Potent and Broad Anti-HIV-1 Activity Exhibited by a...

JOURNAL OF VIROLOGY, Sept. 2011, p. 8467–8476 Vol. 85, No. 170022-538X/11/$12.00 doi:10.1128/JVI.00520-11Copyright © 2011, American Society for Microbiology. All Rights Reserved.

Potent and Broad Anti-HIV-1 Activity Exhibited by aGlycosyl-Phosphatidylinositol-Anchored Peptide

Derived from the CDR H3 of BroadlyNeutralizing Antibody PG16�†‡

Lihong Liu,1 Michael Wen,1 Weiming Wang,1 Shumei Wang,1 Lifei Yang,1 Yong Liu,3 Mengran Qian,1Linqi Zhang,2 Yiming Shao,3 Jason T. Kimata,4 and Paul Zhou1*

Unit of Anti-Viral Immunity and Genetic Therapy, Key Laboratory of Molecular Virology and Immunology, Institut Pasteur ofShanghai, Chinese Academy of Sciences, Shanghai 200025, China1; Comprehensive AIDS Research Center, School of

Medicine, Tsinghua University, Beijing 100084, China2; State Key Laboratory for Infectious Disease Prevention andControl, National Center for AIDS/STD Control and Prevention, Chinese Center for Disease Control and

Prevention, Beijing 102206, China3; and Department of Molecular Virology and Microbiology,Baylor College of Medicine, Houston, Texas 770304

Received 15 March 2011/Accepted 13 June 2011

PG9 and PG16 are two recently isolated quaternary-specific human monoclonal antibodies that neutralize70 to 80% of circulating HIV-1 isolates. The crystal structure of PG16 shows that it contains an exceptionallylong CDR H3 that forms a unique stable subdomain that towers above the antibody surface to confer finespecificity. To determine whether this unique architecture of CDR H3 itself is sufficient for epitope recognitionand neutralization, we cloned CDR H3 subdomains derived from human monoclonal antibodies PG16, PG9,b12, E51, and AVF and genetically linked them to a glycosyl-phosphatidylinositol (GPI) attachment signal.Each fusion gene construct is expressed and targeted to lipid rafts of plasma membranes through a GPIanchor. Moreover, GPI-CDR H3(PG16, PG9, and E51), but not GPI-CDR H3(b12 and AVF), specificallyneutralized multiple clades of HIV-1 isolates with a great degree of potency when expressed on the surface oftransduced TZM-bl cells. Furthermore, GPI-anchored CDR H3(PG16), but not GPI-anchored CDR H3(AVF),specifically confers resistance to HIV-1 infection when expressed on the surface of transduced human CD4� Tcells. Finally, the CDR H3 mutations (Y100HF, D100IA, and G7) that were previously shown to compromisethe neutralization activity of antibody PG16 also abolished the neutralization activity of GPI-CDR H3(PG16).Thus, we conclude that the CDR H3 subdomain of PG16 neutralizes HIV-1 when targeted to the lipid raft ofthe plasma membrane of HIV-1-susceptible cells and that GPI-CDR H3 can be an alternative approach fordetermining whether the CDR H3 of certain antibodies alone can exert epitope recognition and neutralization.

During human immunodeficiency virus type 1 (HIV-1) in-fection, a proportion of individuals develop broadly neutraliz-ing sera over time (32). From a few such individuals, a numberof potent and broadly cross-neutralizing monoclonal antibod-ies (MAbs) have also been isolated (36, 38, 40). Among them,PG9 and PG16 are recently isolated quaternary-specific neu-tralizing MAbs from a subtype A HIV-1-infected individual inAfrica that neutralize 70 to 80% of circulating HIV-1 isolates(36). PG9 and PG16 bind to overlapping, but distinct, gp120epitopes composed of conserved elements from the second andthird variable regions (V2 and V3, respectively). The quater-nary epitopes are glycosylated (6) and are preferentially dis-played on envelope trimers on the surface of virions and trans-fected cells but not on recombinant monomeric gp120 orsoluble trimers (36).

To gain insight into the molecular features of antibody bind-ing and neutralizing activities, Pancera et al. (23) and Pejchalet al. (24) recently determined the crystal structures of the Fabfragment of PG16. Antibodies PG9 and PG16 were found tobe sulfated (24). The fine specificity of the antibodies is con-ferred by an exceptionally long third-heavy-chain complemen-tarity-determining region (CDR H3) that forms a unique sta-ble subdomain towering above the antibody surface (23, 24).

The lipid raft is a specialized dynamic microdomain of theplasma membrane that is rich in cholesterol, sphingolipids, andglycerophospholipids (31). The lipid raft has been shown to bea gateway for HIV-1 budding (4, 17) as well as for HIV-1 entryinto T cells and macrophages (2, 26, 27). Interestingly, CD4,the receptor for HIV-1 entry, was found to be located in thelipid raft of the plasma membrane (14, 25). Previously, weshowed that by genetically linking single-chain Fv (scFv) ofhuman anti-HIV-1 envelope antibodies with a glycosyl-phos-phatidylinositol (GPI) attachment signal derived from decay-accelerating factor (DAF) (18), scFvs are targeted into thelipid raft of the plasma membrane. GPI-anchored scFvs (X5,48d, and 4E10) exhibit greater neutralization against diverseHIV-1 strains than do their soluble counterparts (37).

Therefore, the exceptionally long and unique structure ofthe CDR H3 subdomain of PG16 led us to postulate that the

* Corresponding author. Mailing address: Laboratory of Anti-ViralImmunity and Genetic Therapy, Institut Pasteur of Shanghai, ChineseAcademy of Sciences, Shanghai 200025, China. Phone: 862163851390.Fax: 862163843571. E-mail: [email protected].

† Supplemental material for this article may be found at http://jvi.asm.org/.

� Published ahead of print on 29 June 2011.‡ The authors have paid a fee to allow immediate free access to

this article.

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CDR H3 subdomain itself may bind to the epitope of gp120and that the targeting of this subdomain to the lipid raft of theplasma membrane of HIV-1-susceptible cells could neutralizeHIV-1 infection efficiently.

