A workflow for rapid SARS-CoV-2 epitope discovery on peptide microarrays is herein reported. The process started with a proteome-wide screening of immunoreactivity based on the use of a high-density microarray followed by a refinement and validation phase on a restricted panel of probes using microarrays with tailored peptide immobilization through a click-based strategy. Progressively larger, independent cohorts of Covid-19 positive sera were tested in the refinement processes, leading to the identification of immunodominant regions on SARS-CoV-2 spike (S), nucleocapsid (N) protein and Orf1ab polyprotein. A summary study testing 50 serum samples highlighted an epitope of the N protein (region 155–71) providing good diagnostic performance in discriminating Covid-19 positive vs. healthy individuals. Using this epitope, 92% sensitivity and 100% specificity were reached for IgG detection in Covid-19 samples, and no cross-reactivity with common cold coronaviruses was detected. Likewise, IgM immunoreactivity in samples collected within the first month after symptoms onset showed discrimination ability. Overall, epitope 155–171 from N protein represents a promising candidate for further development and rapid implementation in serological tests.

Since the outbreak of the COVID-19 crisis, the handling of biological samples from confirmed or suspected SARS-CoV-2-positive individuals demanded the use of inactivation protocols to ensure laboratory operators’ safety. While not standardized, these practices can be roughly divided into two categories, namely heat inactivation and solvent-detergent treatments. These routine procedures should also apply to samples intended for Extracellular Vesicles (EVs) analysis. Assessing the impact of virus-inactivating pre-treatments is therefore of pivotal importance, given the well-known variability introduced by different pre-analytical steps on downstream EVs isolation and analysis. Arguably, shared guidelines on inactivation protocols tailored to best address EVs-specific requirements will be needed among the analytical community, yet deep investigations in this direction have not yet been reported. We here provide insights into SARS-CoV-2 inactivation practices to be adopted prior to serum EVs analysis by comparing solvent/detergent treatment vs. heat inactivation. Our analysis entails the evaluation of EVs recovery and purity along with biochemical, biophysical and biomolecular profiling by means of a set of complementary analytical techniques: Nanoparticle Tracking Analysis, Western Blotting, Atomic Force Microscopy, miRNA content (digital droplet PCR) and tetraspanin assessment by microarrays. Our data suggest an increase in ultracentrifugation (UC) recovery following heat treatment; however, it is accompanied by a marked enrichment in EVs-associated contaminants. On the other hand, solvent/detergent treatment is promising for small EVs (<150 nm range), yet a depletion of larger vesicular entities was detected. This work represents a first step towards the identification of optimal serum inactivation protocols targeted to EVs analysis.

The human parasitic disease Schistosomiasis is caused by the Schistosoma trematode flatworm that infects freshwaters in tropical regions of the world, particularly in Sub-Saharan Africa, South America, and the Far-East. It has also been observed as an emerging disease in Europe, due to increased immigration. In addition to improved therapeutic strategies, it is imperative to develop novel, rapid, and sensitive diagnostic tests that can detect the Schistosoma parasite, allowing timely treatment. Present diagnosis is difficult and involves microscopy-based detection of Schistosoma eggs in the feces. In this context, we present the 3.22 Å resolution crystal structure of the circulating antigen Serine protease inhibitor from S. mansoni (SmSPI), and we describe it as a potential serodiagnostic marker. Moreover, we identify three potential immunoreactive epitopes using in silico-based epitope mapping methods. Here, we confirm effective immune sera reactivity of the recombinant antigen, suggesting the further investigation of the protein and/or its predicted epitopes as serodiagnostic Schistosomiasis biomarkers.

Mesoscale interactions—such as biomolecular coronas, transient associations, aggregation, and fusion—are increasingly recognized for their biological significance and, under certain conditions, are essential for extracellular nanoparticles to fulfill their functions. Among these interactions, the binding between extracellular vesicles and lipoproteins has recently gained attention for its potential impact on extracellular vesicle function and fate in vivo. These interactions must be understood to clarify and possibly engineer and exploit the biological modes of action of EVs. A bottom-up simplified system consisting of red blood cell-derived extracellular vesicles and purified human lipoproteins was used to investigate extracellular vesicles-lipoprotein binding in saline buffer and in human plasma. A customized toolbox of orthogonal analytical techniques was developed to characterize these interactions at multiple scales, also using label-free materials, while preserving their natural binding states. This toolbox includes fluorescence cross-correlation spectroscopy, super-resolution microscopy, flow cytometry, and Single Molecule Array assay. Our findings reveal that lipoproteins bind to red blood cell EVs with affinities ranging from 10 nM to 1 µM. The percentage of individual extracellular vesicles interacting with lipoproteins is dependent on the specific lipoprotein class and the incubation conditions, and is always considerable, with up to 100% EV interacting with High Density Lipoproteins in the presence of plasma proteins. Such binding is stable, proving resistant also to several washing steps. Our data depict the EV – lipoprotein interaction as a generalizable phenomenon, which is shared among all the lipoprotein classes to different degrees. This implies that in physiological conditions, EVs may be constantly associated with a certain number of lipoproteins in the bloodstream, with possible impacts on EV surface identity and, therefore, function. This finding advances our understanding of extracellular nanoparticle interactome, and provides a step forward in deciphering the physicochemical foundations of biodistribution and clearance mechanisms of natural, synthetic, and hybrid nanoparticles.

To be profitably exploited in medicine, nanosized systems must be endowed with biocompatibility, targeting capability, the ability to evade the immune system, and resistance to clearance. Currently, biogenic nanoparticles, such as Extracellular Vesicles (EVs), are intensively investigated as the platform that naturally recapitulates these highly needed characteristics. EV native targeting properties and pharmacokinetics can be further augmented by decorating the EV surface with specific target ligands as antibodies. However, up to date, works dealing with the functionalization of EV surface with proteins have never considered the biomolecular corona (BC) “variable”, namely the fact that extrinsic biomolecules, mainly proteins, may spontaneously adsorb on the EV surface in biofluids, contributing to determine the biological identity of the EV. In this work, we explore and compare EVs modified with the antibody Cetuximab (CTX) by chemisorption (covalent binding of CTX via biorthogonal click-chemistry) and by physisorption (formation of a CTX corona). Results (surprisingly) indicate that (i) no differences exist between the two formulations in terms of binding affinity imparted by molecular recognition of CTX versus its natural binding partner (epidermal growth factor receptor, EGFR), but (ii) significative differences emerge at the cellular level, where CTX-EVs prepared by click chemistry display superior binding and uptake toward target cells in Fetal Bovine Serum (FBS)-supplied culture medium, very likely due to the higher robustness of the CTX anchorage that resists to the formation of a BC due to interaction with the FBS proteins.

Initially observed on synthetic nanoparticles, biomolecular corona existence and role in determining nanoparticle identity and function are now beginning to be acknowledged in biogenic nanoparticles, particularly in extracellular vesicles. We have developed here a methodology based on Fluorescence Correlation Spectroscopy to track biomolecular corona formation on extracellular vesicles derived from red blood cells and placental mesenchymal stromal cells when these vesicles are dispersed in human plasma. The methodology allows for the study of corona dynamics in situ in physiological conditions. Results evidence that the two extracellular vesicle populations feature distinct corona dynamics, with red blood cell-derived extracellular vesicles exchanging a higher number of proteins. These findings indicate that the dynamics of the biomolecular corona may ultimately be linked to the cellular origin of the extracellular vesicles, revealing an additional level of heterogeneity, and possibly of bionanoscale identity, that characterizes circulating extracellular vesicles.