Snezana Vasiljevic

Envelope glycans of immunodeficiency virions are almost entirely oligomannose antigens

The envelope spike of HIV is one of the most highly N-glycosylated structures found in nature. However, despite extensive research revealing essential functional roles in infection and immune evasion, the chemical structures of the glycans on the native viral envelope glycoprotein gp120—as opposed to recombinantly generated gp120—have not been described. Here, we report on the identity of the N-linked glycans from primary isolates of HIV-1 (clades A, B, and C) and from the simian immunodeficiency virus. MS analysis reveals a remarkably simple and highly conserved virus-specific glycan profile almost entirely devoid of medial Golgi-mediated processing. In stark contrast to recombinant gp120, which shows extensive exposure to cellular glycosylation enzymes (>70% complex type glycans), the native envelope shows barely detectable processing beyond the biosynthetic intermediate Man5GlcNAc2 (<2% complex type glycans). This oligomannose (Man5–9GlcNAc2) profile is conserved across primary isolates and geographically divergent clades but is not reflected in the current generation of gp120 antigens used for vaccine trials. In the context of vaccine design, we also note that Manα1→2Man-terminating glycans (Man6–9GlcNAc2) of the type recognized by the broadly neutralizing anti-HIV antibody 2G12 are 3-fold more abundant on the native envelope than on the recombinant monomer and are also found on isolates not neutralized by 2G12. The Manα1→2Man residues of gp120 therefore provide a vaccine target that is physically larger and antigenically more conserved than the 2G12 epitope itself. This study revises and extends our understanding of the glycan shield of HIV with implications for AIDS vaccine design.

Composition and Antigenic Effects of Individual Glycan Sites of a Trimeric HIV-1 Envelope Glycoprotein

Summary The HIV-1 envelope glycoprotein trimer is covered by an array of N-linked glycans that shield it from immune surveillance. The high density of glycans on the trimer surface imposes steric constraints limiting the actions of glycan-processing enzymes, so that multiple under-processed structures remain on specific areas. These oligomannose glycans are recognized by broadly neutralizing antibodies (bNAbs) that are not thwarted by the glycan shield but, paradoxically, target it. Our site-specific glycosylation analysis of a soluble, recombinant trimer (BG505 SOSIP.664) maps the extremes of simplicity and diversity of glycan processing at individual sites and reveals a mosaic of dense clusters of oligomannose glycans on the outer domain. Although individual sites usually minimally affect the global integrity of the glycan shield, we identify examples of how deleting some glycans can subtly influence neutralization by bNAbs that bind at distant sites. The network of bNAb-targeted glycans should be preserved on vaccine antigens.

Dissecting the molecular mechanism of IVIg therapy: the interaction between serum IgG and DC-SIGN is independent of antibody glycoform or Fc domain.

Intravenous immunoglobulin (IVIg) therapy is used to treat a wide range of autoimmune conditions and consists of pooled immunoglobulin G (IgG) from healthy donors. The immunosuppressive effects of IVIg are, in part, attributed to terminal α2,6-linked sialic acid residues on the N-linked glycans of the IgG Fc (fragment crystallizable) domain. This α2,6-sialylated Fc (sFc) has been reported to bind to the carbohydrate recognition domain (CRD) of the cell-surface lectin DC-SIGN (dendritic cell-specific intercellular adhesion molecule-3-grabbing non-integrin) and its murine orthologue SIGN-R1 (specific intracellular adhesion molecule-grabbing non-integrin R1) and, via this interaction, to signal the downstream expression of immunosuppressive cytokines and receptors. Consistent with this model, the antiinflammatory effect of IVIg treatment is abolished in a murine knock-out of SIGN-R1 and can be restored by a knock-in with human DC-SIGN. In contrast, however, existing glycan array and X-ray crystallographic studies indicate that the CRDs of both SIGN-R1 and DC-SIGN bind to a restricted set of primarily oligomannose-type glycans that does not include the glycans found on sFc. We attempted to reconcile these immunological and biophysical observations. We first generated hypersialylated, desialylated, deglycosylated and untreated serum IgG and found that the affinity for the complete extracellular region of the DC-SIGN tetramer was similar for all antibody glycoforms. Moreover, the binding could be attributed to cross-reactive, polyclonal Fab (fragment antigen-binding) specificities in serum as neither recombinant Fc nor sFc bound to DC-SIGN. In addition, serum IgG exhibited no competition against known ligands of the DC-SIGN CRD. These findings lead us to suggest that IVIg therapy does not involve binding of IgG Fc to DC-SIGN and that alternative cell-surface lectins are required for the antiinflammatory activity of sFc.

Ion Mobility Mass Spectrometry for Extracting Spectra of N-Glycans Directly from Incubation Mixtures Following Glycan Release: Application to Glycans from Engineered Glycoforms of Intact, Folded HIV gp120

The analysis of glycosylation from native biological sources is often frustrated by the low abundances of available material. Here, ion mobility combined with electrospray ionization mass spectrometry have been used to extract the spectra of N-glycans released with PNGase F from a serial titration of recombinantly expressed envelope glycoprotein, gp120, from the human immunodeficiency virus (HIV). Analysis was also performed on gp120 expressed in the α-mannosidase inhibitor, and in a matched mammalian cell line deficient in GlcNAc transferase I. Without ion mobility separation, ESI spectra frequently contained no observable ions from the glycans whereas ions from other compounds such as detergents and residual buffer salts were abundant. After ion mobility separation on a Waters T-wave ion mobility mass spectrometer, the N-glycans fell into a unique region of the ion mobility/m/z plot allowing their profiles to be extracted with good signal:noise ratios. This method allowed N-glycan profiles to be extracted from crude incubation mixtures with no clean-up even in the presence of surfactants such as NP40. Furthermore, this technique allowed clear profiles to be obtained from sub-microgram amounts of glycoprotein. Glycan profiles were similar to those generated by MALDI-TOF MS although they were more susceptible to double charging and fragmentation. Structural analysis could be accomplished by MS/MS experiments in either positive or negative ion mode but negative ion mode gave the most informative spectra and provided a reliable approach to the analysis of glycans from small amounts of glycoprotein.