To test this hypothesis, we constructed CDR H3 derivedfrom five human monoclonal antibodies, PG16, PG9, b12, E51,and AVF. Antibody AVF recognizes the influenza virus hem-agglutinin, which is used here as a negative control (33). An-tibody b12 is a well-known broadly neutralizing antibody witha protruding, fingerlike, long CDR H3 that penetrates therecessed CD4 binding site of gp120 (1, 29, 41). In addition, aTyr residue in the CDR H2 loop and a number of Arg residuesin CDR L1 are also important for b12 binding (42). Neverthe-less, a soluble b12 CDR H3 peptide exhibits relatively weakneutralization (42). Antibody E51 is another sulfated antibodythat recognizes the CCR5 binding site of gp120 (39). A sul-fated peptide derived from CDR H3 of E51 binds gp120 andinhibits HIV-1 infection (7). In addition, we constructed threeCDR H3 mutants (Y100HF, D100IA, and G7) of PG16. TheseCDR H3 mutants were previously shown to compromise theneutralization activity of antibody PG16 (24). Here, we reportthat by genetically linking the CDR H3 of PG16, PG9, AVF,b12, and E51 and the CDR H3 mutants of PG16 with a GPIattachment signal of DAF, CDR H3 and the CDR H3 mutantsare targeted to lipid rafts of plasma membranes through a GPIanchor. Moreover, GPI-CDR H3 derived from PG16, PG9,and E51 [GPI-CDR H3(PG16, PG9, and E51)], but not GPI-CDR H3(AVF and b12), neutralizes multiple clades of HIV-1isolates with a high degree of potency. Furthermore, the trans-duction of human CD4� T cells with GPI-CDR H3(PG16), butnot GPI-CDR H3(AVF), confers resistance to HIV-1 infec-tion. Finally, the CDR H3 mutations (Y100HF, D100IA, andG7) that were previously shown to compromise the neutraliza-tion activity of antibody PG16 also abolished the neutralizationactivity of GPI-CDR H3(PG16). Thus, we conclude that theCDR H3 subdomain of PG16, when targeted to the lipid raft ofthe plasma membrane of HIV-1-susceptible cells, efficientlyneutralizes HIV-1 infection.

MATERIALS AND METHODS

Gene construction and cells. Fusion gene fragments encoding the CDR H3subdomain of antibodies PG16, PG9, E51, b12, and AVF and the CDR H3mutants (Y100HF, D100IA, and G7) of PG16 along with an IgG3 hinge, a Histag, and a GPI attachment signal were generated by overlapping PCR, ligatedinto the TA vector system (Invitrogen Life Technologies, San Diego, CA), andsequenced as described previously (28). The gene fragments with the correctsequences were religated between the BamHI and SalI sites of a third-generationlentiviral transfer vector, pRRLsin-18.PPT.hPGK.Wpre (8). The resulting lenti-viral transfer constructs were designated pRRL-CDR H3(PG16, PG9, E51, b12,and AVF)/IgG3 hinge/His tag/DAF (Fig. 1A) and pRRL-CDR H3 mutants(Y100HF, D100IA, and G7) of PG16/IgG3 hinge/His tag/DAF (see Fig. 4A),respectively.

The packaging cell line 293T was purchased from Invitrogen Life Technologiesand was maintained in complete Dulbecco’s modified Eagle’s medium (DMEM)(i.e., high-glucose DMEM supplemented with 10% fetal bovine serum [FBS], 2mM L-glutamine, 1 mM sodium pyruvate, penicillin [100 U/ml], and streptomycin[100 �g/ml]) plus G418 (500 �g/ml) (Invitrogen Life Technologies). The humanCD4� T cell line CEMss-CCR5 was generated as described previously (37).TZM-bl cells were obtained from the NIH AIDS Research and ReferenceReagent Program (ARRRP; Germantown, MD), contributed by J. Kappes andX. Wu (16). CEMss-CCR5 and TZM-bl cells were maintained in completeDMEM.

Generation of recombinant lentiviral vectors. Recombinant lentiviral vectorswere generated as described previously (35, 37). Briefly, 4 � 106 293T cells were

seeded onto a P-100 dish in 10 ml complete DMEM. After culturing overnight,cells were cotransfected with 20 �g transfer construct (one of the above-men-tioned pRRL-CDR H3/IgG3 hinge/His tag/DAF or pRRL-CDR H3 mutants ofPG16/IgG3 hinge/His tag/DAF constructs), 10 �g packaging construct encodingHIV-1 Gag/Pol (pLP1), 7.5 �g plasmids encoding the vesicular stomatitis virus Gprotein (VSV-G) envelope (pLP/VSVG), and 7.5 �g HIV-1 Rev protein (pLP2)(Invitrogen), using a calcium phosphate precipitation method. Sixteen hourslater, culture supernatants were removed and replaced with fresh completeDMEM plus 1 mM sodium butyrate (Sigma). Eight hours later, supernatantswere again removed and replaced with fresh DMEM plus 4% FBS. After another20 h, the culture supernatants were harvested and concentrated by ultracentrif-ugation as described previously (35, 37). The vector pellets were resuspended ina small volume of DMEM and stored in aliquots in a �80°C freezer. Vector titerswere determined as previously described (35, 37). The amount of HIV-1 Gag p24in concentrated vector stocks was determined by an enzyme-linked immunosor-bent assay (ELISA) (see below).

Generation of stable cell lines expressing GPI-anchored CDR H3. To trans-duce CEMss-CCR5 cells, 1 � 105 CEMss-CCR5 cells and 2 � 106 transducingunits (TU) of one of the lentiviral vectors containing pRRL-CDR H3(PG16 andAVF)/IgG3 hinge/His tag/DAF were added to 24-well tissue culture plate in thepresence of 8 �g/ml of Polybrene (Sigma). Twenty-four hours later cells wereextensively washed and cultured in complete DMEM.

