Detection of N-glycolyl-neuraminic acid-containing glycolipids in human skin

Humans lack the enzyme that produces the sialic acid N-glycolyl neuraminic acid (Neu5Gc), but several lines of evidence have shown that Neu5Gc can be taken up by mammalian food sources and replace the common human sialic acid N-acetyl neuraminic acid (Neu5Ac) in glycans. Cancer tissue has been shown to have increased the presence of Neu5Gc and Neu5Gc-containing glycolipids such as the ganglioside GM3, which have been proposed as tumor-specific antigens for antibody treatment. Here, we show that a previously described antibody against Neu5Gc-GM3 is binding to Neu5GC-containing gangliosides and is strongly staining different cancer tissues. However, we also found a strong intracellular staining of keratinocytes of healthy skin. We confirmed this staining on freshly isolated keratinocytes by flow cytometry and detected Neu5Gc by mass spectrometry. This finding implicates that non-human Neu5Gc can be incorporated into gangliosides in human skin, and this should be taken into consideration when targeting Neu5Gc-containing gangliosides for cancer immunotherapy

Structure-Guided Redesign Improves NFL HIV Env Trimer Integrity and Identifies an Inter-Protomer Disulfide Permitting Post-Expression Cleavage

Soluble HIV-1 envelope glycoprotein (Env) trimers are under active investigation as vaccine candidates in relevant pre-clinical models. Like SOSIPs, the cleavage-independent native flexibly linked (NFL) trimers are faithful mimics of the Env spike. Here, we analyzed multiple new designs to explore alternative modifications, informing tertiary interactions, while maintaining NFL trimer homogeneity and integrity. Accordingly, we performed a proline (P) substitution screen in the gp41 heptad repeat 1 region, identifying other trimer-enhancing Ps, including L555P. This P improved trimer integrity compared to I559P in selected properties. Next, we screened 15 structure-guided potential cysteine pairs in gp140 and found that A501C-L663C (“CC2”) forms an inter-protomer disulfide bond that demonstrably increased NFL trimer thermostability. We combined these two approaches with trimer-derived substitutions, coupled with glycine substitutions at helix-to-coil transitions, developed by our group. To increase the exposure of the fusion peptide (FP) N-terminus, we engineered an enterokinase (EK) cleavage site upstream of the FP for controlled post-expression cleavage. In combination, the redesigns resulted in highly stable and homogeneous NFL mimics derived from different clades. Following recombinant EK cleavage, the NFL trimers retained covalent linkage, maintaining a native-like structure while displaying enhanced stability and favorable antigenic features. These trimers also displayed increased exposure of neutralizing epitopes in the FP and gp120/gp41 interface, while retaining other neutralizing epitopes and occluding non-neutralizing elements. This array of Env-structure-guided designs reveals additional interactive regions in the prefusion state of the HIV Env spike, affording the development of novel antigens and immunogens

Improving the immunogenicity of native-like HIV-1 envelope trimers by hyperstabilization

The production of native-like recombinant versions of the HIV-1 envelope glycoprotein (Env) trimer requires overcoming the natural flexibility and instability of the complex. The engineered BG505 SOSIP.664 trimer mimics the structure and antigenicity of native Env. Here, we describe how the introduction of new disulfide bonds between the glycoprotein (gp)120 and gp41 subunits of SOSIP trimers of the BG505 and other genotypes improves their stability and antigenicity, reduces their conformational flexibility, and helps maintain them in the unliganded conformation. The resulting next-generation SOSIP.v5 trimers induce strong autologous tier-2 neutralizing antibody (NAb) responses in rabbits. In addition, the BG505 SOSIP.v6 trimers induced weak heterologous NAb responses against a subset of tier-2 viruses that were not elicited by the prototype BG505 SOSIP.664. These stabilization methods can be applied to trimers from multiple genotypes as components of multivalent vaccines aimed at inducing broadly NAbs (bNAbs)

Fine structure of the HIV-1 glycan shield - Supplementary information

Data were generated in the course of a DPhil thesis at the Department of Biochemistry, University of Oxford, PI: Dr. Max Crispi

Fine structure of the HIV-1 glycan shield - Supplementary information

Data were generated in the course of a DPhil thesis at the Department of Biochemistry, University of Oxford, PI: Dr. Max Crispi

