Dr. Leonardo Alonso from Argentina, with support from Instruct-ERIC and EMBL Hamburg, led a project to explore the effect of deamidation at position N481 in the SARS-CoV-2 Spike protein’s Receptor-Binding Domain (RBD) on the binding of neutralizing antibodies. SARS-CoV-2’s capacity to evade immune responses and vaccine-induced protection is a critical challenge, and this project aimed to examine whether deamidation—a spontaneous post-translational modification—could contribute to antigenic drift and affect immune recognition.
The team used insect cell-produced RBD, which was allowed to age naturally to induce deamidation at N481. They also created a specific N481D mutant, replacing the neutral asparagine with a charged aspartic acid to mimic the deamidation process in a controlled manner. After protein purification via IMAC and SEC methods, the samples were analyzed at the EMBL Hamburg facility using biophysical tools such as OCTET to measure affinity constants.
Key findings included:
- There were no significant differences in binding affinity between the wild-type RBD and the N481D mutant with the hACE2 receptor, indicating that N481 is not directly involved in ACE2 binding.
- The affinity constants for two therapeutic antibodies, Imdevimab and Casirivimab, which target non-overlapping regions of the RBD, also showed no substantial differences between the wild-type and N481D mutant RBDs. However, deamidation slightly decreased the binding affinity of Imdevimab in naturally aged RBD samples, suggesting a potential role in immune evasion over time.
- Interestingly, reduction of disulfide bonds in the RBD led to a stronger effect on antibody binding than deamidation, hinting at the importance of maintaining protein structure for efficient antibody interaction.
The study highlights deamidation as a subtle but potentially significant factor in antigenic drift and immune escape, though it may not dramatically affect direct receptor binding. Future steps will focus on expanding the analysis to other regions of the Spike protein, using molecular dynamics simulations to predict structural changes caused by deamidation, and exploring the broader implications for viral entry and vaccine efficacy.