Research ArticleHIV

Mimicry of an HIV broadly neutralizing antibody epitope with a synthetic glycopeptide

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Science Translational Medicine  15 Mar 2017:
Vol. 9, Issue 381, eaai7521
DOI: 10.1126/scitranslmed.aai7521
  • Fig. 1. Design of gp120 V3 domain broadly neutralizing epitope mimics.

    Structure of the chemically synthesized Man9-V3-biotin glycopeptide and of aglycone V3-biotin. See procedures for synthesis in Supplementary Text and data set S1.

  • Fig. 2. Man9-V3 glycopeptide binding to V3-glycan bnAbs PGT128 and PGT125.

    (A) ELISA binding analysis to calculate EC50 of PGT128 and PGT125 binding to Man9-V3 glycopeptide. OD, optical density. (B and C) BLI binding analyses for Kd measurements for the binding of PGT128 (B) and PGT125 (C) to Man9-V3 glycopeptide. Kinetics rates (ka1 and kd1) of the faster components (figs. S7 and S8) were derived from global curve fitting analysis to a bivalent avidity model and used to derive the apparent Kd values. Binding analysis for affinity measurements was carried out by BLI, as described in Materials and Methods. Data are representative of three and two independent experiments, respectively, for PGT128 and PGT125.

  • Fig. 3. Isolation and characterization of N332-dependent antibodies isolated using native-like SOSIP trimers and synthetic Man9-V3 glycopeptide.

    (A) ELISA binding analyses of plasma from the HIV-infected donor CH765 to Man9-V3 and aglycone V3 peptides. Donor plasma was screened in duplicate assays, and binding is represented as mean values. (B) Memory B cells from donor CH765 were decorated with either fluorophore-conjugated BG505.T332N.SOSIP and DU156.12.SOSIP in phycoerythrin (PE) and allophycocyanin (APC), or Man9-V3 tetramers tagged to SA–AF647 (Alexa Fluor 647) and SA–BV421 (Brilliant Violet 421). The B cells from which CH765-VRC41.01, VRC41.02, and DH563 were cloned are indicated as green, red, and blue dots, respectively. (C) Immunogenetics and phylogeny of the DH563-VRC41 clonal lineage were inferred using Clonanalyst (33, 34). See also fig. S3. nt, nucleotide. (D) Reactivity of CH765-VRC41.01, CH765-VRC41.02, and DH563 to Man5, Man6, Man7-D1, Man7-D3, Man8-D1D3, and Man9 glycans, depicted on the right, printed on an array and detected via immunofluorescence. See also figs. S5 and S6. (E) Affinity measurements of newly isolated V3-glycan mAbs to Man9-V3 and BG505.T332N.SOSIP trimer were determined by BLI. Binding curves and data analysis are shown in figs. S7 and S8. (F) CH765-VRC41.01, VRC41.02, and DH563 were tested for neutralization breadth against a diverse panel of Env pseudoviruses using the TZM-bl assay. See also table S2 and fig. S9.

  • Fig. 4. Immunogenicity of Man9-V3 glycopeptide in rhesus macaques.

    (A) Study design to assess immunogenicity of Man9-V3 in rhesus macaques. Monomeric Man9-V3 was formulated in GLA-SE adjuvant and injected intramuscularly in sequentially increasing doses, as indicated. (B) Plasma from immunized macaques was tested for binding to biotinylated Man9-V3 and aglycone V3 in ELISA, as described in Materials and Methods. (C) Man9-V3 reactive antibodies were isolated via antigen-specific memory B cell sorts and tested for binding to recombinant HIV-1 Envs in ELISA. See also fig. S11 and table S3. AUC, area under the curve.

Supplementary Materials

  • www.sciencetranslationalmedicine.org/cgi/content/full/9/381/eaai7521/DC1

    Supplementary Text.

    Fig. S1. Man9-V3 glycopeptide binding to glycan-dependent V3 bnAbs.

    Fig. S2. PGT128-like specificity in HIV-1–chronically infected donor CH765.

    Fig. S3. Sort gates to isolate V3-glycan bnAbs from HIV-1–infected donor CH765.

    Fig. S4. Epitope mapping of newly isolated V3-glycan mAbs.

    Fig. S5. Binding of DH563-VRC41 V3-glycan bnAb family to Man9-V3 and aglycone V3.

    Fig. S6. Autoreactivity of newly isolated V3-glycan mAbs.

    Fig. S7. Affinity measurement of V3 bnAb binding to Man9-V3 glycopeptide.

