Natural variation in Fc glycosylation of HIV-specific antibodies impacts antiviral activity

Abstract

While the induction of a neutralizing antibody response against HIV remains a daunting goal, data from both natural infection and vaccine-induced immune responses suggest that it may be possible to induce antibodies with enhanced Fc effector activity and improved antiviral control via vaccination. However, the specific features of naturally induced HIV-specific antibodies that allow for the potent recruitment of antiviral activity and the means by which these functions are regulated are poorly defined. Because antibody effector functions are critically dependent on antibody Fc domain glycosylation, we aimed to define the natural glycoforms associated with robust Fc-mediated antiviral activity. We demonstrate that spontaneous control of HIV and improved antiviral activity are associated with a dramatic shift in the global antibody-glycosylation profile toward agalactosylated glycoforms. HIV-specific antibodies exhibited an even greater frequency of agalactosylated, afucosylated, and asialylated glycans. These glycoforms were associated with enhanced Fc-mediated reduction of viral replication and enhanced Fc receptor binding and were consistent with transcriptional profiling of glycosyltransferases in peripheral B cells. These data suggest that B cell programs tune antibody glycosylation actively in an antigen-specific manner, potentially contributing to antiviral control during HIV infection.

Figure 1. Effector function and global antibody glycosylation profiles.

(A) Antibodies from HIV-infected subjects were examined for their ability to inhibit viral replication via neutralization (AB) or ability to recruit NK cells as effectors (NK + AB) in an ADCVI assay. Colored bars correspond to unique glycan structures. (B) NK cell degranulation (dot plot) and cytokine secretion were determined in the ADCVI assay. Pie charts depict the polyfunctional profile of antibody-mediated NK cell recruitment, including degranulating NK cells (CD107a, 1 function), IFN-γ secretion plus degranulation (2 functions), or IFN-γ plus TNF-α plus degranulation (3 functions). (C) Glycan dependence of ADCVI activity. Removal of antibody glycan with the endoglycosidase PNGaseF did not affect neutralization (AB), but ablated viral inhibition resulting from recruitment of NK cells as effectors (AB+). (A–C) Individual symbols represent each subject tested, and horizontal lines represent the median for a given group of clinically related subjects. (D) Global plasma IgG glycosylation profiles for all glycan structures (first panel), galactose content (second panel), and fucose content (third panel). neg, healthy controls; acute, acutely HIV-infected subjects; untx, untreated chronic progressors; tx, treated chronic progressors; ctr, controllers; F, fucosylated; nonF, nonfucosylated.

Figure 2. Complete glycan prevalence data for bulk plasma IgG.

Relative prevalences (%) of all observed glycoforms in HIV-negative, acute-infected, treated, and untreated subjects and HIV controllers. Colored underlays represent Tukey’s bars and whiskers. In box-and-whisker plots, horizontal bars indicate the medians, boxes indicate 25th to 75th percentiles, and whiskers indicate 10th and 90th percentiles.

Figure 3. HIV-specific antibodies exhibit dramatically inflammatory glycosylation profiles.

(A) Relative prevalence of glycans on bulk plasma and antigen-specific antibodies. Statistical analysis was performed using Wilcox matched-pairs signed-rank test. NF, nonfucosylated. **P < 0.005; ***P < 0.0005. (B) Glycosylation profiles of HIV-specific antibodies for all glycan structures (first panel), galactose content (second panel), and fucose content (third panel).

Figure 4. Comparative glycan profiles of bulk plasma and HIV-specific antibodies in each HIV-positive subject class.

(A) Significant differences observed between bulk and HIV-specific antibodies within each subject group. (B) Significant differences observed in HIV-specific antibodies among subject groups. In box-and-whisker plots, horizontal bars indicate the medians, boxes indicate 25th to 75th percentiles, and whiskers indicate 10th and 90th percentiles. Dots outside of box plots represent outliers. *P < 0.05.

Figure 5. Antiviral activity is associated with specific glycoforms and FCGR3A binding.

(A) Total agalactosylated (G0 class) glycans prevalence correlates positively with ADCVI activity. (B) Bigalactosylated (G2 class) glycans prevalence correlates negatively with NK cell ADCVI activity. (C) Bulk plasma antibody affinity for FCGR3A, measured as Biacore response units, correlates positively with ADCVI activity. (D) Impact of enzymatic treatment with a galactoside on agalactosylated glycan and (E) bigalactosylated glycan content. (F) Binding of treated and untreated antibody samples to FCGR3A beads. (A–C and F) Individual symbols represent each subject tested, and horizontal lines represent the median for a given group of clinically related subjects.

Figure 6. Transcriptional profiling of GTs and glycosidases relevant to IgG glycans in peripheral B cells.

(A) GTs involved in synthesis of all antibody glycans, the MGAT1 and MGAT2 enzymes involved in the conversion of oligomannose to complex N-glycans and ALG2 involved in initiating N-linked glycosylation, were upregulated in untreated subjects. (B) Fucosyltransferase FUT8 expression was increased in untreated progressors, while fucosidase FUCA2 was increased in controllers and untreated progressors. (C) Galactose transferases B4GALT1 and B4GALT3 were decreased in HIV-infected subjects. The y axis represents transcript expression relative to a housekeeping gene. Individual symbols represent each subject tested, and horizontal lines represent the median for a given group of clinically related subjects. *P < 0.05; **P < 0.005; ***P < 0.0005.