistent withtheir high

levels of somatic hypermutation (Fig. 4e). To investigate the target of

the anti-rh-eCD4-Ig responses, we increasedthe sensitivity of our assayand compared longitudinally the reactivity of inoculated rhesus sera toa series of antigens. rh-eCD4-Ig (Fig. 4f) and rh-CD4-Ig (without theCCR5mim2 sulfopeptide; Fig. 4g) were recognized by rhesus sera withnearlythe same reactivity, whereas CCR5mim2 fused to a human IgG1Fc domain was not (Fig. 4h), indicating that the sulfopeptide was notimmunogenic.Rhesus CD4domains1 and2 fused to a human IgG1 Fcwasmuch less reactive than thesame CD4domains fused to therhesus

IgG2 Fc,withoutor withthe Ile39Asnmutation(Extended Data Fig.8e,f),whereas an unrelated construct bearing the rhesus IgG2 Fc domainshowed no reactivity (Extended Data Fig. 8g), suggesting that a neo-epitope formed by the rhesus CD4 and Fc domains was recognized bymost anti-rh-eCD4-Ig antibodies. Thus eCD4-Ig is less immunogenicthan bNAbs, and can be expressed for at least 40 weeks at concentra-tionsthat are welltolerated and protectiveagainstseveral robust SHIV-AD8 challenges.

A key question is whether eCD4-Ig or a similar construct could beused to prevent new HIV-1 infections in a population, and whether itmightdo so more effectivelythana bNAb. Weshow that AAV-deliveredrhesus eCD4-Igprotectedall inoculated macaquesfrom multiple infec-tious doses that are probably higher than those present in most humantransmission events, although we have not yet tested protection from

mucosalchallenges.Protection lasted at least 34 weeks afterinoculation(Fig. 4b), and other studies indicate that these protective titres can besustained for several years2. Previous studies of CD4-Ig indicate that itis safe when passively administered12,14, and in particular it does notengage MHC II or otherwiseinterfere with immunefunction13, althoughfurther safety studies of eCD4-Ig are warranted. eCD4-Ig has fewernon-self B- and T-cell epitopes thanheavily hypermutatedbNAbs, andthus elicits fewer endogenous antibodies that can impair its expressionand activity (Fig. 4e). Its most prominent non-self element is its sulfo-peptide, whichdid notelicitany measurableantibodyresponses(Fig.4fh).However, the clearest advantage of eCD4-Ig over bNAbs is its potencyand its unmatched breadth (Fig. 3 and Extended Data Figs 47). Thebreadth of eCD4-Ig arises from the necessary conservation of its bind-ingsites on Env, suggesting that emergenceof eCD4-Ig escapevariantsinapopulationislesslikelythanwithbNAbs.Moreover,anyvirusthatdoes bypassprophylaxis is likelyto bind CD4and CCR5 less efficientlyin the continued presence of eCD4-Ig, and may therefore be less effi-ciently retransmitted.Its potency suggests thatrelatively lower concen-trations of eCD4-Igwillbe sufficientto protect against most circulating

viruses, a feature that may be critical to its use with AAV in humans.Although there are remaining challenges, these observations suggestthat AAV-expressed eCD4-Ig could provide effective, long-term andnear universal protection from HIV-1.

Online ContentMethods, along with any additional Extended Data display itemsandSourceData,are available in theonline versionof thepaper; references uniqueto these sections appear only in the online paper.

Received 29 June 2013; accepted 27 January 2015.

Published online 18 February 2015.

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Figure 4| AAV-rh-eCD4-Ig protects rhesus macaques from SHIV-AD8.a, Infection analysis comparing four male Indian-origin rhesus macaquesinoculated intramuscularly with 23 1013 AAV particlesdelivering rh-eCD4-Ig(blue)and four age- andgender-matchedcontrols(red). At 8, 11,16, 20,26 and34 weeks after inoculation, macaques were challenged with the indicatedp27 titres of SHIV-AD8. Significant protection (P5 0.006; MantelCox test)

was observed in the AAV-rh-eCD4-Ig-treated group.b, Viral loads ofinoculated (blue)and control (red) macaques areshown, with thetime andtitreof challenge indicated above the graph.c, Concentrations of rh-eCD4-Ig inthe sera of inoculated macaques were measured by ELISA to week 40 afterinoculation. d, Theneutralizingpotencyof macaque sera obtained4 weeksafterAAV-inoculation was compared to pre-inoculation sera (pre-sera), and pre-sera mixed with laboratory-produced rh-eCD4-Ig, as in Fig. 2b.e, Anti-transgene antibody responses in AAV-rh-eCD4-Ig-inoculated macaques werecompared to those in macaques inoculated with AAV expressing the indicatedbNAbs bearing constant regions of rhesus IgG2. Sera from 4weeks afterinoculation were analysed. Plates were coated with equivalent amounts ofrh-eCD4-Ig or rhesusformsof bNAbs andincubated with sera andanti-rhesuslambda chain (left) or kappa chain (right) antibody conjugated to horseradishperoxidase. Note that 3BNC117 and NIH45-46 bear a kappa light chain,whereas PGT121 and 10-1074 bear a lambda light chain, so that only hostantibody responses were detected. Values indicate absorbance at 450nM.Pvalues (Students two-tailedt-test) are indicated above the figures.f, Thesensitivity of the assay in e was increased to measure longitudinally the anti-rh-eCD4-Ig activity in the sera of inoculated macaques. Both anti-kappa andanti-lambda secondary antibodies were used.Values are scaled for comparisonto values ine.g,h, The same assay as infexcept that responses to rh-CD4-Ig,lacking CCR5mim2 (g) or to CCR5mim2 fused to a human IgG1 Fcdomain (h) were measured. Experiments inchwere performed at least twicewith similar results. Errors bars denote one s.e.m. of duplicates.