To transduce TZM-bl cells, 5 � 104 TMZ-bl cells per well were seeded ontoa 24-well plate. After culturing overnight, 2 � 106 TU of one of the lentiviralvectors containing pRRL-CDR H3(PG16, PG9, E51, b12, and AVF)/IgG3 hinge/His tag/DAF or pRRL-CDR H3 mutants (Y100 HF, D100IA, and G7) ofPG16/IgG3 hinge/His tag/DAF was added onto a 24-well tissue culture plate inthe presence of 8 �g/ml of Polybrene. Twenty-four hours later cells were exten-sively washed and cultured in complete DMEM. The expressions of pRRL-CDRH3/IgG3 hinge/His tag/DAF and pRRL-CDR H3 mutant/IgG3 hinge/His tag/DAF constructs were measured by fluorescence-activated cell sorter (FACS)analysis (see below). We usually found that after a single round of transduction,over 98% of cells express transgenes (data not shown). After transduced cellswere generated, cells were continuously cultured in complete DMEM and splitevery 3 or 4 days. Periodically, the expression of transgenes was measured. Wefound that the level of transgene expression was very stable in the transduced celllines (data not shown).

FACS analysis. To study the cell surface expression of pRRL-CDR H3/IgG3hinge/His tag/DAF and pRRL-CDR H3 mutant/IgG3 hinge/His tag/DAF, 2 �105 mock-transduced and pRRL-CDR H3(PG16, PG9, E51, b12 and AVF)/IgG3hinge/His tag/DAF-transduced or pRRL-CDR H3 mutant (Y100 HF, D100IA,and G7) of PG16/IgG3 hinge/His tag/DAF-transduced TZM-bl cells and mock-transduced and pRRL-CDR H3(PG16 and AVF)/IgG3 hinge/His tag/DAF-transduced CEMss-CCR5 cells were incubated with a mouse anti-His tag anti-body (Sigma) for 45 min on ice. Cells were then washed twice with FACS buffer(phosphate-buffered saline [PBS] containing 1% bovine serum albumin [BSA]and 0.02% NaN3) and stained with phycoerythrin (PE)-conjugated goat anti-mouse IgG antibody (Sigma) for another 45 min on ice. Cells then were washedtwice with FACS buffer and fixed with 1% formaldehyde in 0.5 ml of FACSbuffer. FACS analysis was performed with a FACScan instrument (Becton Dick-inson, Mountain View, CA).

To determine whether the expression of pRRL-CDR H3/IgG3 hinge/His tag/DAF or pRRL-CDR H3 mutant/IgG3 hinge/His tag/DAF is truly targetedthrough a GPI anchor, 8 � 105 mock- and pRRL-CDR H3(PG16, E51, b12, andAVF)/IgG3 hinge/His tag/DAF- or pRRL-CDR H3 mutant (Y100HF, D100IA,and G7) of PG16/IgG3 hinge/His tag/DAF-transduced TMZ-bl cells were firstincubated with or without 6 U/ml phosphatidylinositol-specific phospholipase C(PI-PLC) (Invitrogen) in 0.5 ml 1� PBS and rocked at 4°C for 20 min. After theincubation, cells were washed twice to remove the remaining PI-PLC and thenstained with a mouse anti-His tag antibody as described above.

Generation of pseudotypes of the HIV-1 vector and a single-cycle infectivityassay. To generate pseudotypes with the HIV-1 vector, 4 � 106 293T packagingcells were cotransfected with 10 �g of an HIV-1–luciferase transfer vector (10)and 1 �g of a DNA plasmid encoding one of several HIV-1 envelopes (Q168,AD8, ADA, Yu2, JRFL, HXBc2, Mj4, consensus C, CNE3, CNE5, CNE6,CNE8, CNE11, CNE15, CNE17, CNE30, CNE50, CNE55, CH091.9, CH110.2,CH114.8, CH119.10, CH120.6, and CH181.12) or control retroviral envelope10A1 (19) and VSV-G (37) by using a calcium phosphate precipitation method.DNA plasmids encoding HIV-1 envelopes Q168, AD8, ADA-M, JRFL, HXBc2,and Mj4 were obtained from the ARRRP. The DNA plasmid encoding consen-sus C was a generous gift of B. H. Hahn at the University of Alabama. Q168 isderived from an R5-tropic clade A virus (22). AD8, Yu2, JRFL, and ADA-M arederived from R5-tropic clade B viruses (12, 13). HXBc2 is derived from an

8468 LIU ET AL. J. VIROL.

X4-tropic clade B virus (9). Consensus C is an artificial HIV-1 envelope (12). Mj4is derived from R5-tropic clade C viruses (21). CNE3, CNE5, CNE8, and CNE55are derived from R5-tropic CRF01_AE viruses. The HIV-1 envelopes CNE6 andCNE11 are derived from a R5-tropic clade B� viruses. HIV-1 envelopes CNE15,CNE17, CNE30, CNE50, CH091.9, CH110.2, CH114.8, CH119.10, CH120.6, andCH181.12 are derived from CRF07_B�C viruses (30). The pseudotype-contain-ing supernatants were harvested and stored in aliquots in a freezer at �80°C. Theamount of HIV-1 p24 in collected supernatants was measured by an ELISA.

In a single-cycle assay to measure the infectivity of pseudotypes, 10,000 mock-,pRRL-CDR H3/IgG3 hinge/His tag/DAF-, or pRRL-CDR H3 mutant/IgG3hinge/His tag/DAF-transduced TZM-bl or CEMss-CCR5 cells were transducedwith HIV-1, 10A1, or VSV-G pseudotype-containing supernatants equivalent toa relative luciferase activity (RLA) of 179,000 to 1,800,000 overnight. Cells werethen washed twice with PBS and cultured in complete DMEM for 2 days. Cellswere then washed once with PBS and lysed in 100 �l of lysis buffer. The luciferaseactivity in 50-�l cell suspensions was measured by a BrightGlo luciferase assayaccording to the manufacturer’s instructions (Promega).

