Engineering immunogens that select for specific mutations in HIV broadly neutralizing antibodies

Abstract

Vaccine development targeting rapidly evolving pathogens such as HIV-1 requires induction of broadly neutralizing antibodies (bnAbs) with conserved paratopes and mutations, and in some cases, the same Ig-heavy chains. The current trial-and-error search for immunogen modifications that improve selection for specific bnAb mutations is imprecise. Here, to precisely engineer bnAb boosting immunogens, we use molecular dynamics simulations to examine encounter states that form when antibodies collide with the HIV-1 Envelope (Env). By mapping how bnAbs use encounter states to find their bound states, we identify Env mutations predicted to select for specific antibody mutations in two HIV-1 bnAb B cell lineages. The Env mutations encode antibody affinity gains and select for desired antibody mutations in vivo. These results demonstrate proof-of-concept that Env immunogens can be designed to directly select for specific antibody mutations at residue-level precision by vaccination, thus demonstrating the feasibility of sequential bnAb-inducing HIV-1 vaccine design.

Fig. 1. Molecular dynamics simulation of DH270.6 association with a truncated CH848 HIV-1 Env gp120.

A Schematic representation of an antibody F_v and gp120 antigen associating through an intermediate encounter ensemble. Transition rates k₁ and k_-1 represent the ensemble rate of forming a collision-induced encounter state and dissociating to the unbound conformation, respectively, while k₂ describes the conversion rate from an encounter intermediate to the bound complex. B (left) An HIV-1 Env trimer ectodomain structure. The protomer on the left highlights gp120 (blue) allosteric elements V1/V2 (green), V3 (red), β20- β21 (yellow), as well as gp41 (orange). The protomer to the right highlights the gp120 region that is extracted (gp120_T) from the prefusion closed state structure (orange) and gp41 (purple). The third protomer of the trimer is represented in a gray surface. (middle) The Man9 glycosylated gp120_T highlights the position of the N332-glycan (green surface). (right) The Man9 glycosylated gp120_T with the DH270.6 antibody F_v positioned near the N332-glycan. C RMSD trajectories (light blue) of three representative transitions from encounter states to the bound state. Distances between DH270.6 heavy chain R57 and gp120 D325 (black) show close interactions (<3.0 Å, red line) before reaching the bound state. D Representative encounter to bound transition showing rotation of the DH270.6 F_v (fade from red to white to blue) around the N332-glycan (purple/red spheres). E Frames from the representative simulations in an R57 to D325 interactive encounter transition (top) and the final, non-interactive R57 to D325 bound state. Source data are provided as a Source Data file.

Fig. 2. The association pathway for the DH270.6 Fv identifies design targets.

A Association pathway for DH270.6 determined from an MSM prepared from the simulated set. Important regions of the gp120_T (blue) include the V1 loop (purple), the V4 loop (light orange), the GDIK motif (gray), and the antibody HCDR3 (bright green). A single gp120 state is used throughout, with 50 representative samples of the antibody shown for each state (ensemble of line structures). Arrows show transition paths with sizes adjusted to indicate each transition’s relative flux. Structures are positioned based on the probability that each state will move forward to the bound state. Transitions targeted for design are highlighted (pink). B Graphical representation of lineage-based design depicting a simplified phylogenetic tree leading from the inferred UCA and intermediates to the mature DH270.6 bnAb. Design targets D1-D4, each targeting low probability mutations (red), with the I3.6 targeting D2 identified as the primary focus for design. The I3.6 intermediate contains two key improbable mutations, the V_H R98T and V_L L48Y. C (left) Representative state from State 1 highlighting the position of the N408-glycan and the contacts between the design target D2 mutation sites V_H T98 and V_L Y48. (middle) Representative state from State 3 highlighting the position of the N442-glycan relative to the N301-glycan and its bound state interactive Y27 residue sidechain. (right) The bound state conformation. D (left) Association rates determined by SPR for the parent CH848 Env SOSIP ectodomain and each design (right) Association kinetics for a CH848 parent and design in which two glycans in the V1 loop have been eliminated via mutation (referred to as DT). Arrows and values indicate the fold change in k_a between the parent and combined mutations. Three replicate measures are shown with the mean and error bars indicating the standard deviation. E (lower left) Simplified graphical representation of encounter transition in the DH270.6 interaction with gp120_T. (upper right) Shapes placed in their relative positions on the gp120_T (orange) map encounter states to the structure. Encounter state 1 maps to the cyan V4 block while Encounter state 2 and 3 map to the orange and yellow. Source data are provided as a Source Data file.

Fig. 3. DH270.6 encounter state design improves the selection of target mutations.

