Short CDRL1 in intermediate VRC01-like mAbs is not sufficient to overcome key glycan barriers on HIV-1 Env

Abstract

VRC01-class broadly neutralizing antibodies (bnAbs) have been isolated from people with HIV-1, but they have not yet been elicited by vaccination. They are extensively somatically mutated and sometimes accumulate CDRL1 deletions. Such indels may allow VRC01-class antibodies to accommodate the glycans expressed on a conserved N276 N-linked glycosylation site in loop D of the gp120 subunit. These glycans constitute a major obstacle in the development of VRC01-class antibodies, as unmutated antibody forms are unable to accommodate them. Although immunizations of knock-in mice expressing human VRC01-class B-cell receptors (BCRs) with specifically designed Env-derived immunogens lead to the accumulation of somatic mutations in VRC01-class BCRs, CDRL1 deletions are rarely observed, and the elicited antibodies display narrow neutralizing activities. The lack of broad neutralizing potential could be due to the absence of deletions, the lack of appropriate somatic mutations, or both. To address this point, we modified our previously determined prime-boost immunization with a germline-targeting immunogen nanoparticle (426c.Mod.Core), followed by a heterologous core nanoparticle (HxB2.WT.Core), by adding a final boost with a cocktail of various stabilized soluble Env trimers. We isolated VRC01-like antibodies with extensive somatic mutations and, in one case, a seven-amino acid CDRL1 deletion. We generated chimeric antibodies that combine the vaccine-elicited somatic mutations with CDRL1 deletions present in human mature VRC01 bnAbs. We observed that CDRL1 indels did not improve the neutralizing antibody activities. Our study indicates that CDRL1 length by itself is not sufficient for the broadly neutralizing phenotype of this class of antibodies.

Importance: HIV-1 broadly neutralizing antibodies will be a key component of an effective HIV-1 vaccine, as they prevent viral acquisition. Over the past decade, numerous broadly neutralizing antibodies (bnAbs) have been isolated from people with HIV. Despite an in-depth knowledge of their structures, epitopes, ontogenies, and, in a few rare cases, their maturation pathways during infection, bnAbs have, so far, not been elicited by vaccination. This necessitates the identification of key obstacles that prevent their elicitation by immunization and overcoming them. Here we examined whether CDRL1 shortening is a prerequisite for the broadly neutralizing potential of VRC01-class bnAbs, which bind within the CD4 receptor binding site of Env. Our findings indicate that CDRL1 shortening by itself is important but not sufficient for the acquisition of neutralization breadth, and suggest that particular combinations of amino acid mutations, not elicited so far by vaccination, are most likely required for the development of such a feature.

Keywords: BCR sequencing; CDRL1; HIV-1; VRC01-class antibodies; neutralization.

Fig 1

Antibody responses 2 weeks following the final boost immunization. (A) Mice (n = 5) were primed with 426c.Mod.Core ferritin nanoparticles with GLA-LSQ at week 0, followed by adjuvanted HxB2.WT.Core ferritin nanoparticle boost at week 10, and adjuvanted SOSIP cocktail as the final boost at week 18. Mice were bled at the indicated time points (red circles) and sacked for blood, spleen, and lymph node tissues, at week 20 (green circle). (B) Plasma was assayed by ELISA for binding against 426.Mod.Core (blue solid line), eOD-GT8 (green solid line), HxB2.WT.Core (red solid line), as well as their corresponding antigens with CD4-BS or VRC01 epitope knock-out (KO) (dotted lines); mean endpoint titers against the indicated proteins with SEM values are shown over time. Black dotted lines indicate the time of the booster immunizations. (C) CD4-BS-specific EC50 values against the indicated proteins are shown post each boost where open bars represent response post Boost 1 and filled bars, post Boost 2. See also Table S1 and Fig. S1.