SIV and HIV-1 infections and luciferase and p24 assays. Mock-, pRRL-CDRH3/IgG3 hinge/His tag/DAF-, or pRRL-CDR H3 mutant/IgG3 hinge/His tag/DAF-transduced TZM-bl cells at 5,000 cells per well were seeded onto a 96-wellplate. After culturing overnight, cells were infected with HIV-1 strains Bru-3,Bru-Yu2, and AD8 (34) as well as a simian immunodeficiency virus (SIV) strainSIVMne027 as a control (11) at a multiplicity of infection (MOI) of 2 in a finalvolume of 0.2 ml overnight. Cells were then washed three times with Hanks’balanced salt solution (HBSS), cultured in 0.2 ml of complete DMEM, andincubated at 37°C for 2 days. The infectivity of HIV-1 was determined by aBrightGlo luciferase assay (see above).

To test the effect of GPI-CDR H3(PG16) on HIV-1 infection and replicationin human CD4� T cells, 1 � 106 CEMss-CCR5 cells transduced with GPI-

CDR3(PG16 and AVF) were infected with HIV-1 strains Bru-3 and Bru-Yu2 atan MOI of 0.01 in a final volume of 0.5 ml overnight. Cells were then extensivelywashed with HBSS, resuspended in 6 ml of complete DMEM, and cultured for15 days. Every 3 days, 4.5 ml of cell suspensions was harvested and replaced withfresh medium. The supernatants were then collected. HIV-1 p24 in the super-natants was measured by an ELISA (Beckman Coulter) according to the man-ufacturer’s instructions.

Immunofluorescent staining and confocal analysis. Parental and TZM-bl-GPI-CDR3(PG16 and AVF) cells were seeded (5,000 cells per well) onto a tissueculture-treated glass slide (BD Biosciences) and incubated at 37°C in 5% CO2

for 2 days. Cells were then washed twice with 500 �l PBS and fixed by fixationbuffer (4% formaldehyde in PBS plus 1% BSA) for 15 min. Cells were washedtwice with 500 �l PBS and blocked with blockage buffer (5% goat serum in PBSplus 1% BSA) for 1 h. Cells were stained with Alexa 555-conjugated choleratoxin subunit B (CtxB) (Invitrogen Life Technologies) at 4°C for 45 min. Afterbeing washed 3 times with PBS, cells were stained with mouse anti-His tagantibody (Sigma) at 4°C for 45 min and then stained with Alexa 488-conjugatedgoat anti-mouse IgG antibody (Invitrogen) at 4°C. After cells were washed 3times with PBS, cells were stained with 4�,6-diamidino-2-phenylindole (DAPI) inpermeabilization buffer (blockage buffer plus 0.5% saponin) for 5 min. The slideswere mounted before being analyzed under a confocal fluorescence microscope(Zeiss model LSM 510).

RESULTS

Expression of the CDR H3 subdomain in the lipid raft of theplasma membrane through a GPI anchor. To generate GPI-anchored CDR H3, the sequences encoding CDR H3 derived

FIG. 1. Expression of GPI-anchored CDR H3 in transduced TZM-bl cells. (A) Schematic diagram of the lentiviral vectors pRRL-CDRH3/hinge/His tag/DAF. CDR H3s were derived from four human monoclonal antibodies, PG16, b12, E51, and AVF. Hinge, a human IgG3 hingeregion; His tag, a six-histidine-residue tag; DAF, the C-terminal 34 amino acid residues of decay-accelerating factor; RSV, Roys sarcoma virus;PGK, human phosphoglycerate kinase gene promoter; RRE, Rev-responsive element; cPPT, central polypurine tract and termination sequence.(B) List of CDR H3 peptide sequences of PG16, b12, E51, and AVF. (C) FACS analysis of cell surface expression of CDR H3/hinge/His tag/DAFin mock- and CDR H3(PG16, b12, E51, and AVF)/hinge/His tag/DAF-transduced TZM-bl cells with or without PI-PLC treatment.

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from four different human antibodies, PG16, b12, E51, andAVF, were genetically linked with the sequence encoding aHis-tagged IgG3 hinge region and the sequence encoding aGPI attachment signal of DAF (18). The CDR H3/IgG3 hinge/His tag/DAF (PG16, b12, E51, and AVF) fusion genes wereinserted into a third-generation lentiviral vector, pRRL (Fig.1A). The peptide sequences of human antibodies PG16, b12,E51, and AVF CDR H3 are shown in Fig. 1B. The recombi-nant viruses were then generated as described previously (35)and used to transduce TZM-bl cells and human CEMss-CCR5CD4� T cells (see below).

To determine if CDR H3/hinge/His tag/DAF was expressedon the cell surface through a GPI anchor, four TZM-bl cells—one transduced with lentiviral vector expressing CDRH3(PG16)/hinge/His tag/DAF, one transduced with lentiviralvector expressing CDR H3(b12)/hinge/His tag/DAF, onetransduced with lentiviral vector expressing CDR H3(E51)/hinge/His tag/DAF, and one transduced with lentiviral vectorexpressing CDR H3(AVF)/hinge/His tag/DAF—were treatedwith or without phosphatidylinositol-specific phospholipase

C (PI-PLC) and stained with an anti-His tag antibody, fol-lowed by FACS analysis. Figure 1C shows that all 4 CDRH3/hinge/His tag/DAFs were highly expressed on the sur-face of the cells, and their expression was substantially re-duced with PI-PLC treatment, indicating that the attach-ment of CDR H3/hinge/His tag/DAF to the cell surface isindeed through a GPI anchor. Thus, for the sake of simplic-ity, in the remaining text we will refer to CDR H3/hinge/Histag/DAF as GPI-CDR H3.

To determine if GPI-CDR H3 is located in the lipid rafts ofplasma membranes, mock- and GPI-CDR H3(PG16 andAVF)-transduced TZM-bl cells were cultured overnight andcostained with (i) mouse anti-His tag antibody followed byAlexa 488-conjugated goat anti-mouse IgG antibody, (ii) Alexa555-conjugated cholera toxin subunit B (CtxB), and (iii) DAPI.CtxB interacts with GM1 (a lipid raft marker) on the cellsurface. Figure 2 shows that both GPI-CDR H3(PG16) andGPI-CDR H3(AVF) are colocalized with GM1 on the cellsurface, implying that they are located in the lipid raft of theplasma membrane.

