Delicate balance among thermal stability, binding affinity, and conformational space explored by single-domain VHH antibodies.

The high binding affinities and specificities of antibodies have led to their use as drugs and biosensors. Single-domain VHH antibodies exhibit high specificity and affinity but have higher stability and solubility than conventional antibodies as they are single-domain proteins. In this work, based on physicochemical measurements and molecular dynamics (MD) simulations, we have gained insight that will facilitate rational design of single-chain VHH antibodies. We first assessed two homologous VHH antibodies by differential scanning calorimetry (DSC); one had a high (64.8 °C) and the other a low (58.6 °C) melting temperature. We then generated a series of the variants of the low stability antibody and analyzed their thermal stabilities by DSC and characterized their structures through MD simulations. We found that a single mutation that resulted in 8.2 °C improvement in melting temperature resulted in binding affinity an order of magnitude lower than the parent antibody, likely due to a shift of conformational space explored by the single-chain VHH antibody. These results suggest that the delicate balance among conformational stability, binding capability, and conformational space explored by antibodies must be considered in design of fully functional single-chain VHH antibodies.

Placental syncytium forms a biophysical barrier against pathogen invasion.

Fetal syncytiotrophoblasts form a unique fused multinuclear surface that is bathed in maternal blood, and constitutes the main interface between fetus and mother. Syncytiotrophoblasts are exposed to pathogens circulating in maternal blood, and appear to have unique resistance mechanisms against microbial invasion. These are due in part to the lack of intercellular junctions and their receptors, the Achilles heel of polarized mononuclear epithelia. However, the syncytium is immune to receptor-independent invasion as well, suggesting additional general defense mechanisms against infection. The difficulty of maintaining and manipulating primary human syncytiotrophoblasts in culture makes it challenging to investigate the cellular and molecular basis of host defenses in this unique tissue. Here we present a novel system to study placental pathogenesis using murine trophoblast stem cells (mTSC) that can be differentiated into syncytiotrophoblasts and recapitulate human placental syncytium. Consistent with previous results in primary human organ cultures, murine syncytiotrophoblasts were found to be resistant to infection with Listeria monocytogenes via direct invasion and cell-to-cell spread. Atomic force microscopy of murine syncytiotrophoblasts demonstrated that these cells have a greater elastic modulus than mononuclear trophoblasts. Disruption of the unusually dense actin structure--a diffuse meshwork of microfilaments--with Cytochalasin D led to a decrease in its elastic modulus by 25%. This correlated with a small but significant increase in invasion of L. monocytogenes into murine and human syncytium. These results suggest that the syncytial actin cytoskeleton may form a general barrier against pathogen entry in humans and mice. Moreover, murine TSCs are a genetically tractable model system for the investigation of specific pathways in syncytial host defenses.

T cell responses to antigen: hasty proposals resolved through long engagements.

T cells discriminate between peptide-MHC complexes on the surfaces of antigen presenting cells to enact appropriate downstream responses. Great progress has been made over the last 15 years in understanding varied aspects of T cell activation on short timescales (minutes), yet the mechanics and significance of long term T cell receptor signaling (hours or days) remain unclear. Furthermore, there remain some controversies regarding the correlation of the biophysical parameters of ligand-receptor interactions with the scaling of downstream effector functions. Here we review recent studies that emphasize the importance of long-term engagement of antigens to fine-tuning the activation of T cells over the duration of the complete immune response. We discuss how T cells dynamically regulate T cell receptor signaling via antigen crosstalk, competition and consumption to accurately counter antigenic challenges.

Blurred line between chemotactic chase and phagocytic consumption: an immunophysical single-cell perspective.

An innate immune cell can sense a pathogen, either from a distance by recognizing chemoattractant stimuli or by direct physical contact. The pathogen is subsequently neutralized, which usually occurs through its phagocytic internalization. By investigating chemotaxis and phagocytosis from an immunophysical single-cell perspective, it now appears that the demarcation between these two processes is less distinct than originally thought. Several lines of evidence support this notion. First, chemotactic stimulation does not cease at the moment of initial contact between the cell and the pathogenic target. Second, even when classical chemotaxis of neutrophils is suppressed, the early cell response to contact with typical chemoattractant targets, such as zymosan, fungal spores or chemokine-coated particles, can still involve morphological attributes of chemotaxis. Recognizing that the changing morphology of motile cells is inextricably linked to physical cell behavior, this Commentary focuses on the mechanical aspects of the early response of innate immune cells to chemotactic and phagocytic stimuli. On the basis of this perspective, we propose that the combined study of chemotaxis and phagocytosis will, potentially, not only advance our grasp of the mechanisms underlying immune-cell motility but also open new lines of research that will promote a deeper understanding of the innate recognition of pathogens.

Comparison of the results obtained by ELISA and surface plasmon resonance for the determination of antibody affinity.

The aim of this study was to compare the affinity values obtained for a monoclonal antibody/antigen complex using two different techniques, surface plasmon resonance (SPR) and an enzyme linked immunosorbent assay (ELISA) approach recently described by Bobrovnik S.A. and by Stevens F.J. These two techniques can be used in particular to determine the equilibrium dissociation constant, K(D), of the complex in solution or on a surface. Bobrovnik's method gives two K(D) values that differ by a factor of 100, demonstrating that two populations of complexes are present in solution. In an initial step, one protein binds relatively weakly to the other (high K(D)) and this is followed by a conformational change in the most flexible portion of the antigen, which increases the affinity (low K(D)). Only the higher of the two K(D) values can be detected when complex formation in solution is investigated using SPR, because the interaction measured concerns the fibronectin/antibody complexes of lowest affinity. In contrast, when measuring association at the sensor surface, SPR gives an average result between the two K(D) values because complexes corresponding to both affinities can form in this situation. The constants that characterise the kinetics of the fibronectin-antibody interaction obtained by SPR and ELISA are therefore different, because the methods do not allow the same phenomena to be observed. However they are consistent and complementary.