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18. Farzan, M. etal. Tyrosine sulfation of theamino terminus of CCR5 facilitates HIV-1entry.Cell96,667676 (1999).

19. Farzan, M.et al.A tyrosine-sulfated peptide based on the N terminus of CCR5interacts with a CD4-enhanced epitopeof theHIV-1 gp120 envelopeglycoproteinand inhibits HIV-1 entry.J. Biol. Chem.275, 3351633521 (2000).

20. Dorfman, T., Moore, M. J., Guth, A. C., Choe, H. & Farzan, M. A tyrosine-sulfatedpeptide derived from the heavy-chain CDR3 region of an HIV-1-neutralizingantibody binds gp120 and inhibits HIV-1 infection.J. Biol. Chem.281,2852928535 (2006).

21. Choe,H. etal. Tyrosine sulfation of human antibodies contributesto recognitionofthe CCR5 binding region of HIV-1 gp120.Cell 114, 161170 (2003).

22. Chiang, J. J. et al.Enhanced recognition and neutralization of HIV-1 by antibody-derived CCR5-mimetic peptide variants.J. Virol.86,1241712421 (2012).

23. Ridgway,J. B.,Presta, L. G. & Carter,P. Knobs-into-holes engineering of antibodyCH3domains for heavychain heterodimerization. ProteinEng. 9, 617621 (1996).

24. Kwong, J. A.et al.A tyrosine-sulfated CCR5-mimetic peptide promotesconformational transitions in the HIV-1 envelope glycoprotein.J. Virol.85,75637571 (2011).

25. Seaman, M. S. et al.Tiered categorization of a diverse panel of HIV-1 Envpseudoviruses for assessment of neutralizing antibodies.J. Virol. 84, 14391452(2010).

26. Alpert,M. D. etal. A novel assay for antibody-dependent cell-mediated cytotoxicityagainst HIV-1- or SIV-infected cells reveals incomplete overlap with antibodiesmeasured by neutralization and binding assays.J. Virol. 86,1203912052

(2012).27. Humes, D., Emery, S., Laws, E. & Overbaugh, J. A species-specific amino acid

difference in the macaque CD4 receptor restricts replication by global circulatingHIV-1 variants representing viruses from recent infection.J. Virol. 86,1247212483 (2012).

AcknowledgementsThisproject was supported by National Institutes of Health (NIH)grants R01 AI091476 and R01 AI080324 (M.F.), P01 AI100263 (G.G., R.C.D., M.F.),RR000168 (M.R.G., L.M.K., D.T.E., R.C.D., M.F.), R01 AI058715 (B.H.H.), by theIntramural Research program of the Vaccine Research Center, NIAID, NIH (J.G., B.Z.,P.D.K.),and by federalfundsfrom theNational CancerInstitute,NIH under contractno.HHSN261200800001E. The authors would like to thank H. Choe and M. Martin forcritical advice.

Author ContributionsM.R.G. andL.M.K.contributed equallyto thiswork.M.R.G., L.M.K.,H.R.K.,M.V.S., T.D., J.J.C.,M.D.A.,M.P.,J.D.L.,R.C.D., D.T.E.,B.H.H.,P.M.C., M.S.S.,A.P. andM.F. designed experiments. M.R.G., L.M.K., H.R.K., M.V.S., T.D., J.J.C., K.G.H., J.M.D.,M.D.A., C.C.B.,C.H.F., V.R.J.,B.D.Q. and A.Y.Y.performed experiments.L.M.K.conductedall non-humanprimatestudies. J.G.and P.D.K. assistedwithmodelling.J.M.M.-N.,H.M.,B.Z., P.P., M.S.S., M.C.N. and D.R.B. contributed advice and critical reagents. M.F.conceived the study and, with important assistance from M.R.G. and L.M.K., wrote themanuscript.

Author InformationReprints and permissions information is available atwww.nature.com/reprints. The authors declare no competing financial interests.Readersare welcome to commenton the online version of thepaper. Correspondenceand requests for materials should be addressed to M.F. ([email protected]) .

