Influence of glycosylation on the immunogenicity and antigenicity of viral immunogens

Abstract

A key aspect of successful viral vaccine design is the elicitation of neutralizing antibodies targeting viral attachment and fusion glycoproteins that embellish viral particles. This observation has catalyzed the development of numerous viral glycoprotein mimetics as vaccines. Glycans can dominate the surface of viral glycoproteins and as such, the viral glycome can influence the antigenicity and immunogenicity of a candidate vaccine. In one extreme, glycans can form an integral part of epitopes targeted by neutralizing antibodies and are therefore considered to be an important feature of key immunogens within an immunization regimen. In the other extreme, the existence of peptide and bacterially expressed protein vaccines shows that viral glycosylation can be dispensable in some cases. However, native-like glycosylation can indicate native-like protein folding and the presence of conformational epitopes. Furthermore, going beyond native glycan mimicry, in either occupancy of glycosylation sites or the glycan processing state, may offer opportunities for enhancing the immunogenicity and associated protection elicited by an immunogen. Here, we review key determinants of viral glycosylation and how recombinant immunogens can recapitulate these signatures across a range of enveloped viruses, including HIV-1, Ebola virus, SARS-CoV-2, Influenza and Lassa virus. The emerging understanding of immunogen glycosylation and its control will help guide the development of future vaccines in both recombinant protein- and nucleic acid-based vaccine technologies.

Keywords: Glycoprotein; Glycosylation; Immunogen; Vaccine; Virus.

Figure 1 –. Factors shaping viral glycosylation.

A) Structural inhibition of glycan processing can induce elevated levels of oligomannose-type glycans. B) The rate of ER-Golgi trafficking and localization of viral glycoproteins can be impacted by N- or C-terminal signal sequences. C) The number of glycan sites across viral glycoproteins can change in response to consistent exposure to the host immune system. D) Viral glycan processing and composition can differ depending on the viral host. E) The differential glycosyltransferase expression in different cell types can result in differential glycan processing. Schematics of the factors influencing viral glycosylation are arranged across the ER/Golgi trafficking pathway according to the extent of glycan processing that they are each most closely associated with, across a scale of least- to most-processed (oligomannose-to-complex).

Figure 2 –. Methods to stabilize soluble viral glycoproteins.

A) Example protein schematic demonstrating commonly used protein engineering approaches to stabilize the non-covalent trimeric conformation following loss of the transmembrane domain (TMD), colored light blue. TMD replacement using foldon domains is shown in yellow. The dashed boxes represent regions of viral glycoproteins where the highlighted engineering approaches can be employed. B) Summary of key innovations around recombinant protein stabilization including covalent linkers, proline stabilization (red circles), the use of foldon domains and the introduction of additional components to promote nanoparticle display. Truncation of the protein sequence upstream of the TMD is represented using scissors.

Figure 3 –. Glycan-dependent neutralizing antibody epitopes of HIV-1 Env and LASV GPC.

A) Examples of complete glycan dependence of glycan-targeting HIV-1 and LASV bnAbs are mapped on the trimers. Glycan sites displaying an integral epitope for glycan-dependent bnAbs are individually colored. Anti-glycan HIV-1 bnAbs include V2-apex bnAbs, BG18, and PGT135. Anti-glycan LASV bnAbs include 12.1F, 19.7E and LAVA01. Hydrogen bond interactions between HIV-1 bnAbs, PG9 and BG18 (B), LASV bnAbs, 19.7E and LAVA01 (C), and N-linked glycans that form part of their composite epitopes. N-linked glycans are colored according to those in panel A. Hydrogen bonds are colored cyan, and nAb Fab regions are colored gray. PDB accession codes: 7T77, 6DFG, 8EJI, 7SGF.

Figure 4 –. Glycoengineering techniques to improve nAb engagement by viral immunogens.

A) Inhibition of ER α-mannosidase I using kifunensine converts all N-linked glycans on the protein to Man₉GlcNAc₂. Treatment with endoglycosidase H (Endo H) cleaves the glycans between the core GlcNAc residues, yielding a single GlcNAc that remains attached to the Asn. B) The generation GnTI KO cell lines truncate glycan processing at Man₅GlcNAc₂, thereby inhibiting complex- and hybrid-type glycan processing. C) Endo H treatment of viral glycoproteins with dense glycan shields. Only glycosidase-accessible high mannose glycans will be cleaved, leaving those which are glycosidase-inaccessible intact.

Figure 5 –. Antibody responses to glycan holes in HIV-1 Env and Ebola GP.

A) An undesirable antibody response to the N611 glycan hole on gp41. RM20E1, a macaque glycan hole non-nAb, binds to the N611 site in the absence of glycans, which dominates mAb responses following immunization with immunogens possessing low N611 glycan occupancy. High glycan occupancy at N611 sterically inhibits RM20E1 binding. B) Mature VRC01-class bnAbs use glycans at N276 as a scaffold to bind the proteinaceous epitopes near the CD4bs. C) A simplified view of the germline-targeting approach using HIV-1 Env mimetics created using BioRender.com. Germline-targeting priming immunogens that engage VRC01-class germlines are devoid of the N276 site to enhance VRC01 precursor access to its epitope. Subsequent guiding immunogens reintroduce the N276 glycan site to further guide VRC01 bnAb maturation. D) Glycan cap bnAbs EBOV-293 and -442 bound to EBOV GP. The CDR contacts of both bnAbs with their epitopes within the MLD-anchor are based upon the data presented in Murin et al. (2021) (Murin et al., 2021). PBD accession codes used: 6VLR; 6V8X; 7KEX; 7KFB.