Antibodies to neutralising epitopes synergistically block the interaction of the receptor‐binding domain of SARS‐CoV‐2 to ACE 2

Abstract

Objectives A major COVID‐19 vaccine strategy is to induce antibodies that prevent interaction between the Spike protein's receptor‐binding domain (RBD) and angiotensin‐converting enzyme 2 (ACE2). These vaccines will also induce T‐cell responses. However, concerns were raised that aberrant vaccine‐induced immune responses may exacerbate disease. We aimed to identify minimal epitopes on the RBD that would induce antibody responses that block the interaction of the RBD and ACE2 as a strategy leading to an effective vaccine with reduced risk of inducing immunopathology. Methods We procured a series of overlapping 20‐amino acid peptides spanning the RBD and asked which were recognised by plasma from COVID‐19 convalescent patients. Identified epitopes were conjugated to diphtheria‐toxoid and used to vaccinate mice. Immune sera were tested for binding to the RBD and for their ability to block the interaction of the RBD and ACE2. Results Seven putative vaccine epitopes were identified. Memory B‐cells (MBCs) specific for one of the epitopes were identified in the blood of convalescent patients. When used to vaccinate mice, six induced antibodies that bound recRBD and three induced antibodies that could partially block the interaction of the RBD and ACE2. However, when the sera were combined in pairs, we observed significantly enhanced inhibition of binding of RBD to ACE2. Two of the peptides were located in the main regions of the RBD known to contact ACE2. Of significant importance to vaccine development, two of the peptides were in regions that are invariant in the UK and South African strains. Conclusion COVID‐19 convalescent patients have SARS‐CoV‐2‐specific antibodies and MBCs, the specificities of which can be defined with short peptides. Epitope‐specific antibodies synergistically block RBD–ACE2 interaction.

Chemical Conjugation Strategies for the Development of Protein-Based Subunit Nanovaccines

“…However, the vaccine elicited much higher IgG titers against DT than for J8 in clinical trials, showing the risk associated with the use of protein carrier systems [146]. In addition, DT-based conjugates have been used for screening SARS-CoV-2 spike protein-derived peptide epitopes for the development of a potential peptide-based SARS vaccine by the same group [147].…”

Detection and Quantification of SARS-CoV-2 Receptor Binding Domain Neutralization by a Sensitive Competitive ELISA Assay

“…Human IgG-Fc-conjugated ACE2 (hFc-ACE2) was applied, followed by 2 ry Abs and substrate. This strategy is similar to our approach (Figure 1D) [8,25], although the initial anti-His coating step is not included in our strategy since RBD or S-protein binds well to ACE2 when coated directly onto the plate [8]; however, no additional controls were employed, in strategy (C), to determine the amount of indirectly bound S-protein. Assays that use anti-His coating require extra time and effort due to addition of competitor ACE2 protein, which is generally longer than standard ELISA.…”

“…Currently, the most common cELISA strategy [6,7], which has also been reported by our group [8], involves allowing RBD-or S-protein-coated plates to interact with immune sera, i.e., become neutralized. Thereafter, hFc-ACE2 (or biotinylated-ACE2, for testing human sera) is added, followed by anti-hFc 2 ry Ab-HRP (or streptavidin-HRP for human serum) and substrate.…”

“…To fast-track the development of effective vaccines against SARS-CoV-2, surrogate high-throughput efficacy tests are urgently needed. Neutralization efficacy screening of vaccine candidates can be expedited by abstracting/distilling neutralization evaluation into a binding study between ACE2 and S-protein, or its RBD domain, in the presence or absence of vaccinated animal or human immune serum [6][7][8].…”

“…Binding is then detected by a chromogen-labeled secondary (2 ry ) Ab/probe [9][10][11]. ELISA is not only used to evaluate antigen-specific immune responses, it is also used to study binding affinity and kinetics, singularly or competitively, to extract meaningful binding values, e.g., the binding dissociation constant (K D ) or binding extent [6][7][8].…”