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METHODSPlasmidsand cells. Plasmid expressing CD4-Ig was previously described20. Fusionconstructswere createdby adding sequences encoding CCR5mim1 and tetra-glycinelinker to N terminus (fusion1) or between domain 2 and human Fc (fusion2) ofCD4-Ig by inverse PCR.eCD4-Ig(fusion3) and eCD4-Igmim2 were created by add-ing sequence encoding a tetra-glycine linker and CCR5mim1 or CCR5mim2, re-spectively, tothe C terminusofCD4-Ig byinversePCR. TheGln40 toAla mutationwas introduced in eCD4-Ig and eCD4-Igmim2 by Quickchange PCR.The eCD4-Ig/CD4-Ig heterodimer was generated as previously described23 andanalysedby SDS

PAGE under reducing and non-reducing conditions. rh-eCD4-Ig, consisting ofrhesus CD4 domains 1 and 2 bearing an Ile39Asn mutation, rhesus IgG2 Fc andCCR5mim2, was synthesized and clonedinto a previouslydescribed single-strandedAAV plasmid2. AAV expression plasmids for HIV-1 antibodies were created bysynthesizingthe variable heavy andlightchains of 3BNC117, NIH45-46,PGT121and 10-1074 with the rhesus heavy and light constant regions, and cloning thesegenes into a previously described ssAAV plasmid2. The following reagent wasobtained throughtheNIH AIDS ReagentProgram (Divisionof AIDS, NIAID,NIH):CMVR-VRC01-H, CMVR-VRC01-L, from J. Mascola28,29, pNL4-3.Luc.R-.E- fromN. Landeau30,31, TZM-blcellsfromJ. C. Kappes, X.Wu andTranzymeInc3236, SF162gp160from L. Stamatatos andC. Cheng-Mayer37, andGHOST-CCR5 and-CXCR4-cells from V. KewalRamani and D. Littman. Human embryonic kidney HEK293Tcells were obtained from ATCC. Cf2Th-CD41.CCR51 and CfTh-CCR51 cells werea gift from H. Choe. No testing for mycoplasma contamination was performed inanycell lineafter theirreceipt fromthese contributors.The variable heavyand lightchains of IgG-b12, NIH45-46, 3BNC117, 10-1074 and PGT121 were cloned intothe CMVR-VRC01-H and -L plasmids. Plasmids encoding TPST-2 or the enve-lopeglycoproteins pNL4-3Denv, 89.6, ADA, SG3, SA32,YU2, JRFL, KB9, VSV-G,HIV-2ST, SIVmac239,SIVmac316and replicative 89.6or SG3 viruses wereprevi-ously described20,21,3840.Purification of antibodies, CD4Ig and eCD4-Ig variants.Production of CD4-Ig, eCD4-Ig variants and antibodies was performed as previously described41. Inbrief, HEK293T cellsin 140mm plates were transfectedwith25 mg per plate at50%confluency by the calcium phosphatetransfectionmethod. Plasmids encodingsul-fatedproteins werecotransfectedwith a plasmidencodinghuman tyrosine proteinsulfotranserase2 (TPST2). At 12h after transfection, 10% FBS-DMEM media wasreplaced withserum-free293 Freestyle media(Invitrogen).Mediawas collectedafter48 h, debris was cleared by centrifugation for 10 min at 1,500gand filtered using0.45-mm filter flasks (Millipore).Completeproteaseinhibitorcocktail (Roche) wasaddedto the filtered supernatants. A 500-ml bedvolume ofProtein A sepharosebeads(GE Healthcare) was added and agitated at 4 uC overnight. The beadmedia mix-

ture wascollected bygravityflow column(Biorad) andwaswashedwith 30ml PBS(Lonza) plus 0.5M NaCl (0.65 M NaCl final) followed by 10 ml PBS. Protein waselutedwith3 M MgCl2 inPBS. Bufferwas exchangedfor PBSand protein wascon-centrated to 1 mg ml21 by Ultrafiltration (Amicon Ultra) at 4,000g.Flowcytometry analysis of CD4-Ig and eCD4-Igbinding to cell-expressed enve-lope glycoprotein.HEK293T cells were transfected with plasmids expressing en-

velope glycoprotein lacking cytoplasmic residues 732 to 876 (HXBc2 numbering)together withplasmidencoding the tat protein.Transfectionmediumwas replacedafter an overnight incubation and cells were collected 48 h after transfection. Col-lected cells were washed twice in flow cytometry buffer (PBS with 2% goat serum,0.01% sodium azide). Cells were incubated with CD4-Ig or eCD4-Ig on ice for 1 hand thenwashed twice with flow cytometry buffer. A secondary antibodyrecogniz-inghumanFc (Jackson ImmunoResearch) wasaddedto thecellsfor 30min. Cellswere washed twice with flow cytometry buffer, twice with PBS, and resuspendedin 1% paraformaldehyde solution. Binding was analysed with an Accuri C6 FlowCytometer (BDBiosciences)and dataanalysed withthe C6 Software(BD Biosciences).

Viral entry enhancement assay.HIV-1 pseudovirus expressing firefly luciferasewaspre-incubatedwith titrated amounts of CD4-Ig or eCD4-Ig variantsin DMEM(10% FBS) for 1 h at 37 uC. CD4-negative Cf2Th-CCR5 cells were collected anddiluted in DMEM (10% FBS) to 100,000 cells ml21 and added to the pseudovirus/inhibitormixture.Cells were then incubated for 48 h at 37 uC. Viral entry wasana-lysed using Britelite Plus (Perkin Elmer) and luciferase activity of cell lysates wasread using a Victor X3 plate reader (Perkin Elmer).HIV-1 neutralization assays. GHOST-CCR5 or -CXCR4 cells were plated into12-well platesat 50,000cellsper well. HIV-1 pseudovirus wasdilutedin RPMI andtitrated amounts of CD4-Ig, fusion1, fusion2 or eCD4-Ig were added. Virus andinhibitorwereincubated at room temperaturefor 20min andadded to thecellsfor2 h at 37 uC. Cells were then washed with serum-free medium and then incubatedin 1 ml of DMEM (10% FBS) for 48 h at 37 uC. Cells were collected by trypsiniza-tion,fixedin 1% paraformaldehyde inPBS,and viralentrywas determined byflowcytometry based on GFP expression.

