Induction of HIV Neutralizing Antibody Lineages in Mice with Diverse Precursor Repertoires

Abstract

The design of immunogens that elicit broadly reactive neutralizing antibodies (bnAbs) has been a major obstacle to HIV-1 vaccine development. One approach to assess potential immunogens is to use mice expressing precursors of human bnAbs as vaccination models. The bnAbs of the VRC01-class derive from the IGHV1-2 immunoglobulin heavy chain and neutralize a wide spectrum of HIV-1 strains via targeting the CD4 binding site of the envelope glycoprotein gp120. We now describe a mouse vaccination model that allows a germline human IGHV1-2(∗)02 segment to undergo normal V(D)J recombination and, thereby, leads to the generation of peripheral B cells that express a highly diverse repertoire of VRC01-related receptors. When sequentially immunized with modified gp120 glycoproteins designed to engage VRC01 germline and intermediate antibodies, IGHV1-2(∗)02-rearranging mice, which also express a VRC01-antibody precursor light chain, can support the affinity maturation of VRC01 precursor antibodies into HIV-neutralizing antibody lineages.

Figure 1. Generation and characterization of V_H1-2 mouse model

(A) Illustration of genetic modifications in the V_H1-2 mouse mode (see text, Figure S1A and S1B for details). (B) Illustration of Rag2 deficient blastocyst complementation (See text for details). (C) HTGTS-rep-seq analysis of V_H usage in V_H1-2 mouse model and in control 129/Sv mouse. The X-axis represents V_H locus from the distal to the D-proximal ends; a subset of mouse V_Hs are labeled for comparison between 129/Sv control and V_H1-2 mice. The histogram displays the percent usage of each V_H of all productive V_H(D)J_H rearrangements. Data for the V_H1-2 mouse model were average of three experiments with error bars representing standard deviations; data for control 129/Sv mouse is consistent with prior studies (Lin et al. 2016). (D) Pie chart Illustration of D segment usage in productive IGHV1-2*02 rearrangements in V_H1-2 mouse model, average D usage frequency with standard deviation for three biological repeats is shown in adjacent panel. (E) Length distribution of IGHV1-2*02 CDR H3 in V_H1-2 mouse model derived from data in (C). The number of total reads and unique reads for IGHV1-2*02 associated CDR H3’s are shown and Venn diagram, which reveals tremendous CDR3 complexity since there is little overlap in CDR H3 sequences in three technical repeats of a single B cell sample (see Lin et al. 2016). (F) Length distribution of CDR L3 of mouse IgL chains in V_H1-2 mice based on three biological replicates with error bars displayed and frequency of 5-amino acid CDR L3 indicated. Other details are in Methods.

Figure 2. Elicitation of VRC01-class germline antibodies in V_H1-2 mouse model

(A) Immunization Schema. (B) Sera binding to eOD-GT8 (solid line) and its CD4bs-knock-out (Δ) mutant (dashed line) assessed by ELISA (mean with standard deviation is shown). (C, D) Sorting and frequency of CD4bs-specific eOD-GT8⁺/ΔeOD-GT8⁻ splenic IgG+ B-cells. Statistical comparisons were performed using a two-tailed t-test. (E) Distribution of CDR L3 amino acid (aa) length of the cloned VH1-2 antibodies from CD4bs-specific B-cells. Mouse IDs are shown on top of each pie chart and the number of total sequences is shown at the center. VRC01-class germline antibodies were defined as antibodies containing a VH1-2 heavy chain and a light chain with a 5aa CDR L3 (in red). (F) Sequence conservation at each position of the 5aa CDR L3 in the cloned VRC01-class germline antibodies compared to known VRC01-class antibodies and VRC01. (G) Binding affinity of synthesized antibodies compared to VRC01 and its germline-V-gene-revertant (VRC01gl) as assayed with Biolayer Interferometry (BLI) Octet. Dashed line indicates the detection limit at 5μM. (H) Heavy and light chain paired sequencing of the B-cell repertoire of naïve and eOD-GT8 60mer-immunized mice. The bar represents the mean of the samples in C and H.

