Research ArticleHIV

Initiation of immune tolerance–controlled HIV gp41 neutralizing B cell lineages

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Science Translational Medicine  27 Apr 2016:
Vol. 8, Issue 336, pp. 336ra62
DOI: 10.1126/scitranslmed.aaf0618
  • Fig. 1. Immune tolerance in 2F5 mature and UA dKI mice.

    (A to D) B cell development in 2F5 mature and UA dKI mice, compared to wild-type (WT) (B6). (A) Percentages in progenitor/precursor (pro/pre), immature (imm), transitional (trans), and mature (mat) subsets. (B) Total numbers of B cells in spleen. (C) Ratios of mature to transitional splenic B cells. (D) BCR densities on newly formed/transitional (CD21CD23) splenic B cells, measured by median fluorescence intensity (MFI) of κLC or IgM expression [see table S8 for summary of statistical test results for (B) to (D)]. (E) Ex vivo proximal signaling responses to BCR crosslinking in transitional splenic B cells from naïve 2F5 mature or UA dKI and control B6 mice based on Ca2+ levels (Fluo-4 MFI) before/after anti-IgM stimulation. (F) Expansion of MPER-reactive splenic transitional B cells in 2F5 mature and UA dKI mice immunized six times with MPER peptide-liposomes. Control, saline-injected mice; SP62, MPER peptide QQEKNEQELLELDKWASLWN-biotin. (G) Comparison of IgG-switched splenic B cells in immunized 2F5 mature and UA dKI mice. (H) Total and MPER-specific serum IgG/IgM levels in immunized 2F5 mature and UA dKI mice, compared to B6. X axis arrows, number of MPER peptide-liposome injections after which serum was collected; 0, pre-bleed in naïve mice. Error bars represent means ± SEM [see figs. S7 and S8 for replicate images for (A) and (E), respectively].

  • Fig. 2. DH570 clonal lineage members neutralize tier 1 and 2 HIV isolates in TZM-bl/FcγRI assay.

    (A) Phylogenetic relationship of 32 mAbs in DH570 clonal lineage. The mAbs neutralizing tier 1 and/or tier 2 HIV isolates in TZM-bl/FcγRI assay are highlighted in red. The mutation frequency of the most mutated antibody is 21.3% (DH570.20). (B) Immunogenetics of DH570 mAb. (C) Neutralization activity of representative observed and inferred IAs in the DH570 clonal lineage. Neutralization assays were performed in both TZM-bl and TZM-bl/FcγRI cell lines. IC50s reported as median values of at least two independent experiments except where noted by asterisks.

  • Fig. 3. Crystal structures of DH570 clonal lineage antibodies.

    (A) Structure of unliganded DH570 (blue), DH570.4 (green), DH570.9 (red), and DH570 (cyan) in complex with gp41660–670 peptide (orange). (B) Superposition of liganded DH570 (cyan) and liganded 2F5 (magenta)(28) with insets respectively highlighting the different peptide antigen conformations (orange) as well as the different antibody-antigen contacts between the two complexes. C ter, C terminus; N ter, N terminus.

  • Fig. 4. DH570 members exhibit slower association rates relative to 2F5.

    (A) Computed lipid insertion propensity scores ΔGwif (kcal/mol) of HCDR3 and LCDR3 (summed) are more favorable for neutralizing than nonneutralizing DH570 clonal members (*P = 0.0054, Wilcoxon-Mann-Whitney test with Benjamini-Hochberg correction; n = 23 and 15; lines denote means ± SEM). (B to D) Association rate (Ka, on-rate) and dissociation rate (Kd, off-rate) constants of DH570 clonal lineage branch I (B) and IV (C) antibodies binding to MPER peptide-liposome with greater detail of branch I antibodies shown in (D). Dashed lines represent association rate ceiling. Arrows show maturation pathways derived from phylogenetic tree of the DH570 clonal lineage in Fig. 2A. Data in (B) to (D) were obtained by BLI and were representative of two independent association and dissociation analyses. Kinetic rates data plotted here are listed in table S6 and each BLI curve is plotted in fig. S6.

  • Fig. 5. DH570 mutant acquires high affinity binding and neutralization capacity in the TZM-bl neutralization assay.

    (A and B) BLI sensorgrams of MPER peptide-liposome binding by DH570 Fab (A) and DH570.Mut58 Fab (B) at different antibody concentrations (2, 5, 10, and 20 μg/ml). (C) Measured kinetic rates (Ka and Kd) and dissociation (Kd) constants of DH570 and DH570.Mut58 binding to MPER peptide-liposomes. (D) Neutralization activity of whole IgG1 DH570 and DH570.Mut58 against W61D in the TZM-bl assay. BLI measurements in (A) to (C) are representative of two independent experiments (see fig. S9 for replicate plot and kinetic data).

