Measurement of Antibody Binding Affinity on Bacterial Surfaces Using Flow Cytometry.

Antibody binding to bacterial surfaces plays a crucial role in immunity, and a key characteristic of this protein-protein interaction is the binding affinity. Determining the affinity of an antibody binding to its antigen is the first step in predicting the function in a physiological environment where other competing protein interactions may be present. Antibody-antigen affinity is often evaluated with isolated proteins. It is informative to also be able to assess antibody binding to a bacterial surface where many antigens might be present, including multiple copies of the specific antigen the antibody recognizes, and in a context where the antigen might be in a more natural conformation. In this chapter, we present a flow cytometry-based assay to measure and calculate the cell surface binding affinity or avidity of any mono- or polyclonal antibody solution.

A human monoclonal antibody bivalently binding two different epitopes in streptococcal M protein mediates immune function.

Group A streptococci have evolved multiple strategies to evade human antibodies, making it challenging to create effective vaccines or antibody treatments. Here, we have generated antibodies derived from the memory B cells of an individual who had successfully cleared a group A streptococcal infection. The antibodies bind with high affinity in the central region of the surface-bound M protein. Such antibodies are typically non-opsonic. However, one antibody could effectively promote vital immune functions, including phagocytosis and in vivo protection. Remarkably, this antibody primarily interacts through a bivalent dual-Fab cis mode, where the Fabs bind to two distinct epitopes in the M protein. The dual-Fab cis-binding phenomenon is conserved across different groups of M types. In contrast, other antibodies binding with normal single-Fab mode to the same region cannot bypass the M protein's virulent effects. A broadly binding, protective monoclonal antibody could be a candidate for anti-streptococcal therapy. Our findings highlight the concept of dual-Fab cis binding as a means to access conserved, and normally non-opsonic regions, regions for protective antibody targeting.

Invasive Streptococcal Infection Can Lead to the Generation of Cross-Strain Opsonic Antibodies.

The human pathogen Streptococcus pyogenes causes substantial morbidity and mortality. It is unclear if antibodies developed after infections with this pathogen are opsonic and if they are strain specific or more broadly protective. Here, we quantified the opsonic-antibody response following invasive S. pyogenes infection. Four patients with S. pyogenes bacteremia between 2018 and 2020 at Skåne University Hospital in Lund, Sweden, were prospectively enrolled. Acute- and convalescent-phase sera were obtained, and the S. pyogenes isolates were genome sequenced (emm118, emm85, and two emm1 isolates). Quantitative antibody binding and phagocytosis assays were used to evaluate isolate-dependent opsonic antibody function in response to infection. Antibody binding increased modestly against the infecting isolate and across emm types in convalescent- compared to acute-phase sera for all patients. For two patients, phagocytosis increased in convalescent-phase serum both for the infecting isolate and across types. The increase was only across types for one patient, and one had no improvement. No correlation to the clinical outcomes was observed. Invasive S. pyogenes infections result in a modestly increased antibody binding with differential opsonic capacity, both nonfunctional binding and broadly opsonic binding across types. These findings question the dogma that an invasive infection should lead to a strong type-specific antibody increase rather than a more modest but broadly reactive response, as seen in these patients. Furthermore, our results indicate that an increase in antibody titers might not be indicative of an opsonic response and highlight the importance of evaluating antibody function in S. pyogenes infections. IMPORTANCE The bacterium Streptococcus pyogenes is a common cause of both mild and severe human diseases resulting in substantial morbidity and mortality each year. No vaccines are available, and our understanding of the antibody response to this human pathogen is still incomplete. Here, we carefully analyzed the opsonic antibody response following invasive infection in four patients. Unexpectedly, the patients did not always generate opsonic antibodies against the specific infecting strain. Instead, we found that some patients could generate cross-opsonic antibodies, leading to phagocytosis of bacteria across strains. The emergence of cross-opsonic antibodies is likely important for long-term immunity against S. pyogenes. Our findings question the dogma that mostly strain-specific immunity is developed after infection and add to our overall understanding of how immunity to S. pyogenes can evolve.

