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. 2013 Feb;87(3):1899–1905. doi: 10.1128/JVI.01038-12

Trimeric Glycosylphosphatidylinositol-Anchored HCDR3 of Broadly Neutralizing Antibody PG16 Is a Potent HIV-1 Entry Inhibitor

Lihong Liu a, Weiming Wang a, Lifei Yang a, Huanhuan Ren a, Jason T Kimata b, Paul Zhou a,✉
PMCID: PMC3554188  PMID: 23152526

Abstract

PG9 and PG16 are two quaternary-structure-specific broadly neutralizing antibodies with unique HCDR3 subdomains. Previously, we showed that glycosylphosphatidylinositol (GPI)-anchored HCDR3 subdomains (GPI-HCDR3) can be targeted to lipid rafts of the plasma membrane, bind to the epitope recognized by HCDR3 of PG16, and neutralize diverse HIV-1 isolates. In this study, we further developed trimeric GPI-HCDR3s and demonstrated that trimeric GPI-HCDR3 (PG16) dramatically improves anti-HIV-1 neutralization, suggesting that a stoichiometry of recognition of 3 or 2 HCDR3 molecules (PG16) to 1 viral spike is possible.

TEXT

PG9 and PG16 are two recently isolated quaternary-structure-specific human monoclonal antibodies that neutralize 70 to 80% of circulating HIV-1 isolates (1). The crystal structure of PG16 shows that it contains an exceptionally long HCDR3 that forms a unique stable subdomain that towers above the antibody surface to confer fine specificity (2, 3). Previously, we showed that monomeric glycosylphosphatidylinositol (GPI)-anchored HCDR3 (GPI-HCDR3) (PG9 and PG16) neutralizes HIV-1 (4). We postulate that since the antibodies preferentially bind to assembled viral spikes (1, 5), multimeric GPI-HCDR3 may have better binding avidity than monomeric GPI-HCDR3, resulting in a potent entry inhibitor.

To test this hypothesis, sequences encoding HCDR3 (PG16 and AVF), the IgG3 hinge region, foldon, and a histidine tag were genetically linked to the sequence encoding a GPI attachment signal of delay-accelerating factor (DAF) (6). The antibody AVF recognizes influenza virus hemagglutinin (7) and was therefore used as a negative control. Foldon is a 27-residue trimerization domain at the C-terminal bacteriophage T4 fibritin (8). Polypeptides that are fused to foldon form trimers (9–11). The fusion genes HCDR3/hinge/foldon/His tag/DAF (PG16 and AVF) were inserted into a lentiviral vector, pRRL (12) (Fig. 1A). The recombinant viruses were generated to transduce TZM-bl and CEMss-CCR5 cells (13, 14).

Fig 1.

Fig 1

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Expression of GPI-HCDR3 and GPI-HCDR3-foldon in transduced TZM-bl cells. (A) Schematic diagram of the lentiviral vectors pRRL-HCDR3/hinge/his-tag/DAF and pRRL-HCDR3/hinge/foldon/his-tag/DAF. HCDR3s were derived from the human monoclonal antibodies PG16 and AVF; hinge, a human IgG3 hinge region; foldon, a 27-residue β-propeller-like trimeric structure at the C terminus of bacteriophage T4 fibritin; His tag, 6-histidine-residue tag; DAF, the C-terminal 34 amino acid residues of decay-accelerating factor. (B) FACS analysis of cell surface expression of GPI-HCDR3 and GPI-HCDR3-foldon in mock-, GPI-HCDR3-, and GPI-HCDR3-foldon (PG16 and AVF)-transduced TZM-bl cells with or without PI-PLC treatment. (C) Confocal analysis of mock- or GPI-HCDR3-foldon (PG16 and AVF)-transduced TZM-bl cells. CtxB, cells were stained with Alexa 555-conjugated cholera toxin B subunit; anti-His, cells were stained with mouse anti-His-tag antibody followed by Alexa 488-conjugated goat anti-mouse IgG antibody.

To determine transgene expression, HCDR3/hinge/foldon/His tag/DAF (PG16 and AVF)-transduced TZM-bl cells and previously generated TZM-bl-GPI-HCDR3 (PG16 and AVF) (4) were treated with or without phosphatidylinositol phospholipase C (PI-PLC) and stained with anti-His-tag antibody, followed by fluorescence-activated cell sorting (FACS) analysis. Figure 1B shows that like GPI-HCDR3, HCDR3/hinge/foldon/His tag/DAFs were highly expressed, and their expression was substantially reduced with PI-PLC treatment, indicating that a majority of HCDR3/hinge/foldon/His tag/DAF is attached to the cell surface through a GPI anchor. Thus, we refer to the HCDR3/hinge/foldon/His tag/DAF as GPI-HCDR3-foldon.

