Qualitative monitoring of SARS-CoV-2 mRNA vaccination in humans using droplet microfluidics.

SARS-CoV-2 mRNA vaccination generates protective B cell responses targeting the SARS-CoV-2 spike glycoprotein. Whereas anti-spike memory B cell responses are long lasting, the anti-spike humoral antibody response progressively wanes, making booster vaccinations necessary for maintaining protective immunity. Here, we qualitatively investigated the plasmablast responses by measuring from single cells within hours of sampling the affinity of their secreted antibody for the SARS-CoV-2 spike receptor binding domain (RBD) in cohorts of BNT162b2-vaccinated naive and COVID-19-recovered individuals. Using a droplet microfluidic and imaging approach, we analyzed more than 4,000 single IgG-secreting cells, revealing high interindividual variability in affinity for RBD, with variations over 4 logs. High-affinity plasmablasts were induced by BNT162b2 vaccination against Hu-1 and Omicron RBD but disappeared quickly thereafter, whereas low-affinity plasmablasts represented more than 65% of the plasmablast response at all time points. Our droplet-based method thus proves efficient at fast and qualitative immune monitoring and should be helpful for optimization of vaccination protocols.

High-affinity autoreactive plasma cells disseminate through multiple organs in patients with immune thrombocytopenic purpura.

The major therapeutic goal for immune thrombocytopenic purpura (ITP) is to restore normal platelet counts using drugs to promote platelet production or by interfering with mechanisms responsible for platelet destruction. Eighty percent of patients with ITP possess anti-integrin αIIbß3 IgG autoantibodies that cause platelet opsonization and phagocytosis. The spleen is considered the primary site of autoantibody production by autoreactive B cells and platelet destruction. The immediate failure in approximately 50% of patients to recover a normal platelet count after anti-CD20 rituximab-mediated B cell depletion and splenectomy suggests that autoreactive, rituximab-resistant, IgG-secreting B cells (IgG-SCs) reside in other anatomical compartments. We analyzed more than 3,300 single IgG-SCs from spleen, bone marrow, and/or blood of 27 patients with ITP, revealing high interindividual variability in affinity for αIIbß3, with variations over 3 logs. IgG-SC dissemination and range of affinities were, however, similar for each patient. Longitudinal analysis of autoreactive IgG-SCs upon treatment with the anti-CD38 mAb daratumumab demonstrated variable outcomes, from complete remission to failure with persistence of high-affinity anti-αIIbß3 IgG-SCs in the bone marrow. This study demonstrates the existence and dissemination of high-affinity autoreactive plasma cells in multiple anatomical compartments of patients with ITP that may cause the failure of current therapies.

Deep phenotypic characterization of immunization-induced antibacterial IgG repertoires in mice using a single-antibody bioassay.

Antibodies with antibacterial activity need to bind to the bacterial surface with affinity, specificity, and sufficient density to induce efficient elimination. To characterize the anti-bacterial antibody repertoire, we developed an in-droplet bioassay with single-antibody resolution. The assay not only allowed us to identify whether the secreted antibodies recognized a bacterial surface antigen, but also to estimate the apparent dissociation constant (KD app) of the interaction and the density of the recognized epitope on the bacteria. Herein, we found substantial differences within the KD app/epitope density profiles in mice immunized with various species of heat-killed bacteria. The experiments further revealed a high cross-reactivity of the secreted IgG repertoires, binding to even unrelated bacteria with high affinity. This application confirmed the ability to quantify the anti-bacterial antibody repertoire and the utility of the developed bioassay to study the interplay between bacteria and the humoral response.

Dynamic single-cell phenotyping of immune cells using the microfluidic platform DropMap.

Characterization of immune responses is currently hampered by the lack of systems enabling quantitative and dynamic phenotypic characterization of individual cells and, in particular, analysis of secreted proteins such as cytokines and antibodies. We recently developed a simple and robust microfluidic platform, DropMap, to measure simultaneously the kinetics of secretion and other cellular characteristics, including endocytosis activity, viability and expression of cell-surface markers, from tens of thousands of single immune cells. Single cells are compartmentalized in 50-pL droplets and analyzed using fluorescence microscopy combined with an immunoassay based on fluorescence relocation to paramagnetic nanoparticles aligned to form beadlines in a magnetic field. The protocol typically takes 8-10 h after preparation of microfluidic chips and chambers, which can be done in advance. By contrast, enzyme-linked immunospot (ELISPOT), flow cytometry, time-of-flight mass cytometry (CyTOF), and single-cell sequencing enable only end-point measurements and do not enable direct, quantitative measurement of secreted proteins. We illustrate how this system can be used to profile downregulation of tumor necrosis factor-α (TNF-α) secretion by single monocytes in septic shock patients, to study immune responses by measuring rates of cytokine secretion from single T cells, and to measure affinity of antibodies secreted by single B cells.

The Quantitative Assessment of the Secreted IgG Repertoire after Recall to Evaluate the Quality of Immunizations.

One of the major goals of vaccination is to prepare the body to rapidly secrete specific Abs during an infection. Assessment of the vaccine quality is often difficult to perform, as simple measurements like Ab titer only partly correlate with protection. Similarly, these simple measurements are not always sensitive to changes in the preceding immunization scheme. Therefore, we introduce in this paper a new, to our knowledge, method to assay the quality of immunization schemes for mice: shortly after a recall with pure Ag, we analyze the frequencies of IgG-secreting cells (IgG-SCs) in the spleen, as well as for each cells, the Ag affinity of the secreted Abs. We observed that after recall, appearance of the IgG-SCs within the spleen of immunized mice was fast (<24 h) and this early response was free of naive IgG-SCs. We further confirmed that our phenotypic analysis of IgG-SCs after recall strongly correlated with the different employed immunization schemes. Additionally, a phenotypic comparison of IgG-SCs presented in the spleen during immunization or after recall revealed similarities but also significant differences. The developed approach introduced a novel (to our knowledge), quantitative, and functional highly resolved alternative to study the quality of immunizations.

Adaptive eyeglasses for presbyopia correction: an original variable-focus technology.

We propose an original variable-focus technology specially designed for presbyopia-correcting adaptive eyeglasses. It has been thought through to offer vision comfort without cutting on aesthetics. It relies on a fluid-filled variable-focus lens (presenting 2 liquids and 1 ultra-thin membrane) assisted by a low-power, high-volume microfluidic actuator. It also features a distance-sensing system to provide automatic focusing. We demonstrate the qualities of this novel technology on our first prototype. Our prototype achieves the necessary 3-diopter-high power variation on a 20-millimeter-wide variable zone with low actuation pressures (~200 Pa at most), and the preliminary optical quality analysis shows the spatial resolution is much better than the one specified by classic eye charts. We discuss further improvements in terms of optics, aesthetics and portability. In particular, we point out that this variable technology is compatible with standard base curves, and we highlight an optimal configuration where the power consumption of our opto-fluidic engine is about 25 mW peak.