Structural properties of immune complexes formed by viral antigens and specific antibodies shape the inflammatory response of macrophages.

Data on the course of viral infections revealed severe inflammation as a consequence of antiviral immune response. Despite extensive research, there are insufficient data on the role of innate immune cells in promoting inflammation mediated by immune complexes (IC) of viral antigens and their specific antibodies. Recently, we demonstrated that antigens of human polyomaviruses (PyVs) induce an inflammatory response in macrophages. Here, we investigated macrophage activation by IC. We used primary murine macrophages as a cell model, virus-like particles (VLPs) of PyV capsid protein as antigens, and a collection of murine monoclonal antibodies (mAbs) of IgG1, IgG2a, IgG2b subclasses. The inflammatory response was investigated by analysing inflammatory chemokines and activation of NLRP3 inflammasome. We observed a diverse pattern of chemokine secretion in macrophages treated with different IC compared to VLPs alone. To link IC properties with cell activation status, we characterised the IC by advanced optical and acoustic techniques. Ellipsometry provided precise real-time kinetics of mAb-antigen interactions, while quartz crystal microbalance measurements showed changes in conformation and viscoelastic properties during IC formation. These results revealed differences in mAb-antigen interaction and mAb binding parameters of the investigated IC. We found that IC-mediated cell activation depends more on IC characteristics, including mAb affinity, than on mAb affinity for the activating Fc receptor. IC formed by the highest affinity mAb showed a significant enhancement of inflammasome activation. This may explain the hyperinflammation related to viral infection and vaccination. Our findings demonstrate that IC promote the viral antigen-induced inflammatory response depending on antibody properties.

Revealing the SARS-CoV-2 Spike Protein and Specific Antibody Immune Complex Formation Mechanism for Precise Evaluation of Antibody Affinity.

The profound understanding and detailed evaluation of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) spike (SCoV2-S) protein and specific antibody interaction mechanism is of high importance in the development of immunosensors for COVID-19. In the present work, we studied a model system of immobilized SCoV2-S protein and specific monoclonal antibodies by molecular dynamics of immune complex formation in real time. We simultaneously applied spectroscopic ellipsometry and quartz crystal microbalance with dissipation to reveal the features and steps of the immune complex formation. We showed direct experimental evidence based on acoustic and optical measurements that the immune complex between covalently immobilized SCoV2-S and specific monoclonal antibodies is formed in two stages. Based on these findings it was demonstrated that applying a two-step binding mathematical model for kinetics analysis leads to a more precise determination of interaction rate constants than that determined by the 1:1 Langmuir binding model. Our investigation showed that the equilibrium dissociation constants (KD) determined by a two-step binding model and the 1:1 Langmuir model could differ significantly. The reported findings can facilitate a deeper understanding of antigen-antibody immune complex formation steps and can open a new way for the evaluation of antibody affinity towards corresponding antigens.

Study of SARS-CoV-2 Spike Protein Wild-Type and the Variants of Concern Real-Time Interactions with Monoclonal Antibodies and Convalescent Human Serum.

The spike (S) protein and its receptor-binding domain (RBD) of the coronavirus SARS-CoV-2 have been continually evolving, yielding the majority of significant missense mutations and new variants of concern. In this study, we examined how monoclonal antibodies against RBD (mAbs-SCoV2-RBD) and polyclonal antibodies present in convalescent human serum specifically interact with the S protein of wild-type and SARS-CoV-2 variants of concern (VOCs) in real time and how this can be reflected through surface mass density. Moreover, we combined two distinct, label-free measurement techniques: one based on changes in surface electromagnetic waves after reflection from the surface, and the other on changes in acoustic waves. The results demonstrated that dry surface mass density (ΓSE) of mAbs-SCoV2-RBD attached to the RBD of the S protein decreases three-fold, from 148 ng/cm2 to 46 ng/cm2, due to the B.1.351 or so-called beta mutation of coronavirus and its S protein (SCoV2-ß). Consequently, the obtained wet mass ΓQCM-D resulted in values two times lower, from 319 ng/cm2 to 158 ng/cm2, and the hydration of mAbs-SCoV2-RBD/SCoV2-ß immune complex was 70.88%. Conversely, when polyclonal antibodies present in convalescent human serum form immune complexes with the S protein of SARS-CoV-2 variants of concern, the ΓSE decreased from 279 ng/cm2 to 249 ng/cm2, and ΓQCM-D from 1545 ng/cm2 to 1366 ng/cm2. These results can give insights into the differences between the interaction of monoclonal and polyclonal antibodies with SARS-CoV-2 VOCs.

Investigation of SARS-CoV-2 nucleocapsid protein interaction with a specific antibody by combined spectroscopic ellipsometry and quartz crystal microbalance with dissipation.

Detailed evaluations of the antigen and antibody interaction rate and strength of the immune complex formed are very important for medical and bioanalytical applications. These data are crucial for the development of sensitive and fast immunosensors suitable for continuous measurements. Therefore, combined spectroscopic ellipsometry (SE) and quartz crystal microbalance with dissipation (QCM-D) technique (SE/QCM-D) was used for the evaluation: (i)of covalent immobilization of SARS-CoV-2 nucleocapsid protein (SCoV2-N) on QCM-D sensor disc modified by self-assembled monolayer based on 11-mercaptoundecanoic acid and (ii)interaction of immobilized SCoV2-N with specific polyclonal anti-SCoV2-N antibodies followed by immune complex formation process. The results show that the SCoV2-N monolayer is rigid due to the low energy dissipation registered during the QCM-D measurement. In contrast, the anti-SCoV2-N layer produced after interaction with the immobilized SCoV2-N formed a soft and viscous layer. It was determined, that the sparse distribution of SCoV2-N on the surface affected the spatial arrangement of the antibody during the formation of immune complexes. The hinge-mediated flexibility of the antibody Fab fragments allows them to reach the more distantly located SCoV2-N and establish a bivalent binding between proteins in the formed SCoV2-N/anti-SCoV2-N complex. It was noted that the SE/QCM-D method can provide more precise quantitative information about the flexibility and conformational changes of antibody during the formation of the immune complex on the surface over time.

Investigation and Comparison of Specific Antibodies' Affinity Interaction with SARS-CoV-2 Wild-Type, B.1.1.7, and B.1.351 Spike Protein by Total Internal Reflection Ellipsometry.

SARS-CoV-2 vaccines provide strong protection against COVID-19. However, the emergence of SARS-CoV-2 variants has raised concerns about the efficacy of vaccines. In this study, we investigated the interactions of specific polyclonal human antibodies (pAb-SCoV2-S) produced after vaccination with the Vaxzevria vaccine with the spike proteins of three SARS-CoV-2 variants of concern: wild-type, B.1.1.7, and B.1.351. Highly sensitive, label-free, and real-time monitoring of these interactions was accomplished using the total internal reflection ellipsometry method. Thermodynamic parameters such as association and dissociation rate constants, the stable immune complex formation rate constant (kr), the equilibrium association and dissociation (KD) constants and steric factors (Ps) were calculated using a two-step irreversible binding mathematical model. The results obtained show that the KD values for the specific antibody interactions with all three types of spike protein are in the same nanomolar range. The KD values for B.1.1.7 and B.1.351 suggest that the antibody produced after vaccination can successfully protect the population from the alpha (B.1.1.7) and beta (B.1.351) SARS-CoV-2 mutations. The steric factors (Ps) obtained for all three types of spike proteins showed a 100-fold lower requirement for the formation of an immune complex when compared with nucleocapsid protein.