Immunization for HIV-1 Broadly Neutralizing Antibodies in Human Ig Knockin Mice

Abstract

A subset of individuals infected with HIV-1 develops broadly neutralizing antibodies (bNAbs) that can prevent infection, but it has not yet been possible to elicit these antibodies by immunization. To systematically explore how immunization might be tailored to produce them, we generated mice expressing the predicted germline or mature heavy chains of a potent bNAb to the CD4 binding site (CD4bs) on the HIV-1 envelope glycoprotein (Env). Immunogens specifically designed to activate B cells bearing germline antibodies are required to initiate immune responses, but they do not elicit bNAbs. In contrast, native-like Env trimers fail to activate B cells expressing germline antibodies but elicit bNAbs by selecting for a restricted group of light chains bearing specific somatic mutations that enhance neutralizing activity. The data suggest that vaccination to elicit anti-HIV-1 antibodies will require immunization with a succession of related immunogens.

Figure 1. Characterization of B cells in GLV_H and MuV_H mice

(A) Representative FACS plots show binding of eOD-GT8 and eOD-GT8 CD4bs knock-out (KO) proteins by mature naive B cells in naive wild type C57Bl6 (WT), GLV_H and MuV_H mice. (B) Graph shows frequency of eOD-GT8-binding B cells in naive WT, GLV_H and MuV_H mice. Each dot represents one mouse. (C) Pie charts show heavy (HC) and light chain (LC) sequences cloned from purified single cell sorted naive B cells from naive mice. The number in the center of the pie chart is the number of sequences analyzed, each colored slice represents one clone (identical V gene and CDRL3) and its size is proportional to the size of the clone. White indicates unique sequences. (D) CDRL3 amino acid (aa) lengths from LCs in C. See also Figure S1.

Figure 2. Antibody responses to immunization by GLV_H mice

(A) Graphs show ELISA results of serum (1/900 dilution) for individual mice against eOD-GT8 (blue) and eOD-GT8 CD4bs knock-out (KO, red) from wild type C57Bl6 (WT) and GLV_H mice. Naive serum (0) and serum after one, two or three (1, 2 or 3) immunizations with eOD-GT8 60mers. (B) Graphs show ELISA results of serum (1/900 dilution) for individual mice against 426c.TM₄ΔV1-3 (blue) or 426c.TM₄ΔV1-3 CD4bs knock-out (KO, red) from wild type C57Bl6 (WT) and GLV_H mice. Naive serum (0) and serum after one (1) immunization with multimerized 426c.TM₄ΔV1-3. (C) Graphs show ELISA results of serum (1/300 dilution) for individual mice against BG505 SOSIP and YU2gp140-F in naive mice (0) or after three immunizations (3) with eOD-GT8 60mers. (D) Heavy (HC) and light chain (LC) sequences from single antigen binding B cells isolated from four eOD-GT8 60mer-immunized GLV_H mice. Pie charts as in Figure 1A. A clone is defined by identical V gene and similar CDRL3. (E) CDRL3 amino acid (aa) lengths from LCs in (D). (F) Graph shows ELISA results for monoclonal antibodies (5μg/ml) cloned from eOD-GT8 60mer-immunized GLV_H mice against eOD-GT8 (blue) and eOD-GT8 CD4bs knock-out (KO, red). (G) Graphs show ELISA results of serum (1/300 dilution) for individual mice against BG505 SOSIP or YU2 SOSIP in wild type C57Bl6 (WT) and GLV_H mice after immunization with BG505 SOSIP or YU2 SOSIP respectively. Naive serum (0) and serum after two-(2) or three (3) immunizations. See also Figure S2, Figure S3 and Table S1.

Figure 3. Antibody responses to immunization by MuV_H mice

(A) Graphs show ELISA results of serum (1/300 dilution) for individual mice against eOD-GT8 (blue) and eOD-GT8 CD4bs knock-out (KO, red), 2cc core and YU2 gp140-F. Naive serum (0) and serum after three (3) immunizations with eOD-GT8 60mer. (B) As in (A) but after BG505 SOSIP-immunization. ELISA against BG505 SOSIP, 2cc core and YU2 gp140-F (blue) and YU2 gp140-F CD4bs knock-out (KO, red). (C) Neutralizing activity in TZM-bl assays of serum from individual naive, eOD-GT8 60mer- or BG505 SOSIP -immunized mice (M) against a panel of HIV-1 viruses. Numbers indicate the reciprocal dilution of serum at the median inhibitory concentration (IC50): red, >1000; orange, 100–1000; yellow, 50–100 and white, was not neutralized at any dilution tested. Grey indicates background activities against the control MuLV virus. See also Figure S4.