    Fig. S8. Affinity measurements of isolated V3-glycan bnAbs to BG505 SOSIP trimers.

    Fig. S9. Neutralization of V3-glycan bnAbs against a diverse panel of viruses.

    Fig. S10. Flow cytometry sort gates to isolate DH706-DH710 from Man9-V3 immunized macaques.

    Fig. S11. Binding of vaccine-induced antibodies isolated from Man9-V3 immunized rhesus macaques to HIV-1 antigens.

    Table S1. Immunogenetics of DH563-VRC41 V3-glycan bnAb family.

    Table S2. Neutralization of N332-dependent mAbs isolated with Man9-V3 glycopeptide and native-like SOSIP trimers.

    Table S3. Immunogenetics of vaccine-induced antibodies isolated from Man9-V3 immunized rhesus macaques.

    Data set S1. Mass spectrophotometry spectra for Man9-V3 and synthetic intermediates.

    Data set S2. IC50 and IC80 neutralization values for V3-glycan bnAbs against a diverse panel of HIV-1 Env pseudoviruses.

    Data set S3. Primers used to amplify DH563 Ig genes.

    References (40, 41)

  • Supplementary Material for:

    Mimicry of an HIV broadly neutralizing antibody epitope with a synthetic glycopeptide

    S. Munir Alam,* Baptiste Aussedat, Yusuf Vohra, R. Ryan Meyerhoff, Evan M. Cale, William E. Walkowicz, Nathan A. Radakovich, Kara Anasti, Lawrence Armand, Robert Parks, Laura Sutherland, Richard Scearce, M. Gordon Joyce, Marie Pancera, Aliaksandr Druz, Ivelin S. Georgiev, Tarra Von Holle, Amanda Eaton, Christopher Fox, Steven G. Reed, Mark Louder, Robert T. Bailer, Lynn Morris, Salim S. Abdool-Karim, Myron Cohen, Hua-Xin Liao, David C. Montefiori, Peter K. Park, Alberto Fernández-Tejada, Kevin Wiehe, Sampa Santra, Thomas B. Kepler, Kevin O. Saunders, Joseph Sodroski, Peter D. Kwong, John R. Mascola, Mattia Bonsignori, M. Anthony Moody, Samuel Danishefsky, Barton F. Haynes*

    *Corresponding author. Email: barton.haynes{at}dm.duke.edu (B.F.H.); munir.alam{at}dm.duke.edu (S.M.A.)

    Published 15 March 2017, Sci. Transl. Med. 9, eaai752 (2017)
    DOI: 10.1126/scitranslmed.aai7521

    This PDF file includes:

    • Supplementary Text
    • Fig. S1. Man9-V3 glycopeptide binding to glycan-dependent V3 bnAbs.
    • Fig. S2. PGT128-like specificity in HIV-1–chronically infected donor CH765.
    • Fig. S3. Sort gates to isolate V3-glycan bnAbs from HIV-1–infected donor CH765.
    • Fig. S4. Epitope mapping of newly isolated V3-glycan mAbs.
    • Fig. S5. Binding of DH563-VRC41 V3-glycan bnAb family to Man9-V3 and aglycone V3.
    • Fig. S6. Autoreactivity of newly isolated V3-glycan mAbs.
    • Fig. S7. Affinity measurement of V3 bnAb binding to Man9-V3 glycopeptide.
    • Fig. S8. Affinity measurements of isolated V3-glycan bnAbs to BG505 SOSIP trimers.
    • Fig. S9. Neutralization of V3-glycan bnAbs against a diverse panel of viruses.
    • Fig. S10. Flow cytometry sort gates to isolate DH706-DH710 from Man9-V3 immunized macaques.
    • Fig. S11. Binding of vaccine-induced antibodies isolated from Man9-V3 immunized rhesus macaques to HIV-1 antigens.
    • Table S1. Immunogenetics of DH563-VRC41 V3-glycan bnAb family.
    • Table S2. Neutralization of N332-dependent mAbs isolated with Man9-V3 glycopeptide and native-like SOSIP trimers.
    • Table S3. Immunogenetics of vaccine-induced antibodies isolated from Man9-V3 immunized rhesus macaques.
    • Legends for data sets S1 to S3
    • Data set S1. Mass spectrophotometry spectra for Man9-V3 and synthetic intermediates.
    • Data set S2. IC50 and IC80 neutralization values for V3-glycan bnAbs against a diverse panel of HIV-1 Env pseudoviruses.
    • Data set S3. Primers used to amplify DH563 Ig genes.
    • References (40, 41)

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