FIG. 2. Localization of GPI-anchored CDR H3 in transduced TZM-bl cells. Shown are data for confocal analysis of mock- or GPI-CDRH3(PG16 and AVF)-transduced TZM-bl cells. Cells were stained with Alexa 555-conjugated cholera toxin B subunit (CtxB), and cells were stainedwith a mouse anti-His tag antibody followed by Alexa 488-conjugated goat anti-mouse IgG antibody.


GPI-CDR H3(PG16) exhibits a remarkable degree ofbreadth and potency against HIV-1. Next, we tested the neu-tralization activity of GPI-CDR H3 against HIV-1. Becausethe ability of GPI-CDR H3 to neutralize virus could be im-pacted by differences in the level of expression of CD4,CXCR4, or CCR5 on the surface of transduced TZM-bl cells,we first assessed the expression levels of these receptors inmock-transduced parental TZM-bl cells as well as GPI-CDRH3-transduced TZM-bl cells. In all cases, the values were sim-ilar (see Fig. S1 in the supplemental material), suggesting thatthe expression of the GPI-CDR H3 transgenes does not alterthe expression of the receptor and the coreceptors for HIV-1in transduced cells. In addition, the expression of the trans-genes did not alter cell growth (data not shown).

To test the neutralization activity of GPI-CDR H3 againstHIV-1, a panel of 24 virions pseudotyped with envelopes rep-resenting different HIV-1 clades or a control retroviral enve-lope, 10A1, was used to infect GPI-CDR H3(PG16, b12, E51,and AVF)-transduced TZM-bl cells in a single-round infectionexperiment (37). The retroviral envelope 10A1 recognizes ei-ther Ram-1 or Glvr-1 as a receptor for cell entry (19) and wasused here as negative control. The panel of HIV-1 pseudotypesconsists of HIV-1 envelopes derived from clade A (Q168),clade B (ADA-M, AD8, HXBc2, JRFL, and Yu2), clade B�(CNE6 and CNE11), clade C (Mj4 and consensus C),CRF07_B�C (CNE15, CNE17, CNE30, CNE50, CH091.9,CH110.2, CH114.8, CH119.10, CH120.6, and CH181.12), andCRF01_AE (CNE3, CNE5, CNE8, and CNE55). Figure 3Ashows the percentage of the reduction of relative luciferaseactivity (RLA) in four GPI-CDR H3(PG16, b12, E51, andAVF)-transduced cells infected with these pseudotypes com-pared to mock-transduced parental cells infected with thesame panel of pseudotypes. Compared to mock-transducedparental TZM-bl cells, cells transduced with GPI-CDRH3(AVF, PG16, b12, and E51) did not show any significantneutralization activity against the 10A1 pseudotyped controlvirus. Furthermore, compared to mock-transduced parentalTZM-bl cells, cells transduced with GPI-CDR H3(AVF) didnot show any significant neutralization activity against any ofthe 24 HIV-1 pseudotypes, and cells transduced with GPI-CDR H3(b12) had minimum neutralization activity against 2(Q168 and Yu2) of the 24 HIV-1 pseudotypes with a lowdegree of potency. In contrast, cells transduced with both GPI-CDR H3(PG16) and GPI-CDR H3(E51) neutralized all 24HIV-1 pseudotypes with remarkable degrees of potency. GPI-CDR H3(PG16) reduced the infection of 17 HIV-1 pseu-dotypes by over 99%, inhibited the infection of the other 6HIV-1 pseudotypes by over 90%, and reduced the infection ofJRFL by 70%. GPI-CDR H3(E51) conferred over 99% inhi-bition of 11 HIV-1 pseudotypes, over 90% inhibition of theother 12 HIV-1 pseudotypes, and 83% inhibition of JRFL.Both GPI-CDR H3(PG16) and GPI-CDR H3(E51) inhibitedHIV-1 pseudotypes expressing envelopes derived from cladesA, B, and B� with similar degrees of potency, whereas for mostof the HIV-1 pseudotypes derived from clades B�C, C, and AE,GPI-CDR H3(PG16) inhibited infection better than did GPI-CDR H3(E51).

We next tested the neutralization activity of GPI-CDR H3against three replication-competent HIV-1 strains (Bru-3, Bru-Yu2, and AD8) as well as a SIVMne027 control. Figure 3B

shows means and standard deviations of RLA in parentalTZM-bl cells and GPI-CDR H3(AVF, b12, PG16, and E51)-transduced TZM-bl cells infected with these HIV-1 and SIVstrains. As expected, compared to mock-transduced parentalTZM-bl cells, cells transduced with GPI-CDR H3(AVF, b12,PG16, and E51) did not show any significant neutralizationactivity against the SIVMne027 control. Compared to mock-transduced parental TZM-bl cells, cells transduced with GPI-CDR H3(AVF and b12) did not show any significant neutral-ization activity against any of 3 HIV-1 strains. In contrast,similar to what was observed with HIV-1-pseudotyped virions,cells transduced with GPI-CDR H3(PG16 and E51) neutral-ized all 3 viruses.

Since antibodies PG9 and PG16 are somatic variants and a7-amino-acid region in CDR H3 is responsible for the finespecificity of these antibodies (24), we also constructed GPI-CDR H3(PG9) and transduced the construct into TZM-blcells. We then compared the cell surface expressions and anti-HIV-1 activities of GPI-CDR H3(PG9) and GPI-CDRH3(PG16). Figure 3C shows similar degrees of cell surfaceexpression of GPI-CDR H3(PG9) compared with those of theother GPI-CDR H3 constructs (PG16, AVF, and E51). Figure3D shows that GPI-CDR H3(PG9) exhibited the same degreeof inhibition against 5 representative HIV-1 pseudotypes asthat of GPI-CDR H3(PG16 and E51). Again, none of theseGPI-CDR H3 constructs exhibited any neutralization activityagainst the SIVMne027 control.

