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.

Biologically active conformations of the IgG1 Fc homodimer are maintained by multiple hydrophobic interactions between the protein surface and the N-glycan. The Fc glycan modulates biological effector functions, including antibody-dependent cellular cytotoxicity (ADCC) which is mediated in part through the activatory Fc receptor, FcγRIIIA. Consistent with previous reports, we found that site-directed mutations disrupting the protein–carbohydrate interface (F241A, F243A, V262E, and V264E) increased galactosylation and sialylation of the Fc and, concomitantly, reduced the affinity for FcγRIIIA. We rationalized this effect by crystallographic analysis of the IgG1 Fc F241A mutant, determined here to a resolution of 1.9 Å, which revealed localized destabilization of this glycan–protein interface. Given that sialylation of Fc glycans decreases ADCC, one explanation for the effect of these mutants on FcγRIIIA binding is their increased sialylation. However, a glycan-engineered IgG1 with hypergalactosylated and hypersialylated glycans exhibited unchanged binding affinity to FcγRIIIA. Moreover, when we expressed these mutants as a chemically uniform (Man5GlcNAc2) glycoform, the individual effect of each mutation on FcγRIIIA affinity was preserved. This effect was broadly recapitulated for other Fc receptors (FcγRI, FcγRIIA, FcγRIIB, and FcγRIIIB). These data indicate that destabilization of the glycan–protein interactions, rather than increased galactosylation and sialylation, modifies the Fc conformation(s) relevant for FcγR binding. Engineering of the protein–carbohydrate interface thus provides an independent parameter in the engineering of Fc effector functions and a route to the synthesis of new classes of Fc domain with novel combinations of affinities for activatory and inhibitory Fc receptors.

The N-glycan of the IgG constant region (Fc) plays a central role in tuning and directing multiple antibody functions in vivo, including antibody-dependent cellular cytotoxicity, complement deposition, and the regulation of inflammation, among others. However, traditional methods of N-glycan analysis, including HPLC and mass spectrometry, are technically challenging and ill suited to handle the large numbers of low concentration samples analyzed in clinical or animal studies of the N-glycosylation of polyclonal IgG. Here we describe a capillary electrophoresis-based technique to analyze plasma-derived polyclonal IgG-glycosylation quickly and accurately in a cost-effective, sensitive manner that is well suited for high-throughput analyses. Additionally, because a significant fraction of polyclonal IgG is glycosylated on both Fc and Fab domains, we developed an approach to separate and analyze domain-specific glycosylation in polyclonal human, rhesus and mouse IgGs. Overall, this protocol allows for the rapid, accurate, and sensitive analysis of Fc-specific IgG glycosylation, which is critical for population-level studies of how antibody glycosylation may vary in response to vaccination or infection, and across disease states ranging from autoimmunity to cancer in both clinical and animal studies.

Protein endoglycosidases are useful for biocatalytic alteration of glycans on protein surfaces, but the currently limited selectivity of endoglycosidases has prevented effective manipulation of certain N-linked glycans widely found in nature. Here we reveal that a bacterial endoglycosidase from Streptococcus pyogenes , EndoS, is complementary to other known endoglycosidases (EndoA, EndoH) used for current protein remodeling. It allows processing of complex-type N-linked glycans +/- core fucosylation but does not process oligomannose- or hybrid-type glycans. This biocatalytic activity now addresses previously refractory antibody glycoforms.

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