For studies of infectious virus, unstimulated PBMCs were collected and resus-

pendedinRPMImedium(15%FBS,20Uml21 IL-2).Cells were platedina 12-well

plate at 106 cells per well. HIV-1 was diluted in RPMI and varying amounts of in-hibitor wereadded.The virus andinhibitor was incubatedat roomtemperaturefor20min andaddedto the cells for3 h at 37 uC. Cells were then washed with serum-free mediumand resuspended in fresh RPMI medium(15%FBS, 20U ml21 IL-2).At 3-dayintervals afterinfection, supernatants were collectedand freshRPMI me-dium (15% FBS, 20 U ml21 IL-2) was added to the cells. Supernatants were ana-lysed forviralinfection byELISAwith Alliance HIV-1 p24antigenELISAkit (PerkinElmer).

TZM-bl neutralizationassayswere performedas previously described42.Inbrief,

HIV-1 pseudoviruses werepre-incubatedwith titratedamounts of CD4-Igor eCD4-Ig variants in DMEM (10% FBS) for 1 h at 37 uC. TZM-bl cells were collected anddiluted in DMEM (10% FBS) to 100,000 cells ml21 andaddedto thepseudovirus/inhibitormixture.Cellswerethen incubatedfor 48 h at 37uC.Viralentrywas ana-lysed using Britelite Plus (Perkin Elmer) and luciferase activity was read using aVictor X3 plate reader (Perkin Elmer). All neutralization and enhancement stud-ies of Figs 14 were performed at least twice in triplicate. All error bars represents.e.m.

Antibody-dependent cell-mediated cytotoxicityassays. ADCCactivity was per-formed as previously described43. In brief, CEM.NKR CCR5 CD41 T cells wereinfected 4 days with infectious HIV-1 NL4.3,SHIV-KB9or SIVmac239.After 4 days,KHYG-1 effector cells were co-incubated with infected cells in the presence of ti-trated CD4-Ig, eCD4-Ig variants, or the b12 antibody for 8 h. ADCC activity wasmeasured by luciferase activity as above.

Production of HIV-1NL4-3 stocks and SHIV-AD8-EO stocks forin vivostudies.A molecular clone of HIV-1NL4-3was obtained from the AIDS Research and Re-

ference Reagent Program (ARRRP),Divisionof AIDS,NIAID, NIH frommaterialdeposited byS. Gartner,M. Popovic, R. Gallo andM. Martin.Virusstockswere pro-duced in 293T cells by transient transfection using TurboFect (Thermo Scientific)and 12mg of proviral plasmid. Supernatants were collected at 40 h, filtered through0.45-mm filters, anddispensed into singleuse doses andfrozen at280 uC. Viruseswere quantified by p24 ELISA (Zeptometrix) and by GHOST cell titer44 to deter-mine infectious units per millilitre (IU ml21). Titering was performed per theGHOST cell line protocol obtained through ARRRP. The molecular clone of SHIV-AD8-EO was a gift from M. Martin45. 293T cells were plated in140 mm flasks andtransfected with 80mg DNA per plate by calcium phosphate technique. At 12 haftertransfection,flasks were replacedwith fresh DMEM(10% FBS).Mediumwascollected at 48 h after transfection, frozen at280 uC, and titred using an SIV p27ELISA kit (ABL).

Haematopoietic stem cell isolation and NSG mouse transplantation. HumanCD341 haematopoietic stem cells were isolated from fetal livers obtained from

Advanced Bioscience Resources, INC (ABR). Tissue was disrupted and incubatedwith 1mgml21 collagenase/dispase (Roche Applied Sciences) for 15 min at 37 uC.Cells wereisolated by passingthe disrupted tissuethrougha 70-mmfilter.Redbloodcells were lysed in BD Pharm Lyse (BD Biosciences), with CD341 cells being iso-lated using CD34 MACS microbeads (Miltenyi) according to manufacturers in-structions with an additional purification step using a second column. NOD.Cg-Prkdc scid Il2rc tm1Wj/Szj (NOD/SCID/Il2rg2/2, NSG) mice were obtained fromJackson Laboratories. Neonatal mice received 150 cGy radiation, and 24 h later13 106 CD341 haematopoietic stemcells in 1% heparin (Celgene) via intrahepaticinjection. Mice were monitored for engraftment levels of human CD451 cells anddevelopment of T cells and B cells at 8, 10 and 12 weeks after engraftment.

