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

Abstract

A goal for an HIV-1 vaccine is to overcome virus variability by inducing broadly neutralizing antibodies (bnAbs). One key target of bnAbs is the glycan-polypeptide at the base of the envelope (Env) third variable loop (V3). We have designed and synthesized a homogeneous minimal immunogen with high-mannose glycans reflective of a native Env V3-glycan bnAb epitope (Man₉-V3). V3-glycan bnAbs bound to Man₉-V3 glycopeptide and native-like gp140 trimers with similar affinities. Fluorophore-labeled Man₉-V3 glycopeptides bound to bnAb memory B cells and were able to be used to isolate a V3-glycan bnAb from an HIV-1-infected individual. In rhesus macaques, immunization with Man₉-V3 induced V3-glycan-targeted antibodies. Thus, the Man₉-V3 glycopeptide closely mimics an HIV-1 V3-glycan bnAb epitope and can be used to isolate V3-glycan bnAbs.

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 K_d measurements for the binding of PGT128 (B) and PGT125 (C) to Man₉-V3 glycopeptide. Kinetics rates (k_a1 and k_d1) 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 K_d 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.