Supplementary Materials

  • www.sciencetranslationalmedicine.org/cgi/content/full/8/336/336ra62/DC1

    Fig. S1. Rhesus macaque immunization regimen, reactivity of serum and mucosal fluids, and neutralization of HIV isolates.

    Fig. S2. B cell repertoires of macaques 584 and 585.

    Fig. S3. Rhesus macaque clonal lineages using VH and VL genes similar to 2F5VH/VL.

    Fig. S4. Neutralization by members of the DH570 clone.

    Fig. S5. Amino acid and lipid insertion propensity differences within the three DH570 lineage members for which crystal structures were determined.

    Fig. S6. Binding of DH570 lineage antibodies to MPER liposomes.

    Fig. S7. Replicate images for Fig. 1A.

    Fig. S8. Replicate images for Fig. 1E.

    Fig. S9. Replicate plots for Fig. 5.

    Fig. S10. Representative mouse flow cytometry gating strategies.

    Fig. S11. Representative rhesus flow cytometry gating strategies.

    Table S1. Neutralization of HIV isolates by DH573.

    Table S2. Neutralization of HIV isolates by the DH570 clonal lineage.

    Table S3. Characteristics of the DH570 clonal lineage isolated from rhesus macaque 585.

    Table S4. Time course for HIV neutralization in the TZM-bl/FcγRI assay.

    Table S5. Data collection and refinement statistics.

    Table S6. Kinetic rates of DH570 lineage members from branch I and IV binding to MPER peptide-liposomes by BLI.

    Table S7. CDR3 lipid insertion propensity for select DH570 lineage members.

    Table S8. Statistical analysis summary for Fig. 1.

  • Supplementary Material for:

    Initiation of immune tolerance–controlled HIV gp41 neutralizing B cell lineages

    Ruijun Zhang, Laurent Verkoczy, Kevin Wiehe, S. Munir Alam, Nathan I. Nicely, Sampa Santra, Todd Bradley, Charles W. Pemble IV, Jinsong Zhang, Feng Gao, David C. Montefiori, Hilary Bouton-Verville, Garnett Kelsoe, Kevin Larimore, Phillip D. Greenberg, Robert Parks, Andrew Foulger, Jessica N. Peel, Kan Luo, Xiaozhi Lu, Ashley M. Trama, Nathan Vandergrift, Georgia D. Tomaras, Thomas B. Kepler, M. Anthony Moody, Hua-Xin Liao, Barton F. Haynes*

    *Corresponding author. Email: barton.haynes{at}duke.edu

    Published 27 April 2016, Sci. Transl. Med. 8, 336ra62 (2016)
    DOI: 10.1126/scitranslmed.aaf0618

    This PDF file includes:

    • Fig. S1. Rhesus macaque immunization regimen, reactivity of serum and mucosal fluids, and neutralization of HIV isolates.
    • Fig. S2. B cell repertoires of macaques 584 and 585.
    • Fig. S3. Rhesus macaque clonal lineages using VH and VL genes similar to 2F5VH/VL.
    • Fig. S4. Neutralization by members of the DH570 clone.
    • Fig. S5. Amino acid and lipid insertion propensity differences within the three DH570 lineage members for which crystal structures were determined.
    • Fig. S6. Binding of DH570 lineage antibodies to MPER liposomes.
    • Fig. S7. Replicate images for Fig. 1A.
    • Fig. S8. Replicate images for Fig. 1E.
    • Fig. S9. Replicate plots for Fig. 5.
    • Fig. S10. Representative mouse flow cytometry gating strategies.
    • Fig. S11. Representative rhesus flow cytometry gating strategies.
    • Table S1. Neutralization of HIV isolates by DH573.
    • Table S2. Neutralization of HIV isolates by the DH570 clonal lineage.
    • Table S3. Characteristics of the DH570 clonal lineage isolated from rhesus macaque 585.
    • Table S4. Time course for HIV neutralization in the TZM-bl/FcγRI assay.
    • Table S5. Data collection and refinement statistics.
    • Table S6. Kinetic rates of DH570 lineage members from branch I and IV binding to MPER peptide-liposomes by BLI.
    • Table S7. CDR3 lipid insertion propensity for select DH570 lineage members.
    • Table S8. Statistical analysis summary for Fig. 1.

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