Sequential Immunization Elicits Broadly Neutralizing Anti-HIV-1 Antibodies in Ig Knockin Mice

Abstract

A vaccine that elicits broadly neutralizing antibodies (bNAbs) against HIV-1 is likely to be protective, but this has not been achieved. To explore immunization regimens that might elicit bNAbs, we produced and immunized mice expressing the predicted germline PGT121, a bNAb specific for the V3-loop and surrounding glycans on the HIV-1 spike. Priming with an epitope-modified immunogen designed to activate germline antibody-expressing B cells, followed by ELISA-guided boosting with a sequence of directional immunogens, native-like trimers with decreasing epitope modification, elicited heterologous tier-2-neutralizing responses. In contrast, repeated immunization with the priming immunogen did not. Antibody cloning confirmed elicitation of high levels of somatic mutation and tier-2-neutralizing antibodies resembling the authentic human bNAb. Our data establish that sequential immunization with specifically designed immunogens can induce high levels of somatic mutation and shepherd antibody maturation to produce bNAbs from their inferred germline precursors.

Figure 1

ELISA-guided sequential immunization. (A) ELISAs against 10MUT gp120 and a mutated version of the same protein that is missing the PGT121 epitope (10MUT gp120 KO) using serum (1/100 starting dilution) from GL_HL121 and Mut_HGL_L121 mice primed with monomeric 10MUT (10MUT gp120, left) or trimeric 10MUT (10MUT SOSIP, right). (B) ELISA using human PGT121 (5μg/ml) against 10MUT gp120 and 10MUT gp120 KO. (C) Diagram shows the sequential immunization protocol delivered over a period of 5–6 months. (D) ELISAs of serum from a control and a sequentially immunized GL_HL121 mouse after immunization steps 1–4. Test proteins are indicated above each column. ELISAs are ordered from left to right according to level of capture antigen modification compared to wtBG505. When indicated, the specificity of the serologic response for the PGT121 epitope was also evaluated by ELISA against the 10MUT gp120 KO protein. Each row of ELISA graphs represents the indicated step in the immunization protocol. A model of the corresponding immunogen is shown below each row (Steichen et al., submitted). Blue spheres represent glycans and red spheres indicate mutated residues. IgH (H1-3) and IgL (L1-3) loops are represented in the model of wtBG505. Red asterisks indicate the boosting immunogens. VLC= 5 native-like SOSIPs differing in the variable loops (Steichen et al, submitted). See also Figure S1 and S2.

Figure 2

Longitudinal analysis of the serum from sequentially immunized GL_HL121 and Mut_HGL_L121 mice by ELISA. (A) Diagram of the sequential immunization protocol. (B) Graphs show the results of ELISAs on serum (1/100 starting dilution) collected from naïve and sequentially immunized GL_HL121 (3 mice) and Mut_HGL_L121 (3 mice). ELISAs show reactivity against the immunogen and the boosting protein after each step. When indicated, ELISA binding of the human PGT121 bNAb is also shown (1/100 starting dilution of a 500μg/ml solution=5μg/ml). VLC= 5 different native-like SOSIPs. See also Figure S2.

Figure 3

Neutralization in the serum of immunized GL_HL121 and Mut_HGL_L121 knock-in mice. (A) TZM-bl neutralization activity (IC₅₀ or 1/ID50 in mice M8 and M10) in the serum of naïve and sequentially immunized GL_HL121 (M1–M7, top) and Mut_HGL_L121 (M8–12, bottom) mice against a panel of 12 Tier 2 and 2 tier 1B HIV-1 isolates. Samples with asterisk correspond to step 5 in the immunization, all others were measured after complete protocol. The activity of the human PGT121 bNAb and the inferred intermediate 3H+3L against the same panel of viruses is shown. (B) Longitudinal analysis of the neutralization activity in the serum of sequentially immunized GL_HL121 and Mut_HGL_L121 mice. Table shows TZM-bl neutralization activity (1/ID₅₀) against MuLV control and 3 Tier2/1B HIV-1 isolates for 1/50 dilutions of serum from 2 GL_HL121 (M1–M2) and 1 Mut_HGL_L121 (M9) mice collected after each immunization as indicated (left). Samples with an asterisk correspond to purified Igs and not serum. (C) Diagrammatic representation of the immunization protocol (top). Table shows TZM-bl neutralization activity (IC₅₀) in the serum of GL_HL121 and Mut_HGL_L121 mice immunized 7 times with 10MUT. (D) Diagrammatic representation of the immunization protocol (top). Table shows TZM-bl neutralization activity (IC₅₀) in the serum of GL_HL121 and Mut_HGL_L121 mice immunized with 10MUT followed by a boost with wtBG505. IC₅₀ < 0.096 in red; 0.096- 0.5 in orange; 0.5- 4 in yellow; > 4 green; 1/ID₅₀ > 5000 in red; 5000-1000 in orange; 1000-100 in yellow; <100 in green; not detectable (ND) in gray; not tested indicated by a dash. Red asterisk indicates the time point of analysis. See also Figure S3 and S4.