Mouse infections, treatmentand analysis. Humanizedmice withevidence of humanCD41T-cell development in blood were infected with 53 104 IU of HIV-1NL4.3by intraperitoneal injection. Mice were administered with 65mg of eCD4-Ig onceweeklyfor thefirst2 weeks,startingat 8 days beforethe HIV-1 challenge,and thentwice weekly starting at week 3 by retro-orbital injection while under anaesthet-

ization by 2.5% isoflurane. Mock-treatedmice received a retro-orbital injection ofPBS 1 and 8 days before HIV-1 challenge, and were anaesthetized in parallel witheCD4-Ig mice throughout.Every weekafter infection the mice were anaesthetizedby inhalation of 2.5% isoflurane and blood was collected retro-orbitally for ana-lysis. At week 6, three eCD4-Ig-treated mice and one mock-treated mouse (whohad not become infected) were challenged a second time with 53 104 IU HIV-1NL4-3. Mouse blood wasblockedfor 20 minat roomtemperature in FBS(Denville)andstainedwith appropriateantibodiesfor 15 minat roomtemperature. Redbloodcells were removed by incubation in BD FACS Fix/Lysing Solution (BD Biosci-ences), which was removed by dilutionwith PBS before analysisby flowcytometry.HIV-1 levels in peripheral blood were determined by extracting viral RNA frommouseplasmaat eachblood drawusinga viral RNAisolationkit (Qiagen)followedby Taqman One-Step RTPCR (Life Technologies) using a primer and probe settargeting the HIV-1 LTR region, as previously described46,47. Reactions were per-formed and analysed using a 7500 Fast Realtime PCR System (Life Technologies).To analyse engrafted T cells by flow cytometry, stained cells were acquired on a

FACS Canto II (BD Biosciences) and analysed using FlowJo software v7.6.5 (Tree

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Star Inc.). Blood samples were stained using human-specific antibodies at a 1:20dilution for CD4-V450 (RPA-T4), CD8-APC (RPAT8), CD3-PE (UCHT1) andCD45-PerCP(TUI16)(BD Bioscience). Upto 10,000eventswererecordedfor viablecell populations and gated based on fluorescence minus one controls as previouslydescribed46. All mouse studies were performed in accordance with the ScrippsResearch Institute Institutional Review Board, protocol number 14-018. No stat-istical methods were used to predetermine sample size.AAV inoculation of rhesus macaques.Eight 4-year-old AAV1-negative maleIndian-origin rhesus macaques werehousedat the New EnglandPrimate Research

Center in accordance with standards set forth by the American Association forAccreditation of Laboratory Animal Care. Theirweights at the time of AAV inoc-ulation ranged from 5.2 to 8.2 kg. Macaques were separated into age- and weight-matchedcontrol groups,but blindingandrandomization werenot performed. Fourmacaques were inoculated with 1 ml RPMI containing 2.53 1013 AAV1 particlesdelivering 80% of a single-strandrh-eCD4-Ig transgene (IgG2isotype) and20% ofa single-strand rhesusTPST-2 transgene into each quadriceps muscle (two 0.5 mlper injections per quadriceps muscle). Five millilitres of sera was obtained every12weeksafter AAVinoculation beginningat week 4. Animals were challengedatweek 8 after inoculation with 2 pg p27of SHIV-AD8-EO. SHIV-negative animalswere repeatedly challenged with escalating doses of SHIV-AD8-EO up to 800 pgp27. Plasma viral loads were quantified as previously described 45.

For AAV studies of bNAbs, six 2-year-oldAAV1-negativeIndian-origin rhesusmacaques (two males and four females) were housed at the New England PrimateResearch Center in accordance with standards set forth by the American Associ-ation for Accreditation of Laboratory Animal Care. Three macaques were inocu-

lated with 1 ml RPMI containing 13 1013

AAV1particles delivering single-strandrh-3BNC117-IgG2 transgeneinto one quadriceps (two0.5-ml injections) and 1 mlRPMIcontaining 13 1013 AAV1particlesdelivering single-stranded rh-10-1074-IgG2transgene intothe contralateral quadriceps (two0.5-ml injections).The otherthree macaques were inoculated with 1 ml RPMI containing 13 1013 AAV1 part-icles delivering single-strand rh-NIH45-46-IgG2 transgene into one quadriceps(two 0.5-ml injections) and 1 ml RPMI containing 13 1013 AAV1 particles deli-

vering single-strand rh-PGT121-IgG2 transgene into the contralateral quadriceps(two 0.5-ml injections). Five millilitres of sera was obtained every 2 weeks begin-ning at week 2 and analysed by ELISA. All primate studies were performed inaccordance with the Harvard Medical School Standing Committee on Animalsprotocol number 04888.eCD4-Ig, rh-eCD4-Ig and anti-transgene antibody concentrations in NSGmice and rhesus macaque sera. In vivoconcentrations of eCD4-Ig, rh-eCD4-Igweremeasuredby ELISAas previously described2. Inbrief, to measure NSGmouseand macaque serum concentrations, ELISA plates (Costar) were coated with 5 mg