Figure 4. Monoclonal antibodies from immunized MuV_H mice

(A) Pie charts show heavy (HC) and light (LC) sequences cloned from two eODGT8 60mer-immunized (M#7 and 9) and three BG505 SOSIP-immunized (M#16, 17 and 18) MuV_H mice. V_L gene usage and CDRL3 sequences are shown for each clone, colors correspond to colors in pie charts. The same color indicate clones shared by different mice. A clone is defined by identical V gene and similar CDRL3. (B) CDRL3 amino acid (aa) lengths of the LCs in (A). (C) Graphs show ELISA results for individual monoclonal antibodies with the indicated CDRL3s cloned from eOD-GT8 60mer-immunized MuV_H mice against eOD-GT8 (blue) and eOD-GT8 CD4bs knock-out (KO, red) (at 0.55 μg/ml) or against YU2 gp140-F (at 5 μg/ml). (D) Neutralizing activity of a set of representative eOD-GT8 60mer- or BG505 SOSIP- elicited monoclonal antibodies (mAbs) against 7 HIV-1 tier-2 viruses (1=MuLV control, 2=BG505/T 332N, 3=T247-23, 4=62357.14.D3.3489, 5=Q842.d12, 6=3365.V2.c30, 7=YU2.DG, 8=0815.v3.c3) compared to the serum of the mouse they were cloned from. Colors for mAbs indicate concentration of mAb at the median inhibitory concentration (IC50): orange, 0.1 to 1 μg/ml; yellow, 1 to 10 μg/ml; green, 10–25 or 50 μg/ml and white, not neutralized at any concentration tested. Lowest dilution tested for serum was 1:50, highest concentration tested for mAbs were between 10–50 μg/ml (see Table S3 and S5). X indicates sample not tested. The numbering of the antibodies corresponds to the numbering of the antibodies in Table S3 and S5. Colors and numbering for serum as in Figure 3C. (E) Graphs show ELISA results for individual mAbs with the indicated CDRL3s cloned from BG505 SOSIP-immunized MuV_H mice against eOD-GT8 (blue) and eOD-GT8 CD4bs knock-out (KO, red) (at 0.55 μg/ml) or against YU2 gp140-F (at 5 μg/ml). See also Figure S4 and Table S2, S3, S4 and S5.

Figure 5. Somatic mutations in immunized mice

(A) Graph shows number of light chain (LC) somatic hypermutations in individual sequences obtained after eOD-GT8 60mer- or BG505 SOSIP immunization in GLV_H or MuV_H mice. (B) LC somatic hypermutations in V_L10-94*01 in individual sequences obtained after eOD-GT8 60mer- or BG505 SOSIP immunization in MuV_H mice. (C) V_L10-94*01 germline (GL) and 17 monoclonal antibodies (mAbs) from immunized MuV_H mice sorted by neutralization strength. Neutralization data for each antibody tested against virus 1=T247-23, 2=62357.14.D3.3489, 3=BG505/T332N, 4=3365.v2.c30, 5=YU2.DG, 6=0815.v3.c3, 7=HxBC2.DG and 8=MuLV. Colors for mAbs indicate concentration of mAb at the median inhibitory concentration (IC50): red, <0.1 μg/ml; orange, 0.1 to 1 μg/ml; yellow, 1 to 10 μg/ml; green, 10–25 or 50 μg/ml and white, not neutralized at any concentration tested (see also Table S3 and 5). Position of amino acid mutations in the LCs compared to germline are highlighted in red. X indicates sample not tested. (D) Graph shows neutralization score for V_L10-94*01 cloned antibodies with Q₉₀Q or Q₉₀H CDRL3 from immunized MuV_H mice. Neutralization score: sum of viruses tested IC50 0.1–1 μg/ml: 3, 1–10 μg/ml: 2, 10–50 μg/ml: 1 and >50 μg/ml: 0. (E) Somatic hypermutations in individual heavy chain sequences obtained after eOD-GT8 60mer- or BG505 SOSIP-immunization in GLV_H or MuV_H mice. Data in (A), (B) and (E) are pooled from 9 mice (M#1, 2, 3, 4, 7, 9, 16, 17 and 18). (F) Somatic hypermutations in individual germinal center B cell heavy chain (HC) sequences obtained from naive GLV_H or MuV_H mice. (G) Somatic hypermutations in individual germinal center B cell J_H4 intron sequences obtained from naive GLV_H or MuV_H mice. Data in (F) and (G) are pooled from 2 independent experiments with 2–3 mice each. 80 clones were sequenced for GLV_H or MuV_H germinal center B cells HC and J_H4. See also Figure S5, Table S3 and 5.