Evolution of HIV-1 Env glycosylation and the resistance to neutralisation by broadly neutralising antibodies

Abstract

Broadly neutralising antibodies (bnAbs) against the HIV-1 Envelope glycoprotein (Env) target conserved sites of vulnerability to prevent infection. Vaccine strategies aim to elicit bnAbs through antigenic mimicry of Env, which not only includes protein components but also incorporates N-linked glycans. The extensive immunologically ‘self’ glycan shield forms an integral part of the antigenic surface of Env, both shaping bnAb epitopes and effectively impeding their development in vivo. As current leading vaccine candidates focus on eliciting specific bnAbs, understanding the mechanisms by which HIV-1 evades neutralisation is essential. Insights from passive bnAb therapy trials highlight the rate at which escape mutations are selected for, and the nature of these mutations. Beyond sequence variation, modulation of the glycan shield offers HIV-1 a potent means of evading neutralisation.
This thesis aims to identify, characterise, and test glycan-mediated mechanisms of resistance to bnAbs. A workflow was developed to produce and purify non-replicating HIV-1 pseudoviruses for site-specific glycosylation analysis using liquid chromatography-mass spectrometry (LC-MS). Utilising a pseudovirus system allows native-like glycosylation to be captured. Analyses of HIV-1 pseudovirus expressed in T-lymphocyte and B-lymphocyte cell lines, as well as the prototypical HEK 293 cell line, revealed broad conservation of glycosylation signatures at key bnAb epitopes. These observations substantiate the analysis of globally representative pseudovirus panels produced in a manner similar to those used for virus neutralisation assays. This revealed correlations between bnAb resistance and suboptimal glycan site occupancy, both in a direct manner and through the enhancement of glycan processing at key epitope sites. Additionally, the investigation of CD4 binding site-resistant pseudoviruses revealed that proximal glycans contribute to a bnAb resistance phenotype that can be partially restored following their removal.
Collectively, this thesis demonstrates that non-replicating HIV-1 pseudovirus formats can be flexibly harnessed to assess the site-specific glycosylation of a range of different Env stains. This reveals that, while the Env glycan shield is broadly conserved, site-specific alterations can modulate bnAb recognition and facilitate immune escape. This work substantially expands upon the current available knowledge pertaining to the site-specific glycan analysis of HIV-1 pseudoviruses.

More information

Identifiers

ORCID for Maddy Newby: orcid.org/0000-0003-0624-245X

ORCID for Max Crispin: orcid.org/0000-0002-1072-2694

ORCID for Bill Keevil: orcid.org/0000-0003-1917-7706

Catalogue record

Export record