ml21 SIVgp120 overnightat 4 uC.Plates were washedwithPBS-T (PBS plus 0.05%Tween-20) twice and blocked with 5% milk in PBS for 1 h at 37 uC. Sera seriallydiluted in 5% milk in PBS were added to the plate and incubated for 1 h at 37 uC.Samples were washedfivetimeswithPBS-T anda horseradish peroxidase second-ary antibody (Jackson Immuno Research) recognizing human IgG1. Plates wereincubated for 1 h at 37 uC and then washed ten times with PBS-T. TMB solution(Fisher) was added for 10 min at room temperature and then stopped with TMBStop Solution (Southern Biotech). Absorbance was read at 450 nm by a Victor X3platereader (Perkin Elmer) and comparedwith a standard curve generatedusing arh-eCD4-Igmixed withpre-inoculation sera. Anti-rh-eCD4-Igantibodies and anti-bNAb antibodies were measured in the same way except that ELISA plates werecoated with 5 mg ml21 of various constructs. Constructs so assayed included rh-eCD4-Ig, rh-CD4-IgI39N, rh-CD4-Ig domains 1 and 2 (with or without Ile39Asn)bearing a human IgG1 Fc and hinge domain, C-terminal CCR5mim2-Ig (humanIgG1 Fc and hinge, no CD4 domains), NIH45-46 bearing the rhesus IgG2 Fc do-

main andhinge, or HIV-1 bNAbs.Serumsamples were diluted 10-or 20-foldandblocked in 5% milkin PBS. Anti-transgene antibodieswere measured usingsecon-daryantibodiesdetectingeitherthe kappa or lambda light chain (Southern Biotech)thatwas oppositeof the antibodybeingassayedwhen comparing theanti-bNAb re-sponse to thatto rh-eCD4-Ig. Bothanti-kappa andanti-lambdasecondary antibodies

were used when measuring anti-rh-eCD4-Ig responses alone. TMB solution wasadded for 1015 min at room temperature and measured as described above.

28. Wu, X.et al. Rational design of envelope identifies broadly neutralizing humanmonoclonal antibodies to HIV-1.Science329,856861 (2010).

29. Barouch, D. H. et al. A human T-cell leukemia virus type 1 regulatory elementenhances the immunogenicity of human immunodeficiency virus type 1 DNAvaccines in mice and nonhuman primates.J. Virol.79,88288834 (2005).

30. He, J. etal. Humanimmunodeficiencyvirustype1 viral proteinR (Vpr) arrestscellsin the G2 phase of the cell cycle by inhibiting p34cdc2activity.J. Virol.69,67056711 (1995).

31. Connor, R. I., Chen, B. K., Choe, S. & Landau, N. R. Vpr is required for efficientreplication of humanimmunodeficiency virus type-1in mononuclear phagocytes.Virology206, 935944 (1995).

32. Platt, E. J., Bilska, M., Kozak, S. L., Kabat, D. & Montefiori, D. C. Evidence thatecotropic murine leukemiaviruscontamination in TZM-blcells doesnot affect theoutcome of neutralizing antibody assays with human immunodeficiency virustype 1.J. Virol.83,82898292 (2009).

33. Takeuchi, Y., McClure, M. O. & Pizzato, M. Identification of gammaretrovirusesconstitutively released from cell lines used for human immunodeficiency virusresearch.J. Virol.82,1258512588 (2008).

34. Wei, X. et al.Emergence of resistant human immunodeficiency virus type 1 inpatients receiving fusion inhibitor (T-20) monotherapy.Antimicrob. AgentsChemother.46,18961905 (2002).

35. Derdeyn, C. A. et al. Sensitivity of human immunodeficiency virus type 1 to thefusion inhibitorT-20 is modulated by coreceptorspecificitydefined bythe V3loopof gp120.J. Virol. 74,83588367 (2000).

36. Platt, E.J., Wehrly, K.,Kuhmann,S. E., Chesebro,B. & Kabat,D. Effectsof CCR5 andCD4 cell surface concentrations on infections by macrophagetropic isolates ofhuman immunodeficiency virus type 1.J. Virol. 72,28552864 (1998).

37. Harouse,J. M. etal. Mucosal transmissionand induction of simian AIDSby CCR5-specific simian/human immunodeficiency virus SHIV(SF162P3).J. Virol. 75,19901995 (2001).

38. Choe,H. et al. The orphan seven-transmembranereceptor apj supports the entryof primary T-cell-line-tropic and dualtropic human immunodeficiency virustype 1.J. Virol.72,61136118 (1998).

39. Choe,H. et al.The beta-chemokine receptors CCR3 and CCR5 facilitate infectionby primary HIV-1 isolates.Cell85,11351148 (1996).

40. Farzan, M. et al.A tyrosine-rich region in the N terminus of CCR5 is importantforhuman immunodeficiency virus type 1 entry and mediates an associationbetween gp120 and CCR5.J. Virol.72,11601164 (1998).

41. Quinlan,B. D.,Gardner,M. R.,Joshi,V.R., Chiang,J. J.& Farzan,M. Directexpressionand validation of phage-selected peptide variants in mammalian cells.J. Biol.Chem.288,1880318810 (2013).

42. Li,M. etal. Humanimmunodeficiencyvirustype 1 Envclonesfromacuteand earlysubtypeB infections for standardized assessments of vaccine-elicitedneutralizingantibodies. J. Virol.79,1010810125 (2005).

