Research ArticleHIV

Antigen-specific antibody Fc glycosylation enhances humoral immunity via the recruitment of complement

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Science Immunology  17 Aug 2018:
Vol. 3, Issue 26, eaat7796
DOI: 10.1126/sciimmunol.aat7796
  • Fig. 1 Unique Fc functional profiling of HIV-specific Abs from neutralizers.

    (A to C) Serum Abs from neutralizers (N; purple) and non-neutralizers (NN; yellow) were evaluated for their ability to promote NK-dependent ADCC against gp120-pulsed CD4+ T cells (A), monocyte-directed phagocytosis of gp120-functionalized fluorescent beads (B), or complement deposition (C3b) on the surface of gp120-pulsed CD4+ target cells (C). All assays were performed in duplicate or triplicate across all 131 individuals. An unpaired t test was used for statistical analysis. *P < 0.05 and **P < 0.01. The horizontal bars in all panels indicate mean.

  • Fig. 2 Enhanced, but selective, FcR and complement binding by HIV-specific Abs in neutralizers.

    (A) The rainbow heat map shows the breadth of neutralization and sample ID number across the 26 neutralizers and 12 non-neutralizers that were profiled more deeply in this and subsequent figures. (B) The larger heat map illustrates the binding capacity (MFI) of gp140-, gp120-, gp41-, and p24-specific serum Abs to FcγRIIA, FcγRIIB, FcγRIIIA, FcγRIIIB, and C1q protein. Row 19 represents the breadth of neutralization. Each additional row represents the binding level for a single antigen specificity to a single FcR/C1q. Each column represents one individual (sample ID number is below the heat map), either a neutralizer (red box) or a non-neutralizer (blue box). Data were normalized across rows. The scale bar represents the Z-normalized scores. (C) The dot plot represents the representative univariate binding of gp120-specific Abs from each group to FcγRIIA, FcγRIIB, FcγRIIIA, FcγRIIIB, and C1q. (D) The heat map strip highlights the correlation between gp140-, gp120-, gp41-, and p24-specific Ab binding to each FcR/C1q and the breadth of neutralization across all neutralizers and non-neutralizers. (E) The hybrid dot/bar plot shows the evolution of gp120-specific FcγRIIA binding Abs in the first year after infection in a group of acutely infected HIV participants, half of which went on to develop bNAbs (purple) or not (yellow). Each dot represents one individual, with neutralizers (N) in violet and non-neutralizers (NN) in yellow. A Mann-Whitney test was used for statistical analysis to compare groups in (C). An ANOVA, with a post hoc Tukey’s test, was used to compare between groups across time points in (E). A Spearman correlation with a post hoc Bonferroni correction was used to examine the relationship between Fc profiles and breadth of neutralization (D). *P < 0.05 and **P < 0.01. The horizontal bars in all panels indicate mean. Error bars represent standard error of mean (SEM) in (C) and standard deviation (SD) in (E).

  • Fig. 3 Neutralizer ICs drive higher-avidity Abs and expanded GC B cell responses.

    (A to C) BALB/c mice were immunized twice, 3 weeks apart, with alum-adjuvanted gp120 ICs (ICs-alum) generated with serum Abs from four neutralizers (N) or four non-neutralizers (NN) (n = 5 to 8 mice per group, two experiments). Control groups received alum-adjuvanted gp120 or adjuvant alone. Ten days after the last immunization, we measured the titers of gp120-specific IgG Abs (A), the titers of high-avidity gp120-specific IgG Abs (B) in sera, and the percentage of GC B cells (C), defined as live CD3CD19+CD38CD95+ cells, in draining lymph nodes of immune mice. All assays were run in duplicate, and a Mann-Whitney test was used to compare responses induced by neutralizer (N) or non-neutralizer (NN) immunized animals. *P < 0.05 and **P < 0.01. The horizontal bars in all panels indicate mean, and error bars in all panels represent SEM.

  • Fig. 4 Sialylated IgG1 gp120-specific Abs are increased in neutralizers.

    (A) The radar plot shows the relative differences in gp120-specific IgG1, IgG2, IgG3, and IgG4 Ab titers in sera from neutralizers (N; red; n = 26) or non-neutralizers (NN; blue; n = 12). (B) The dot plot shows the gp120-specific IgG1 Ab titer differences across the neutralizers and non-neutralizers. (C) The radar plot describes the relative distribution of gp120-specific IgG Ab glycan levels in sera from neutralizers (N; red) or non-neutralizers (NN; blue). (D) The dot plot illustrates the percentage of sialylated gp120-specific IgG Abs among the groups. (E) The PLS-DA, using all Fc profile data including gp120-specific Ab titers, glycan profiles, binding to FcRs and complement proteins, ADCP, ADCD, and ADCC, was used to separate the two groups. Each dot represents a neutralizer (red) or non-neutralizer (blue). (F) The bar graph shows the variable importance in projection (VIP) score rank for the minimal Ab features that were used in the PLS-DA to separate the groups. As few as 17 of the total features collected across the cohort were required to nearly completely separate out the neutralizers and non-neutralizers. The variables were ranked in their importance in driving separation in the model, with the largest bars representing the most important contributions and the smallest bars representing more minimal contributions. The direction and color of the bars indicate whether the feature was enriched in the neutralizers (red) or in the non-neutralizers (blue). A Mann-Whitney test was used for statistical analysis in (B) and (D). *P < 0.05 and ***P < 0.001. The horizontal bars in all panels indicate mean, and error bars in all panels represent SEM.