A Association rates for the parent CH8483 Env SOSIP (black) and CH848 DE3 design (red) for the DH270 lineage UCA, intermediates, and DH270.6. B Affinities for the parent CH8483 Env SOSIP (black) and CH848 DE3 design (red) for the DH270 lineage UCA, intermediates, and DH270.6. Log scaling is used for visual clarity. C DH270.UCA Ramos B cell BCR triggering was measured as a percentage of calcium flux relative to the maximum flux observed for an anti-IgM Fab₂. D cryo-EM map of the DE3 design bound to DH270.6 (gray) and a difference map between the parent CH848 bound to DH270.6 and the DE3 design bound to DH270.6 highlighting the N442-glycan missing density (green). Contour levels 1.64 and 0.57, respectively. E Panel of pseudo-virus IC50 neutralization titers for sera from IA4 knock-in mice immunized with either the glycan hole-filled CH848-d949 SOSIP trimer (N = 12 mice in group) or the DE3 design (N = 13 mice in group). Significance was calculated using a two-tailed, non-parametric Mann-Whitney test and is marked by an asterisk (*p < 0.05; CH848.10.17, p = 0.203; CH848.10.17 Holes Filled, p = 0.320; CH848.10.17.N332T, p = 0.220; CH848.10.17.DT, p = 0.019; CH848.10.17.DT.N332T, p = 0.964; Q23, p = 0.011; JFRL, p = 0.220; TRO.11, p = 0.055; DU156.12, p > 0.999; ZM106F, p = 0.096; CAAN5342, p > 0.999; 92RW20, p = 0.264; 6101.1, p = 0.024; MLV-SVA, p > 0.999). F Mutation frequencies for the IA4 key improbable mutations heavy chain R98T and light chain L48Y. Bars indicate the geometric mean frequencies. Significance was calculated using a two-tailed, non-parametric Mann-Whitney test and is marked by an asterisk (*p < 0.05, N = 11 mice in the CH848-d949 immunized group and N = 13 mice in the DE3 immunized group). Source data are provided as a Source Data file.

Fig. 4. The association pathway for the CH235.12 Fv identifies design targets.

A Graphical representation of lineage-based design depicting a simplified phylogenetic tree leading from the inferred UCA and intermediates to the mature CH235.12 bnAb. Design targets D1-D4, each targeting low probability mutations (red), that are key for neutralization breadth. B Association pathway for CH235.12 determined from an MSM prepared from the simulated set. Important regions of the gp120_T (blue) include the CD4bs loop (cyan), the V5 loop (red), the loop D (magenta), and the antibody HCDR3 (bright green). A single gp120 state is used throughout, with 50 representative samples of the antibody shown for each state (ensemble of line structures). Arrows show transition paths with sizes adjusted to indicate the relative flux through each transition. Structures are positioned based on the probability that each state will move forward to the bound state along the x-axis. Transitions targeted for design are highlighted (pink). C Representative state from encounter State 2 highlighting the position of the N276-glycan and contact of the HCDR3 with loop D. D Representative state from encounter State 3 highlighting the position of the N386-glycan and contact of the HCDR2 proximal sheet residues with the CD4bs loop. E Mature CH235.12 association rates for the CH505.M5.G458Y parent and the CH505.M5.G458Y CE0 and CE1 designs. Error bars are standard deviations from the mean of three replicate measures. F Aligned structures of the CH505.M5 trimer bound to the early intermediate CH235.I60 Fab or mature bnAb CH235.12 Fab bound to either CH505.M5 or CH505.M5.G458Y highlighting the difference in the V5 loop. The Y458 sidechain is shown for reference. G CH235 UCA, intermediate I60, and mature CH235.12 Fab affinities for the CH505.M5.G458Y parent and the CH505.M5.G458Y CE2 design. Error bars are standard deviations from the mean of three replicate measures. Log scaling is used for visual clarity. Source data are provided as a Source Data file.

Fig. 5. CH235.12 encounter state design improves the selection of target mutations.

A Aligned structures of the CH505.M5.G458Y trimer bound to either early intermediate CH235.I60 Fab (dark blue gp120, purple V_H, dark gray V_L) or mature bnAb CH235.12 Fab (dark red gp120, green V_H, light gray V_L) highlighting the position of loop D residue K279. B (left) Aligned gp120 cores unliganded (light blue; PDB ID 3TGT) and bound (light green; PDB ID 5F96) to CH235.12 (V_H orange, V_L light gray). (right) View of loop D in the unliganded and CH235.12 bound state showing displacement of residue L277. C Kinetics (left, ka, and middle, kd) and affinity (right) for the CH505.M5.G458Y CE3 design (N386R + V5 disulfide+N197D) with either K279 or N279 (log scaling used for visual clarity). D Mutations frequencies for human heavy (left) and light (right) chain genes in CH235 UCA knock-in mice. Significance was calculated using a two-tailed, non-parametric Mann-Whitney test and is marked by an asterisk (*p < 0.05; Heavy Chain p = 0.056, Light Chain p > 0.999, N = 4 mice per group). E CH235 UCA knock-in mouse serum neutralization titers for CH505 isolate-derived pseudo-viruses. Significance was calculated using a two-tailed, non-parametric Mann-Whitney test and is marked by an asterisk (*p < 0.05; CH505TF N279K p = 0.016, CH505TF N279K + G458Y p = 0.008, N = 4 mice per group). F (left) Representative structure of encounter State 3 highlighting the position of the design mutations (red spheres) and the encounter state contacts (pink spheres) in the CH235.12 HCDR2 proximal sheet. (right) mutation frequencies for key I60 mutations from immunized CH235 UCA knock-in mice. Significance was calculated using a two-tailed, non-parametric Mann-Whitney test and is marked by an asterisk (*p < 0.05; N = 4 mice per group). Source data are provided as a Source Data file.