Fine structure of the HIV-1 glycan shield

The HIV-1 envelope glycoprotein trimer (Env) is covered by an extensive array of glycans that shield it from immune surveillance. The high density of glycans on the trimer surface imposes steric constraints that limit the actions of glycan processing enzymes, such that multiple under-processed structures remain on specific locations. These oligomannose-type glycans are recognized by broadly neutralizing antibodies (bNAbs) that are not thwarted by the glycan shield but, perhaps paradoxically, target it. In multiple studies, bNAbs have been shown to be capable of providing passive protection from viral challenge, making Env a focus of antibody-mediated vaccine design. Here, the development of a workflow for the semi-quantitative, site-specific N-glycosylation analysis of a soluble recombinant, native-like trimer mimic (BG505 SOSIP.664) is reported. The resulting data reveal a mosaic of dense clusters on the outer domain of Env and allow mapping the extremes of simplicity and diversity of glycan processing. Although individual sites usually minimally affect the global integrity of the glycan shield, examples are identified of how deleting certain glycans can subtly influence neutralization by bNAbs that bind at distant sites. Env is a trimer of heterodimers of gp120 and gp41, which is generated by cleavage of an endogenous protease. In this thesis, the detailed effect of protease cleavage on glycan processing is examined by comparing the site-specific N-glycosylation profiles of the native-like trimer mimic to the corresponding uncleaved pseudotrimer and the matched gp120 monomer. Trimer-associated glycan remodeling forms a localized subdomain of the native mannose patch. Furthermore, the glycosylation analysis of further Env immunogens â a glycan-depleted trimer and a flexibly-linked, uncleaved trimer (both based on BG505 SOSIP.664) â provides important insights into the robustness of the HIV-1 glycan shield and the Env maturation pathway. Overall, this thesis reveals how structural constraints shape Env glycosylation and the network of bNAb-targeted glycans that should be preserved on recombinant vaccine candidates.</p

Structural principles controlling HIV envelope glycosylation

The heavily glycosylated, trimeric HIV-1 envelope (Env) protein is the sole viral protein exposed on the HIV-1 virion surface and is thus a main focus of antibody-mediated vaccine development. Dense glycosylation at the outer domain of Env constrains normal enzymatic processing, stalling the glycans at immature oligomannose-type structures. Furthermore, native trimerization imposes additional steric constraints, which generate an extensive ‘trimer-induced mannose patch’. Importantly, the immature glycans present a highly conserved feature of the virus that is targeted by broadly neutralizing antibodies. Quantitative mass spectrometry of glycopeptides together with structures of the trimeric viral-spike define the steric principles controlling processing and provide a detailed map of the glycan shield.</p

Targeting glycans of HIV envelope glycoproteins for vaccine design

The surface of the envelope spike of the human immunodeficiency virus (HIV) is covered with a dense array of glycans, which is sufficient to impede the host antibody response while maintaining a window for receptor recognition. The glycan density significantly exceeds that typically observed on self glycoproteins and is sufficiently high to disrupt the maturation process of glycans, from oligomannose- to complex-type glycosylation, that normally occurs during glycoprotein transit through the secretory system. It is notable that this generates a degree of homogeneity not seen in the highly mutated protein moiety. The conserved, close glycan packing and divergences from default glycan processing give a window for immune recognition. Encouragingly, in a subset of individuals, broadly neutralizing antibodies (bNAbs) have been isolated that recognize these features and are protective in passive-transfer models. Here, we review the recent advances in our understanding of the glycan shield of HIV and outline the strategies that are being pursued to elicit glycan-binding bNAbs by vaccination

Global N-Glycan site occupancy of HIV-1 gp120 by metabolic engineering and high-resolution intact mass spectrometry

A vital step in HIV vaccine development strategies has been the observation that some infected individuals generate broadly neutralizing antibodies that target the glycans on the surface of HIV-1 gp120. These antibodies target glycan epitopes on viral envelope spikes, and yet the positions and degree of occupancy of glycosylation sites is diverse. Therefore, there is a need to understand glycosylation occupancy on recombinant immunogens. The sheer number of potential glycosylation sites and degree of chemical heterogeneity impedes assessing the global sequon occupancy of gp120 glycoforms. Here, we trap the glycan processing of recombinant gp120 to generate homogeneous glycoforms, facilitating occupancy assessment by intact mass spectrometry. We show that gp120 monomers of the BG505 strain contain either fully occupied sequons or missing the equivalent of one and sometimes two glycans across the molecule. This biosynthetic engineering approach enables the analysis of therapeutically important glycoproteins otherwise recalcitrant to analysis by native mass spectrometry.</p