Glycan masking of a non-neutralising epitope enhances neutralising antibodies targeting the RBD of SARS-CoV-2 and its variants

Abstract

The accelerated development of the first generation COVID-19 vaccines has saved millions of lives, and potentially more from the long-term sequelae of SARS-CoV-2 infection. The most successful vaccine candidates have used the full-length SARS-CoV-2 spike protein as an immunogen. As expected of RNA viruses, new variants have evolved and quickly replaced the original wild-type SARS-CoV-2, leading to escape from natural infection or vaccine induced immunity provided by the original SARS-CoV-2 spike sequence. Next generation vaccines that confer specific and targeted immunity to broadly neutralising epitopes on the SARS-CoV-2 spike protein against different variants of concern (VOC) offer an advance on current booster shots of previously used vaccines. Here, we present a targeted approach to elicit antibodies that neutralise both the ancestral SARS-CoV-2, and the VOCs, by introducing a specific glycosylation site on a non-neutralising epitope of the RBD. The addition of a specific glycosylation site in the RBD based vaccine candidate focused the immune response towards other broadly neutralising epitopes on the RBD. We further observed enhanced cross-neutralisation and cross-binding using a DNA-MVA CR19 prime-boost regime, thus demonstrating the superiority of the glycan engineered RBD vaccine candidate across two platforms and a promising candidate as a broad variant booster vaccine.

Inducing enhanced neutralizing antibodies against broad SARS-CoV-2 variants through glycan-shielding multiple non-neutralizing epitopes of RBD

“…In another study, an RBD subunit vaccine with a single glycan mask at N519 (via H519N/P521T or H519N/P521S mutation) demonstrated a significant enhancement of neutralizing antibodies against a broad panel of SARS-CoV-2 variants ( 52 ). Glycan masking at a closely neighboring residue, N521 (via the P521N mutation), also led to increased neutralizing antibodies against the Delta (B.1.617.2) and Gamma (P.1) variants when mice were immunized with DNA/Modified Vaccinia Ankara (MVA)-delivered RBD ( 51 ). However, when the glycan mask at N519 (via the H519N/P521T mutation) was introduced to the full-length S gene, a decrease in neutralizing ability against the SARS-CoV-2 prototype, and Alpha (B.1.1.7) and Delta (B.1.617.2) variants was observed ( 50 ).…”

Engineering a SARS-CoV-2 vaccine targeting the RBD cryptic-face via immunofocusing

“…Previous efforts to create an immunofocused-RBD vaccine have utilized techniques such as epitope dissection, 39,40 mosaic display, 41,42 epitope masking via glycosylation, [43][44][45][46][47] and crossstrain boosting. [48][49][50] In this work, we employ the immunofocusing technique called protect, modify, deprotect (PMD) 51 to create a cryptic face-targeting RBD vaccine.…”

Contemporaneous SARS-CoV-2-Neutralizing Antibodies Mediated by N-glycan Shields

“…One proposed theory for this phenomenon is that glycans shield the protein surface so that the protein folding deviates from its native form [ 30 ] ( Figure 1 B). The receptor binding domain (RBD) of the SARS-CoV-2 S protein, which is responsible for recognizing and binding to the ACE2 receptor, is found in a closed or inaccessible receptor conformation, preventing it from being recognized by immune cells [ 35 ]. However, the receptor binding motif (RBM), the most crucial component for attachment to the ACE2 receptor, remains untouched and stable [ 35 ].…”

“…The receptor binding domain (RBD) of the SARS-CoV-2 S protein, which is responsible for recognizing and binding to the ACE2 receptor, is found in a closed or inaccessible receptor conformation, preventing it from being recognized by immune cells [ 35 ]. However, the receptor binding motif (RBM), the most crucial component for attachment to the ACE2 receptor, remains untouched and stable [ 35 ]. Glycan masking is also implicated in this immune shifting, or a shift in the immune response away from specific epitopes while focusing on other epitopes to decrease the extent of off-target antibody production.…”

“…A new approach introduces specific glycosylation sites onto the RBD to counteract this issue and thus elicit more robust immune responses to both previous and mutated variants. A novel study by Carnell et al proposed a modified RBD spike subunit that triggers more broad neutralizing immune responses as a potentially improved booster vaccine [ 35 ]. Their methods were based on studies conducted with MERS and influenza viruses and involved removing and adding new glycan sites.…”