Fig 2

Heavy-chain/light-chain sequence analysis after the final boost immunization at week 20. Pie charts indicate HC (A, D) and LC (B, C, E) characteristics from individually sorted B cells from pooled mouse samples collected 2 weeks post final immunization. The number of HC and LC sequences analyzed is shown in the middle of each pie chart. (A) VH-gene usage, (B) aa length of the CDRL3 domains in the LC, and (C) LC-gene usage, where shades of gray/black slices represent non 5-aa-long CDRL3s. (D–F) Analysis of paired sequences: HCs with the H35N mutation are shown in (D), presence or absence of Glu₉₆LC within the LC sequences with 5-aa-long CDRL3 domains are shown in (E), and (F) number of amino acid changes in the HC and LC of paired sequences at week 20, where each circle represents a paired sequence (see also Table S2).

Fig 3

Binding properties of VRC01-like mAbs generated after the final boost immunization. (A) Fifteen VRC01-like mAbs were evaluated against the indicated soluble monomeric Envs. 8-7 mAb with fastest off rate for 426c.Mod.Core and eOD-GT8 is shown in black. (B) Binding of 15 mAbs against the indicated autologous and heterologous WT.Cores, where the mAbs that displayed cross-reactivity are shown in different colors. (C) Binding of 15 mAbs against the indicated variants of 426c.SOSIP are shown. mVRC01 (solid red line) and glVRC01 (dotted red line) were included as internal controls in all assays. Black dotted lines indicate end of association and dissociation phases (see also Tables S3 and S4).

Fig 4

Neutralizing activities of VRC01-like mAbs. The neutralizing potential of mAbs against the indicated viruses either grown in 293T or 293S/GnTI− cells is shown in (A) and (B). Values represent IC50 neutralization in µg/mL. Bold/shaded values indicate samples displaying neutralizing activity. Neutralization IC50 values of these viruses with the mature VRC01 and germline VRC01 mAb are included for reference.

Fig 5

Binding and neutralizing properties of 8–24 chimeric mAb (8–24 LCC). (A) Sequence alignment of germline κ8–30*01 LC sequence, mAb 8–24, 8–27, and mVRC01 is shown. Red box/shaded region highlights the CDRL1 region; CDRL2 and CDRL3 residues on germline κ8–30*01 LC sequence are highlighted in red. (B) Binding of 8–24 LCC, 8–24, 8–27, mature, and germline VRC01 Abs against the indicated SOSIPs by BLI is shown. Black dotted lines indicate the end of association and dissociation phases. Neutralizing activities of 8–24 LCC against the indicated N276Q viruses either grown in 293T or 293S/GnTI- cells (C) or fully glycosylated WT viruses grown in 293T cells (D) is shown. IC50 neutralization values are provided in µg/mL, and the bold/shaded region indicates samples displaying neutralizing activity. Neutralization IC50 values of the same viruses with 824 mAb, mature VRC01, and germline VRC01 mAb are included for reference.

Fig 6

Cryo-EM structure of mAb 8–24 bound to 426c.WITO.TM.SOSIP. (A) Cryo-EM map of 426c.WITO.TM.SOSIP with 8–24 Fab shown in two orientations. SOSIP protomers are colored in shades of gray, HC of mAb 8–24 is shown in teal, and LC in light green. (B) Alignment of mAb 8–24 and glVRC01 (from PDB ID 6MYY) on a protomer of 426c.WITO.TM.SOSIP shows nearly identical angle of approach and epitope. (C) View of mAb 8–24 LC highlighting matured residues potentially involved in binding (dark blue) and matured residues in the framework regions (red). The CDRL1 comes in close proximity to the gp120 α-helix 2 (shown in blue). N276 is shown in purple. (D) View of mAb 8–24 HC highlighting matured residues potentially involved in binding (dark blue) and matured residues in the framework regions (red). gp120 α-helix 2 is shown in blue. N276 is shown in purple. (E) Sequence alignments of mAb 8–24 HC with human VH1-2*02 KI sequence, and mAb 8–24 LC with glVRC01 LC and mouse 8–30*01 LC. For mAb 8–24, affinity matured residues are colored to reflect if they are potentially involved in binding (dark blue) or not involved (red). Residues of glVRC01 that are involved in binding to 426c.WITO.TM.SOSIP are indicated by stars. Residues not resolved in the CDRL1 of mAb 8–24 are underlined. (F) mAb 8–24 bound to 426c.WITO.TM.SOSIP model and cryo-EM map show a narrow channel that could accommodate short glycans at the N276 position.