Immunogen glycosylation in protein and RNA delivery systems

Abstract

Extensive glycosylation of viral glycoproteins is a key feature of the antigenic surface of viruses, yet glycan processing can be influenced by the assembly of the protein and production systems. These are important factors to consider in vaccine design, as non-native like assembly and the presence of artificial glycan holes can potentially influence the immune response. To address these concerns, various stabilizing approaches, including the incorporation of proline, disulphide-bonds, glycine-rich linker,and many more, have been employed in the design of recombinant protein immunogens to ensure correct assembly and enhanced expression. Additionally, post-expression purification is typically implemented in recombinant-based immunogens to capture properly folded protein and minimize non-native material. A common strategy that has been employed in the stabilization of HIV-1 immunogens, the introduction of proline substitutions, has been adopted in SARS-CoV-2 vaccine development. This thesis reveals the influence of these mutations on SARS-CoV-2 Spike (S) protein conformations and glycan compositions. Furthermore, this study compares the glycan compositions presented by S protein and receptor binding domain (RBD). Additionally, a serology study of chemically treated S protein with kifunensine (ER-mannosidase I inhibitor), aimed at eliciting all oligomannose-type glycans, highlights that the immune response following SARS-CoV-2 infection, with respect to immunoglobin binding, is not dictated by the glycan processing state of the S protein. This is, however, not the case in HIV-1 vaccine design, in which glycans play a crucial role in structural integrity and act as an epitope for many broadly neutralizing antibodies. In contrast to traditional protein-based immunogens, RNA-based vaccines have emerged as a highly effective delivery platform due to its rapid adaptability. However, one potential limitation is that the immunogen is entirely encoded by nucleotide sequence, and thus, there is no opportunity at the post-expression level to control immunogen assembly. In HIV-1 vaccine research, this is of particular importance because non-native epitopes can compromise the desired immune response, and native immunogen assembly is essential for the presentation of glycan-based epitopes targeted by broadly neutralizing antibodies. The work presented here reveals the archetypal protein architecture and glycosylation of the HIV-1 Env expressed via an RNA-based immunogen. Furthermore, this thesis explores the native-like signatures of the Env and the glycan compositions of the material derived from cell-types, muscle, and dendritic cells, which likely produce immunogens near the sites of intramuscular RNA injection. Finally, this study shows that nucleotide editing can enhance the glycan occupancy of RNA-derived immunogens. The data presented in this thesis emphasize the significance of considering glycan site occupancy and glycan heterogeneity when developing and assessing vaccine candidates.

More information

Identifiers

ORCID for Himanshi Chawla: orcid.org/0000-0001-9828-6593

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

ORCID for Paul Skipp: orcid.org/0000-0002-2995-2959

Catalogue record

Export record