To localize GPI-HCDR3-foldon, mock-, GPI-HCDR3-, and GPI-HCDR3-foldon (PG16)-transduced TZM-bl cells were seeded onto a glass slide (BD Biosciences) and costained with the following: (i) anti-His-tag antibody followed by Alexa 488-conjugated anti-mouse IgG antibody, (ii) Alexa 555-conjugated cholera toxin subunit B (CtxB), and (iii) 4′,6-diamidino-2-phenylindole (DAPI). CtxB interacts with GM1 (a lipid raft marker). Figure 1C shows that a majority of GPI-HCDR3-foldon, like GPI-HCDR3, colocalized with GM1 on the cell surface, implying that a majority of GPI-HCDR3 (as in Fig. 1B) is associated with lipid rafts, as expected for GPI anchoring.

We next assessed the levels of CD4, CXCR4, or CCR5 in transduced and control TZM-bl cells by flow cytometry. In all cases, the values were similar (Fig. 2A). To compare neutralization activities, a panel of 14 pseudotypes, including 13 human immunodeficiency virus type 1 (HIV-1) envelopes of subtypes A, B, B′, C, and A/E (15–19) and a 10A1 retroviral envelope (20), and a panel of HIV and simian immunodeficiency virus (SIV) were used to infect GPI-HCDR3- and GPI-HCDR3-foldon-transduced TZM-bl cells in a single-round infectivity assay (14, 21). Figure 2B shows the means and standard deviations of relative luciferase activity (RLA) in GPI-HCDR3 and GPI-HCDR3-foldon (PG16 and AVF)-transduced cells. Compared to cells transduced with GPI-HCDR3 and GPI-HCDR3-foldon (AVF), cells transduced with GPI-HCDR3 (PG16) neutralized 12 of 13 HIV-1 pseudotypes with various degrees of potency. In contrast, cells transduced with GPI-HCDR3-foldon (PG16) were completely resistant to the same 12 HIV-1 pseudotypes. Interestingly, GPI-HCDR3 (PG16) did not neutralize the HIV-1 SF162 pseudotype, whereas GPI-HCDR3-foldon (PG16) reduced infection by only 60%. Figure 2C and D show that although all transduced cells were equally infected with SIVmne027 (22), cells transduced with GPI-HCDR3-foldon (PG16) neutralized all three HIV-1 strains (23, 24) with significantly higher potency than cells transduced with GPI-HCDR3 (PG16). No neutralization was observed in cells transduced with the GPI-HCDR3 and GPI-HCDR3-foldon (AVF) controls.

Fig 2.

Fig 2

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Effects of GPI-HCDR3 and GPI-HCDR3-foldon (PG16 and AVF) on infection of HIV-1 viruses and pseudotypes. (A) Summary of mean and median fluorescent intensities of CD4, CCR5, and CXCR4 expression in mock-, GPI-HCDR3-, and GPI-HCDR3-foldon (PG16 and AVF)-transduced TZM.bl cells. (B) Effects of GPI-HCDR3 and GPI-HCDR3-foldon (PG16 and AVF) on efficiencies of transduction of HIV-1 and 10A1 pseudotypes into GPI-HCDR3- and GPI-HCDR3/foldon (PG16 and AVF)-transduced TZM-bl cells, as shown by the means and standard deviations of relative luciferase activity. Green, ≥50% inhibition; yellow, ≥90% inhibition; red, ≥99% inhibition. The percentage of inhibition was based on the following calculation: (RLA in virus alone to a given transduced cell − RLA in no virus to the same transduced cell)/(RLA in virus alone to parental cells − RLA in no virus to parental cell). (C) Effects of GPI-HCDR3 and GPI-HCDR3-foldon (PG16 and AVF) on wild-type HIV-1 and SIVMne027 infection in GPI-HCDR3 and GPI-HCDR3-foldon (PG16 and AVF)-transduced TZM-bl cells measured by RLA. white, parental TZM-bl cells. (D) Effect of GPI-HCDR3 and GPI-HCDR3-foldon (PG16 and AVF) on wild-type HIV-1 and SIVMne027 infection in GPI-HCDR3 and GPI-HCDR3-foldon (PG16 and AVF)-transduced TZM-bl cells measured by blue-colony staining. First column, parental TZM-bl cells. (E) FACS analysis of cell surface expression of GPI-HCDR3 and GPI-HCDR3-foldon in mock-, GPI-HCDR3-, and GPI-HCDR3-foldon (AVF, PG9, and PG16)-transduced TZM-bl cells. Dashed line, mock-transduced cells; solid line: GPI-HCDR3 (AVF, PG9, and PG16)-transduced cells; gray shadow, GPI-HCDR3-foldon (AVF, PG9, and PG16)-transduced cells. (F) Effect of GPI-HCDR3 and GPI-HCDR3-foldon (AVF, PG9, and PG16) on efficiency of transduction of HIV-1 and 10A1 pseudotypes into GPI-HCDR3 and GPI-HCDR3/foldon (AVF, PG9, and PG16)-transduced TZM-bl cells as shown by the means and standard deviations of relative luciferase activity. Yellow, ≥90% inhibition; red, ≥99% inhibition.