Figure 3. Generation and characterization of V_H1-2/LC model

(A) Illustration of genetic modifications involved in the V_H1-2/LC model. See text, Figure S3A and S3B for details. (B) HTGTS-rep-seq analysis of V_H usage in splenic B cells from V_H1-2/LC mice performed as in Fig. 1C. (C) D segment usage in productive IGHV1-2*02 rearrangements in V_H1-2/LC mouse model. (D) Length distribution of IGHV1-2*02-associated CDR H3s in V_H1-2/LC mouse model. (E) Single cell analysis of VRC01 IgL chain expression in splenic B cells of V_H1-2/LC mouse model. Primers for VRC01LC cDNA in the leader exon (L) of IGKV3-20*01 and the Cκ exon are represented by arrows. Gel images are representative of results from two different V_H1-2/LC mice. Numbers of VRC01LC⁺ and VRC01LC⁻ cells are indicated on the pie chart. Other details are in Experimental Methods.

Figure 4. Stepwise immunization of the V_H1-2/LC mice elicited VRC01-class CD4bs-specific antibodies with neutralizing activity

(A) Immunization Schema. (B) Characterization of immunogens (varied symbols) for ELISA binding to VRC01 and its germline reverted variant (VRC01gl). (C) ELISA endpoint titers of post-immune sera to three germline-binding VRC01-class probes and their respective CD4bs-knock out (Δ) mutants. Error bars indicate standard deviations. (D) Splenocytes were sorted with eOD-GT6 and ΔeOD-GT6 probes to isolate CD4bs-specific IgG+ B cells. (E) Neutralizing activity of post-immune sera to various HIV-1 Env-pseudoviruses (ID₅₀ titers). n.d., not determined.

Figure 5. Stepwise immunization of V_H1-2/LC mice elicited VRC01-class CD4 binding site-specific antibodies with increased SHM

(A) Nucleotide mutation frequency in paired VH1-2 and VK3-20 genes, derived from CD4bs-specific IgG+ B cells of the BG505-immunized mice (G1) and stepwise-immunized mice (G2). Each dot represents one VH1-2 or VK3-20 chain in a VH1-2/VK3-20 paired antibody (n, number of VH1-2/VK3-20 pairs in each animal at indicated time points). The median with interquartile range is plotted. (B) Numbers of total, VRC01-class mimicking, Env-contacting, and both mimicking and contacting amino acid mutations in each VH1-2 HC amplified from specified mice plotted with median and interquartile range and statistically assessed using an unpaired t-tests. VRC01-class mimicking mutations are present in at least two of 9 published VRC01-class bnAb lineages. Env-contacting sites are based on VRC01-gp120 or gp160 crystal structures. (C) Amino acid mutations in all VH1-2 HCs from each specified mouse shown in sequence logo profiles. “n” is the number of all VH1-2 chains amplified from each animal. For reference, 9 published VRC01-class HC lineages are represented below panel G2. VRC01-envelope contact sites are highlighted in pink. The mutation profile of IGHV1-2 constructed from 1080 non-HIV-1 neutralizing antibody lineages (from three healthy donors) is also shown. The red asterisk marks significantly enriched S54R mutations in the G2:wk22 sample. (D) Enriched and depleted amino acid substitutions in VH1-2 cloned from the sequential immunization group G2 compared to non-HIV-1 neutralizing antibodies. The calculated mean occurrence and standard deviations of each mutation and 95% CI are depicted in black. The frequency of enriched (red diamond) or depleted (blue diamond) mutations locates outside of the calculated 95% CI.

Figure 6. Selected IGHV1-2*02/IGKV3-20*01 IgGs cloned from the stepwise immunized mice displayed CD4bs-specific binding to multiple Env proteins

ELISA endpoint binding titers of 27 IGHV1-2*02/IGKV3-20*01 paired IgGs cloned from the stepwise immunized mice 1540 (wk2), 1536 (wk6), 1539 (wk10) and 1538 (wk22) to various Env antigens and their CD4bs-KO mutants (Δ) were compared to the binding titers of VRC01 class antibodies and their germlines (gl). The number of VH1-2 AA mutations in each IgG is listed in the right-most column, and they are reversely correlated with the binding titers to C13 and 426c mutants (p<0.0001).

Figure 7. Selected IGHV1-2*02/IGKV3-20*01 IgGs cloned from the stepwise immunized mice neutralized the same viruses sensitive to the corresponding mouse sera

Neutralization IC₅₀ and IC₈₀ of the 27 IgGs against Env-pseudoviruses 45-01dG5, 426c, the mutant 426c viruses lacking 1, 2 or all 3 glycans at aa 276, 460 and 463, and 247-23 were listed. The presence (+) or absence (−) of N276/460/463 glycans in each pseudovirus is indicated. The neutralizing titers of VRC01 were shown as positive controls.