A Cellular Assay for Spike/ACE2 Fusion: Quantification of Fusion-Inhibitory Antibodies after COVID-19 and Vaccination.

Not all antibodies against SARS-CoV-2 inhibit viral entry, and hence, infection. Neutralizing antibodies are more likely to reflect real immunity; however, certain tests investigate protein/protein interaction rather than the fusion event. Viral and pseudoviral entry assays detect functionally active antibodies but are limited by biosafety and standardization issues. We have developed a Spike/ACE2-dependent fusion assay, based on a split luciferase. Hela cells stably transduced with Spike and a large fragment of luciferase were co-cultured with Hela cells transduced with ACE2 and the complementary small fragment of luciferase. Cell fusion occurred rapidly allowing the measurement of luminescence. Light emission was abolished in the absence of Spike and reduced in the presence of proteases. Sera from COVID-19-negative, non-vaccinated individuals or from patients at the moment of first symptoms did not lead to a significant reduction of fusion. Sera from COVID-19-positive patients as well as from vaccinated individuals reduced the fusion. This assay was more correlated to pseudotyped-based entry assay rather than serology or competitive ELISA. In conclusion, we report a new method measuring fusion-inhibitory antibodies in serum, combining the advantage of a complete Spike/ACE2 interaction active on entry with a high degree of standardization, easily allowing automation in a standard bio-safety environment.

Alpha-1 Antitrypsin Reduces Disease Progression in a Mouse Model of Charcot-Marie-Tooth Type 1A: A Role for Decreased Inflammation and ADAM-17 Inhibition.

Charcot-Marie-Tooth disease type 1 (CMT1A) is a hereditary peripheral neuropathy for which there is no available therapy. Alpha-1 antitrypsin (AAT) is an abundant serine protease inhibitor with anti-inflammatory and immunomodulating properties. Here, we tested whether treatment with human AAT (hAAT) would have a therapeutic effect on CMT1A in a PMP22 transgenic mouse model. Our results show that hAAT significantly improved compound muscle action potential and histopathological features and decreased circulating IL-6 in CMT1A mice. We also investigated some of the possible underlying mechanisms in vitro. We confirmed that hAAT inhibits ADAM-17, a protease that has been implicated in blocking myelination. Furthermore, both hAAT and recombinant human AAT (rhAAT) were able to attenuate the activation of a macrophage/microglia cell line, markedly decreasing the activation of the MHC class II promoter and the expression of pro-inflammatory genes such as IL-1ß and the endoplasmic reticulum (ER) stress marker ATF3. Taken together, our results demonstrate for the first time that hAAT is able to reduce the progression of CMT1A, possibly by dampening inflammation and by regulating ADAM-17. Given the already well-established safety profile of hAAT, specifically in AAT deficiency disease (AATD), we suggest that the findings of our study should be promptly investigated in CMT1A patients.

Proteomics, nanotechnology and molecular diagnostics.

Sequencing of the human genome opened the way to the exploration of the proteome and this has lead to the identification of large numbers of proteins in complex biological samples. The identification of diagnostic patterns in samples taken from patients to aid diagnosis is in the early stages of development. The solution to many of the technical challenges in proteomics and protein based molecular diagnostics will be found in new applications of nanomaterials. This review describes some of the physical and chemical principles underlying nanomaterials and devices and outlines how they can be used in proteomics; developments which are establishing nanoproteomics as a new field. Nanoproteomics will provide the platform for the discovery of next generation biomarkers. The field of molecular diagnostics will then come of age.