In addition, we synthesized three CDR H3 peptides (AVF,E51, and PG16) and constructed four new lentiviral vectorsexpressing the secretory form of CDR H3 [CDR H3(AVF),CDR H3(PG16), CDR H3(PG9), and CDR H3(E51)]. Wethen compared the anti-HIV-1 activities of TZM-bl cells ex-pressing GPI-CDR H3(AVF, PG9, PG16, and E51) andTZM-bl cells expressing the secretory form of CDR H3(AVF,PG9, PG16, and E51). We found that only cells expressingGPI-CDR H3(PG9, PG16, and E51) but not cells expressingthe secretory form of CDR H3(PG9, PG16, and E51) exhibitedsignificant inhibition against five representative HIV-1 pseu-dotypes tested (data not shown). Similarly, soluble AVF,PG16, and E51 CDR H3 peptides at concentrations of 0.1, 1,and 10 �M also did not have any inhibitory effect on HIV-1(data not shown). At this time, it is not clear whether the lackof an inhibitory effect of secreted CDR H3(PG9 and PG16) isdue to a limited amount of CDR H3 peptides present in theculture supernatants and whether the lack of an inhibitoryeffect of synthetic CDR H3 peptides (E51 and PG16) is due tothe lack of sulfation of these synthetic peptides.

Pejchal et al. showed previously that CDR H3 mutants(Y100HF, D100IA, and G7) of antibody PG16 reduced neu-tralization activity against JR-CSF, resulting in 2.2-, 200-, and�500-fold increases in the 50% inhibitory concentration (IC50)compared to that of wild-type antibody PG16 (24). Therefore,to test whether such CDR H3 mutants would also affect theneutralization activity of GPI-anchored CDR H3 of PG16, weconstructed transfer vectors expressing three GPI-CDR H3mutants (Y100HF, D100IA, and G7) of PG16 (Fig. 4A). Re-combinant lentiviruses generated from these transfer vectorswere used to transduce TZM-bl cells. Figure 4B shows that thelevel of cell surface expression of three GPI-CDR H3 mutantsis similar to that of GPI-CDR H3(PG16). Figure S2 in the


supplemental material shows that the expression of GPI-CDRH3 mutants did not alter the cell surface expressions of CD4,CCR5, and CXCR4. We then compared the susceptibilities ofTZM-bl cells transduced with GPI-CDR H3 mutants (Y100HF, D100IA, and G7) with those of TZM-bl cells transducedwith GPI-CDR H3(PG16) by infecting these cells with HIV-1pseudotypes generated with envelope proteins from Q168(clade A), Yu-2 (clade B), CNE11 (clade B�), CNE15(CRF07_B�C), and CNE55 (CRF01_AE) as well as replica-tion-competent HIV-1 Bru-3 and Bru-Yu2. A pseudotypedvirus expressing retroviral envelope 10A1 was also used as anegative control. Figure 4C shows the fold increase in RLA inTZM-bl cells transduced with these GPI-CDR H3 mutantscompared with GPI-CDR H3(PG16) after the infection. As

expected, no significant difference in susceptibility to the 10A1pseudotype control was found among TZM-bl cells transducedwith the wild type and mutants of GPI-CDR H3(PG16). Incontrast, TZM-bl cells transduced with GPI-CDR H3 mutants(G7, D100IA, and Y100HF) were significantly more suscepti-ble to all HIV-1 pseudotypes and replication-competent HIV-1than cells transduced with GPI-CDR H3(PG16). Among thethree mutants, TZM-bl cells transduced with GPI-CDR H3 mu-tant D100IA were more susceptible (i.e., higher fold increases ininfection) than cells transduced with GPI-CDR H3 mutant G7 orY100HF, and cells transduced with GPI-CDR H3 mutantY100HF was less susceptible (i.e., smaller fold increases) thancells transduced with GPI-CDR H3 mutant G7.

FIG. 3. Effect of GPI-CDR H3(PG16, PG9, b12, E51, and AVF) on infection of HIV-1 viruses and pseudotypes. (A) Effect of GPI-CDR H3son efficiency of transduction of HIV-1 and 10A1 pseudotypes into GPI-CDR H3-transduced TZM-bl cells. Shown are percentages of reductionof the relative luciferase activities in TZM-bl cells transduced with GPI-CDR H3(AVF, b12, PG16, and E51) compared with mock-transducedparental TZM-bl cells. Green, �50% inhibition; yellow, �90% inhibition; red, �99% inhibition. The percentage of inhibition was based on thefollowing calculation: (RLA in virus alone in a given transduced cell � RLA in no virus in the same transduced cell)/(RLA in virus alone in theparental cell � RLA in no virus in the parental cell). (B) Effect of GPI-CDR H3s on wild-type HIV-1 and SIVMne027 infection in GPI-CDRH3-transduced TZM-bl cells. w/o, parental TZM-bl cells. (C) FACS analysis of cell surface expression of CDR H3/hinge/His tag/DAF in CDRH3(PG16, PG9, E51, and AVF)/hinge/His tag/DAF-transduced TZM-bl cells. (D) Percentage of the reduction of relative luciferase activity inTZM-bl cells transduced with GPI-CDR H3(AVF, PG9, PG16, and E51) compared with mock-transduced parental TZM-bl cells. Yellow, �90%inhibition; red, �99% inhibition.

FIG. 4. Relative susceptibilities to 10A1 and HIV-1 pseudotypes and replication-competent HIV-1 of TZM-bl cells transduced with GPI-CDRH3(PG16) and with GPI-CDR H3 mutants (G7, D100HI, and Y100HF). (A) Amino acid sequence comparison of wild-type CDR H3(PG16) andits mutants. (B) FACS analysis of cell surface expression of TZM-bl cells transduced with GPI-CDR H3(PG16) and with GPI-CDR H3 mutants(G7, D100HI, and Y100HF). (C) Relative susceptibility to pseudotypes and replication-competent HIV-1 of TZM-bl cells transduced withGPI-CDR H3 mutants (G7, D100HI, and Y100HF) compared to cells transduced with GPI-CDR H3(PG16).


