Abstract
Determining how antibodies interact with the spike (S) protein of the SARS-CoV-2 virus is critical for combating COVID-19. Structural studies typically employ simplified, truncated constructs that may not fully recapitulate the behavior of the original complexes. Here, we combine two single particle mass analysis techniques (mass photometry and charge-detection mass spectrometry) to enable the measurement of full IgG binding to the trimeric SARS-CoV-2 S ectodomain. Our experiments reveal that antibodies targeting the S-trimer typically prefer stoichiometries lower than the symmetry-predicted 3:1 binding. We determine that this behavior arises from the interplay of steric clashes and avidity effects that are not reflected in common antibody constructs (i.e., Fabs). Surprisingly, these substoichiometric complexes are fully effective at blocking ACE2 binding despite containing free receptor binding sites. Our results highlight the importance of studying antibody/antigen interactions using complete, multimeric constructs and showcase the utility of single particle mass analyses in unraveling these complex interactions.
Highlights
The emergence of the SARS-CoV-2 coronavirus and subsequent onset of the coronavirus disease 2019 (COVID19) pandemic have necessitated the rapid development of vaccines and other treatments.[1−3] The primary focus of these countermeasures is the SARS-CoV-2 spike (S) protein present on the viral surface, which is responsible for initiating host infection via complexation to the human ACE2 receptor and subsequent fusion of the viral and host cell membranes.[4]
Introduced as interferometric scattering mass spectrometry,[38] mass photometry (MP) is a light scattering-based, label-free, mass analysis technique that determines the mass of a single particle in solution from its scattering intensity.[41]
We demonstrate here the unique application of two single particle approaches, MP and CD-mass spectrometry (MS), for interrogating the interaction stoichiometries between full Abs, the ACE2 receptor, and the SARS-CoV-2 S protein ectodomain
Summary
The emergence of the SARS-CoV-2 coronavirus and subsequent onset of the coronavirus disease 2019 (COVID19) pandemic have necessitated the rapid development of vaccines and other treatments.[1−3] The primary focus of these countermeasures is the SARS-CoV-2 spike (S) protein present on the viral surface, which is responsible for initiating host infection via complexation to the human ACE2 receptor and subsequent fusion of the viral and host cell membranes.[4]. Understanding how exactly antibodies (Abs) interact with the SARS-CoV-2 S protein is a crucial component for both continuing vaccine development as well as the rational design of target biotherapeutics (e.g., monoclonal Abs).[9,10]. Like the spike proteins of many other viruses, the SARSCoV-2 S protein is present in a trimeric, membrane-embedded state.[11] Effective neutralizing Abs for SARS-CoV-2 often target the receptor binding domain (RBD) of the S protein.[12−15] As the RBD is the site of initial ACE2 receptor binding, these Abs are thought to achieve neutralization largely by sterically preventing interactions between the S protein and host receptor.[16] Due to its trimeric nature, each individual spike contains three copies of the RBD
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