Pejchal et al. have shown that an HCDR3 mutant (G7) of antibody PG16 significantly reduced neutralization compared to that of the wild type antibody PG16 (3). Therefore, to further test the specificity of GPI-HCDR3-foldon (PG16), we constructed a lentiviral vector expressing the G7 mutant of GPI-HCDR3-foldon (PG16) and transduced TZM.bl cells with it. We found that although the expression levels of the G7 mutant and the wild type of GPI-HCDR3-foldon (PG16) on the cell surface are similar, cells transduced with the G7 mutant of GPI-HCDR3-foldon (PG16) were significantly more susceptible to HIV-1 Bru-Yu2 and AD8 than cells transduced with GPI-HCDR3-foldon (PG16) (data not shown).

Since the antibodies PG9 and PG16 are somatic variants (1), we also constructed a lentiviral vector expressing GPI-HCDR3-foldon (PG9) and transduced it into TZM-bl cells. We found that although the cell surface expression of GPI-HCDR3 and GPI-HCDR3-foldon (AVF, PG16, and PG9) is very similar (Fig. 2E), cells transduced with GPI-HCDR3 (PG9), like cells transduced with GPI-HCDR3 (PG16), neutralized 3 HIV-1 pseudotypes tested with various degrees of potency. In contrast, cells transduced with GPI-HCDR3-foldon (PG9), like cells transduced with GPI-HCDR3-foldon (PG16), were completely resistant to the same 3 HIV-1 pseudotypes tested (Fig. 2F).

We next evaluated the inhibitory activities of the GPI-HCDR3-foldon constructs in CEMss-CCR5 cells against replication-competent HIV-1. After transduction, the expression of CD4, CXCR4, or CCR5 (Fig. 3A) and that of GPI-HCDR3 or GPI-HCDR3-foldon (AVF and PG16) (Fig. 3B) on the surface of transduced CEMss-CCR5 cells were similar. Strikingly, inhibition of replication of both HIV-1 Bru-3 and Bru-Yu2 was more profound and prolonged in cells transduced with GPI-HCDR3-foldon (PG16) than in those transduced with GPI-HCDR3 (PG16) (Fig. 3C).

Fig 3.

Fig 3

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Effects of GPI-HCDR3 and GPI-HCDR3-foldon (PG16 and AVF) on anti-HIV-1 activities of transduced human CD4+ T cells. (A) Summary of mean and median fluorescent intensities of CD4, CCR5, and CXCR4 expression in mock-, GPI-HCDR3-, and GPI-HCDR3-foldon (PG16 and AVF)-transduced CEMss-CCR5 cells. (B) FACS analysis of cell surface expression of GPI-HCDR3 and GPI-HCDR3-foldon in mock-, GPI-HCDR3-, and GPI-HCDR3-foldon (PG16 and AVF)-transduced CEMss-CCR5 cells. (C) GPI-HCDR3 and GPI-HCDR3-foldon (PG-16) confer resistance to HIV-1 Bru-3 and Bru-Yu in human CD4+ T cells as measured by HIV-1 Gag p24 in supernatants.