43. Alpert, M. D.et al. ADCC develops over time during persistent infection with live-attenuated SIV and is associated with complete protection against SIVmac251challenge. PLoS Pathog.8,e1002890 (2012).

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Extended Data Figure 1 | Sequences of CD4-Ig and eCD4-Ig variants. Theamino-acid sequences of CD4-Ig, eCD4-Ig, fusion1, fusion2, eCD4-Igmim2,

eCD4-IgQ40A

, eCD4-IgQ40A,mim2

and rh-eCD4-Ig (rh-eCD4-IgG2I39N,mim2

)are

shown. Leader peptides are underlined, CD4 domains 1 and 2 are indicated inred, Fc domains are indicated in cyan, CCR5-mimetics peptides are indicated

in green, and linker sequences are shown in black.

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Extended Data Figure 2| Additional characterization of eCD4-Ig.a,b, Experiments similar to those of Fig. 1b except that CD4-Ig (red), fusion1(grey), fusion2 (green) and fusion3 (eCD4-Ig; blue) are compared usingHIV-1pseudotyped with the envelope glycoproteins of the 89.6 ( a) or ADA (b)isolates.c,d, Experiments similar to those in Fig. 1e except that CD4-Ig (red),eCD4-Ig (blue) or heterodimers thereof (grey) are compared. e, CD4-Ig, eCD4-Ig and the CD4-Ig/eCD4-Ig heterodimer assayed in c,dand Fig. 1e wereanalysed by SDSPAGE andstained with Coomassieblue under reducing (left)

and non-reducing (right) conditions.f,g, Infectious 89.6 (f) or SG3 (g) HIV-1

was incubated with human PBMC in the presence of the indicatedconcentrations of CD4-Ig (red) or eCD4-Ig (blue), or without either inhibitor(grey). Culture supernatants were collected on the indicated day and viralp24 levels were measured by ELISA.h, Viral loads in RNA copies ml21 areshown for each humanized mouse of Fig. 1f. Mice treated with eCD4-Ig areindicatedwith blue lines andmice treated with PBSare indicatedwith redlines.The800 copiesml21 limit of detectionof this assay is indicatedby a dashedline.Experiments inagwere performed at least twice with similar results. Error

bars denote s.e.m. of triplicates.

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Extended Data Figure 3| A model of eCD4-Ig bound to the HIV-1 Envtrimer. a, Thestructure (2QAD)of gp120 (YU2 isolate) bound to thetyrosine-sulfated CD4i antibody 412d andCD4 domains 1 and2 (ref. 10), wasfitted intoa cryoelectron micrograph of the HIV-1 envelope glycoprotein trimer (Env;Bal isolate)bound toCD4 (ref. 48).gp120and CD4 domains 1 and 2 are shownin blue and red, respectively. 412d sulfotyrosines are represented as green(carbon), red (oxygen) and yellow (sulphur) spheres. The remainder of 412dwas excluded for clarity. b, The same structure shown in arotated 90u aboutthehorizontal axis. Note that the sulfotyrosine-binding pockets are proximal tothe trimer axis, whereas the C terminus of CD4 domain 2 is distal from thetrimer axis, preventing both CD4 domains of CD4-Ig from simultaneouslybinding the same Env trimer.c, A model of how eCD4-Ig may associate withEnvis presented.The Fc domainof human IgG1 (1FCC,cyan)49 was positionedto be proximal to the gp120 sulfopeptide-binding pocket occupied bysulfotyrosine 100 (Tys 100) of the 412d heavy chain while avoiding stericinteraction with Env. Tys100 occupiesa pocketin gp120 thought to bind CCR5

sulfotyrosine 10 (ref. 50). This pocket is also critical for binding of CCR5mim1

andCCR5mim 2 (refs 20,22). In this model, the Fc domainis orientedto alloweach eCD4-Ig sulfopeptide to engage a different gp120 protomer24. A singleCD4 domain also binds one of the sulfopeptide-bound protomers. Distancesbetween the C terminus of CD4 and the N terminus of one Fc domainmonomer (38.1 A), between the C terminus of the Fc domain and Tys 100pocket of the CD4-bound gp120 protomer (30.6 A), and between the Cterminus of the Fc domain and Tys 100 pocket of an adjacent gp120 protomer(33.3A), are indicated.d, Residues not visible in the crystal structures used toconstruct this model are shown between brackets. In the model shown inc, these residues span the distancesindicated. Note that these distances are wellunder the extension of a typical beta strand. CD4-, IgG1- and CCR5mim1-derived residues are shown in red, cyan, and green, respectively, with linkerregions shown in black. Residues visible in the crystal structures, includingthe CCR5mim1 sulfotyrosine presumed to fill the Tys 100 pocket, arehighlighted in grey. Modelling was performed using UCSF Chimeraversion 1.8.