Engineering hydrophobic protein-carbohydrate interactions to fine-tune monoclonal antibodies.

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.

Glycan Microheterogeneity at the PGT135 Antibody Recognition Site on HIV-1 gp120 Reveals a Molecular Mechanism for Neutralization Resistance

ABSTRACT Broadly neutralizing antibodies have been isolated that bind the glycan shield of the HIV-1 envelope spike. One such antibody, PGT135, contacts the intrinsic mannose patch of gp120 at the Asn332, Asn392, and Asn386 glycosylation sites. Here, site-specific glycosylation analysis of recombinant gp120 revealed glycan microheterogeneity sufficient to explain the existence of a minor population of virions resistant to PGT135 neutralization. Target microheterogeneity and antibody glycan specificity are therefore important parameters in HIV-1 vaccine design.

HIV-1 Glycan Density Drives the Persistence of the Mannose Patch within an Infected Individual.

ABSTRACT The HIV envelope glycoprotein (Env) is extensively modified with host-derived N-linked glycans. The high density of glycosylation on the viral spike limits enzymatic processing, resulting in numerous underprocessed oligomannose-type glycans. This extensive glycosylation not only shields conserved regions of the protein from the immune system but also acts as a target for anti-HIV broadly neutralizing antibodies (bnAbs). In response to the host immune system, the HIV glycan shield is constantly evolving through mutations affecting both the positions and numbers of potential N-linked glycosylation sites (PNGSs). Here, using longitudinal Env sequences from a clade C-infected individual (CAP256), we measured the impact of the shifting glycan shield during HIV infection on the abundance of oligomannose-type glycans. By analyzing the intrinsic mannose patch from a panel of recombinant CAP256 gp120s displaying high protein sequence variability and changes in PNGS number and positioning, we show that the intrinsic mannose patch persists throughout the course of HIV infection and correlates with the number of PNGSs. This effect of the glycan density on the processing state was also supported by the analysis of a cross-clade panel of recombinant gp120 glycoproteins. Together, these observations underscore the importance of glycan clustering for the generation of carbohydrate epitopes for anti-HIV bnAbs. The persistence of the intrinsic mannose patch over the course of HIV infection further highlights this epitope as an important target for HIV vaccine strategies. IMPORTANCE Development of an HIV vaccine is critical for control of the HIV pandemic, and elicitation of broadly neutralizing antibodies (bnAbs) is likely to be a key component of a successful vaccine response. The HIV envelope glycoprotein (Env) is covered in an array of host-derived N-linked glycans often referred to as the glycan shield. This glycan shield is a target for many of the recently isolated anti-HIV bnAbs and is therefore under constant pressure from the host immune system, leading to changes in both glycan site frequency and location. This study aimed to determine whether these genetic changes impacted the eventual processing of glycans on the HIV Env and the susceptibility of the virus to neutralization. We show that despite this variation in glycan site positioning and frequency over the course of HIV infection, the mannose patch is a conserved feature throughout, making it a stable target for HIV vaccine design.

Isomer Information from Ion Mobility Separation of High-Mannose Glycan Fragments

AbstractExtracted arrival time distributions of negative ion CID-derived fragments produced prior to traveling-wave ion mobility separation were evaluated for their ability to provide structural information on N-linked glycans. Fragmentation of high-mannose glycans released from several glycoproteins, including those from viral sources, provided over 50 fragments, many of which gave unique collisional cross-sections and provided additional information used to assign structural isomers. For example, cross-ring fragments arising from cleavage of the reducing terminal GlcNAc residue on Man8GlcNAc2 isomers have unique collision cross-sections enabling isomers to be differentiated in mixtures. Specific fragment collision cross-sections enabled identification of glycans, the antennae of which terminated in the antigenic α-galactose residue, and ions defining the composition of the 6-antenna of several of the glycans were also found to have different cross-sections from isomeric ions produced in the same spectra. Potential mechanisms for the formation of the various ions are discussed and the estimated collisional cross-sections are tabulated. Graphical Abstractᅟ

Signature of Antibody Domain Exchange by Native Mass Spectrometry and Collision-Induced Unfolding

The development of domain-exchanged antibodies offers a route to high-affinity targeting to clustered multivalent epitopes, such as those associated with viral infections and many cancers. One strategy to generate these antibodies is to introduce mutations into target antibodies to drive domain exchange using the only known naturally occurring domain-exchanged anti-HIV (anti-human immunodeficiency virus) IgG1 antibody, 2G12 , as a template. Here, we show that domain exchange can be sensitively monitored by ion-mobility mass spectrometry and gas-phase collision-induced unfolding. Using native 2G12 and a mutated form that disrupts domain exchange such that it has a canonical IgG1 architecture ( 2G12 I19R ), we show that the two forms can be readily distinguished by their unfolding profiles. Importantly, the same signature of domain exchange is observed for both intact antibody and isolated Fab fragments. The development of a mass spectrometric method to detect antibody domain exchange will enable rapid screening and selection of candidate antibodies engineered to exhibit this and other unusual quaternary antibody architectures.