We next constructed a GPI-HCDR3-foldon mutant (E5R/W20L) of PG16 (Fig. 4A). Structural study has predicted that the E5R/W20L mutations may adversely affect foldon trimerization (8, 25). Figure 4B shows similar expression of GPI-HCDR3, GPI-HCDR3-foldon, and the GPI-HCDR3-foldon mutant on the surface of cells.

Fig 4.

Fig 4

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Trimerization of GPI-HCDR3-foldon and GPI-HCDR3-foldon mutant (E5R/W20L) and their relative susceptibilities to HIV-1 pseudotypes and replication-competent HIV-1. (A) Amino acid sequence comparison between foldon and its E5R/W20L mutant. (B) FACS analysis of cell surface expression of TZM-bl cells transduced with GPI-HCDR3, with GPI-HCDR3-foldon, and with GPI-HCDR-foldon mutant. (C) Western blot analysis of monomeric versus trimeric forms of GPI-HCDR3, GPI-HCDR3-foldon, and GPI-HCDR-foldon mutant detected by anti-His-tag antibody. Antitubulin antibody was used as a control to show equal sample loading. (D) Relative susceptibilities to pseudotypes and replication-competent HIV-1 in mock-, GPI-HCDR3 (PG16)-, GPI-HCDR3-foldon (PG16)- and GPI-HCDR-foldon mutant (E5R/W20L) (PG16)-transduced TZM-bl cells. Here, the amounts of RLA measured in cells transduced with GPI-HCDR3-foldon (PG16) infected with various HIV-1 pseudotypes and wild-type HIV-1 strains are considered to be 1. The amounts of RLA measured in cells transduced with GPI-HCDR3 or GPI-HCDR3-foldon mutant (E5R/W20L) infected with various HIV-1 pseudotypes and wild-type HIV-1 strains were compared with the amounts of RLA measured in cells transduced with GPI-HCDR3-foldon (PG16) infected with the same pseudotypes and wild-type HIV-1 strains and are presented as a fold increase (i.e., higher number, more susceptible). (E) Western blot analysis of secreted trimeric HCDR3-foldon (AVF and PG16) with or without boiling treatment detected by anti-His-tag antibody. (F) Effects of secreted trimeric HCDR3-foldon (PG16 and AVF) on infection with various HIV-1 pseudotypes in secreted HCDR3-foldon (PG16 and AVF)-transduced TZM-bl cells, measured by RLA.

To determine whether GPI-HCDR3-foldon and the GPI-HCDR3-foldon mutant form trimers, mock-, GPI-HCDR3-, GPI-HCDR3-foldon-, and GPI-HCDR3-foldon mutant (E5R/W20L)-transduced TZM-bl cells were solubilized with lysis buffer (10 mM Tris-HCI [pH 7.4], 150 mM NaCl, 1 mM EDTA, 1% [vol/vol] NP-40, and 1% [vol/vol] Triton X-114) at 4°C for 1 h. Samples were either treated with loading dye containing 2-mercaptoethanol (2-ME) and heated at 95°C for 5 min or treated with loading dye without 2-ME and without heat treatment. The samples were then fractionated in 15% Next Gel (Ameresco, Solon, OH), and transferred membranes were then stained with anti-His tag and antitubulin antibodies followed by horseradish peroxidase (HRP)-conjugated anti-mouse IgG Fc. Figure 4C shows that with or without 2-ME and heat treatment, GPI-HCDR3 is a monomer. In contrast, without 2-ME and heat treatment, almost all GPI-HCDR3-foldon is a trimer, whereas with 2-ME and heat treatment, all GPI-HCDR3-foldon becomes a monomer. Interestingly, without 2-ME and heat treatment, about 50% GPI-HCDR3-foldon mutants are trimers and the remaining are monomers, whereas with 2-ME and heat treatment, all GPI-HCDR3-foldon mutants become monomers.

To test whether the GPI-HCDR3-foldon mutant (E5R/W20L) compromises its anti-HIV-1 potency, we compared the susceptibilities of cells transduced with GPI-HCDR3-foldon mutant (E5R/W20L) (PG16) and of cells transduced with GPI-HCDR3 (PG16) and GPI-HCDR3-foldon (PG16). Figure 4D shows that cells transduced with the GPI-HCDR3-foldon mutant (E5R/W20L) (PG16) were significantly more susceptible to HIV-1 pseudotypes and HIV-1 than cells transduced with GPI-HCDR3-foldon (PG16).