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Extended Data Figure 4| IC50values of eCD4-Ig variants againstneutralization-resistant isolates. a, The IC50values (mg ml

21) of CD4-Ig,eCD4-Ig, eCD4-Igmim2 (mim2), eCD4-IgQ40A (Q40A) and eCD4-IgQ40A,mim2

(Q40A,mim2) against 24 HIV-1 and SIV isolates selected for theirneutralization resistance are shown. The clade and tier of each isolate is listed.HIV-1 pseudotyped with the indicated envelope glycoprotein was incubatedin triplicate with TZM-bl cells and varying concentrations of CD4-Ig or eCD4-Ig variant. Luciferase activity was determined 2 days after infection. Foldindicatesthe ratio of theIC50value of CD4-Ig to the geometric mean of the IC50

values of the assayed eCD4-Ig variants. The geometric mean of eCD4-Igvariants and the CD4bs antibodies 3BCN117, NIH45-46 and VRC01 calculatedfrom values reported in previously4,6 are shown in the two right-most columns.b, Neutralization studies similar to those in aexcept that the IC50valuesof CD4-Ig, eCD4-Igmim2 (mim2), eCD4-IgQ40A,mim2 (Q40A,mim2) andNIH45-46 were determined for a panel of 40 viral isolates selected for theirresistance to the CD4bs bNAbs 3BNC117 and NIH45-46. IC50values of theCD4bs antibodies VRC01 and 3BNC117 listed in the two right-most columnswere previously reported4,6.

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Extended Data Figure 5| IC80values of eCD4-Ig variants against neutralization-resistant isolates. a,b, The IC80values (mg ml21) of the experiments

described in Extended Data Fig. 5a (a) and Extended Data Fig. 5b (b) are shown.

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Extended Data Figure 6| Further comparison of eCD4-Ig and HIV-1neutralizing antibodies. a, IC90values for the same experiments shown inFig. 2a, presented in the same format. b, Numeric IC50and IC90values of theexperiment shown inaand Fig. 2a are shown, using the same colour coding ofExtended Data Figs 4 and 5. The s.e.m. of triplicates are indicated below

their IC50and IC90values.c, Experiments similar to those in Fig. 2b exceptthat HIV-1 pseudotyped with the Env of the HIV-2 isolate ST was incubatedwith the indicated concentrations of CD4-Ig, eCD4-Ig variants or theCD4bs antibodies IgG-b12, VRC01 or NIH45-46. Error bars denote s.e.m.of triplicates.

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Extended Data Figure 7| Summary of IC80values for HIV-1, HIV-2 andSIV neutralization studies.The IC80values from studies of Figs 1b, 2a, b, andExtended Data Figs 46 are plotted. The number of isolates resistant to

50mg ml21 of the indicated inhibitors are indicated at the top. Geometricmeansare calculatedfor neutralized isolates andindicatedwith horizontallines.

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Extended Data Figure 8| Additional characterization of rh-eCD4-Ig.a, An experiment similar to that in Fig. 2b, except that rhesus and humanCD4-Ig and eCD4-Ig are compared for their ability to neutralize HIV-1pseudotyped with the SF162envelope glycoprotein.All variants have wild-typerhesus or human CD4 domains. Note that variants bearing rhesus CD4 aremarkedly less potent at neutralizing HIV-1.b, Experiment similar toaandFig. 2b except that human eCD4-Igmim2 and its rhesus analogue bearing or notbearing the Ile39Asn mutation are compared using SHIV-AD8. Note that theIle39Asn mutation largely restores the neutralization activity of rhesuseCD4-Igmim2.c, A representation of the AAV vectors used in the non-humanprimate studies of Fig. 4. Rh-eCD4-Ig (rh-eCD4-IgG2I39N,mim2; blue) andrhesus tyrosine protein sulfotransferase 2 (TPST2; green) were introduced into

a single-stranded AAV vector downstream of a CMV promoter. A woodchuckresponse element (WPRE), used to promote expression, and the SV40polyadenylation signal (SV40pA) were also included. AAV inverted terminalrepeats (ITR) are indicated in grey arrows. d, An experiment similar to thatin Fig. 4dexcept that sera from week6 were analysed. eg, Experiments similarto those in Fig. 4fh except that the reactivity of rhesus sera was examinedfor a construct bearing wild-type rhesus CD4 domains 1 and 2 fused to thehuman IgG1 Fc domain (e), one bearing rhesus CD4 domains 1 and 2 with theIle39Asn mutation, again fused to the human IgG1 Fc domain (f), or theantibody NIH45-46 fused to the rhesus IgG2 constant regions, used here topresent the rhesus IgG2 Fc domain (g). Experiments ina,banddgrepresentat least two with similar results. Error bars denote s.e.m. of triplicates.

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Extended Data Table 1| Potencies and breadth of well-characterized broadly neutralizing antibodies

A summaryof antibodyneutralizationpotenciescompiledusingtheLos AlamosNationalLaboratoryDatabase CATNAPtool (http://www.hiv.lanl.gov/components/sequence/HIV/neutralization/main.comp).The

geometric mean IC50and IC80values are listed for the indicated bNAbs against allreported isolates, excluding those with values greater than 50mg ml21. The percentage of isolates neutralized with IC50values

less than 50mg ml21, or with IC80values less than 5 mg ml21 are shown. bNAbs are ranked bytheir geometric mean IC50values. See Fig. 3 and Extended Data Fig. 7 for comparisons of eCD4-Ig variants with the

bNAbs NIH45-46, 3BNC117 and VRC01.

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