GPI-anchored CDR H3(PG16) renders human CD4� T cellsresistant to HIV-1 but not to transduction of VSV-G-pseu-dotyped lentivirus. Next, we evaluated whether GPI-CDRH3(PG16) would confer resistance to HIV-1 in human CD4�

T cells. Human CD4� CEMss-CCR5 cells (37) were trans-duced with GPI-CDR H3(AVF and PG16). The expression ofGPI-CDR H3(AVF and PG16) as well as the expressions ofCD4, CCR5, and CXCR4 in transduced CEMss-CCR5 cellswere tested by immunostaining followed by FACS analysis asdescribed above. In all cases, the levels of expression of GPI-CDR H3(AVF and PG16) as well as the levels of expression ofCD4, CCR5, and CXCR4 between mock-transduced parentalCEMss-CCR5 cells and GPI-CDR H3-transduced CEMss-CCR5 cells were similar (see Fig. S3 in the supplemental ma-terial). Transduced CEMss-CCR5 cells were then infectedwith HIV-1 strains Bru-3 and Bru-Yu2 at a multiplicity ofinfection of 0.01. As shown in Fig. 5A, the replication of HIV-1Bru-3 was significantly inhibited in cells transduced with GPI-CDR H3(PG16) compared to cells transduced with GPI-CDRH3(AVF). At 6, 9, and 12 days postinfection there was a 2-logreduction in the amounts of HIV-1 Gag p24 produced by cellstransduced with GPI-CDR H3(PG16) compared to theamounts produced by cells transduced with GPI-CDRH3(AVF). However, at 15 days postinfection, there was nosignificant difference in the amounts of HIV-1 Gag p24 pro-

duced by cells transduced with GPI-CDR H3(PG16) and cellstransduced with GPI-CDR H3(AVF). As shown in Fig. 5B,compared to that in cells transduced with GPI-CDRH3(AVF), the replication of HIV-1 Bru-Yu2 was also signifi-cantly inhibited in cells transduced with GPI-CDR H3(PG16)throughout the experiments. At 6, 9, and 12 days postinfectionthere was a 1- or 2-log reduction in the amounts of HIV-1 Gagp24 produced by cells transduced with GPI-CDR H3(PG16)compared to cells transduced with GPI-CDR H3(AVF). At 15days postinfection, there was still a 98% reduction in theamount of HIV-1 Gag p24 produced by cells transduced withGPI-CDR H3(PG16) compared to that produced by cellstransduced with GPI-CDR H3(AVF).

Finally, we transduced parental CEMss-CCR5 cells andCEMss-CCR5-GPI-CDR H3(AVF and PG16) cells with aVSV-G-pseudotyped HIV-1 vector expressing enhanced greenfluorescent protein (EGFP) as described previously (37). TheVSV-G envelope interacts with the lipid moiety in the lipidbilayer of the plasma membrane. Because of this, the VSV-G-pseudotyped lentiviral vector bypasses the requirement for theinteraction between the HIV-1 envelope and its receptor andcoreceptor to enter cells. We found that at all doses tested, thetransduction of parental CEMss-CCR5 cells and CEMss-CCR5-GPI-CDR H3(AVF and PG16) cells with a VSV-G-pseudotyped lentiviral vector results in similar vector dose-

FIG. 6. EGFP expression in parental CEMss-CCR5 cells and CEMss-CCR5 cells expressing GPI-CDR H3(PG16 and AVF) transduced witha VSV-G-pseudotyped HIV-1 vector. (A) Percent EGFP-positive cells; (B) MFI (mean fluorescence intensity).

FIG. 5. Effect of GPI-CDR H3(PG16) on anti-HIV-1 activity of transduced human CD4� T cells. (A) GPI-CDR H3(PG16) confers resistanceto HIV-1 Bru-3 in human CD4� T cells. (B) GPI-CDR H3(PG16) confers resistance to HIV-1 Bru-Yu2 in human CD4� T cells.


dependent transduction efficiencies and transgene expressions(Fig. 6). These results demonstrate that GPI-CDR H3(PG16)does not inhibit VSV-G envelope-mediated viral entry, reversetranscription, integration, or the postintegration protein ex-pression of the HIV-1 vector, indicating that the potent inhi-bition of HIV-1 replication seen for GPI-CDR H3(PG16)-transduced CEMss-CCR5 cells (Fig. 5) is HIV-1 envelopespecific and at the level of viral entry.

DISCUSSION

The crystal structure of quaternary-specific antibody PG16reveals a unique stable hammerhead structure of the CDR H3subdomain that rises above the antibody surface to confer finespecificity (23, 24). Thus, a crucial question is whether thisunique architecture of CDR H3 itself is sufficient to recognizethe quaternary epitope on the trimeric HIV-1 spike (gp1203/gp413) and exert neutralization. In this study, we constructedCDR H3s derived from five human monoclonal antibodies,PG16, PG9, b12, E51, and AVF, and genetically linked themwith a GPI attachment signal of DAF. We demonstrate thatfusion gene constructs are expressed and targeted to lipid raftsof plasma membranes through a GPI anchor (Fig. 1 and 2).GPI-anchored CDR H3s derived from PG16, PG9, and E51,but not GPI-anchored CDR H3s derived from AVF and b12,on the surface of transduced TZM-bl cells specifically neutral-ize multiple clades of HIV-1 isolates with a great degree ofpotency (Fig. 3). Moreover, GPI-anchored CDR H3(PG16),but not GPI-anchored CDR H3(AVF), on the surface of trans-duced human CD4� T cells specifically confers resistance toHIV-1 infection (Fig. 5). Finally, the CDR H3 mutants(Y100HF, D100IA, and G7) that were previously shown tocompromise the neutralization activity of antibody PG16 alsoabolish the neutralization activity of GPI-CDR H3(PG16)(Fig. 4). Thus, we conclude that the CDR H3 subdomain ofPG16 itself, when targeted to the lipid raft of the plasmamembrane of HIV-1-susceptible cells, exerts HIV-1 neutral-ization.

