Summary
The surge of SARS‐CoV‐2 has challenged health systems worldwide and efficient tests to detect viral particles, as well as antibodies generated against them, are needed. Specificity, ...sensitivity, promptness or scalability are the main parameters to estimate the final performance, but rarely all of them match in a single test. We have developed SCOVAM, a protein microarray with several viral antigens (spike, nucleocapsid, main protease Nsp5) as capturing probes in a fluorescence immunoassay for COVID‐19 serological testing. SCOVAM depicts IgG and IgM antibody responses against each of these proteins of 22 individuals in a single microscope slide. It detects specific IgM (0.094 μg ml‐1) and IgG (~0.017 μg ml‐1) and is scalable and cost‐effective. We validated SCOVAM by comparing with a widely used chemiluminescent commercial serological test (n = 742). SCOVAM showed twice the sensitivity and allowed following seroconversion in a single assay. By analysing the prevalence 4 months later in a subset of 76 positive sera, we still detected 93.42% of positives, almost doubling the detection of the commercial assay. The higher sensitivity of SCOVAM is especially relevant to screen sera for convalescent plasma‐based treatments, high‐throughput antibody response monitoring after vaccination or evaluation of vaccine efficiency.
We have developed a test to discriminate antibodies against SARS‐CoV‐2 in the blood serum. It detects IgM and IgG antibodies against the Spike, Nucleocapsid and Main protease of the virus, simultaneously. We printed these viral proteins on slides forming microarrays and incubated them with human sera for fluorescent detection. We established the limit of detection and an algorithm for automated diagnostics. Our test resulted twice as sensitive as a commercial test, being able to better follow antibody response through time, what will be very useful to track the epidemiology of COVID‐19 and the effectivity of the developed vaccines.
Recent computational advancements in the simulation of biochemical processes allow investigating the mechanisms involved in protein regulation with realistic physics-based models, at an atomistic ...level of resolution. These techniques allowed us to design a drug discovery approach, named Pharmacological Protein Inactivation by Folding Intermediate Targeting (PPI-FIT), based on the rationale of negatively regulating protein levels by targeting folding intermediates. Here, PPI-FIT was tested for the first time on the cellular prion protein (PrP), a cell surface glycoprotein playing a key role in fatal and transmissible neurodegenerative pathologies known as prion diseases. We predicted the all-atom structure of an intermediate appearing along the folding pathway of PrP and identified four different small molecule ligands for this conformer, all capable of selectively lowering the load of the protein by promoting its degradation. Our data support the notion that the level of target proteins could be modulated by acting on their folding pathways, implying a previously unappreciated role for folding intermediates in the biological regulation of protein expression.
Deformed templating is the process by which self-replicating protein conformations with a given cross-β folding pattern can seed formation of an alternative self-replicating state with different ...cross-β folding pattern. In particular, uninfectious but propagative PrP amyloid can transform into a
bona fide
infectious conformer, PrP
Sc
through deformed templating. The process can take many rounds of replication (if taking place
in vitro
) or even several passages of the evolving PrP conformers through successive brains if
in vivo
, through experimental transmission. In all cases, deformed templating involves a forced conversion in which there is a mismatch between the template and the substrate and/or the templating environment, typically a recombinant PrP amyloid, adept at converting recombinant PrP under denaturing conditions (e.g., presence of chaotropic agents), encountering a glycosylated, GPI-anchored PrP
C
substrate under physiological conversion conditions. Deformed templating is characterized by emergence of intermediate conformers that exhibit biochemical characteristics that are intermediate between those of the initial PrP amyloid and the final PrP
Sc
conformers. Here, we took advantage of the recent elucidation of the structure of a PrP amyloid by cryo-EM and the availability of a physically plausible atomistic model of PrP
Sc
that we have recently proposed. Using modeling and Molecular Dynamics (MD) approaches, we built a complete molecular modelization of deformed templating, including an atomistic model of a glycosylated intermediate conformer and a modified model of PrP
Sc
. Among other unanticipated outcomes, our results show that fully glycosylated PrP can be stacked in-register, and how 4-rung β-solenoid (4RβS) PrP architectures can share key structural motifs with parallel-in register intermolecular sheet (PIRIBS) PrP amyloids. Our results shed light on the mechanisms of prion replication.
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