Rational HIV immunogen design to target specific germline B cell receptors

Abstract

Vaccine development to induce broadly neutralizing antibodies (bNAbs) against HIV-1 is a global health priority. Potent VRC01-class bNAbs against the CD4 binding site of HIV gp120 have been isolated from HIV-1-infected individuals; however, such bNAbs have not been induced by vaccination. Wild-type gp120 proteins lack detectable affinity for predicted germline precursors of VRC01-class bNAbs, making them poor immunogens to prime a VRC01-class response. We employed computation-guided, in vitro screening to engineer a germline-targeting gp120 outer domain immunogen that binds to multiple VRC01-class bNAbs and germline precursors, and elucidated germline binding crystallographically. When multimerized on nanoparticles, this immunogen (eOD-GT6) activates germline and mature VRC01-class B cells. Thus, eOD-GT6 nanoparticles have promise as a vaccine prime. In principle, germline-targeting strategies could be applied to other epitopes and pathogens.

Fig. 1

Development of a germline (GL)-targeted HIV immunogen. (A) VRC01-class bNAbs bind to gp120 primarily through paratope residues encoded by VH 1-2*02. Residues on gp120 that interact with CD4 are colored yellow on an Env trimer model and on the surface of core gp120 (green) in complex with VRC01 (PDBID: 3GNB). The region encoded by VH 1-2*02 on VRC01 is colored red and shown as secondary structure rendering. Glycans are shown as blue spheres, except for the critical N276 glycan that is shown in magenta. (B) Engineering of a modified gp120-based nanoparticle capable of activating GL VRC01-class B-cells. (C) This nanoparticle can be used in an HIV-1 vaccine GL-prime-boost strategy to bridge this initial recognition gap and initiate clonal expansion and start somatic hypermutation of VRC01-class bNAbs precursors.

Fig. 2. Structural analysis of GL-VRC01 and eOD-GT6

(A) Comparison between the crystal structure of an unliganded predicted GL-VRC01 antibody (heavy and light chains colored blue and yellow, respectively) and VRC01 bound to gp120 (gray). Variable regions of GL-VRC01 are rendered as a surface. Areas of GL-VRC01 and VRC01 that contact gp120, shown as secondary structure cartoons, are similar in conformation. (B) Comparison between the crystal structures of unliganded eOD-GT6 (green) and unliganded gp120 core from HIV-1 strain 93TH057 (PDBID: 3TGT, gray). Structures are rendered according to B-values, with thin and thick lines representing areas possessing low and high flexibility, respectively. (C) Comparison between the crystal structures of GL-VRC01+eOD-GT6 and VRC01+gp120 core. Components are colored as in (A) and (B) and shown as tubes. The positions of the recovered mutations in eOD-GT6 that enable binding of GL-VRC-class Abs are shown as space-fill magenta spheres. The angle of approach of GL-VRC01 and VRC01 to the CD4bs is nearly identical. Key areas where interactions are different between VRC01 on gp120 (upper panels) and GL-VRC01 on eOD-GT6 (bottom panels) are shown in insets. eOD-GT6 confers germline reactivity by removing a potential clash with the N276 glycan, as well as by creating additional contacts with loop D (inset I), the OD exit loop (inset II) and V5 (fig. S12). (D) gp120 residues involved in the VRC01+gp120 and GL-VRC01+eOD-GT6 interfaces are compared in sequence, H-bond (stars), surface buried area and RMSD. Interfaces were calculated using PDBePISA (29) and Cα rmsd using Chimera (30).

Fig. 3

A 60-mer eOD-GT6 nanoparticle activates GL and mature VRC01-class B cells. (A) Model representation of the 60-mer eOD-GT6 nanoparticle. eOD-GT6 is colored in green, with residues that interact with CD4 colored yellow. Glycans are shown as blue spheres and the self-assembling 60mer Lumazine Synthase to which eOD-GT6 is fused is colored red. (B) Raw negative stain electron microscopy images of the 60-mer eOD-GT6 nanoparticle. (C) Calcium flux experiments with various gp120 constructs show that the 60-mer eOD-GT6 nanoparticle activates B cell lines engineered to express either GL or mature VRC01 IgM, 12A12 IgM or NIH45-46 IgG, while a recombinant soluble gp140 trimer activates the B cells expressing mature but not GL VRC01-class Abs. Data for each antibody are representative of at least two separate experiments.