Changela et al. (3) recently determined the crystal structureof another quaternary-specific human antibody, 2909, whichhas limited neutralization breadth against HIV-1. To test therole of CDR H3 in neutralization, the CDR H3 sequencesbetween 2909 and PG16 were swapped, and the chimeras wereassayed for neutralization activity. Intriguingly, a chimera com-posed of antibody 2909 with PG16-derived CDR H3 does notneutralize any HIV-1 isolates tested, while a reciprocal chi-mera composed of antibody PG16 with the 2909-derived CDRH3 does not express neutralization activity. However, when theCDR H3 from each chimera was paired with its native lightchain, neutralization activity was recovered although at a muchreduced level and at a limited breadth (3). It was not clear whatrole the light chain plays. However, in light of the finding in ourpresent study that the CDR H3 of PG16 itself, when targetedto the lipid raft of the plasma membrane of HIV-1-susceptiblecells, exerts HIV-1 neutralization, it is unlikely that the lightchain of PG16 could play a functional role in antigen recogni-tion. Its structural role in maintaining the conformation ofCDR H3, if any, may be found only at the whole-antibodylevel.

Although in transduced TZM-bl cells GPI-CDR H3(PG16)

exhibited remarkable breadth and potency against diverseHIV-1 isolates tested (Fig. 3), in transduced CEMss-CCR5cells GPI-CDR H3(PG16) showed significant inhibition only inthe first 12 or 15 days postinfection (Fig. 5). This level ofneutralization is quite different from what we previously re-ported for CEMss-CCR5 cells transduced with GPI-scFv (X5)(37). The transduction of CEMss-CCR5 cells with GPI-scFv(X5) conferred long-term resistance to HIV-1 infection (37).Although the difference may be attributed to the differentforms, scFv versus CDR H3, it is more likely due to the dif-ferent epitopes that the two antibodies (PG16 and X5) direct.In fact, we compared the anti-HIV-1 activities of TZM-bl cellstransduced with GPI-CDR H3(PG16) and TZM-bl cells trans-duced with GPI-scFv(PG16) and found that cells transducedwith GPI-scFv(PG16) exhibited less inhibition against HIV-1pseudotypes than did cells transduced with GPI-CDRH3(PG16) (data not shown). The X5 epitope is a soluble CD4-inducible epitope, which resides within a conserved region(amino acid residues 417 to 434) of the gp120 core, particularlyin the vicinity of amino acid residues at positions 423 and 432,which is in proximity to the CD4 and coreceptor binding sites(20). It was previously shown that scFv (X5) neutralizes HIV-1better than Fab and whole IgG (15). The epitope recognizedby PG16, however, is a glycosylated quaternary epitope (6) ina region on gp120 that includes the V2 and V3 loops. It forms,appropriately, only in the trimeric HIV-1 spike (36). Becauseof this, antibody PG16 can simultaneously interact with twoepitopes present only in the oligomeric (gp1203/gp413) HIV-1spike. As a result, antibody PG16 may have a much betteravidity of binding to gp120 than GPI-CDR H3(PG16), whichinteracts with only one epitope at a time, even though the latteris present in the lipid raft domain of the plasma membrane.Therefore, a redesigning of fusion gene constructs that expressdimeric and trimeric forms of GPI-CDR H3(PG16) may resultin more potent and broad entry inhibitors of HIV-1.

GPI-CDR H3(PG16, PG9, and E51), with such a remark-able breadth of neutralization activity, should have potentialeither alone or in combination with other anti-HIV-1 geneconstructs, such as GPI-scFv (X5), to be developed into anti-viral agents for HIV-1 prevention and therapy. For example,GPI-CDR H3(PG16) and GPI-scFv (X5), due to their differentspecificities, could be codelivered into hematopoietic progen-itor cells of HIV-1 patients ex vivo through a lentiviral vector,and transduced cells could then be transfused into patients, asrecently described by DiGiusto et al. (5). However, in order toachieve clinical efficacy with this gene therapy approach, manydetails, such as efficient transduction and engraftment, main-tenance of self-renewal, hematopoietic linage cell differentia-tion of transduced hematopoietic progenitor cells, sustainabletransgene expression, and avoidance of potential insertion mu-tagenesis, have to be worked out.

Finally, it was previously shown that a fusion protein com-prised of a sulfated peptide derived from CDR H3 of antibodyE51 with the Fc domain of human IgG1 binds gp120 andinhibits HIV-1 infection (7). In the present study we show thatin transduced TZM-bl cells, GPI-CDR H3(E51), like GPI-CDR H3(PG16 and PG9), also specifically neutralizes diverseHIV-1 isolates with a remarkable potency. Thus, it appearsthat GPI-CDR H3 can be an alternative approach for deter-


mining whether CDR H3 of certain antibodies alone can exertepitope recognition and neutralization.

ACKNOWLEDGMENTS

We thank L. Naldini at the University Torino Medical School,Torino, Italy, for the lentiviral transfer vector and B. H. Hahn at theUniversity of Alabama for the DNA plasmid encoding the consensus CHIV-1 envelope protein. The cell line TZM-bl; HIV-1 molecularclones pMJ4, pBru-3, pBru-Yu2, and pAD8; as well as expressionvectors pADA, pAD8, pQ168ENVa2, and pNL4-3.luc.R�E� wereobtained through the AIDS Research and Reference Reagent Pro-gram, Division of AIDS, National Institute of Allergy and InfectiousDiseases, National Institutes of Health, Germantown, MD. These re-agents were originally developed and contributed by J. Kappes, X. Wu,N. Landau, R. Risser, J. Overbaugh, E. Freed, M. Essex, T. Ndung’u,A. Adachi, M. A. Martin, I. R. Chen, G. W. Shaw, and B. H. Hahn.

This work was supported by research grants from the Chinese Na-tional Science Foundation (grant no. 30740008), the Chinese Scienceand Technology Ministry 973 Program Project (grant no.2006CB504308), National Science and Technology Major Projects(grant no. 2008ZX10001-010, 2009ZX10004-105, and 2009ZX10004-016), the Shanghai Pasteur Foundation (grant no. SPHRF2007001),and French Energy Company Areva to P.Z. and by research grantsfrom the National Institutes of Health to J.T.K. (grant AI47725) andthe Baylor-UTHouston CFAR (grant P30AI036211).

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