Furthermore, we constructed lentiviral vectors expressing a secreted form of HCDR3-foldon (AVF and PG16) and transduced them into TZM.bl cells. We found that although cells transduced with a secreted form of HCDR3-foldon (AVF and PG16) released HCDR3-foldon trimers into culture supernatants (Fig. 4E), soluble HCDR3-foldon trimers (PG16) did not neutralize 6 HIV-1 pseudotypes tested (Fig. 4F).

Recently, the structure of V1/V2 scaffolds in the complex with antibody PG9 was elucidated by cocrystallization (26). The V1/V2 structure in the context of scaffold and PG9 folds as a single topological entity consisting of four antiparallel β-strands. This four-stranded V1/V2 sheet has two faces: concave and convex. The concave face is glycan free and hydrophobic, while the convex face is cationic, with the conserved strands forming stripes on the V1/V2 surface and the N-linked glycan 160 situated at its center. Interestingly, the HCDR3 of PG9 makes several contacts with the convex face of the V1/V2 sheet. First, the HCDR3 grasps the protein-proximal N-acetylglucosamine of 160 glycan, with Asp 100 and Arg 100B making four hydrogen bonds and with Asn 100P and His 100R at the base of the HCDR3 making additional hydrogen bonds to terminal mannose residues. Second, the HCDR3 binds 156 or 173 glycan (dependent upon the HIV-1 strain), with Tyr 100K making a hydrogen bond. And third, the HCDR3 forms intermolecular parallel β-sheet-like hydrogen bonds to strand C of V1/V2. Specific electrostatic interaction is also formed between cationic residues of strand C and acidic residues on PG9. Several of these occur with sulfated tyrosines on HCDR3 (26). Furthermore, both antibodies PG9 and PG16 are shown to preferentially recognize the viral spike. For PG9, the binding affinity with the viral spike is at least 10-fold higher than that with monomeric gp120, whereas for PG16, the difference is at least 100-fold higher (26). Thus, our present finding that trimeric GPI-HCDR3 (PG16) neutralizes more potently than monomeric GPI-HCDR3 not only supports the quaternary preference of PG9 and PG16 but also suggests that trimeric GPI-HCDR3 may indeed simultaneously grasp two or three epitopes present in the viral spike. This may also explain why even though monomeric GPI-HCDR3 (PG16) does not inhibit an HIV-1 SF162 pseudotype that lacks the N-linked glycan 160, trimeric GPI-HCDR3-foldon (PG16) still exhibits 60% neutralization (Fig. 2B).

Finally, trimeric GPI-HCDR3 (PG16) with such remarkable neutralization breadth and potency should have the potential to be developed into anti-HIV-1 agents either alone or in combination with other anti-HIV-1 gene constructs. For example, trimeric GPI-HCDR3 (PG16) and GPI-scFv (X5) (14), due to their different specificities, could be codelivered into hematopoietic progenitor cells of HIV-1 patients ex vivo using a lentiviral vector, and the modified cells could then be transfused into the patients, as recently described by DiGiusto et al. (27). Future studies could test the therapeutic potential of the GPI-HCDR3-foldon (PG16) and GPI-scFv (X5) in a relevant animal model, such as the simian-human immunodeficiency virus (SHIV)-macaque model.

ACKNOWLEDGMENTS

We thank L. Naldini at the University Torino Medical School, Torino, Italy, for a lentiviral transfer vector, B. H. Hahn at the University of Alabama for a DNA plasmid encoding the consensus C HIV-1 envelope protein, and Lu Lu and Wei Xu at Fudan University for technical assistance. Cell line TZM-bl and the HIV-1 molecular clones pBru-3, pBru-Yu2, and pAD8, as well as the expression vectors pADA, pAD8, pQ168ENVa2, and pNL4-3.luc.R-E-, were obtained through the AIDS Research and Reference Reagent Program, Division of AIDS, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Germantown, MD. These reagents were originally developed and contributed by J. Kappes, X. Wu, N. Landau, R. Risser, J. Overbaugh, E. Freed, 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 National Science Foundation (31170871 and 81161160569), the National Science and Technology Major Project (2012ZX10001-008), the Shanghai Pasteur Foundation (SPHRF2007001), and French Energy Company Areva to P.Z. and by research grants from the National Institutes of Health to J.T.K. (AI47725) and Baylor-UT Houston CFAR (P30AI036211).

Footnotes

Published ahead of print 14 November 2012

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