A generalized HIV vaccine design strategy for priming of broadly neutralizing antibody responses

Abstract

Vaccine induction of broadly neutralizing antibodies (bnAbs) to HIV remains a major challenge. Germline-targeting immunogens hold promise for initiating the induction of certain bnAb classes; yet for most bnAbs, a strong dependence on antibody heavy chain complementarity-determining region 3 (HCDR3) is a major barrier. Exploiting ultradeep human antibody sequencing data, we identified a diverse set of potential antibody precursors for a bnAb with dominant HCDR3 contacts. We then developed HIV envelope trimer-based immunogens that primed responses from rare bnAb-precursor B cells in a mouse model and bound a range of potential bnAb-precursor human naïve B cells in ex vivo screens. Our repertoire-guided germline-targeting approach provides a framework for priming the induction of many HIV bnAbs and could be applied to most HCDR3-dominant antibodies from other pathogens.

Fig. 1.. Engineering germline-targeting trimers for an HCDR3-dependent bnAb.

(A) Cryo-EM structure of BG18 (HC, purple; LC, cyan) bound to BG505 MD39 Env trimer (gray, with N332 and N392 glycans shown as green sticks), and conserved residues near the base of V3 [G324, D325, I326, R327, Q328, A329, H330, T415, L416 and P417] colored red). (B) Cryo-EM structure of BG18 iGL₀ in complex with N332-GT2 Env trimer with MD64 stabilizing mutations (23); coloring as in (A). (C) Schematic of the directed evolution process to design N332-GT1, -GT2 and -GT5. (D) N332-GT binding affinities (K_D) for BG18 iGL_0-2 (red), BG18 iGL₁ with alternate germline V_L (blue open symbols) or V_H genes (blue filled symbols), and BG18 iGL containing NGS-derived HCDR3s (pre1-15) (black). MD39 is the reference Env trimer with no germline-targeting mutations. Pre8 was found to be highly polyreactive and was not included in the analysis. Solid black, blue, and red lines indicate the geomean K_Ds for NGS-derived precursors, alternate V_H/V_L precursors, and inferred germline precursors, respectively. Dashed line indicates the limit of detection.

Fig. 2.. Immunization of BG18^gH B cell adoptive transfer recipient mice with N332-GT2 Env nanoparticles.

(A) Gating strategy to identify epitope-specific (N332-GT2⁺⁺/N332-GT2-KO⁻) B cells in BG18^gH and WT mice. Each symbol represents a different mouse. Bars indicate mean ± SD from experiments in 3 mice in each model. (B) Frequency of epitope-specific B cells in non-immunized BG18^gH and WT mice. (C) Distribution of V_H and V_L genes in epitope-specific naive B cells in non-immunized BG18^gH mice. (D) Frequency of GC B cells (left) or CD45.2⁺ GC B cells (right) in four immunization conditions. Each symbol represents a different mouse. Bars indicate mean ± SD from experiments in the following number of mice in each condition: BG18gH (GT2), n = 6; WT (GT2), n = 5; BG18gH (MD39), n = 3; WT (MD39), n = 3. (E) Frequency of CD45.2⁺ (left) or CD45.1⁺ (right) epitope-specific B cells in four immunization conditions. Each symbol represents a different mouse. Bars indicate mean ± SD from experiments in the following number of mice in each condition: BG18gH (GT2), n = 6; WT (GT2), n = 5; BG18gH (MD39), n = 3; WT (MD39), n = 3. (F) Serum ELISA 50% equilibrium dilution values for N332-GT2 and N332-GT2-KO at day 14 after immunization for four immunization conditions. Each symbol represents a different mouse. Bars indicate geometric mean and geometric SD from experiments in the following number of mice in each condition: BG18gH (GT2), n = 5; WT (GT2), n = 5; BG18gH (MD39), n = 3; WT (MD39), n = 3. Student’s t-test; ns, P > 0.05; *, P < 0.05; **, P < 0.01. Data in (A)-(F) are from one of three representative experiments with three or more animals in each group. (G) Distribution of V_H and V_L genes in epitope-specific GC (CD38^lowCD95⁺) B cells 8 and 42 days after immunization of BG18^gH B cell adoptive transfer recipient mice. (H) SPR dissociation constants (K_D) for N332-GT2 trimer binding to epitope-specific Fabs derived from naive B cells in non-immunized BG18^gH mice and GC B cells 8 and 42 days after immunization of BG18^gH B cell adoptive transfer recipient mice. Each symbol corresponds to a different Fab and represents one or two measurements. Bars indicate geometric mean and geometric SD. (I) Phylogenetic trees of BCR HCs isolated from epitope-specific CD45.2⁺ B cells 8 and 42 days after immunization with N332-GT2 NPs. Tree scale indicates the number of substitutions per site. (J) SPR dissociation constants (K_D) for the five highest affinity naive Fabs from (H) binding to the V1 loop-modified BG505 trimer (BG505_V1_mod), and for nine of the high affinity day 42 Fabs from (H) and five inferred-germline variants of the high-affinity day 42 Fabs (Day42.iGL) binding to V1 loop-modified trimers from BG505 and three other HIV isolates (SF162P3, AC10, AD8) as well as a BG505 trimer with a less modified V1 loop (BG505_7mut), a native-like trimer (BG505_MD39) and an epitope-KO trimer (N332-GT2_KO). Each symbol corresponds to a different Fab and represents one or two measurements. Bars indicate geometric mean and geometric SD. Dashed line indicates limit of detection. (K) Neutralization potency (IC50) against native (BG505 T332N) and V1 loop-modified (BG505-V1mod) pseudoviruses, for the BG18 bnAb, the five highest affinity naive Fabs from (H), five inferred-germline variants of the high-affinity day 42 Fabs (Day42.iGL), and five high affinity day 42 Fabs. Each IC50 is an average from two measurements. ND indicates not determined.