Nanoscale binding site localization by molecular distance estimation on native cell surfaces using topological image averaging.

Antibody binding to cell surface proteins plays a crucial role in immunity, and the location of an epitope can altogether determine the immunological outcome of a host-target interaction. Techniques available today for epitope identification are costly, time-consuming, and unsuited for high-throughput analysis. Fast and efficient screening of epitope location can be useful for the development of therapeutic monoclonal antibodies and vaccines. Cellular morphology typically varies, and antibodies often bind heterogeneously across a cell surface, making traditional particle-averaging strategies challenging for accurate native antibody localization. In the present work, we have developed a method, SiteLoc, for imaging-based molecular localization on cellular surface proteins. Nanometer-scale resolution is achieved through localization in one dimension, namely, the distance from a bound ligand to a reference surface. This is done by using topological image averaging. Our results show that this method is well suited for antibody binding site measurements on native cell surface morphology and that it can be applied to other molecular distance estimations as well.

Antibodies play a key role in the immune system. These proteins stick to harmful substances, such as bacteria and other disease-causing pathogens, marking them for destruction or blocking their attack. Antibodies are highly selective, and this ability has been used to target particular molecules in research, diagnostics and therapies. Typically, antibodies need to stick to a particular segment, or 'epitope', on the surface of a cell in order to trigger an immune response. Knowing where these regions are can help explain how these immune proteins work and aid the development of more effective drugs and diagnostic tools. One way to identify these sites is to measure the nano-distance between antibodies and other features on the cell surface. To do this, researchers take multiple images of the cell the antibody is attached to using light microscopy. Various statistical methods are then applied to create an 'average image' that has a higher resolution and can therefore be used to measure the distance between these two points more accurately. While this approach works on fixed shapes, like a perfect circle, it cannot handle human cells and bacteria which are less uniform and have more complex surfaces. Here, Kumra Ahnlide et al. have developed a new method called SiteLoc which can overcome this barrier. The method involves two fluorescent probes: one attached to a specific site on the cell's surface, and the other to the antibody or another molecule of interest. These two probes emit different colours when imaged with a fluorescent microscope. To cope with objects that have uneven surfaces, such as cells and bacteria, the two signals are transformed to 'follow' the same geometrical shape. The relative distance between them is then measured using statistical methods. Using this approach, Kumra Ahnlide et al. were able to identify epitopes on a bacterium, and measure distances on the surface of human red blood cells. The SiteLoc system could make it easier to develop antibody-based treatments and diagnostic tools. Furthermore, it could also be beneficial to the wider research community who could use it to probe other questions that require measuring nanoscale distances.

A Predictive Model of Antibody Binding in the Presence of IgG-Interacting Bacterial Surface Proteins.

Many bacteria can interfere with how antibodies bind to their surfaces. This bacterial antibody targeting makes it challenging to predict the immunological function of bacteria-associated antibodies. The M and M-like proteins of group A streptococci (GAS) exhibit IgGFc-binding regions, which they use to reverse IgG binding orientation depending on the host environment. Unraveling the mechanism behind these binding characteristics may identify conditions under which bound IgG can drive an efficient immune response. Here, we have developed a biophysical model for describing these complex protein-antibody interactions. We show how the model can be used as a tool for studying the binding behavior of various IgG samples to M protein by performing in silico simulations and correlating this data with experimental measurements. Besides its use for mechanistic understanding, this model could potentially be used as a tool to aid in the development of antibody treatments. We illustrate this by simulating how IgG binding to GAS in serum is altered as specified amounts of monoclonal or pooled IgG is added. Phagocytosis experiments link this altered antibody binding to a physiological function and demonstrate that it is possible to predict the effect of an IgG treatment with our model. Our study gives a mechanistic understanding of bacterial antibody targeting and provides a tool for predicting the effect of antibody treatments in the presence of bacteria with IgG-modulating surface proteins.