Figure 4

Somatic mutations in antibody sequences from immunized mice. (A) Diagram of the sequential immunization protocol (Protocol 1). (B) Number of nucleotide mutations in the IgL of antigen specific single B cells isolated from sequentially immunized GL_HL121 (left) and Mut_HGL_L121 (right) mice. Each column represents one mouse and each dot one B cell. (C) Number of nucleotide mutations in the IgH sequences of antigen specific single B cells isolated from sequentially immunized GL_HL121 (left) and Mut_HGL_L121 (right) mice. Each column represents one mouse and each dot one B cell. (D) Number of nucleotide mutations in the IgL obtained from sequentially immunized GL_HL121 and Mut_HGL_L121 mice. Data represent pooled sequences for each genotype. **p< 0.01. (E) Number of nucleotide mutations in the heavy chain sequences obtained from sequentially immunized GL_HL121 and Mut_HGL_L121 mice. Data represent pooled sequences for each genotype. ****p< 0.0001. Nt= Nucleotide. See also Figure S5 and S7.

Figure 5

Somatic mutations after repeated or sequential immunization. (A) Diagram of the sequential or repeated immunization protocols (Protocol 1 and 2 respectively). (B) Number of nucleotide mutations in the IgL of single antigen specific B cells isolated from individual GL_HL121 mice immunized repeatedly with 10mut. Each column represents one mouse and each dot one B cell. (C) Number of nucleotide mutations in the IgH of single antigen specific B cells isolated from individual GL_HL121 mice immunized repeatedly with 10MUT. Each column represents one mouse and each dot one B cell. (D) Comparison of the number of nucleotide mutations in IgL obtained from GL_HL121 mice immunized sequentially (7 mice pooled, protocol 1) or repeatedly with 10MUT (3 mice pooled, protocol 2) ****p< 0.0001. Each dot represents one B cell (E) Comparison of the number of nucleotide mutations in heavy chains obtained from GL_HL121 mice immunized sequentially (7 mice pooled) or repeatedly (3 mice pooled) ****p< 0.0001. Each column represents one mouse and each dot one B cell. (F) Frequency of mutation per amino acid position in IgL (top) and IgH (bottom) of two representative mice immunized sequentially (M2, protocol 1) or repeatedly (M16, protocol 2). Framework regions (FWR) are shaded in gray and white areas correspond to complementarity determining regions (CDRs). Nt= Nucleotide; Aa= Amino acid. See also Figure S5 and S7.

Figure 6

Monoclonal antibody neutralizing activity. (A) 3D plot showing neutralization activity (color coded), total number of amino acid mutations (X axis) and the number of mutations that are identical or chemically equivalent to mutations in the human PGT121, PGT122, PGT123 or 10-1074 bNAbs (bNAb-like) (Y axis) for all monoclonal antibodies from GL_HL121 mice immunized according to Protocol 1 and 2. Chemical equivalence is as in (B). Each antibody was assigned a neutralization score (see Materials and Methods). Red indicates higher neutralization scores. Correlation coefficients (r²) are indicated. r² (X vs. Y) refers to the correlation between total amino acid mutations and the number of bNAb-like mutations; r² (X vs. Neut) refers to the correlation between total amino acid mutations and neutralization score: r² (Y vs. Neut) refers to the correlation between bNAb-like mutations and neutralization score. (B) Graphs show the total number of amino acid mutations (X axis) in IgH (left) and IgL (right) of all antibody sequences from GL_HL121 mice immunized with protocols 1, 2 or 3 vs. the number of mutations that are identical or chemically equivalent to mutations in the human PGT121, PGT122, PGT123 or 10-1074 bNAbs (bNAb-like) (Y axis). 292 and 405 IgH and IgL sequences respectively were analyzed. The size of the dot is proportional to the number of sequences. Chemical equivalence was as follows: Group 1: G/A/V/L/I; Group 2: S/T; Group 3: C/M; Group 4: D/N/E/Q; Group 5: R/K/H; Group 6: F/Y/W; Group 7: P. Correlation coefficient (r²) is indicated. (C) Table shows the results of TZM-bl assays on monoclonal antibodies from GL_HL121 mice immunized according to Protocol 1 and 2 as indicated in the diagrams on top. The protocol, mouse, antibody name, and the 14 tier 2/1B HIV-1 isolates are indicated at left and top respectively. Neutralization activity code: IC₅₀ <0.01 in red; 0.01–0.1 in orange; 0.1–1 in yellow; >1 in green; not detectable (ND) in gray; not tested indicated by a dash. See also Figure S6.

Figure 7

Alignment of the monoclonal IgH and IgL to the inferred germline and mature mutated PGT121, PGT122, PGT123 and 10-1074 bNAbs. (A) Alignment of IgH sequences from the monoclonal antibodies in Figure 6. (B) Alignment of IgL sequences from monoclonal antibodies in Figure 6. The name of the antibody is indicated. Colors represent different amino acids. Dots represent identity to the germline reference sequence. Color coded asterisks next to the antibody name indicate neutralizing antibodies and their neutralization score (see Materials and Methods). Red indicates higher neutralization score. Amino acid substitutions that were previously identified as critical for binding to Env and neutralization of HIV-1 isolates are enclosed in a red rectangle. Antibody regions are indicated in gray. FR= framework region; CDR= complementarity determining region. Amino acid position is indicated with numbers on top of each reference sequence.