  • Fig. 5 Sialylated Ab-ICs promote enhanced humoral immune responses.

    (A) The dot plots show gp120-specific Ab titers (left) or avid Ab titers (right) 10 days after the second immunization in BALB/c mice receiving alum alone (green), alum-adjuvanted gp120 (blue), agalactosylated PGT121 (G0-ICs) with alum (aqua), nonsialylated galactosylated PGT121 (G1/G2 NS-ICs) with alum (lavender), or sialylated galactosylated PGT121 (G1/G2 S-ICs) with alum (pink) (n = 3 to 8 mice, two experiments). (B) The dot plots highlight the number of fluorescently labeled ICs on noncognate B cells (gp120+ B cells), macrophages (gp120+ SSM), or FDCs (gp120+ FDCs) from draining lymph node at 1 and 3 hours after injection with nonsialylated IC (NS-IC) or sialylated IC (S-IC) (n = 4 to 8 mice, two experiments). (C) The confocal microscopy images depict the level of IC deposition in a B cell follicle 1 hour after a mouse is immunized with NS-ICs (left) or S-ICs (right) and stained for g120 (blue), macrophages (green), and GL-7 (red). Representative image of three images from two to three mice per condition. (D) The bar graphs show the number (left) and area occupied (right) of gp120+bead+ FDCs in a draining lymph node. (E) The confocal microscopy images depict IC deposition in a B cell follicle 3 hours after immunization and stained with the same targets as (C). Representative image of three images from two to three mice per condition. (F and G) The line graphs demonstrate the percentage of bound Raji or naïve human B cells after incubation with APC bead–labeled NS-ICs (yellow) or S-ICs (pink), with (colored line) or without complement (gray line), with (blue) or without complement (light blue) alone, or α-FcγRIIB blocking Abs (hatched lines) (F, n = 2; G, n =3). (H) The dot plots show the gp120-specific IgG Ab titers (left) and high-avidity gp120-specific Ab titers (right) after immunizing WT or C1q−/− C57BL/6 mice (n = 2 to 8, two experiments) with alum (white), gp120-alum (blue), NS-ICs with alum (yellow), or S-ICs with alum (pink). (I) The dot plot highlights the ratio of high-avidity gp120-specific Abs to total gp120-specific Abs in WT (dark pink) versus C1q−/− (light pink) mice after immunization with S-ICs. All ELISAs were performed in triplicate, and a one-way ANOVA followed by Tukey’s post test was used for (A), (B), and (D). Mann-Whitney and one-way ANOVA with Holm-Sidak’s multiple comparisons test were used for (H). Two-way ANOVA with Dunnett’s correction comparing samples with the negative control (no Ab, no complement) was used for (G). Mann-Whitney test was used in (I). *P < 0.05, **P < 0.01, and ***P < 0.001. The horizontal bars in all panels indicate mean. Error bars represent SEM in (B), (D), (H), and (I) and SD in (A).

Supplementary Materials

  • immunology.sciencemag.org/cgi/content/full/3/26/eaat7796/DC1

    Fig. S1. Process of selection of plasma samples and dilutions for IC immunization.

    Fig. S2. Titers of serum HIV-specific and flu-specific Ab subclasses.

    Fig. S3. Correlation of IgG gp120-specific titers and neutralization breadth to CD4 count, days after diagnosis, viral load, or controller status.

    Fig. S4. Binding and glycosylation profiles of modified PGT121 monoclonals.

    Table S1. Cohort neutralization profiles.

    Table S2. Raw data sets.

  • Supplementary Materials

    The PDF file includes:

    • Fig. S1. Process of selection of plasma samples and dilutions for IC immunization.
    • Fig. S2. Titers of serum HIV-specific and flu-specific Ab subclasses.
    • Fig. S3. Correlation of IgG gp120-specific titers and neutralization breadth to CD4 count, days after diagnosis, viral load, or controller status.
    • Fig. S4. Binding and glycosylation profiles of modified PGT121 monoclonals.
    • Table S1. Cohort neutralization profiles.

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    Other Supplementary Material for this manuscript includes the following:

    • Table S2 (Microsoft Excel format). Raw data sets.

    Files in this Data Supplement:

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