Fig. 3.. Naive human B cells sorted with N332-GT Env trimers.

(A) Gating strategy for N332-GT epitope-specific sorting of naive human B cells. (B) Frequency of epitope-specific B cells among IgG-negative B cells. Each symbol represents a different human subject. Bars indicate geometric mean and geometric mean SD from the following number of independent subjects: N332-GT1, n = 9; N332-GT2, n = 11; N332-GT5, n = 4. (C) HCDR3 length distribution from epitope-specific sorted cells compared to control B cells. (D) Frequency of V_L3-25 or V_L3-1 LCs from epitope-specific sorted cells relative to control B cells. Significance of differences from control was evaluated by a Chi-sqaured test. *, P=0.01; **, P=0.005; ****, P=0.0001. (E) SPR-derived binding specificities for 46 HMP Fabs corresponding to epitope-specific naive human B cells isolated by N332-GT1 or N332-GT2 (top), with light chain V gene usage for non-binding Fabs (bottom left) and for N332-epitope-specific Fabs (bottom right). (F) SPR dissociation constants for HMP epitope-specific Fabs isolated with N332-GT1 and N332-GT2 Env trimers. Dashed line indicates the limit of detection.

Fig. 4.. Sequence and structural characterization of type I and type II BG18-like naive antibodies isolated by N332-GT Env trimers.

(A) HCDR3 sequence and gene segment assignments for three type I BG18-like naive human precursor antibodies. V, D, and J gene segments are colored blue, red, and green, respectively. (B) HCDR3 sequence and gene segment assignments for 20 type II BG18-like precursor antibodies. (C) Cryo-EM structural analysis of type I (HMP1) and type II (HMP42) precursor antibody LC interactions with N332-GT5 compared to BG18 iGL LC interactions with N332-GT2. Gp120 is colored gray, and the LCs are colored cyan, yellow, and blue for BG18 iGL, HMP1 and HMP42, respectively. (D) Cryo-EM structural analysis of type I and type II precursor HCDR3 interactions with N332-GT5 compared to HCDR3 interactions for BG18+MD39 and BG18iGL+N332-GT2 complexes. Gp120 is colored gray with conserved residues (or the corresponding germline-targeting amino acids) near the base of V3 in red as in Fig. 1A. Glycans are shown as green sticks.